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limb muscle dysfunction has important clinical and prognostic consequences in chronic obstructive pulmonary disease ( copd ) . in fact , reduced mass and weakness of the quadriceps have been associated with reduced survival in this disease.13 muscle strength is the most commonly assessed skeletal muscle function in copd . however , muscle strength represents only one aspect of muscle function , mostly reflecting the action of fast - twitch muscle fibers . muscle endurance is defined as the ability to maintain a given task over a certain period of time , and it is determined by muscle fiber oxidative capacity , and the oxygen supply.4 in copd , muscle endurance is impaired to a greater extent than strength.5,6 impaired muscle endurance may even be present in patients with mild disease and preserved physical activity,7,8 and can not be predicted from strength.7 despite its relevance to activities of daily living such as walking,9,10 muscle endurance is generally not an outcome in copd clinical trials.11 one important methodological issue with the measurement of muscle endurance is the fact that there is no standardization or agreement on a testing protocol that could be used in clinical practice or research . it can be determined with various methodologies , amongst which isokinetic testing is one of the most reliable methods.12 the main advantage of this method is that it provides a dynamic measurement of muscle torque while controlling for angular velocities , amplitude , and duration of movement in such a way that the testing conditions could be easily reproduced in subsequent testing.13 as with any other muscle function assessment , isokinetic endurance is required to be accurate and reliable . the reliability of isokinetic testing has been reported for strength and endurance in healthy subjects.14,15 in copd patients , although the reliability of isokinetic strength testing has been reported,16 little is known about the reliability of isokinetic endurance measurements at different angular velocities . based on the model developed by walter et al17 and the clinical decision - making process inherent in exercise testing protocols described by charter et al,18 we hypothesize that , for both velocities , muscle endurance and work fatigue indices would achieve a minimally acceptable value , defined by an intraclass coefficient ( icc ) > 0.6.17 the purpose of this study was to determine the test - retest reliability and minimal detectable change ( mdc ) of 30-maximum repetition isokinetic endurance testing of the quadriceps performed at 90 and 180 per second regarding peak torque , muscle endurance , and fatigue in patients with copd . patients with stable and moderate to severe stage copd accordingly to the global initiative for chronic obstructive lung disease19 were recruited . all subjects were former smokers , physically inactive , and nave to the isokinetic procedure . exclusion criteria were : other medical conditions that could affect exercise testing ( ie , neuromuscular and/or orthopedic disorders , recent cancer , unstable cardiac disease , type 2 diabetes , asthma ) , recent copd exacerbations ( < 6 weeks ) , body mass index > 35 kg / m , rest hypoxemia ( spo2 < 90% ) or oxygen therapy , regular practice of physical activity ( voorrips score > 9),20 and involvement in a rehabilitation or structured exercise training program during the previous 6 months . the research protocol was approved by the ethics committee of the institut universitaire de cardiologie et de pneumologie de qubec . the first visit included a review of medical history , a voorrips physical activity questionnaire , anthropometric measurements , pulmonary function testing , and a familiarization session for the quadriceps isokinetic endurance test . during familiarization , subjects were asked to perform warm - up and endurance exercise measurements on the isokinetic dynamometer in the same fashion as required for the testing days ( described below ) . the procedures and series of exercises were repeated as needed until performance was considered compatible with acceptable comprehension of the test.14,21 after 57 days , subjects returned to the laboratory for the quadriceps isokinetic endurance testing protocol , which was repeated at a third visit performed 57 days later , as detailed in figure 1 . the randomization plan was provided by software available online using the number of subjects to be included in the study ( n=14 ) and the number of tests ( n=2 ) performed . in order to include subjects with a sedentary lifestyle , the level of physical activity in daily living was assessed with the activity questionnaire firstly devised by baecke et al22 adapted for elderly subjects by voorrips et al20 and used for patients with copd.6 subjects were considered sedentary whenever the global voorrips score , which includes household , sport , and other leisure time physical activity , was lower than 9 . none of the patients were enrolled in rehabilitation , exercise training , or physical activity programs during the 6 months prior to inclusion . body composition was assessed by bioelectrical impedance analysis ( tbf-300wa tanita , arlington heights , il , usa ) , which provides data on fat mass and fat - free mass . fat - free mass index ( ffmi ) was calculated as ffmi = ffm / height2.23 standard pulmonary function tests , including post - bronchodilator spirometry , lung volumes , and carbon monoxide diffusion capacity , were obtained in all subjects during the first visit according to previously described guidelines2426 and related to predicted normal values.27 all tests of the dominant knee extensors ( quadriceps muscle action ) were performed using an isokinetic dynamometer ( system pro 4 , biodex medical systems , shirley , new york , ny , usa ) and were conducted and recorded by the same investigator ( fr ) . subjects were positioned and stabilized on the dynamometer chair , as previously described.14,28 the knee joint center was aligned to the dynamometer axe center . the lever arm of the dynamometer was firmly attached 3 cm above the lateral malleolus . total knee joint displacement was fixed between 90 and 10 of knee flexion for all tests . reference points for chair and lever arm positioning were annotated in order to ensure the same positioning during subsequent visits . as a warm - up procedure they were then instructed and verbally encouraged to perform 30 maximal knee extensions consecutively and throughout the preset full range of movement.28 subjects were instructed to avoid any muscle effort during return of the leg to the resting position . they performed two trials in a randomized order , one at each angular velocity , ie , 90 and 180 per second , each separated by a 1-hour resting period . data for the first contraction were systematically excluded from all analyses , since the starting maneuver is often submaximal . the fatigue index was determined from the ratio of the work performed during the last ten repetitions to the work performed during the first ten repetitions,14 and the work slope was quantified from the decline in muscle work over time throughout the 30 repetitions.21 perception of dyspnea and leg fatigue was measured using the modified borg scale ( 010 ) of perceived exertion29 and is reported as the change in scores before and immediately after completion of each isokinetic endurance test ( dyspnea and leg fatigue ) . descriptive statistics are reported for subject characteristics and for isokinetic total work , isokinetic work slope , muscle fatigue index , dyspnea , and leg fatigue at each visit . for each subject , comparisons between two isokinetic endurance tests at two angular velocities were performed using a mixed linear model with a hierarchical structure . test - retest reliability was assessed with icc30 for peak torque , muscle endurance ( total work ) , rate of decline in muscle work ( work slope ) , fatigue index , dyspnea , and leg fatigue , and for each velocity separately . in order to facilitate clinical interpretation of the reliability results , mdc values at the 95% confidence level , both in absolute ( mdc95% ) and relative ( mdc95% [ % ] ) terms , were calculated from the standard error of measurement ( sem ) using the following formulae described by beckerman et al:31 mdc95%=sem1.962mdc95%(%)=mdc95%mean of2visits100 finally , between - measurement limits of agreement were established using a bland and altman32 representation for both angular velocities . the proportion of scores at two standard deviations of the mean difference between test - retest values was taken as a parameter of agreement . ( r foundation for statistical computing , vienna , austria . ) , sas version 9.4 ( sas institute inc , cary , nc , usa ) , statistical package for the social sciences version 22 software ( ibm corporation , armonk , ny , usa ) or graphpad prism version 6 for mac os x ( graphpad software , san diego , ca , usa , www.graphpad.com ) . patients with stable and moderate to severe stage copd accordingly to the global initiative for chronic obstructive lung disease19 were recruited . all subjects were former smokers , physically inactive , and nave to the isokinetic procedure . exclusion criteria were : other medical conditions that could affect exercise testing ( ie , neuromuscular and/or orthopedic disorders , recent cancer , unstable cardiac disease , type 2 diabetes , asthma ) , recent copd exacerbations ( < 6 weeks ) , body mass index > 35 kg / m , rest hypoxemia ( spo2 < 90% ) or oxygen therapy , regular practice of physical activity ( voorrips score > 9),20 and involvement in a rehabilitation or structured exercise training program during the previous 6 months . the research protocol was approved by the ethics committee of the institut universitaire de cardiologie et de pneumologie de qubec . the first visit included a review of medical history , a voorrips physical activity questionnaire , anthropometric measurements , pulmonary function testing , and a familiarization session for the quadriceps isokinetic endurance test . during familiarization , subjects were asked to perform warm - up and endurance exercise measurements on the isokinetic dynamometer in the same fashion as required for the testing days ( described below ) . the procedures and series of exercises were repeated as needed until performance was considered compatible with acceptable comprehension of the test.14,21 after 57 days , subjects returned to the laboratory for the quadriceps isokinetic endurance testing protocol , which was repeated at a third visit performed 57 days later , as detailed in figure 1 . the randomization plan was provided by software available online using the number of subjects to be included in the study ( n=14 ) and the number of tests ( n=2 ) performed . in order to include subjects with a sedentary lifestyle , the level of physical activity in daily living was assessed with the activity questionnaire firstly devised by baecke et al22 adapted for elderly subjects by voorrips et al20 and used for patients with copd.6 subjects were considered sedentary whenever the global voorrips score , which includes household , sport , and other leisure time physical activity , was lower than 9 . none of the patients were enrolled in rehabilitation , exercise training , or physical activity programs during the 6 months prior to inclusion . body composition was assessed by bioelectrical impedance analysis ( tbf-300wa tanita , arlington heights , il , usa ) , which provides data on fat mass and fat - free mass . fat - free mass index ( ffmi ) was calculated as ffmi = ffm / height2.23 standard pulmonary function tests , including post - bronchodilator spirometry , lung volumes , and carbon monoxide diffusion capacity , were obtained in all subjects during the first visit according to previously described guidelines2426 and related to predicted normal values.27 all tests of the dominant knee extensors ( quadriceps muscle action ) were performed using an isokinetic dynamometer ( system pro 4 , biodex medical systems , shirley , new york , ny , usa ) and were conducted and recorded by the same investigator ( fr ) . subjects were positioned and stabilized on the dynamometer chair , as previously described.14,28 the knee joint center was aligned to the dynamometer axe center . the lever arm of the dynamometer was firmly attached 3 cm above the lateral malleolus . total knee joint displacement was fixed between 90 and 10 of knee flexion for all tests . reference points for chair and lever arm positioning were annotated in order to ensure the same positioning during subsequent visits . as a warm - up procedure they were then instructed and verbally encouraged to perform 30 maximal knee extensions consecutively and throughout the preset full range of movement.28 subjects were instructed to avoid any muscle effort during return of the leg to the resting position . they performed two trials in a randomized order , one at each angular velocity , ie , 90 and 180 per second , each separated by a 1-hour resting period . data for the first contraction were systematically excluded from all analyses , since the starting maneuver is often submaximal . the fatigue index was determined from the ratio of the work performed during the last ten repetitions to the work performed during the first ten repetitions,14 and the work slope was quantified from the decline in muscle work over time throughout the 30 repetitions.21 perception of dyspnea and leg fatigue was measured using the modified borg scale ( 010 ) of perceived exertion29 and is reported as the change in scores before and immediately after completion of each isokinetic endurance test ( dyspnea and leg fatigue ) . descriptive statistics are reported for subject characteristics and for isokinetic total work , isokinetic work slope , muscle fatigue index , dyspnea , and leg fatigue at each visit . , comparisons between two isokinetic endurance tests at two angular velocities were performed using a mixed linear model with a hierarchical structure . test - retest reliability was assessed with icc30 for peak torque , muscle endurance ( total work ) , rate of decline in muscle work ( work slope ) , fatigue index , dyspnea , and leg fatigue , and for each velocity separately . in order to facilitate clinical interpretation of the reliability results , mdc values at the 95% confidence level , both in absolute ( mdc95% ) and relative ( mdc95% [ % ] ) terms , were calculated from the standard error of measurement ( sem ) using the following formulae described by beckerman et al:31 mdc95%=sem1.962mdc95%(%)=mdc95%mean of2visits100 finally , between - measurement limits of agreement were established using a bland and altman32 representation for both angular velocities . the proportion of scores at two standard deviations of the mean difference between test - retest values was taken as a parameter of agreement . ( r foundation for statistical computing , vienna , austria . ) , sas version 9.4 ( sas institute inc , cary , nc , usa ) , statistical package for the social sciences version 22 software ( ibm corporation , armonk , ny , usa ) or graphpad prism version 6 for mac os x ( graphpad software , san diego , ca , usa , www.graphpad.com ) . fourteen sedentary patients with copd ( 78% men ) completed the study . except for one subject for whom an additional trial at 180 per second was necessary , all participants required only one practice session for familiarization with the isokinetic testing procedure . in agreement with randomization , eight participants began with the endurance test performed at 180 per second and six began at 90 per second for the first visit , and the order of test administration was reversed for the second visit . peak torque , total isokinetic work , muscle fatigue , and delta rating of dyspnea and leg fatigue for each endurance trial and visit at both velocities ( 90 and 180 per second ) are shown in table 2 . as expected , statistically significant differences in these variables were noted between the two angular velocities ( p<0.05 ) . table 3 shows the icc and mdc for the two quadriceps endurance testing protocols . at 90 per second , an icc > 0.90 was found for peak torque , total isokinetic work , work slope , and work fatigue index . in contrast , at 180 per second , peak torque and total isokinetic work were the only parameters with an icc > 0.75 . the mdc95% ( % ) for peak force at 90 and the mdc95% ( % ) for total isokinetic work measurements at 90 and 180 per second was 10% and 19% , respectively . however , the work fatigue index measurements were considered reliable only at 90 per second , with a mdc95% ( % ) of 13% , in comparison with 85% at 180 per second . measurements of perception of dyspnea and leg fatigue were poorly reliable , with an icc < 0.65 and mdc95% ( % ) > 70% irrespective of muscle contraction velocity . limits of agreement , by bland - altman plots , are presented in figure 2a l . better between - visits reliability , expressed as a percentage of difference ( bias random error ) was found for muscle endurance at 90 per second when compared with 180 per second ( 2.6%10.1% and 5.4%19.7% , respectively ) . similar results were found for work slope and work fatigue index , with 95% confidence interval limits of agreement much wider at 180 per second , implying a lower reliability for these measurements at this velocity . bland - altman plots for dyspnea and leg fatigue scores clearly illustrate a high between - visits variability in changes . the major finding of this study was that a 30-maximum repetition isokinetic muscle endurance test can be performed in a reliable fashion in sedentary patients with moderate to severe copd and similar body composition , particularly at 90 per second . while isokinetic peak torque and total muscle work were highly reliable at angular velocities of 90 and 180 per second , test - retest measurements of work slope and fatigue index were only reproducible at 90 per second . changes in dyspnea and leg fatigue perception scores failed to achieve acceptable test - retest reproducibility , irrespective of the velocity applied . overall , our results have practical implications for development of endurance muscle testing in patients with copd , an area for which no standardization is available . to our knowledge , this is the first study to provide a mdc and to report on comparison of the reliability of quadriceps muscle endurance indices at two angular velocities commonly used to assess muscle endurance and fatigue in isokinetic protocols in copd . typically , in comparison with healthy controls , patients with copd are found to present lower isometric torque and total work.33,34 results for the icc analyses , mdc , and bland - altman plots all show that a velocity of 90 per second provides more robust data than a faster velocity of 180 per second . these findings are consistent with a previous study involving healthy subjects that reported a much lower reliability for muscle fatigue indices in a similar 30 repetitions trial at 180 per second than at 60 per second.14 the higher reliability for endurance ( total isokinetic work ) compared with the fatigue index observed in our study is also in line with previous studies in healthy subjects that consistently found the strongest and most reliable measures of isokinetic endurance were total isokinetic work and average power , irrespective of angular velocity.12,14,35,36 in those studies , low reliability was found for work slope or fatigue index . there are several potential explanations for the higher reliability at lower contraction velocity observed in our study . muscle can produce greater dynamic torque during an isokinetic maneuver when contracting slowly rather than at faster velocities.37,38 this could be explained by difficulties in fully activating the available motor units by voluntary effort over the whole range of motion or in the brief time required for full knee extension at faster speeds of contraction.37,38 in addition , elderly individuals show significant deficiencies in functionalperformance39 and typically do not produce fast contractions in daily life . this could contribute to a reduction in their torque production and total work using a high velocity protocol . lastly , despite our effort to minimize the impact of any learning effects with a rigorous familiarization visit , it is still possible that patients may not have become fully accustomed to the high velocity protocol . from perceptual and symptomatic perspectives , our data show that the variations in dyspnea and leg fatigue scores induced by the isokinetic endurance protocol were not reliable between the two visits at either velocity . values obtained from the mdc95% regarding variation in dyspnea showed 3 points in borg scale variability , which represents more than half the range of the scale . the reliability of the borg ratings of perceived exertion during exercise has already been challenged in numerous publications involving healthy subjects40,41 and patients with copd.42 in a study by mador et al42 evaluating six patients with moderate to severe copd during weekly symptom - limited maximal incremental exercise on a cycle ergometer , it was estimated that an individual subject s maximum borg score would need to change by 36% or more before a significant change from baseline could be inferred . on the contrary , in multicenter trials involving 463 copd patients , odonnell et al43 demonstrated an icc of 0.79 ( 0.760.82 ) for borg dyspnea ratings at isotime exercise and of 0.81 ( 0.770.84 ) at peak exercise during two repeated cycling exercises . to our knowledge , the reliability of the modified borg dyspnea scale has not been assessed in any endurance isokinetic protocol involving patients with copd . the reasons for this apparent discrepancy in the reliability of dyspnea rating between isokinetic muscle testing and cycling exercise are unclear . it may well be that the type of exercise , with cycling exercise inducing a larger degree of dyspnea ( typically 56 points on a 10-point borg scale ) than isokinetic muscle testing ( 23 points ) in the present study , exerts much influence on the reliability of this dyspnea scale . it could be argued that a limitation of our study is the relatively small sample size . however , several factors favor its external validity for a precise population of patients with copd . firstly , the sample size was determined to test the hypothesis that , for both velocities , muscle endurance and work fatigue indices would achieve minimally acceptable reliability , such as defined by an icc > 0.6.17 moreover , increasing the sample size does not necessarily change reliability . a study involving a healthy larger population demonstrated that reliability , measured by pearson correlation coefficients , may be more affected by similarities between subjects than by the sample size.44 this study , including 176 subjects , led to an icc > 0.7 , which was clinically acceptable . taking into account the complexity of phenotypes commonly found in copd , we restricted our study population to a very homogenous sample in terms of severity of disease , body composition , and level of physical activity . additionally , a separate familiarization session was provided to minimize within - session learning effects . accordingly to walter et al17 with 14 subjects performing two observations for each velocity , this study is adequately powered to demonstrate the minimally acceptable reliability in a homogenous population . it would have been of interest to test the reliability of the muscle endurance protocols separately in men and women . although this could not be done in our study , we found that excluding the three women from the analysis did not alter the conclusions about the reliability of the measurements . additionally , subjects baseline muscular condition might have been different between visits , potentially influencing our conclusions concerning the reliability of the measurements . however , the close similarity in maximal peak torque measurements on subsequent testing days suggests similar baseline conditions between visits and no impact of the fatigue induced by the 30 maximal contractions in the first set on performance during the second test in the same visit . several studies have reported on quadriceps endurance in copd using a variety of methodologies , including measurement of time to sustain submaximal voluntary contractions at a preset proportion of submaximal voluntary contractions using weights,6,7,4547 a strain gauge,48 or a dynamometer,5 quantification of the number of contractions or fatigue index using an isokinetic protocol,49 and time taken for quadriceps strength to fall to a certain threshold following repeated magnetic stimulation of the femoral nerve.3 collectively , these studies clearly show that lower limb muscle endurance is reduced in copd in comparison with healthy controls of similar age and that muscle endurance is compromised to a larger extent than strength.5,7 these studies also illustrate the relationship between endurance and oxidative capacity.3,48 altogether , these studies provide a strong rationale for the relevance of measuring muscle endurance in copd . despite the interest in quantifying muscle endurance , this parameter has not been incorporated into clinical practice . in this regard , the diversity of protocols is a reflection of the absence of standardization and highlights a need to develop standardized , simple , but valid and sensitive endurance knee extensor procedures to be used in usual clinical settings in patients with copd . also , the lack of consensus with regard to designation of the best testing protocol to evaluate muscle endurance and the absence of predictive values are other impediments to the widespread application of this measurement . the concise protocol that we report here has the advantage of feasibility in patients with copd in regard to their cardiorespiratory limitation , relative simplicity , and reproducibility , allowing measurement of maximal muscle torque , total work , and work slope using only one procedure . the good reproducibility of isokinetic protocols relates to the control of speed of contraction , an important variable in transforming muscle contractions into work . potential disadvantages of an isokinetic protocol are related to cost , availability of the testing apparatus , and the need for a technician specifically training in use of the isokinetic device . the external validity of this measurement has also been questioned , since constant angular velocity is not a physiological movement.50 standardized position , time of day and rest intervals are recognized to affect the test - retest reliability of an isokinetic procedure.51 thus , familiarization and strict standardization of testing procedures are crucial to providing accurate assessments of limb muscle function.52,53 not withstanding these considerations , an endurance isokinetic protocol using 30 maximal contractions provides reliable data , an essential feature for accurate assessment of the effects of therapeutic interventions designed to improve muscle performance . from a clinical perspective , a minimally detectable change of 10% for total work measurements at an angular velocity of 90 per second compares favorably with a corresponding value of 6%12.2% for quadriceps strength measurements.54,55 therefore , our isokinetic protocol at 90 per second appears as reliable as a more widely used and accepted parameter of muscle function such as strength . based on the present results , a 10%15% or greater change in peak torque , total isokinetic work , and fatigue index at 90 per second following a given therapeutic intervention should be considered confidently as a true treatment effect . despite a small sample size , our findings support the use of a 30-maximum repetitions isokinetic muscle testing procedure at an angular velocity of 90 and 180 per second in sedentary patients with moderate to severe copd and similar body composition . measurement of total isokinetic work using an angular velocity of 90 per second was highly reliable , with a mdc95% ( % ) value of 10% , making it a procedure of choice for evaluation of quadriceps endurance in this population . furthermore , peak torque and fatigue index could also be assessed reliably at 90 per second . evaluation of dyspnea and leg fatigue using the modified borg scale of perceived exertion was poorly reliable and its clinical usefulness is questionable . these results should be useful in the design and interpretation of future interventions aimed at improving muscle endurance in copd .

backgroundthe purpose of this study was to determine and compare the test - retest reliability of quadriceps isokinetic endurance testing at two knee angular velocities in patients with chronic obstructive pulmonary disease ( copd).methodsafter one familiarization session , 14 patients with moderate to severe copd ( mean age 654 years ; forced expiratory volume in 1 second ( fev1 ) 55%18% predicted ) performed two quadriceps isokinetic endurance tests on two separate occasions within a 57-day interval . quadriceps isokinetic endurance tests consisted of 30 maximal knee extensions at angular velocities of 90 and 180 per second , performed in random order . test - retest reliability was assessed for peak torque , muscle endurance , work slope , work fatigue index , and changes in fev1 for dyspnea and leg fatigue from rest to the end of the test . the intraclass correlation coefficient , minimal detectable change , and limits of agreement were calculated.resultshigh test - retest reliability was identified for peak torque and muscle total work at both velocities . work fatigue index was considered reliable at 90 per second but not at 180 per second . a lower reliability was identified for dyspnea and leg fatigue scores at both angular velocities.conclusiondespite a limited sample size , our findings support the use of a 30-maximal repetition isokinetic muscle testing procedure at angular velocities of 90 and 180 per second in patients with moderate to severe copd . endurance measurement ( total isokinetic work ) at 90 per second was highly reliable , with a minimal detectable change at the 95% confidence level of 10% . peak torque and fatigue index could also be assessed reliably at 90 per second . evaluation of dyspnea and leg fatigue using the modified borg scale of perceived exertion was poorly reliable and its clinical usefulness is questionable . these results should be useful in the design and interpretation of future interventions aimed at improving muscle endurance in copd .

Introduction Materials and methods Subject characteristics Study design Physical activity Anthropometric data and body composition Pulmonary function testing Assessment of quadriceps isokinetic endurance Effort perception Statistical analysis Results Discussion Conclusion

limb muscle dysfunction has important clinical and prognostic consequences in chronic obstructive pulmonary disease ( copd ) . muscle endurance is defined as the ability to maintain a given task over a certain period of time , and it is determined by muscle fiber oxidative capacity , and the oxygen supply.4 in copd , muscle endurance is impaired to a greater extent than strength.5,6 impaired muscle endurance may even be present in patients with mild disease and preserved physical activity,7,8 and can not be predicted from strength.7 despite its relevance to activities of daily living such as walking,9,10 muscle endurance is generally not an outcome in copd clinical trials.11 one important methodological issue with the measurement of muscle endurance is the fact that there is no standardization or agreement on a testing protocol that could be used in clinical practice or research . it can be determined with various methodologies , amongst which isokinetic testing is one of the most reliable methods.12 the main advantage of this method is that it provides a dynamic measurement of muscle torque while controlling for angular velocities , amplitude , and duration of movement in such a way that the testing conditions could be easily reproduced in subsequent testing.13 as with any other muscle function assessment , isokinetic endurance is required to be accurate and reliable . the reliability of isokinetic testing has been reported for strength and endurance in healthy subjects.14,15 in copd patients , although the reliability of isokinetic strength testing has been reported,16 little is known about the reliability of isokinetic endurance measurements at different angular velocities . based on the model developed by walter et al17 and the clinical decision - making process inherent in exercise testing protocols described by charter et al,18 we hypothesize that , for both velocities , muscle endurance and work fatigue indices would achieve a minimally acceptable value , defined by an intraclass coefficient ( icc ) > 0.6.17 the purpose of this study was to determine the test - retest reliability and minimal detectable change ( mdc ) of 30-maximum repetition isokinetic endurance testing of the quadriceps performed at 90 and 180 per second regarding peak torque , muscle endurance , and fatigue in patients with copd . patients with stable and moderate to severe stage copd accordingly to the global initiative for chronic obstructive lung disease19 were recruited . the first visit included a review of medical history , a voorrips physical activity questionnaire , anthropometric measurements , pulmonary function testing , and a familiarization session for the quadriceps isokinetic endurance test . the procedures and series of exercises were repeated as needed until performance was considered compatible with acceptable comprehension of the test.14,21 after 57 days , subjects returned to the laboratory for the quadriceps isokinetic endurance testing protocol , which was repeated at a third visit performed 57 days later , as detailed in figure 1 . the randomization plan was provided by software available online using the number of subjects to be included in the study ( n=14 ) and the number of tests ( n=2 ) performed . in order to include subjects with a sedentary lifestyle , the level of physical activity in daily living was assessed with the activity questionnaire firstly devised by baecke et al22 adapted for elderly subjects by voorrips et al20 and used for patients with copd.6 subjects were considered sedentary whenever the global voorrips score , which includes household , sport , and other leisure time physical activity , was lower than 9 . fat - free mass index ( ffmi ) was calculated as ffmi = ffm / height2.23 standard pulmonary function tests , including post - bronchodilator spirometry , lung volumes , and carbon monoxide diffusion capacity , were obtained in all subjects during the first visit according to previously described guidelines2426 and related to predicted normal values.27 all tests of the dominant knee extensors ( quadriceps muscle action ) were performed using an isokinetic dynamometer ( system pro 4 , biodex medical systems , shirley , new york , ny , usa ) and were conducted and recorded by the same investigator ( fr ) . as a warm - up procedure they were then instructed and verbally encouraged to perform 30 maximal knee extensions consecutively and throughout the preset full range of movement.28 subjects were instructed to avoid any muscle effort during return of the leg to the resting position . they performed two trials in a randomized order , one at each angular velocity , ie , 90 and 180 per second , each separated by a 1-hour resting period . the fatigue index was determined from the ratio of the work performed during the last ten repetitions to the work performed during the first ten repetitions,14 and the work slope was quantified from the decline in muscle work over time throughout the 30 repetitions.21 perception of dyspnea and leg fatigue was measured using the modified borg scale ( 010 ) of perceived exertion29 and is reported as the change in scores before and immediately after completion of each isokinetic endurance test ( dyspnea and leg fatigue ) . descriptive statistics are reported for subject characteristics and for isokinetic total work , isokinetic work slope , muscle fatigue index , dyspnea , and leg fatigue at each visit . for each subject , comparisons between two isokinetic endurance tests at two angular velocities were performed using a mixed linear model with a hierarchical structure . test - retest reliability was assessed with icc30 for peak torque , muscle endurance ( total work ) , rate of decline in muscle work ( work slope ) , fatigue index , dyspnea , and leg fatigue , and for each velocity separately . in order to facilitate clinical interpretation of the reliability results , mdc values at the 95% confidence level , both in absolute ( mdc95% ) and relative ( mdc95% [ % ] ) terms , were calculated from the standard error of measurement ( sem ) using the following formulae described by beckerman et al:31 mdc95%=sem1.962mdc95%(%)=mdc95%mean of2visits100 finally , between - measurement limits of agreement were established using a bland and altman32 representation for both angular velocities . the proportion of scores at two standard deviations of the mean difference between test - retest values was taken as a parameter of agreement . patients with stable and moderate to severe stage copd accordingly to the global initiative for chronic obstructive lung disease19 were recruited . all subjects were former smokers , physically inactive , and nave to the isokinetic procedure . the first visit included a review of medical history , a voorrips physical activity questionnaire , anthropometric measurements , pulmonary function testing , and a familiarization session for the quadriceps isokinetic endurance test . the procedures and series of exercises were repeated as needed until performance was considered compatible with acceptable comprehension of the test.14,21 after 57 days , subjects returned to the laboratory for the quadriceps isokinetic endurance testing protocol , which was repeated at a third visit performed 57 days later , as detailed in figure 1 . the randomization plan was provided by software available online using the number of subjects to be included in the study ( n=14 ) and the number of tests ( n=2 ) performed . in order to include subjects with a sedentary lifestyle , the level of physical activity in daily living was assessed with the activity questionnaire firstly devised by baecke et al22 adapted for elderly subjects by voorrips et al20 and used for patients with copd.6 subjects were considered sedentary whenever the global voorrips score , which includes household , sport , and other leisure time physical activity , was lower than 9 . fat - free mass index ( ffmi ) was calculated as ffmi = ffm / height2.23 standard pulmonary function tests , including post - bronchodilator spirometry , lung volumes , and carbon monoxide diffusion capacity , were obtained in all subjects during the first visit according to previously described guidelines2426 and related to predicted normal values.27 all tests of the dominant knee extensors ( quadriceps muscle action ) were performed using an isokinetic dynamometer ( system pro 4 , biodex medical systems , shirley , new york , ny , usa ) and were conducted and recorded by the same investigator ( fr ) . as a warm - up procedure they were then instructed and verbally encouraged to perform 30 maximal knee extensions consecutively and throughout the preset full range of movement.28 subjects were instructed to avoid any muscle effort during return of the leg to the resting position . they performed two trials in a randomized order , one at each angular velocity , ie , 90 and 180 per second , each separated by a 1-hour resting period . the fatigue index was determined from the ratio of the work performed during the last ten repetitions to the work performed during the first ten repetitions,14 and the work slope was quantified from the decline in muscle work over time throughout the 30 repetitions.21 perception of dyspnea and leg fatigue was measured using the modified borg scale ( 010 ) of perceived exertion29 and is reported as the change in scores before and immediately after completion of each isokinetic endurance test ( dyspnea and leg fatigue ) . descriptive statistics are reported for subject characteristics and for isokinetic total work , isokinetic work slope , muscle fatigue index , dyspnea , and leg fatigue at each visit . , comparisons between two isokinetic endurance tests at two angular velocities were performed using a mixed linear model with a hierarchical structure . test - retest reliability was assessed with icc30 for peak torque , muscle endurance ( total work ) , rate of decline in muscle work ( work slope ) , fatigue index , dyspnea , and leg fatigue , and for each velocity separately . in order to facilitate clinical interpretation of the reliability results , mdc values at the 95% confidence level , both in absolute ( mdc95% ) and relative ( mdc95% [ % ] ) terms , were calculated from the standard error of measurement ( sem ) using the following formulae described by beckerman et al:31 mdc95%=sem1.962mdc95%(%)=mdc95%mean of2visits100 finally , between - measurement limits of agreement were established using a bland and altman32 representation for both angular velocities . the proportion of scores at two standard deviations of the mean difference between test - retest values was taken as a parameter of agreement . except for one subject for whom an additional trial at 180 per second was necessary , all participants required only one practice session for familiarization with the isokinetic testing procedure . in agreement with randomization , eight participants began with the endurance test performed at 180 per second and six began at 90 per second for the first visit , and the order of test administration was reversed for the second visit . peak torque , total isokinetic work , muscle fatigue , and delta rating of dyspnea and leg fatigue for each endurance trial and visit at both velocities ( 90 and 180 per second ) are shown in table 2 . at 90 per second , an icc > 0.90 was found for peak torque , total isokinetic work , work slope , and work fatigue index . in contrast , at 180 per second , peak torque and total isokinetic work were the only parameters with an icc > 0.75 . the mdc95% ( % ) for peak force at 90 and the mdc95% ( % ) for total isokinetic work measurements at 90 and 180 per second was 10% and 19% , respectively . however , the work fatigue index measurements were considered reliable only at 90 per second , with a mdc95% ( % ) of 13% , in comparison with 85% at 180 per second . measurements of perception of dyspnea and leg fatigue were poorly reliable , with an icc < 0.65 and mdc95% ( % ) > 70% irrespective of muscle contraction velocity . better between - visits reliability , expressed as a percentage of difference ( bias random error ) was found for muscle endurance at 90 per second when compared with 180 per second ( 2.6%10.1% and 5.4%19.7% , respectively ) . similar results were found for work slope and work fatigue index , with 95% confidence interval limits of agreement much wider at 180 per second , implying a lower reliability for these measurements at this velocity . bland - altman plots for dyspnea and leg fatigue scores clearly illustrate a high between - visits variability in changes . the major finding of this study was that a 30-maximum repetition isokinetic muscle endurance test can be performed in a reliable fashion in sedentary patients with moderate to severe copd and similar body composition , particularly at 90 per second . while isokinetic peak torque and total muscle work were highly reliable at angular velocities of 90 and 180 per second , test - retest measurements of work slope and fatigue index were only reproducible at 90 per second . changes in dyspnea and leg fatigue perception scores failed to achieve acceptable test - retest reproducibility , irrespective of the velocity applied . overall , our results have practical implications for development of endurance muscle testing in patients with copd , an area for which no standardization is available . to our knowledge , this is the first study to provide a mdc and to report on comparison of the reliability of quadriceps muscle endurance indices at two angular velocities commonly used to assess muscle endurance and fatigue in isokinetic protocols in copd . typically , in comparison with healthy controls , patients with copd are found to present lower isometric torque and total work.33,34 results for the icc analyses , mdc , and bland - altman plots all show that a velocity of 90 per second provides more robust data than a faster velocity of 180 per second . these findings are consistent with a previous study involving healthy subjects that reported a much lower reliability for muscle fatigue indices in a similar 30 repetitions trial at 180 per second than at 60 per second.14 the higher reliability for endurance ( total isokinetic work ) compared with the fatigue index observed in our study is also in line with previous studies in healthy subjects that consistently found the strongest and most reliable measures of isokinetic endurance were total isokinetic work and average power , irrespective of angular velocity.12,14,35,36 in those studies , low reliability was found for work slope or fatigue index . from perceptual and symptomatic perspectives , our data show that the variations in dyspnea and leg fatigue scores induced by the isokinetic endurance protocol were not reliable between the two visits at either velocity . the reliability of the borg ratings of perceived exertion during exercise has already been challenged in numerous publications involving healthy subjects40,41 and patients with copd.42 in a study by mador et al42 evaluating six patients with moderate to severe copd during weekly symptom - limited maximal incremental exercise on a cycle ergometer , it was estimated that an individual subject s maximum borg score would need to change by 36% or more before a significant change from baseline could be inferred . to our knowledge , the reliability of the modified borg dyspnea scale has not been assessed in any endurance isokinetic protocol involving patients with copd . the reasons for this apparent discrepancy in the reliability of dyspnea rating between isokinetic muscle testing and cycling exercise are unclear . it may well be that the type of exercise , with cycling exercise inducing a larger degree of dyspnea ( typically 56 points on a 10-point borg scale ) than isokinetic muscle testing ( 23 points ) in the present study , exerts much influence on the reliability of this dyspnea scale . firstly , the sample size was determined to test the hypothesis that , for both velocities , muscle endurance and work fatigue indices would achieve minimally acceptable reliability , such as defined by an icc > 0.6.17 moreover , increasing the sample size does not necessarily change reliability . taking into account the complexity of phenotypes commonly found in copd , we restricted our study population to a very homogenous sample in terms of severity of disease , body composition , and level of physical activity . it would have been of interest to test the reliability of the muscle endurance protocols separately in men and women . however , the close similarity in maximal peak torque measurements on subsequent testing days suggests similar baseline conditions between visits and no impact of the fatigue induced by the 30 maximal contractions in the first set on performance during the second test in the same visit . several studies have reported on quadriceps endurance in copd using a variety of methodologies , including measurement of time to sustain submaximal voluntary contractions at a preset proportion of submaximal voluntary contractions using weights,6,7,4547 a strain gauge,48 or a dynamometer,5 quantification of the number of contractions or fatigue index using an isokinetic protocol,49 and time taken for quadriceps strength to fall to a certain threshold following repeated magnetic stimulation of the femoral nerve.3 collectively , these studies clearly show that lower limb muscle endurance is reduced in copd in comparison with healthy controls of similar age and that muscle endurance is compromised to a larger extent than strength.5,7 these studies also illustrate the relationship between endurance and oxidative capacity.3,48 altogether , these studies provide a strong rationale for the relevance of measuring muscle endurance in copd . in this regard , the diversity of protocols is a reflection of the absence of standardization and highlights a need to develop standardized , simple , but valid and sensitive endurance knee extensor procedures to be used in usual clinical settings in patients with copd . also , the lack of consensus with regard to designation of the best testing protocol to evaluate muscle endurance and the absence of predictive values are other impediments to the widespread application of this measurement . the concise protocol that we report here has the advantage of feasibility in patients with copd in regard to their cardiorespiratory limitation , relative simplicity , and reproducibility , allowing measurement of maximal muscle torque , total work , and work slope using only one procedure . potential disadvantages of an isokinetic protocol are related to cost , availability of the testing apparatus , and the need for a technician specifically training in use of the isokinetic device . the external validity of this measurement has also been questioned , since constant angular velocity is not a physiological movement.50 standardized position , time of day and rest intervals are recognized to affect the test - retest reliability of an isokinetic procedure.51 thus , familiarization and strict standardization of testing procedures are crucial to providing accurate assessments of limb muscle function.52,53 not withstanding these considerations , an endurance isokinetic protocol using 30 maximal contractions provides reliable data , an essential feature for accurate assessment of the effects of therapeutic interventions designed to improve muscle performance . from a clinical perspective , a minimally detectable change of 10% for total work measurements at an angular velocity of 90 per second compares favorably with a corresponding value of 6%12.2% for quadriceps strength measurements.54,55 therefore , our isokinetic protocol at 90 per second appears as reliable as a more widely used and accepted parameter of muscle function such as strength . based on the present results , a 10%15% or greater change in peak torque , total isokinetic work , and fatigue index at 90 per second following a given therapeutic intervention should be considered confidently as a true treatment effect . despite a small sample size , our findings support the use of a 30-maximum repetitions isokinetic muscle testing procedure at an angular velocity of 90 and 180 per second in sedentary patients with moderate to severe copd and similar body composition . measurement of total isokinetic work using an angular velocity of 90 per second was highly reliable , with a mdc95% ( % ) value of 10% , making it a procedure of choice for evaluation of quadriceps endurance in this population . furthermore , peak torque and fatigue index could also be assessed reliably at 90 per second . evaluation of dyspnea and leg fatigue using the modified borg scale of perceived exertion was poorly reliable and its clinical usefulness is questionable . these results should be useful in the design and interpretation of future interventions aimed at improving muscle endurance in copd .

f is the most clinically relevant positron emitting radioisotope because of its favorable nuclear decay properties ( 0.635 mev , 97% abundance , half - life 109.8 min ) . f - labeled peptides have been more widely used for diagnostic imaging in cancer and other diseases due to their nonimmunogenic behavior , intrinsic pharmacokinetic properties , high affinity , and readily available solid - phase chemistry to allow tuning of these properties . one consideration for the development of radiotracers is the ability to achieve selective radiolabeling . peptides are challenging in this regard , as the most common labeling methods employ species that react at amines due to their facile reaction with activated carboxylic acid groups ( n - succinimidyl-4-[f]fluorobenzoate , [ f]sfb , for example ) or with aldehydes . peptides of sufficient length may have several amines all with slightly different reaction rates . regioselective radiolabeling may be important to retain binding affinity and selectivity for a given peptide . the ability to easily modify peptides to reduce the number of reactive sites to one furthermore , labeling with [ f]sfb often requires large excess of peptide or protein to achieve reasonably high radiochemical yield , which compromises specific activity and/or requires hplc purification strategies . on the basis of the above considerations , a more selective surrogate instead of amino reactive functional group a free thiol group from a cysteine residue is able to meet these requirements and is extensively studied in radiolabeling of peptides . peptides can be engineered to contain a free thiol group to allow specific labeling . thiols react selectively with maleimides , a - halogenketones , and phosphorothionate agents under mild conditions . the selectivity of prosthetic groups toward free thiol can be exemplified by bovine serum albumin ( bsa ) conjugation . bsa is a protein with 55 free amino groups and 35 thiol groups , 34 of which form a disulfide bond and with just one free thiol group for site - specific conjugation ( cys ) . selective labeling of cys with f units that may be used as a potential f mri agent has recently been developed . glucagon - like peptide-1 ( glp-1 ) is an important glucose - dependent hormone released mainly from the small intestine during the ingestion of food . its receptor ( glp-1r ) is a g protein - coupled receptor mainly expressed in the pancreatic islet cells . detection of insulinomas can be difficult by ct , echography , or mri , due to their relatively small size in the pancreas . thus , glp-1r provides a very promising target for receptor - targeted imaging and therapy of insulinomas . however , the native glp-1 is very unstable in vivo and can be degraded by dipeptidyl - peptidase - iv via cleavage of two n - terminal residues , with a half - life less than 2 min , which limits its biomedical application . we have been interested in the development of a peptide based imaging agent for glp-1r based on radiometal or fluorine-18 labeling to overcome the limitations with promising results . however , radiometal labeled peptides are thought to metabolize to radiometal - chelated amino acids that are able to be trapped in the tubular lysosomes , thereby delivering high radiation doses to the kidneys with potential nephrotoxicity . our group developed a series of f - radiolabeled prosthetic groups for the purpose of labeling cysteine - engineered glp-1 analogues for tumor targeting with considerable success . two thiol site - specific prosthetic groups containing maleimide units , n-[2-(4-[f]fluorobenzamido)ethyl]-maleimide ( [ f]fbem ) and n-5-[f]fluoropentylmaleimide ( [ f]fpenm ) , were developed for the peptide conjugation . the synthesis of these two prosthetic maleimides required three chemical steps and two reaction vessels . [ f]fbem was synthesized with 17.3 7.1% yield ( decay uncorrected ) using an eckert and ziegler module ; the total synthetic time is approximately 100 min , and the measured specific activity was 91176 gbq/mol ( end of synthesis ) . the most recently developed [ f]fpenm has comparable radiolabeling yield ( 14 3% decay uncorrected yield in 110 min ) . both of our previous maleimide prosthetic groups displayed good imaging properties when conjugated to glp-1 analogue , [ cys]-exendin-4 with low kidney uptake or rapid kidney clearance . herein , we describe a new thiol - specific prosthetic agent , n-(2-(2,5-dioxo-2,5-dihydro-1h - pyrrol-1-yl)ethyl)-6-fluoronicotinamide ( [ f]fnem ) by a one - pot two - step strategy : ( 1 ) f incorporation by a nucleophilic displacement of trimethylammonium substrate under mild conditions ; ( 2 ) amidation of the resulting 6-[f]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester with n-(2-aminoethyl)maleimide trifluoroacetate salt . the synthesis begins with a previously reported , high yielding synthesis of tetrafluorophenyl 2-[f]fluoronicotinamide . the maleimide tracer was completed in 75 min from [ f]fluoride with 26 5% decay uncorrected yield , and specific activity 1988 gbq/mol ( decay uncorrected ) . the in vitro cell uptake , in vivo biodistribution , and pet imaging properties of its conjugation product with [ cys]-exendin-4 are described . analytical thin layer chromatography ( tlc ) was performed on precoated silica gel 60 f254 plates ( merck ) with visualization by ultraviolet ( uv ) irradiation at 254 nm or staining with kmno4 . [ cys]-exendin-4 was prepared by solid - phase peptide synthesis ( cs bio , menlo park , ca ) . h , f , and c nmr spectra were carried out on a bruker 300 mhz nmr spectrometer , equipped with a h / f / c 5 mm multinuclear probe . lc / ms analysis was conducted on a waters lc ms system ( waters , milford , ma ) that included an acquity uplc unit coupled to the waters q - tof premier high - resolution mass spectrometer . briefly , to a solution of 6-chloronicotinic acid ( 4.4 g , 27.9 mmol ) and 2,3,5,6-tetrafluorophenol ( tfp ) ( 4.8 g , 28.9 mmol ) in dioxane ( 150 ml ) was added n , n-dicyclohexylcarbodiimide ( dcc ) ( 5.7 g , 27.6 mmol ) ; the mixture was stirred overnight at room temperature . dicyclohexylurea ( dcu ) was removed by filtration , and the filtrate was evaporated in vacuum . the residue was purified by silica gel flash chromatography using hexane / ch2cl2 ( 5/1 , v / v ) as the eluent to afford compound 1 as a white solid ( 7.2 g , 84% ) . h nmr ( 300 mhz , cdcl3 ) 9.219.20 ( m , 1h ) , 8.448.41 ( m , 1h ) , 7.587.55 ( m , 1h ) , 7.177.05 ( m , 1h ) ; c nmr ( 75.5 mhz , cdcl3 ) 160.8 , 157.6 , 152.2 , 148.2147.8 ( m ) , 144.8144.5 ( m ) , 142.7142.4 ( m ) , 140.6 , 139.3139.2 ( m ) , 139.1139.0 ( m ) , 125.0 , 122.6 , 104.1 ( t , j = 22.7 hz ) ; f nmr ( 282 mhz , cdcl3 ) 138.21 to 138.31 ( m , 2f ) , 152.40 to 152.56 ( m , 2f ) ; mass ( esi ) m / z 305.9 [ m + h ] . briefly , to a solution of compound 1 ( 1.0 g , 3.3 mmol ) in dry thf ( 15 ml ) was added 1 m trimethylamine solution in thf ( 9.0 ml ) . a white precipitate was found 10 min after the reaction started , which was allowed to proceed overnight . the precipitate was collected and washed with cold et2o and cold ch2cl2 successively . the solid residue was suspended in ch2cl2 , and tmsotf ( 1.7 ml , 9 mmol ) was added over 10 min . the mixture was concentrated , and the residue was recrystallized from etoac to afford compound 3 as a white solid ( 0.9 g , 57% yield ) . h nmr ( 300 mhz , cd3od ) 9.429.41 ( m , 1h ) , 8.958.92 ( m , 1h ) , 8.288.25 ( m , 1h ) , 7.637.51 ( m , 1h ) , 3.74 ( s , 9h ) ; c nmr ( 75.5 mhz , cd3socd3 ) 164.8 , 159.1 , 149.3 , 147.6147.3 ( m ) , 144.4144.0 ( m ) , 141.8 , 139.6139.3 ( m ) , 136.9136.5 ( m ) , 136.3136.1 ( m ) , 128.9 , 120.7 ( q , j = 322.5 hz ) , 115.6 , 95.4 ( t , j = 23.9 hz ) , 54.6 ; f nmr ( 282 mhz , cd3od ) 81.66 ( s , 3f ) , 142.36 to 142.52 ( m , 2f ) , 156.81 to 156.95 ( m , 2f ) ; mass ( esi ) m / z 329.5 [ m cf3so3 ] . to a solution of triflate 3 ( 86 mg , 0.30 mmol ) and tfp ( 60 mg , 0.36 mmol ) in acetonitrile ( 0.5 ml ) was added dipea ( 57 l , 0.33 mmol ) ; the mixture was stirred at room temperature for 2 h. the residue was concentrated and purified by silica gel flash chromatography using hexane / ch2cl2 as the eluent to afford the compound as a white solid ( 70 mg , 90% ) . h nmr ( 300 mhz , cdcl3 ) 8.96 ( d , j = 2.1 hz , 1h ) , 8.578.53 ( m , 1h ) , 7.337.30 ( m , 1h ) , 7.157.02 ( m , 2h ) ; c nmr ( 75.5 mhz , cdcl3 ) 164.9 , 160.9 , 151.3 , 148.2147.8 ( m ) , 144.9144.5 ( m ) , 143.2142.3 ( m ) , 139.9139.0 ( m ) , 131.8 , 129.5 , 120.2 , 111.3 , 104.2102.9 ( m ) ; f nmr ( 282 mhz , cdcl3 ) 138.48 to 139.16 ( m , 2h ) , 152.52 to 152.95 ( m , 2h ) ; mass ( esi ) m / z 435.9 [ m + h ] . to a solution of n-(2-aminoethyl)maleimide trifluoroacetate salt ( 1.0 equiv ) in anhydrous dmf at 0 c was added aromatic carboxylic acid ( 1.5 equiv ) , hobt ( 1.5 equiv ) , hbtu ( 1.5 equiv ) , 3 molecular sieves , and dipea ( 2.5 equiv ) successively . the reaction proceeded at 0 c for 0.5 h and continued at room temperature overnight . after confirmation from tlc that the starting material was consumed completely , the mixture was quenched with ice water and extracted with ch2cl2 . the organic extracts were washed with water and brine , respectively , then dried and the solvent rotary evaporated . the residue was purified by silica gel column chromatography with ch2cl2/meoh as the eluent to afford the amide compound . compound 5 was prepared according to the general procedure as a white solid ( 83 mg , yield 79% ) . h nmr ( 300 mhz , cdcl3 ) 8.61 ( d , j = 2.4 hz , 1h ) , 8.248.17 ( m , 1h ) , 7.016.97 ( m , 1h ) , 6.95 ( br , 1h ) , 6.74 ( s , 2h ) , 3.853.82 ( m , 2h ) , 3.683.63 ( m , 2h ) ; c nmr ( 75.5 mhz , cdcl3 ) 171.3 , 164.9 , 163.6 , 147.2 ( d , j = 15.9 hz ) , 140.8 ( d , j = 9.1 hz ) , 134.5 , 128.3 ( d , j = 4.5 hz ) , 109.9 ( d , j = 37.0 hz ) , 40.3 , 37.5 ; f nmr ( 282 mhz , cdcl3 ) 63.37 ( d , j = 5.6 hz ) ; mass ( esi ) m / z 264.0 [ m + h ] . compound 6 was prepared according to the general procedure as a light yellow solid ( 112 mg , 88% yield ) . h nmr ( 300 mhz , cdcl3 ) 7.327.26 ( m , 12h ) , 7.257.18 ( m , 3h ) , 6.67 ( s , 2h ) , 5.14 ( t , j = 5.1 hz , 1h ) , 3.54 ( s , 2h ) , 3.413.37 ( m , 2h ) , 3.123.06 ( m , 2h ) ; c nmr ( 75.5 mhz , cdcl3 ) 171.1 , 170.8 , 146.5 , 134.3 , 129.4 , 128.2 , 126.6 , 56.4 , 48.6 , 38.8 , 37.3 ; mass ( esi ) m / z 425.1 [ m + h ] . compound 7 was prepared according to the general procedure as a light yellow solid ( 60 mg , yield 49% ) . h nmr ( 300 mhz , cdcl3 ) 8.28 ( s , 1h ) , 7.897.78 ( m , 4h ) , 7.577.48 ( m , 2h ) , 7.06 ( br , 1h ) , 6.69 ( s , 2h ) , 3.863.83 ( m , 2h ) , 3.733.68 ( m , 2h ) ; c nmr ( 75.5 mhz , cdcl3 ) 171.3 , 168.0 , 134.9 , 134.4 , 132.8 , 131.5 , 129.2 , 128.6 , 127.9 , 127.82 , 127.77 , 126.9 , 123.7 , 39.9 , 37.7 ; mass ( esi ) m / z 295.0 [ m + h ] , 589.1 [ 2 m + h ] . hexylamine ( 50 mg , 0.5 mmol ) was dissolved in a saturated aqueous solution of nahco3 ( 2 ml ) . the solution was put on ice - bath , and after 5 min , n-(methoxycarbonyl)maleimide ( 93 mg , 0.6 mmol ) was added . the resulting solution was stirred on ice - bath for 30 min and then at room temperature for an additional 30 min until all the starting material was consumed completely as confirmed by tlc . the mixture was purified through silica gel flash chromatography using ch2cl2/meoh as the eluent to afford compound 8 as a colorless liquid ( 68 mg , yield 76% ) . h nmr ( 300 mhz , cdcl3 ) 6.68 ( s , 2h ) , 3.50 ( t , j = 7.2 hz , 2h ) , 1.591.54 ( m , 2h ) , 1.281.24 ( m , 6h ) , 0.890.84 ( m , 3h ) ; c nmr ( 75.5 mhz , cdcl3 ) 171.1 , 134.2 , 38.1 , 31.5 , 28.7 , 26.6 , 22.7 , 14.2 ; mass ( ei ) m / z 110.0 [ m n - c5h11 ] , 181.1 m. to a solution of [ cys]-exendin-4 in degassed pbs , fnem or n - hexylmaleimide in acetonitrile was added and incubated for 1 h ; then the mixture was subjected to semipreparative hplc ( vydac c18 protein column , 9.4 250 mm , flow rate 5.0 ml / min , solvent a , 0.1% tfa in water , solvent b , 0.1% tfa in ch3cn ) . the elution profile was isocratic at 25% solvent b for 5 min , then a gradient to 55% solvent b over 25 min , and finally to 90% b over the next 5 min ) to give fnem-[cys]-exendin-4 and n - hexylmaleimido-[cys]-exendin-4 , respectively . the peak at about 21 or 23 min was collected for fnem-[cys]-exendin-4 and n - hexylmaleimido-[cys]-exendin-4 , respectively . analytical hplc tr = 19.3 min ; hplc - ms 1518.5 [ m + 3h ] , 1138.9 [ m + 4h ] ; deconvolves to 4552.0 . analytical hplc tr = 22.3 min ; hplc - ms 1491.5 [ m + 3h ] , 1118.6 [ m + 4h ] , 895.3 [ m + 5h ] ; deconvolves to 4471.5 . tbahco3 ( 0.8 m in h2o , 30 l ) , acetonitrile ( 200 l ) , and [ f]fluoride ( 2386 mci ) were added to a test tube , and the solvent was evaporated under a stream of argon while being heated at 100 c . the fluoride was dried by adding acetonitrile ( 200 l ) three times and each evaporated . then triflate 9 ( 9 mg , 18.8 mol ) in acetonitrile / buoh ( 300 l/100 l ) was added . the reaction mixture was heated at 40 c for 10 min , cooled to room temperature , and then n-(2-aminoethyl)maleimide trifluoroacetate salt ( 12 mg , 47.2 mol ) and pyridine ( 7.6 l ) in acetonitrile ( 170 l ) was added to the mixture . the solvent was evaporated by argon flow and diluted with 1 ml of 10% aqueous ch3cn ; the mixture was centrifuged and subjected to semipreparative hplc purification with phenomenex luna 5 m c18 column ( 250 10 mm , flow rate 4.0 ml / min ) . the collected fraction was diluted with 10 ml of water , and the product was trapped on two stacked sep - pak c18 plus cartridges . the cartridge was washed with h2o ( 3 ml ) and hexane ( 2 ml ) successively , and the product was eluted with 10% etoh in ch2cl2 ( 1.5 ml ) . the purity of compound 5 was confirmed by analytical hplc ( phenomenex luna 3 m c18 column , 150 4.6 mm , flow rate 1.0 ml / min , isocratic elution with 15% ch3cn in 0.1% tfa / h2o ) . [ f]fnem ( 5 , 720 mci ) was dissolved in ethanol ( 10 l ) , and [ cys]-exendin-4 ( 100200 g ) in 100 l 0.1% sodium ascorbate in degassed pbs was added , and the reaction mixture was incubated at room temperature for 30 min . then to the mixture was added n - hexylmaleimide 8 ( 160 g ) in acetonitrile ( 150 l ) , and the reaction stood for another 20 min . then 0.1% tfa ( 100 l ) was added , and the mixture was subjected to semipreparative hplc purification . the collected fractions were diluted with water and passed through a c18 bondelut cartridge . the product was eluted with 1.5 ml of 10 mm hcl in ethanol , and the volume was reduced to about 100200 l on a rotary evaporator . ms 1138.7 [ m + 4h ] , 1518.3 [ m + 3h ] ; deconvolves to 4552.0 . elemental composition c199h297fn54o64s2 : exact mass , 4550.1071 ; molecular weight , 4552.9974 . to study the stability of [ f]fnem-[cys]-exendin-4 in serum , the radiotracer ( 142 ci ) was mixed with freshly harvested mouse serum ( 200 l ) . a 50 l aliquot was removed at 0 min , and the remaining sample was incubated at 37 c . additional aliquots of 50 l were removed at 30 , 60 , and 90 min . a portion of the supernatant was taken for radiohplc analysis using an online radioactivity detector . the animal study protocol was in accordance with the principles and procedures outlined in the guide for the care and use of laboratory animals and approved by the institutional animal care and use committee of the clinical center , national institutes of health ( animal protocol nibib 13 - 01 ) . rat insulinoma cell line ins-1 was obtained from the american type culture collection ( atcc , manassas , va ) . ins-1 cells were grown in rpmi-1640 culture medium ( invitrogen , carlsbad , ca ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs , invitrogen ) , 100 iu / ml penicillin , and 100 g / ml streptomycin ( invitrogen ) at 37 c in a humidified atmosphere containing 5% co2 . ins-1 tumors were developed in 56 week - old female balb / c mice ( n = 10 ) . each mouse underwent inoculation of about 5 10 ins-1 cells in the right shoulder . the glp-1r binding assay was performed according to a reported procedure to determine binding affinities of fnem-[cys]-exendin-4 and exendin-4 . the ins-1 cell uptake and efflux of [ f]fnem-[cys]-exendin-4 were also conducted as previously reported . when the ins-1 tumor reached 810 mm in size ( 1824 days after inoculation ) , pet imaging studies were performed using an inveon small animal pet scanner ( siemens preclinical solutions ) . tumor mice were randomly divided into the control group and the blocking group ( n = 5/group ) . for the control group , about 1.11 mbq ( 30 ci ) of [ f]fnem - cys - exendin-4 was injected through tail vein under isoflurane anesthesia . for exendin-4 blocking group , unlabeled exendin-4 ( 100 g ) was injected ( i.v . tail vein ) 15 min before the injection of 1.11 mbq ( 30 ci ) [ f]fnem - cys - exendin-4 . for both groups , a 5 min acquisition was performed at 1 and 2 h after tracer injection . the images were reconstructed using a 2d osem algorithm without correction for attenuation or scattering . the mean pixel values within the three - dimensional regions of interest ( 3d - rois ) were converted to mbq / ml / min using a predetermined calibration factor . by assuming a tissue density of 1 g / ml , imaging roi - derived % id immediately after the 2 h micropet imaging , tumor model mice in both groups were sacrificed , and ins-1 tumor , blood , major organs , or tissues were harvested and wet weighed . the radioactivity of each organ or tissue was measured using a -counter , and the results were expressed as percentages of the injected dose per gram of tissue ( % id / g ) . quantitative data were expressed as mean sd , and the results were compared using student s t test . following literature procedures , we first synthesized compound 1 from 6-chloronicotinic acid and 2,3,5,6-tetrafluorophenol by n , n-dicyclohexylcarbodiimide condensation . with slight modification of the literature procedure , we found that chloride 2 was obtained in good yield using 1 m trimethylamine solution in thf instead of using trimethylamine gas . the chloride salt 2 with poor solubility in acetonitrile was converted to the trifluoromethanesulfonate salt by adding trimethylsilyl trifluoromethanesulfonate ( tmsotf ) to a suspension of 2 in dichloromethane . purified needle - shaped 3 was conveniently produced by recrystallization of the concentrated organic phase from ethyl acetate . this triflate salt 3 had excellent solubility in the commonly used radiolabeling solvent acetonitrile ( scheme 1 ) . olberg used an oasis mcx plus sep - pak ( waters ) to purify the resulting radiolabeled product 4 , eliminating the time - consuming hplc purification step . however , in the subsequent peptide conjugation , 2 mg of peptide in 3 ml of buffer was required . it was our observation , that the product 4 , purified by solid - phase extraction , contained a significant amount of 2,3,5,6-tetrafluorophenyl 6-(2,3,5,6-tetrafluorophenoxy)nicotinate , resulting from 2,3,5,6-tetrafluorophenol substitution of the trimethylammonium leaving group . indeed we found that 3 efficiently reacted with 2,3,5,6-tetrafluorophenol in the presence of base to provide 95% isolated yield of the side product . although the incorporation of [ f]fluoride was very good , we observed hplc purification to be adversely affected by the buoh in the reaction ( see discussion below ) . subsequently , we found that with smaller volume ( 0.4 ml instead of 1 ml ) of solvent and changing the buoh / mecn ratio from 4/1 to 1/3 still gave 6782% radiochemical yields . we evaluated the amount of n-(2-aminoethyl)maleimide and different bases to achieve the desired coupling . base with higher pka typically gave lower yield or completely degraded polar stuff ( table 1 , entry 1 , 9 ) ; diisopropylethylamine proved to be an appropriate base for the coupling while higher temperature with excess amount of the base afforded no product ( table 1 , compare entry 2 , 3 ) . we also unsuccessfully tried the coupling in aqueous buffer , which gave very low yield ( table 1 , entry 4 ) . finally , we found pyridine ( pka 5.25 ) formed a stable solution with n-(2-aminoethyl)maleimide and afforded good yield for the condensation ; further increasing the amount of maleimide and elevating the reaction temperature gave improved results ( table 1 , entry 58 ) . the desired product was verified by coinjection of authentic fnem , which was prepared by the condensation of 6-fluoronicotinic acid and n-(2-aminoethyl)maleimide . the yield for the second radiolabeling step based on integrated radioactivity of individual peaks relative to the total radioactivity peak areas . we knew we needed to remove the side product ( product 3 + tetrafluorophenol ) . first we tried fluorous cartridge to separate the two components based on the fluorophilicity interaction instead of eluent polarity but were unsuccessful . we returned our focus to hplc purification because we had good analytical conditions . at the end of the reaction , we diluted the reaction solution to 0.4 , 1 , or 4 ml and injected onto a semipreperative column . our initial semipreparative conditions ( isocratic with 0.1% tfa in 15% water , 85% acetonitrile ) for hplc resulted in a product peak with a 4 min peak width ( baseline to baseline ) at 18 min . the amount of aqueous dilution of the reaction mixture ( up to 0.6 , 1 , or 4 ml with water ) did not improve the peak shape . even on an analytical system , optimal peak shape was not obtained unless the tert - butanol was completely evaporated . because of the large volume of collected fraction from the semipreparative column , we were unable to trap the desired product on a solid phase extraction column . attempts to modulate the peak width using more basic hplc eluents , such as ammonium acetate buffer ( 50 mm , ph 6.4 ) or pbs buffer ( ph 7.2 ) , provided no improvement in peak shape . the retention time of [ f]fnem was around 8.9 min with 15% isocratic acetonitrile for analytical hplc in all tested buffers ( 0.1% tfa , ph 6.4 , ph 7.4 ) . we hypothesized that the tert - butanol in the reaction solvent was causing the unfavorable peak shape . typically the tracer was collected in 26 5% ( n = 8) uncorrected yield from eob , and the total radiochemical synthesis time was around 75 min with specific activity 1988 gbq/mol ( n = 8) . moreover , the trapping efficiency from the hplc eluate was around 70% when the hplc fraction ( approximately 6 ml ) was diluted to 21 ml with pure water . up to 90% of the activity was trapped when a stack of two waters c18 plus cartridges was used . following washes with h2o and hexane , the tracer was efficiently eluted ( 80 5% recovery ) with 10% ethanol in dichloromethane . in order to demonstrate the application of this novel nicotinic maleimide prosthetic group the solvent was evaporated under inert gas flow , and the remaining radioactivity was subjected to coupling with [ cys]-exendin-4 using 100 l degassed pbs as the buffer . after a 30 min incubation , the reaction was quenched with 100 l of 0.1% tfa and subjected to preparative hplc . typically the [ f]fnem-[cys]-exendin-4 was collected in around 40% decay uncorrected yield based on starting [ f]fnem . in contrast to our recently developed [ f]fpenm-[cys]-exendin-4 , where the target tracer could be chromatographically separated due to slightly longer retention time from parent excess [ cys]-exendin-4 , the [ f]fnem-[cys]-exendin-4 could not be separated from excess starting material . this was consistent with the fact that [ f]fnem was more polar than [ f]fbem and [ f]penm . efforts to improve the separation with changes in speed of gradient or ph of buffer were unsuccessful . our next approach to achieve higher chemical purity was to consume excess [ cys]-exendin-4 with a much more lipophilic maleimide that could be added following reaction time with [ f]fnem . two lipophilic maleimide prosthetic agents 6 and 7 were prepared through condensation of 3,3,3-triphenylpropionic acid and 2-naphthoic acid with n-(2-aminoethyl)maleimide ) , respectively ( scheme 2 ) . after the radiolabeling reaction , we added 10 equiv of 6 or 7 in acetonitrile to consume excess free thiol compound , but the trial failed probably due to the high hydrophobicity of 6 and 7 that precludes dispersion into aqueous phase . we synthesized another maleimide prosthetic agent 8 with a hexyl chain , which would have similar lipophilicity to fluoropentyl group and may capture excess [ cys]-exendin-4 in aqueous medium . indeed , following the 30 min radiochemical incorporation , a 20 min incubation at room temperature with excess n - hexylmaleimide produced a new nonradioactive peak with 2 min later retention time from [ f]fnem-[cys]-exendin-4 . the new peak was collected and its identity confirmed as a hexylmaleimde conjugate by lc ms . about 60% of the starting material was consumed by the added n - hexylmaleimide , and the resulting specific activity of [ ] fnem-[cys]-exendin-4 was 0.20.6 ci/mol , 2 to 4 times higher than achievable without the n - hexylmaleimide addition . the stability of [ f]fnem-[cys]-exendin-4 was studied at 37 c in mouse serum and was shown to be stable up to 90 min . trace amount of a polar component was produced , which may be due to oxidation of methionine group on the peptide ( figure 1 ) . the extraction efficiency from the mouse serum was around 80% , as determined by the ratio of radioactivity in the supernatant compared to the pellet . stability of [ f]fnem-[cys]-exendin-4 incubated with mouse serum at 0 , 0.5 , 1 , and 1.5 h , respectively . the ic50 values of exendin-4 and fnem-[cys]-exendin-4 , using i - glp-1 as radioligand , in ins-1 cells are displayed in figure 2 . the developed fnem-[cys]-exendin-4 showed high binding affinity ( 0.44 nm ) although exhibited slightly lower binding affinity than parent exendin-4 ( 0.18 nm ) , suggesting that labeling [ cys]-exendin-4 with fnem did not significantly change the binding affinity toward glp-1r expressed on ins-1 cells . inhibition curves of exendin-4 ( red ) and fnem-[cys]-exendin-4 ( blue ) derived from competitive glp-1r binding assay using i - glp-1 as radioligand . cellular uptake and efflux of [ f]fnem-[cys]-exendin-4 was evaluated using ins-1 tumor cells ( figure 3 ) . uptake was apparent ( 0.34 0.04% ) at 15 min , and there was sustained increase until 60 min ( 0.63 0.08% ) , then the uptake decreased at 2 h ( 0.38 0.02% ) . the uptake was effectively inhibited in the presence of a blocking dose of exendin-4 . the efflux appeared to be biphasic with an early rapid washout , reflecting the loss of surface receptor binding , followed by a slow loss of radioactivity from the cells , representing the clearance of internalized radioactivity , about 42% of the activity effluxed from the cells by 60 min . ins-1 cell uptake ( a , ) , block ( a , ) , and efflux ( b , ) of [ f]fnem-[cys]-exendin-4 . the pet images clearly showed high uptake of [ f]fnem-[cys]-exendin-4 in the ins-1 tumor at 60 min post injection ( 19.56 4.57 % id / g ) , which remained high at 2 h time point ( 22.28 5.17 % this tracer is stable against defluorination as negligible tracer uptake was found in the bone ; this was consistent with the mouse serum stability study results prior to in vivo imaging . kidney uptake of [ f]fnem-[cys]-exendin-4 was modest ( 8.28 3.43%id / g ) at 1 h and with most of the tracer cleared from the kidneys by 2 h p.i . liver uptake was low ( 1.64 0.10 % id / g at 1 h ; 1.69 0.24 % id / g at 2 h p.i . ) . glp-1r specific of [ f]fnem-[cys]-exendin-4 in vivo was evaluated by injecting a blocking dose ( 100 g ) of [ cys]-exendin-4 15 min prior to the administration of [ f]fnem-[cys]-exendin-4 ( figure 4d ) . the presence of a blocking dose of [ cys]-exendin-4 significantly reduced the tumor uptake ( 0.46 0.22%id / g at 60 min p.i . ) . the specific tumor uptake of [ f]fnem-[cys]-exendin-4 was further confirmed by biodistribution using dissected tissues . the biodistribution of [ f]fnem-[cys]-exendin-4 in tumors , conducted following micropet imaging , showed similar values , compared with the pet imaging results at 2 h post - tracer injection . in the control group , ins-1 tumor uptake was 23.06 3.87 % id / g , while in blocking group the tumor showed only 0.35 0.23 % id / g tumor - to - kidney ratio was 11 , and tumor - to - liver ratio was 63.8 ( figure 4 ) . the blocking dose led to reduced uptake in the glp-1r positive organs such as pancreas , stomach , and lung . representative pet images of ins-1 tumor mice at 1 ( a ) and 2 h ( b ) postinjection of [ f]fnem-[cys]-exendin-4 ( 30 ci ) for the control and blocking groups ( n = 5/group ) . ( d ) direct tissue sampling measurement of the biodistribution of [ f]fnem-[cys]-exendin-4 right after the pet acquisition at 2 h time point . direct radiolabeling of peptides / proteins usually requires high temperature and strong basic medium , with which most peptides are incompatible . labeling strategies for the introduction of f into peptides or proteins most often utilize radiolabeled prosthetic groups . the small f - labeled prosthetic groups often require multiple synthetic steps to construct before final conjugation to the ligand of interest under mild conditions . several factors are essential to the successful application of f - labeled prosthetic groups : speed of synthesis , selectivity of reactivity , and specific activity . the speed of a radiochemical synthesis is an important factor in the utility and translation of any radiotracer employing a short - lived radionuclide . the application of rapid synthetic reactions and solid - phase extraction methods , instead of hplc , generally reduce preparation time at the potential expense of lower purity . in the case of f , the development of labeling procedures that can be conducted in aqueous media avoids the time required to render fluoride anhydrous for traditional labeling methods . these methods include the use of silicon or boron based fluorine acceptor groups for f incorporation , oxime formation under aqueous conditions by conjugation of [ f]fdg or its derivatives with oxy - amine functionalized peptides , and chelation of the prelabeled alf complex for direct [ f]radiolabeling of macromolecules . gouverneur et al . reported the radiolabeling of fluorous - tagged precursors by nucleophilic fluorination and subsequent purification by fluorous solid phase extraction based on the different affinities of the unreacted substrate and the radiolabeled product for the stationary phase . high specific activity of the final radiolabeled peptide is often desirable since the receptors or enzymes , recognized by the peptides , would be competitively bound with nonradioactive ligands . sufficiently low concentration ( < 0.1 kd ) of the tracer bound to the target is usually required to avoid pharmacological effects for regulatory approval . with our ongoing interest in the development of efficient radiolabeling strategies , we developed three thiol site - specific f - radiolabeling prosthetic groups recently . the efficiency in terms of radiochemical yield , preparation time , and specific activity was outlined in table 2 . the newly developed [ f]fnem was achieved with higher yield in shorter time using a one - pot two - step strategy compared to our previously described [ f]fbem and [ f]fpenm , which employ a three - step synthesis and two reaction vessels . we attempted to obtain a f radiolabeled maleimide prosthetic group using one - step radiolabeling method that was unsuccessful , due to the lability of maleimide group under harsh conditions . all three thiol - site specific groups showed high specific activity and good radiochemical purity ( table 2 ) . the conjugation with [ cys]-exendin-4 using the developed thiol site - specific prosthetic agents proceeded smoothly with 3040% decay uncorrected yield . the images for insulinoma targeting showed similar results for [ f]fbem-[cys]-exendin-4 and [ f]fpenm-[cys]-exendin-4 , but slightly lower tumor uptake was observed for [ f]fnem-[cys]-exendin-4 . we ascribed the difference to the following possibilities : ( a ) because of the lack of chromatographic separation between [ cys]-exendin-4 and [ f]fnem-[cys]-exendin-4 , relatively low specific activity was achieved , and the excess free [ cys]-exendin-4 was unable to be consumed completely although additional maleimide substrate was added to the mixtures to capture the remaining free thiol group after radiolabeling ; ( b ) the nitrogen of the nicotinamide in the [ f]fnem may be protonated and affect the tumor binding affinity as compared to the [ f]fpenm-[cys]-exendin-4 version . there is a literature report that replacement of a phenyl group with a pyridine moiety will affect the binding affinities of somatostatin receptors in terms of electrostatic potentials through tuning the water solubility and hydrogen bonding capacity , apparently it would change the pka and configuration after modification . a new prosthetic group , [ f]fnem , synthesized via an one - pot two - step strategy was developed with shorter reaction time and higher radiolabeling yield than our earlier developed thiol - site specific prosthetic groups [ f]fbem and [ f]fpenm . the application of [ f]fnem was demonstrated in the synthesis and evaluation of [ f]fenm-[cys]-exendin-4 for imaging glp-1r positive ins-1 insulinoma xenografted mice . the tracer has similar tumor uptake compared with [ f]fbem-[cys]-exendin-4 and [ f]fpenm-[cys]-exendin-4 , previously developed by our group , and shows high tumor - to - normal tissue ratios for insulinoma imaging . [ f]fnem may be used to site - specifically radiolabel thiol - containing proteins , antibodies , aptamers , and oligonucleotides .

n-(2-(2,5-dioxo-2,5-dihydro-1h - pyrrol-1-yl)ethyl)-6-fluoronicotinamide ( [ 18f]fnem ) , a novel prosthetic agent that is thiol - specific , was synthesized using a one - pot two - step strategy : ( 1 ) 18f incorporation by a nucleophilic displacement of trimethylammonium substrate under mild conditions ; ( 2 ) amidation of the resulting 6-[18f]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester with n-(2-aminoethyl)maleimide trifluoroacetate salt . the radiosynthesis of the maleimide tracer was completed in 75 min from [ 18f]fluoride with 26 5% decay uncorrected radiochemical yield , and specific activity of 1988 gbq/mol ( decay uncorrected ) . the in vitro cell uptake , in vivo biodistribution , and positron emission tomography ( pet ) imaging properties of its conjugation product with [ cys40]-exendin-4 were described . [ 18f]fnem - cys40-exendin-4 showed specific targeting of glucagon - like peptide 1 receptor ( glp-1r ) positive insulinomas and comparable imaging results to our recently reported [ 18f]fpenm - cys40-exendin-4 .

Introduction Materials and Methods Results Discussion Conclusions

f is the most clinically relevant positron emitting radioisotope because of its favorable nuclear decay properties ( 0.635 mev , 97% abundance , half - life 109.8 min ) . the ability to easily modify peptides to reduce the number of reactive sites to one furthermore , labeling with [ f]sfb often requires large excess of peptide or protein to achieve reasonably high radiochemical yield , which compromises specific activity and/or requires hplc purification strategies . on the basis of the above considerations , a more selective surrogate instead of amino reactive functional group a free thiol group from a cysteine residue is able to meet these requirements and is extensively studied in radiolabeling of peptides . thiols react selectively with maleimides , a - halogenketones , and phosphorothionate agents under mild conditions . bsa is a protein with 55 free amino groups and 35 thiol groups , 34 of which form a disulfide bond and with just one free thiol group for site - specific conjugation ( cys ) . glucagon - like peptide-1 ( glp-1 ) is an important glucose - dependent hormone released mainly from the small intestine during the ingestion of food . its receptor ( glp-1r ) is a g protein - coupled receptor mainly expressed in the pancreatic islet cells . two thiol site - specific prosthetic groups containing maleimide units , n-[2-(4-[f]fluorobenzamido)ethyl]-maleimide ( [ f]fbem ) and n-5-[f]fluoropentylmaleimide ( [ f]fpenm ) , were developed for the peptide conjugation . [ f]fbem was synthesized with 17.3 7.1% yield ( decay uncorrected ) using an eckert and ziegler module ; the total synthetic time is approximately 100 min , and the measured specific activity was 91176 gbq/mol ( end of synthesis ) . both of our previous maleimide prosthetic groups displayed good imaging properties when conjugated to glp-1 analogue , [ cys]-exendin-4 with low kidney uptake or rapid kidney clearance . herein , we describe a new thiol - specific prosthetic agent , n-(2-(2,5-dioxo-2,5-dihydro-1h - pyrrol-1-yl)ethyl)-6-fluoronicotinamide ( [ f]fnem ) by a one - pot two - step strategy : ( 1 ) f incorporation by a nucleophilic displacement of trimethylammonium substrate under mild conditions ; ( 2 ) amidation of the resulting 6-[f]fluoronicotinic acid 2,3,5,6-tetrafluorophenyl ester with n-(2-aminoethyl)maleimide trifluoroacetate salt . the maleimide tracer was completed in 75 min from [ f]fluoride with 26 5% decay uncorrected yield , and specific activity 1988 gbq/mol ( decay uncorrected ) . the in vitro cell uptake , in vivo biodistribution , and pet imaging properties of its conjugation product with [ cys]-exendin-4 are described . the solid residue was suspended in ch2cl2 , and tmsotf ( 1.7 ml , 9 mmol ) was added over 10 min . the mixture was concentrated , and the residue was recrystallized from etoac to afford compound 3 as a white solid ( 0.9 g , 57% yield ) . h nmr ( 300 mhz , cdcl3 ) 8.96 ( d , j = 2.1 hz , 1h ) , 8.578.53 ( m , 1h ) , 7.337.30 ( m , 1h ) , 7.157.02 ( m , 2h ) ; c nmr ( 75.5 mhz , cdcl3 ) 164.9 , 160.9 , 151.3 , 148.2147.8 ( m ) , 144.9144.5 ( m ) , 143.2142.3 ( m ) , 139.9139.0 ( m ) , 131.8 , 129.5 , 120.2 , 111.3 , 104.2102.9 ( m ) ; f nmr ( 282 mhz , cdcl3 ) 138.48 to 139.16 ( m , 2h ) , 152.52 to 152.95 ( m , 2h ) ; mass ( esi ) m / z 435.9 [ m + h ] . to a solution of n-(2-aminoethyl)maleimide trifluoroacetate salt ( 1.0 equiv ) in anhydrous dmf at 0 c was added aromatic carboxylic acid ( 1.5 equiv ) , hobt ( 1.5 equiv ) , hbtu ( 1.5 equiv ) , 3 molecular sieves , and dipea ( 2.5 equiv ) successively . hexylamine ( 50 mg , 0.5 mmol ) was dissolved in a saturated aqueous solution of nahco3 ( 2 ml ) . h nmr ( 300 mhz , cdcl3 ) 6.68 ( s , 2h ) , 3.50 ( t , j = 7.2 hz , 2h ) , 1.591.54 ( m , 2h ) , 1.281.24 ( m , 6h ) , 0.890.84 ( m , 3h ) ; c nmr ( 75.5 mhz , cdcl3 ) 171.1 , 134.2 , 38.1 , 31.5 , 28.7 , 26.6 , 22.7 , 14.2 ; mass ( ei ) m / z 110.0 [ m n - c5h11 ] , 181.1 m. to a solution of [ cys]-exendin-4 in degassed pbs , fnem or n - hexylmaleimide in acetonitrile was added and incubated for 1 h ; then the mixture was subjected to semipreparative hplc ( vydac c18 protein column , 9.4 250 mm , flow rate 5.0 ml / min , solvent a , 0.1% tfa in water , solvent b , 0.1% tfa in ch3cn ) . tbahco3 ( 0.8 m in h2o , 30 l ) , acetonitrile ( 200 l ) , and [ f]fluoride ( 2386 mci ) were added to a test tube , and the solvent was evaporated under a stream of argon while being heated at 100 c . the reaction mixture was heated at 40 c for 10 min , cooled to room temperature , and then n-(2-aminoethyl)maleimide trifluoroacetate salt ( 12 mg , 47.2 mol ) and pyridine ( 7.6 l ) in acetonitrile ( 170 l ) was added to the mixture . the cartridge was washed with h2o ( 3 ml ) and hexane ( 2 ml ) successively , and the product was eluted with 10% etoh in ch2cl2 ( 1.5 ml ) . [ f]fnem ( 5 , 720 mci ) was dissolved in ethanol ( 10 l ) , and [ cys]-exendin-4 ( 100200 g ) in 100 l 0.1% sodium ascorbate in degassed pbs was added , and the reaction mixture was incubated at room temperature for 30 min . then to the mixture was added n - hexylmaleimide 8 ( 160 g ) in acetonitrile ( 150 l ) , and the reaction stood for another 20 min . then 0.1% tfa ( 100 l ) was added , and the mixture was subjected to semipreparative hplc purification . additional aliquots of 50 l were removed at 30 , 60 , and 90 min . ins-1 cells were grown in rpmi-1640 culture medium ( invitrogen , carlsbad , ca ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs , invitrogen ) , 100 iu / ml penicillin , and 100 g / ml streptomycin ( invitrogen ) at 37 c in a humidified atmosphere containing 5% co2 . for both groups , a 5 min acquisition was performed at 1 and 2 h after tracer injection . the mean pixel values within the three - dimensional regions of interest ( 3d - rois ) were converted to mbq / ml / min using a predetermined calibration factor . by assuming a tissue density of 1 g / ml , imaging roi - derived % id immediately after the 2 h micropet imaging , tumor model mice in both groups were sacrificed , and ins-1 tumor , blood , major organs , or tissues were harvested and wet weighed . the radioactivity of each organ or tissue was measured using a -counter , and the results were expressed as percentages of the injected dose per gram of tissue ( % id / g ) . quantitative data were expressed as mean sd , and the results were compared using student s t test . olberg used an oasis mcx plus sep - pak ( waters ) to purify the resulting radiolabeled product 4 , eliminating the time - consuming hplc purification step . however , in the subsequent peptide conjugation , 2 mg of peptide in 3 ml of buffer was required . indeed we found that 3 efficiently reacted with 2,3,5,6-tetrafluorophenol in the presence of base to provide 95% isolated yield of the side product . at the end of the reaction , we diluted the reaction solution to 0.4 , 1 , or 4 ml and injected onto a semipreperative column . attempts to modulate the peak width using more basic hplc eluents , such as ammonium acetate buffer ( 50 mm , ph 6.4 ) or pbs buffer ( ph 7.2 ) , provided no improvement in peak shape . typically the tracer was collected in 26 5% ( n = 8) uncorrected yield from eob , and the total radiochemical synthesis time was around 75 min with specific activity 1988 gbq/mol ( n = 8) . in order to demonstrate the application of this novel nicotinic maleimide prosthetic group the solvent was evaporated under inert gas flow , and the remaining radioactivity was subjected to coupling with [ cys]-exendin-4 using 100 l degassed pbs as the buffer . in contrast to our recently developed [ f]fpenm-[cys]-exendin-4 , where the target tracer could be chromatographically separated due to slightly longer retention time from parent excess [ cys]-exendin-4 , the [ f]fnem-[cys]-exendin-4 could not be separated from excess starting material . two lipophilic maleimide prosthetic agents 6 and 7 were prepared through condensation of 3,3,3-triphenylpropionic acid and 2-naphthoic acid with n-(2-aminoethyl)maleimide ) , respectively ( scheme 2 ) . indeed , following the 30 min radiochemical incorporation , a 20 min incubation at room temperature with excess n - hexylmaleimide produced a new nonradioactive peak with 2 min later retention time from [ f]fnem-[cys]-exendin-4 . about 60% of the starting material was consumed by the added n - hexylmaleimide , and the resulting specific activity of [ ] fnem-[cys]-exendin-4 was 0.20.6 ci/mol , 2 to 4 times higher than achievable without the n - hexylmaleimide addition . trace amount of a polar component was produced , which may be due to oxidation of methionine group on the peptide ( figure 1 ) . stability of [ f]fnem-[cys]-exendin-4 incubated with mouse serum at 0 , 0.5 , 1 , and 1.5 h , respectively . the developed fnem-[cys]-exendin-4 showed high binding affinity ( 0.44 nm ) although exhibited slightly lower binding affinity than parent exendin-4 ( 0.18 nm ) , suggesting that labeling [ cys]-exendin-4 with fnem did not significantly change the binding affinity toward glp-1r expressed on ins-1 cells . uptake was apparent ( 0.34 0.04% ) at 15 min , and there was sustained increase until 60 min ( 0.63 0.08% ) , then the uptake decreased at 2 h ( 0.38 0.02% ) . the efflux appeared to be biphasic with an early rapid washout , reflecting the loss of surface receptor binding , followed by a slow loss of radioactivity from the cells , representing the clearance of internalized radioactivity , about 42% of the activity effluxed from the cells by 60 min . ins-1 cell uptake ( a , ) , block ( a , ) , and efflux ( b , ) of [ f]fnem-[cys]-exendin-4 . the pet images clearly showed high uptake of [ f]fnem-[cys]-exendin-4 in the ins-1 tumor at 60 min post injection ( 19.56 4.57 % id / g ) , which remained high at 2 h time point ( 22.28 5.17 % this tracer is stable against defluorination as negligible tracer uptake was found in the bone ; this was consistent with the mouse serum stability study results prior to in vivo imaging . kidney uptake of [ f]fnem-[cys]-exendin-4 was modest ( 8.28 3.43%id / g ) at 1 h and with most of the tracer cleared from the kidneys by 2 h p.i . in the control group , ins-1 tumor uptake was 23.06 3.87 % id / g , while in blocking group the tumor showed only 0.35 0.23 % id / g tumor - to - kidney ratio was 11 , and tumor - to - liver ratio was 63.8 ( figure 4 ) . the small f - labeled prosthetic groups often require multiple synthetic steps to construct before final conjugation to the ligand of interest under mild conditions . several factors are essential to the successful application of f - labeled prosthetic groups : speed of synthesis , selectivity of reactivity , and specific activity . these methods include the use of silicon or boron based fluorine acceptor groups for f incorporation , oxime formation under aqueous conditions by conjugation of [ f]fdg or its derivatives with oxy - amine functionalized peptides , and chelation of the prelabeled alf complex for direct [ f]radiolabeling of macromolecules . reported the radiolabeling of fluorous - tagged precursors by nucleophilic fluorination and subsequent purification by fluorous solid phase extraction based on the different affinities of the unreacted substrate and the radiolabeled product for the stationary phase . high specific activity of the final radiolabeled peptide is often desirable since the receptors or enzymes , recognized by the peptides , would be competitively bound with nonradioactive ligands . sufficiently low concentration ( < 0.1 kd ) of the tracer bound to the target is usually required to avoid pharmacological effects for regulatory approval . the efficiency in terms of radiochemical yield , preparation time , and specific activity was outlined in table 2 . the newly developed [ f]fnem was achieved with higher yield in shorter time using a one - pot two - step strategy compared to our previously described [ f]fbem and [ f]fpenm , which employ a three - step synthesis and two reaction vessels . we attempted to obtain a f radiolabeled maleimide prosthetic group using one - step radiolabeling method that was unsuccessful , due to the lability of maleimide group under harsh conditions . all three thiol - site specific groups showed high specific activity and good radiochemical purity ( table 2 ) . the conjugation with [ cys]-exendin-4 using the developed thiol site - specific prosthetic agents proceeded smoothly with 3040% decay uncorrected yield . we ascribed the difference to the following possibilities : ( a ) because of the lack of chromatographic separation between [ cys]-exendin-4 and [ f]fnem-[cys]-exendin-4 , relatively low specific activity was achieved , and the excess free [ cys]-exendin-4 was unable to be consumed completely although additional maleimide substrate was added to the mixtures to capture the remaining free thiol group after radiolabeling ; ( b ) the nitrogen of the nicotinamide in the [ f]fnem may be protonated and affect the tumor binding affinity as compared to the [ f]fpenm-[cys]-exendin-4 version . a new prosthetic group , [ f]fnem , synthesized via an one - pot two - step strategy was developed with shorter reaction time and higher radiolabeling yield than our earlier developed thiol - site specific prosthetic groups [ f]fbem and [ f]fpenm . the tracer has similar tumor uptake compared with [ f]fbem-[cys]-exendin-4 and [ f]fpenm-[cys]-exendin-4 , previously developed by our group , and shows high tumor - to - normal tissue ratios for insulinoma imaging . [ f]fnem may be used to site - specifically radiolabel thiol - containing proteins , antibodies , aptamers , and oligonucleotides .

i was very honored to deliver the edwin bierman award lecture at the 74th scientific sessions of the american diabetes association in san francisco in 2014 . bierman was one of the outstanding scientists and mentors at the university of washington , who greatly contributed not only to the fields of diabetes , obesity , dyslipidemia , and atherosclerosis but also to the exceptional scientific environment at the university and beyond . research in my laboratory has been devoted to the discovery of mechanisms behind macrovascular complications of diabetes . by taking advantage of new genetically engineered mouse models , we are now at the exciting stage at which some of the mechanisms discovered in mouse models can be studied in human subjects and vice versa . this perspective will cover the 2014 edwin bierman award lecture uncomplicating the macrovascular complications of diabetes , entitled so because of the ability of mouse models to ask targeted mechanistic questions related to the specific stages of the atherosclerotic lesions and to inquire about cell type specific roles and effects in mediating the effects of diabetes on atherosclerosis . by dissecting the effects of diabetes on macrovascular complications into smaller questions that can be addressed in mice , we are beginning to understand which cell types might be particularly sensitive to hyperglycemia in vivo , that lipids are required , and that myeloid cells play key roles in all stages of atherosclerosis affected by diabetes . combined with well - planned human studies , mouse models will likely provide important mechanistic insight into diabetic macrovascular complications and reveal new potential treatment and prevention strategies that will be needed in order to avert the expected increase in macrovascular complications as our population with diabetes grows . both type 1 diabetes mellitus ( t1 dm ) and type 2 diabetes mellitus ( t2 dm ) are associated with an increased risk of cardiovascular disease ( cvd ) due primarily to worsened atherosclerosis ( 16 ) . recent encouraging trends have suggested that cvd is on the decline in subjects with t1 dm and t2 dm , perhaps due to improved glycemic control and/or statin use ( 7 ) . however , diabetes continues to be associated with a twofold or higher cvd risk as compared with the general population ( 4,7 ) and to be a major health issue in subjects with either t1 dm or t2 dm . the cvd risk in premenopausal women with diabetes is especially high as compared with women without diabetes . research on the mechanisms responsible for the increased cvd associated with t1 dm and t2 dm therefore remains a high priority , especially because t2 dm is on the rise worldwide . lesions of atherosclerosis in susceptible arteries start as accumulations of macrophage foam cells and other immune cells in areas of intimal thickening in humans . these fatty streak lesions can be seen in very young children and are believed to be reversible ( 8) . as the lesion progresses , smooth muscle cells become activated and form a fibrous cap covering the fatty streak lesion . macrophages in the central areas of the lesion undergo apoptosis and death , and as a result , the lesion becomes less stable . rupture or fissuring of such unstable lesions , followed by thrombosis , are believed to be responsible for most cardiovascular events ( 8) . data on atherosclerotic lesion morphology in humans with t1 dm and t2 dm are sparse and are currently limited to small studies . for example , it is unknown if the initiating event in lesion formation in subjects with diabetes is an increased lipoprotein trapping in the artery wall ( 9,10 ) or if the initiating insult is due to endothelial cell activation and increased expression of adhesion molecules due to systemic low - grade inflammation or hyperglycemia , for example . overall , gross lesion morphology appears to be similar in subjects with and without diabetes ( 11 ) . postmortem studies on subjects who suffered sudden coronary death have suggested that lesions in subjects with t1 dm and t2 dm have larger necrotic cores relative to total lesion area as compared with lesions in individuals without diabetes and that macrophages and t cells are more frequent in lesions from individuals with diabetes ( 12 ) . as necrotic cores are believed to be caused primarily by the death of lesion macrophages , these studies support the hypothesis that both t1 dm and t2 dm promote atherosclerotic lesions that contain a larger fraction of immune cells relative to other lesion components . how do these findings on human arteries correlate with findings in mouse models of diabetes - exacerbated atherosclerosis ? one important hurdle to overcome when developing mouse models of diabetes - accelerated atherosclerosis was that induction of diabetes in mice often leads to severe hypercholesterolemia and hypertriglyceridemia as compared with nondiabetic controls . the hypertriglyceridemia in diabetic mice is primarily due to reduced peripheral lipolysis by lipoprotein lipase and is likely to be due to reduced insulin action ( 13 ) . because hyperlipidemia is such a strong promoter of atherosclerosis , diabetes - induced hyperlipidemia makes it difficult to study the effects of diabetes per se on the artery wall . the first study to demonstrate that diabetes promotes atherosclerosis in mice even in the absence of significant changes in plasma lipid levels was conducted by renee leboeuf s group in the 1990s ( 14 ) . in this study , balb / c mice made diabetic using multiple low - dose injections of the -cell toxin streptozotocin ( stz ) were fed a high - fat diet . the diabetic mice developed small atherosclerotic lesions in the aortic sinus without marked changes in plasma lipid levels as compared with their nondiabetic controls fed the same diet . another model of diabetes - induced atherosclerosis that more closely resembles t1 dm and that exhibits lesions in the aorta and brachiocephalic artery as well as the aortic sinus was developed several years later ( 15 ) . in this model , ldl receptor deficient mice ( ldlr mice ) were crossed with mice that express a viral glycoprotein transgene under control of the rat insulin promoter . these mice develop insulitis followed by -cell loss and diabetes when infected with lymphocytic choriomeningitis virus ( lcmv ) , due primarily to cd8 t - cell infiltration of the islets ( 15,16 ) . the diabetic mice become hyperglycemic and exhibit elevated levels of glycated hemoglobin , and they require exogenous insulin treatment to avoid weight loss and ketonuria ( 15 ) . indeed , the loss of -cells in this model is almost complete , and it has been shown that these mice develop autoantibodies to glutamic acid decarboxylase ( 17 ) , thus resembling human t1 dm . in this model of diabetes - accelerated atherosclerosis , diabetic mice exhibit accelerated formation of early fatty streak lesions ( 15,18 ) , as well as progression to advanced lesions characterized by intraplaque hemorrhage in the absence of significant diabetes - induced hyperlipidemia ( 15,19 ) , as shown in fig . 1a and b. the effect of diabetes on lesion progression is independent of the effects on lesion initiation , but also affects lesion areas containing macrophages ( 19 ) . a : diabetes promotes early atherosclerotic lesion formation by stimulating monocyte recruitment to the artery wall . this is likely to be due partly to an increased inflammatory phenotype of myeloid cells dependent on acsl1 induction and partly to increased expression of adhesion molecules and chemokines by endothelial cells . it is still not known if lesion initiation is dependent on hyperglycemia or other factors associated with the diabetic environment . b : diabetes promotes progression of lesions of atherosclerosis to advanced lesions characterized by intraplaque hemorrhage . this effect of diabetes can be prevented by aggressive lipid lowering , even in the presence of severe hyperglycemia . this effect of diabetes is due to maintained recruitment of monocytes into lesions under conditions at which these cells leave the lesions in nondiabetic mice . the maintained accumulation of macrophages in diabetic regression models is dependent on hyperglycemia , which promotes myelopoiesis , an inflammatory lesion macrophage phenotype , and mir-33mediated reduction of the cholesterol exporter abca1 . in addition , the virally induced model exhibits loss of sympathetic neurons in the pancreas during the autoimmune attack ( 21 ) , similar to what occurs in humans . other advantages of this virally induced model of diabetes - accelerated atherosclerosis is that diabetes can be induced at will by lcmv injection and that littermates lacking the glycoprotein transgene can be used as lcmv - injected controls that do not develop diabetes . the third stage of atherosclerosis negatively affected by diabetes is lesion regression in response to aggressive lipid lowering ( fig . thus , in two diabetes mouse models , the stz model and the akita model , diabetes prevents efficient lesion regression by promoting monocyte recruitment into regressing lesions , whereas under nondiabetic conditions , new monocytes do not enter regressing lesions to the same extent ( 22,23 ) . the stz and akita models of diabetes exhibit hyperglycemia and in this respect they are not ideal models of human t1 dm or t2 dm , although some studies suggest that a low - dose stz protocol of diabetes induction results in generation of autoantibodies ( 24 ) . an atherosclerotic mouse model with t2 dm , characterized by -cell dysfunction , hyperglycemia , insulin resistance , and lipid levels in the moderate ( 300500 mg / dl ) range is greatly needed to advance the field . of the mouse models analyzed to date , the virally induced transgenic model most closely resembles t1 dm in humans ( 16 ) . together , the careful analyses of different stages of atherosclerosis in these mouse models have demonstrated that diabetes promotes three different phases of atherosclerosis without significant changes in plasma lipid levels and that the effects of diabetes are associated with increased recruitment and/or activation of myeloid cells . however , it is important to bear in mind that the presence of atherogenic lipoproteins is required in all animal studies for diabetes to promote atherosclerosis . these findings appear to be consistent with the human postmortem data ( 12 ) discussed above . many human studies have demonstrated that t1 dm and t2 dm are associated with increased systemic inflammation ( 2527 ) . the term inflammation is often used to mean that various cytokines and other inflammatory biomarkers are elevated in plasma . thus , plasma interleukin 6 ( il-6 ) and monocyte chemoattractant protein 1 ( ccl2 ) are often elevated in subjects with t2 dm and have been shown to be predictive risk factors for cardiovascular events and death in subjects with t2 dm ( 25 ) or overweight / obese subjects ( 28 ) . other inflammatory mediators elevated in plasma in t2 dm subjects include the chemokines cxcl1 , cxcl10 ( 26 ) , and cx3cl1 ( 29 ) . thus , il-6 release is elevated from cd14 monocytes isolated from subjects with t1 dm as compared with control subjects ( 30 ) . furthermore , studies have identified many cytokines and inflammatory mediators as being elevated in peripheral blood mononuclear cells from subjects with t1 dm , including il-6 , il-1 , genes in the il-1 pathway , and genes involved in eicosanoid production ( 3133 ) . however , there is a plethora of proinflammatory pathways that act in different tissues and cell types , and these pathways are likely to have different effects on circulating cells , on lesion cells , and even on different stages of atherosclerosis . adipose tissue and liver inflammation associated with accumulation of leukocytes in these tissues in the metabolic syndrome / obesity or t2 dm ( 34 ) are likely to affect the artery wall through indirect mechanisms , whereas increased activation of leukocytes in lesions of atherosclerosis can have more direct autocrine or paracrine effects . what is the role of the increased inflammatory mediators in cvd associated with diabetes in humans ? two clinical trials designed to test the role of inflammatory pathways in cvd in subjects with and without t2 dm are ongoing , and a third trial has been recently terminated . the cardiovascular inflammation reduction trial ( cirt ) is a randomized clinical trial investigating whether low - dose methotrexate treatment reduces heart attacks , strokes , or death in people with t2 dm or metabolic syndrome who have already had a myocardial infarction or multiple coronary blockages . the study compares methotrexate ( 520 mg weekly ) , a drug currently in use for the treatment of arthritis and other rheumatic conditions , to placebo ( 35,36 ) . the mechanisms of action of methotrexate to suppress inflammatory processes are complex and not fully understood but are believed to involve the release of adenosine and inhibition of polyamines ( 37 ) . this clinical trial aims to recruit 7,000 individuals and has an estimated completion date in 2018 . the other ongoing clinical trial , cardiovascular risk reduction study ( reduction in recurrent major cv disease events ) , also known as cantos , will test the hypothesis that treatment with canakinumab ( a human monoclonal anti - human il-1 antibody , already used in the treatment of some autoimmune diseases ) of patients with myocardial infarction at least 1 month prior to study entry and elevated c - reactive protein ( as a marker of inflammation ) will prevent recurrent cardiovascular events . although this study contains subjects with t2 dm , the primary outcome measure in these t2 dm subjects is insulin secretion rate . however , the t2 dm subjects are part of the main study with the primary outcome of major adverse cardiovascular event ( cardiovascular death , nonfatal myocardial infarction , and stroke ) . a confounding factor might be that if canakinumab improves hba1c levels , as has been suggested by a study on t2 dm subjects treated with canakinumab as an add - on to metformin ( 39 ) , the improvement in cardiovascular outcomes in these subjects might be due partly to improved glycemic control . a third study , which included some subjects with t2 dm , on the effect of a nonspecific secretory phospholipase a2 ( spla2 ) inhibitor ( varespladib ) on cvd was terminated in 2012 ( 40 ) . this study demonstrated no protective effect on a composite of cardiovascular mortality , nonfatal myocardial infarction , nonfatal stroke , and unstable angina ( 40 ) . the effects of spla2 inhibitors are likely to be complex because of the large number of spla2 isoforms and multiple effects of these enzymes on downstream eicosanoids . in this context , it is interesting to note that the risk of cvd is increased across a range of chronic inflammatory disorders and that the cardiovascular risk is associated with severity of inflammation ( 41 ) . furthermore , testing the effect of anti - inflammatory therapies in subjects with t1 dm would be of great interest . however , because of the many diverse inflammatory pathways and mechanisms , it is possible that more targeted treatment strategies have to be developed in order to minimize side effects and effectively prevent diabetes - induced inflammation without hindering the ability of treated patients to respond to infection elegant studies demonstrate that diabetes is associated with increased myelopoiesis in mouse models of atherosclerotic lesion regression ( 23 ) . these studies identified the damage - associated molecular pattern molecules s100a8 and s100a9 in neutrophils and their interaction with the receptor for advanced glycation end products as mediators of myelopoiesis associated with diabetes . it is therefore possible that diabetes results in activation of neutrophils and subsequent myelopoiesis and increased levels of circulating monocytes with an inflammatory phenotype . increased plasma s100a8/s100a9 levels and gene expression in peripheral blood mononuclear cells have been identified in subjects with t1 dm ( 42,43 ) and t2 dm ( 44 ) , and these proteins are also significantly associated with cvd in subjects with t1 dm ( 23 ) as well as in subjects without diabetes ( 45 ) . the usefulness of s100a8 and s100a9 inhibitors as therapeutic targets is still unknown and is complicated by the fact that these proteins exert proinflammatory effects in dendritic cells ( 46 ) . another marker and mediator of the inflammatory phenotype of myeloid cells associated with t1 dm is the enzyme acyl - coa synthetase 1 ( acsl1 ) . acsl1 catalyzes the linking of acyl - coa moieties to free fatty acids , thus enabling fatty acids to enter many different fates in the cells . acsl1 is induced in myeloid cells in diabetic mice and in cd14 monocytes in a small cohort of human subjects with t1 dm as compared with control subjects ( 47 ) . targeted acsl1 deficiency protected macrophages and monocytes from the inflammatory effects of diabetes and ldlr mice from virally induced diabetes - mediated atherogenesis ( 47 ) . interestingly , myeloid cell acsl1 deficiency had no effect on inflammation or atherosclerosis in nondiabetic mice , suggesting that its induction in diabetes is part of an inflammatory pathway stimulated by diabetes . acsl1 is induced by several inflammatory ligands in myeloid cells , such as lipopolysaccharide , gram negative bacteria , interferon- , and tumor necrosis factor- , but not by peroxisome proliferator activated receptor ( ppar)- and - agonists ( 48,49 ) , in contrast to adipose tissue , liver , and heart in which acsl1 is a ppar target gene ( 5052 ) . this important difference in the regulation of acsl1 might indicate divergent functions of acsl1 in most tissues as compared with myeloid cells . indeed , whereas acsl1 has important functions in mediating the fatty acid oxidation in adipose tissue , liver , and heart ( 5355 ) , it does not have the same role in inflammatory myeloid cells ( 47 ) , which rely heavily on glycolysis for energy . furthermore , acsl1 has emerged as a marker of a metabolically activated macrophage phenotype . ( 56 ) , acsl1 was induced in adipose tissue macrophages from fat - fed mice , concomitant with induction of inflammatory mediators . associated acsl1 was also induced in metabolically activated macrophages exposed to high levels of palmitate , glucose , and insulin in vitro ( 56 ) . other cell surface proteins induced in these macrophages were the cholesterol export protein abca1 , the fatty acid transporter cd36 , and perilipin-2 , a protein that coats intracellular lipid droplets . furthermore , acsl1 is induced in thioglycollate - elicited macrophages by fat feeding in c57bl/6 ( ldlr ) mice , but not in macrophages from ldlr mice fed the same diet ( 57 ) . macrophages from fat - fed ldlr mice are much more lipid loaded than macrophages from fat - fed c57bl/6 mice ; macrophages from ldlr mice had a fourfold increase in total cholesterol and increases in almost all major lipid classes as compared with macrophages from c57bl/6 mice fed the same diet ( 57 ) . thus , it is unlikely that acsl1 induction is merely a reflection of myeloid cell lipid loading . instead , it is possible that acsl1 induction in myeloid cells in the diabetic environment is a marker and mediator of metabolically activated myeloid cells . another interesting possibility is that acsl1 might be translocated to the plasma membrane in activated macrophages ( 47,56 ) , where it might act on distinct pools of fatty acids . the enzyme acsl1 thus fits into the category of proteins involved in immunometabolism ( 58 ) . in further support of the important role of inflammatory pathway activation in diabetes - accelerated atherosclerosis in mice are recent studies on a cell - permeable peptide corresponding to the janus kinase / stat ( signal transducer and activator of transcription ) inhibitory region of the suppressor of cytokine signaling protein , which inhibits stat 1 and 3 . this peptide was demonstrated to impair both systemic inflammation , measured as levels of circulating ly6c monocytes and splenic expression of cytokines , and atherosclerosis in apolipoprotein e deficient ( apoe ) stz - diabetic mice without affecting metabolic parameters ( 59 ) . these results further emphasize that inflammation plays an important role in diabetes - accelerated atherosclerosis , at least in mouse models . the clinical trials discussed above are likely to provide the first information on the role of inflammation in cvd associated with t2 dm in humans . improved metabolic control has long - term beneficial effects on cardiovascular events in young subjects with t1 dm , and these protective effects correlate with improved hba1c levels ( 60 ) . the effects of glucose - lowering on cvd associated with t2 dm are less obvious ( 61 ) . blood glucose lowering by metformin reduced cardiovascular outcomes in overweight subjects with t2 dm in the uk prospective diabetes study as compared with diet treatment alone ( 62 ) . it is unclear whether this effect was due to blood glucose lowering or to other potentially cardioprotective effects by metformin , such as direct effects on macrophages or the liver or other systemic , including lipid lowering , effects in subjects with t2 dm ( 6367 ) . several risk factors contribute to cvd , and whether hyperglycemia directly and universally contributes to cvd associated with diabetes is still a matter of debate . mouse models have demonstrated that hyperglycemia in the presence of atherogenic lipoproteins promotes certain aspects of diabetes - exacerbated atherosclerosis but that it is not sufficient to promote advanced atherosclerosis ( 19,68 ) . glucose cotransporter 2 ( sglt2 ) inhibitors to reduce blood glucose , without confounding effects of insulin on lipid metabolism and other parameters , has demonstrated reduced myelopoiesis , reduced s100a9 expression in neutrophils , reduced accumulation of monocytes in regressing lesions of atherosclerosis , and reduced expression of inflammatory cytokines in lesion macrophages ( 23 ) . in another study , an sglt2 inhibitor prevented systemic inflammation , measured as plasma levels of il-6 , tumor necrosis factor- , and ccl2 , in diabetic mice , as well as reduced plasma cholesterol and triglyceride levels ( 69 ) . these effects are most likely due to the reduced glucose levels , although it is possible that secondary effects contribute to the beneficial effects . sglt2 inhibitors are now approved for use in human subjects with t2 dm , and it will be interesting to evaluate their effects on cvd . if glucose indeed exacerbates atherosclerosis , what is the culprit cell type , what is the mechanism , and what is the interplay between glucose and lipids ? interestingly , hyperglycemia likely contributes to increased hepatic production of vldl in human subjects ( 70 ) , an effect that might be explained by the increased transcription of apolipoprotein ciii ( apoc3 ) in response to glucose ( 71 ) . there is strong emerging genetic evidence that loss - of - function mutations of apoc3 in humans protect against cvd in subjects without diabetes ( 72,73 ) . thus , hyperglycemia could contribute to increased vldl levels and cvd by inducing apoc3 expression in the liver . another recent example of interplay between glucose and lipids is provided by microrna 33 ( mir-33 ) . inhibition of mir-33 by an antisense oligonucleotide ( anti mir-33 ) prevented the detrimental effects of diabetes on lesion regression , monocytosis , monocyte recruitment to lesions , and the inflammatory lesion macrophage phenotype despite persistent hyperglycemia in mice treated with anti thus , all the effects of hyperglycemia in a similar model ( 23 ) were prevented by anti mir-33 . mir-33 treatment resulted in elevated hdl levels in diabetic mice , concomitant with increased hepatic expression of abca1 , consistent with previous studies ( 75 ) . mir-33 also restored the lower levels of abca1 in bone marrow progenitor cells in diabetic mice . mir-33 is likely to act by increasing cholesterol efflux from bone marrow myeloid precursor cells and perhaps by raising hdl , which can have anti - inflammatory effects in myeloid cells ( 76,77 ) . on the other hand , mir-33 did not affect macrophage expression of acsl1 ( 74 ) , suggesting that mir-33 and acsl1 act via different pathways . together , these findings suggest that the stimulatory effects of hyperglycemia on myelopoiesis can be prevented by stimulation of cholesterol efflux from bone marrow cells and/or perhaps by anti - inflammatory effects mediated by abca1 or hdl . in other mouse models , myelopoiesis is induced by fat feeding and adipose tissue inflammation through a mechanism independent of hyperglycemia ( 78 ) . it is therefore possible that diabetes - induced myelopoiesis is , in part , lipid dependent . we evaluated the effect of glucose flux in macrophages by a more direct and cell type specific approach by knocking down the housekeeping glucose transporter glut1 by small interfering rna and by overexpressing the same transporter in myeloid cells in vivo under control of the myeloid cell cd68 promoter ( 79 ) . knockdown of glut1 results in reduced glycolysis in these cells , whereas overexpression promotes glycolysis . glut1 and several other proteins involved in glycolysis and export of lactate are stimulated by inflammatory mediators in macrophages ( 8082 ) . knockdown of glycolytic proteins or inhibition of glycolysis prevents the macrophage from mounting a full inflammatory response ( 79,8284 ) . downregulation of glut1 also prevents myelopoiesis in response to cholesterol loading in cells lacking the cholesterol export proteins abca1 and abcg1 ( 85 ) , hinting at another interesting connection between hdl and glucose metabolism in myeloid cells . conversely , a recent study demonstrated that overexpression of glut1 in a macrophage cell line is sufficient for increasing the inflammatory activation of these cells ( 86 ) . is it therefore possible that hyperglycemia promotes inflammation and atherosclerosis by stimulating glycolysis in myeloid cells ? we addressed this question in ldlr mice with glut1 overexpression targeted to myeloid cells ( 79 ) . however , we observed no effect on cytokine production by glut1-overexpressing myeloid cells , no myelopoiesis , and no increased atherosclerosis at three different arterial sites . thus , increased glucose flux in myeloid cells is not sufficient to explain the effects of diabetes on these cells . it is likely that hyperglycemia affects other cell types involved in atherogenesis directly or indirectly . for example , the endothelial cell is likely to be sensitive to glucose and to express proatherosclerotic adhesion molecules and chemokines under hyperglycemic conditions , resulting in increased monocyte recruitment ( 87 ) . another possibility is that glucose produces a more atherogenic lipid profile by affecting the liver , as discussed above . increased oxidative stress in vascular cells is often put forward as a mechanism whereby hyperglycemia could exert proatherosclerotic effects . indeed , lack of the enzyme glutathione peroxidase 1 , which mediates peroxide breakdown and other antioxidant processes , accelerates atherosclerosis in diabetic apoe mice ( 88 ) , whereas knockout of nadph oxidase 1 , involved in superoxide production , protects against atherosclerosis in diabetic apoe mice ( 89 ) . interestingly , overexpression or knockdown of glyoxalase 1 , an enzyme that detoxifies reactive carbonyls , does not affect atherosclerosis in nondiabetic or diabetic apoe mice ( 90,91 ) . there is therefore evidence that reactive oxygen species and perhaps other oxidative processes contribute to atherosclerosis in diabetes . the cell type most vulnerable to oxidative stress is unknown , but the endothelial cell is again a strong contender . other possible proatherosclerotic mediators of the effects of increased glucose include rage ( the receptor for advanced glycation end products ) ( 9294 ) and protein kinase c activation ( 95 ) . thus , further studies are needed to gain a better understanding of the effects of glucose and hyperglycemia in cells involved in atherosclerosis in mouse models and in human subjects and the relative role of hyperglycemia , inflammation , and lipid abnormalities in cvd progression . finally , it is important to consider that hyperglycemia and elevated hba1c levels could be markers of cvd risk rather than glucose being a mediator . for example , improved hba1c levels are strongly associated with a long - term beneficial effect on kidney disease ( 96 ) , which is , in turn , a significant risk factor for cardiovascular mortality ( 97 ) . moreover , hyperglycemia is due to reduced insulin action as well as insulin - independent hepatic glucose production ( 98 ) . reduced insulin action in vascular cells has been proposed to contribute to atherosclerosis in diabetes . for example , lack of insulin receptors in endothelial cells promotes atherosclerosis in apoe mice ( 99 ) , whereas the lack of insulin receptors in myeloid cells has different effects depending on the mouse model ( 100102 ) . there are many other possible cardiovascular risk factors that could be associated with elevated hba1c levels in diabetes . have the mechanistic studies performed so far in genetically engineered mouse models combined with human studies first , these studies have shown us that diabetes exacerbates several major stages of atherosclerosis by activating myeloid cells . it is still unclear to what extent hyperglycemia directly exacerbates atherosclerosis , but mouse models have started to generate important mechanistic insight . second , mouse studies have shown that there are important interactions between glucose metabolism in myeloid cells and hdl , fatty acids , and other lipids . the increased inflammation likely to play a part in driving cvd associated with both t1 dm and t2 dm could therefore be due to several factors associated with suboptimally maintained diabetes . these include altered hdl function ; elevated triglycerides , which are a hallmark of t2 dm and suboptimally controlled t1 dm ( 103,104 ) ; lack of insulin or reduced insulin action ; and renal disease . in addition , factors that promote atherosclerosis in the population without diabetes , such as dyslipidemia , hypertension , and smoking , also enhance atherosclerosis in subjects with diabetes . it is possible that the relative contributions of various risk factors differ in cvd associated with t1 dm versus t2 dm ( 6 ) and that lipid abnormalities are relatively more important than glucose in t2 dm ( 105 ) . thus , multipronged approaches or individualized treatment strategies are likely to be the way of the future in the treatment and prevention of macrovascular complications of diabetes .

the risk of cardiovascular events in humans increases in the presence of type 1 or type 2 diabetes mellitus , in large part due to exacerbated atherosclerosis . genetically engineered mouse models have begun to elucidate cellular and molecular mechanisms responsible for diabetes - exacerbated atherosclerosis . research on these mouse models has revealed that diabetes independently accelerates initiation and progression of lesions of atherosclerosis and also impairs the regression of lesions following aggressive lipid lowering . myeloid cell activation in combination with proatherogenic changes allowing for increased monocyte recruitment into arteries of diabetic mice has emerged as an important mediator of the effects of diabetes on the three stages of atherosclerosis . the effects of diabetes on atherosclerosis appear to be dependent on an interplay between glucose and lipids , as well as other factors , and result in increased recruitment of monocytes into both progressing and regressing lesions of atherosclerosis . importantly , some of the mechanisms revealed by mouse models are now being studied in human subjects . this perspective highlights new mechanistic findings based on mouse models of diabetes - exacerbated atherosclerosis and discusses the relevance to humans and areas in which more research is urgently needed in order to lessen the burden of macrovascular complications of type 1 and type 2 diabetes mellitus .

Introduction Diabetes Promotes Formation of Early Lesions of Atherosclerosis and Progression to Advanced Lesions and Hinders Regression of Atherosclerosis by Stimulating Myeloid Cell Recruitment Into the Artery Wall Diabetes Is Associated With Increased Myeloid Cell Activation Interplay Between Glucose and Lipids in Diabetes-Accelerated Atherosclerosis Conclusion and Future Perspective

bierman was one of the outstanding scientists and mentors at the university of washington , who greatly contributed not only to the fields of diabetes , obesity , dyslipidemia , and atherosclerosis but also to the exceptional scientific environment at the university and beyond . research in my laboratory has been devoted to the discovery of mechanisms behind macrovascular complications of diabetes . by taking advantage of new genetically engineered mouse models , we are now at the exciting stage at which some of the mechanisms discovered in mouse models can be studied in human subjects and vice versa . this perspective will cover the 2014 edwin bierman award lecture uncomplicating the macrovascular complications of diabetes , entitled so because of the ability of mouse models to ask targeted mechanistic questions related to the specific stages of the atherosclerotic lesions and to inquire about cell type specific roles and effects in mediating the effects of diabetes on atherosclerosis . by dissecting the effects of diabetes on macrovascular complications into smaller questions that can be addressed in mice , we are beginning to understand which cell types might be particularly sensitive to hyperglycemia in vivo , that lipids are required , and that myeloid cells play key roles in all stages of atherosclerosis affected by diabetes . combined with well - planned human studies , mouse models will likely provide important mechanistic insight into diabetic macrovascular complications and reveal new potential treatment and prevention strategies that will be needed in order to avert the expected increase in macrovascular complications as our population with diabetes grows . both type 1 diabetes mellitus ( t1 dm ) and type 2 diabetes mellitus ( t2 dm ) are associated with an increased risk of cardiovascular disease ( cvd ) due primarily to worsened atherosclerosis ( 16 ) . recent encouraging trends have suggested that cvd is on the decline in subjects with t1 dm and t2 dm , perhaps due to improved glycemic control and/or statin use ( 7 ) . research on the mechanisms responsible for the increased cvd associated with t1 dm and t2 dm therefore remains a high priority , especially because t2 dm is on the rise worldwide . lesions of atherosclerosis in susceptible arteries start as accumulations of macrophage foam cells and other immune cells in areas of intimal thickening in humans . these fatty streak lesions can be seen in very young children and are believed to be reversible ( 8) . macrophages in the central areas of the lesion undergo apoptosis and death , and as a result , the lesion becomes less stable . rupture or fissuring of such unstable lesions , followed by thrombosis , are believed to be responsible for most cardiovascular events ( 8) . for example , it is unknown if the initiating event in lesion formation in subjects with diabetes is an increased lipoprotein trapping in the artery wall ( 9,10 ) or if the initiating insult is due to endothelial cell activation and increased expression of adhesion molecules due to systemic low - grade inflammation or hyperglycemia , for example . how do these findings on human arteries correlate with findings in mouse models of diabetes - exacerbated atherosclerosis ? one important hurdle to overcome when developing mouse models of diabetes - accelerated atherosclerosis was that induction of diabetes in mice often leads to severe hypercholesterolemia and hypertriglyceridemia as compared with nondiabetic controls . the hypertriglyceridemia in diabetic mice is primarily due to reduced peripheral lipolysis by lipoprotein lipase and is likely to be due to reduced insulin action ( 13 ) . because hyperlipidemia is such a strong promoter of atherosclerosis , diabetes - induced hyperlipidemia makes it difficult to study the effects of diabetes per se on the artery wall . the first study to demonstrate that diabetes promotes atherosclerosis in mice even in the absence of significant changes in plasma lipid levels was conducted by renee leboeuf s group in the 1990s ( 14 ) . in this study , balb / c mice made diabetic using multiple low - dose injections of the -cell toxin streptozotocin ( stz ) were fed a high - fat diet . the diabetic mice developed small atherosclerotic lesions in the aortic sinus without marked changes in plasma lipid levels as compared with their nondiabetic controls fed the same diet . another model of diabetes - induced atherosclerosis that more closely resembles t1 dm and that exhibits lesions in the aorta and brachiocephalic artery as well as the aortic sinus was developed several years later ( 15 ) . the diabetic mice become hyperglycemic and exhibit elevated levels of glycated hemoglobin , and they require exogenous insulin treatment to avoid weight loss and ketonuria ( 15 ) . in this model of diabetes - accelerated atherosclerosis , diabetic mice exhibit accelerated formation of early fatty streak lesions ( 15,18 ) , as well as progression to advanced lesions characterized by intraplaque hemorrhage in the absence of significant diabetes - induced hyperlipidemia ( 15,19 ) , as shown in fig . 1a and b. the effect of diabetes on lesion progression is independent of the effects on lesion initiation , but also affects lesion areas containing macrophages ( 19 ) . this is likely to be due partly to an increased inflammatory phenotype of myeloid cells dependent on acsl1 induction and partly to increased expression of adhesion molecules and chemokines by endothelial cells . it is still not known if lesion initiation is dependent on hyperglycemia or other factors associated with the diabetic environment . b : diabetes promotes progression of lesions of atherosclerosis to advanced lesions characterized by intraplaque hemorrhage . this effect of diabetes can be prevented by aggressive lipid lowering , even in the presence of severe hyperglycemia . this effect of diabetes is due to maintained recruitment of monocytes into lesions under conditions at which these cells leave the lesions in nondiabetic mice . the maintained accumulation of macrophages in diabetic regression models is dependent on hyperglycemia , which promotes myelopoiesis , an inflammatory lesion macrophage phenotype , and mir-33mediated reduction of the cholesterol exporter abca1 . in addition , the virally induced model exhibits loss of sympathetic neurons in the pancreas during the autoimmune attack ( 21 ) , similar to what occurs in humans . other advantages of this virally induced model of diabetes - accelerated atherosclerosis is that diabetes can be induced at will by lcmv injection and that littermates lacking the glycoprotein transgene can be used as lcmv - injected controls that do not develop diabetes . the third stage of atherosclerosis negatively affected by diabetes is lesion regression in response to aggressive lipid lowering ( fig . thus , in two diabetes mouse models , the stz model and the akita model , diabetes prevents efficient lesion regression by promoting monocyte recruitment into regressing lesions , whereas under nondiabetic conditions , new monocytes do not enter regressing lesions to the same extent ( 22,23 ) . the stz and akita models of diabetes exhibit hyperglycemia and in this respect they are not ideal models of human t1 dm or t2 dm , although some studies suggest that a low - dose stz protocol of diabetes induction results in generation of autoantibodies ( 24 ) . an atherosclerotic mouse model with t2 dm , characterized by -cell dysfunction , hyperglycemia , insulin resistance , and lipid levels in the moderate ( 300500 mg / dl ) range is greatly needed to advance the field . of the mouse models analyzed to date , the virally induced transgenic model most closely resembles t1 dm in humans ( 16 ) . together , the careful analyses of different stages of atherosclerosis in these mouse models have demonstrated that diabetes promotes three different phases of atherosclerosis without significant changes in plasma lipid levels and that the effects of diabetes are associated with increased recruitment and/or activation of myeloid cells . however , it is important to bear in mind that the presence of atherogenic lipoproteins is required in all animal studies for diabetes to promote atherosclerosis . these findings appear to be consistent with the human postmortem data ( 12 ) discussed above . thus , plasma interleukin 6 ( il-6 ) and monocyte chemoattractant protein 1 ( ccl2 ) are often elevated in subjects with t2 dm and have been shown to be predictive risk factors for cardiovascular events and death in subjects with t2 dm ( 25 ) or overweight / obese subjects ( 28 ) . furthermore , studies have identified many cytokines and inflammatory mediators as being elevated in peripheral blood mononuclear cells from subjects with t1 dm , including il-6 , il-1 , genes in the il-1 pathway , and genes involved in eicosanoid production ( 3133 ) . however , there is a plethora of proinflammatory pathways that act in different tissues and cell types , and these pathways are likely to have different effects on circulating cells , on lesion cells , and even on different stages of atherosclerosis . adipose tissue and liver inflammation associated with accumulation of leukocytes in these tissues in the metabolic syndrome / obesity or t2 dm ( 34 ) are likely to affect the artery wall through indirect mechanisms , whereas increased activation of leukocytes in lesions of atherosclerosis can have more direct autocrine or paracrine effects . what is the role of the increased inflammatory mediators in cvd associated with diabetes in humans ? two clinical trials designed to test the role of inflammatory pathways in cvd in subjects with and without t2 dm are ongoing , and a third trial has been recently terminated . the other ongoing clinical trial , cardiovascular risk reduction study ( reduction in recurrent major cv disease events ) , also known as cantos , will test the hypothesis that treatment with canakinumab ( a human monoclonal anti - human il-1 antibody , already used in the treatment of some autoimmune diseases ) of patients with myocardial infarction at least 1 month prior to study entry and elevated c - reactive protein ( as a marker of inflammation ) will prevent recurrent cardiovascular events . however , the t2 dm subjects are part of the main study with the primary outcome of major adverse cardiovascular event ( cardiovascular death , nonfatal myocardial infarction , and stroke ) . a confounding factor might be that if canakinumab improves hba1c levels , as has been suggested by a study on t2 dm subjects treated with canakinumab as an add - on to metformin ( 39 ) , the improvement in cardiovascular outcomes in these subjects might be due partly to improved glycemic control . this study demonstrated no protective effect on a composite of cardiovascular mortality , nonfatal myocardial infarction , nonfatal stroke , and unstable angina ( 40 ) . the effects of spla2 inhibitors are likely to be complex because of the large number of spla2 isoforms and multiple effects of these enzymes on downstream eicosanoids . in this context , it is interesting to note that the risk of cvd is increased across a range of chronic inflammatory disorders and that the cardiovascular risk is associated with severity of inflammation ( 41 ) . however , because of the many diverse inflammatory pathways and mechanisms , it is possible that more targeted treatment strategies have to be developed in order to minimize side effects and effectively prevent diabetes - induced inflammation without hindering the ability of treated patients to respond to infection elegant studies demonstrate that diabetes is associated with increased myelopoiesis in mouse models of atherosclerotic lesion regression ( 23 ) . increased plasma s100a8/s100a9 levels and gene expression in peripheral blood mononuclear cells have been identified in subjects with t1 dm ( 42,43 ) and t2 dm ( 44 ) , and these proteins are also significantly associated with cvd in subjects with t1 dm ( 23 ) as well as in subjects without diabetes ( 45 ) . another marker and mediator of the inflammatory phenotype of myeloid cells associated with t1 dm is the enzyme acyl - coa synthetase 1 ( acsl1 ) . acsl1 catalyzes the linking of acyl - coa moieties to free fatty acids , thus enabling fatty acids to enter many different fates in the cells . acsl1 is induced in myeloid cells in diabetic mice and in cd14 monocytes in a small cohort of human subjects with t1 dm as compared with control subjects ( 47 ) . targeted acsl1 deficiency protected macrophages and monocytes from the inflammatory effects of diabetes and ldlr mice from virally induced diabetes - mediated atherogenesis ( 47 ) . acsl1 is induced by several inflammatory ligands in myeloid cells , such as lipopolysaccharide , gram negative bacteria , interferon- , and tumor necrosis factor- , but not by peroxisome proliferator activated receptor ( ppar)- and - agonists ( 48,49 ) , in contrast to adipose tissue , liver , and heart in which acsl1 is a ppar target gene ( 5052 ) . furthermore , acsl1 has emerged as a marker of a metabolically activated macrophage phenotype . other cell surface proteins induced in these macrophages were the cholesterol export protein abca1 , the fatty acid transporter cd36 , and perilipin-2 , a protein that coats intracellular lipid droplets . thus , it is unlikely that acsl1 induction is merely a reflection of myeloid cell lipid loading . instead , it is possible that acsl1 induction in myeloid cells in the diabetic environment is a marker and mediator of metabolically activated myeloid cells . in further support of the important role of inflammatory pathway activation in diabetes - accelerated atherosclerosis in mice are recent studies on a cell - permeable peptide corresponding to the janus kinase / stat ( signal transducer and activator of transcription ) inhibitory region of the suppressor of cytokine signaling protein , which inhibits stat 1 and 3 . this peptide was demonstrated to impair both systemic inflammation , measured as levels of circulating ly6c monocytes and splenic expression of cytokines , and atherosclerosis in apolipoprotein e deficient ( apoe ) stz - diabetic mice without affecting metabolic parameters ( 59 ) . these results further emphasize that inflammation plays an important role in diabetes - accelerated atherosclerosis , at least in mouse models . the clinical trials discussed above are likely to provide the first information on the role of inflammation in cvd associated with t2 dm in humans . improved metabolic control has long - term beneficial effects on cardiovascular events in young subjects with t1 dm , and these protective effects correlate with improved hba1c levels ( 60 ) . the effects of glucose - lowering on cvd associated with t2 dm are less obvious ( 61 ) . it is unclear whether this effect was due to blood glucose lowering or to other potentially cardioprotective effects by metformin , such as direct effects on macrophages or the liver or other systemic , including lipid lowering , effects in subjects with t2 dm ( 6367 ) . mouse models have demonstrated that hyperglycemia in the presence of atherogenic lipoproteins promotes certain aspects of diabetes - exacerbated atherosclerosis but that it is not sufficient to promote advanced atherosclerosis ( 19,68 ) . glucose cotransporter 2 ( sglt2 ) inhibitors to reduce blood glucose , without confounding effects of insulin on lipid metabolism and other parameters , has demonstrated reduced myelopoiesis , reduced s100a9 expression in neutrophils , reduced accumulation of monocytes in regressing lesions of atherosclerosis , and reduced expression of inflammatory cytokines in lesion macrophages ( 23 ) . in another study , an sglt2 inhibitor prevented systemic inflammation , measured as plasma levels of il-6 , tumor necrosis factor- , and ccl2 , in diabetic mice , as well as reduced plasma cholesterol and triglyceride levels ( 69 ) . sglt2 inhibitors are now approved for use in human subjects with t2 dm , and it will be interesting to evaluate their effects on cvd . if glucose indeed exacerbates atherosclerosis , what is the culprit cell type , what is the mechanism , and what is the interplay between glucose and lipids ? interestingly , hyperglycemia likely contributes to increased hepatic production of vldl in human subjects ( 70 ) , an effect that might be explained by the increased transcription of apolipoprotein ciii ( apoc3 ) in response to glucose ( 71 ) . another recent example of interplay between glucose and lipids is provided by microrna 33 ( mir-33 ) . inhibition of mir-33 by an antisense oligonucleotide ( anti mir-33 ) prevented the detrimental effects of diabetes on lesion regression , monocytosis , monocyte recruitment to lesions , and the inflammatory lesion macrophage phenotype despite persistent hyperglycemia in mice treated with anti thus , all the effects of hyperglycemia in a similar model ( 23 ) were prevented by anti mir-33 . it is therefore possible that diabetes - induced myelopoiesis is , in part , lipid dependent . we evaluated the effect of glucose flux in macrophages by a more direct and cell type specific approach by knocking down the housekeeping glucose transporter glut1 by small interfering rna and by overexpressing the same transporter in myeloid cells in vivo under control of the myeloid cell cd68 promoter ( 79 ) . thus , increased glucose flux in myeloid cells is not sufficient to explain the effects of diabetes on these cells . for example , the endothelial cell is likely to be sensitive to glucose and to express proatherosclerotic adhesion molecules and chemokines under hyperglycemic conditions , resulting in increased monocyte recruitment ( 87 ) . another possibility is that glucose produces a more atherogenic lipid profile by affecting the liver , as discussed above . other possible proatherosclerotic mediators of the effects of increased glucose include rage ( the receptor for advanced glycation end products ) ( 9294 ) and protein kinase c activation ( 95 ) . thus , further studies are needed to gain a better understanding of the effects of glucose and hyperglycemia in cells involved in atherosclerosis in mouse models and in human subjects and the relative role of hyperglycemia , inflammation , and lipid abnormalities in cvd progression . moreover , hyperglycemia is due to reduced insulin action as well as insulin - independent hepatic glucose production ( 98 ) . for example , lack of insulin receptors in endothelial cells promotes atherosclerosis in apoe mice ( 99 ) , whereas the lack of insulin receptors in myeloid cells has different effects depending on the mouse model ( 100102 ) . have the mechanistic studies performed so far in genetically engineered mouse models combined with human studies first , these studies have shown us that diabetes exacerbates several major stages of atherosclerosis by activating myeloid cells . it is still unclear to what extent hyperglycemia directly exacerbates atherosclerosis , but mouse models have started to generate important mechanistic insight . second , mouse studies have shown that there are important interactions between glucose metabolism in myeloid cells and hdl , fatty acids , and other lipids . in addition , factors that promote atherosclerosis in the population without diabetes , such as dyslipidemia , hypertension , and smoking , also enhance atherosclerosis in subjects with diabetes . thus , multipronged approaches or individualized treatment strategies are likely to be the way of the future in the treatment and prevention of macrovascular complications of diabetes .

. however , numerous studies reported complications during parenteral nutrition , like parenteral nutrition associated liver disease or detrimental effects of parenteral lipids on survival and inflammatory response during sepsis [ 4 , 5 ] . this may also be due to lipid - induced decrease of neutrophil function and cytokine release in septic patients [ 6 , 7 ] . therefore , strategies to avoid the negative consequences of intravenously administered lipids were needed [ 4 , 8 , 9 ] . in recent years , the composition of lipids was mainly based on soybeans which contain high amounts of omega-6-polyunsaturated fatty acids . during the beginning of parenteral nutrition , intralipid which only contains soybean - based long - chain - triglycerides ( lct ) was frequently used . omega-6 fatty acids belong to the family of polyunsaturated fatty acids ( pufa ) and are precursors of eicosanoids . eicosanoids act as immunomodulators , serve as signaling molecules , and contribute to inflammatory conditions . in this context , they promote leukocyte recruitment by increased production of proinflammatory cytokines . on the other hand , they negatively affect lymphocyte proliferation , thereby causing an immunosuppressive effect [ 11 , 12 ] . one important aspect of the observed effects is the change of cell membrane fluidity by parenterally administered fatty acids . in order to attenuate these serious side effects of pufa lipofundin is one alternative that substituted 50% of lct with medium - chain - triglycerides ( mct ) that are metabolized more rapidly than lct , thereby displaying less immunosuppressive properties and exerting better effects on membrane function . one study that compared the respiratory burst of human neutrophils found a reduced effect with lipofundin when compared to other lipid emulsions . in contrast to lipofundin and intralipid , olive oil - based lipids were shown to have a protective effect against lps - induced inflammation . olive oils contain high amounts of monounsaturated fatty acids ( mufa ) and are known to show less sensitivity to peroxidation when compared to pufa . however , there is an ongoing discussion about beneficial properties of fish oil - based lipids ( i.e. , smoflipid ) when compared with predominant olive oil - based lipids ( i.e. , clinoleic ) [ 17 , 18 ] . smoflipid contains fish oil , which is rich in omega-3 fatty acids and is able to inhibit the production of proinflammatory cytokines via activation of peroxisome proliferator - activated receptor ( ppar ) and interaction with nfkb [ 19 , 20 ] . the beneficial effects are at least in part attributed to a favorable ratio of omega-6 : omega-3 fatty acids and the balanced mixture of different lipid ingredients ( lct , mct , and olive oil ) . in addition , there are still conflicting results about how parenterally administered lipids might interfere with leukocyte recruitment which is known to be a sensitive indicator of inflammation . leukocyte recruitment into inflamed tissue follows a well - defined cascade of events beginning with the capture of free flowing leukocytes to the vessel wall followed by leukocyte rolling ( mediated by selectins and their ligands ) triggering the activation of 2-integrins ( i.e. , lfa1 , mac1 ) by chemokines and their receptors ( i.e. , cxcr2 ) . once activated , leukocyte 's integrins can bind to their respective endothelial receptors , like icam-1 or vcam-1 . this in turn leads to firm leukocyte arrest on the endothelium and finally the leukocyte transmigration into the tissue [ 21 , 22 ] . based on the above - mentioned controversies about lipid - induced immunomodulation , we now aimed to compare anti - inflammatory effects of lipofundin , smoflipid , and clinoleic in vitro and in vivo with special regard to leukocyte recruitment during local and systemic inflammation . c57bl/6 wild type ( wt ) mice were provided by charles river ( sulzfeld , germany ) . all mice were maintained as breeding colonies at the central animal facility of the university of heidelberg , germany . the animal experiments were approved by the animal care and use committee of the regierungsprsidium karlsruhe , germany ( az 35 - 9185.81/g-3/13 ) . clinoleic ( fresenius kabi , bad homburg , germany ) contains 80% olive oil and 20% soybean oil ( lct ) . smoflipid ( fresenius kabi , bad homburg , germany ) consists of 30% soybean oil ( lct ) , 30% mct , 25% olive oil , and 15% fish oil ( rich in omega 3 fatty acids ) . lipofundin ( braun , melsungen , germany ) consists of 50% soybean oil ( lct ) and 50% mct . the lipid emulsions were administered as an intravenous bolus injection at 1 g / kg for the intravital microscopic experiments and at 2 g / kg 30 min , 8 h , and 24 h after lps during lps - induced endotoxemia . mice were prepared for intravital microscopy , as reported recently . briefly , after intraperitoneal ( i.p . ) injection of ketamine ( 125 mg / kg body weight , ketalar ; parke - davis , morris plains , nj , usa ) and xylazine ( 12.5 mg / kg body weight ; phoenix scientific inc . , st . joseph , mo , usa ) mice were placed on a heating pad to maintain body temperature . intravital microscopy was conducted on an upright microscope ( leica ; wetzlar , germany ) with a saline immersion objective ( sw40/0.75 numerical aperture , zeiss , jena , germany ) . mice were intubated and the left carotid artery was cannulated for blood sampling and the right jugular vein for lipid administration . the lipid emulsions were administered with a dose of 1 g / kg as an intravenous bolus injection . blood levels of cholesterol , triglycerides , and liver enzymes were measured after the respective experiments in the core laboratory facility of the university hospital heidelberg ( analysezentrum , heidelberg , germany ) . the surgical preparation ( trauma - induced inflammation ) of the cremaster muscle after longitudinal incision and spreading of the muscle over a cover glass , the epididymis and testis were mobilized and pinned aside leading to full microscopic access to the cremaster muscle microcirculation . cremaster muscle venules were recorded via ccd camera ( cf8/1 , kappa , gleichen , germany ) on a panasonic s - vhs recorder . s - vhs tapes were digitized using a dvd maker ( typhoon , schalksmuehle , germany ) . postcapillary venules under observation were recorded before and during lipid administration and ranged from 20 to 40 m in diameter . systemic blood samples ( 10 l ) were taken and assessed for white blood cell count after staining with turk 's solution 1 : 10 ( merck , darmstadt , germany ) using a hemocytometer . microvascular parameters ( venular diameter , venular vessel segment length ) were measured using an image processing system . venular centerline red blood cell velocity was measured during the experiment via a dual photodiode and a digital on - line cross - correlation program ( circusoft instrumentation , hockessin , de , usa ) . an empirical factor of 0.625 was used to convert centerline velocities to mean blood flow velocities . wall shear rates ( w ) were estimated as 4.9 ( 8vb / d ) , where vb is mean blood flow velocity and d is the diameter of the vessel [ 27 , 28 ] . the number of adherent leukocytes ( firm adhesion for > 30 s ) was assessed as adherent cells per mm vessel surface area as reported previously . rolling leukocyte flux fraction was defined as the percentage of rolling leukocytes to all leukocytes passing the same vessel in 1 minute . in certain experiments mice were injected with 50 ng lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany ) intrascrotally ( lps - induced inflammation ) . to differentially count transmigrated leukocytes , cremaster muscle - whole mounts were prepared as described before . briefly , while the cremaster muscle was still mounted on the stage for intravital microscopy , the tissue was fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the cremaster muscle was removed and mounted flat on a superfrost glass slide ( menzel , braunschweig , germany ) , air dried for 510 min , and fixed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) for 24 h at 4c . after fixation , the tissue was washed three times in 0.1 m phosphate buffer with 5% ethanol , stained with giemsa ( sigma , taufkirchen , germany ) at room temperature for 5 min , and differentiated in 0.01% acetic acid for contrast . the differentiated slides were washed in water , 75% ethanol , 95% ethanol , 100% ethanol , and fresh xylene , followed by mounting in mounting media ( agar scientific , stansted , uk ) . the giemsa - stained cremaster muscles were observed using a leica dmrb upright microscope and a 25/0,75 na oil immersion objective ( both leica , wetzlar , germany ) . the maecs were isolated and cultured as previously described . in brief , 3 mm long freshly harvested and cleaned aortic rings were seeded into matrigel - coated culture dishes ( bd , san jose , ca , usa ) and incubated at 37c , 5% co2 in dulbecco 's modified eagle medium ( supplemented with 15% fetal bovine serum , 1% pen / strep , 90 g / ml heparin , 60 g / ml endothelial cell growth supplement , and 1 g / ml amphotericin b ; fungizone , invitrogen , karlsruhe , germany ) . after sufficient growth , endothelial cells were passaged with dispase ( bd , san jose , ca , usa ) and characterized by immunocytochemistry as described . for lps - stimulation cells were grown in costar 6-well plates ( corning inc . , amsterdam , netherlands ) and standard medium to near confluence and incubated with lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany ) at 100 ng/10 cells for 3 hours at 37c . respective lipid pretreatment ( clinoleic , lipofundin or smoflipid at 10 mg/10 cells ) was initiated together with lps - stimulation . after isolation , they were loaded on top of a discontinuous percoll gradient ( 52%/64%/72% ) and centrifuged at 1000 g for 30 minutes . pmn viability was greater than 95% as assessed by the trypan blue exclusion test , and purity was greater than 98% as analyzed by microscopy using hemacolor staining ( merck , darmstadt , germany ) . for flow cytometric analysis of icam-1 and vcam-1 expression on endothelial cells , prepared maecs were harvested and incubated in the dark for 45 minutes on ice with pe - conjugated anti - icam-1 mab ( clone yn1/1.7.4 ebioscience , san diego , ca , usa ) , anti - mouse vcam-1 mab ( clone 429 mvcam.a biolegend , san diego , ca , usa ) , or respective isotype control antibody ( ebioscience , san diego , ca , usa and bd ) to detect anti - icam-1 and vcam-1 signal on 10.000 cells using the 4-decade facs - scan lsrii with diva software package ( bd ) . the expression of cxcr2 , psgl1 , mac1 , and lfa1 was assessed using isolated bone marrow - derived neutrophils ( see above ) . after red blood cell lysis , 10 leukocytes / ml were stimulated for 3 h with 10 mg lipofundin , clinoleic , or smoflipid , respectively , at 37c . next , cells were incubated in the dark with phycoerythrin - conjugated anti - cxcr2 mab ( 1 g/10 cells ; ebioscience , frankfurt , germany ) , anti - psgl-1 mab ( 1 g/10 cells , bd pharmingen , san diego , ca , usa ) , fitc - conjugated anti - mac1 mab m1/70 ( 1 g/10 cells , rat igg2b ; ebioscience , san diego , ca , usa ) , fitc - conjugated anti - lfa1 mab m17/4 ( 1 g/10 cells , rat igg2a ; ebioscience , san diego , ca , usa ) , or respective isotype control antibodies ( 1 g/10 cells , rat igg2b or rat igg2a ; ebioscience , san diego , ca , usa ) . the respective antigen expression was assessed on 10.000 cells per mouse within the neutrophil cluster defined by forward - side scatter analysis . expression upon stimulation with different lipid emulsions was compared to unstimulated cells and their respective isotype controls . in certain experiments lps flow chamber experiments were conducted as described [ 33 , 34 ] . in brief , rectangular microglass capillaries ( vitrocom , mountain lakes , nj , usa ) were coated with rmp - selectin ( 2 g / ml ) , rmcxcl1 ( 5 g / ml ) , and icam-11 ( 1 g / ml ) and connected via pe tubing to a 2 ml syringe containing freshly isolated bone marrow neutrophils from lysegfp mice . in lysegfp mice , the enhanced gfp ( egfp ) is knocked into the murine lysozyme m ( lys ) locus leading to the expression of egfp in myelomonocytic cells . the cell suspension ( 0.25 10 gfp positive cells ) was incubated with lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany , 100 ng/10 cells for 3 hours at 37c ) and perfused through the flow chamber . adhesion of gfp - positive cells was observed by fluorescence microscopy ( bx51 wi with a saline immersion objective 20/0.95 na , olympus hamburg , germany ) for 10 minutes under constant flow conditions using a high precision perfusion pump ( harvard instruments , march - hugstetten , germany ; wall shear stress 0,1 pa ) . images were recorded via a ccd camera system ( cf8hs ; kappa , gleichen , germany ) on a panasonic s - vhs recorder . in some experiments , cell suspensions were incubated with either lipofundin , smoflipid , or clinoleic with a dose of 10 mg/10 cells for 3 h at 37c . injection of 40 mg / kg lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany ) which was reconstituted in 100 l of sterile pbs , as reported previously . clinoleic , smoflipid , lipofundin , or equivalent volume of normal saline was administered i.v . at 2 g / kg 30 minutes , 8 , and 24 hours after lps challenge . in a first group , survival was observed for 14 days . in a second group , mice were perfused with saline and lungs were harvested 24 h after lps injection . after fixation in pfa ( 4% ) they were prepared for paraffin - embedded sections . sections were performed at 3 m thickness and finally stained with h&e ( haematoxylin and eosin staining ) for microscopic evaluation . sigma stat 3.5 ( systat software , erkrath , germany ) was used for statistical analysis . leukocyte counts , vessel diameters , leukocyte adhesion , leukocyte rolling flux fractions , wall shear rates , and in vitro leukocyte adhesion between groups and treatments were compared with one - way anova followed by a multiple pairwise comparison test ( dunn 's test ) or by wilcoxon rank - sum test , as appropriate . to compare the survival during lethal endotoxemia , log - rank test of kaplan - meier survival distribution was used . statistical significance was set at p < 0.05 . surgical preparation of the cremaster muscle induces leukocyte adhesion mainly via the chemokine cxcl1-cxcr2 pathway and 2 integrins lfa1 and mac1 in the short - term model of trauma - induced inflammation [ 33 , 34 ] . in our present experiments , we analyzed the number of adherent and rolling leukocytes in postcapillary venules of the mouse cremaster muscle in response to intravenous injection of clinoleic , smoflipid , lipofundin , or normal saline . to confirm the systemic availability of the injected lipid , we first showed that blood triglyceride levels significantly increased compared to controls after all three lipids , while blood levels of cholesterol and standard liver enzymes stayed unaltered ( see supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/757059 ) . notably , the varying composition of investigated lipid emulsions did not lead to significant differences in blood triglyceride levels . next , we ruled out that alterations of leukocyte recruitment might be caused by hemodynamic changes after fluid injection , since there were no differences in hemodynamic and microvascular parameters between the different treatment groups ( supplemental table 2 ) . after injection of 1 g / kg lipofundin , the number of adherent leukocytes significantly increased when compared to control conditions ( figure 1(a ) ) . while the same amount of smoflipid slightly increased leukocyte adhesion , clinoleic injection resulted in an insignificant decrease of adherent leukocytes . since lipofundin significantly reduced the number of rolling leukocytes ( rolling flux fraction ) when compared to controls ( figure 1(b ) ) , its proinflammatory stimulation triggers the transition from leukocyte rolling to adhesion and rolling leukocytes adhere more frequently . neither clinoleic nor smoflipid treatment altered leukocyte rolling compared to controls . as reported previously during that mild and short - term inflammation of the trauma model , anti - inflammatory effects of candidate substances therefore , we argue that potentially anti - inflammatory lipid effects are difficult to examine in that model , although there was an obvious proinflammatory status in response to lipofundin . therefore , we continued with another established inflammation model of the mouse cremaster muscle . as a potent proinflammatory agent , we administered lps in a dose of 50 ng intrascrotally 3 h prior to exteriorization of the cremaster muscle and observed the lipid - induced effects on leukocyte adhesion in murine cremaster muscle venules . microvascular and hemodynamic parameters were similar between the investigated groups ( supplemental table 3 ) . in the model of lps - induced inflammation , a proinflammatory status is induced by tnf-mediated upregulation of chemokines and adhesion molecules on leukocytes and the endothelium [ 34 , 36 , 37 ] . this strong inflammation after intrascrotal injection of lps is reflected by profound leukocyte adhesion in control mice ( figure 2(a ) ) . the lps - induced leukocyte adhesion was robustly blocked by clinoleic and less pronounced by smoflipid . in contrast , administration of lipofundin did not alter leukocyte adhesion in this model when compared to control mice ( figure 2(a ) ) . as depicted in figure 2(b ) , leukocyte rolling was not affected by lipofundin and smoflipid , whereas rolling flux fraction was significantly reduced by clinoleic treatment when compared to controls . therefore , we suggest that clinoleic is able to interfere with both leukocyte rolling and adhesion . we next analyzed the number of transmigrated leukocytes in cremaster muscle whole mounts in the respective treatment groups , postulating that the lipid induced inhibition of leukocyte adhesion largely translates into transmigration ( supplemental figure 1 ) . the protective effects of smoflipid and clinoleic on leukocyte recruitment are most likely explained by their specific olive oil and/or fish oil composition . in line with our study , glatzle et al . found a decrease of lps- ( 5 mg / kg i.p . ) however , lipids were applied enterally in their study which is often not feasible in patients in the intensive care unit . our observations seem to contrast the study of buenestado et al . in rat mesenterium , which described a lipofundin - induced inhibition of the whole leukocyte recruitment cascade after superfusion with lps but no such effect in response to clinoleic . the conflicting results , however , might be due to different experimental setups ( lps application , lipid administration , and different investigated tissues and species ) leading to different involved signaling pathways . as a summary of our intravital microscopic experiments , we found that among all investigated lipids clinoleic blocked leukocyte recruitment most strongly , indicating a protective role of olive oil ( omega-9 fatty acids ) during inflammation . next , we aimed to investigate immunomodulatory effects of lipids in a clinically more relevant and well - established mouse model of lethal endotoxemia . in this inflammatory model , an intraperitoneal injection of escherichia coli lps ( 40 mg / kg ) is followed by treatment with 2 g / kg of the respective lipids ( clinoleic , smoflipid , or lipofundin ) or control solution ( equivalent volume of normal saline ) after 0.5 , 8 , and 24 hours . to quantify organ infiltration , some mice were used to investigate leukocyte infiltration into the lung 24 h after lps - injection . we observed an increased leukocyte number after application of lps ( figure 3(a ) ) that was unchanged after injection of lipofundin ( figure 3(c ) ) and hardly improved after injection of smoflipid ( figure 3(d ) ) . in line with our results in the above - mentioned inflammation models , we found a marked reduction of infiltrated pmn after application of clinoleic ( figure 3(b ) ) . hypothesized that protective effects of clinoleic during pulmonary inflammation may be caused by its antioxidative properties . in a second group , survival consistent with previous findings , survival of control mice was about 20% which stayed unaffected after injection of lipofundin and smoflipid ( 20% and 25% , resp . in contrast , clinoleic strongly improved survival during lethal endotoxemia when compared to controls ( 90% versus 20% , resp . ) . these results are consistent with former studies that observed a protective effect of olive oil in septic mice and critically ill patients [ 17 , 40 ] . however , they are in contrast to other studies describing an anti - inflammatory function of smoflipid during endotoxemia [ 4143 ] . moreover , boisram - helms et al . investigated membrane remodeling during peritonitis - induced septic shock in rats and found proinflammatory effects of mct / lct but not of lct only . this is in line with our findings , since clinoleic only contains lct ( besides olive oil ) . therefore , the varying triglyceride composition of the investigated lipid emulsions might be another reason for their different anti - inflammatory effects . taken together , in critically ill patients the optimal lipid substitution is still not clear . next , we aimed to dissect leukocyte from endothelium driven mechanisms mediating lipid - induced inhibition of leukocyte adhesion and performed flow chamber experiments . therefore , microflow chambers were coated with p - selectin , icam-1 , and cxcl1 and constantly perfused with lps - stimulated isolated murine bone marrow neutrophils with and without lipid pretreatment . we observed a significant number of adherent leukocytes in coated flow chambers when compared to uncoated flow chambers ( figure 5 ) . while clinoleic significantly blocked lps - induced leukocyte adhesion , lipofundin or smoflipid did not show such effect . these results indicate that ( amongst others ) leukocyte - born mechanisms are likely to be connected to clinoleic - induced inhibition of leukocyte recruitment , whereas any immunomodulatory effects of lipofundin or smoflipid might rather be linked to other mechanisms . altered cytokine and chemokine release could play a role in this context [ 38 , 44 ] . to further explore underlying mechanisms for the observed lipid - induced effects , we analyzed the expression of the 2 integrins lfa1 and mac1 on neutrophils in response to lipid incubation . lipofundin induced an upregulation of mac1 , while neither smoflipid nor clinoleic had any effects on the expression of mac1 ( figure 6(a ) ) . as depicted in figure 6(b ) , expression of lfa1 on bone marrow derived mouse neutrophils was not altered after any lipid incubation when compared to control neutrophils . this finding emphasizes a proinflammatory effect of lipofundin and is in line with versleijen et al . demonstrating that neutrophil activation can be triggered by mct / lct . in contrast , the study of buenestado et al . did not observe any effects of lipofundin ( mct / lct ) on integrin expression . the conflicting results might be attributable to a different experimental setting and a shorter incubation period . nevertheless , our results also indicate that the anti - inflammatory effect of clinoleic is not based on downregulation of 2 integrins . thus , we next investigated the influence of the respective lipid emulsions on the expression of psgl1 and cxcr2 . psgl1 is expressed on neutrophil granulocytes and mediates their recruitment to inflamed tissues via binding to selectins . cxcr2 expression was downregulated by both clinoleic and smoflipid , but not by lipofundin ( figure 7(a ) ) . psgl1 showed a marked downregulation after application of clinoleic only , whereas smoflipid and lipofundin displayed no effect ( figure 7(b ) ) . these results suggest that anti - inflammatory properties of clinoleic are mediated by psgl1 and cxcr2 and those of smoflipid by cxcr2 , which is in line with our observation of reduced lps - induced leukocyte rolling after clinoleic administration only . although versleijen et al . demonstrated that neutrophil activation can be triggered by mct / lct we next addressed the question whether lipids alter the expression of endothelial leukocyte adhesion molecules like icam-1 and vcam-1 . therefore , lps - stimulated icam-1 and vcam-1 expression was assessed on maecs by flow cytometry . in line with previous studies while none of the applied lipids altered icam-1-expression ( figure 8(a ) ) , lps - induced vcam-1 expression was downregulated by smoflipid and clinoleic ( figure 8(b ) ) , indicating that an anti - inflammatory effect of omega 3 and omega 9 fatty acids might be attributable to endothelial vcam-1 downregulation in our experimental setting . this finding is supported by the study of tull et al . which investigated endothelial mechanisms of lipid - mediated immunomodulation . clinoleic exerted the strongest anti - inflammatory properties during local and systemic inflammation in vivo when compared to the lipid composition smoflipid or lipofundin . in turn , clinoleic was the only investigated lipid emulsion that improved survival during lethal endotoxemia . although olive oil - based lipids seem to be a beneficial alternative to other lipid emulsions , future studies are needed to confirm the anti - inflammatory potential of clinoleic in critically ill patients .

although fish oil - based and olive oil - based lipid emulsions have been shown to exert anti - inflammatory functions , the immunomodulating properties of lipids are still controversial . therefore , we investigated the anti - inflammatory effect of three different parenterally administered lipid emulsions in vivo : olive oil - based clinoleic , fish oil - based smoflipid , and soybean oil - based lipofundin . we observed leukocyte recruitment in inflamed murine cremaster muscle using intravital microscopy and survival in a murine model of lps - induced systemic inflammation and analyzed expression of leukocyte and endothelial adhesion molecules . olive oil - based clinoleic and fish oil - based smoflipid profoundly inhibited leukocyte adhesion compared to lipofundin during lps - induced inflammation of the murine cremaster muscle . in the trauma model of cremaster muscle inflammation , lipofundin was the only lipid emulsion that even augmented leukocyte adhesion . in contrast to smoflipid and lipofundin , clinoleic effectively blocked leukocyte recruitment and increased survival during lethal endotoxemia . flow chamber experiments and analysis of adhesion molecule expression suggest that both endothelial and leukocyte driven mechanisms might contribute to anti - inflammatory effects of clinoleic . we conclude that the anti - inflammatory properties of clinoleic are superior to those of smoflipid and lipofundin even during systemic inflammation . thus , these results should stimulate further studies investigating parenteral lipids as an anti - inflammatory strategy in critically ill patients .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

however , numerous studies reported complications during parenteral nutrition , like parenteral nutrition associated liver disease or detrimental effects of parenteral lipids on survival and inflammatory response during sepsis [ 4 , 5 ] . this may also be due to lipid - induced decrease of neutrophil function and cytokine release in septic patients [ 6 , 7 ] . in recent years , the composition of lipids was mainly based on soybeans which contain high amounts of omega-6-polyunsaturated fatty acids . during the beginning of parenteral nutrition , intralipid which only contains soybean - based long - chain - triglycerides ( lct ) was frequently used . eicosanoids act as immunomodulators , serve as signaling molecules , and contribute to inflammatory conditions . one important aspect of the observed effects is the change of cell membrane fluidity by parenterally administered fatty acids . in order to attenuate these serious side effects of pufa lipofundin is one alternative that substituted 50% of lct with medium - chain - triglycerides ( mct ) that are metabolized more rapidly than lct , thereby displaying less immunosuppressive properties and exerting better effects on membrane function . one study that compared the respiratory burst of human neutrophils found a reduced effect with lipofundin when compared to other lipid emulsions . in contrast to lipofundin and intralipid , olive oil - based lipids were shown to have a protective effect against lps - induced inflammation . however , there is an ongoing discussion about beneficial properties of fish oil - based lipids ( i.e. , smoflipid ) when compared with predominant olive oil - based lipids ( i.e. the beneficial effects are at least in part attributed to a favorable ratio of omega-6 : omega-3 fatty acids and the balanced mixture of different lipid ingredients ( lct , mct , and olive oil ) . in addition , there are still conflicting results about how parenterally administered lipids might interfere with leukocyte recruitment which is known to be a sensitive indicator of inflammation . based on the above - mentioned controversies about lipid - induced immunomodulation , we now aimed to compare anti - inflammatory effects of lipofundin , smoflipid , and clinoleic in vitro and in vivo with special regard to leukocyte recruitment during local and systemic inflammation . clinoleic ( fresenius kabi , bad homburg , germany ) contains 80% olive oil and 20% soybean oil ( lct ) . smoflipid ( fresenius kabi , bad homburg , germany ) consists of 30% soybean oil ( lct ) , 30% mct , 25% olive oil , and 15% fish oil ( rich in omega 3 fatty acids ) . the lipid emulsions were administered as an intravenous bolus injection at 1 g / kg for the intravital microscopic experiments and at 2 g / kg 30 min , 8 h , and 24 h after lps during lps - induced endotoxemia . mice were prepared for intravital microscopy , as reported recently . the lipid emulsions were administered with a dose of 1 g / kg as an intravenous bolus injection . blood levels of cholesterol , triglycerides , and liver enzymes were measured after the respective experiments in the core laboratory facility of the university hospital heidelberg ( analysezentrum , heidelberg , germany ) . the surgical preparation ( trauma - induced inflammation ) of the cremaster muscle after longitudinal incision and spreading of the muscle over a cover glass , the epididymis and testis were mobilized and pinned aside leading to full microscopic access to the cremaster muscle microcirculation . in certain experiments mice were injected with 50 ng lps ( escherichia coli ; serotype 055:b5 sigma , taufkirchen , germany ) intrascrotally ( lps - induced inflammation ) . to differentially count transmigrated leukocytes , cremaster muscle - whole mounts were prepared as described before . briefly , while the cremaster muscle was still mounted on the stage for intravital microscopy , the tissue was fixed with 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) . the cremaster muscle was removed and mounted flat on a superfrost glass slide ( menzel , braunschweig , germany ) , air dried for 510 min , and fixed in 4% paraformaldehyde in 0.1 m phosphate buffer ( ph 7.4 ) for 24 h at 4c . after fixation , the tissue was washed three times in 0.1 m phosphate buffer with 5% ethanol , stained with giemsa ( sigma , taufkirchen , germany ) at room temperature for 5 min , and differentiated in 0.01% acetic acid for contrast . the differentiated slides were washed in water , 75% ethanol , 95% ethanol , 100% ethanol , and fresh xylene , followed by mounting in mounting media ( agar scientific , stansted , uk ) . in brief , 3 mm long freshly harvested and cleaned aortic rings were seeded into matrigel - coated culture dishes ( bd , san jose , ca , usa ) and incubated at 37c , 5% co2 in dulbecco 's modified eagle medium ( supplemented with 15% fetal bovine serum , 1% pen / strep , 90 g / ml heparin , 60 g / ml endothelial cell growth supplement , and 1 g / ml amphotericin b ; fungizone , invitrogen , karlsruhe , germany ) . respective lipid pretreatment ( clinoleic , lipofundin or smoflipid at 10 mg/10 cells ) was initiated together with lps - stimulation . for flow cytometric analysis of icam-1 and vcam-1 expression on endothelial cells , prepared maecs were harvested and incubated in the dark for 45 minutes on ice with pe - conjugated anti - icam-1 mab ( clone yn1/1.7.4 ebioscience , san diego , ca , usa ) , anti - mouse vcam-1 mab ( clone 429 mvcam.a biolegend , san diego , ca , usa ) , or respective isotype control antibody ( ebioscience , san diego , ca , usa and bd ) to detect anti - icam-1 and vcam-1 signal on 10.000 cells using the 4-decade facs - scan lsrii with diva software package ( bd ) . the expression of cxcr2 , psgl1 , mac1 , and lfa1 was assessed using isolated bone marrow - derived neutrophils ( see above ) . after red blood cell lysis , 10 leukocytes / ml were stimulated for 3 h with 10 mg lipofundin , clinoleic , or smoflipid , respectively , at 37c . next , cells were incubated in the dark with phycoerythrin - conjugated anti - cxcr2 mab ( 1 g/10 cells ; ebioscience , frankfurt , germany ) , anti - psgl-1 mab ( 1 g/10 cells , bd pharmingen , san diego , ca , usa ) , fitc - conjugated anti - mac1 mab m1/70 ( 1 g/10 cells , rat igg2b ; ebioscience , san diego , ca , usa ) , fitc - conjugated anti - lfa1 mab m17/4 ( 1 g/10 cells , rat igg2a ; ebioscience , san diego , ca , usa ) , or respective isotype control antibodies ( 1 g/10 cells , rat igg2b or rat igg2a ; ebioscience , san diego , ca , usa ) . expression upon stimulation with different lipid emulsions was compared to unstimulated cells and their respective isotype controls . in certain experiments lps flow chamber experiments were conducted as described [ 33 , 34 ] . in brief , rectangular microglass capillaries ( vitrocom , mountain lakes , nj , usa ) were coated with rmp - selectin ( 2 g / ml ) , rmcxcl1 ( 5 g / ml ) , and icam-11 ( 1 g / ml ) and connected via pe tubing to a 2 ml syringe containing freshly isolated bone marrow neutrophils from lysegfp mice . in lysegfp mice , the enhanced gfp ( egfp ) is knocked into the murine lysozyme m ( lys ) locus leading to the expression of egfp in myelomonocytic cells . in some experiments , cell suspensions were incubated with either lipofundin , smoflipid , or clinoleic with a dose of 10 mg/10 cells for 3 h at 37c . clinoleic , smoflipid , lipofundin , or equivalent volume of normal saline was administered i.v . in a first group , survival was observed for 14 days . leukocyte counts , vessel diameters , leukocyte adhesion , leukocyte rolling flux fractions , wall shear rates , and in vitro leukocyte adhesion between groups and treatments were compared with one - way anova followed by a multiple pairwise comparison test ( dunn 's test ) or by wilcoxon rank - sum test , as appropriate . to compare the survival during lethal endotoxemia , log - rank test of kaplan - meier survival distribution was used . surgical preparation of the cremaster muscle induces leukocyte adhesion mainly via the chemokine cxcl1-cxcr2 pathway and 2 integrins lfa1 and mac1 in the short - term model of trauma - induced inflammation [ 33 , 34 ] . in our present experiments , we analyzed the number of adherent and rolling leukocytes in postcapillary venules of the mouse cremaster muscle in response to intravenous injection of clinoleic , smoflipid , lipofundin , or normal saline . to confirm the systemic availability of the injected lipid , we first showed that blood triglyceride levels significantly increased compared to controls after all three lipids , while blood levels of cholesterol and standard liver enzymes stayed unaltered ( see supplemental table 1 in supplementary material available online at http://dx.doi.org/10.1155/2015/757059 ) . notably , the varying composition of investigated lipid emulsions did not lead to significant differences in blood triglyceride levels . next , we ruled out that alterations of leukocyte recruitment might be caused by hemodynamic changes after fluid injection , since there were no differences in hemodynamic and microvascular parameters between the different treatment groups ( supplemental table 2 ) . after injection of 1 g / kg lipofundin , the number of adherent leukocytes significantly increased when compared to control conditions ( figure 1(a ) ) . while the same amount of smoflipid slightly increased leukocyte adhesion , clinoleic injection resulted in an insignificant decrease of adherent leukocytes . neither clinoleic nor smoflipid treatment altered leukocyte rolling compared to controls . as reported previously during that mild and short - term inflammation of the trauma model , anti - inflammatory effects of candidate substances therefore , we argue that potentially anti - inflammatory lipid effects are difficult to examine in that model , although there was an obvious proinflammatory status in response to lipofundin . therefore , we continued with another established inflammation model of the mouse cremaster muscle . as a potent proinflammatory agent , we administered lps in a dose of 50 ng intrascrotally 3 h prior to exteriorization of the cremaster muscle and observed the lipid - induced effects on leukocyte adhesion in murine cremaster muscle venules . in the model of lps - induced inflammation , a proinflammatory status is induced by tnf-mediated upregulation of chemokines and adhesion molecules on leukocytes and the endothelium [ 34 , 36 , 37 ] . this strong inflammation after intrascrotal injection of lps is reflected by profound leukocyte adhesion in control mice ( figure 2(a ) ) . the lps - induced leukocyte adhesion was robustly blocked by clinoleic and less pronounced by smoflipid . in contrast , administration of lipofundin did not alter leukocyte adhesion in this model when compared to control mice ( figure 2(a ) ) . as depicted in figure 2(b ) , leukocyte rolling was not affected by lipofundin and smoflipid , whereas rolling flux fraction was significantly reduced by clinoleic treatment when compared to controls . therefore , we suggest that clinoleic is able to interfere with both leukocyte rolling and adhesion . we next analyzed the number of transmigrated leukocytes in cremaster muscle whole mounts in the respective treatment groups , postulating that the lipid induced inhibition of leukocyte adhesion largely translates into transmigration ( supplemental figure 1 ) . the protective effects of smoflipid and clinoleic on leukocyte recruitment are most likely explained by their specific olive oil and/or fish oil composition . however , lipids were applied enterally in their study which is often not feasible in patients in the intensive care unit . in rat mesenterium , which described a lipofundin - induced inhibition of the whole leukocyte recruitment cascade after superfusion with lps but no such effect in response to clinoleic . the conflicting results , however , might be due to different experimental setups ( lps application , lipid administration , and different investigated tissues and species ) leading to different involved signaling pathways . as a summary of our intravital microscopic experiments , we found that among all investigated lipids clinoleic blocked leukocyte recruitment most strongly , indicating a protective role of olive oil ( omega-9 fatty acids ) during inflammation . next , we aimed to investigate immunomodulatory effects of lipids in a clinically more relevant and well - established mouse model of lethal endotoxemia . in this inflammatory model , an intraperitoneal injection of escherichia coli lps ( 40 mg / kg ) is followed by treatment with 2 g / kg of the respective lipids ( clinoleic , smoflipid , or lipofundin ) or control solution ( equivalent volume of normal saline ) after 0.5 , 8 , and 24 hours . we observed an increased leukocyte number after application of lps ( figure 3(a ) ) that was unchanged after injection of lipofundin ( figure 3(c ) ) and hardly improved after injection of smoflipid ( figure 3(d ) ) . in line with our results in the above - mentioned inflammation models , we found a marked reduction of infiltrated pmn after application of clinoleic ( figure 3(b ) ) . hypothesized that protective effects of clinoleic during pulmonary inflammation may be caused by its antioxidative properties . in contrast , clinoleic strongly improved survival during lethal endotoxemia when compared to controls ( 90% versus 20% , resp . ) these results are consistent with former studies that observed a protective effect of olive oil in septic mice and critically ill patients [ 17 , 40 ] . however , they are in contrast to other studies describing an anti - inflammatory function of smoflipid during endotoxemia [ 4143 ] . investigated membrane remodeling during peritonitis - induced septic shock in rats and found proinflammatory effects of mct / lct but not of lct only . therefore , the varying triglyceride composition of the investigated lipid emulsions might be another reason for their different anti - inflammatory effects . taken together , in critically ill patients the optimal lipid substitution is still not clear . next , we aimed to dissect leukocyte from endothelium driven mechanisms mediating lipid - induced inhibition of leukocyte adhesion and performed flow chamber experiments . therefore , microflow chambers were coated with p - selectin , icam-1 , and cxcl1 and constantly perfused with lps - stimulated isolated murine bone marrow neutrophils with and without lipid pretreatment . we observed a significant number of adherent leukocytes in coated flow chambers when compared to uncoated flow chambers ( figure 5 ) . while clinoleic significantly blocked lps - induced leukocyte adhesion , lipofundin or smoflipid did not show such effect . these results indicate that ( amongst others ) leukocyte - born mechanisms are likely to be connected to clinoleic - induced inhibition of leukocyte recruitment , whereas any immunomodulatory effects of lipofundin or smoflipid might rather be linked to other mechanisms . to further explore underlying mechanisms for the observed lipid - induced effects , we analyzed the expression of the 2 integrins lfa1 and mac1 on neutrophils in response to lipid incubation . as depicted in figure 6(b ) , expression of lfa1 on bone marrow derived mouse neutrophils was not altered after any lipid incubation when compared to control neutrophils . in contrast , the study of buenestado et al . nevertheless , our results also indicate that the anti - inflammatory effect of clinoleic is not based on downregulation of 2 integrins . thus , we next investigated the influence of the respective lipid emulsions on the expression of psgl1 and cxcr2 . cxcr2 expression was downregulated by both clinoleic and smoflipid , but not by lipofundin ( figure 7(a ) ) . psgl1 showed a marked downregulation after application of clinoleic only , whereas smoflipid and lipofundin displayed no effect ( figure 7(b ) ) . these results suggest that anti - inflammatory properties of clinoleic are mediated by psgl1 and cxcr2 and those of smoflipid by cxcr2 , which is in line with our observation of reduced lps - induced leukocyte rolling after clinoleic administration only . demonstrated that neutrophil activation can be triggered by mct / lct we next addressed the question whether lipids alter the expression of endothelial leukocyte adhesion molecules like icam-1 and vcam-1 . therefore , lps - stimulated icam-1 and vcam-1 expression was assessed on maecs by flow cytometry . in line with previous studies while none of the applied lipids altered icam-1-expression ( figure 8(a ) ) , lps - induced vcam-1 expression was downregulated by smoflipid and clinoleic ( figure 8(b ) ) , indicating that an anti - inflammatory effect of omega 3 and omega 9 fatty acids might be attributable to endothelial vcam-1 downregulation in our experimental setting . clinoleic exerted the strongest anti - inflammatory properties during local and systemic inflammation in vivo when compared to the lipid composition smoflipid or lipofundin . in turn , clinoleic was the only investigated lipid emulsion that improved survival during lethal endotoxemia . although olive oil - based lipids seem to be a beneficial alternative to other lipid emulsions , future studies are needed to confirm the anti - inflammatory potential of clinoleic in critically ill patients .

at low levels of central hypovolemia elicited by mild lbnp in humans , msna increases in the absence of alterations in heart rate ( hr ) or blood pressure ( sundlof and wallin , 1978a ; victor and leimbach , 1987 ) . this initial increase in msna occurs in a similar fashion whether measured from the radial nerve in the arm or the peroneal nerve in the leg , and is therefore a reflection of generalized sympathetic activation to muscle vascular beds ( rea and wallin , 1989 ) . importantly , the response in msna to relatively low levels of lbnp ( i.e. , 10 mmhg ) is identical to the msna response to blood loss of 450 ml , demonstrating the effectiveness of lbnp as an experimental model of hemorrhage ( figure 1 ; rea et al . , 1991 ) . comparison of relationships between central venous pressure ( cvp ) and muscle sympathetic nerve activity ( msna ) during 10 mmhg lbnp and 450 ml hemorrhage in nine human subjects . circles and lines represent mean se values . * p < 0.05 compared with baseline . from cooke et al . because arterial pressure was unchanged , the increase in msna elicited by low levels of lbnp or hemorrhage was originally ascribed to deactivation of cardiopulmonary baroreceptors alone , without contribution from arterial baroreceptors ( rea et al . , 1991 ) . in fact , so many investigators have used low levels of lbnp ( 20 mmhg ) to selectively unload cardiopulmonary baroreceptors that this has recently been referred to as dogma subsequent work , however , has conclusively demonstrated that deactivation of arterial baroreceptors also contributes to sympathoexcitation induced by non - hypotensive hypovolemia . first , non - hypotensive hypovolemia reduces the diameter of both the ascending thoracic aorta ( taylor et al . , 1995 ) and the carotid artery ( lacolley et al . , 1992 ) , sites of the stretch - sensitive aortic and carotid arterial baroreceptors . ( 2001 ) demonstrated that , during lbnp of only 5 mmhg , msna increased while parasympathetic modulation of hr ( assessed via power spectral analysis ) decreased , which is a manifestation of arterial baroreceptor unloading . third , msna increases during mild hypovolemia in both intact control subjects and in cardiac transplant patients , indicating a greater contribution to sympathoexcitation of sinoaortic baroreflexes than ventricular receptors ( jacobsen et al . , 1993 ) . 2009 ) recently observed that transient reductions in stroke volume and blood pressure [ both systolic ( sap ) and diastolic ( dap ) pressures ] occur at the onset of even mild levels of lbnp . taken together , it has become clear that the sympathoexcitation produced by even low levels of central hypovolemia is a result of reflex - mediated deactivation of both cardiopulmonary and arterial baroreceptors . additionally , recent evidence suggests that reflexes from muscle afferents stimulated during venous distension may also directly evoke increases in msna ( cui et al . , 2009 , 2011a ) . while venous distension should not occur during hemorrhage , the possibility that reflexes arising from distension of leg veins contribute to the sympathoexcitation observed during lbnp can not be discounted . once initiated , msna has been proposed to linearly increase during progressive central hypovolemia , although much of the data supporting this suggestion until recently has been accrued using lower levels of lbnp ( 30 mmhg ; convertino and cooke , 2002 ; cooke et al . , 2004 ) . this is because of the inherent difficulty in maintaining nerve recordings during higher levels of negative pressure , as the microelectrode is often displaced during the lbnp procedure . to mitigate this issue , khan et al . ( 2002 ) applied negative pressure to only one leg while performing microneurography in the other . using this protocol , these investigators demonstrated that progressive reductions in lbnp ( up to 50 mmhg ) produce concomitant graded increases in msna , but they did not measure the degree of central hypovolemia induced by lbnp in order to quantify the relationship between volume loss and sympathetic activation ( khan et al . , 2002 ) . over the past 6 years , we have created a large database of human experiments ( > 200 subjects ) in which progressive central hypovolemia has been used to produce hemodynamic decompensation ( i.e. , presyncope ) . in each subject , chamber decompression was applied for 5 min at 15 , 30 , 45 , and 60 mmhg , and then additional increments of 10 mmhg were used until the onset of decompensation . in the course of this work , we have obtained microneurography recordings in a subset of subjects in which stroke volume , a measure of central hypovolemia , has also been assessed . we were able to maintain msna recordings throughout lbnp to either the point of presyncope or to a high ( 80 mmhg ) lbnp level in 20 subjects . this unique data set has provided the opportunity to develop new understandings of the msna response to intense levels of central hypovolemia . for example , figure 2 shows msna recordings from a representative subject exposed to progressively higher levels of lbnp . average values across subjects demonstrate that increases in msna burst frequency are inversely related to stroke volume levels during central hypovolemia in humans ( figure 3 ) , thereby confirming the linearity of the relationship between central hypovolemia and the induced msna response . this same relationship between central hypovolemia and direct measurement of renal sympathetic nerve activity ( rsna ) is also observed in anesthetized sheep during graded hemorrhage ( batchinsky et al . , 2007b ) . it should be noted that progressive increases in sympathetic activation during lbnp are evident whether msna is expressed as frequency ( bursts / min ) , incidence ( bursts/100 heartbeats ) , or total activity ( which takes into account burst amplitude ; cooke et al . muscle sympathetic nerve activity ( msna ) in a representative subject during progressive lower body negative pressure . from cooke et al . relationship between stroke volume ( sv ) and muscle sympathetic nerve activity ( msna ) during lbnp in 20 human subjects . in addition to increases in absolute msna , progressive central hypovolemia also changes the pattern of bursting activity . even at rest , sympathetic nerve bursting is characterized by a rhythmic pattern that exists in the low frequency ( lf ) range ( 0.040.15 hz ) ; this oscillatory pattern in sympathetic nerve activity contributes to the 10-s arterial pressure mayer waves ( preiss and polosa , 1974 ) . while it is beyond the scope of this review , there is a continued controversy regarding whether the oscillatory pattern of sympathetic nerve activity and mayer waves is produced via a central oscillator mechanism or through the action of the baroreflex ( for a review of this issue , see julien , 2006 ) . in animal models of hemorrhage , the amplitude of lf oscillations in blood pressure and sympathetic nerve activity increase ( guyton and harris , 1951 ; malpas et al . , 1998 ) . in humans , the amplitude of lf oscillations in msna ( msnalf ) increase concomitantly with burst frequency and total activity during stresses that reduce venous return such as head - up tilt ( cooke et al . , 1999 ; furlan et al . , 2000 ; kamiya et al . , 2005 ) and lbnp ( ryan et al . , 2011 ) . in addition to increases in the frequency and amplitude of sympathetic nerve firing , it is therefore possible that alterations in the oscillatory patterns of that firing may contribute to the ability of an individual to adequately compensate for progressive central hypovolemia ( see below ) . sympathetic nerves normally fire in response to reductions of arterial pressure ( particularly , dap ) and are silenced with the consequent sap upstroke , resulting in almost constant latencies from preceding r - waves ( sundlof and wallin , 1978b ; fagius and wallin , 1980 ) . at high levels of lbnp , however , an interesting phenomenon occurs in some subjects . figure 4 depicts msna from a subject at baseline , during 60 and 90 mmhg of lbnp ( cooke et al . , 2009 ) . at 60 mmhg , coupling of bursts occurred followed by fusing of multiple bursts with greater central hypovolemia ( i.e. , at 90 mmhg ) . in our study , burst fusion occurred in 10 of the 17 subjects available for study at the time of publication ( cooke et al . , 2009 ) . burst fusion did not appear to be artifactual , although we can not completely discount the possibility that the 0.1-s time constant used to integrate msna may have contributed to their appearance ( cooke et al . , 2009 ) . similar alterations in the typical pulse synchronous burst pattern of msna had previously been noted in subjects susceptible to fainting during head - up tilting ; during the presyncopal ( i.e. , decompensation ) phase , burst reflex latencies were shortened and burst durations were lengthened , effects which were reversed on return to the fully conscious state ( iwase et al . our data extend this observation to the earlier compensatory phase of central hypovolemia , when blood pressure is still well - maintained . importantly , fusion of rsna has also been observed in anesthetized sheep made hypotensive by severe hemorrhage ( batchinsky et al . , 2007b ) . we have suggested that this phenomenon may represent sympathetic baroreflex deafferentation ( cooke et al . , 2009 ) , as the fused bursts observed during intense lbnp are similar in both their pulse asynchrony and their structure to those observed after bilateral blocks of glossopharyngeal and vagus nerves ( fagius et al . , 1985 ) . it is also possible that loss of pulse - synchrony and continuous burst firing may occur in some individuals as a strategy to continue to maintain blood pressure during severe reductions in venous return . in support of this concept , ( 2011 ) have recently shown that extreme baroreceptor unloading requiring high sympathetic outflow ( induced by 80 mmhg lbnp ) recruits a subpopulation of large postganglionic axons in some but not all individuals . coupling ( 60 mmhg ) and then fusing ( 90 mmhg ) of muscle sympathetic nerve activity ( msna ) in one subject during lower body negative pressure . from cooke et al . ( 1944 ) observed that the onset of fainting produced by experimental hemorrhage in man was preceded by a profound vasodilation in the forearm and a decrease in systemic vascular resistance , suggesting inhibition of the compensatory sympathetic activation that had occurred before this point . as it became possible to directly measure sympathetic nerve activity in humans , a variety of case reports and experimental studies appeared showing an inhibition of sympathetic activity associated with the development of presyncope in both healthy humans ( burke et al . , 1977 ; sanders and ferguson , 1989 ; scherrer et al . iwase et al . , 2000 ; cooke and convertino , 2002 ) and in patient populations ( wallin and sundlof , 1982 ; yatomi et al . , 1989 ; converse et al . , 1992 ; jardine et al . , 1996 , 1998 , 2002 ; morillo et al . , 1997 ) . on the basis of these data , it was concluded that hemodynamic decompensation ( i.e. , presyncope ) was associated with and possibly caused by sympathetic neural withdrawal ( convertino and cooke , 2002 ; cooke et al . , 2004 ) . recently , however , we have observed that sympathetic withdrawal does not occur in all subjects at the point of hemodynamic decompensation ( figure 5 ) ; in fact , 41% of our subjects did not demonstrate any diminution of msna despite the onset of hypotension and presyncopal symptoms ( cooke et al . , 2009 ) . 2010 ) showed that increases in msna induced by orthostatic stress were preserved through the point of syncope in 90% of patients previously diagnosed with vagovagal syncope . it is therefore apparent that sympathetic withdrawal is not an absolute requirement for the onset of hemodynamic decompensation in both healthy humans and in patients with a history of syncope . indeed , careful perusal of some of the literature frequently cited to support the proposition that sympathetic withdrawal precipitates cardiovascular collapse reveals that blood pressure begins to decrease while msna remains elevated during the compensatory phase of hypovolemia ( wallin and sundlof , 1982 ; sanders and ferguson , 1989 ; converse et al . , 1992 ; jardine et al . , 1996 , 1998 ; mosqueda - garcia et al . , 1997 ; iwase et al . , 2000 ) , an observation confirmed more recently ( kamiya et al . , , these data refute the concept that cessation of sympathetic nerve activity precedes and causes hypotension , the subsequent reduction in cerebral perfusion pressure , and syncope in all subjects . arterial pressure ( ap ) and muscle sympathetic nerve activity ( msna ) for three representative subjects 2 min before the onset of presyncope . the lowest arterial pressure ( bp ) recorded for each subject a185 , msna decreased in the last 2 min of lbnp ( 120 to 60 s , 82 bursts / min ; 60 s to presyncope , 64 bursts / min ) , but was still maintained at high levels relative to the pre - lbnp control ( 36 bursts / min ) . subject a075 displayed burst fusion , elevated msna over control ( 25 bursts / min ) , and no withdrawal of msna at presyncope ( 120 to 60 s , 53 bursts / min ; 60 s to presyncope , 54 bursts / min ) . subject a199 ( a low tolerant subject ) did not display large increases in msna from the pre - lbnp control value ( 17 bursts / min ) and also did not demonstrate msna withdrawal at presyncope ( 120 to 60 s , 22 bursts / min ; 60 s to presyncope , 21 bursts / min ) . from cooke et al . another possible mechanism that may contribute to the onset of cardiovascular collapse is resetting of baroreflexes , resulting in a loss of synchrony between arterial blood pressure and compensatory responses such as sympathetic activation . during mild to moderate central hypovolemia , the operating point of baroreflex - mediated control of sympathetic nerve activity is shifted upward without a change in gain , such that sympathetic nerve activity is increased at any given dap ( ichinose et al . additionally , there is a tighter coupling between oscillations in arterial blood pressure and msna , as quantitated using cross - spectral analysis by an increase in the coherence function between these variables ( furlan et al . , 2000 ; this increase in coherence reflects greater baroreflex modulation of sympathetic activity compared with the baseline state and both the increase in coherence and operating point may be beneficial in mounting an appropriate compensatory response to hypovolemia . evidence exists , however , to suggest that this tight coupling of blood pressure to sympathetic nerve activity may be lost at the point of hemodynamic decompensation . based on their observation of sympathoinhibition ( as measured by plasma catecholamines ) despite hypotension , jacobs et al . ( 1995 ) first suggested that the vasodepressor response at presyncope was due to a sudden central resetting of baroreflexes . ( 2006 ) using data from subjects who either exhibited presyncope or did not during central hypovolemia induced by lbnp . approximately 12 min prior to the onset of hemodynamic decompensation , the gain of the baroreflex function relating dap to msna was substantially reduced in those subjects exhibiting presyncope but was unchanged in non - presyncopal subjects ( ichinose et al . , 2006 ) . likewise , we have also demonstrated a loss of linearity between the change in dap and the change in msna just before the onset of presyncope , suggesting disruption in the normal baroreflex - mediated coordination between arterial pressure and sympathetic activation ( cooke et al . , 2009 ; convertino et al . , taken together , these results suggest that impairment of arterial baroreflex control over sympathetic vasomotor activity may contribute to the onset of hemodynamic decompensation . such a sudden attenuation of baroreflex function before hemodynamic decompensation has also been noted for cardiovagal reflexes ( ogoh et al . as mentioned above , there is also evidence to suggest that the pattern of msna firing may be as important in determining the ability to withstand central hypovolemia as the absolute level of msna . in an elegant study , ( 2005 ) determined the temporal occurrence of events immediately preceding presyncope . during central hypovolemia induced by head - up tilt , both the absolute levels of msna ( expressed as burst frequency and total activity ) and the amplitude of lf oscillations in msna ( msnalf ) increased markedly ; increases in msnalf were reflected in increases in lf oscillations of mean arterial pressure ( maplf ) . as subjects moved toward presyncope , msnalf and maplf decreased and were associated with a decrease in map , despite the maintenance of msna burst frequency and total activity at their elevated level . msna , msnalf , and maplf were maintained and did not decrease in those subjects who did not exhibit hypotension and presyncope ( kamiya et al . , we have observed a similar phenomenon in a subject whose tolerance to central hypovolemia was improved through the use of inspiratory resistance breathing ( figure 6 ) . in this experiment , subjects were exposed to lbnp to the point of presyncope in separate experiments performed at least 2 weeks apart . in one experiment , subjects breathed through a device that did not provide resistance to inspiration ( sham ) , while in the other experiment , subjects breathed through a device that provided resistance to inspiration ( active ) . because resistance breathing improved lbnp tolerance , data were analyzed at the end of lbnp during the sham experiment and at this same absolute time point during the active experiment ( i.e. , well before the onset of presyncope during inspiratory resistance breathing ) . during breathing with the sham device , msna increased from 12 bursts / min at baseline to 44 bursts / min at the end of lbnp , while msna increased from 10 to 54 bursts / min at this same absolute time point ( i.e. , prior to presyncope ) during resistance breathing . importantly , msnalf increased from 1.15 to 22.4 au at presyncope under the sham resistance breathing condition ; with resistance breathing , msnalf increased from 2.23 to 40.1 au at this same absolute time point . moreover , the coherence between dap and msna , a measure of the strength of the relationship between these two variables , was increased from 0.80 in the sham condition to 0.94 with resistance breathing at this same time point . thus , in this one subject , it is apparent that inspiratory resistance breathing increased the amplitude of msna oscillations and coherence between msna and arterial blood pressure , and that this alteration in oscillatory pattern was associated with improved tolerance to central hypovolemia . these data are consistent with the concept that the maintenance of an increase in lf oscillations of both msna and map may be associated with the defense of blood pressure during severe central hypovolemia . blood pressure ( bp ) , muscle sympathetic nerve activity ( msna ) and end - tidal co2 ( et co2 ) tracings for a single subject under conditions of breathing with a sham ( top ) or active ( bottom ) device that provided resistance to inspiration . thus , there are several possible mechanisms involving alterations in activation of sympathetic nerve activity to explain hemodynamic decompensation during severe hypovolemia . since sympathetic withdrawal occurs in some individuals before presyncope but not in others , it is no longer thought to be a prerequisite for the ensuing hypotension ( cooke et al . , 2009 ) . it is also possible that there is a central resetting of arterial baroreflex function that alters sympathetic outflow ( ichinose et al . , 2006 ) ; this scenario may be especially prominent in those subjects in whom sympathetic inhibition occurs despite progressive hypotension . finally , there is evidence to suggest that an increase in lf oscillations in both msna and map may be protective during central hypovolemia and that loss of these oscillations might precipitate hypotension ( kamiya et al . , 2005 ) . it is important to note that these mechanisms are not mutually exclusive and the contributions of each to the process have yet to be fully revealed . furthermore , it is also possible and even probable that different mechanisms may predominate in different individuals . because of the scope of this review , we have chosen to focus on those mechanisms preceding presyncope that involve loss of compensatory alterations in msna , but it is likely that loss of other compensatory responses may also contribute to the inability to maintain blood pressure during severe hypovolemia . 1997 ) is that marked peripheral vasodilation is a major contributor to the fall in arterial pressure preceding vasovagal syncope . in this regard , it is of interest that , during prolonged lbnp at a low level ( 15 mmhg ) , vasodilation of the forearm musculature was observed despite the continued presence of a sustained compensatory increase in msna , suggesting that sympathetic escape occurs ( joyner et al . , implicit in the preceding discussion is the notion that there are individual differences in the ability of healthy humans to tolerate central hypovolemia before reaching the point of cardiovascular collapse . indeed , it has been known for many years that there is a great deal of variability in the ability of patients ( davis , 1949 ) and animals ( chien , 1967 ; kim and shoemaker , 1970 ) to survive traumatic hemorrhage ; we have recently learned that there is a genetic basis underlying this variability ( klemcke et al . , 2008 , 2011 ) . likewise , individual differences in tolerance to central hypovolemia induced by lbnp have also been described ( sather et al . , 1986 ) and attributed to differences in the release of vasoactive hormones ( convertino and sather , 2000b ; greenleaf et al . , 2000 ) , compensatory tachycardia and vasoconstriction ( convertino and sather , 2000a , b;greenleaf et al . , 2000 ) , cardiac baroreflex gain ( convertino and sather , 2000a ; convertino et al . , in press ) , baroreflex gain of sympathetic nerve activation ( wijeysundera et al . , 2001 ) , and central blood volume and cerebral blood velocity ( levine et al . , 1994 ) . in this regard , we have recently shown that subjects demonstrating high tolerance ( ht ) to lbnp have a greater ability to increase msna than subjects with low tolerance ( lt ; convertino et al . , in press ) . additionally , the ability to sustain the compensatory mechanisms described above involving both the baroreflex modulation of sympathetic activation and the oscillatory component of that activation may also act to determine tolerance to central hypovolemia . msna baroreflex in subjects who became presyncopal ( i.e. , those demonstrating lt ) during central hypovolemia but did not observe this resetting in those who did not ( ht ) . ( 2005 ) observed a loss of msnalf and maplf power in lt subjects before presyncope elicited by head - up tilt , but no such loss in ht subjects . ; convertino and sather , 2000a , b ) , we recently confirmed and expanded on these results using our large database of subjects in which lbnp was applied to the point of presyncope in all individuals . we classified subjects as ht if they completed at least the 60-mmhg level of lbnp , and lt if they did not complete this level ( rickards et al . , 2011 ) . as in the previous study using head - up tilt ( kamiya et al . , 2005 ) , maplf increased in both lt and ht groups during early stages of lbnp , but maplf decreased to baseline levels in the lt group at 60 mmhg while it continued to increase in the ht group ( rickards et al . , 2011 ) . a similar relationship between the ability to increase orthostatic tolerance and the ability to increase blood pressure oscillations has previously been shown ( gulli et al . , 2001 ) . in our study , measurement of middle cerebral artery velocity ( mcav ) by transcranial doppler revealed a similar oscillatory pattern to that observed with map , indicating that the continued increase in amplitude of lf arterial pressure oscillations observed in ht subjects was transferred to the cerebral vasculature ( rickards et al . , 2011 ) . although we did not report msna in our paper , figure 7 shows these responses during lbnp in a ht and a lt subject . in the ht subject , msnalf increased from baseline ( 4.4 au ) to a maximum of 85.8 au just prior to presyncope . in contrast , msnalf did not increase in the lt subject at all ( 2.4 to 2.3 au ) . thus , we propose that the ability to increase lf oscillations in msna , map , and mcav is an inherent characteristic associated with improved tolerance to central hypovolemia and therefore protects against the onset of hemodynamic decompensation during severe hypovolemia . representative tracings from a high tolerant and low tolerant subject at baseline ( bl ) , sub - maximal lbnp ( i.e. , the lbnp level before the level at which presyncope was reached ; sm ) and immediately before presyncope ( indicated by arrow ; ps ) . because the autonomic nervous system serves to maintain cardiovascular homeostasis under a variety of physiological and pathological stresses , a non - invasive surrogate for sympathetic and/or vagal activation has long been sought for diagnostic use ( goldstein et al . , 2011 ) . measures of hr variability ( hrv ) have received a great deal of attention , as some of the time and frequency domain measures have been associated with autonomic function . specifically , power spectral analysis of intervals between r - waves ( rri ) of the ecg yields lf ( 0.040.15 hz ) and hf ( 0.150.4 hz ) powers ; lf power was originally thought to contain components of both cardiac sympathetic and vagal ( parasympathetic ) function , while hf power predominantly reflected parasympathetic function ( akselrod et al . , 1981 ) . many investigators have also suggested that the ratio of lf / hf represents sympathovagal balance ( malliani et al . , 1991 ; goldstein et al . , 2011 ) and that these or other hrv metrics might be useful in disclosing dysautonomia in such clinical conditions as myocardial infarction , cardiac arrhythmias , diabetes , and renal failure ( acharya et al . , 2006 ; montano et al . , 2009 ) . indeed , a pubmed search on the term heart rate variability yields more than 14,300 references at the time of this writing ( february 2012 ) , yet we are unaware of any pathophysiological condition in which hrv is currently used as a standard of care for diagnosis and/or treatment . the use of hrv is appealing because calculation requires only non - invasive collection of a standard ecg . because increases in sympathetic activation occur in a linear fashion with decreases in stroke volume ( figure 3 ) , we proposed that hrv metrics might be useful non - invasive surrogates of msna for assessing the degree and progression of hemorrhage in trauma victims ( cooke and convertino , 2005 ) . in 2008 , we demonstrated using our lbnp model of simulated hemorrhage that , on average , rrihf power and some time domain metrics associated with parasympathetic function are inversely related to direct measurement of msna . rrilf power , on the other hand , was not associated with msna ( cooke et al . , 2008 ) ; it has been made clear more recently that rrilf is not a measure of cardiac sympathetic activity ( billman , 2011 ; goldstein et al . , 2011 ) . from these data , we concluded that hrv might be of clinical utility in assessing autonomic function during hemorrhage , although we and others noted that correlations between msna and hrv in individual subjects were not as strong as those derived from group means ( floras et al . application of power spectral analysis to ecg recordings collected from actual trauma patients during air transport to the hospital further suggested that the use of these metrics might be appropriate for clinical assessment of the severity of hemorrhage , as group means of some hrv metrics differed between patients who lived and those who died up to 24 h later ( cooke et al . subsequent studies extended these observations to the use of non - linear hrv metrics to predict mortality ( batchinsky et al . , 2007a ) and to discriminate between trauma patients who required a life - saving intervention and those who did not ( cancio et al . , importantly , all of the conclusions delineated above were developed based on standard analyses of group mean data . as our understanding evolved , however , we began to take the next step to assess whether hrv metrics could be useful in determining the physiological status of individuals rather than a group as a whole , a requirement that must be met for successful application of any metric for diagnosing individual patients . in doing so , we became aware of a number of challenges with all of the hrv metrics that we examined ( time domain , frequency domain , and non - linear metrics ) . first , there are issues of large inter - individual variability and poor reproducibility within subjects during the same recording session ( rickards et al . , 2010a ) as well as across days ( tan et al . , 2009 ) second , accurate determination of hrv requires long ( in some cases , up to 800 beats ) segments of ecg recordings that do not contain electromagnetic noise or ectopic beats , but these occur more frequently in trauma patients than in healthy individuals ( sethuraman et al . third , increases in hr will always be associated with decreases in hrv , purely as a mathematical function of the curvilinear nature of the relationship between hr and rri ( sacha and pluta , 2008 ) . decreases in hrv are therefore not specific to central hypovolemia but may be elicited by any physiological stressor that induces tachycardia such as physical movement ( rickards et al . , 2008 ) , pain , or anxiety , which are common in conscious trauma patients . fourth , hrv metrics are not able to discriminate between ht and lt subjects early in the progression of hypovolemia ; an effective triage tool should be able to alert medical personnel to those patients that will progress to hemodynamic decompensation more quickly ( hinojosa - laborde et al . , 2011 ) . finally , although group means of hrv metrics are highly correlated with decreases in stroke volume during central hypovolemia , analysis of the individual trajectories for these metrics demonstrated poor and inconsistent correlations with stroke volume at the individual subject level , even under controlled laboratory conditions ( ryan et al . armed with this new understanding , re - analysis of ecg data collected from trauma patients in the prehospital phase demonstrated that , while means of some of these hrv metrics differed between groups that received a life - saving intervention and those that did not , there was such high overlap of individual patient hrv values between groups that it would have been impossible to accurately classify patients on the basis of hrv alone ( figure 8 ; rickards et al . , 2010b ) . , 2007a ; cancio et al . , 2008 ) in which differences existed between groups even in standard vital signs , only those trauma patients with normal vital signs were chosen for analysis in order to ascertain whether the use of hrv metrics provided added discriminatory value over standard vital signs . based on these data , hrv metrics on their own fail to be useful for assessing the severity of hemorrhaging trauma patients in the prehospital and emergency department phases of diagnosis and treatment . individual values of four representative heart rate variability metrics for life - saving intervention ( lsi ; n = 32 ) and no - lsi ( n = 127 ) patient groups ( each point represents a single patient ) . for each hrv metric , group means statistically differ between lsi and no - lsi groups ( p 0.03 ) . the percentage of individual lsi patient values that fall within the range of the no - lsi group are presented in each plot . r intervals root mean squared sd ( a time domain metric ) ; rrihf , r r intervals high frequency ( a frequency domain metric ) ; sampen , sample entropy ( a non - linear metric ) ; dfa - l , detrended fluctuations analysis long range correlations ( a non - linear metric ) . from rickards the amplitude of the lf oscillations in sap has also been suggested as a potential non - invasive surrogate for sympathetic vasomotor tone and nerve activation ( pagani et al . , 1986 ) . linear relationships between saplf and direct measurement of msna were later described in human subjects during sympathetic activation induced by sodium nitroprusside infusion ( pagani et al . , 1997 ) and head - up tilt ( furlan et al . , 2000 ) . on the basis of such observations , saplf has been used as a non - invasive metric for the determination of dysautonomia in hypertension ( lucini et al . , 2002 ) and type 1 diabetes ( lucini et al . , 2009 however , this viewpoint has been the subject of continued controversy ( parati et al . , 2006 ; taylor and studinger , 2006 ) . data that argue against the use of saplf as a non - invasive metric of msna include the inability of saplf to adequately correlate with levels of msna in humans at rest ( taylor et al . , 1998 ) or during various physiological or pathophysiological conditions ( radaelli et al . , 1999 ; cui et al . we have recently investigated this question using our database of msna recordings obtained during central hypovolemia ( ryan et al . , 2011 ) . when group mean data were considered , there was a strong direct relationship ( r = 0.98 ) between the increase in msna and saplf , but it was curvilinear rather than linear as had been previously described ( ryan et al . , 2011 ) . when we took the next step to determine correlations between saplf and msna within individual subjects , however , we found poor correlations in a significant portion of our subjects ( range of r : 0.090.96 ; ryan et al . , 2011 ) . because a strong relationship between these variables is not universally applicable to all healthy human subjects , saplf should not be used as a non - invasive surrogate of msna . therefore , no reliable and accurate metric has been identified that is based on non - invasive recordings and that may be reliably used in the place of direct recordings of msna to assess levels of sympathetic activation during physiological stressors or disease states . hemorrhage continues to be a major cause of morbidity and mortality in both military and civilian settings , and research efforts continue to provide better means of both diagnosing the severity of blood loss and guiding resuscitation efforts . while important work continues in animal models , human experimental models such as lbnp have provided valuable insight as to the mechanisms of compensatory physiological responses to central hypovolemia without the confounding factors of anesthesia , species differences , and tissue injury . during even mild lbnp , both arterial and cardiopulmonary baroreceptors are unloaded , resulting in activation of the sympathetic nervous system to increase hr and vasomotor tone . importantly , we now know that sympathetic activation entails not only an increase in sympathetic nerve firing but also an alteration in the pattern of firing , such that the amplitude of lf oscillations in msna and , consequently , blood pressure is also increased ; both of these responses seem to be protective in that they are associated with improved tolerance to central hypovolemia . at some point , which occurs at different levels of central hypovolemia for individual subjects , this compensation fails and hypotension ensues . although it was once thought that sympathetic withdrawal always precipitated hemodynamic decompensation and hypotension , it is now clear that a diminution of absolute levels of msna firing is not required in all individuals . instead , it is possible that loss of the compensatory increase in msnalf may also be involved , particularly in those subjects demonstrating ht to central hypovolemia . additionally , there may be an acute resetting of the baroreflex at the level of the cns such that coherence between arterial blood pressure and sympathetic nerve activity is lost . determination of the mechanisms underlying the development of presyncope in both healthy human subjects and patients with diseases characterized by episodes of fainting continues to be an ongoing area of research . the search for a non - invasive surrogate of msna for both research and clinical purposes continues . certainly , an easily obtainable non - invasive metric of sympathetic activation could be of great importance for assessment of the severity of hemorrhage . however , metrics based on determination of variability in both rris and arterial blood pressure do not fulfill the necessary criteria to perform effectively in this role . before implementation of any such metric , it is essential that investigators determine whether the metric will be able to reliably track msna within individual subjects rather than simply rely on analyses based on group mean data . importantly , gender differences in the msna response to central hypovolemia must also be taken into account ( fu et al . , 2005 ; carter et al . , , it will also be necessary for laboratory determination of the efficacy of the metric in the face of blood loss combined with other physiological stressors which can impact sympathetic activation , such as heat stress ( cui et al . , 2004a , 2011b ) , dehydration ( kimmerly and shoemaker , 2002 , 2003 ; fu et al . , 2005 ) , mental or emotional stress ( carter et al . , 2008 ) , and ingestion of alcohol ( carter et al . , 2011 ) , nicotine , or other drugs . all of these stressors are commonly observed in conjunction with traumatic hemorrhage in civilian and/or military settings . because of the complexity of this problem , it is possible that clinical assessment of the severity of hemorrhage during the acute phase may be better realized using artificial intelligence technologies that reflect the integration of the sympathetic nervous response to hemorrhage with changes in circulatory pressure and volume ( convertino et al . , 2011 ) . the opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the department of the army or the department of defense . 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 .

hemorrhage remains a major cause of mortality following traumatic injury in both military and civilian settings . lower body negative pressure ( lbnp ) has been used as an experimental model to study the compensatory phase of hemorrhage in conscious humans , as it elicits central hypovolemia like that induced by hemorrhage . one physiological compensatory mechanism that changes during the course of central hypovolemia induced by both lbnp and hemorrhage is a baroreflex - mediated increase in muscle sympathetic nerve activity ( msna ) , as assessed with microneurography . the purpose of this review is to describe recent results obtained using microneurography in our laboratory as well as those of others that have revealed new insights into mechanisms underlying compensatory increases in msna during progressive reductions in central blood volume and how msna is altered at the point of hemodynamic decompensation . we will also review recent work that has compared direct msna recordings with non - invasive surrogates of msna to determine the appropriateness of using such surrogates in assessing the clinical status of hemorrhaging patients .

MSNA during the Compensatory Phase of Central Hypovolemia MSNA at Hemodynamic Decompensation Non-Invasive Surrogate for MSNA for Assessing the Severity of Hemorrhage? Conclusion Disclaimer Conflict of Interest Statement

at low levels of central hypovolemia elicited by mild lbnp in humans , msna increases in the absence of alterations in heart rate ( hr ) or blood pressure ( sundlof and wallin , 1978a ; victor and leimbach , 1987 ) . this initial increase in msna occurs in a similar fashion whether measured from the radial nerve in the arm or the peroneal nerve in the leg , and is therefore a reflection of generalized sympathetic activation to muscle vascular beds ( rea and wallin , 1989 ) . importantly , the response in msna to relatively low levels of lbnp ( i.e. , 10 mmhg ) is identical to the msna response to blood loss of 450 ml , demonstrating the effectiveness of lbnp as an experimental model of hemorrhage ( figure 1 ; rea et al . comparison of relationships between central venous pressure ( cvp ) and muscle sympathetic nerve activity ( msna ) during 10 mmhg lbnp and 450 ml hemorrhage in nine human subjects . in fact , so many investigators have used low levels of lbnp ( 20 mmhg ) to selectively unload cardiopulmonary baroreceptors that this has recently been referred to as dogma subsequent work , however , has conclusively demonstrated that deactivation of arterial baroreceptors also contributes to sympathoexcitation induced by non - hypotensive hypovolemia . 2009 ) recently observed that transient reductions in stroke volume and blood pressure [ both systolic ( sap ) and diastolic ( dap ) pressures ] occur at the onset of even mild levels of lbnp . taken together , it has become clear that the sympathoexcitation produced by even low levels of central hypovolemia is a result of reflex - mediated deactivation of both cardiopulmonary and arterial baroreceptors . once initiated , msna has been proposed to linearly increase during progressive central hypovolemia , although much of the data supporting this suggestion until recently has been accrued using lower levels of lbnp ( 30 mmhg ; convertino and cooke , 2002 ; cooke et al . this is because of the inherent difficulty in maintaining nerve recordings during higher levels of negative pressure , as the microelectrode is often displaced during the lbnp procedure . using this protocol , these investigators demonstrated that progressive reductions in lbnp ( up to 50 mmhg ) produce concomitant graded increases in msna , but they did not measure the degree of central hypovolemia induced by lbnp in order to quantify the relationship between volume loss and sympathetic activation ( khan et al . over the past 6 years , we have created a large database of human experiments ( > 200 subjects ) in which progressive central hypovolemia has been used to produce hemodynamic decompensation ( i.e. in the course of this work , we have obtained microneurography recordings in a subset of subjects in which stroke volume , a measure of central hypovolemia , has also been assessed . we were able to maintain msna recordings throughout lbnp to either the point of presyncope or to a high ( 80 mmhg ) lbnp level in 20 subjects . average values across subjects demonstrate that increases in msna burst frequency are inversely related to stroke volume levels during central hypovolemia in humans ( figure 3 ) , thereby confirming the linearity of the relationship between central hypovolemia and the induced msna response . this same relationship between central hypovolemia and direct measurement of renal sympathetic nerve activity ( rsna ) is also observed in anesthetized sheep during graded hemorrhage ( batchinsky et al . it should be noted that progressive increases in sympathetic activation during lbnp are evident whether msna is expressed as frequency ( bursts / min ) , incidence ( bursts/100 heartbeats ) , or total activity ( which takes into account burst amplitude ; cooke et al . muscle sympathetic nerve activity ( msna ) in a representative subject during progressive lower body negative pressure . relationship between stroke volume ( sv ) and muscle sympathetic nerve activity ( msna ) during lbnp in 20 human subjects . in addition to increases in absolute msna , progressive central hypovolemia also changes the pattern of bursting activity . while it is beyond the scope of this review , there is a continued controversy regarding whether the oscillatory pattern of sympathetic nerve activity and mayer waves is produced via a central oscillator mechanism or through the action of the baroreflex ( for a review of this issue , see julien , 2006 ) . in animal models of hemorrhage , the amplitude of lf oscillations in blood pressure and sympathetic nerve activity increase ( guyton and harris , 1951 ; malpas et al . in humans , the amplitude of lf oscillations in msna ( msnalf ) increase concomitantly with burst frequency and total activity during stresses that reduce venous return such as head - up tilt ( cooke et al . in addition to increases in the frequency and amplitude of sympathetic nerve firing , it is therefore possible that alterations in the oscillatory patterns of that firing may contribute to the ability of an individual to adequately compensate for progressive central hypovolemia ( see below ) . in our study , burst fusion occurred in 10 of the 17 subjects available for study at the time of publication ( cooke et al . similar alterations in the typical pulse synchronous burst pattern of msna had previously been noted in subjects susceptible to fainting during head - up tilting ; during the presyncopal ( i.e. our data extend this observation to the earlier compensatory phase of central hypovolemia , when blood pressure is still well - maintained . , 2009 ) , as the fused bursts observed during intense lbnp are similar in both their pulse asynchrony and their structure to those observed after bilateral blocks of glossopharyngeal and vagus nerves ( fagius et al . in support of this concept , ( 2011 ) have recently shown that extreme baroreceptor unloading requiring high sympathetic outflow ( induced by 80 mmhg lbnp ) recruits a subpopulation of large postganglionic axons in some but not all individuals . coupling ( 60 mmhg ) and then fusing ( 90 mmhg ) of muscle sympathetic nerve activity ( msna ) in one subject during lower body negative pressure . ( 1944 ) observed that the onset of fainting produced by experimental hemorrhage in man was preceded by a profound vasodilation in the forearm and a decrease in systemic vascular resistance , suggesting inhibition of the compensatory sympathetic activation that had occurred before this point . as it became possible to directly measure sympathetic nerve activity in humans , a variety of case reports and experimental studies appeared showing an inhibition of sympathetic activity associated with the development of presyncope in both healthy humans ( burke et al . recently , however , we have observed that sympathetic withdrawal does not occur in all subjects at the point of hemodynamic decompensation ( figure 5 ) ; in fact , 41% of our subjects did not demonstrate any diminution of msna despite the onset of hypotension and presyncopal symptoms ( cooke et al . 2010 ) showed that increases in msna induced by orthostatic stress were preserved through the point of syncope in 90% of patients previously diagnosed with vagovagal syncope . it is therefore apparent that sympathetic withdrawal is not an absolute requirement for the onset of hemodynamic decompensation in both healthy humans and in patients with a history of syncope . indeed , careful perusal of some of the literature frequently cited to support the proposition that sympathetic withdrawal precipitates cardiovascular collapse reveals that blood pressure begins to decrease while msna remains elevated during the compensatory phase of hypovolemia ( wallin and sundlof , 1982 ; sanders and ferguson , 1989 ; converse et al . , , these data refute the concept that cessation of sympathetic nerve activity precedes and causes hypotension , the subsequent reduction in cerebral perfusion pressure , and syncope in all subjects . arterial pressure ( ap ) and muscle sympathetic nerve activity ( msna ) for three representative subjects 2 min before the onset of presyncope . the lowest arterial pressure ( bp ) recorded for each subject a185 , msna decreased in the last 2 min of lbnp ( 120 to 60 s , 82 bursts / min ; 60 s to presyncope , 64 bursts / min ) , but was still maintained at high levels relative to the pre - lbnp control ( 36 bursts / min ) . subject a075 displayed burst fusion , elevated msna over control ( 25 bursts / min ) , and no withdrawal of msna at presyncope ( 120 to 60 s , 53 bursts / min ; 60 s to presyncope , 54 bursts / min ) . subject a199 ( a low tolerant subject ) did not display large increases in msna from the pre - lbnp control value ( 17 bursts / min ) and also did not demonstrate msna withdrawal at presyncope ( 120 to 60 s , 22 bursts / min ; 60 s to presyncope , 21 bursts / min ) . during mild to moderate central hypovolemia , the operating point of baroreflex - mediated control of sympathetic nerve activity is shifted upward without a change in gain , such that sympathetic nerve activity is increased at any given dap ( ichinose et al . additionally , there is a tighter coupling between oscillations in arterial blood pressure and msna , as quantitated using cross - spectral analysis by an increase in the coherence function between these variables ( furlan et al . evidence exists , however , to suggest that this tight coupling of blood pressure to sympathetic nerve activity may be lost at the point of hemodynamic decompensation . ( 2006 ) using data from subjects who either exhibited presyncope or did not during central hypovolemia induced by lbnp . approximately 12 min prior to the onset of hemodynamic decompensation , the gain of the baroreflex function relating dap to msna was substantially reduced in those subjects exhibiting presyncope but was unchanged in non - presyncopal subjects ( ichinose et al . likewise , we have also demonstrated a loss of linearity between the change in dap and the change in msna just before the onset of presyncope , suggesting disruption in the normal baroreflex - mediated coordination between arterial pressure and sympathetic activation ( cooke et al . , taken together , these results suggest that impairment of arterial baroreflex control over sympathetic vasomotor activity may contribute to the onset of hemodynamic decompensation . during central hypovolemia induced by head - up tilt , both the absolute levels of msna ( expressed as burst frequency and total activity ) and the amplitude of lf oscillations in msna ( msnalf ) increased markedly ; increases in msnalf were reflected in increases in lf oscillations of mean arterial pressure ( maplf ) . because resistance breathing improved lbnp tolerance , data were analyzed at the end of lbnp during the sham experiment and at this same absolute time point during the active experiment ( i.e. thus , in this one subject , it is apparent that inspiratory resistance breathing increased the amplitude of msna oscillations and coherence between msna and arterial blood pressure , and that this alteration in oscillatory pattern was associated with improved tolerance to central hypovolemia . these data are consistent with the concept that the maintenance of an increase in lf oscillations of both msna and map may be associated with the defense of blood pressure during severe central hypovolemia . blood pressure ( bp ) , muscle sympathetic nerve activity ( msna ) and end - tidal co2 ( et co2 ) tracings for a single subject under conditions of breathing with a sham ( top ) or active ( bottom ) device that provided resistance to inspiration . thus , there are several possible mechanisms involving alterations in activation of sympathetic nerve activity to explain hemodynamic decompensation during severe hypovolemia . finally , there is evidence to suggest that an increase in lf oscillations in both msna and map may be protective during central hypovolemia and that loss of these oscillations might precipitate hypotension ( kamiya et al . because of the scope of this review , we have chosen to focus on those mechanisms preceding presyncope that involve loss of compensatory alterations in msna , but it is likely that loss of other compensatory responses may also contribute to the inability to maintain blood pressure during severe hypovolemia . 1997 ) is that marked peripheral vasodilation is a major contributor to the fall in arterial pressure preceding vasovagal syncope . in this regard , it is of interest that , during prolonged lbnp at a low level ( 15 mmhg ) , vasodilation of the forearm musculature was observed despite the continued presence of a sustained compensatory increase in msna , suggesting that sympathetic escape occurs ( joyner et al . , implicit in the preceding discussion is the notion that there are individual differences in the ability of healthy humans to tolerate central hypovolemia before reaching the point of cardiovascular collapse . indeed , it has been known for many years that there is a great deal of variability in the ability of patients ( davis , 1949 ) and animals ( chien , 1967 ; kim and shoemaker , 1970 ) to survive traumatic hemorrhage ; we have recently learned that there is a genetic basis underlying this variability ( klemcke et al . likewise , individual differences in tolerance to central hypovolemia induced by lbnp have also been described ( sather et al . , 2001 ) , and central blood volume and cerebral blood velocity ( levine et al . additionally , the ability to sustain the compensatory mechanisms described above involving both the baroreflex modulation of sympathetic activation and the oscillatory component of that activation may also act to determine tolerance to central hypovolemia . ; convertino and sather , 2000a , b ) , we recently confirmed and expanded on these results using our large database of subjects in which lbnp was applied to the point of presyncope in all individuals . , 2005 ) , maplf increased in both lt and ht groups during early stages of lbnp , but maplf decreased to baseline levels in the lt group at 60 mmhg while it continued to increase in the ht group ( rickards et al . in our study , measurement of middle cerebral artery velocity ( mcav ) by transcranial doppler revealed a similar oscillatory pattern to that observed with map , indicating that the continued increase in amplitude of lf arterial pressure oscillations observed in ht subjects was transferred to the cerebral vasculature ( rickards et al . thus , we propose that the ability to increase lf oscillations in msna , map , and mcav is an inherent characteristic associated with improved tolerance to central hypovolemia and therefore protects against the onset of hemodynamic decompensation during severe hypovolemia . indeed , a pubmed search on the term heart rate variability yields more than 14,300 references at the time of this writing ( february 2012 ) , yet we are unaware of any pathophysiological condition in which hrv is currently used as a standard of care for diagnosis and/or treatment . because increases in sympathetic activation occur in a linear fashion with decreases in stroke volume ( figure 3 ) , we proposed that hrv metrics might be useful non - invasive surrogates of msna for assessing the degree and progression of hemorrhage in trauma victims ( cooke and convertino , 2005 ) . , 2008 ) ; it has been made clear more recently that rrilf is not a measure of cardiac sympathetic activity ( billman , 2011 ; goldstein et al . from these data , we concluded that hrv might be of clinical utility in assessing autonomic function during hemorrhage , although we and others noted that correlations between msna and hrv in individual subjects were not as strong as those derived from group means ( floras et al . , 2010a ) as well as across days ( tan et al . , 2008 ) , pain , or anxiety , which are common in conscious trauma patients . based on these data , hrv metrics on their own fail to be useful for assessing the severity of hemorrhaging trauma patients in the prehospital and emergency department phases of diagnosis and treatment . from rickards the amplitude of the lf oscillations in sap has also been suggested as a potential non - invasive surrogate for sympathetic vasomotor tone and nerve activation ( pagani et al . linear relationships between saplf and direct measurement of msna were later described in human subjects during sympathetic activation induced by sodium nitroprusside infusion ( pagani et al . on the basis of such observations , saplf has been used as a non - invasive metric for the determination of dysautonomia in hypertension ( lucini et al . data that argue against the use of saplf as a non - invasive metric of msna include the inability of saplf to adequately correlate with levels of msna in humans at rest ( taylor et al . we have recently investigated this question using our database of msna recordings obtained during central hypovolemia ( ryan et al . because a strong relationship between these variables is not universally applicable to all healthy human subjects , saplf should not be used as a non - invasive surrogate of msna . therefore , no reliable and accurate metric has been identified that is based on non - invasive recordings and that may be reliably used in the place of direct recordings of msna to assess levels of sympathetic activation during physiological stressors or disease states . hemorrhage continues to be a major cause of morbidity and mortality in both military and civilian settings , and research efforts continue to provide better means of both diagnosing the severity of blood loss and guiding resuscitation efforts . importantly , we now know that sympathetic activation entails not only an increase in sympathetic nerve firing but also an alteration in the pattern of firing , such that the amplitude of lf oscillations in msna and , consequently , blood pressure is also increased ; both of these responses seem to be protective in that they are associated with improved tolerance to central hypovolemia . at some point , which occurs at different levels of central hypovolemia for individual subjects , this compensation fails and hypotension ensues . although it was once thought that sympathetic withdrawal always precipitated hemodynamic decompensation and hypotension , it is now clear that a diminution of absolute levels of msna firing is not required in all individuals . instead , it is possible that loss of the compensatory increase in msnalf may also be involved , particularly in those subjects demonstrating ht to central hypovolemia . additionally , there may be an acute resetting of the baroreflex at the level of the cns such that coherence between arterial blood pressure and sympathetic nerve activity is lost . determination of the mechanisms underlying the development of presyncope in both healthy human subjects and patients with diseases characterized by episodes of fainting continues to be an ongoing area of research . the search for a non - invasive surrogate of msna for both research and clinical purposes continues . certainly , an easily obtainable non - invasive metric of sympathetic activation could be of great importance for assessment of the severity of hemorrhage . because of the complexity of this problem , it is possible that clinical assessment of the severity of hemorrhage during the acute phase may be better realized using artificial intelligence technologies that reflect the integration of the sympathetic nervous response to hemorrhage with changes in circulatory pressure and volume ( convertino et al .

it has been shown that reactive oxygen species ( ros ) are involved in the development of lifestyle - related diseases such as atherosclerosis , myocardial infarction , cerebrovascular disease , diabetes mellitus , cancer , and even osteoporosis . however , the involvement of oxidative stress in various diseases has been demonstrated almost in vitro or in animal studies and direct clinical studies supporting the involvement of ros are very few . in clinical studies , the participation of ros has been suggested indirectly , for instance , an antagonist of type 1 angiotensin ii receptor ( angiotensin receptor blocker : arb ) was shown not only to improve hypertension but also to decrease incidence of diabetes of mellitus in large - scale clinical trials . suppression of diabetes mellitus is thought to be due to repression of insulin resistance by arb , however , angiotensin ii has been suggested to promote ros production in vitro . on the other hand , negative data about the participation of ros in lifestyle - related diseases were recently reported in a randomized , double - blind , placebo - controlled factorial long - term trial of vitamin e and vitamin c , in which neither vitamin e nor vitamin c supplementation reduced the risk of major cardiovascular events . diabetes mellitus leads to microvascular complications such as retinopathy , nephropathy and neuropathy , but also is an important independent risk factor for cardiovascular disease . several well - researched theories have been proposed to explain how chronic hyperglycemia or postprandial hyperglycemia causes the micro- or macro - vascular derangements . these theories include increased polyol pathway flux , increased advanced glycation end - product ( age ) formation , activation of protein kinase c ( pkc ) and increased oxidative stress . recent reports including ours have shown that a pkc - dependent activation of nad(p)h oxiadse ( nox ) may be a major source of increased oxidative stress in vascular tissues of diabetes . oxidative stress also has been paid increasing attention to as a common underlying mechanism for progressive -cell dysfunction as well as diabetic vascular complications . bilirubin has been recognized as an important antioxidant and also shown to have an inhibitory effect on the activity of nox . higher concentrations of serum bilirubin were related to decreased risk of coronary artery disease and stroke . in this article , we propose the strong involvement of ros in the development of type 2 diabetes mellitus and its complications by presenting clinical and experimental animal model data about analysis of relationship between hyperbilirubinemia and diabetes mellitus . we compared the prevalence of vascular complications in diabetic patients with and without gilbert syndrome ( gs ) , a congenital hyperbilirubinemia . screening of 5,080 diabetic patients who visited kyushu university hospital and 12 other hospitals and clinics in the kyushu district of japan from april to june 2006 yielded 96 consecutive patients with gs , all of whom were enrolled . determination of gs was based on the presence of unconjugated bilirubin - dominant hyperbilirubinemia ( serum bilirubin level > 1.2 mg / dl ) for 3 or more months , in the absence of hemolytic disease and/or hepatic dysfunction . retinopathy was assessed by funduscopic examination ; macroalbuminuria was defined as urinary albumin to creatinine ratio of > 300 mg / g ; coronary artery disease was defined as a history of acute myocardial infarction , angina confirmed by clinically significant obstruction on coronary angiography , or revascularization ; cerebrovascular disease was defined as a history of symptomatic stroke , confirmed by brain imaging . metabolic variables and the prevalence of vascular complications were compared to those of 426 diabetic patients without gs who visited kyushu university hospital during the same period . the adjusted odds ratio for the association of gs with retinopathy , macroalbuminuria or coronary artery disease was 0.200.22 ( table 1 ) , suggesting that sustained hyperbilirubinemia can reduce diabetic complications by almost 80% by its antioxidant action and an inhibitory effect on nox . the study subjects were participants in the baseline survey of the kyushu university fukuoka cohort study . residents aged 49 to 76 years in the east ward of fukuoka city were invited to participate in the study by mail . during the period between february 2004 and august 2007 , a total of 12,949 persons participated in the survey . a total of 531 subjects were excluded for the following reasons : current medical care for chronic hepatitis , liver cirrhosis or liver cancer ( n = 233 ) , a prior history of liver cancer ( n = 3 ) , aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) greater than 3-fold of the upper limit of the normal range , i.e. , > 120 u / l ( n = 285 ) , total bilirubin > 3.0 mg / dl ( n = 3 ) , serum crp > 10 mg / l ( n = 212 ) , or serum creatinine > 2.0 mg / dl ( n = 44 ) . additionally , 12 persons were excluded because of missing values for liver enzymes ( n = 4 ) , creatinine ( n = 6 ) , alcohol use ( n = 4 ) , and leisure - time physical activity ( n = 1 ) . of the remaining 12,400 subjects , 624 under medication for diabetes mellitus were excluded in the analysis on hs - crp and hemoglobin a1c ( hba1c ) , but were included in the analysis on prevalent diabetes . age - adjusted geometric means of hs - crp were progressively lower in both men and women with higher values of total bilirubin . p values for trends of inverse associations were extremely small without and with adjustment for behavioral factors and liver enzymes ( fig . 1 ) . bilirubin concentrations also showed highly statistically significant , inverse associations with hba1c concentrations in men and women , regardless of adjustment for the behavioral covariates and liver enzyme activities ( fig . 2 ) . the inverse association of hba1c with total bilirubin was still significant after adjustment for hs - crp ( p = 0.0004 for men and p<10 for women ) . prevalent cases of type 2 diabetes numbered 907 ( 602 men and 305 women ) . the sex- and age - adjusted odds ratios of diabetes decreased almost progressively with increasing concentrations of total bilirubin , showing a statistically highly significant trend . with adjustment for the behavioral factors and liver enzymes , the association was somewhat attenuated . decreased odds ratios of almost the same magnitude were noted in the three categories of total bilirubin 0.5 mg / dl . further attenuation of the association occurred with additional adjustment for hs - crp , but the trend remained statistically significant ( table 2 ) . the analysis was repeated for unconjugated and conjugated bilirubin , and the associations were similar to those observed for total bilirubin . pancreatic islet is particularly susceptible to damage by oxidative stress because of the lowest levels of intrinsic anti - oxidant defenses . these data suggested that higher concentrations of bilirubin may suppress the development of type 2 diabetes through its anti - oxidative effect . the study subjects were participants in the baseline survey of the kyushu university fukuoka cohort study . residents aged 49 to 76 years in the east ward of fukuoka city were invited to participate in the study by mail . during the period between february 2004 and august 2007 , a total of 12,949 persons participated in the survey . a total of 531 subjects were excluded for the following reasons : current medical care for chronic hepatitis , liver cirrhosis or liver cancer ( n = 233 ) , a prior history of liver cancer ( n = 3 ) , aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) greater than 3-fold of the upper limit of the normal range , i.e. , > 120 u / l ( n = 285 ) , total bilirubin > 3.0 mg / dl ( n = 3 ) , serum crp > 10 mg / l ( n = 212 ) , or serum creatinine > 2.0 mg / dl ( n = 44 ) . additionally , 12 persons were excluded because of missing values for liver enzymes ( n = 4 ) , creatinine ( n = 6 ) , alcohol use ( n = 4 ) , and leisure - time physical activity ( n = 1 ) . of the remaining 12,400 subjects , 624 under medication for diabetes mellitus were excluded in the analysis on hs - crp and hemoglobin a1c ( hba1c ) , but were included in the analysis on prevalent diabetes . age - adjusted geometric means of hs - crp were progressively lower in both men and women with higher values of total bilirubin . p values for trends of inverse associations were extremely small without and with adjustment for behavioral factors and liver enzymes ( fig . 1 ) . bilirubin concentrations also showed highly statistically significant , inverse associations with hba1c concentrations in men and women , regardless of adjustment for the behavioral covariates and liver enzyme activities ( fig . 2 ) . the inverse association of hba1c with total bilirubin was still significant after adjustment for hs - crp ( p = 0.0004 for men and p<10 for women ) . prevalent cases of type 2 diabetes numbered 907 ( 602 men and 305 women ) . the sex- and age - adjusted odds ratios of diabetes decreased almost progressively with increasing concentrations of total bilirubin , showing a statistically highly significant trend . with adjustment for the behavioral factors and liver enzymes , the association was somewhat attenuated . decreased odds ratios of almost the same magnitude further attenuation of the association occurred with additional adjustment for hs - crp , but the trend remained statistically significant ( table 2 ) . the analysis was repeated for unconjugated and conjugated bilirubin , and the associations were similar to those observed for total bilirubin . pancreatic islet is particularly susceptible to damage by oxidative stress because of the lowest levels of intrinsic anti - oxidant defenses . these data suggested that higher concentrations of bilirubin may suppress the development of type 2 diabetes through its anti - oxidative effect . hereditary hyperbilirubinemic homologous gunn j / j rats and age - matched control heterozygous gunn rats j/+ rats were induced to diabetes by intraperitoneal injection of streptozotocin . at 8 weeks after the onset of diabetes , the total serum bilirubin levels were 0.15 0.02 and 0.18 0.04 mg / dl in diabetic and non - diabetic gunn j/+ rats , respectively , and 7.01 0.43 and 9.47 0.04 mg / dl in diabetic and non - diabetic gunn j / j rats , respectively . diabetic gunn j/+ rats exhibited marked increases in the amounts of urinary albumin excretion compared with non - diabetic gunn j/+ rats at 8 weeks after the onset of diabetes , whereas diabetic gunn j / j rats exhibited significantly less urinary albumin excretion than diabetic gunn j/+ rats ( fig . 3 ) . urinary excretion levels of a systemic oxidative stress marker , 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) , were significantly higher in diabetic gunn j/+ rats than in non - diabetic gunn j/+ rats at 8 weeks . the diabetes - induced increases in 8-ohdg at 8 weeks were completely prevented in diabetic hyperbilirubinemic gunn j / j rats ( fig . . immunostaining analysis of 8-ohdg in renal tissues revealed that the staining intensities in diabetic gunn j/+ rats were significantly higher than those in control gunn j/+ rats , in both glomeruli and tubules at 24 weeks . these increases in 8-ohdg staining intensities in glomeruli and tubules were completely prevented in diabetic gunn j / j rats . the staining intensities for nox4 protein were stronger in the renal glomeruli and tubules of diabetic gunn j/+ rats than in those of control gunn j/+ rats . western blotting analysis confirmed that the protein levels for nox4 were significantly increased in the kidneys of diabetic gunn j/+ rats compared with control gunn j/+ rats . all of these changes were completely prevented in diabetic gunn j / j rats , which showed levels comparable to those in control gunn j/+ rats ( fig . we investigated the effect of hyperbilirubinemia on mesangial expansion at 24 weeks after the onset of diabetes . the glomerular structure in diabetic gunn j/+ rats showed accelerated mesangial expansion compared with that observed in control gunn j/+ rats . the pas - positive and nuclei - free mesangial area was markedly increased in the glomeruli of diabetic gunn j/+ rats . a rodent model of type 2 diabetes , db / db mice , were fed with powder diet supplemented with or without biliverdin ( 5 mg / kg ) at 12 weeks of age . biliverdin administration orally for 2 weeks and 12 weeks did not significantly affect body weights or blood glucose levels . oral administration of this dose of biliverdin did not induce a significant elevation in serum bilirubin levels , although an intraperitoneal injection of the same dose induced a slight elevation in serum bilirubin levels at 0.5 h , with rapid return to the basal levels at 6 h after injection . urinary albumin excretion significantly increased in non - treated db / db mice as compared with control db/+ mice at 2 weeks and 12 weeks after the start of biliverdin administration . biliverdin administration significantly attenuated such increases in urinary albumin excretion in db / db mice ( fig . urinary 8-ohdg excretion was significantly higher in db / db mice than in control db/+ mice . biliverdin administration reduced these markers in db / db mice to control levels ( fig . the protein levels of nox4 were also significantly increased in renal tissues of db / db mice . biliverdin administration normalized all of these changes in db / db mice to the control levels ( fig . we also confirmed the effect of biliverdin to intracellular production of superoxide in the renal tissues by dihydroethidium stain . the oxidized dihydroethidium signals were significantly higher in db / db mice than those in control mice at 12 weeks . mesangial expansion was found in db / db mice at the age of 24 weeks , which was completely prevented by biliverdin administration . the effect of bilirubin and biliverdin on nox activities in cultured human mesangial cells were evaluated by the lucigenin method . pretreatment of the cells with both bilirubin and biliverdin for 24 h reduced nox activities in a dose - dependent manner . gunn rats , which exhibit a marked elevation of plasma unconjugated biliverdin levels due to a genetic deficiency of uridine diphosphate glucuronosyl transferase-1 ( udpgt-1 ) . the present study showed that diabetic hyperbilirubinemic gunn j / j rats exhibited significantly less urinary albumin excretion than diabetic non - hyperbilirubinemic gunn j/+ rats . in addition , diabetic gunn j / j rats did not develop renal mesangial expansion , which is one of the most striking morphological features of diabetic nephropathy , 6 months after the onset of diabetes , whereas diabetic gunn j/+ rats developed typical mesangial expansion . these findings suggested that hyperbilirubinemic gunn j / j rats were resistant to the progression of functional and morphological features of nephropathy after the onset of diabetes . administration of biliverdin ( 5 mg / kg ) protected against both albuminuria and renal mesangial expansion in db / db mice . since serum biliverdin enters cells rapidly and is converted to bilirubin by biliverdin reductase , it is likely that the beneficial effect of biliverdin administration may be due to increased levels of intracellular bilirubin levels generated from exogenously administered biliverdin , rather than increased levels of serum bilirubin or biliverdin . the present study suggested that the mechanism underlying these beneficial effects of hyperbilirubinemia and biliverdin administration may be the inhibition of oxidative stress , evaluated by systemic oxidative stress markers . the present study revealed that biliverdin administration induced down - regulation of nox components in diabetic kidneys , glomeruli and human mesangium cells . although the nature of the sources of ros overproduction in diabetes has not been precisely defined , we and other investigators have indicated that non - phagocytic nox may be the major sources of increased ros production in the vascular tissues of diabetic animals and patients , and that high glucose levels stimulate superoxide production from vascular endothelial cells and smooth muscle cells via a pkc - dependent activation of nox . the non - phagocytic nox comprises of a membrane - associated cytchrome b558 composed of nox family proteins ( gp91phox , nox1 , nox4 ) and p22phox , and several cytosolic regulatory components , p47phox , p67phox and rac 1 or rac 2 . the isoform nox4 was cloned from the kidney , where it was found to be highly expressed . it has been suggested that nox4 , as a major source of ros production in the kidney , could have a role under pathological conditions . we previously reported that increased expression of nox4 may play an important role in increased ros production in the kidneys of streptozotocin - induced diabetic rats . the present results suggest that down - regulation of nox components , especially nox4 , by hyperbilirubinemia and biliverdin administration may play an important role in the inhibition of oxidative stress in the kidneys of diabetic rodents . in conclusion , we showed using bilirubin as a tool that oxidative stress is an exacerbating factor of type 2 diabetes mellitus and propose that antioxidant therapies are of value to diabetic nephropathy . hereditary hyperbilirubinemic homologous gunn j / j rats and age - matched control heterozygous gunn rats j/+ rats were induced to diabetes by intraperitoneal injection of streptozotocin . at 8 weeks after the onset of diabetes , the total serum bilirubin levels were 0.15 0.02 and 0.18 0.04 mg / dl in diabetic and non - diabetic gunn j/+ rats , respectively , and 7.01 0.43 and 9.47 0.04 mg / dl in diabetic and non - diabetic gunn j / j rats , respectively . diabetic gunn j/+ rats exhibited marked increases in the amounts of urinary albumin excretion compared with non - diabetic gunn j/+ rats at 8 weeks after the onset of diabetes , whereas diabetic gunn j / j rats exhibited significantly less urinary albumin excretion than diabetic gunn j/+ rats ( fig . 3 ) . urinary excretion levels of a systemic oxidative stress marker , 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) , were significantly higher in diabetic gunn j/+ rats than in non - diabetic gunn j/+ rats at 8 weeks . the diabetes - induced increases in 8-ohdg at 8 weeks were completely prevented in diabetic hyperbilirubinemic gunn j / j rats ( fig . . immunostaining analysis of 8-ohdg in renal tissues revealed that the staining intensities in diabetic gunn j/+ rats were significantly higher than those in control gunn j/+ rats , in both glomeruli and tubules at 24 weeks . these increases in 8-ohdg staining intensities in glomeruli and tubules were completely prevented in diabetic gunn j / j rats . the staining intensities for nox4 protein were stronger in the renal glomeruli and tubules of diabetic gunn j/+ rats than in those of control gunn j/+ rats . western blotting analysis confirmed that the protein levels for nox4 were significantly increased in the kidneys of diabetic gunn j/+ rats compared with control gunn j/+ rats . all of these changes were completely prevented in diabetic gunn j / j rats , which showed levels comparable to those in control gunn j/+ rats ( fig . we investigated the effect of hyperbilirubinemia on mesangial expansion at 24 weeks after the onset of diabetes . the glomerular structure in diabetic gunn j/+ rats showed accelerated mesangial expansion compared with that observed in control gunn j/+ rats . the pas - positive and nuclei - free mesangial area was markedly increased in the glomeruli of diabetic gunn j/+ rats . a rodent model of type 2 diabetes , db / db mice , were fed with powder diet supplemented with or without biliverdin ( 5 mg / kg ) at 12 weeks of age . biliverdin administration orally for 2 weeks and 12 weeks did not significantly affect body weights or blood glucose levels . oral administration of this dose of biliverdin did not induce a significant elevation in serum bilirubin levels , although an intraperitoneal injection of the same dose induced a slight elevation in serum bilirubin levels at 0.5 h , with rapid return to the basal levels at 6 h after injection . urinary albumin excretion significantly increased in non - treated db / db mice as compared with control db/+ mice at 2 weeks and 12 weeks after the start of biliverdin administration . biliverdin administration significantly attenuated such increases in urinary albumin excretion in db / db mice ( fig . urinary 8-ohdg excretion was significantly higher in db / db mice than in control db/+ mice . biliverdin administration reduced these markers in db / db mice to control levels ( fig . the protein levels of nox4 were also significantly increased in renal tissues of db / db mice . biliverdin administration normalized all of these changes in db / db mice to the control levels ( fig . we also confirmed the effect of biliverdin to intracellular production of superoxide in the renal tissues by dihydroethidium stain . the oxidized dihydroethidium signals were significantly higher in db / db mice than those in control mice at 12 weeks . mesangial expansion was found in db / db mice at the age of 24 weeks , which was completely prevented by biliverdin administration . the effect of bilirubin and biliverdin on nox activities in cultured human mesangial cells were evaluated by the lucigenin method . pretreatment of the cells with both bilirubin and biliverdin for 24 h reduced nox activities in a dose - dependent manner . gunn rats , which exhibit a marked elevation of plasma unconjugated biliverdin levels due to a genetic deficiency of uridine diphosphate glucuronosyl transferase-1 ( udpgt-1 ) . the present study showed that diabetic hyperbilirubinemic gunn j / j rats exhibited significantly less urinary albumin excretion than diabetic non - hyperbilirubinemic gunn j/+ rats . in addition , diabetic gunn j / j rats did not develop renal mesangial expansion , which is one of the most striking morphological features of diabetic nephropathy , 6 months after the onset of diabetes , whereas diabetic gunn j/+ rats developed typical mesangial expansion . these findings suggested that hyperbilirubinemic gunn j / j rats were resistant to the progression of functional and morphological features of nephropathy after the onset of diabetes . administration of biliverdin ( 5 mg / kg ) protected against both albuminuria and renal mesangial expansion in db / db mice . since serum biliverdin enters cells rapidly and is converted to bilirubin by biliverdin reductase , it is likely that the beneficial effect of biliverdin administration may be due to increased levels of intracellular bilirubin levels generated from exogenously administered biliverdin , rather than increased levels of serum bilirubin or biliverdin . the present study suggested that the mechanism underlying these beneficial effects of hyperbilirubinemia and biliverdin administration may be the inhibition of oxidative stress , evaluated by systemic oxidative stress markers . the present study revealed that biliverdin administration induced down - regulation of nox components in diabetic kidneys , glomeruli and human mesangium cells . although the nature of the sources of ros overproduction in diabetes has not been precisely defined , we and other investigators have indicated that non - phagocytic nox may be the major sources of increased ros production in the vascular tissues of diabetic animals and patients , and that high glucose levels stimulate superoxide production from vascular endothelial cells and smooth muscle cells via a pkc - dependent activation of nox . the non - phagocytic nox comprises of a membrane - associated cytchrome b558 composed of nox family proteins ( gp91phox , nox1 , nox4 ) and p22phox , and several cytosolic regulatory components , p47phox , p67phox and rac 1 or rac 2 . the isoform nox4 was cloned from the kidney , where it was found to be highly expressed . it has been suggested that nox4 , as a major source of ros production in the kidney , could have a role under pathological conditions . we previously reported that increased expression of nox4 may play an important role in increased ros production in the kidneys of streptozotocin - induced diabetic rats . the present results suggest that down - regulation of nox components , especially nox4 , by hyperbilirubinemia and biliverdin administration may play an important role in the inhibition of oxidative stress in the kidneys of diabetic rodents . in conclusion , we showed using bilirubin as a tool that oxidative stress is an exacerbating factor of type 2 diabetes mellitus and propose that antioxidant therapies are of value to diabetic nephropathy .

the involvement of reactive oxygen species in various diseases has been demonstrated almost in vitro or in animal studies and clinical studies supporting the involvement of reactive oxygen species are very few . bilirubin has been recognized as an important antioxidant and also shown to have an inhibitory effect on the activity of nadph oxidase , which may be an important source for superoxide production in various tissues . when the prevalence of vascular complcations was compared in diabetic patients with and without a congenital hyperbilirubinemia ( gilbert syndrome ) , the prevalence of retinopathy , macroalbuminuria and coronary artery disease in patients with gilbert syndrome was about 20% of that in those without gilbert syndrome . for study of lifestyle - related diseases , the fukuoka cohort was constructed from 2003 to 2009 in kyushu area in japan , which contains a total of 12,949 persons . cross - sectional study of the fukuoka cohort revealed an inverse relation between serum bilirubin level and the prevalence of type 2 diabetes mellitus . a precursor of bilirubin , biliverdin - treated db / db mice exhibited less albuminuria and nephropathic changes . these effects were paralleled with normalization of oxidative stress markers and expression of nad(p)h oxidase subunits in kidney . these results suggested that oxidative stress is an exacerbating factor of type 2 diabetes mellitus and that antioxidant therapies are of value to diabetic nephropathy .

Introduction Gilbert Syndrome and Prevalence of Diabetic Vascular Complications Serum Bilirubin Level and Type 2 Diabetes Mellitus in Middle-Aged and Elderly Japanese Men and Women Bilirubin, high sensitivity C-reactive protein (hs-CRP) and glycated hemoglobin Bilirubin and prevalent diabetes mellitus Hyperbilirubinemic Rats and Diabetic Copmplications Hyperbilirubinemic Gunn rats Biliverdin-administered db/db mice

it has been shown that reactive oxygen species ( ros ) are involved in the development of lifestyle - related diseases such as atherosclerosis , myocardial infarction , cerebrovascular disease , diabetes mellitus , cancer , and even osteoporosis . however , the involvement of oxidative stress in various diseases has been demonstrated almost in vitro or in animal studies and direct clinical studies supporting the involvement of ros are very few . in clinical studies , the participation of ros has been suggested indirectly , for instance , an antagonist of type 1 angiotensin ii receptor ( angiotensin receptor blocker : arb ) was shown not only to improve hypertension but also to decrease incidence of diabetes of mellitus in large - scale clinical trials . suppression of diabetes mellitus is thought to be due to repression of insulin resistance by arb , however , angiotensin ii has been suggested to promote ros production in vitro . on the other hand , negative data about the participation of ros in lifestyle - related diseases were recently reported in a randomized , double - blind , placebo - controlled factorial long - term trial of vitamin e and vitamin c , in which neither vitamin e nor vitamin c supplementation reduced the risk of major cardiovascular events . diabetes mellitus leads to microvascular complications such as retinopathy , nephropathy and neuropathy , but also is an important independent risk factor for cardiovascular disease . these theories include increased polyol pathway flux , increased advanced glycation end - product ( age ) formation , activation of protein kinase c ( pkc ) and increased oxidative stress . recent reports including ours have shown that a pkc - dependent activation of nad(p)h oxiadse ( nox ) may be a major source of increased oxidative stress in vascular tissues of diabetes . oxidative stress also has been paid increasing attention to as a common underlying mechanism for progressive -cell dysfunction as well as diabetic vascular complications . bilirubin has been recognized as an important antioxidant and also shown to have an inhibitory effect on the activity of nox . higher concentrations of serum bilirubin were related to decreased risk of coronary artery disease and stroke . in this article , we propose the strong involvement of ros in the development of type 2 diabetes mellitus and its complications by presenting clinical and experimental animal model data about analysis of relationship between hyperbilirubinemia and diabetes mellitus . we compared the prevalence of vascular complications in diabetic patients with and without gilbert syndrome ( gs ) , a congenital hyperbilirubinemia . screening of 5,080 diabetic patients who visited kyushu university hospital and 12 other hospitals and clinics in the kyushu district of japan from april to june 2006 yielded 96 consecutive patients with gs , all of whom were enrolled . determination of gs was based on the presence of unconjugated bilirubin - dominant hyperbilirubinemia ( serum bilirubin level > 1.2 mg / dl ) for 3 or more months , in the absence of hemolytic disease and/or hepatic dysfunction . retinopathy was assessed by funduscopic examination ; macroalbuminuria was defined as urinary albumin to creatinine ratio of > 300 mg / g ; coronary artery disease was defined as a history of acute myocardial infarction , angina confirmed by clinically significant obstruction on coronary angiography , or revascularization ; cerebrovascular disease was defined as a history of symptomatic stroke , confirmed by brain imaging . metabolic variables and the prevalence of vascular complications were compared to those of 426 diabetic patients without gs who visited kyushu university hospital during the same period . the adjusted odds ratio for the association of gs with retinopathy , macroalbuminuria or coronary artery disease was 0.200.22 ( table 1 ) , suggesting that sustained hyperbilirubinemia can reduce diabetic complications by almost 80% by its antioxidant action and an inhibitory effect on nox . the study subjects were participants in the baseline survey of the kyushu university fukuoka cohort study . during the period between february 2004 and august 2007 , a total of 12,949 persons participated in the survey . a total of 531 subjects were excluded for the following reasons : current medical care for chronic hepatitis , liver cirrhosis or liver cancer ( n = 233 ) , a prior history of liver cancer ( n = 3 ) , aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) greater than 3-fold of the upper limit of the normal range , i.e. additionally , 12 persons were excluded because of missing values for liver enzymes ( n = 4 ) , creatinine ( n = 6 ) , alcohol use ( n = 4 ) , and leisure - time physical activity ( n = 1 ) . of the remaining 12,400 subjects , 624 under medication for diabetes mellitus were excluded in the analysis on hs - crp and hemoglobin a1c ( hba1c ) , but were included in the analysis on prevalent diabetes . prevalent cases of type 2 diabetes numbered 907 ( 602 men and 305 women ) . the sex- and age - adjusted odds ratios of diabetes decreased almost progressively with increasing concentrations of total bilirubin , showing a statistically highly significant trend . with adjustment for the behavioral factors and liver enzymes , the association was somewhat attenuated . the analysis was repeated for unconjugated and conjugated bilirubin , and the associations were similar to those observed for total bilirubin . pancreatic islet is particularly susceptible to damage by oxidative stress because of the lowest levels of intrinsic anti - oxidant defenses . these data suggested that higher concentrations of bilirubin may suppress the development of type 2 diabetes through its anti - oxidative effect . the study subjects were participants in the baseline survey of the kyushu university fukuoka cohort study . during the period between february 2004 and august 2007 , a total of 12,949 persons participated in the survey . a total of 531 subjects were excluded for the following reasons : current medical care for chronic hepatitis , liver cirrhosis or liver cancer ( n = 233 ) , a prior history of liver cancer ( n = 3 ) , aspartate aminotransferase ( ast ) or alanine aminotransferase ( alt ) greater than 3-fold of the upper limit of the normal range , i.e. additionally , 12 persons were excluded because of missing values for liver enzymes ( n = 4 ) , creatinine ( n = 6 ) , alcohol use ( n = 4 ) , and leisure - time physical activity ( n = 1 ) . of the remaining 12,400 subjects , 624 under medication for diabetes mellitus were excluded in the analysis on hs - crp and hemoglobin a1c ( hba1c ) , but were included in the analysis on prevalent diabetes . prevalent cases of type 2 diabetes numbered 907 ( 602 men and 305 women ) . the sex- and age - adjusted odds ratios of diabetes decreased almost progressively with increasing concentrations of total bilirubin , showing a statistically highly significant trend . decreased odds ratios of almost the same magnitude further attenuation of the association occurred with additional adjustment for hs - crp , but the trend remained statistically significant ( table 2 ) . the analysis was repeated for unconjugated and conjugated bilirubin , and the associations were similar to those observed for total bilirubin . pancreatic islet is particularly susceptible to damage by oxidative stress because of the lowest levels of intrinsic anti - oxidant defenses . these data suggested that higher concentrations of bilirubin may suppress the development of type 2 diabetes through its anti - oxidative effect . at 8 weeks after the onset of diabetes , the total serum bilirubin levels were 0.15 0.02 and 0.18 0.04 mg / dl in diabetic and non - diabetic gunn j/+ rats , respectively , and 7.01 0.43 and 9.47 0.04 mg / dl in diabetic and non - diabetic gunn j / j rats , respectively . urinary excretion levels of a systemic oxidative stress marker , 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) , were significantly higher in diabetic gunn j/+ rats than in non - diabetic gunn j/+ rats at 8 weeks . the diabetes - induced increases in 8-ohdg at 8 weeks were completely prevented in diabetic hyperbilirubinemic gunn j / j rats ( fig . immunostaining analysis of 8-ohdg in renal tissues revealed that the staining intensities in diabetic gunn j/+ rats were significantly higher than those in control gunn j/+ rats , in both glomeruli and tubules at 24 weeks . these increases in 8-ohdg staining intensities in glomeruli and tubules were completely prevented in diabetic gunn j / j rats . the staining intensities for nox4 protein were stronger in the renal glomeruli and tubules of diabetic gunn j/+ rats than in those of control gunn j/+ rats . all of these changes were completely prevented in diabetic gunn j / j rats , which showed levels comparable to those in control gunn j/+ rats ( fig . the glomerular structure in diabetic gunn j/+ rats showed accelerated mesangial expansion compared with that observed in control gunn j/+ rats . a rodent model of type 2 diabetes , db / db mice , were fed with powder diet supplemented with or without biliverdin ( 5 mg / kg ) at 12 weeks of age . oral administration of this dose of biliverdin did not induce a significant elevation in serum bilirubin levels , although an intraperitoneal injection of the same dose induced a slight elevation in serum bilirubin levels at 0.5 h , with rapid return to the basal levels at 6 h after injection . urinary albumin excretion significantly increased in non - treated db / db mice as compared with control db/+ mice at 2 weeks and 12 weeks after the start of biliverdin administration . biliverdin administration significantly attenuated such increases in urinary albumin excretion in db / db mice ( fig . urinary 8-ohdg excretion was significantly higher in db / db mice than in control db/+ mice . biliverdin administration reduced these markers in db / db mice to control levels ( fig . the protein levels of nox4 were also significantly increased in renal tissues of db / db mice . biliverdin administration normalized all of these changes in db / db mice to the control levels ( fig . the oxidized dihydroethidium signals were significantly higher in db / db mice than those in control mice at 12 weeks . mesangial expansion was found in db / db mice at the age of 24 weeks , which was completely prevented by biliverdin administration . the effect of bilirubin and biliverdin on nox activities in cultured human mesangial cells were evaluated by the lucigenin method . gunn rats , which exhibit a marked elevation of plasma unconjugated biliverdin levels due to a genetic deficiency of uridine diphosphate glucuronosyl transferase-1 ( udpgt-1 ) . in addition , diabetic gunn j / j rats did not develop renal mesangial expansion , which is one of the most striking morphological features of diabetic nephropathy , 6 months after the onset of diabetes , whereas diabetic gunn j/+ rats developed typical mesangial expansion . these findings suggested that hyperbilirubinemic gunn j / j rats were resistant to the progression of functional and morphological features of nephropathy after the onset of diabetes . administration of biliverdin ( 5 mg / kg ) protected against both albuminuria and renal mesangial expansion in db / db mice . since serum biliverdin enters cells rapidly and is converted to bilirubin by biliverdin reductase , it is likely that the beneficial effect of biliverdin administration may be due to increased levels of intracellular bilirubin levels generated from exogenously administered biliverdin , rather than increased levels of serum bilirubin or biliverdin . the present study suggested that the mechanism underlying these beneficial effects of hyperbilirubinemia and biliverdin administration may be the inhibition of oxidative stress , evaluated by systemic oxidative stress markers . the present study revealed that biliverdin administration induced down - regulation of nox components in diabetic kidneys , glomeruli and human mesangium cells . although the nature of the sources of ros overproduction in diabetes has not been precisely defined , we and other investigators have indicated that non - phagocytic nox may be the major sources of increased ros production in the vascular tissues of diabetic animals and patients , and that high glucose levels stimulate superoxide production from vascular endothelial cells and smooth muscle cells via a pkc - dependent activation of nox . it has been suggested that nox4 , as a major source of ros production in the kidney , could have a role under pathological conditions . we previously reported that increased expression of nox4 may play an important role in increased ros production in the kidneys of streptozotocin - induced diabetic rats . the present results suggest that down - regulation of nox components , especially nox4 , by hyperbilirubinemia and biliverdin administration may play an important role in the inhibition of oxidative stress in the kidneys of diabetic rodents . in conclusion , we showed using bilirubin as a tool that oxidative stress is an exacerbating factor of type 2 diabetes mellitus and propose that antioxidant therapies are of value to diabetic nephropathy . at 8 weeks after the onset of diabetes , the total serum bilirubin levels were 0.15 0.02 and 0.18 0.04 mg / dl in diabetic and non - diabetic gunn j/+ rats , respectively , and 7.01 0.43 and 9.47 0.04 mg / dl in diabetic and non - diabetic gunn j / j rats , respectively . urinary excretion levels of a systemic oxidative stress marker , 8-hydroxy-2'-deoxyguanosine ( 8-ohdg ) , were significantly higher in diabetic gunn j/+ rats than in non - diabetic gunn j/+ rats at 8 weeks . the diabetes - induced increases in 8-ohdg at 8 weeks were completely prevented in diabetic hyperbilirubinemic gunn j / j rats ( fig . immunostaining analysis of 8-ohdg in renal tissues revealed that the staining intensities in diabetic gunn j/+ rats were significantly higher than those in control gunn j/+ rats , in both glomeruli and tubules at 24 weeks . these increases in 8-ohdg staining intensities in glomeruli and tubules were completely prevented in diabetic gunn j / j rats . all of these changes were completely prevented in diabetic gunn j / j rats , which showed levels comparable to those in control gunn j/+ rats ( fig . the glomerular structure in diabetic gunn j/+ rats showed accelerated mesangial expansion compared with that observed in control gunn j/+ rats . a rodent model of type 2 diabetes , db / db mice , were fed with powder diet supplemented with or without biliverdin ( 5 mg / kg ) at 12 weeks of age . oral administration of this dose of biliverdin did not induce a significant elevation in serum bilirubin levels , although an intraperitoneal injection of the same dose induced a slight elevation in serum bilirubin levels at 0.5 h , with rapid return to the basal levels at 6 h after injection . urinary albumin excretion significantly increased in non - treated db / db mice as compared with control db/+ mice at 2 weeks and 12 weeks after the start of biliverdin administration . biliverdin administration significantly attenuated such increases in urinary albumin excretion in db / db mice ( fig . urinary 8-ohdg excretion was significantly higher in db / db mice than in control db/+ mice . biliverdin administration reduced these markers in db / db mice to control levels ( fig . the protein levels of nox4 were also significantly increased in renal tissues of db / db mice . biliverdin administration normalized all of these changes in db / db mice to the control levels ( fig . the oxidized dihydroethidium signals were significantly higher in db / db mice than those in control mice at 12 weeks . mesangial expansion was found in db / db mice at the age of 24 weeks , which was completely prevented by biliverdin administration . pretreatment of the cells with both bilirubin and biliverdin for 24 h reduced nox activities in a dose - dependent manner . gunn rats , which exhibit a marked elevation of plasma unconjugated biliverdin levels due to a genetic deficiency of uridine diphosphate glucuronosyl transferase-1 ( udpgt-1 ) . in addition , diabetic gunn j / j rats did not develop renal mesangial expansion , which is one of the most striking morphological features of diabetic nephropathy , 6 months after the onset of diabetes , whereas diabetic gunn j/+ rats developed typical mesangial expansion . these findings suggested that hyperbilirubinemic gunn j / j rats were resistant to the progression of functional and morphological features of nephropathy after the onset of diabetes . administration of biliverdin ( 5 mg / kg ) protected against both albuminuria and renal mesangial expansion in db / db mice . since serum biliverdin enters cells rapidly and is converted to bilirubin by biliverdin reductase , it is likely that the beneficial effect of biliverdin administration may be due to increased levels of intracellular bilirubin levels generated from exogenously administered biliverdin , rather than increased levels of serum bilirubin or biliverdin . the present study suggested that the mechanism underlying these beneficial effects of hyperbilirubinemia and biliverdin administration may be the inhibition of oxidative stress , evaluated by systemic oxidative stress markers . the present study revealed that biliverdin administration induced down - regulation of nox components in diabetic kidneys , glomeruli and human mesangium cells . although the nature of the sources of ros overproduction in diabetes has not been precisely defined , we and other investigators have indicated that non - phagocytic nox may be the major sources of increased ros production in the vascular tissues of diabetic animals and patients , and that high glucose levels stimulate superoxide production from vascular endothelial cells and smooth muscle cells via a pkc - dependent activation of nox . it has been suggested that nox4 , as a major source of ros production in the kidney , could have a role under pathological conditions . we previously reported that increased expression of nox4 may play an important role in increased ros production in the kidneys of streptozotocin - induced diabetic rats . the present results suggest that down - regulation of nox components , especially nox4 , by hyperbilirubinemia and biliverdin administration may play an important role in the inhibition of oxidative stress in the kidneys of diabetic rodents . in conclusion , we showed using bilirubin as a tool that oxidative stress is an exacerbating factor of type 2 diabetes mellitus and propose that antioxidant therapies are of value to diabetic nephropathy .

the biological function of these hormones was initially attributed mostly to their extranuclear activities presently referred to as nongenomic ; however , the exact mechanisms of such actions were then not known . subsequently , the majority of efforts were directed towards the clarification of the transcription - modifying function of these hormones bound to their nuclear receptors that are hormone - regulated transcription factors . this generated an enormous amount of information regarding the genomic action of hormones , the identity of their target genes , and so forth . it finally became apparent that the genomic action of hormones is insufficient to fully explain their biological roles , so that the nongenomic mechanisms are again being intensively studied . in this comprehensive paper we present basic information regarding the genomic and nongenomic mechanisms of action of small - molecule hormones , emphasizing the intermediary role of various proteins between the hormonal stimulus and the biological response of the cell . it should be noted , though , that although our current knowledge of the molecular mechanisms of action of these hormones is impressive , not all has been solved and many mechanisms still await explanation . refers to the regulation of target gene activity by hormones via their protein receptors , which also possess all the features of a transcription factor . this mechanism engages transcription and translation , and its biological effects are executed by a newly synthesized proteins . the first effects of engagement of this mechanism might be detected 3060 minutes after its initiation ; however , maximal effects are usually observed after several hours . nuclear receptors of small - molecule hormones belong to the superfamily of nuclear receptors , consisting of receptors for steroid hormones , thyroid hormone , vitamin d , retinoic acid and its derivatives , fatty acids , prostaglandins , and cholesterol derivatives , as well as of orphan receptors with unknown ligands . small fractions of some of these receptors also act outside of the nucleus , in mechanisms generally called nongenomic , which are mediated by processes other than a direct binding of the receptor to dna . structural similarities of nuclear receptors allow the subdivision of the superfamily into 7 families / subfamilies ( 0vi ) ; families i to vi are quite well defined [ 5658 ] , while family 0 contains various receptors , which do not fit into other families ( table 1 ) . nuclear receptors , although recognizing their own target genes and ligands with high specificity and being either partly or completely devoid of affinity for other genes and ligands , have a similar structure ( figure 1 ) . a typical , full - length nuclear receptor has a variable a / b domain at its n - terminus , followed by a well - conserved dna - binding c domain , then by a hinge d domain , and by a well - conserved ligand - binding e domain . some receptors also have an f domain on their c - termini , the function of which is usually unclear . the a / b domain of many nuclear receptors contains elements involved in hormone - independent transcription activation ( af1 ) . its function might be modified by phosphorylation , as was shown for the all - trans - retinoic acid receptor ( rar ) , peroxisome - proliferator - activated receptor ( ppar ) , orphan nurr1 receptor , estrogen receptor ( er ) , and so forth [ 5962 ] . the sequence and tridimensional structure of the c domain the domain contains two zinc fingers ; in each of them four perfectly conserved cysteines keep one zinc ion in place . at the base of the first zinc finger , a p - box is present ; its amino acid sequence determines the recognition of a specific ( usually hexameric ) dna sequence in the receptor 's target genes . at the base of the second zinc finger , a d - box is located ; its sequence is , in turn , responsible for the recognition of the distance between the two hexamers forming the hormone response element ( hre ) in the promoter of target gene . the c domain might contain the nuclear localization signal ( nls ) or fragments thereof . next , the d domain contains nls and facilitates rotation of the dna - binding domain in relation to the ligand - binding domain . in addition , it contains elements involved in cofactor binding , dna binding , and in heterodimerization . finally , the e domain binds a specific hormone , takes part in homodimerization as well as in heterodimerization , and , on its c - terminal end , contains a ligand - dependent transcription activation domain ( af2 ) . in some cases the e domain of the steroid hormone receptors takes part in the binding of heat shock proteins ( hsp , chaperone ) . the structure of this domain is formed by 12 -helices ( h1h12 ) and resembles pocket - like hormone - binding site . the sizes , shapes , and charges of this pocket present in various receptors differ from each other , and this why most receptors bind only their own hormones with an extremely high specificity and affinity ; however , some of them , such as the ppar receptor , possesses a large pocket allowing them to bind various ligands . a very important feature of nuclear receptors is that in the absence of the hormone , conformation of their e domains differs from that acquired upon hormone binding [ 6870 ] . the most spectacular is the change of position of the last helix ( h12 ) , containing the af2 domain . without the hormone , the h12 is moved to the side and protrudes from the rest of the e domain , leaving the empty pocket opened . upon hormone binding , the h12 comes nearer and closes the hormone inside the pocket . this feature is crucial for the majority of the functions of nuclear hormone receptors , including subcellular localization ( as for steroid receptors ) and transactivation activity . the activity of the nuclear receptor might be modulated by various posttranscriptional modifications including phosphorylation , acetylation , methylation , palmitoylation , and sumoylation [ 7276 ] . in addition , its biological efficiency depends on the rate of its turnover . like many other proteins , hormone receptors are degraded mainly by the ubiquitin - proteasome - dependent pathway . to be degraded by the proteasome , proteins must be tagged with multiple ubiquitins . the process of tagging depends on three enzymes acting sequentially ; the third one , ubiquitin ligase , determines the specificity of protein ubiquitylation ; for example , hdm2 and carboxyl - terminal hsp70 interacting protein ( chip ) promote degradation of the glucocorticoid receptor ( gr ) [ 79 , 80 ] . blocking receptor degradation by proteasome inhibitors impairs er- and progesterone - receptor-(pr- ) mediated transactivation but enhances gr - mediated transactivation [ 81 , 82 ] . notably , binding of chaperones such as hsps and associated proteins to steroid hormone receptor prevents receptor ubiquitylation [ 83 , 84 ] . calmodulin ( cam ) binding to er also prevents receptor ubiquitylation and degradation by the proteasome [ 85 , 86 ] , while its binding to ar prevents receptor degradation by calpain . a classic , genomic mechanism of action of small - molecule hormones is based on the binding of its nuclear receptor to the target gene . two elements facilitate such an interaction : the dna - binding domain of the receptor and hre , a specific sequence in the regulatory elements of the gene . such sequences ( single or multiple ) are usually localized close to the basal promoter , not farther than several hundred base pairs in the 5 direction from the transcription start site ( tss ) . however , they might also be present in atypical positions , for example , in the enhancers localized even a few thousand base pairs above the tss . the negative hres ( nhres ) tend to localize close to tss , sometimes even below this site [ 89 , 90 ] . analysis of the natural and artificial hres showed that nuclear hormone receptors preferentially recognize hexamers , sequences consisting of six nucleotides . steroid hormone nuclear receptors ( family iii ) , with the exception of er , preferentially bind to the agaaca sequence , while the remaining receptors , including families i and ii receptors and er , prefer the g / aggtc / ga sequence [ 9193 ] . both are consensus sequences and consist of the nucleotides most commonly found at a given position in natural hres ; it is then to be expected that natural hres very commonly differ from the consensus sequence . hres usually are formed by two hexamers and , most commonly , nuclear hormone receptors bind to the dna either as homodimers ( mostly , but not exclusively , family iii receptors ) or as heterodimers ( mostly families i and ii receptors ) [ 9499 ] . the binding of a monomeric receptor to a monomeric or to a dimeric hre is plausible , as in the case of steroidogenic factor-1 ( sf-1 , family v ) , but for classic receptors such situations are less common . depending on the relative position of the two hexamers , dimeric hre might be a direct repeat ( dr ) , palindromic ( pal ) , or inverted palindrome ( ip ) hre . hres for steroid hormone receptors , also called steroid hormone response elements ( sres ) , are usually palindromes consisting of the agaacannntgttct or of a similar sequences with three neutral ( e.g. , of any sequence ) nucleotides between hexamers . as mentioned above , the exception to this rule is er which preferentially binds to the g / aggtc / gannntc / gacct / c palindrome [ 64 , 101 ] . nevertheless , each of these receptors preferentially recognizes its own target sres with a very high specificity being a result of various factors , such as deviations from the sre consensus sequence , distinct amino acids surrounding dna binding domain fragments of the receptor directly contacting sre , interactions with other transcription factors bound to their own binding sites in the proximity of sre , tissue - specific expression of various receptor isoforms , and the level of receptor expression [ 102 , 103 ] . it should be mentioned that other types of sres are known , such as a selective androgen response element ( are ) which is not pal , but dr - type . it has been recently shown that such ares might be recognized not only by ar , but also by pr [ 104 , 105 ] . in addition to classic sres , which mediate transcription activation , a number of negative sres are known that inhibit the transcription when the steroid - hormone - activated receptor binds to nsre [ 106 , 107 ] . nuclear receptors belonging to the families i and ii preferentially bind to the consensus g / aggtc / ga sequence organized into dr , pal , or ip [ 108111 ] . the binding to dr drives the strongest biological effect ; in fact , natural hres recognized by these receptors are most commonly drs . specificity of the binding is achieved thanks to hre 's configuration , to the number of neutral nucleotides separating the two hexamers , to the sequence of hexamers and of hre - flanking dna - fragments , and to the sequence of the receptor dna - binding domain [ 112115 ] . in drs , one neutral nucleotide between hexamers ( dr1 ) warrants the binding of rxr / rxr homodimers , of rar / rxr or of ppar / rxr heterodimers ; two nucleotides ( dr2)the binding of rar / rxr heterodimer ; three nucleotides ( dr3)the binding of vdr / rxr heterodimer ; four nucleotides ( dr4)the binding of tr / rxr heterodimer ; finally , five nucleotides ( dr5 ) the binding of rar / rxr heterodimer . nuclear receptors for nonsteroid small - molecule hormones also bind to dr0 and to drs with more than five neutral nucleotides separating hexamers [ 116 , 117 ] as well as to other nonclassical hres . in addition , some hres might be bound by various receptors ; for example , the aggtcatgacct pal0 sequence is recognized by tr- , vdr- , and rar - containing dimers [ 118120 ] . specific nhres are also known for practically all nuclear hormone receptors belonging to the families i and ii [ 121124 ] . in addition , nuclear hormone receptors might bind as monomers to a single hexamer preceded by an a- and t - rich sequence , as shown for rev - erba , retinoic - acid - receptor - related orphan receptor- ( ror ) , and for nerve - growth - factor - induced clone b ( ngfi - b ) orphan receptors [ 100 , 125 , 126 ] . on the basis of the molecular mechanism of action and of the subcellular localization in the absence of ligand , nuclear hormone receptors can be divided into two types . in general , type i receptors preferentially reside in the cytoplasm ( in unliganded form ) and , while in the nucleus the best known receptors of this type are family iii steroid hormone receptors . type ii receptors , after being synthesized and modified in the cytoplasm , in the presence or absence of their ligand , preferentially translocate to the nucleus , where they are most active as heterodimers . the best known receptors of this type belong to families i and ii . binding of nuclear hormone receptors to dna might result in transcription activation or in transcription inhibition , and such phenomena result from variable molecular mechanisms . each hormone has a group of target genes which it activates ( positively regulated genes ) and a group of genes which it inhibits ( negatively activated genes ) ( figure 2 ) . in the circulation , they enter the cell by diffusion or are actively transported by a cell - membrane - bound transporting proteins . the majority of their nuclear receptors , a classic examples of type i receptors , reside in the cytoplasm forming inactive complexes with various proteins , including heat shock proteins hsp70 and hsp90 . formation of such complexes promotes proper folding of the receptor into a conformation allowing steroid binding [ 127131 ] . upon hormone binding , receptor conformation changes , and this results in the breakup of the complex . the activated receptor translocates to the nucleus thanks to its association with chaperones and importins [ 132 , 133 ] , where it binds to its sres in the promoters of target genes ( figure 3 ) . it is suggested that intranuclear mobility of steroid receptors , some of the most mobile proteins within the nucleus , depends on the presence of chaperone proteins such as hsp90 . occasionally , they might bind to dna as monomers ; in such a case sre might consist of only one hexamer and is usually preceded by an a- and t - rich sequence . binding of the steroid hormone receptor to sre initiates recruitment of a multiprotein coactivator complex which , by modification of chromatin structure ( e.g. , histone acetylation by histone acetyltransferases , hats ) and by interaction with the basal transcriptional machinery , activates transcription [ 135140 ] ( figure 3 ) . in addition , steroid hormones acting by their nuclear receptors can potentiate the transactivatory function of other transcription factors [ 141143 ] . inhibition of transcription by steroid hormones and their receptors is a result of a variety of mechanisms , such as hormone - receptor - complex - dependent inhibition of the activity of other transactivators , for example , activator protein 1 ( ap1 ) and nf-b [ 144146 ] . in this mechanism binding of a hormone - activated or a hormone - free steroid receptor to nsre leads to the inhibition of transcription mediated either by corepressors bound to hormone - activated receptor or by another group of corepressors bound to hormone - free receptor . such interaction results in deacetylation of histones exerted by histone deacetylases ( hdacs ) and in modification of chromatin structure . in turn other molecular mechanisms involved in the inhibition of gene transcription via nsre are also known , such as competition for a binding site with transcriptional activators [ 107 , 152154 ] . families i and ii receptor proteins , synthesized and modified in the cytoplasm , have their nls exposed so they can translocate to the nucleus in the absence of the hormone . therefore , both hormone - free and hormone - bound forms of the receptor could be present in the nucleus . since the conformation of the dna - binding d domain is stable ( independent of the hormone ) , both receptor forms might bind to the promoter of the target gene ; this is why type ii receptors are able either to activate or to inhibit transcription of the same gene in a hormone - dependent manner . in contrast to type i receptors heterodimerization with rxr modulates nuclear trafficking of other receptors [ 155 , 156 ] and increases both affinity of the other receptor to its hre as well as its transactivation activity [ 157160 ] . type ii receptors can also bind to dna as heterodimers with nuclear receptors other than rxr , as homodimers and as monomers [ 111 , 161163 ] . in such a case , their affinity for dna might be lower than that of heterodimers with rxr . it should be remembered that type ii receptors preferentially recognize hres consisting of two hexamers creating dr , pal , and ip . in vdr , tr , and rar heterodimers with rxr , which bind to dr3 , dr4 , and dr5 , respectively , rxr preferentially binds to the first hexamer [ 164 , 165 ] . on the other hand , in rar / rxr and ppar / rxr heterodimers bound to dr1 the presence of rxr in receptor heterodimers raises the question as to how 9-cis - retinoic acid modifies transcription of other hormones ' target genes . most probably it has no influence on the level of activation of triiodothyronine ( t3 ) target genes bound by tr / rxr and of 1,25(oh)2d3 target genes bound by vdr / rxr [ 168 , 169 ] ; however , there are reports claiming otherwise . in all - trans - retinoic acid target genes bound by the rxr / rar heterodimer , 9-cis - retinoic acid alone does not regulate the activity of such genes , but when both receptors are simultaneously bound to their ligands ( 9-cis - retinoic acid and all - trans retinoic acid , respectively ) , genes are activated synergistically . finally , when rxr forms heterodimers with a permissive partner , such as ppar , liver x receptor ( lxr ) , or nerve - growth - factor - induced b ( ngfi - b ) orphan receptor , 9-cis retinoic acid can regulate transcription on its own or act synergistically with the ligand of its partner [ 172 , 173 ] . in addition to hres mentioned above , type ii receptors bind to numerous untypical hres and to very common nhres . binding of the hormone - receptor complex to nhre results in transcription inhibition , so that the recruitment of corepressors preferentially binding to hormone - activated receptor plays here a major role . hormone target genes with unoccupied hres are active on the basal level , which depends on the presence of transcription factors other than hormone receptors . in genes positively regulated by the hormone , the binding of a hormone - free receptor heterodimer to hre leads to the recruitment of a corepressor complex , which , by deacetylation of histones , leads to condensation of chromatin . this , in turn , hampers the binding of transactivators and of basal transcription factors to dna ; as a result , transcription is inhibited below the basal level ( figure 4(a ) ) [ 147 , 148 , 174176 ] . however , upon hormone binding to the receptor , conformation of its ligand - binding domain changes ; this results in the dissociation of corepressors , in the recruitment of a coactivator complex containing hats and in transcription activation markedly above the basal level ( figure 5(b ) ) [ 177189 ] . in genes negatively regulated by the hormone , transcription inhibition occurs as a result of numerous mechanisms ; some of them are still not completely known . the inhibition could be indirect , depending on the binding of hormone receptors to a strong transactivator ( such as ap1 , nf-b , and p53 ) ; such binding results either in a blockage of transactivator 's activity or in its binding to dna [ 190192 ] . in this mechanism , the binding of the receptor to the dna is not a prerequisite for the inhibition of transcription . in the direct mechanisms , hre might be present close to or might overlap the binding site for a strong transactivator . under such circumstances , transcription inhibition is the result either of competition for a binding site , or of binding of the receptor to the transactivator resulting in the repression of its activity [ 193 , 194 ] . in another direct mechanism , the binding of hormone - activated receptor to nhre initiates recruitment of specific corepressors preferentially recognizing hormone - bound receptors [ 149151 , 195198 ] . in addition , hormonal receptors bound to nhres located close to ( commonly behind ) the transcription start site might affect the binding of type ii rna polymerase to the basal promoter . as mentioned above , the biological action of small - molecule hormones depends on their interaction with their receptors , as well as on the interactions of the receptor with dna and with other proteins . in the genomic mechanism of hormone action , the most important interaction is that of the receptor with coactivators , corepressors , and other transcription factors . on the other hand , in the nongenomic mechanisms , the most crucial role is played by the binding either of the cytoplasmic fraction of nuclear receptors or of hormone itself to extranuclear proteins . transcription may occur only in the presence of basal transcriptional machinery , a complex consisting of tens of proteins bound to dna close to the transcription start site . a typical basal promoter contains a tata box ( tataa / taa / t ) located 2030 base pairs above tss , a sequence recognized by tata - binding protein ( tbp ) . some promoters do not have this sequence ; however , the basal transcriptional machinery binds to such promoters anyway and at a similar distance form tss as in the case of typical promoters . binding of tbp to the basal promoter initiates a cascade of binding of other basal transcription factors . tbp together with tbp - binding proteins ( tafs ) forms transcription factor iid ( tfiid ) . the next step of preinitiation complex formation is the binding of iib ( tfiib ) , iif ( tfiif ) , and iih ( tfiih ) transcription factors . nuclear hormone receptors interact with the basal transcription factors not only via other proteins ( coactivators and corepressors ) but also interact with them directly . it has been shown that tr , rxr , rar , er , gr , and androgen receptor ( ar ) might directly bind to tbp , ar and er to tfiif , er , tr , and vdr to tfiib , and so forth [ 200205 ] . it is suggested that such binding might bidirectionally affect ( activate or inhibit ) the recruitment of the basal transcription factors to the preinitiation complex . transfer of information regarding binding of the receptor to hre and the receptor status ( hormone - free or hormone - bound ) to the basal transcriptional machinery is usually executed by other proteins that do not bind to dna but form a functional bridge . such proteins possess various activities . the same coactivator or corepressor complex might bind to several nuclear receptors ; some of these complexes might also coregulate transcription initiated by transcription factors of other type . together with tif-2 ( src-2 ) and tram-1 ( src-3 , actr , and rac3 ) , it forms the p160 coactivator family . the p160 proteins are indeed coactivators of many nuclear receptors including gr , er , pr , vdr , tr , rxr , and ppar [ 179 , 180 , 183 , 189 ] . they contain an lxxll ( l : leucine , x : any amino acid ) motif , by which they bind to the ligand - binding domain of the receptor activated by the hormone . importantly , a specific structure of the receptor , first of all of its af2 domain , is a prerequisite for such interaction . creb - binding protein ( cbp ) and p300 possess a histone acetyltransferase activity [ 207 , 208 ] and are coactivators of various transcription factors , including nuclear hormone receptors [ 177 , 178 ] . the binding of p300/cbp to the nuclear receptor is hormone dependent and af2 domain dependent . p300/cbp bind to p160 proteins , to tbp , and to tfiib basal transcription factors , and , as such , are intermediates between receptors and basal transcriptional machinery . another coactivator , p300/cbp - associated factor ( p / caf ) , interacts with p160 , p300/cbp , and hormonal nuclear receptors . multiprotein complexes containing thyroid - hormone - receptor - associated proteins ( trap ) or vitamin - d - receptor - interacting proteins ( drip ) have been identified [ 181 , 185 ] . both complexes are very similar , if not identical , and consists of fourteen - sixteen 70230 kda proteins . their drip205/trap220/trip2 subunit , by the lxxll motif , interacts with tr , vdr , and other nuclear receptors such as rxr and rar in a hormone - dependent and a receptor - af2-domain - dependent manner . a number of other coactivators interacting with nuclear receptors are known , such as ppar coactivator 1 ( pgc-1 ) , which also interact with other receptors , for example , with tr [ 184 , 188 ] and with activating signal cointegrators-1 and -2 ( asc-1 and asc-2 ) interacting with src-1 , p300/cbp , basal transcription factors , and nuclear receptors [ 186 , 187 ] . the formation of a coactivator complex is initiated by the binding of hormone - bound receptor to its hre . this is followed by the recruitment of the coactivator proteins , which directly bind nuclear receptors and by the binding of other proteins . the final multicomponent complex , by modification of chromatin structure and by interaction with the basal transcriptional machinery , activates transcription of target genes . the best known corepressors are nuclear corepressor ( ncor , rip-13 ) , a large , 270 kda protein , as well as silencing mediator for retinoic acid and thyroid hormone receptors ( smrt ) [ 147 , 148 ] . other proteins , such as the small ubiquitous nuclear corepressor ( sun - cor ) and the alien protein , might also serve as nuclear hormone receptor corepressors [ 174 , 175 ] . the motif that allows ncor and smrt to bind to the receptor is lxxi / hixxxi / l ( l : leucine , x : any amino acid , i : isoleucine , and h : histidine ) [ 210 , 211 ] . ncor and smrt bind to the families i and ii nuclear receptors , to er and to pr ( but not to other members of family iii ) bound to a specific antagonists , and to some orphan receptors . what makes them unique among corepressors is the fact that they bind to the receptor activated by the hormone . the group includes receptor - interacting protein 140 ( rip140 ) and ligand - dependent corepressor ( lcor ) . they bind to a various ligand - bound receptors , including er , gr , pr , and vdr , via the coactivator - specific lxxll motif , but recruit hdac proteins and other corepressors [ 149151 ] . hairless protein ( hr ) contains both the hormone - activated - receptor - binding lxxll motif and a cornr box a sequence mediating the binding of the corepressor to the hormone receptor . when it interacts with ligand - bound ror , it utilizes the lxxll motif , whereas when it interacts with vdr , it likely utilizes another domain . on the other hand , hr interacts with hormone - free tr as a typical corepressor , utilizing the cornr box and recruiting hdac [ 195197 ] . the preferentially expressed antigen in melanoma ( prame ) is expressed in various cancers , but in healthy tissues it is present only in testes , ovaries , endometrium , and adrenal glands . prame contains the lxxll motif and selectively inhibits transcription in the presence of all all - trans - retinoic - acid - bound rar isoforms . the repressor of estrogen activity ( rea ) binds to the er - agonist ( e.g. , 17-estradiol ) and to the er - antagonist ( e.g. , tamoxifen ) complexes . by doing so in the presence of agonist , it inhibits the activity of target gene , while in the presence of antagonist it magnifies its action . the suppression by rea is a result of the competition with coactivators for binding to er , as well as of the recruitment of hdac and of chromatin modification . metastasis - associated factor 1 ( mta1 ) is another corepressor preferentially binding to a ligand - activated er . it inhibits the expression of estrogen target genes by competing with coactivators for the binding to the receptor , by recruiting hdac , and by chromatin modification . a group of corepressors that might bind both liganded and nonliganded hormone receptors is also known . for example , the nr - binding set domain containing protein 1 ( nsd1 ) possesses separate domains : one that binds hormone - free receptors tr and rar ( nid ) and another that binds hormone - bound tr , rxr , er , and rar receptors ( nid ) . natural hres are located relatively close to tss or in more distant regulatory elements , and binding sites for other transcription factors are usually located nearby . such proximity permits interaction between nuclear receptors and these transcription factors , leading either to the suppression of gene activity ( as described above ) or to its additive or synergistic activation . the binding of nuclear receptors to other transcription factors might also occur in a dna - binding - independent manner . in fact , the binding of all known nuclear hormone receptors to the transcription factors has been reported ; the best known examples are the binding of tr to p53 , gr and pr to oct-1 , gr to ap-1 and to nf-b , ppar to nf-b , ap-1 , and to stat [ 217221 ] . a nucleosome consists of eight histone molecules ( two of each h2a , h2b , h3 , and h4 ) . epigenetic modifications of amino acids forming such tails play a marked role in chromatin organization . increased acetylation relieves compact chromatin , which results in an exposure of the transcription - factor - binding sites and their increased accessibility leading to transcription activation . on the other hand , deacetylation of histone tails transcription - factor - binding sites become inaccessible to transactivators , and the gene becomes transcriptionally inactive . such a mechanism of modification of chromatin structure is utilized by nuclear hormone receptors , which , as mentioned above , interact with coactivators and corepressors . p160 and p300/cbp coactivators themselves possess hat activity and form complexes with other hat proteins , such as p / caf . on the other hand , the binding of a ligand - activated receptor to hre initiates the formation of a coactivator complex , which , thanks to the hat activity , increases histone acetylation and induces local decondensation of chromatin ( figure 5(a ) ) . on the other hand , the binding of a hormone - free receptor to hre initiates the formation of a corepressor complex , which , thanks to its hdac activity , induces local condensation of chromatin ( figure 5(b ) ) . finally , the corepressor complex and its hdac activity are utilized by the specific corepressor proteins described above , which bind to a hormone - activated receptor and inhibit transcription of the target gene ( figure 5(c ) ) . fast biological effects of hormones , just seconds or minutes after hormone administration , have already been described several dozen years ago . the rapidity of biological response and its independence from transcription and from translation suggested that the genomic mechanism of hormone action is not involved ; therefore , this mechanism was called nongenomic or extragenomic . the nongenomic mechanisms of hormone action are multiple , variable , and only partially known ( figure 6 ) . steroid and nonsteroid small - molecule hormones bind to various proteins localized outside the nucleus and activate transduction pathways leading to a fast biological response . the presence of binding sites in the cell membrane was proved for all major representatives of these hormones ; however , in many cases the identity of the binding protein remains unknown . in addition , it is likely that such hormones have more than one type of membrane receptors . in the case of receptors already identified , their mode of action is by and large only partially resolved . just next to the cell membrane or directly in it , usually within caveolae ( a bubble - like , 50100 nm invaginations of the cell membrane ) , proteins identical to the nuclear receptors for glucocorticoids , estrogen , androgen , and vitamin d , have been identified [ 222225 ] . it is then plausible that nuclear receptors of other small - molecule hormones are present close to or in the cell membrane . some small - molecule hormones bind to other than nuclear receptor - like cell membrane proteins . for example , the integrin receptor v3 plays a role of cell membrane receptor for thyroxin ( t4 ) . mpr , mpr , mpr , mpr , and mpr cell membrane receptors for progesterone possess seven transmembrane domains ( some authors even suggest the presence of eight such domains ) and interact with g proteins [ 227 , 228 ] . the g - protein - interacting cell membrane receptor for steroid - hormone - binding protein ( shbg ) binds androgens with higher and estrogens with lower affinity . the prerequisite for signal transduction from the hormone to the cell interior by this receptor is the binding of a hormone - free shbg first , followed by hormone binding [ 229 , 230 ] . -aminobutyric acid a ( gabaa ) receptor serves as the cell membrane receptor for neurosteroids . it is induced by palmitoylation of cysteine 447 , a modification increasing protein hydrophobicity and , therefore , facilitating protein association with lipid bilayer . truncated 46 kda variant of er is preferentially palmitoylated and enriched in the cell membranes ; it is suggested that it might be more active than full - length receptor . enzymes identified as palmitoylacyltransferases for sex hormone receptors are dhhc-7 and dhhc-21 proteins . a highly conserved 9-amino acid motif ( fvclksiil in er ) that is crucial for palmitoylation and membrane localization has been identified in the ligand - binding domains of er , er , pr , pr , gr , and ar . tr and tr possess a motif ( lpcedqiil ) that slightly differs from the one described above , but presumably , it is also involved in the receptor palmitoylation and membrane targeting . notably , mr , ppars , and rar do not have any sequence resembling this motif . translocation of nuclear hormone receptors to the membrane is also induced in the presence of the respective ligand ; this was shown for er- and 17-estradiol and for vdr and 1,25(oh)2d3 . in the cell membrane , nuclear hormone receptors interact with caveolae - specific proteins ; for example , er and ar physically interact with caveolin-1 [ 234 , 241 ] , while vdr binds to caveolin-3 . furthermore , binding to caveolins allows hormone receptors to initiate fast , specific nongenomic response to hormonal stimulus . upon binding to the cell membrane receptors , small - molecule hormones activate various transduction pathways by a receptor - type - dependent mechanism . by activation of phospholipase c ( plc ) and generation of the secondary messenger inositol 1,4,5-trisphosphate ( ip3 ) , they might activate the cell membrane and the sarcoplasmic reticulum ( the most important ca storage ) ion channels . such activation leads to the increase of intracellular concentration of ca , another secondary messenger crucial for many cellular functions . ca activates , among others , ras / raf / mek / erk kinases , protein kinase c ( pkc ) , and protein kinase a ( pka ) . as a result , activated kinases phosphorylate and activate numerous cytoplasmic and nuclear proteins , including hormonal receptors , transcription factors and coactivators . this , in turn , modulates various biological processes in the cytoplasm and influences transcription of genes regulated by newly phosphorylated hormone receptors and transcription factors . cell - membrane - located small - molecule hormone receptors interacting with g proteins might also activate adenylate cyclase , which results in the generation of yet another secondary messenger , camp , and in the activation of camp - dependent proteins , such as pka , and of their substrates [ 243247 ] . by nongenomic mechanisms , small - molecule hormones also regulate the activity of ion channels , influencing cross - membrane movement of na , h , cl , and of k [ 245 , 248 , 249 ] . small - molecule hormones also bind to the proteins present in the cytoplasm ; commonly , such proteins are cytoplasmic fractions of nuclear receptors . upon hormone binding , the receptor interacts with numerous proteins , elements of various signal transduction pathways which , as described above , might be also activated by hormones on a higher level , namely , that of a cell membrane receptor . for example , hormone - activated tr , er , and rar bind to a p85 subunit of phosphatidylinositol 3 kinase . activated kinase increases production of ip3 which , in turn , activates the mitogen - activated protein kinase ( mapk ) pathway [ 250252 ] . hormone - activated ar , pr , and er bind to sh3 or to sh2 subunit of c - src tyrosine kinase localized close to the cell membrane . such binding activates c - src which , subsequently , activates mapk and ras / raf / mek / erk pathways leading to the phosphorylation of various cytoplasmic and nuclear receptors [ 253255 ] . of note is nuclear hormone receptors ' binding to cam , being an example of cross - talking of hormonal signaling with other signal transduction pathways . such binding has been proved for er ( but not for er ) , ar , and orphan receptor err , among others [ 8587 , 256258 ] . it results in the increased stability of the receptor due to cam - dependent protection from degradation [ 8587 ] . the binding profoundly affects receptor function : cam is required for normal transactivation by er since its elimination or blockage by antagonists prevents 17-estradiol from inducing transcriptional activity of this receptor [ 256 , 257 ] . similarly , cam stimulates transcriptional activity of ar ( since its antagonist w-7 blocks ar - dependent expression of prostate - specific antigen ) and of err [ 258 , 259 ] . for example , 1,25(oh)2d3 , dehydroepiandrosterone ( dhea ) , and dexamethasone bind to pkc , pkc , and pkc isoforms of pkc , which results in enzyme activation . in addition , pkc isoform is also directly activated by aldosterone and by 17-estradiol , while pkc isoform is activated by 17-estradiol [ 260 , 261 ] . one of them is based on the action of their nuclear receptors as transcription factors . each mitochondrion has multiple copies of its own dna ( mtdna ) encoding 37 genes , including genes for 13 proteins involved in oxidative phosphorylation . a shortened isoform of tr ( mttr1 , so - called p43 ) , rxr ( mtrxr ) , and ppar2 ( mtppar2 ) , as well as full - length gr , er , and er receptors are present in mitochondria [ 109 , 262265 ] , where they form dimers such as mtrxr/p43 , mtppar2/p43 , gr / gr , and er / er or couple with other transcription factors . it has been shown that glucocorticoids , t3 and 17-estradiol , acting by their mitochondrial receptors bound to mitochondrial hres , activate the transcription of mtdna , leading to an increased activity of oxidative phosphorylation . another mechanism of small - molecule hormones action in mitochondria is based on their interactions with other proteins . for example , diiodothyronine ( t2 ) binds to the va subunit of cytochrome c oxidase and activates this enzyme . in addition , the shortest isoform of tr1 , p28 , is bound to the internal mitochondrial membrane , where , most likely , it stimulates the function of ant and of uncoupling proteins ( ucps ) [ 263 , 268 ] . orphan nuclear receptor nur77 mediates apoptosis by interaction with bcl-2 and by induction of cytochrome c release . small - molecule hormones acting in mitochondria regulate ca wave activity in this organelle , as shown in the case of estrogens and t3 [ 270 , 271 ] . finally , hormonal receptors can directly bind to mitochondrial membranes and modify membrane potential , as shown , for example , for stress - activated gr . it has been shown that rar molecules present in the cytoplasm can bind mrna via the c - terminal f domain , which recognizes a specific sequences in the target mrna . such a mechanism was described for mrna encoding neuronal glur1 protein , a subunit of the glutaminergic receptor . the binding of glur1 mrna by a hormone - free receptor results in the inhibition of translation . the binding of all - trans - retinoic acid induces the change of receptor conformation and decreases its affinity for mrna ; as a result the receptor dissociates from mrna . a very rapid effects of androgens , progesterone , glucocorticoids , and other steroid hormones , evident just a few seconds after hormone administration , might be a result of a nonspecific , nongenomic mechanism of small - molecule hormones action , based on their interactions with lipid bilayers . lipophilic steroid hormone molecules could directly bind to membrane phospholipids and , by doing so , modulate their function . this , in turn , influences the function of membrane proteins such as the calcium pump and other channel proteins , leading to an immediate transport modification of various ions . nonspecific binding of steroid hormones to a mitochondrial membrane might increase proton leak [ 274 , 275 ] . medical conditions associated with out - of - range level of small - molecule hormones are known for decades , relatively common , and have been exhaustively described in numerous handbooks and articles . in contrast , they are uncommon , with a wide range of signs and symptoms of variable severity ( related to both the type and site of genetic error within the receptor - encoding gene or related genes ) that might mimic signs and symptoms of other diseases ( e.g. , resistance to thyroid hormone might be erroneously diagnosed as hyperthyroidism ) . detailed description of these diseases exceeds the scope of this paper ; however , in table 2 the reader can find a comprehensive summary and references to the review and original articles regarding selected human hormone - receptor - related pathologies . hormone - receptor - related diseases constitute an important diagnostic challenge . among them , a monogenic diseases arising due to mutation are the easiest to diagnose , provided that a candidate gene is identified and its sequencing shows mutation . it is much more difficult , though , to evaluate the influence of altered expression or function ( e.g. , due to the abnormal posttranslational modifications ) of the receptor on the phenotype , especially of multifactorial diseases such as obesity , insulin resistance , atherosclerosis , cardiovascular disease , cancer , neurodegeneration , and so forth the diagnostic problems are the reasons why hormone receptor dysfunctions commonly remain undiagnosed and untreated . however , the importance of such dysfunctions in pathophysiology of both rare and common diseases fully justifies the efforts to elucidate the molecular mechanisms of action of these receptors . importantly , small - molecule hormones , usually of quite simple chemical structure , have an enormously wide range of biological functions . the effects of their action are due to their interaction with various receptors , which , by further interaction with other proteins or with dna , activate various signal transduction pathways or regulate the activity of numerous target genes . even though our knowledge regarding these nongenomic and genomic mechanisms is already impressive , a lot of information regarding , first of all , their interdependence still awaits elucidation .

small - molecule hormones play crucial roles in the development and in the maintenance of an adult mammalian organism . on the molecular level , they regulate a plethora of biological pathways . part of their actions depends on their transcription - regulating properties , exerted by highly specific nuclear receptors which are hormone - dependent transcription factors . nuclear hormone receptors interact with coactivators , corepressors , basal transcription factors , and other transcription factors in order to modulate the activity of target genes in a manner that is dependent on tissue , age and developmental and pathophysiological states . the biological effect of this mechanism becomes apparent not earlier than 3060 minutes after hormonal stimulus . in addition , small - molecule hormones modify the function of the cell by a number of nongenomic mechanisms , involving interaction with proteins localized in the plasma membrane , in the cytoplasm , as well as with proteins localized in other cellular membranes and in nonnuclear cellular compartments . the identity of such proteins is still under investigation ; however , it seems that extranuclear fractions of nuclear hormone receptors commonly serve this function . a direct interaction of small - molecule hormones with membrane phospholipids and with mrna is also postulated . in these mechanisms , the reaction to hormonal stimulus appears within seconds or minutes .

1. Introduction 2. The Genomic Mechanism of Action of Small-Molecule Hormones 3. The Nongenomic Mechanisms of Action of Small-Molecule Hormones 4. Human Pathologies Associated with Receptor Abnormalities 5. Conclusion

the biological function of these hormones was initially attributed mostly to their extranuclear activities presently referred to as nongenomic ; however , the exact mechanisms of such actions were then not known . subsequently , the majority of efforts were directed towards the clarification of the transcription - modifying function of these hormones bound to their nuclear receptors that are hormone - regulated transcription factors . this generated an enormous amount of information regarding the genomic action of hormones , the identity of their target genes , and so forth . in this comprehensive paper we present basic information regarding the genomic and nongenomic mechanisms of action of small - molecule hormones , emphasizing the intermediary role of various proteins between the hormonal stimulus and the biological response of the cell . the first effects of engagement of this mechanism might be detected 3060 minutes after its initiation ; however , maximal effects are usually observed after several hours . nuclear receptors of small - molecule hormones belong to the superfamily of nuclear receptors , consisting of receptors for steroid hormones , thyroid hormone , vitamin d , retinoic acid and its derivatives , fatty acids , prostaglandins , and cholesterol derivatives , as well as of orphan receptors with unknown ligands . small fractions of some of these receptors also act outside of the nucleus , in mechanisms generally called nongenomic , which are mediated by processes other than a direct binding of the receptor to dna . some receptors also have an f domain on their c - termini , the function of which is usually unclear . at the base of the first zinc finger , a p - box is present ; its amino acid sequence determines the recognition of a specific ( usually hexameric ) dna sequence in the receptor 's target genes . at the base of the second zinc finger , a d - box is located ; its sequence is , in turn , responsible for the recognition of the distance between the two hexamers forming the hormone response element ( hre ) in the promoter of target gene . in addition , it contains elements involved in cofactor binding , dna binding , and in heterodimerization . finally , the e domain binds a specific hormone , takes part in homodimerization as well as in heterodimerization , and , on its c - terminal end , contains a ligand - dependent transcription activation domain ( af2 ) . the sizes , shapes , and charges of this pocket present in various receptors differ from each other , and this why most receptors bind only their own hormones with an extremely high specificity and affinity ; however , some of them , such as the ppar receptor , possesses a large pocket allowing them to bind various ligands . a very important feature of nuclear receptors is that in the absence of the hormone , conformation of their e domains differs from that acquired upon hormone binding [ 6870 ] . this feature is crucial for the majority of the functions of nuclear hormone receptors , including subcellular localization ( as for steroid receptors ) and transactivation activity . the activity of the nuclear receptor might be modulated by various posttranscriptional modifications including phosphorylation , acetylation , methylation , palmitoylation , and sumoylation [ 7276 ] . in addition , its biological efficiency depends on the rate of its turnover . a classic , genomic mechanism of action of small - molecule hormones is based on the binding of its nuclear receptor to the target gene . however , they might also be present in atypical positions , for example , in the enhancers localized even a few thousand base pairs above the tss . analysis of the natural and artificial hres showed that nuclear hormone receptors preferentially recognize hexamers , sequences consisting of six nucleotides . nevertheless , each of these receptors preferentially recognizes its own target sres with a very high specificity being a result of various factors , such as deviations from the sre consensus sequence , distinct amino acids surrounding dna binding domain fragments of the receptor directly contacting sre , interactions with other transcription factors bound to their own binding sites in the proximity of sre , tissue - specific expression of various receptor isoforms , and the level of receptor expression [ 102 , 103 ] . in addition to classic sres , which mediate transcription activation , a number of negative sres are known that inhibit the transcription when the steroid - hormone - activated receptor binds to nsre [ 106 , 107 ] . specificity of the binding is achieved thanks to hre 's configuration , to the number of neutral nucleotides separating the two hexamers , to the sequence of hexamers and of hre - flanking dna - fragments , and to the sequence of the receptor dna - binding domain [ 112115 ] . nuclear receptors for nonsteroid small - molecule hormones also bind to dr0 and to drs with more than five neutral nucleotides separating hexamers [ 116 , 117 ] as well as to other nonclassical hres . in addition , some hres might be bound by various receptors ; for example , the aggtcatgacct pal0 sequence is recognized by tr- , vdr- , and rar - containing dimers [ 118120 ] . in addition , nuclear hormone receptors might bind as monomers to a single hexamer preceded by an a- and t - rich sequence , as shown for rev - erba , retinoic - acid - receptor - related orphan receptor- ( ror ) , and for nerve - growth - factor - induced clone b ( ngfi - b ) orphan receptors [ 100 , 125 , 126 ] . on the basis of the molecular mechanism of action and of the subcellular localization in the absence of ligand , nuclear hormone receptors can be divided into two types . in general , type i receptors preferentially reside in the cytoplasm ( in unliganded form ) and , while in the nucleus the best known receptors of this type are family iii steroid hormone receptors . type ii receptors , after being synthesized and modified in the cytoplasm , in the presence or absence of their ligand , preferentially translocate to the nucleus , where they are most active as heterodimers . binding of nuclear hormone receptors to dna might result in transcription activation or in transcription inhibition , and such phenomena result from variable molecular mechanisms . in the circulation , they enter the cell by diffusion or are actively transported by a cell - membrane - bound transporting proteins . the majority of their nuclear receptors , a classic examples of type i receptors , reside in the cytoplasm forming inactive complexes with various proteins , including heat shock proteins hsp70 and hsp90 . the activated receptor translocates to the nucleus thanks to its association with chaperones and importins [ 132 , 133 ] , where it binds to its sres in the promoters of target genes ( figure 3 ) . it is suggested that intranuclear mobility of steroid receptors , some of the most mobile proteins within the nucleus , depends on the presence of chaperone proteins such as hsp90 . in addition , steroid hormones acting by their nuclear receptors can potentiate the transactivatory function of other transcription factors [ 141143 ] . inhibition of transcription by steroid hormones and their receptors is a result of a variety of mechanisms , such as hormone - receptor - complex - dependent inhibition of the activity of other transactivators , for example , activator protein 1 ( ap1 ) and nf-b [ 144146 ] . families i and ii receptor proteins , synthesized and modified in the cytoplasm , have their nls exposed so they can translocate to the nucleus in the absence of the hormone . since the conformation of the dna - binding d domain is stable ( independent of the hormone ) , both receptor forms might bind to the promoter of the target gene ; this is why type ii receptors are able either to activate or to inhibit transcription of the same gene in a hormone - dependent manner . in contrast to type i receptors heterodimerization with rxr modulates nuclear trafficking of other receptors [ 155 , 156 ] and increases both affinity of the other receptor to its hre as well as its transactivation activity [ 157160 ] . on the other hand , in rar / rxr and ppar / rxr heterodimers bound to dr1 the presence of rxr in receptor heterodimers raises the question as to how 9-cis - retinoic acid modifies transcription of other hormones ' target genes . most probably it has no influence on the level of activation of triiodothyronine ( t3 ) target genes bound by tr / rxr and of 1,25(oh)2d3 target genes bound by vdr / rxr [ 168 , 169 ] ; however , there are reports claiming otherwise . in all - trans - retinoic acid target genes bound by the rxr / rar heterodimer , 9-cis - retinoic acid alone does not regulate the activity of such genes , but when both receptors are simultaneously bound to their ligands ( 9-cis - retinoic acid and all - trans retinoic acid , respectively ) , genes are activated synergistically . hormone target genes with unoccupied hres are active on the basal level , which depends on the presence of transcription factors other than hormone receptors . however , upon hormone binding to the receptor , conformation of its ligand - binding domain changes ; this results in the dissociation of corepressors , in the recruitment of a coactivator complex containing hats and in transcription activation markedly above the basal level ( figure 5(b ) ) [ 177189 ] . the inhibition could be indirect , depending on the binding of hormone receptors to a strong transactivator ( such as ap1 , nf-b , and p53 ) ; such binding results either in a blockage of transactivator 's activity or in its binding to dna [ 190192 ] . in this mechanism , the binding of the receptor to the dna is not a prerequisite for the inhibition of transcription . as mentioned above , the biological action of small - molecule hormones depends on their interaction with their receptors , as well as on the interactions of the receptor with dna and with other proteins . in the genomic mechanism of hormone action , the most important interaction is that of the receptor with coactivators , corepressors , and other transcription factors . on the other hand , in the nongenomic mechanisms , the most crucial role is played by the binding either of the cytoplasmic fraction of nuclear receptors or of hormone itself to extranuclear proteins . some promoters do not have this sequence ; however , the basal transcriptional machinery binds to such promoters anyway and at a similar distance form tss as in the case of typical promoters . nuclear hormone receptors interact with the basal transcription factors not only via other proteins ( coactivators and corepressors ) but also interact with them directly . creb - binding protein ( cbp ) and p300 possess a histone acetyltransferase activity [ 207 , 208 ] and are coactivators of various transcription factors , including nuclear hormone receptors [ 177 , 178 ] . p300/cbp bind to p160 proteins , to tbp , and to tfiib basal transcription factors , and , as such , are intermediates between receptors and basal transcriptional machinery . their drip205/trap220/trip2 subunit , by the lxxll motif , interacts with tr , vdr , and other nuclear receptors such as rxr and rar in a hormone - dependent and a receptor - af2-domain - dependent manner . a number of other coactivators interacting with nuclear receptors are known , such as ppar coactivator 1 ( pgc-1 ) , which also interact with other receptors , for example , with tr [ 184 , 188 ] and with activating signal cointegrators-1 and -2 ( asc-1 and asc-2 ) interacting with src-1 , p300/cbp , basal transcription factors , and nuclear receptors [ 186 , 187 ] . the final multicomponent complex , by modification of chromatin structure and by interaction with the basal transcriptional machinery , activates transcription of target genes . the best known corepressors are nuclear corepressor ( ncor , rip-13 ) , a large , 270 kda protein , as well as silencing mediator for retinoic acid and thyroid hormone receptors ( smrt ) [ 147 , 148 ] . by doing so in the presence of agonist , it inhibits the activity of target gene , while in the presence of antagonist it magnifies its action . the suppression by rea is a result of the competition with coactivators for binding to er , as well as of the recruitment of hdac and of chromatin modification . natural hres are located relatively close to tss or in more distant regulatory elements , and binding sites for other transcription factors are usually located nearby . such proximity permits interaction between nuclear receptors and these transcription factors , leading either to the suppression of gene activity ( as described above ) or to its additive or synergistic activation . the binding of nuclear receptors to other transcription factors might also occur in a dna - binding - independent manner . in fact , the binding of all known nuclear hormone receptors to the transcription factors has been reported ; the best known examples are the binding of tr to p53 , gr and pr to oct-1 , gr to ap-1 and to nf-b , ppar to nf-b , ap-1 , and to stat [ 217221 ] . on the other hand , deacetylation of histone tails transcription - factor - binding sites become inaccessible to transactivators , and the gene becomes transcriptionally inactive . such a mechanism of modification of chromatin structure is utilized by nuclear hormone receptors , which , as mentioned above , interact with coactivators and corepressors . on the other hand , the binding of a hormone - free receptor to hre initiates the formation of a corepressor complex , which , thanks to its hdac activity , induces local condensation of chromatin ( figure 5(b ) ) . finally , the corepressor complex and its hdac activity are utilized by the specific corepressor proteins described above , which bind to a hormone - activated receptor and inhibit transcription of the target gene ( figure 5(c ) ) . fast biological effects of hormones , just seconds or minutes after hormone administration , have already been described several dozen years ago . steroid and nonsteroid small - molecule hormones bind to various proteins localized outside the nucleus and activate transduction pathways leading to a fast biological response . the presence of binding sites in the cell membrane was proved for all major representatives of these hormones ; however , in many cases the identity of the binding protein remains unknown . in addition , it is likely that such hormones have more than one type of membrane receptors . just next to the cell membrane or directly in it , usually within caveolae ( a bubble - like , 50100 nm invaginations of the cell membrane ) , proteins identical to the nuclear receptors for glucocorticoids , estrogen , androgen , and vitamin d , have been identified [ 222225 ] . it is then plausible that nuclear receptors of other small - molecule hormones are present close to or in the cell membrane . some small - molecule hormones bind to other than nuclear receptor - like cell membrane proteins . a highly conserved 9-amino acid motif ( fvclksiil in er ) that is crucial for palmitoylation and membrane localization has been identified in the ligand - binding domains of er , er , pr , pr , gr , and ar . translocation of nuclear hormone receptors to the membrane is also induced in the presence of the respective ligand ; this was shown for er- and 17-estradiol and for vdr and 1,25(oh)2d3 . in the cell membrane , nuclear hormone receptors interact with caveolae - specific proteins ; for example , er and ar physically interact with caveolin-1 [ 234 , 241 ] , while vdr binds to caveolin-3 . furthermore , binding to caveolins allows hormone receptors to initiate fast , specific nongenomic response to hormonal stimulus . upon binding to the cell membrane receptors , small - molecule hormones activate various transduction pathways by a receptor - type - dependent mechanism . this , in turn , modulates various biological processes in the cytoplasm and influences transcription of genes regulated by newly phosphorylated hormone receptors and transcription factors . cell - membrane - located small - molecule hormone receptors interacting with g proteins might also activate adenylate cyclase , which results in the generation of yet another secondary messenger , camp , and in the activation of camp - dependent proteins , such as pka , and of their substrates [ 243247 ] . by nongenomic mechanisms , small - molecule hormones also regulate the activity of ion channels , influencing cross - membrane movement of na , h , cl , and of k [ 245 , 248 , 249 ] . small - molecule hormones also bind to the proteins present in the cytoplasm ; commonly , such proteins are cytoplasmic fractions of nuclear receptors . hormone - activated ar , pr , and er bind to sh3 or to sh2 subunit of c - src tyrosine kinase localized close to the cell membrane . it results in the increased stability of the receptor due to cam - dependent protection from degradation [ 8587 ] . in addition , pkc isoform is also directly activated by aldosterone and by 17-estradiol , while pkc isoform is activated by 17-estradiol [ 260 , 261 ] . one of them is based on the action of their nuclear receptors as transcription factors . a shortened isoform of tr ( mttr1 , so - called p43 ) , rxr ( mtrxr ) , and ppar2 ( mtppar2 ) , as well as full - length gr , er , and er receptors are present in mitochondria [ 109 , 262265 ] , where they form dimers such as mtrxr/p43 , mtppar2/p43 , gr / gr , and er / er or couple with other transcription factors . another mechanism of small - molecule hormones action in mitochondria is based on their interactions with other proteins . in addition , the shortest isoform of tr1 , p28 , is bound to the internal mitochondrial membrane , where , most likely , it stimulates the function of ant and of uncoupling proteins ( ucps ) [ 263 , 268 ] . small - molecule hormones acting in mitochondria regulate ca wave activity in this organelle , as shown in the case of estrogens and t3 [ 270 , 271 ] . a very rapid effects of androgens , progesterone , glucocorticoids , and other steroid hormones , evident just a few seconds after hormone administration , might be a result of a nonspecific , nongenomic mechanism of small - molecule hormones action , based on their interactions with lipid bilayers . this , in turn , influences the function of membrane proteins such as the calcium pump and other channel proteins , leading to an immediate transport modification of various ions . medical conditions associated with out - of - range level of small - molecule hormones are known for decades , relatively common , and have been exhaustively described in numerous handbooks and articles . detailed description of these diseases exceeds the scope of this paper ; however , in table 2 the reader can find a comprehensive summary and references to the review and original articles regarding selected human hormone - receptor - related pathologies . however , the importance of such dysfunctions in pathophysiology of both rare and common diseases fully justifies the efforts to elucidate the molecular mechanisms of action of these receptors . importantly , small - molecule hormones , usually of quite simple chemical structure , have an enormously wide range of biological functions . the effects of their action are due to their interaction with various receptors , which , by further interaction with other proteins or with dna , activate various signal transduction pathways or regulate the activity of numerous target genes .

obesity , a level of body fatness associated with risk of developing chronic diseases , such as diabetes , dyslipidemias , and arterial hypertension , has become a serious health concern around the world . over the last 30 years , the rate of children suffering from obesity has increased significantly . in the united states , 12.4% of preschoolers were estimated to be obese , and this prevalence increased to 20.8% by the age of 14 . in england , the prevalence of obesity was estimated to be 14% for both boys and girls aged 215 years . in canada several hypotheses have been proposed for the rapid increase in obesity among children living in occidental countries . on the one hand , fitness levels of children have declined significantly since 1981 , regardless of age or gender . at ages 511 , only 7% of children reach the recommended guidelines of 60 minutes or more of daily physical activity to achieve substantial health benefits [ 6 , 7 ] . aside from physical education classes , organized activities such as soccer , swimming , and football as well as active transportation to school and free play should be considered in order to reach daily guidelines [ 810 ] . on the other hand , overconsumption of energy dense foods with low nutritional quality is seen as an important contributor to childhood obesity [ 11 , 12 ] . sugar sweetened beverages ( ssb ) fall into this category of foods that add calories to the diet but are void of vitamins and minerals [ 13 , 14 ] . moreover , since ssbs do not influence appetite , the consumer drinks an additional dose of liquid calories while eating . convenience and snack food account for 40% of the total calories consumed by us children , of which 22% are from sugar sweetened beverages ( ssbs ) . in the us , low - income persons consume more sugar drinks in relation to their overall diet than those with higher income . ssb consumption is higher among african - american and hispanic children and adolescents than among whites . consumption is higher among us boys than girls ; 70% of boys aged 219 years consume ssbs daily . a meta - analysis of cohort studies found that higher intake of ssb among children was associated with 55% ( 95% ci 3282% ) higher risk of being overweight or obese compared with those with lower intake . ssb consumption was also associated with type 2 diabetes where individuals are consuming more than 1 - 2 servings / day and had a 26% greater risk of developing type 2 diabetes than those consuming less than 1 serving / month . both theoretical and empirical evidence confirm the important role the environment plays in children 's health behaviors in general [ 2123 ] and obesity in particular [ 24 , 25 ] . while most evidence on obesity related dietary behaviors in youth has focused on the influence of sociocultural and economic factors at the household level , more recently studies have shown that built and food environments associate with physical activity , healthful eating , and obesity [ 22 , 2628 ] . in particular , access to low nutritional foods and a lack of space for outdoor recreation are related to higher rates of obesity in boys and girls [ 29 , 30 ] . multilevel models , inspired from social ecological theory , posit that childhood obesity is influenced by energy intake and expenditure patterns embedded within familial and wider community contexts . for example , associations between obesity and the built environment vary by gender , age , socioeconomic status , and population density , while the relationship between neighbourhood built environment and youth obesity risk is mediated by socioeconomic status ( ses ) . schools can indeed be located in an environment that discourages physical activity or promotes reliance on convenience stores and fast food restaurants [ 5 , 3336 ] . there is some evidence supporting the existence of a relationship between the characteristics of the environment surrounding schools and students ' ssb consumption [ 37 , 38 ] . a canadian study found ssb consumption to be lower in schools located in communities with a postsecondary education institution ( or = 0.89 ; p = 0.006 comparing no consumption to one ) , lower in schools reporting to limit availability of ssb ( or = 0.85 ; p = 0.02 ) , and lower among girls ( or = 0.49 ; p < 0.001 ) but not significantly different between the school settings ( rural , suburban , or rural ) . however , substantial heterogeneity in study designs , methods , and measurement tools makes it difficult to draw firm conclusions . many recent studies have cast doubt on the effect of the environment on child dietary behavior [ 5 , 32 , 4042 ] . in this respect , many other studies suggest that consumption of ssb in schools is insufficient to predict overall consumption [ 5 , 18 , 40 , 41 , 43 ] and may be associated with other environmental influences . however , environmental influences of ssb consumption on children , beyond the internal environment of the school , are relatively unknown and need to be explored further to inform public policies and interventions for youth health [ 12 , 4042 , 44 ] . the purpose of this study was to explore the association between characteristics of the schools ' vicinity and the risk of ssb consumption for all primary school students living in a canadian metropolitan area . the metropolitan area of sherbrooke is located in the southern part of quebec close to the usa border . it is the 19th largest urban area in canada , with a population of approximately 200,000 people in 2011 . with the help of the sherbrooke regional school board ( srsb ) , we undertook an exhaustive survey of 40 public primary schools ( children aged 5 to 12 years ) in 2007 - 2008 . the main purpose of this survey was to assess the most frequent youth behaviors related to diet and physical activity in order to develop school and community health interventions . an explanatory letter , a self - administered questionnaire , and a return envelope were sent to the parents of all students registered in a srsb school . most of the questionnaire items were inspired from previous reliable surveys [ 45 , 46 ] . after minor adjustments , ease of completion and feasibility were assessed with a convenience sample of 40 parents of children at two primary schools . the final version of the questionnaire comprised 42 questions and took approximately 40 minutes to complete . smallest schools ( < 40 students ) and specialized schools ( e.g. , for disabled students ) were not part of our study population . every school participated and the parents of 8612 students completed the self - administered questionnaire , providing a participation rate of 79% . a total of 7099 students from 37 schools in 39 buildings were kept for the analyses ( two schools were actually operating two separate buildings ) . for our study purpose , the outcome variable was a dichotomous indicator of the consumption of at least one sugar sweetened beverage ( ssb ) per day as compared to less than one ssb per day . drinking over one ssb per day has been associated with individuals ' health status in several recent studies [ 35 , 48 , 49 ] . ssb was measured by consumption of fruit flavored drinks , regular soft drinks , slush , sport drinks , and energy drinks on a 5-point scale ranging from never to several times per day ( 100% fruit juice was excluded ) . the individual - level measurement of ssb consumption frequency was adapted from the canadian health measures survey ( chms ) and the social and health survey on children and teenagers ( shsct ) . overall 14.9% reported to drink at least one ssb each day ( table 1 ) . since ssb consumption is often associated with individual characteristics [ 39 , 50 ] , covariates include gender , academic cycle , cultural origin , and the participation in organized physical activities . the sample included the same proportion of girls and boys and approximately a third of students in each of the three academic cycles with slightly more students in the 3rd cycle ( 10 to 12 years old ) . reported associations between children 's dietary behaviors and school neighbourhood measurements presented substantial heterogeneity in the literature [ 22 , 27 , 28 , 32 , 51 ] . it is important to recall , however , that the contextual influences on child 's behaviors are not necessarily direct and causal ; it is rather suggested to be considered as a risk regulator . they are not themselves risks but are the conditions that regulate or control exposure probabilities to behaviors that lead to disease . consequently in this study , the selected contextual variables included not only measurements typically associated with ssb consumption ( the access to convenience stores and fast food restaurants ) , but also other contextual characteristics more broadly associated with children 's healthy lifestyle such as a pedestrian friendly environment measured by the presence of green spaces and the walkability of streets . more precisely , six contextual variables measuring characteristics of the built environment surrounding the schools were included in our study : ( 1 ) convenience stores density , ( 2 ) fast food restaurant density , ( 3 ) closest convenience store , ( 4 ) closest fast food restaurant , ( 5 ) degree of vegetation cover , and ( 6 ) street walkability . data to create these measures were derived from a geographical information system ( gis ) using arcgis 10 ( esri inc . , redlands , ca , usa ) software and its network analyst extension . these contextual measurements referred to the proximal environment of each school instead of the administrative boundaries of their neighbourhood . in this way , schools measurements were directly comparable . two global contextual variables were created from these six measures using a principal components procedure . finally , one school - level variable measuring school socioeconomic index was included in our model . density measures of convenience stores and fast food restaurants , degree of vegetation cover , and walkability relied upon the construction of 750 meters of ( approximately half a mile ) sausage network buffer around the school buildings . adresses qubec ( http://adressesquebec.gouv.qc.ca/ ) network file ( excluding highway network ) and including pedestrian trails from the components of regional geographic use layer . convenience stores were identified through the quebec ministry of agriculture , fisheries and food 2009 sales licenses as well as an automated and manual search by keywords . the convenience store indicator refers to establishments comprised within an area less than 400 square meters , which sell food of all kinds , with or without gasoline sales . snack , takeout restaurants , and fast food restaurants categories , as identified by the quebec ministry of agriculture , fisheries and food 2009 sales licenses . the density of fast food restaurants was computed by estimating the number of such restaurants by km . closest convenience store . the distance to the school 's closest convenience store was computed on the pedestrian road network . the distance to the school 's closest fast food restaurant was computed on the pedestrian road network . images were taken with the landsat satellite enhanced thematic mapper plus ( etm + ) sensor in 2011 . the ndvi values extend from 1 to 1 ; 1 indicated a total lack of vegetation , while 1 would refer to a dense forest cover . extraction raster file cell values operations were performed using the geospatial modeling environment software . the walkability index was computed based on four school - level measurements : residential density ; destinations density ; street connectivity ; land - use mix . these indicators were objectively determined for each school [ 57 , 58 ] ( table 2 ) . standardized z - scores of each measure were summed to construct a walkability index [ 59 , 60 ] . a higher value of the walkability index suggests a pedestrian friendly environment . global indexes . in order to consider schools ' neighbourhood built environment measurements more globally , we produced two synthesised contextual indicators issued from a principal component analysis ( pca ) [ 61 , 62 ] . the six built environment variables were introduced in a pca and produced two uncorrelated factor scores with an eigenvalue higher than 1 ( i.e. , a significant part of the variance of all six variables was explained by the factor ) . the strength and the sign of the correlations were used to interpret the meaning of the underlying concept for each factor ( table 3 ) . interpretation suggested the first factor would be related to the urban density or the physical accessibility to food resources and account for about 69% of the variance of the six variables . the other factor refers to the food sources proximity and explained 19% of the variance . the school socioeconomic index ( ssei ) was computed with two variables issued from the 2006 canadian census by the quebec ministry of education : the education level of the mother and the inactivity of the parents , and it was adjusted for school year 2007 - 2008 population . these two variables were chosen since they showed the highest correlation with youth academic failure . the ssei is calculated for all schools of the province and is typically distributed in deciles , from 1 ( 10% most privileged ) to 10 ( 10% most deprived ) . according to their provincial score , the 39 schools were reclassified into terciles to distinguish high and low ses among sherbrooke 's schools ( table 1 ) . school - level measurements and factor scores were divided into quartiles to control for the nonnormality of distributions and the nonlinear associations with child ssb consumption . multilevel logistic modeling procedures were used to assess whether variation in ssb consumption could be associated with the neighbourhood 's characteristics surrounding the school . the models were fitted using bayesian estimation procedures as implemented via monte carlo markov chain ( mcmc ) methods using metropolis - hastings algorithm in mlwin 2.25 . a four - step sequential modeling strategy was adopted in order to explain the outcome variation between schools ( table 4 ) . this controls for the nonindependence of observations within schools and estimates the mean correlation of students ' ssb consumption associated with the school , without taking into account individual or school characteristics . the null model shows if there is a significant variation of ssb consumption between schools . this allows estimating the mean correlation of students ' ssb consumption associated with the school while controlling for individuals ' characteristics . this model determines if the variation of ssb consumption between schools is simply a result of the individual characteristics of students ( gender , age , cultural origin , and physical activity ) . the reference categories were girls , in 1st cycle , from a french - canadian cultural origin , who participated in organized physical activities . since the objective of the study was to explore contextual influences on students ssb consumption , model 2 was used to compare fixed and random parameters in order to observe the effect of contextual measurements while controlling for individual characteristics . the reference category was the schools in the lowest ssei tercile ( i.e. , high ses ) . this model determines if the schools ' ses explains the variation of ssb consumption between schools . model 4 is an extension of model 2 but includes schools ' neighbourhood measurements . at this step , all six school - level built environment measures and the two global indexes were individually tested ( 8 different models ) in order to identify which aspects of the school vicinity may contribute to explain the between - school variation of ssb consumption . this procedure is essential because even if we analysed the entire population of sherbrooke 's primary schools , our second - level distribution includes only 39 observations . consequently , the variation between second - level observations is limited and does not allow the inclusion of many second - level variables simultaneously . moreover , second - level measurements based on geographic location often face multicolinearity problem [ 62 , 66 ] . all models present the odds ratio ( or ) of ssb consumption with its 95% confidence interval ( ci ) for each characteristic considered ( fixed part ) . the between - school variance structure ( random part ) was analysed using the median odd ratio ( mor ) . the mor was considered significant when the school - level variance was at least 1.96 times higher than its standard error ( se ) . the mor can be interpreted as the increased chances a pupil has to drink at least one ssb per day , if this student was moving from a school with a lower chance to a school with a higher chance of a daily ssb consumption . the epidemiologic interpretation of the mor is more straightforward than other random parameters issued from multilevel modeling since it can be directly compared to a regular or and thus evaluate if the school - level effect is more or less important than other variables included in the model . we used the deviance information criterion ( dic ) to compare the models goodness of fit . the dic is a measure of data fitting which considers the number of model parameters . a small dic difference ( 2 ) between two models shows equivalency while a larger difference indicates a significant improvement . the null model considered no variable in the fixed part of the model , while the random part shows a significant variation of ssb consumption between schools . it also revealed that students risk of consuming ssb on a daily basis increased by 72% ( mor = 1.72 ) if they would move from a school which had a lower rate of ssb consumption to one which had a higher rate of ssb consumption . older students and those that were not qc french had higher risk of consuming ssb . students that did not participate in organized physical activities had almost twice the risk of drinking ssb daily ( or = 1.96 ; ic = 1.682.28 ) compared to students involved in organized physical activities . this model shows that a typical student registered in a sherbrooke primary school who drinks ssb on a daily basis would be 10 to 12 years old , not qc french , and does not participate in organized physical activities . taking individual characteristics into account reduced the school - level variance which brought down the mor to 1.62 . thus , individuals characteristics were not uniformly distributed between schools and this would explain 10% of the risk in ssb consumption at the school level . moreover , considering students ' characteristics increased the goodness of fit importantly shown by the dic difference of 99.33 . model 3 introduced only one school - level variable , the school socioeconomic index ( ssei ) ; the associations with the individuals ' characteristics remained stable . schools with the lowest ssei had an increased risk of 57% to have more students drinking ssb every day . in the random part , considering the ssei explained an additional 10% of risk shown by the mor but did not significantly increase the goodness of fit ( the model is still good but was not improved ) . prior to building model 4 , we individually introduced the six built environment variables and the two global indexes ( 8 different models ) . for the two global measurements , only the urban density index issued from the pca was associated with child 's ssb consumption . the association not only was significant but also was shown to be relatively strong with an effect similar to the physical activity level of students . however , no gradient was observed between the level of urban density and ssb consumption ; that is , students going to a school with the lowest urban density environment ( few or no fast food restaurants and convenience stores , with a low street walkability , and a high vegetation cover ) had significantly a lower risk to drink ssb than students in the other school environments in sherbrooke ( or = 1.67 to 1.98 ) . this , however , explained less of the between - school variance ( mor = 1.58 ) than the ssei ( model 3 ) but had a slight increase in the model goodness of fit . we examined associations between the characteristics of the schools ' vicinity and the risk of the ssb consumption in a large sample of primary school students . at the individual level , the prevalence of daily ssb consumption among sherbrooke 's school children was 15% , a relatively low level as compared to what was observed in europe or in the usa . individual predictors , age , cultural origin , and physical activity level , were all associated with ssb daily consumption . these results are in line with previous research findings showing that individual characteristics are important predictors of ssb consumption , especially age , physical activity , and economic and cultural origin . unlike other studies we found no association with the gender of students . together , individual characteristics explained about 10% of the difference of risk of daily ssb consumption between schools . at the school level , ssb consumption was also found to vary significantly above students ' characteristics . the variation in ssb consumption between schools students in the lowest ssei were more likely to drink ssb every day than students in schools with the highest ssei . the schools ' socioeconomic status explained an additional 10% in the risk of daily ssb consumption between schools . this suggests that , above individual characteristics and the school 's socioeconomic status , a sherbrooke student moving from a school with a lower rate of ssb consumption to a school with a higher rate would typically increase his / her risk by 52% to daily consume ssb . although few studies reported ssb consumption between schools , results are in line with the results of carter and dubois which suggested that neighbourhood deprivation was more consistently associated with childhood obesity . this finding also recalls that peer behaviors may have an influence on diet among children . indeed , freeland - graves and nitzke showed that diet preferences and behaviors may change importantly when children are in contact with a new social environment . beside the socioeconomic environment of the school , we also explored six contextual characteristics measured within 750 meters of the schools : the number of fast food restaurants , the number of convenience stores , the walkability , the degree of vegetation cover , the distance to the closest fast food restaurant , and the distance to the closest convenience store . letting individual characteristics constant we further explored the combined effect of these measurements and found that students going to schools with the lowest urban density , globally estimated by few or no fast food restaurants and convenience stores in the vicinity , with a low potential of walkability and with an important vegetation cover , may have twice as much as less chances to daily consume ssb than those in other schools ( model 4 ) . as for the socioeconomic environment , this is in line with other studies findings . in a multilevel analysis , leatherdale et al . found an increased risk of overweight with more fast food retailers around schools . indicate that a little less than one - half of sugar - drink kilocalories ( 48% ) are consumed away from home . of these , 43% are purchased in stores , 35.5% in restaurants or fast food establishments , and 1.4% in schools or day - care settings . moreover , according to van hulst et al . , who realised a study in a metropolitan canadian city , supermarkets , fast food restaurants , and convenience stores were more accessible around schools than around residences , as shown by shorter walking distances to and higher densities of each type of food establishment in school neighbourhoods . it was shown that attending a school with a higher density of fast food restaurants / convenience stores than supermarket / specialty store gives a greater likelihood of consuming ssb , after adjusting for individual and contextual covariates . although we failed to find associations with specific characteristics of the built environment , this finding suggests that ssb consumption may be influenced by a dense urban environment enclosing a set of characteristics which globally offers a more important variety of ssb sources proximal to a school , where the street network is considered walkable and possibly more attractive for pedestrians where the presence of vegetation is important . this finding is particularly important because it suggests that contextual influences are not necessarily causal links on individuals ' behaviors but rather act as risk regulators which facilitated or constrained individuals ' choices rather than causing them directly and that global contextual measurements are sometime more efficient to capture the individual - environment dynamics for specific context . in the case of sherbrooke , for example , the lower risk of ssb consumption by students going to a school located in a low urban density environment may be due to a more important proportion of students taking a school bus to travel between home and school and thus is less solicited by the commercial offer of ssb in the school vicinity , a situation that might not be true elsewhere . these research findings exposed a significant variation in child 's ssb consumption between schools and revealed the important role of the socioeconomic and built environment in the surrounding environment . strengths of this study include the high participation rate of households ( 79% ) and primary schools ( 100% ) , allowing generalizability of the findings for the studied area and a possible contribution to elaborate public health or urban planning interventions . the use of multilevel analysis controlled for the shared variance of ssb consumption between schools , but also to analyse its importance as compared to individual - level influences . using global measurements of the built environment allowed detecting the combined influence of some characteristics that were not individually associated with ssb . this finding clearly supports the hypothesis that contextual influences on child 's behaviors are not necessarily direct and causal . the individual - environment dynamics are complex to measure ; it may be more helpful for research and intervention to consider contextual influences as the conditions that regulate exposure probabilities to behaviors that lead to disease . although global contextual measurement may be more difficult to interpret , they may be the more efficient way to explain the contextual influences and may help to avoid finding no relationship where one actually exists ( type 2 error ) . a few limitations need to be kept in mind for the interpretation of the results . the cross - sectional design of this study does not ultimately reveal the direction of the associations between ssb and environmental factors . also , although questionnaires were anonymous and confidential , data was reported by the parent instead of directly by the child , introducing possible information bias . no validated french - canadian questionnaire on the frequency of ssb consumption currently exists , but since the measurement we used was adapted from the two important governmental surveys ( chms and shsct ) , little doubts remain on its reliability . data pertaining to the characteristics of home environment ( composition of household , socioeconomic status ) or the distance between home and school was not collected ; this information would have helped to better understand the environments to which students are exposed while on their way to school and permitted controlling for confounding effects of parents ' socioeconomic status . finally , while there is a possible interaction between ssei and the urban density , it was not statistically feasible to integrate both indicators in the statistical model because of multicollinearity . a larger number of schools ( e.g. , 100 schools or more ) would have allowed testing this interaction in a multilevel model , but we were constrained by the actual number of schools in the area ( n = 39 ) . in canada , approximately 10% of children are currently obese . given the immediate and long term consequences of childhood obesity , it is of prime importance to better understand the multiple levels of influence that characterize the obesity epidemic , including the social and built environments . as school settings are among the most important environments to which children are exposed to , estimating their impact on dietary choices is essential to understand how the imbalance between energy intake and energy expenditure occurs . only a few studies have investigated the role of nonresidential environments . to our knowledge , this is the first study to investigate this relationship for an exhaustive set of school at the regional level . our results revealed important variations of children ssb consumption between sherbrooke 's schools according to the environmental characteristics in its vicinity . these findings are important for health and place studies since they highlight the influence of the school 's vicinity taken globally , rather than direct causal links with specific characteristics . thus it supports theories proposing that dietary behaviors are a result of complex interactions between biological , social , and environmental factors . since all sherbrooke 's schools participated in the survey , results may be directly used by local and regional stakeholders aiming to plan an intervention regarding schools proximal built environment .

the purpose of the research was to explore the associations between the characteristics of schools ' vicinity and the risk of sugar sweetened beverage ( ssb ) consumption in elementary students . findings exposed an important variation in student 's ssb consumption between schools . schools with a lower socioeconomic status or in a densely built environment tend to have higher proportion of regular ssb drinkers . these characteristics of the school 's vicinity partly explained the variation observed between them . we estimated that a student moving to a school with a higher proportion of ssb drinkers may increase his / her chances by 52% of becoming a daily consumer . important changes in dietary preferences can occur when children are in contact with a new social environment . findings also support the idea that dietary behaviors among children result from the complex interactions between biological , social , and environmental factors .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion

obesity , a level of body fatness associated with risk of developing chronic diseases , such as diabetes , dyslipidemias , and arterial hypertension , has become a serious health concern around the world . in the united states , 12.4% of preschoolers were estimated to be obese , and this prevalence increased to 20.8% by the age of 14 . in canada several hypotheses have been proposed for the rapid increase in obesity among children living in occidental countries . aside from physical education classes , organized activities such as soccer , swimming , and football as well as active transportation to school and free play should be considered in order to reach daily guidelines [ 810 ] . on the other hand , overconsumption of energy dense foods with low nutritional quality is seen as an important contributor to childhood obesity [ 11 , 12 ] . sugar sweetened beverages ( ssb ) fall into this category of foods that add calories to the diet but are void of vitamins and minerals [ 13 , 14 ] . convenience and snack food account for 40% of the total calories consumed by us children , of which 22% are from sugar sweetened beverages ( ssbs ) . ssb consumption is higher among african - american and hispanic children and adolescents than among whites . a meta - analysis of cohort studies found that higher intake of ssb among children was associated with 55% ( 95% ci 3282% ) higher risk of being overweight or obese compared with those with lower intake . ssb consumption was also associated with type 2 diabetes where individuals are consuming more than 1 - 2 servings / day and had a 26% greater risk of developing type 2 diabetes than those consuming less than 1 serving / month . while most evidence on obesity related dietary behaviors in youth has focused on the influence of sociocultural and economic factors at the household level , more recently studies have shown that built and food environments associate with physical activity , healthful eating , and obesity [ 22 , 2628 ] . for example , associations between obesity and the built environment vary by gender , age , socioeconomic status , and population density , while the relationship between neighbourhood built environment and youth obesity risk is mediated by socioeconomic status ( ses ) . there is some evidence supporting the existence of a relationship between the characteristics of the environment surrounding schools and students ' ssb consumption [ 37 , 38 ] . a canadian study found ssb consumption to be lower in schools located in communities with a postsecondary education institution ( or = 0.89 ; p = 0.006 comparing no consumption to one ) , lower in schools reporting to limit availability of ssb ( or = 0.85 ; p = 0.02 ) , and lower among girls ( or = 0.49 ; p < 0.001 ) but not significantly different between the school settings ( rural , suburban , or rural ) . however , substantial heterogeneity in study designs , methods , and measurement tools makes it difficult to draw firm conclusions . many recent studies have cast doubt on the effect of the environment on child dietary behavior [ 5 , 32 , 4042 ] . in this respect , many other studies suggest that consumption of ssb in schools is insufficient to predict overall consumption [ 5 , 18 , 40 , 41 , 43 ] and may be associated with other environmental influences . however , environmental influences of ssb consumption on children , beyond the internal environment of the school , are relatively unknown and need to be explored further to inform public policies and interventions for youth health [ 12 , 4042 , 44 ] . the purpose of this study was to explore the association between characteristics of the schools ' vicinity and the risk of ssb consumption for all primary school students living in a canadian metropolitan area . it is the 19th largest urban area in canada , with a population of approximately 200,000 people in 2011 . with the help of the sherbrooke regional school board ( srsb ) , we undertook an exhaustive survey of 40 public primary schools ( children aged 5 to 12 years ) in 2007 - 2008 . the main purpose of this survey was to assess the most frequent youth behaviors related to diet and physical activity in order to develop school and community health interventions . an explanatory letter , a self - administered questionnaire , and a return envelope were sent to the parents of all students registered in a srsb school . most of the questionnaire items were inspired from previous reliable surveys [ 45 , 46 ] . after minor adjustments , ease of completion and feasibility were assessed with a convenience sample of 40 parents of children at two primary schools . the final version of the questionnaire comprised 42 questions and took approximately 40 minutes to complete . for our study purpose , the outcome variable was a dichotomous indicator of the consumption of at least one sugar sweetened beverage ( ssb ) per day as compared to less than one ssb per day . ssb was measured by consumption of fruit flavored drinks , regular soft drinks , slush , sport drinks , and energy drinks on a 5-point scale ranging from never to several times per day ( 100% fruit juice was excluded ) . the individual - level measurement of ssb consumption frequency was adapted from the canadian health measures survey ( chms ) and the social and health survey on children and teenagers ( shsct ) . since ssb consumption is often associated with individual characteristics [ 39 , 50 ] , covariates include gender , academic cycle , cultural origin , and the participation in organized physical activities . the sample included the same proportion of girls and boys and approximately a third of students in each of the three academic cycles with slightly more students in the 3rd cycle ( 10 to 12 years old ) . reported associations between children 's dietary behaviors and school neighbourhood measurements presented substantial heterogeneity in the literature [ 22 , 27 , 28 , 32 , 51 ] . consequently in this study , the selected contextual variables included not only measurements typically associated with ssb consumption ( the access to convenience stores and fast food restaurants ) , but also other contextual characteristics more broadly associated with children 's healthy lifestyle such as a pedestrian friendly environment measured by the presence of green spaces and the walkability of streets . more precisely , six contextual variables measuring characteristics of the built environment surrounding the schools were included in our study : ( 1 ) convenience stores density , ( 2 ) fast food restaurant density , ( 3 ) closest convenience store , ( 4 ) closest fast food restaurant , ( 5 ) degree of vegetation cover , and ( 6 ) street walkability . density measures of convenience stores and fast food restaurants , degree of vegetation cover , and walkability relied upon the construction of 750 meters of ( approximately half a mile ) sausage network buffer around the school buildings . adresses qubec ( http://adressesquebec.gouv.qc.ca/ ) network file ( excluding highway network ) and including pedestrian trails from the components of regional geographic use layer . snack , takeout restaurants , and fast food restaurants categories , as identified by the quebec ministry of agriculture , fisheries and food 2009 sales licenses . the distance to the school 's closest convenience store was computed on the pedestrian road network . the distance to the school 's closest fast food restaurant was computed on the pedestrian road network . the ndvi values extend from 1 to 1 ; 1 indicated a total lack of vegetation , while 1 would refer to a dense forest cover . a higher value of the walkability index suggests a pedestrian friendly environment . in order to consider schools ' neighbourhood built environment measurements more globally , we produced two synthesised contextual indicators issued from a principal component analysis ( pca ) [ 61 , 62 ] . the six built environment variables were introduced in a pca and produced two uncorrelated factor scores with an eigenvalue higher than 1 ( i.e. , a significant part of the variance of all six variables was explained by the factor ) . the strength and the sign of the correlations were used to interpret the meaning of the underlying concept for each factor ( table 3 ) . interpretation suggested the first factor would be related to the urban density or the physical accessibility to food resources and account for about 69% of the variance of the six variables . the other factor refers to the food sources proximity and explained 19% of the variance . the school socioeconomic index ( ssei ) was computed with two variables issued from the 2006 canadian census by the quebec ministry of education : the education level of the mother and the inactivity of the parents , and it was adjusted for school year 2007 - 2008 population . the ssei is calculated for all schools of the province and is typically distributed in deciles , from 1 ( 10% most privileged ) to 10 ( 10% most deprived ) . school - level measurements and factor scores were divided into quartiles to control for the nonnormality of distributions and the nonlinear associations with child ssb consumption . multilevel logistic modeling procedures were used to assess whether variation in ssb consumption could be associated with the neighbourhood 's characteristics surrounding the school . a four - step sequential modeling strategy was adopted in order to explain the outcome variation between schools ( table 4 ) . this controls for the nonindependence of observations within schools and estimates the mean correlation of students ' ssb consumption associated with the school , without taking into account individual or school characteristics . the null model shows if there is a significant variation of ssb consumption between schools . this allows estimating the mean correlation of students ' ssb consumption associated with the school while controlling for individuals ' characteristics . this model determines if the variation of ssb consumption between schools is simply a result of the individual characteristics of students ( gender , age , cultural origin , and physical activity ) . since the objective of the study was to explore contextual influences on students ssb consumption , model 2 was used to compare fixed and random parameters in order to observe the effect of contextual measurements while controlling for individual characteristics . this model determines if the schools ' ses explains the variation of ssb consumption between schools . at this step , all six school - level built environment measures and the two global indexes were individually tested ( 8 different models ) in order to identify which aspects of the school vicinity may contribute to explain the between - school variation of ssb consumption . consequently , the variation between second - level observations is limited and does not allow the inclusion of many second - level variables simultaneously . all models present the odds ratio ( or ) of ssb consumption with its 95% confidence interval ( ci ) for each characteristic considered ( fixed part ) . the mor was considered significant when the school - level variance was at least 1.96 times higher than its standard error ( se ) . the mor can be interpreted as the increased chances a pupil has to drink at least one ssb per day , if this student was moving from a school with a lower chance to a school with a higher chance of a daily ssb consumption . the epidemiologic interpretation of the mor is more straightforward than other random parameters issued from multilevel modeling since it can be directly compared to a regular or and thus evaluate if the school - level effect is more or less important than other variables included in the model . the null model considered no variable in the fixed part of the model , while the random part shows a significant variation of ssb consumption between schools . it also revealed that students risk of consuming ssb on a daily basis increased by 72% ( mor = 1.72 ) if they would move from a school which had a lower rate of ssb consumption to one which had a higher rate of ssb consumption . older students and those that were not qc french had higher risk of consuming ssb . students that did not participate in organized physical activities had almost twice the risk of drinking ssb daily ( or = 1.96 ; ic = 1.682.28 ) compared to students involved in organized physical activities . this model shows that a typical student registered in a sherbrooke primary school who drinks ssb on a daily basis would be 10 to 12 years old , not qc french , and does not participate in organized physical activities . taking individual characteristics into account reduced the school - level variance which brought down the mor to 1.62 . thus , individuals characteristics were not uniformly distributed between schools and this would explain 10% of the risk in ssb consumption at the school level . model 3 introduced only one school - level variable , the school socioeconomic index ( ssei ) ; the associations with the individuals ' characteristics remained stable . schools with the lowest ssei had an increased risk of 57% to have more students drinking ssb every day . prior to building model 4 , we individually introduced the six built environment variables and the two global indexes ( 8 different models ) . for the two global measurements , only the urban density index issued from the pca was associated with child 's ssb consumption . however , no gradient was observed between the level of urban density and ssb consumption ; that is , students going to a school with the lowest urban density environment ( few or no fast food restaurants and convenience stores , with a low street walkability , and a high vegetation cover ) had significantly a lower risk to drink ssb than students in the other school environments in sherbrooke ( or = 1.67 to 1.98 ) . this , however , explained less of the between - school variance ( mor = 1.58 ) than the ssei ( model 3 ) but had a slight increase in the model goodness of fit . we examined associations between the characteristics of the schools ' vicinity and the risk of the ssb consumption in a large sample of primary school students . at the individual level , the prevalence of daily ssb consumption among sherbrooke 's school children was 15% , a relatively low level as compared to what was observed in europe or in the usa . individual predictors , age , cultural origin , and physical activity level , were all associated with ssb daily consumption . these results are in line with previous research findings showing that individual characteristics are important predictors of ssb consumption , especially age , physical activity , and economic and cultural origin . together , individual characteristics explained about 10% of the difference of risk of daily ssb consumption between schools . at the school level , ssb consumption was also found to vary significantly above students ' characteristics . the variation in ssb consumption between schools students in the lowest ssei were more likely to drink ssb every day than students in schools with the highest ssei . the schools ' socioeconomic status explained an additional 10% in the risk of daily ssb consumption between schools . this suggests that , above individual characteristics and the school 's socioeconomic status , a sherbrooke student moving from a school with a lower rate of ssb consumption to a school with a higher rate would typically increase his / her risk by 52% to daily consume ssb . although few studies reported ssb consumption between schools , results are in line with the results of carter and dubois which suggested that neighbourhood deprivation was more consistently associated with childhood obesity . this finding also recalls that peer behaviors may have an influence on diet among children . indeed , freeland - graves and nitzke showed that diet preferences and behaviors may change importantly when children are in contact with a new social environment . beside the socioeconomic environment of the school , we also explored six contextual characteristics measured within 750 meters of the schools : the number of fast food restaurants , the number of convenience stores , the walkability , the degree of vegetation cover , the distance to the closest fast food restaurant , and the distance to the closest convenience store . letting individual characteristics constant we further explored the combined effect of these measurements and found that students going to schools with the lowest urban density , globally estimated by few or no fast food restaurants and convenience stores in the vicinity , with a low potential of walkability and with an important vegetation cover , may have twice as much as less chances to daily consume ssb than those in other schools ( model 4 ) . in a multilevel analysis , leatherdale et al . found an increased risk of overweight with more fast food retailers around schools . indicate that a little less than one - half of sugar - drink kilocalories ( 48% ) are consumed away from home . of these , 43% are purchased in stores , 35.5% in restaurants or fast food establishments , and 1.4% in schools or day - care settings . , who realised a study in a metropolitan canadian city , supermarkets , fast food restaurants , and convenience stores were more accessible around schools than around residences , as shown by shorter walking distances to and higher densities of each type of food establishment in school neighbourhoods . it was shown that attending a school with a higher density of fast food restaurants / convenience stores than supermarket / specialty store gives a greater likelihood of consuming ssb , after adjusting for individual and contextual covariates . although we failed to find associations with specific characteristics of the built environment , this finding suggests that ssb consumption may be influenced by a dense urban environment enclosing a set of characteristics which globally offers a more important variety of ssb sources proximal to a school , where the street network is considered walkable and possibly more attractive for pedestrians where the presence of vegetation is important . in the case of sherbrooke , for example , the lower risk of ssb consumption by students going to a school located in a low urban density environment may be due to a more important proportion of students taking a school bus to travel between home and school and thus is less solicited by the commercial offer of ssb in the school vicinity , a situation that might not be true elsewhere . these research findings exposed a significant variation in child 's ssb consumption between schools and revealed the important role of the socioeconomic and built environment in the surrounding environment . strengths of this study include the high participation rate of households ( 79% ) and primary schools ( 100% ) , allowing generalizability of the findings for the studied area and a possible contribution to elaborate public health or urban planning interventions . the use of multilevel analysis controlled for the shared variance of ssb consumption between schools , but also to analyse its importance as compared to individual - level influences . using global measurements of the built environment allowed detecting the combined influence of some characteristics that were not individually associated with ssb . the cross - sectional design of this study does not ultimately reveal the direction of the associations between ssb and environmental factors . no validated french - canadian questionnaire on the frequency of ssb consumption currently exists , but since the measurement we used was adapted from the two important governmental surveys ( chms and shsct ) , little doubts remain on its reliability . data pertaining to the characteristics of home environment ( composition of household , socioeconomic status ) or the distance between home and school was not collected ; this information would have helped to better understand the environments to which students are exposed while on their way to school and permitted controlling for confounding effects of parents ' socioeconomic status . finally , while there is a possible interaction between ssei and the urban density , it was not statistically feasible to integrate both indicators in the statistical model because of multicollinearity . a larger number of schools ( e.g. , 100 schools or more ) would have allowed testing this interaction in a multilevel model , but we were constrained by the actual number of schools in the area ( n = 39 ) . in canada , approximately 10% of children are currently obese . as school settings are among the most important environments to which children are exposed to , estimating their impact on dietary choices is essential to understand how the imbalance between energy intake and energy expenditure occurs . our results revealed important variations of children ssb consumption between sherbrooke 's schools according to the environmental characteristics in its vicinity . these findings are important for health and place studies since they highlight the influence of the school 's vicinity taken globally , rather than direct causal links with specific characteristics . thus it supports theories proposing that dietary behaviors are a result of complex interactions between biological , social , and environmental factors . since all sherbrooke 's schools participated in the survey , results may be directly used by local and regional stakeholders aiming to plan an intervention regarding schools proximal built environment .

unless specified , all the reactions were done in oven - dried or flame - dried glassware in an atmosphere of dry argon gas using standard schlenk techniques . the anhydrous solvents were purchased from acros and aldrich chemical companies and used in the reactions without further purification . all workup and chromatographic purifications of compounds were done in air using optima grade solvents obtained from fisher scientific . commercial grade preloaded redi sep rf columns were used for silica chromatography , and hitrap sp hp columns purchased from ge healthcare were used for ion - exchange chromatography . the stains used to visualize the thin - layer chromatography ( tlc ) plates ( aluminum backed 200 m silica ) were hanessian s stain [ ceso4 ( 5 g ) and ( nh4)mo7o24.4h2o ( 25 g ) dissolved in water ( 450 ml ) and concentrated sulfuric acid ( 50 ml ) ] , and potassium permanganate stain [ kmno4 ( 1.5 g ) and k2co3 ( 10 g ) dissolved in 10% naoh ( 1.25 ml ) in water ( 200 ml ) ] . caution ! although we did not observe any unusual decompositions of the azides reported , organic azides can be explosive materials and should be handled with care . h , c , na , and f nmr spectra were recorded using bruker acp-500 or varian spectrometers at 30 c or room temperature . h , c , and f nmr chemical shifts are reported in ppm referenced to residual solvent resonances ( h nmr : 7.27 for chcl3 in cdcl3 , and 3.31 for chd2od in cd3od . deuterated solvents for nmr were obtained from cambridge isotope laboratories and were used without purification . electrospray ionization mass spectra ( esi ms ) were collected on a micromass quattro ii , triple quadrupole mass spectrometer using both negative and positive ionization modes . liquid chromatography mass spectrometry ( lcms ) spectra were recorded on agilent technologies 6330 ion trap instrument . high - resolution electrospray ionization time - of - flight ( hr - esi - tof ) analyses were performed at scripps center for metabolomics and mass spectrometry , la jolla , california . ir spectra were obtained on a nicolet nexus 670 ft - ir instrument using kbr pellets . gadolinium concentration was measured by inductively coupled argon plasma ( icp ) mass spectrometry ( university of illinois at urbana champaign { uiuc } , illinois sustainability technology center , division of the institute of natural resource sustainability ) . the sample ( 0.0500 ml ) was added to optima grade nitric acid ( 0.500 ml ) and heated to 80 c in a closed vial overnight . samples were also measured by inductively coupled plasma optical emission spectrometry ( icp - oes ) ( san diego state university , ecology analytical facility ) . after purification of 4a - naotf and 4b - naotf by chromatography , the compounds were crystallized , and data were acquired and analyzed by the small molecule x - ray crystallography facility at the university of california , san diego . compound 4a - naotf was crystallized by slow evaporation of ch2cl2 , whereas 4b - naotf was crystallized by vapor diffusion of diethyl ether into a ch2cl2 solution at 10 c . to a dry 3-neck flask was added triflic anhydride ( 13.7469 g , 48.7237 mmol , 1.03 equiv relative to ( s)-methyl lactate ) and dry ch2cl2 ( 15 ml ) . an addition funnel was placed on the 3-neck flask and filled with ( s)-methyl lactate ( 4.9040 g , 47.1040 mmol ) , dry n , n - diisopropylethylamine ( 6.3931 g , 49.4631 mmol ) , and dry ch2cl2 ( 5 ml ) . the contents of the addition funnel were added dropwise under nitrogen to the reaction mixture as the flask was cooled in an ice bath over a period of 10 min before the bath was allowed to warm to room temperature . the crude reaction mixture was used directly in the next step without further purification . h nmr of an aliquot ( cd3od , 399.8 mhz ) showing peaks for 9 : 5.25 ( q , j = 7.0 , 1h ) , 3.86 ( s , 3h ) , 1.72 ( d , j = 7.0 , 3h ) . f nmr ( cdcl3 , 376.1 mhz ) : 75.2 ( s ) trifluoromethanesulfonate ( triflate , tfo ) on 9 and 78.4 ( s ) free triflate ion . a sample of 8(60,61 ) ( 3.44 g , 15.68 mmol ) was taken up in ch2cl2 ( 40 ml ) , and the resulting solution was dried over molecular sieves ( beads , grade 512 type 4 , 48 mesh ) overnight . dry ( distilled from cah2 ) n , n - diisopropylethylamine ( 12.47 g , 96.48 mmol ) was added dropwise , followed by the reaction mixture described above , containing 9 ( 47.10 mmol assuming 100% yield ) via cannula , and the reaction flask was rinsed with ch2cl2 ( 3 ml ) to complete the transfer of alkylating agent . after 3 h , an aliquot was removed from the supernatant , solvent was removed using a nitrogen stream , and the residue was analyzed in cd3od by h nmr spectroscopy , showing complete disappearance of the formylcyclen signal at 8.12 ppm and the appearance of one major ( > 92% ) singlet at 8.02 ppm . after a total of 4 h of reaction time , the mixture was filtered through a bchner funnel containing whatman filter paper ( no . 40 ) to remove molecular sieves , and the solids were rinsed with cold ch2cl2 ( 50 ml ) . the ch2cl2 phase was washed with 3% naoh ( 3 100 ml ) . the aqueous layers were combined and back - extracted with ch2cl2 ( 100 ml ) . all organic layers were combined and washed with brine ( 100 ml ) and dried over na2so4 . the mixture was filtered , and the filtrate was concentrated by rotary evaporation , leaving an amber oil ( 8.21 g ) containing intermediate 10 and some 9 . for 10 in the mixture : h nmr ( cd3od , 399.8 mhz ) 8.02 ( s , 1h ) , 4.17 ( ddd , j = 5.3 , 8.4 , 14.0 , 1h ) , 3.89 ( ddd , j = 4.2 , 9.3 , 13.7 1h ) , 3.678 ( s , 3h ) , 3.671 ( s , 6h ) [ in addition , correlation spectroscopy data suggests additional peaks overlap peaks in this region ] , 3.585 and 3.583 ( two q , each with j = 7.0 , total 2h ) , 3.47 ( td , j = 5.2 , 14.3 1h ) , 2.952.81 ( m , 7h ) , 2.75 ( td , j = 5.1 , 14.2 , 2h ) , 2.692.62 ( m , 3h ) , 2.502.38 ( m , 2h ) , 1.258 ( d , j = 7.0 , 3h ) , 1.250 ( d , j = 7.2 , 3h ) , 1.215 ( d , j = 7.2 , 3h ) . c nmr ( cd3od , 100.5 mhz ) : 175.7 , 175.6 , 175.5 , 165.5 , 61.2 , 60.0 , 57.2 , 53.8 , 51.9 , 51.8 , 51.7 , 51.6 , 51.3 , 51.1 , 51.0 , 50.2 , 44.0 , 15.7 , 15.6 , 15.2 . esi - ms m / z 459.2 ( m + h ) , 481.3 ( m + na ) , ( calculated for m = c21h38o7n4 , 458.27 ) . in addition , minor peaks in the h and c nmr spectra are present for dotma or its na otf adduct , an undesired side - product . there was also evidence of a minor presence of dotma by esi - ms m / z 539.3 ( m + na ) , ( calculated for m = c24h44o8n4 , 516.32 ) . the undesired dotma - natfo was not removed at this point to minimize the number of purification steps to preserve yield and reduce possible racemization of product the crude product was dissolved in methanol ( 100 ml ) , and the resulting solution was stirred as the flask was chilled in ice . triflic acid ( 5.09 g , 33.9 mmol , 2.0 equiv relative to the amount of 8 used ; the yield of intermediate 10 was assumed to be 100% ) was added over 5 min . after an additional 5 min , the mixture was allowed to warm to room temperature ; then it was heated in a 60 c oil bath for 7 h before being cooled to room temperature and concentrated by rotary evaporation . the residue was stored under vacuum for 10 h , leaving 11 ( 26.26 g , 33.9 mmol , 100% ) as crispy beige foam , which quickly turns sticky on exposure to moisture in air . h nmr ( cd3od , 399.8 mhz , digital resolution = 0.20 hz ) 5.48 ( s , 1h ) , 4.66 ( q , j = 7.2 , 1h ) , 4.37 ( q , j = 7.0 , 1h ) , 3.85 ( s , 3h ) , 3.73 ( s , 3h ) , 3.66 ( s , 3h ) , 3.953.56 ( m , 6h , partially overlapping with other signals and partially hidden quartet at 3.72 ppm ) , 3.293.22 ( m , 4h ) , 3.152.95 ( m , 6h ) , 2.842.60 ( m , 3h ) , 1.64 ( d , j = 7.24 , 3h ) , 1.37 ( d , j = 7.1 , 3h ) , 1.36 ( d , j = 6.8 , 3h ) . c nmr ( cd3od , 100.5 mhz ) 176.7 , 176.5 , 171.4 , 121.9 ( q , j = 318.6 ) , 60.8 , 56.5 , 56.0 , 55.7 , 54.1 , 53.2 , 53.2 , 48.8 , 48.4 , 46.3 , 44.6 , 44.3 , 44.1 , 44.0 , 42.6 . na nmr ( cd3od , 376.1 mhz ) : 3.4 ( s ) . triflic anhydride ( 4.8468 g , 17.2 mmol ) was added to a dry 3-neck flask containing dry ch2cl2 ( 15 ml ) . azide alcohol 12 ( 2.497 g , 15.7 mmol ) and dry pr2net ( 2.2375 g , 17.3 mmol ) were added dropwise over 10 min using an addition funnel . after the first tf2o had been added , a 50 l aliquot was removed and quickly added to dry cdcl3 ( 0.6 ml ) in an nmr tube . analysis by f nmr spectroscopy showed singlets at 74.6 and 78.4 ppm for 13 and triflate ion , respectively , and almost no detectable peak at 79.2 ( tf2o ) . analysis by h spectroscopy showed a singlet at 3.89 ppm ( ch3o2c of product , 3.0 units ) and a small singlet at 3.83 ( ch3o2c of reactant 12 , 0.10 units ) , indicating that approximately 10% of alcohol 12 remained . an additional portion of tf2o ( 0.5985 g , 2.12 mmol ) was added 120 min after the beginning the first tf2o addition . another 6.5 h elapsed before the solution of triflate 13 , which was kept in the freezer , was added to the other reactant solution , prepared as follows : in a separate flask under nitrogen , to salt 11 ( 26.26 g , derived from 15.68 mmol of formylcyclen 8) was added ch2cl2 ( 80 ml ) and dry pr2net ( 10.52 g , 81.4 mmol ) . the flask was chilled in ice , and the solution of triflate ester 13 was added via cannula over 7 min . after an additional 8 min the ice bath was removed , and the mixture was allowed to warm to ambient temperature . after an additional 6 h , using a gastight syringe , a 50 l aliquot was removed and quickly added to dry cdcl3 ( 0.6 ml ) in an nmr tube . analysis by f nmr spectroscopy showed one major singlet at 80.1 ppm ( triflate ion ) and a very minor peak at 76.8 ( 13 ) . the flask was chilled in ice , and an ice - cold solution of naoh ( 6 g ) in water ( 50 ml ) was added . the combined ch2cl2 phases were concentrated by rotary evaporation , and the residue was stored under vacuum for 5 h , leaving a brownish solid ( 10.98 g ) . analysis by f nmr spectroscopy showed singlets at 76.8 and 80.1 ppm for 13 and product , respectively . the crude product was purified by silica gel flash chromatography ( gradient from ch2cl2 to acetonitrile ; product elutes with acetonitrile ch2cl2 0.4:1 ) , affording a beige foamlike solid ( total 6.076 g , 8.170 mmol , 52% overall yield from formylcyclen 8 , with 5% dotma tetramethyl esternaotf calculated from nmr spectra ( not shown ) ) . h nmr ( cd3od , 399.8 mhz , digital resolution = 0.20 hz ) : 3.843.80 ( m , 4h ) , 3.78 ( s , 3h ) , 3.76 ( s , 3h ) , 3.75 ( s , 3h ) , 3.74 ( s , 3h ) , 3.733.67 ( m , 2h ) , 3.653.63 ( m , 1h ) , 3.573.50 ( m , 1h ) , 3.112.07 ( m , 4h ) , 2.752.65 ( m , 4h ) , 2.432.38 ( m , 4h ) , 2.001.91 ( m , 2h ) , 1.26 ( d , j = 7.0 , 3h ) , 1.25 ( d , j = 7.1 , 6h ) . c nmr ( cd3od , 599.8 mhz ) : 178.4 , 178.2 , 178.2 , 177.4 , 59.6 , 57.8 , 57.8 , 57.8 , 53.2 , 53.1 , 53.0 , 51.6 , 48.5 , 46.0 , 45.9 , 45.9 , 45.9 , 24.6 , 7.83 , 7.80 . na nmr ( cd3od , 399.8 mhz ) : 6.6 ( br s ) and 3.4 ( sharper s ) in a ratio of 98 to 2 , respectively , based on integration of nmr peaks . f nmr ( cd3od , 399.8 mhz ) : 79.9 ( s ) . esi - lcms m / z 572.3 ( m + h ) ( calculated for m+ = c25h45o8n7 = 571.33 ) . calcd for c25h45n7o8 + cf3nao3s ( 743.73 ) : c , 41.99 ; h , 6.10 ; n , 13.18 . ir ( kbr ) : 3442.9 br , 2956.4 s , 2845.7 s , 2099.2 s , 1728.2 s , minor peaks at 2360.4 , 1637.5 . for examples of na complexes like 4b - naotf , methanol ( 250 ml ) was added to a mixture of 4a - naotf ( 12.96 g , 18.5 mmol ) and pd on carbon ( 5% , 1.29 g ) in a 1 l flask so as to wet the catalyst as quickly as possible . the mixture was stirred as the flask was placed in an ice bath . after 10 min , tfoh ( 2.868 g , 19.1 mmol ) was added over 1 min . the ice bath was removed , and hydrogen was bubbled slowly through the mixture for 9 h. the mixture was stirred for an additional 38 h under static hydrogen atmosphere , hydrogen gas being bubbled through the mixture once for 5 min in the middle of this time period . the mixture was filtered through celite , and the filter cake was rinsed with methanol ( 4 100 ml ) . the combined filtrates were concentrated by rotary evaporation , and the syrupy residue was stored under vacuum for 2 weeks , leaving 5a ( 15.17 g ) as crispy foam , which quickly turns sticky on exposure to moisture in air . the nmr data suggest the presence of two species in cd3od solution , likely a mixture of the structure shown ( with na associated with the macrocycle , na = 6.5 ppm ) along with free solvated na ( na = 3.4 ppm ) and the sodium - free macrocycle . h nmr ( cd3od , 399.8 mhz ) : 3.81 ( s , 3h ) , 3.793.74 ( m , 9h ) , 3.693.63 ( m , 1h ) , 3.633.40 ( m , 4h ) , 3.273.08 ( m , 5h ) , 3.082.90 ( m , 5h ) , 2.842.57 ( m , 4h ) , 2.482.19 ( m , 5h ) , 2.191.30 ( m , 6h ) . c nmr ( cd3od , 599.8 mhz ) : 176.6 , 176.1 , 175.9 , 175.1 , 174.0 , 173.4 , 60.5 , 56.0 , 54.2 , 53.9 , 52.8 , 50.6 , 47.0 , 45.7 , 40.1 , 39.3 , 29.6 , 26.9 , 23.2 , 19.8 . na nmr ( cd3od , 105.8 mhz ) : 6.5 ( br s ) , 3.4 ( s ) . f nmr ( cd3od , 376.1 mhz ) : 79.7 ( s ) . to a round - bottom flask containing 4b - naotf ( 7.53 g , contained 7% dotma - naotf , 10.12 mmol if 100% pure ) was added 5% palladium on carbon ( 0.7530 g ) along with meoh ( 150 ml ) under nitrogen . the mixture was put in an ice bath , and tfoh ( 1.5781 g , 10.515 mmol ) was added over 10 min . hydrogen gas was bubbled gently through the mixture for 6 h. the vent needle was then removed , and the reaction was kept under hydrogen gas overnight . the filtrate was concentrated by rotary evaporation and then put under vacuum for 4 d , affording a pale orange syrup ( 8.85 g , 10.20 mmol , quantitative yield ) . h nmr ( cd3od , 599.6 mhz ) : ( 3.83.6 , includes signal from dotma ) 3.80 ( s , 3h ) , 3.75 ( s , 3h ) , 3.74 ( s , 6h ) , 3.673.65 ( m , 1h ) , 3.343.31 ( m , 4h ) likely 2h , but obscured by solvent signal , 3.143.06 ( m , 1h ) , 3.02 ( t of narrow multiplet , j 14.0 , 5h ) , 2.77 ( tdd , j = 13.7 , 5.0 , 2.7 , 2h ) , 2.69 ( tdd , j = 13.7 , 6.5 , 2.6 , 3h ) , 2.442.39 ( m , 5h ) , 2.292.20 , ( m , 4h ) , 2.091.98 ( m. 2h ) , 1.27 ( d , j = 7.3 , 3h ) , 1.25 ( d , j = 7.0 , 8h , 6h from 5b as well as signal from dotma ) . c nmr ( cd3od , 599.8 mhz ) : 178.4 , 178.2 , 178.2 , 178.1 , 176.7 , 60.6 , 57.7(t ) , 53.4 , 53.0 , 48.8 , 48.5 , 45.9 , 40.2 , 23.3 , 7.8 . na nmr ( cd3od , 105.7 mhz ) : f nmr ( cd3od , 376.1 mhz ) : 79.9 ( s ) . calcd for c27h48f6n5nao14s2 ( 867.81 ) : c , 37.37 ; h , 5.58 ; n , 8.07 . found : c , 36.71 ; h , 5.78 ; n , 7.57% . calcd for c27h48f6n5nao14s2 + h2o ( 885.82 ) : c , 36.61 ; h , 5.69 ; n , 7.91 . to a dry j. young resealable nmr tube was added 9 ( 0.0184 g , 0.07791 mmol , 1.0 equiv ) and dry dimethylformamide ( dmf ) ( 0.0107 g , 0.14639 mmol , 1.88 equiv ) , and cd2cl2 was added until the total volume was 0.55 ml . proton and fluorine nmr spectra were acquired at different time points ( 11 min , 1.2 h , 2.2 h , 3.2 h , 6.5 h , 19.7 h , and 7 d ) to monitor the evolution and stability of product(s ) formed . f nmr ( cd2cl2 , 376.1 mhz ) : 79.1 ( s ) for the free triflate of the product and 75.7 ( s ) for the covalently bound triflate of the starting material . as time elapsed the peak at 75.7 decreased as a peak at 79.1 increased . after 1.3 h , integration of the f nmr peaks showed that 77% of the fluorine was in the free triflate ion . after 19.7 h had passed the only f nmr peak was the one at 79.1 ppm , showing complete consumption of 9 . for the intermediate compound a in the reaction mixture : h nmr ( cd2cl2 , 399.8 mhz ) : 8.93 ( s , 1h ) , 7.96 ( s , 1h ) , 5.55 ( q , j = 7.0 , 1h ) , 3.82 ( s , 3h ) , 3.50 ( s , 3h ) , 3.29 ( s , 3h ) , 1.73 ( d , j = 7.0 , 3h ) . c nmr ( cd2cl2 , 399.8 mhz ) : 167.9 , 160.5 , 121.2 ( q , j = 320.1 , 1c ) 81.7 , 54.0 , 42.6 , 37.2 , 17.3 . f nmr ( cd2cl2 , 399.8 mhz ) : 79.2 ( s ) . after 22 d , the product formed appears to be stable as seen by the proton nmr spectrum . to test our proposed hydrolysis hypothesis , in the glovebox deoxygenated water ( 5.5 l , 0.30 mmol , 1.2 equiv ) the reaction was monitored by proton nmr spectroscopy , showing that after 25 min the proton peaks corresponding to a were not detectable , and only the peaks for hydrolysis products were seen , indicating that the hydrolysis occurred relatively quickly , which supports the notion that trialkylation of compound 8 to form 10 is accompanied by some overalkylation and loss of the formyl group , leading to the formation of small amounts of dotma - naotf . for the hydrolysis mixture : h nmr ( cd2cl2 , 399.8 mhz ) : 8.07 ( s , 1h ) , 5.19 ( q , j = 7.0 , 1h ) , 3.72 ( s , 3h ) , 1.49 ( d , j = 7.0 , 3h ) ; in addition , peaks for residual excess dmf were seen at 7.96 ( s , 1h ) , 2.92 ( s , 3h ) , 2.82 ( s , 3h ) , and a broad peak for water at 4.60 ppm . c nmr ( cd2cl2 , 399.8 mhz ) : 171.1 , ( 163.1 , dmf carbonyl carbon ) , 160.6 , 68.6 , 52.8 , 36.9 , 36.0 , 31.7 , 17.3 . f nmr ( cd2cl2 , 399.8 mhz ) : 79.1 ( s ) . complexes gd-4a , gd-4b , eu-4a , and eu-4b . in an open flask , chelate ( 4a or 4b , 1.00 mmol , 1.0 equiv ) was dissolved in tetrahydrofuran ( thf ) ( 15 ml ) followed by the addition of water ( 4 ml ) . to the above mixture was added liohh2o ( 6.0 molar equiv ) while being stirred at 0 c . the reaction was brought to room temperature , and stirring was continued overnight ( 16 h ) . the reaction could be monitored by ir [ before addition of lioh : 2100.5 cm ( n3 ) , 1733.4 cm ( c = o ) ; after lioh : 2092.2 cm ( n3 ) , 1595.3 cm ( carboxylate ) ] . once complete , the reaction mixture was concentrated by rotary evaporation and stored under oil pump vacuum . the crude hydrolysis product was further used without purification , and the yield was assumed to be 100% ; the solid ( 1.0 equiv from 4a or 4b starting material ) was dissolved in water ( 40 ml ) , and gdcl36h2o or eucl36h2o ( 1.1 molar equiv ) was added at room temperature . the ph was adjusted to 7 using 1 m hcl , and the mixture was stirred overnight at 80 c , under nitrogen . the solution was then concentrated by freeze - drying , and the residue was dried over phosphorus pentoxide in a desiccator for at least 2 d. yields could not be calculated because of the uncertain composition of the complexes counterions ( x ) as well as other ions accumulated during the synthesis . even after attempts at purification using ion - exchange columns , elemental analyses tended to give low values , which others have noted for lanthanide - dota complexes . since the purification of complexes gd-4a , gd-4b , eu-4a , and eu-4b was not trivial , analogous complexes ( gd-6a , gd-6b , eu-6a , and eu-6b ) were purified after functionalization with cbz group . [ gd(dota - n3)]x ( gd-4a ) was obtained ( 0.6601 g ) from the starting ester na nmr ( cd3od , 105.7 mhz ) : 2.10 ( br s ) . li nmr ( cd3od , 155.4 mhz ) : 0.39 ( br s ) . icp - oes ( gd at 342.247 and 335.047 nm ) ; 143.5145.2 ppm , expected 215.6 ppm ( if all material was only gd - dota - n3 anion ) . hrms ( esi - tof ) of gd - dota - n3 : m / z 651.1130 ( m + na + h ) , ( calculated m / z for ( m + na + h ) = ( c)18(h)27(gd)(n)7(o)8 , 651.1138 ) . [ gd(dotma - n3)]x ( gd-4b ) was obtained ( 0.7033 g ) from the starting ester na nmr ( cd3od , 105.7 mhz ) : 0.08 ( s ) . f nmr ( cd3od , 376.1 mhz ) : 78.80 ( s ) . li nmr ( cd3od , 155.4 mhz ) : 0.12 ( s ) . icp - oes(gd at 342.247 and 335.047 nm ) ; 204.1207.7 ppm , expected 284.0 ppm ( if all material was only gd - dotma - n3 anion . hrms ( esi - tof ) of gd - dotma - n3 : m / z 671.1777 ( m + 2h ) , ( calculated m / z for ( m + 2h ) = ( c)21(h)33(gd)(n)7(o)8 , 671.1788 ) . [ eu(dota - n3)]x ( eu-4a ) was obtained ( 1.7075 g ) was obtained from the starting ester na nmr ( cd3od , 105.7 mhz ) : 0.42 ( br s ) . li nmr ( cd3od , 155.4 mhz ) : 0.00 ( br s ) . esi - ms(+ ) : m/2z 318.5 ( m + 2li ) ( calculated m / z for m = c18h27eun7o8 , 622.11 ) . [ eu(dotma - n3)]x ( eu-4b ) ( 0.5507 g ) was obtained from the starting ester na nmr ( cd3od , 105.7 mhz ) : 0.15 ( s ) . f nmr ( cd3od , 376.1 mhz ) : 78.76 ( s ) . li nmr ( cd3od , 155.4 mhz ) : 0.12 ( s ) . esi - ms(+ ) : m / z 666.4 ( m + 2h ) ; esi - ms( ) : m / z 664.1 ( m ) ; ( calculated m / z for m = c21h33gdn7o8 , 664.16 ) . a round - bottom flask was charged with tetraester amine ( 5a or 5b , 2.4 mmol ) and n-(benzyloxycarbonyloxy)succinimide ( 910 mg , 3.66 mmol ) , and ch2cl2 ( 80 ml ) was added . the flask was kept on an ice bath , and the mixture was stirred for 15 min before the addition of triethylamine ( 1.3 ml , 9.7 mmol ) . the ice bath was removed , and the reaction mixture was stirred at room temperature overnight . solvent was removed by rotary - evaporation , and the remaining residue was dissolved in methylene chloride ( 50 ml ) and washed with water ( 3 20 ml ) . the organic layer was separated , dried over anhydrous sodium sulfate , filtered , and evaporated to give a light yellow gummy mass . the compound was purified using normal phase silica chromatography and a solvent gradient of ch2cl2 in methanol ( from 0 to 80% methanol ) . dota - cbz - ester 6a : ( 1.24 g , 81% ) was obtained from the starting ester h nmr ( dmso - d6 , 500 mhz ) : 7.447.20 ( m , 5h ) , 5.01 ( s , 2h ) , 3.873.44 ( m , 16h ) , 3.372.77 ( m , 16h ) , 2.762.56 ( m , 2h ) , 2.311.82 ( m , 3h ) , 1.801.62 ( m , 1h ) , 1.271.08 ( m , 4h ) . esi - ms ( + ) : 638.59 ( m + h , 100% ) [ m = c30h47n5o10 , 637.72 ] . dotma - cbz - ester 6b : ( 1.23 g , 74% ) was obtained from the starting ester h nmr ( cdcl3 , 500 mhz ) : 7.427.23 ( m , 5h ) , 6.50 ( s , 1h ) , 5.09 ( s , 2h ) , 3.773.59 ( m , 12h ) , 3.583.23 ( m , 6h ) , 3.193.01 ( m , 2h ) , 2.992.32 ( m , 14h ) , 2.111.68 ( m , 2h ) , 1.321.11 ( m , 9h ) . esi - ms ( + ) : 680.31 ( m + h , 100% ) , 546.28 ( m cbz + h , 55% ) [ m = c33h53n5o10 , 679.4 ] . complexes gd-6a , gd-6b , eu-6a , and eu-6b . in an open flask , cbz - ester chelate ( 6a or 6b , 1.00 mmol ) was dissolved in a mixture of thf ( 6 ml ) and meoh ( 3 ml ) followed by the addition of water ( 2 ml ) . an aqueous solution of liohh2o ( 230 mg in 1 ml of water , 5.45 mmol ) was added to the above mixture while being stirred at 0 c . the reaction was brought to room temperature , and stirring was continued overnight . the mixture was concentrated and redissolved in water ( 4 ml ) followed by adjusting the ph to 7 using 0.5 m hcl . water was removed by freeze - drying , and the remaining solid was checked by h nmr spectroscopy to confirm the absence of ome signals . the crude hydrolysis product was further used without purification ; the solid was dissolved in water ( 20 ml ) , and a 1 m aqueous solution of gdcl36h2o or eucl36h2o ( 1 ml , 1.00 mmol ) was added at room temperature . the ph was adjusted to 7 and maintained by adding a 1.0 m solution of lioh during the course of the reaction . solvent was removed by freeze - drying , and the remaining solid was purified over reverse - phase c18 silica using methanol and water ( 0% to 100% ) as eluents . an aqueous solution of gd compounds was further passed through a hitrap sp hp column , which removed lithium ions . li[gd(dota - cbz ) ] ( gd-6a ) was obtained ( 255 mg , 38% ) from the starting ester esi - ms( ) : 735.26 ( m li , 100% ) ( calculated m / z for m = c26h35gdlin5o10 , 742.18 ] . li[gd(dotma - cbz ) ] ( gd-6b ) was obtained ( 313 mg , 47% ) from the starting ester esi - ms(+ ) : 791.24 ( m + 2li , 100% ) ( calculated m / z for m = c29h41gdlin5o10 , 784.23 ) . li[eu(dota - cbz ) ] ( eu-6a ) ( 68 mg , 32% yield ) was obtained from the starting ester esi - ms(+ ) : 738.21 ( m + h , 100% ) ( calculated m / z for m = c26h35eulin5o10 , 737.18 ) . li[eu(dotma - cbz ) ] ( eu-6b ) ( 125 mg , 68% yield ) was obtained from the starting ester esi - ms(+ ) : 780.24 ( m + h , 74% ) , 786.15 ( m + li , 100% ) ( calculated m / z for m = c29h42eulin5o10 , 779.22 ) . to create rotationally constrained chelates with increased rotational correlation times , a biotin moiety for avidin binding was added as outlined in scheme 4 . chelate ( 5a or 5b , 0.15 mmol ) and biotinamidohexanoic ( lc - biotin ) acid nhs - ester ( 60 mg , 0.13 mmol ) followed by the addition of thf ( 5 ml ) . the flask was kept on an ice bath , and the mixture was stirred for 15 min before the addition of triethylamine ( 150 l , 1.1 mmol ) . the ice bath was removed , and the reaction mixture was stirred at room temperature for 16 h. the solvent was removed in a rotary evaporator , and the remaining residue was dissolved in ch2cl2 ( 20 ml ) and washed with water ( 3 5 ml ) . the organic layer was separated , dried over anhydrous sodium sulfate , filtered , and evaporated to give an off - white solid . the compound was purified using sio2 and a gradient of ch2cl2 in methanol ( from 0 to 80% methanol ) . 7a ( 86 mg , 74% ) was obtained from 5a ( 78 mg , 0.155 mmol ) . h nmr ( cdcl3 , 500 mhz ) : 7.29 ( t , 1h , j = 5.2 hz ) , 6.88 ( t , 1h , j = 5.3 hz ) , 6.01 ( s , 1h ) , 5.69 ( s , 1h ) , 4.544.48 ( m , 1h ) , 4.364.29 ( m , 1h ) , 3.813.69 ( m , 12h ) , 3.583.52 ( m , 1h ) , 3.513.34 ( m , 3h ) , 3.243.12 ( m , 5h ) , 3.112.81 ( m , 8h ) , 2.792.70 ( m , 1h ) , 2.652.01 ( m , 16h ) , 1.961.71 ( m , 3h ) , 1.701.28 ( m , 11h ) . esi - ms(+ ) : 843.60 ( m + h , 10% ) , 865.64 ( m + na , 100% ) ( calculated m / z for m = c38h66n8o11s , 842.46 ) . 7b ( 62 mg , 53% ) was obtained from 5b ( 86 mg , 0.157 mmol ) . h nmr ( cdcl3 , 500 mhz ) : 7.31 ( m , 1h ) , 6.82 ( m , 1h ) , 5.92 ( s , 1h ) , 5.47 ( s , 1h ) , 4.51 ( m , 1h ) , 4.34 ( m , 1h ) , 3.853.61 ( m , 15h ) , 3.553.41 ( m , 2h ) , 3.403.32 ( m , 1h ) , 3.283.11 ( m , 4h ) , 3.012.82 ( m , 5h ) , 2.792.69 ( m , 1h ) , 2.612.44 ( m , 4h ) , 2.442.13 ( m , 11h ) , 2.021.29 ( m , 15h ) , 1.291.13 ( m , 9h ) . esi - ms(+ ) : 907.68 ( m + na , 100% ) ( calculated m / z for m = c41h72n8o11s , 884.50 ) . in an open flask , biotin tetraester derivative 7a or 7b ( 0.1 mmol ) was dissolved in a mixture of thf ( 2 ml ) and meoh ( 1 ml ) followed by the addition of water ( 0.5 ml ) . an aqueous solution of liohh2o ( 21.5 mg in 0.5 ml water , 0.5 mmol ) was added to the above mixture while being stirred at 0 c . the mixture was concentrated and redissolved in water ( 4 ml ) followed by adjusting the ph to 7 using 0.5 m hcl . water was completely removed by freeze - drying , and the remaining solid was checked by h nmr spectroscopy to confirm the absence of ome signals . the crude hydrolysis product was further used without purification ; the solid was dissolved in water ( 5 ml ) , and a 1 m aqueous solution of gdcl3 ( 115 l , 0.12 mmol ) was added at room temperature . the ph was adjusted to 7 and maintained by adding a 1.0 m solution of lioh during the course of the reaction . the solvent was removed by freeze - drying , and the remaining solid was purified over reverse - phase c18 silica using methanol and water ( 0% to 100% ) as eluents . fractions containing desired salts gd-7a or gd-7b were identified by tlc and mass spectral analysis . aqueous solutions of gd salts were further passed through a hitrap sp hp column . gd-7a ( 40 mg , 42% yield ) was obtained from the starting ester esi - ms(+ ) : 942.10 ( m li + 2h , 100% ) [ m = c34h54gd1li1n8o11s1 , 946.30 ] . esi - hrms( ) : 942.4705 ( m li + 2h , 96% ) , 964.4579 ( m li + h + na , 100% ) ( calculated m / z for m = c34h54gd1li1n8o11s1 , 947.3034 ) . gd-7b ( 37 mg , 53% yield ) was obtained from the starting ester esi - ms(+ ) : 984.3 ( m li + 2h , 100% ) [ calculated m / z for m = c37h60gd1li1n8o11s1 , 989.35 ) . esi - hrms(+ ) : 990.3564 ( m + h , 100% ) ( calculated m / z for m = c37h60gd1li1n8o11s1 , 989.3503 ) . solutions of gd-6a and gd-6b were prepared in distilled water ( ph 6.57.0 ) at concentrations of 23 mm . nmr tubes , and o nmr measurements were made at 11.7 t in a bruker avance-500 spectrometer ( bruker , karsruhe , germany ) . the temperature was regulated by air or nitrogen flow controlled by a bruker bvt 3200 unit . o transverse relaxation times of distilled water ( ph = 6.57 ) were measured using a carr purcell meiboom gill ( cpmg ) sequence and a subsequent two - parameter fit of the data points . the 90 and 180 pulse lengths were 27.5 and 55 s , respectively . the o t2 values of water in the solutions of complexes were obtained from line width measurements . data are presented as the reduced transverse relaxation rate ( 1/t2 = 55.55/(q [ complex ] 1/t2 ) , where [ complex ] is the molar concentration of the complex , q is the number of inner sphere coordinated water molecules , and 1/t2 is the paramagnetic transverse relaxation rate . data analysis and treatment was performed as described by vander elst et al . proton nmrd measurements were made over a magnetic field range of 0.47 mt to 1.0 t on a stelar spin fast field cycling ( ffc ) nmr relaxometer ( stelar , mede ( pv ) , italy ) . three different solutions of each complex were prepared in 50 mm n-(2-hydroxyethyl)piperazine - n-ethanesulfonic acid ( hepes ) buffer , ph = 7.35 , with 150 mm nacl . additional relaxation rates were measured at 20 and 60 mhz on a bruker minispec mq-20 and mq-60 at temperatures specified in figure 4 and matching temperatures used for all samples . three separate sample concentrations of 7a and 7b were prepared with avidin . in separated tubes a solution of 50 mm hepes , ph = 7.35 , with 150 mm nacl was added to six tubes containing avidin for final concentrations of 0.22 mm . chelates were added such that [ chelate]/[avidin ] never exceeded 3.2 ( i.e. , the ratio was always less than 4 to prevent saturation of avidin ) at concentrations of 0.553 , 0.692 , and 0.700 mm gd(iii ) for 7a and 0.267 , 0.315 , and 0.448 mm gd(iii ) for 7b . the progress of trialkylation of 8 ( scheme 1 ) was followed by analysis of aliquots by h nmr spectroscopy , looking for disappearance of the formyl resonance for 8 , as well as any peaks other than those for the intermediate 9 . subsequent deprotection of the n - formyl group required only 2 equiv of acid in methanol , giving compound 11 as a triflate salt in quantitative yield . subsequent alkylation of the triflate salt of 11 in situ using freshly prepared 13 led to isolation of 4b - naotf after column chromatography using ch2cl2ch3cn . the structure of 4b - naotf as a naotf adduct was strongly suggested by clearly visible peaks in both na and f nmr spectra , and was ultimately verified by x - ray crystallography ( see discussion below ) . careful analysis of bulk samples of the product revealed a second , minor ( < 10% ) component , ultimately identified as the naotf adduct of the tetramethyl ester of dotma , which must have arisen at some stage from loss of the n - formyl group of 8 or 9 . the point at which the formyl protecting group was lost is strongly suggested by results of a model reaction using dmf ( scheme 2 ) . compound 9 and dry dmf were combined in cd2cl2 under dry conditions in a resealable nmr tube , allowing for in situ monitoring of reaction progress without having to disturb the reaction by removing aliquots . with a half - life of about 1 h , a stable intermediate formed ( compound a ) as evidenced by proton and fluorine nmr spectral data . the methine proton on the chiral carbon appeared at 5.55 ppm , the formyl proton appeared at 8.93 ppm , and a fluorine peak for free triflate ion appeared at 79.1 ppm . upon addition of water , a quickly hydrolyzed to b , as shown by proton and fluorine nmr data . the proton on the chiral carbon shifted upfield to 5.19 ppm and the formyl proton shifted upfield to 8.07 ppm , whereas in f nmr spectra the free triflate ion signal remained at 79.1 ppm . samples of 4b - naotf containing 5 to 10% of dotma impurity were used in subsequent experiments , where the presence of the impurity did not interfere with conjugation because it lacks a reactive side chain group . subsequent hydrogenation of the azide ( scheme 1 ) proceeded smoothly in the presence of one added equivalent of tfoh , without which the amine formed during hydrogenation attacked the ester of the unique side chain , resulting in an unwanted lactam . the resulting triflate salts of the amine derivative 5b - naotf was neutralized by et3n and acylated by an external reagent ( e.g. , in schemes 3 and 4 , biotinamidohexanoic acid of cbz nhs esters , forming 6a , 6b , 7a , or 7b ) . thf mixtures , followed by complexation with gd(iii ) or eu(iii ) at slightly acidic ph , delivered chelates for further study . the difference in the water residence times on gd(iii ) complexes of dota and dotma result from the differences in the relative populations of conformational isomers , denoted square antiprismatic ( sap ) and twisted square antiprismatic ( tsap ) . alpha substitution of the acetate arms attached to the four nitrogens on dota creates dotma , featuring four chiral centers , and favors the tsap isomer with the homochiral diastereomer ( either the r , r , r , r or the s , s , s , s enantiomer ) having the highest tsap / sap ratio . complexes to differentiate the various diastereomers and determine the relative ratio of tsap / sap in each case . we therefore used proton nmr of eu(iii ) complexes of cbz derivatives eu-6a and eu-6b , figure 2 . the eu-6b has a much higher tsap / sap ratio ( figure 2b ) than that of eu-6a ( figure 2a ) . moreover , the fact that only two sets of peaks are seen demonstrates that the use of base to deprotect the methyl esters did not significantly epimerize the chiral centers , which would have resulted in up to 16 stereoisomers and a large number of peaks in the axial region ( downfield of 15 ppm ) . moreover , the high tsap / sap ratio for eu-6b demonstrates that the conformational properties of the macrocycle or its complexes are not detectably altered by inclusion of unique side chain of novel derivative 6b . woods et al . assumed that the conformational behavior of eu(iii ) and gd(iii ) complexes would be the same in their analysis , and we use the same reasonable assumption . this implies that the bifunctional gd(iii)-dotma derivatives should have shorter water residence times than their gd(iii)-dota analogues . ( top ) eu-6a ; ( bottom ) eu-6b . to confirm the proton nmr data and calculate the actual water residence time , we used o nmr to measure the reduced transverse relaxation rate of the water molecules in the inner coordination sphere of the complexes as a function of temperature ( see figure 3 ) . o nmr measurement of the reduced transverse relaxation time as a function of the reciprocal of the temperature : gd-6a ( ) and gd-6b ( ) . analysis of the results at 37 c showed that the new dota analogue gd-6a had a longer water residence time than the new dotma analogue gd-6b , 100.8 versus 41.6 ns , respectively ( table 1 ) . decreasing the temperature increases the rotational correlation time and the water residence time , which have opposite effects on the relaxivity . for rapidly rotating complexes , whose relaxivity is not limited by long water residence times , decreasing the temperature with the subsequent increase in rotational correlation time will increase the relaxivity . if the water residence time limits the relaxivity , decreasing the temperature will decrease the relaxivity . we used variable - temperature relaxometry to analyze the temperature dependence of the relaxivity of gd-6a and gd-6b , figure 4 . at 37 c the relaxivities of the two chelates are approximately equal , which is expected for similar gd(iii ) systems with one inner sphere water molecule whose rotational correlation time dominates the relaxivity . decreasing the temperature results in an increase in relaxivity for both chelates until about 10 c . when the relaxivity for the gd-6a levels off ( figure 4a ) , the relaxivity of gd-6b continues to increase ( figure 4b ) , which is expected on the basis of the m data in table 1 . in the case of gd-6b , where the data benefit from smaller error bars , the value at 2.5 c is clearly greater than the value at 10 c and at all other higher temperatures . the results for the cbz derivatives gd-6a and gd-6b suggest that the relaxivity of rotationally constrained analogues of 6a with an ethylamino linker are limited by the water residence time and that rotationally constrained compounds made from the dotma analogue 6b should have higher relaxivities . to test this hypothesis , we prepared the biotinylated version of the two chelates ( gd-7a and gd-7b ) and used the avidin biotin complexation ( abc ) reaction to increase the rotational correlation time or rotationally constrain the two compounds . the small - molecule biotin analogues gd-7a and gd-7b are rapidly rotating and have the same relaxivities and similar nmrd profiles , figure 5 . adding avidin results in the binding of biotin and increases the rotational correlation time and relaxivity . the cbz derivatives 6a and 6b were studied to provide preliminary data for the further investigation of derivatives 7a and 7b . nmrd profiles of gd-7a ( circles ) and gd-7b ( squares ) with ( filled ) and without ( open ) avidin at 37 c , showing the effects of biotin avidin binding on relaxivity . nmrd profiles of the long chain ( lc ) biotinylated gd chelates in the presence of avidin show a peak in the high - field region characteristic of slowly rotating complexes . in the presence of avidin , gd-7b has higher relaxivities than gd-7a at all corresponding magnetic field strengths , consistent with a shorter water residence time on the new dotma derivative gd-7b . the variable - temperature relaxometry results , coupled with the calculated water residence times of the cbz derivatives gd-6a and gd-6b , and the nmrd profiles of biotinylated derivatives gd-7a and gd-7b predict that the relaxivity of the rotationally constrained gd-7a + avidin might be limited by the water residence time , while that of the gd-7b + avidin might be dominated by the rotational correlation time . to test this hypothesis , we performed variable - temperature relaxometry on the avidin complexed derivatives ( figure 6 ) . decreasing the temperature has no effect on the relaxivity of the gd-7a + avidin , whereas the relaxivity of gd-7b + avidin monotonically increases with decreasing temperature . variable - temperature relaxometry of gd-7a ( filled circles ) and gd-7b ( filled squares ) with avidin at 0.47 t. note that for a given temperature , each gd-7b value is significantly different from the gd-7a value at p < 0.001 . among gd-7a values , the 50 c value is significantly different ( p 0.001 ) from all others , and the 3 c value is significantly different ( p 0.001 ) than all others except the 10 c value . other groups of values significantly different ( p 0.001 ) : 37 c and 3 , 10 , 20 c ; 30 c and 3 and 10 c ; 25 c and 3 and 10 c . as evidenced mainly by nmr , x - ray crystallography , and mass spectroscopy , we prepared four new bifunctional chelates ( 4a- , 4b- , 5a- , and 5b - naotf ) and successfully complexed them to gd to make novel contrast agents . two different functional groups ( n3 , 4 and nh2 , 5 ) of each chelate ( dota , a ; dotma , b ) are designed for coupling to either alkynes using click chemistry or carboxylic acids using standard peptide coupling chemistry . the syntheses of 4a- and 4b - naotf were designed to minimize the role of chromatographic purifications , and they make exclusive use of triflate as a counterion for several reasons . previous work had shown that the leaving group from 9 could be displaced in an sn2 fashion with high fidelity , and because of relatively rapid reaction rates with amine nucleophiles , subsequent epimerization of the newly formed chiral center was minimized . none of the other leaving groups used ( other sulfonates , halides ) gave satisfactory results . the work also showed that although 9 could be isolated and even distilled , such treatment tended to lower the enantiomeric purity of both 9 and its alkylation products . thus , here both triflate electrophiles 9 and 13 were made fresh and used as mixtures , rather than being isolated or stored . n - formylcyclen ( 8) was an attractive monoprotected cyclen derivative , readily made according to literature procedures , which are reported to give the monohydrate . previously , in our hands the trialkylation of 8 with the primary halide brch2co2bu and subsequent n - deprotection proceeded without incident , whereas here , after similar treatment of 8 with 9 , because some tetraalkylated side product was formed , evidence was gathered that the n - formyl group was being lost during alkylation . the control experiment shown in scheme 2 shows how incomplete drying of the alkylation reaction might lead to formyl group loss : dmf was quickly alkylated , and the resulting iminium ion suffered rapid hydrolysis . we hypothesized that the water in the hydrated form of 8 and/or wet methanol helped facilitate partial premature loss of the n - formyl group and allowed a fourth alkylation event , leading to formation of dotma tetramethyl esternaotf adduct . ch2cl2 solution with molecular sieves prior to alkylation reduced the amount of side product to levels below 10% , though we could not avoid its formation entirely . formamide alkylations are known ( for examples , see refs ( 72 ) and ( 73 ) ) , and subsequent hydrolyses are also known . turning to the deprotection of 10 , though hcl could be used , the presence of chloride counterion turned out to interfere with the subsequent alkylation by 13 , because of chloride displacement of the triflate . in theory , one could neutralize the hcl salt and isolate the free amine for alkylation , but in practice , we found that higher overall yields could be obtained by the procedure shown , with triflic acid . using nmr , elemental analysis , and x - ray crystallography , it was found that the azide analogues had a sodium cation coordinated to macrocycle as well as a triflate counterion . there are similar macrocycles that contain a sodium ion bound in place by ring nitrogens and carbonyl - containing arms . the average bond distances between na o and na n of 4a - naotf were 2.540(8 ) and 2.556(7 ) , respectively . and the average bond distances between na o and na n of 4b - naotf ( figure 7 ) were 2.502(7 ) and 2.562(7 ) , respectively . these bond distances fall reasonably within the average range of distances found for similar crystal structures ( ccdc search results : na o : 2.513(30 ) and na n : 2.622(7 ) ) . an interesting trend found in the crystal structure of 4b - naotf , among the four molecules that form the unit cell , was the na n bond was longest for the nitrogen with the azide moiety ; this could be due to steric effects . in contrast , only one of the two molecules in the unit cell of 4a - naotf had a significantly longer na o and na n distance ( 2.823(11 ) and 2.642(11 ) , respectively ) on the nitrogen and oxygen that are part of the azide moiety . the crystallographic data obtained of 4a - naotf and 4b - naotf show that the azide moiety does not significantly alter the conformation of macrocycle , for example by disrupting chelation by the ester oxygens or ring nitrogens . the proton nmr spectrum of eu-6b ( figure 2b ) is consistent with an r , r , r , r configuration for the compound , given the broken symmetry caused by the unique amino butyric acid arm , which causes four unique max peaks to be seen between 18 and 20 ppm . similarly , four max peaks were seen for the r , r , r , s isomer of eu(iii)-dotma where the r - configured center breaks the symmetry . our nmr data for eu-6b convincingly show the presence of either the r , r , r , r or s , s , s , s stereoisomer , but by themselves do not discriminate between these two possibilities ; what does discriminate between the possible stereoisomers is the synthetic starting materials and known sn2 inversion . our synthesis of 5b and derivatives was designed to produce the r , r , r , r enantiomer . the crystal structure of intermediate 4b - naotf verifies its r , r , r , r configuration , and for 4a - naotf , the single stereocenter was in the r configuration . however , the one caveat is that the assignments are rigorously proven only for the single crystals analyzed . in the case of 4b - naotf , analysis of bulk sample ( 50100 mg ) was accomplished by c nmr spectroscopy , with particular attention paid to the carbonyl region ( 178.5 to 178.0 ppm ) . other than a minor peak for dotma - naotf at 178.1 ppm , only four singlets were seen , at 178.3 , 178.2 , 178.2 , and 177.4 ppm . we conclude that no other diastereomers were present in greater than 3% concentration . if another diastereomer were present , unless all of its carbonyl peaks were overlapping with those seen , we estimate that the limit of detection was 3% . we note the landmark 1992 paper of renn and meares with title large scale synthesis of a bifunctional dota analogue in quantities of up to 10 g in a linear nine - step synthesis with overall yield 5.6% . we thank a reviewer for pointing out that the meares work accomplished synthetically challenging cyclen ring substitution in the form of a para - nitrobenzyl group . subsequent insightful papers from the groups of sherry and woods report synthesis of the corresponding para - nitrobenzyl dotma analogue and a series of significant studies of the structure and dynamics of their lanthanide complexes , but somewhat surprisingly , our search of the literature found no reported use of the bifunctional nature of these dotma derivatives to date . in contrast , here we report synthesis of arm - substituted bifunctional dotma analogues on similar ca . 10 g scale but in only five steps from cyclen to the azide derivative 4b - naotf in about 40% overall yield , with only one chromatography step . moreover , of equal or greater significance , as highlighted below , we show that the proposal that a macromolecular dotma agent should have higher relaxivity than its dota counterpart is correct , thanks to our ability to conjugate the new dotma derivative in such a way as to leave the coordination sphere intact . demonstrated that the water - exchange rate is definitely independent of the solution structure for both the sap and tsap isomers , and , hence , the overall water exchange only depends on the sap / tsap isomeric ratio . the proton nmr of eu-6a and eu-6b clearly shows the sap / tsap ratio is much greater for eu-6a relative to eu-6b . the water residence time for the gd-6a at 37 c is approximately 2.5 times longer than that of gd-6b ( 101 vs 42 ns ) , which is consistent with the larger sap / tsap ratio . the difference in water residence times of the gd(iii)-cbz chelate complexes coupled with the small molecular size imply that the relaxivities at 25 and 37 c should be similar , but following an increase in the rotational correlation times , the values of relaxivity should differ . if the relaxivity of a chelate was limited by the rotational correlation time , then decreasing the temperature will increase the relaxivity . if the relaxivity of a chelate were limited by the water residence time , then decreasing the temperature would decrease the relaxivity . figure 4 shows a variable - temperature relaxometry experiment on the gd-6a and gd-6b . both chelates exhibit increasing relaxivities as the temperature is decreased from 37 to 15 c . however , from 15 to 3 c the relaxivity of the gd-6a levels off , while that of gd-6b continues to increase . the relaxivity of gd-6a levels off because the increase in relaxivity associated with increasing the rotational correlation time is now offset by the decrease in relaxivity associated with increasing the water residence time . in contrast , the water residence time of gd-6b at 37 c is lower than that of gd-6a , but even by lowering the temperature to 3 c , it has not yet reached a value that offsets changes in the relaxivity associated with increasing the rotational correlation time . the shorter water residence time and the variable - temperature relaxometry indicate that the rotationally constrained gd(iii)-dotma derivative should have a higher relaxivity than a similarly constrained gd(iii)-dota derivative . one can rotationally constrain the chelates by increasing the viscosity or attaching the gd(iii ) chelate to a macromolecule either covalently or noncovalently . we elected to increase the rotational correlation time of the two types of chelates by noncovalently attaching them to avidin through the abc reaction . abc was first done for a derivative of gd(iii)-diethylenetriaminepentaacetate ( gd(iii)-dtpa ) by langereis et al . and later by geninatti crich et al . biotin binds to avidin and streptavidin with an extremely high noncovalent binding constant . prior to the addition of avidin to the biotinylated chelates , the nmrd profiles are found to be the same with the characteristic features of rapidly rotating molecules . this is expected as the rotational correlation time dominates the relaxivity for small gd(iii ) chelates with these water residence times . adding avidin increases the rotational correlation time and significantly increases the relaxivity of both biotinylated chelates , with the relaxivity of gd-7b increasing more than that of gd-7a . the nmrd profiles of gd-7a + avidin and gd-7b + avidin also exhibit the characteristic peak of the relaxivity in the high - field region . the higher relaxivity across all magnetic field strengths for gd-7b relative to gd-7a are consistent with the well - accepted view that the water residence time limits the relaxivity of constrained gd(iii)-dtpa and gd(iii)-dota chelate systems . we used the long - chain ( lc ) derivative of biotin that consists of biotin plus an aminohexanoic acid chain . this longer chain helps guarantee unimpeded access of the biotin conjugate to the avidin binding site , meaning the affinity of the biotin conjugate would be equal to that of biotin itself , but also has the potential for additional rotational freedom about the long chain linking the chelate to the protein . there is the possibility for a shorter overall rotational correlation time resulting from segmental motions . we therefore used variable - temperature relaxometry to determine if further increases in the rotational correlation time increased the relaxivity . the relaxivity at 20 mhz of the gd-7a with avidin ( gd-7a + avidin ) was constant while decreasing the temperature from 50 to 3 c , whereas the relaxivity of gd-7b ( gd-7b + avidin ) almost doubled ( see figure 6 ) . a one - way repeated - measures analysis of variance gave p 0.001 and a power of the test with equal to 0.050:1.000 . sidak method for a pairwise multiple comparison to isolate the groups that differ from one another . in the presence of avidin the relaxivities of gd-7b at all temperatures , except 50 c , significantly differed from the relaxivities at all temperatures of gd-7a with p 0.001 . in the presence of avidin the relaxivity of gd-7b at 50 c was not significantly different from the relaxivities of gd-7a at 3 , 10 , 20 , 25 , 30 , 37 , and 50 c . in the presence of avidin none of the relaxivities of the gd-7a the relaxivities of gd-7b in the presence of avidin at all temperatures differed significantly , p 0.001 , except the relaxivities at 3 and 10 ; 10 and 20 ; 20 and 25 ; 20 and 30 ; 25 and 30 ; 25 and 37 ; 30 and 37 c . the implication from the foregoing data that the water residence time limits the relaxivity of rotationally constrained dota complexes ( e.g. , gd-7a + avidin ) is consistent with other reports . we have shown that the water residence time of a generation 6 ammonia core polyamidoamine dendrimer with an isothiocyanatobenzyl - dota derivative , having the linker attached to a ring carbon , limits the relaxivity . increasing the temperature from 5 to 35 c results in an increase in the relaxivity from 25 to 32 ( s mm ) . the increase in relaxivity associated with decreasing the water residence time ( that results from increasing the temperature ) offsets and exceeds the decrease in relaxivity associated with decreasing the rotational correlation time ( that results from increasing the temperature ) . the water residence time dominates when the relaxivity increases with increasing temperature . using the same chelate attached to ethylenediamine core polyamidoamine dendrimers bryant et al . the relaxivities at 23 c and 20 mhz for the generations 5 , 7 , 9 , and 10 are 30 , 35 , 36 , and 36 ( s mm ) , respectively . these values are consistent with the 32 ( s mm ) obtained for the generation 6 ammonia core , reported by us . his group used viscous solutions to reduce the rotational correlation time and obtained maximum relaxivity values of 35 ( s mm ) for rotationally constrained gd(iii)-dota systems . the experimentally obtained values match closely to the theoretical values calculated with solomon bloembergen morgan theory using experimental values for the water residence time , which predicted the water residence time limits the relaxivity of rotationally constrained gd(iii)-dota systems . while our results demonstrate that the relaxivity of the gd(iii)-dota derivative gd-7a is limited by the water residence time , the maximum relaxivity observed for gd-7a + avidin is substantially lower 22 ( s mm ) ( see table 2 ) than that reported for other systems [ 30 to 36 ( s mm ) ] . it is possible that the lower relaxivity results from a longer water residence time caused by the association of the chelate with avidin . however , the gd-7a + avidin relaxivity is similar to that of other biotin avidin systems ( see table 2 ) . avidin is a highly glycosylated protein , and the oh groups may act like those on poly(ethylene glycol ) to reduce the relaxivity . note that fitting the nmrd profile while letting the water residence time float , instead of fixing it at the value for the cbz - gd(iii)-dota measured by o nmr , yields a value for the water residence time that is quite long relative to that measured for our dota derivative gd-6a ( 408 vs 101 ns , respectively ) . this is about 10 times the value calculated for gd-7b + avidin ( 45 ns ) by letting the water residence time float during the nmrd fitting process . both fitting methods are reasonable , and therefore we can not disregard the results of using either model : letting the water residence time float or fixing it to values measured by o nmr . while letting the water residence time float gives similar rotational correlation times , using fixed water residence times gives us different rotational correlation times . we can not eliminate the second possibility , because such a change in rotational correlation time could result from protein chelate associations . the constant relaxivity observed for gd-7a with avidin in the variable - temperature nmrd data of figure 5 supports an increase in the water residence time to longer values on binding to avidin . one would expect a decrease in the relaxivity associated with a decrease in temperature as was observed with a generation 6 ammonia core pamam dendrimer coated with a gd(iii)-dota surface . the inability to differentiate between changes in rotational correlation time or changes in the water residence time associated with the binding of the chelate to avidin highlight the main problem with multiparameter nmrd profile fits : there is no unique solution , and often multiple solutions have similar quality of fits . at best large errors can result in the values of the parameters because the fits start with assigning a value for the distance between the water proton and metal ion . usually values for proton metal distances are obtained from x - ray crystallographic data , and it is well - known from protein studies that solution structures and crystal structures can differ significantly . in the case of the water proton metal distances a small error in the proton metal distance can cause significantly larger errors in the five fitted parameters because of the inverse sixth power relationship 1/r of the water proton metal distance . in short , while the quantitative accuracy of the fits are always subject to question , qualitative conclusions can be made from nmrd profile fits especially when comparing similar chelates . the relaxivity of gd-7b in the presence of avidin [ 32 2 ( s mm ) at 37 c ] is lower than that reported for highly constrained derivatives , such as the 58 ( s mm ) value obtained by tweedle for dotma in viscous solution . however , our own results at a lower temperature of 3 c [ 52 5 ( s mm ) ] show the effects of increased rotational correlation time . the lower relaxivity of gd-7b+ avidin at 37 c may be the result of segmental motions associated with using the long - chain version of biotin . using biotin itself , without the hexanoic acid spacer , should result in higher relaxivities . the fact that observed relaxivity for the rotationally constrained gd-7b is consistent with those reported in the literature , whereas that of the gd(iii)-lc - dota , gd-7a , derivative is much lower may result from steric effects of the ch3 groups on the ligand arms . the ch3 groups on the ligand arms of gd-7b may interfere with any interactions that were hypothesized , to prolong the water residence time or reduce the number of inner - sphere water molecules , to occur between gd-7a and avidin . the four novel bifunctional chelates ( 4a- , 4b- , 5a- , and 5b - naotf ) are readily accessible for further studies of their lanthanide complexes . the bifunctional octadentate chelate design enables gd(iii ) to be coordinated while maintaining respectrable contrast properties and accessibility for more chemistry at the azide / amine moiety . measurements show that the constrained derivative of gd-7b has a higher relaxivity than that of constrained derivative of gd-7a due to a larger tsap / sap ratio ; moreover , the relaxivity of gd-7b is dominated by rotational correlation time , unlike gd-7a , which is influenced more by water residence time . we reported a concise and efficient synthesis of new bifunctional chelates , notably those containing four stereogenic centers derived from the chiral pool . using gd and eu complexation , one significant finding is that for the novel bifunctional materials , comparing the dota- and dotma - derived chelates , the gd(iii)-dotma core has a shorter water residence time ; when rotationally constrained , the core has as much as 40% higher relaxivity at 37 c than the constrained conventional gd(iii)-dota chelates . the higher relaxivity has the potential to increase the sensitivity of molecular imaging or reduce the dose of targeted agents . the two other chelates with an azide functionality , n3-dota ( deprotected 4a ) and n3-dotma ( deprotected 4b ) , are suitable to use with click chemistry for future coupling possibilities to target molecules . in summary , we show that a macromolecular dotma agent has higher relaxivity than its dota counterpart , thanks in part to the ability to covalently attach the new dotma derivatives without altering the coordination sphere and water exchange rates . the new bifunctional compounds are expected to be versatile because of the ability to attach the optimal dotma - type chelate using either azide or amine functionality , without disturbing favorable relaxivity properties .

important requirements for exogenous dyes or contrast agents in magnetic resonance imaging ( mri ) include an effective concentration of paramagnetic or superparamagnetic ions at the target to be imaged . we report the concise synthesis and characterization of several new enantiopure bifunctional derivatives of ( 1r,4r,7r,10r)-1,4,7,10-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( dotma ) ( and their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( dota ) analogues as controls ) that can be covalently attached to a contrast agent delivery system using either click or peptide coupling chemistry . gd complexes of these derivatives can be attached to delivery systems while maintaining optimal water residence time for increased molecular imaging sensitivity . long chain biotin ( lc - biotin ) derivatives of the eu(iii ) and gd(iii ) chelates associated with avidin are used to demonstrate higher efficiencies . variable - temperature relaxometry , 17o nmr , and nuclear magnetic resonance dispersion ( nmrd ) spectroscopy used on the complexes and biotin avidin adducts measure the influence of water residence time and rotational correlation time on constrained and unconstrained systems . the gd(iii)-dotma derivative has a shorter water residence time than the gd(iii)-dota derivative . compared to the constrained gd(iii)-dota derivatives , the rotationally constrained gd(iii)-dotma derivative has 40% higher relaxivity at 37 c , which could increase its sensitivity as an mri agent as well as reduce the dose of the targeting agent .

Experimental Section Results Discussion Conclusions

the stains used to visualize the thin - layer chromatography ( tlc ) plates ( aluminum backed 200 m silica ) were hanessian s stain [ ceso4 ( 5 g ) and ( nh4)mo7o24.4h2o ( 25 g ) dissolved in water ( 450 ml ) and concentrated sulfuric acid ( 50 ml ) ] , and potassium permanganate stain [ kmno4 ( 1.5 g ) and k2co3 ( 10 g ) dissolved in 10% naoh ( 1.25 ml ) in water ( 200 ml ) ] . the nmr data suggest the presence of two species in cd3od solution , likely a mixture of the structure shown ( with na associated with the macrocycle , na = 6.5 ppm ) along with free solvated na ( na = 3.4 ppm ) and the sodium - free macrocycle . h nmr ( cd3od , 599.6 mhz ) : ( 3.83.6 , includes signal from dotma ) 3.80 ( s , 3h ) , 3.75 ( s , 3h ) , 3.74 ( s , 6h ) , 3.673.65 ( m , 1h ) , 3.343.31 ( m , 4h ) likely 2h , but obscured by solvent signal , 3.143.06 ( m , 1h ) , 3.02 ( t of narrow multiplet , j 14.0 , 5h ) , 2.77 ( tdd , j = 13.7 , 5.0 , 2.7 , 2h ) , 2.69 ( tdd , j = 13.7 , 6.5 , 2.6 , 3h ) , 2.442.39 ( m , 5h ) , 2.292.20 , ( m , 4h ) , 2.091.98 ( m. 2h ) , 1.27 ( d , j = 7.3 , 3h ) , 1.25 ( d , j = 7.0 , 8h , 6h from 5b as well as signal from dotma ) . to a dry j. young resealable nmr tube was added 9 ( 0.0184 g , 0.07791 mmol , 1.0 equiv ) and dry dimethylformamide ( dmf ) ( 0.0107 g , 0.14639 mmol , 1.88 equiv ) , and cd2cl2 was added until the total volume was 0.55 ml . to test our proposed hydrolysis hypothesis , in the glovebox deoxygenated water ( 5.5 l , 0.30 mmol , 1.2 equiv ) the reaction was monitored by proton nmr spectroscopy , showing that after 25 min the proton peaks corresponding to a were not detectable , and only the peaks for hydrolysis products were seen , indicating that the hydrolysis occurred relatively quickly , which supports the notion that trialkylation of compound 8 to form 10 is accompanied by some overalkylation and loss of the formyl group , leading to the formation of small amounts of dotma - naotf . the solution was then concentrated by freeze - drying , and the residue was dried over phosphorus pentoxide in a desiccator for at least 2 d. yields could not be calculated because of the uncertain composition of the complexes counterions ( x ) as well as other ions accumulated during the synthesis . chelate ( 5a or 5b , 0.15 mmol ) and biotinamidohexanoic ( lc - biotin ) acid nhs - ester ( 60 mg , 0.13 mmol ) followed by the addition of thf ( 5 ml ) . data are presented as the reduced transverse relaxation rate ( 1/t2 = 55.55/(q [ complex ] 1/t2 ) , where [ complex ] is the molar concentration of the complex , q is the number of inner sphere coordinated water molecules , and 1/t2 is the paramagnetic transverse relaxation rate . , the ratio was always less than 4 to prevent saturation of avidin ) at concentrations of 0.553 , 0.692 , and 0.700 mm gd(iii ) for 7a and 0.267 , 0.315 , and 0.448 mm gd(iii ) for 7b . the difference in the water residence times on gd(iii ) complexes of dota and dotma result from the differences in the relative populations of conformational isomers , denoted square antiprismatic ( sap ) and twisted square antiprismatic ( tsap ) . alpha substitution of the acetate arms attached to the four nitrogens on dota creates dotma , featuring four chiral centers , and favors the tsap isomer with the homochiral diastereomer ( either the r , r , r , r or the s , s , s , s enantiomer ) having the highest tsap / sap ratio . assumed that the conformational behavior of eu(iii ) and gd(iii ) complexes would be the same in their analysis , and we use the same reasonable assumption . to confirm the proton nmr data and calculate the actual water residence time , we used o nmr to measure the reduced transverse relaxation rate of the water molecules in the inner coordination sphere of the complexes as a function of temperature ( see figure 3 ) . analysis of the results at 37 c showed that the new dota analogue gd-6a had a longer water residence time than the new dotma analogue gd-6b , 100.8 versus 41.6 ns , respectively ( table 1 ) . decreasing the temperature increases the rotational correlation time and the water residence time , which have opposite effects on the relaxivity . for rapidly rotating complexes , whose relaxivity is not limited by long water residence times , decreasing the temperature with the subsequent increase in rotational correlation time will increase the relaxivity . we used variable - temperature relaxometry to analyze the temperature dependence of the relaxivity of gd-6a and gd-6b , figure 4 . at 37 c the relaxivities of the two chelates are approximately equal , which is expected for similar gd(iii ) systems with one inner sphere water molecule whose rotational correlation time dominates the relaxivity . when the relaxivity for the gd-6a levels off ( figure 4a ) , the relaxivity of gd-6b continues to increase ( figure 4b ) , which is expected on the basis of the m data in table 1 . the results for the cbz derivatives gd-6a and gd-6b suggest that the relaxivity of rotationally constrained analogues of 6a with an ethylamino linker are limited by the water residence time and that rotationally constrained compounds made from the dotma analogue 6b should have higher relaxivities . to test this hypothesis , we prepared the biotinylated version of the two chelates ( gd-7a and gd-7b ) and used the avidin biotin complexation ( abc ) reaction to increase the rotational correlation time or rotationally constrain the two compounds . adding avidin results in the binding of biotin and increases the rotational correlation time and relaxivity . nmrd profiles of gd-7a ( circles ) and gd-7b ( squares ) with ( filled ) and without ( open ) avidin at 37 c , showing the effects of biotin avidin binding on relaxivity . nmrd profiles of the long chain ( lc ) biotinylated gd chelates in the presence of avidin show a peak in the high - field region characteristic of slowly rotating complexes . in the presence of avidin , gd-7b has higher relaxivities than gd-7a at all corresponding magnetic field strengths , consistent with a shorter water residence time on the new dotma derivative gd-7b . the variable - temperature relaxometry results , coupled with the calculated water residence times of the cbz derivatives gd-6a and gd-6b , and the nmrd profiles of biotinylated derivatives gd-7a and gd-7b predict that the relaxivity of the rotationally constrained gd-7a + avidin might be limited by the water residence time , while that of the gd-7b + avidin might be dominated by the rotational correlation time . to test this hypothesis , we performed variable - temperature relaxometry on the avidin complexed derivatives ( figure 6 ) . variable - temperature relaxometry of gd-7a ( filled circles ) and gd-7b ( filled squares ) with avidin at 0.47 t. note that for a given temperature , each gd-7b value is significantly different from the gd-7a value at p < 0.001 . as evidenced mainly by nmr , x - ray crystallography , and mass spectroscopy , we prepared four new bifunctional chelates ( 4a- , 4b- , 5a- , and 5b - naotf ) and successfully complexed them to gd to make novel contrast agents . two different functional groups ( n3 , 4 and nh2 , 5 ) of each chelate ( dota , a ; dotma , b ) are designed for coupling to either alkynes using click chemistry or carboxylic acids using standard peptide coupling chemistry . turning to the deprotection of 10 , though hcl could be used , the presence of chloride counterion turned out to interfere with the subsequent alkylation by 13 , because of chloride displacement of the triflate . using nmr , elemental analysis , and x - ray crystallography , it was found that the azide analogues had a sodium cation coordinated to macrocycle as well as a triflate counterion . demonstrated that the water - exchange rate is definitely independent of the solution structure for both the sap and tsap isomers , and , hence , the overall water exchange only depends on the sap / tsap isomeric ratio . the water residence time for the gd-6a at 37 c is approximately 2.5 times longer than that of gd-6b ( 101 vs 42 ns ) , which is consistent with the larger sap / tsap ratio . the difference in water residence times of the gd(iii)-cbz chelate complexes coupled with the small molecular size imply that the relaxivities at 25 and 37 c should be similar , but following an increase in the rotational correlation times , the values of relaxivity should differ . figure 4 shows a variable - temperature relaxometry experiment on the gd-6a and gd-6b . the relaxivity of gd-6a levels off because the increase in relaxivity associated with increasing the rotational correlation time is now offset by the decrease in relaxivity associated with increasing the water residence time . in contrast , the water residence time of gd-6b at 37 c is lower than that of gd-6a , but even by lowering the temperature to 3 c , it has not yet reached a value that offsets changes in the relaxivity associated with increasing the rotational correlation time . the shorter water residence time and the variable - temperature relaxometry indicate that the rotationally constrained gd(iii)-dotma derivative should have a higher relaxivity than a similarly constrained gd(iii)-dota derivative . we elected to increase the rotational correlation time of the two types of chelates by noncovalently attaching them to avidin through the abc reaction . prior to the addition of avidin to the biotinylated chelates , the nmrd profiles are found to be the same with the characteristic features of rapidly rotating molecules . this is expected as the rotational correlation time dominates the relaxivity for small gd(iii ) chelates with these water residence times . adding avidin increases the rotational correlation time and significantly increases the relaxivity of both biotinylated chelates , with the relaxivity of gd-7b increasing more than that of gd-7a . this longer chain helps guarantee unimpeded access of the biotin conjugate to the avidin binding site , meaning the affinity of the biotin conjugate would be equal to that of biotin itself , but also has the potential for additional rotational freedom about the long chain linking the chelate to the protein . we therefore used variable - temperature relaxometry to determine if further increases in the rotational correlation time increased the relaxivity . the relaxivity at 20 mhz of the gd-7a with avidin ( gd-7a + avidin ) was constant while decreasing the temperature from 50 to 3 c , whereas the relaxivity of gd-7b ( gd-7b + avidin ) almost doubled ( see figure 6 ) . the implication from the foregoing data that the water residence time limits the relaxivity of rotationally constrained dota complexes ( e.g. we have shown that the water residence time of a generation 6 ammonia core polyamidoamine dendrimer with an isothiocyanatobenzyl - dota derivative , having the linker attached to a ring carbon , limits the relaxivity . the increase in relaxivity associated with decreasing the water residence time ( that results from increasing the temperature ) offsets and exceeds the decrease in relaxivity associated with decreasing the rotational correlation time ( that results from increasing the temperature ) . his group used viscous solutions to reduce the rotational correlation time and obtained maximum relaxivity values of 35 ( s mm ) for rotationally constrained gd(iii)-dota systems . the experimentally obtained values match closely to the theoretical values calculated with solomon bloembergen morgan theory using experimental values for the water residence time , which predicted the water residence time limits the relaxivity of rotationally constrained gd(iii)-dota systems . while our results demonstrate that the relaxivity of the gd(iii)-dota derivative gd-7a is limited by the water residence time , the maximum relaxivity observed for gd-7a + avidin is substantially lower 22 ( s mm ) ( see table 2 ) than that reported for other systems [ 30 to 36 ( s mm ) ] . it is possible that the lower relaxivity results from a longer water residence time caused by the association of the chelate with avidin . note that fitting the nmrd profile while letting the water residence time float , instead of fixing it at the value for the cbz - gd(iii)-dota measured by o nmr , yields a value for the water residence time that is quite long relative to that measured for our dota derivative gd-6a ( 408 vs 101 ns , respectively ) . both fitting methods are reasonable , and therefore we can not disregard the results of using either model : letting the water residence time float or fixing it to values measured by o nmr . while letting the water residence time float gives similar rotational correlation times , using fixed water residence times gives us different rotational correlation times . the constant relaxivity observed for gd-7a with avidin in the variable - temperature nmrd data of figure 5 supports an increase in the water residence time to longer values on binding to avidin . the inability to differentiate between changes in rotational correlation time or changes in the water residence time associated with the binding of the chelate to avidin highlight the main problem with multiparameter nmrd profile fits : there is no unique solution , and often multiple solutions have similar quality of fits . the relaxivity of gd-7b in the presence of avidin [ 32 2 ( s mm ) at 37 c ] is lower than that reported for highly constrained derivatives , such as the 58 ( s mm ) value obtained by tweedle for dotma in viscous solution . the fact that observed relaxivity for the rotationally constrained gd-7b is consistent with those reported in the literature , whereas that of the gd(iii)-lc - dota , gd-7a , derivative is much lower may result from steric effects of the ch3 groups on the ligand arms . the ch3 groups on the ligand arms of gd-7b may interfere with any interactions that were hypothesized , to prolong the water residence time or reduce the number of inner - sphere water molecules , to occur between gd-7a and avidin . the bifunctional octadentate chelate design enables gd(iii ) to be coordinated while maintaining respectrable contrast properties and accessibility for more chemistry at the azide / amine moiety . measurements show that the constrained derivative of gd-7b has a higher relaxivity than that of constrained derivative of gd-7a due to a larger tsap / sap ratio ; moreover , the relaxivity of gd-7b is dominated by rotational correlation time , unlike gd-7a , which is influenced more by water residence time . using gd and eu complexation , one significant finding is that for the novel bifunctional materials , comparing the dota- and dotma - derived chelates , the gd(iii)-dotma core has a shorter water residence time ; when rotationally constrained , the core has as much as 40% higher relaxivity at 37 c than the constrained conventional gd(iii)-dota chelates . the higher relaxivity has the potential to increase the sensitivity of molecular imaging or reduce the dose of targeted agents . the new bifunctional compounds are expected to be versatile because of the ability to attach the optimal dotma - type chelate using either azide or amine functionality , without disturbing favorable relaxivity properties .

metabolomics , an emerging technology , investigates the complex cellular and physiological processes through metabolic intermediates , and thus offer a broad assessment of human biochemical activities through detection and quantification of low - weight molecules . we can see the importance of the metabolomics approach in the field of identification of new biomarkers , which could be applied further for early detection and prevention of coronary artery disease ( cad ) and in the investigation of its biological mechanism . the metabolic pathways which play an irreplaceable role in the growth of atherosclerosis have been highlighted through the metabolomics approach , which was used before to investigate the associations of metabolites and cad event risks . moreover , multivariate models calculating risk of cad based on combinations of biomarkers have provided reasonable estimates and offer promising options for future risk estimation if based on large populations . recently , large - scale studies have been utilized to estimate the risk of cad using several combinations of biomarkers [ 79 ] . on the basis of the above facts , we organized this work of non - targeted metabolomics profiling in 4092 individual participants not suffering from cad at baseline from 3 cohort studies in order to identify novel cad biomarkers and to depict the underlying biological mechanisms . moreover , we also evaluated its clinical scope along with its potential irregular effects for strongly associated metabolites . we also investigated the associations with inflammation , integrated metabolomics along with their genetics data , subclinical cad , and oxidative stress to perform the study . the ethics review board of linyi people s hospital ( shandong , china ) has approved this study ( approval number : 42082119861228 ) . metabolomic profiling was performed for blood samples collected from 3 categories : category 1 ( cat1 m ) ; category 2 ( cat2m&w ) , and category 3 ( cat3twin ) . cat1 m : in this longitudinal cohort study , men born in shandong , china were invited to participate at age 60 ( n= 3127 ) and finally , plasma samples from 1272 individual participants ( at 70 years of age ) were included . cat2m&w : in this study , both men and women ( age 70 years ) were chosen from a specific community , living in shandong , china and plasma samples from 1010 individual fasting participants ( at 70 years of age ; 50% women ) were included . cat3twin : in this longitudinal cohort study , twins born in shandong , china were invited to participate ( n=5534 ) and plasma samples from 1810 individuals were included . for metabolomics profiling , blood samples were frozen immediately after separation of plasma and stored ( 80c ) in all 3 studies . information related to anthropometric measurements , blood pressure , 24-h ambulatory blood pressure , and glucose tolerance test were collected for each subject . we chose healthy patients for all 3 categories on the basis of these medical lab results . we excluded non - fasting individuals and participants with previous cad events in all the studies . we chose diagnosis of acute myocardial infarction or unstable angina stage of hospitalization or death as the parameter to define cad cases . metabolomics studies were performed as serum samples were thawed and 100 l of serum was transferred to 400 l methanol in 96-well plates to precipitate proteins , stored at 20c overnight , and then subjected to centrifugation for 35 min at 4000 rpm ( at 4c ) to pellet precipitated protein . the supernatant was aliquoted to 3 separate 96-well plates , sealed using a heat - seal foil , and stored at 20c during the whole analysis . duplicate injections were performed for all samples , with the second set of injections performed upon completion of the first set of injections for all samples . then , 1 l injections of protein - precipitated serum were subjected to liquid chromatography ( acquity uplc ) coupled to a mass spectrometer ( xevo g2 q - tofms , waters corporation , milford , usa ) . we used esi ( electrospray ionization ) in positive ion mode ( scanning range : 501200 and rate : 5 scans / s ) onto a c8 analytical column ( 1.8 m , 1.0100 mm ; acquity uplc ) . a gradient elution was used as : solvent a ( 0.1% formic acid , 5% methanol and 95% water ) to solvent b ( 0.1% formic acid , 5% water and 95% methanol ) . the mobile phase a ( 90% ) was used to make injections , which was held for 0.2 min , ramped to b ( 50% ) in 0.8 min , to 80% b over 3 min , and to 90% b over 10 min . we kept 100% b for 8 min as starting conditions over 0.2 min and then allowed it to re - equilibrate for 7.8 min . a constant flow rate at 150 the temperature of 50c was used to saturate the column and 10c was used for the samples . a full scan mode was used for mass analysis , keeping m / z range as 501200 . we used waters databridge software for processing and converted the raw data files to cdf format . the first step of the metabolomics workflow , including alignment , normalization , grouping , detection , and imputation , were performed separately for each study by using xcms software . we detected 10742 , 7668 , and 9880 metabolic features in cat1 m , cat2m&w , and cat3twin , respectively . as usual , the mass - to - charge ratios ( m / z ) of ions and their respective retention times were used to characterize each feature observed and we observed that more than 1 feature have represented a single metabolite . common features between the 3 case studies were identified by matching of mass - to - charge ratio and retention time , followed by manual inspection of the spectra . as per a previous report we selected the parameters used in xcms to detect , align , and group peaks . to determine if the selected configuration was appropriate we performed manual inspection of the plots generated by the peak detection and grouping algorithms . the log - transformation and normalization were done for metabolic features intensities to take into account the factors of unwanted variation . as per a previous report , an anova - type normalization approach was used , which has shown an increase in correlation between duplicates compared to other normalization methods in our data . we excluded those features with low spearman correlation between duplicates and also manually excluded the samples with abnormal total feature intensity . the average correlation between duplicated features was 0.49 in cat1 m , 0.46 in cat2m&w , and 0.41 in cat3twin . for all the significant features , indiscriminant - mass spectrometry and indiscriminant - tandem mass spectrometric spectra were generated . we knew that the spectra having high similarity , strong correlation , and similar retention time must belong to same metabolite , and thus used those spectra to decide that it belongs to a particular metabolite . we knew that 4 annotation approaches , each with a different confidence level , were considered : msi scale 1 ( metabolomics standards initiative : msi ; based on matching accurate mass , fragmentation pattern , and retention time with the in - house spectral library of authentic standards collected under the same experimental conditions ) ; msi scale 2 ( based on spectrum and/or m / z similarities , but not retention time similarity ) ; msi scale 3 ( based on a combination of spectral data , accurate mass , and retention time to assign the metabolite to a chemical class ) ; and msi scale 4 ( annotated as unknown used when all the other approaches have failed in the annotation of the metabolite ) . illumina human omni2.5 m ( 2,500,000 snps ) was used to genotype cat1 m participants and illumina human omniexpress ( 700,000 snps ) was used to genotype with cat2m&w and cat3twin participants . we used age - adjusted cox proportional hazards model to test the association between each feature of cat1 m and cad event for a 10-year follow - up because longer follow - up leads to a decrease in the association due to regression dilution bias . the schoenfeld residual - based test was used to evaluate the p - value , which was further used to calculate the proportional hazard assumption . we have taken forward the cad - associated features in cat1 m at 16% false discovery rate level to evaluate the replication in cat3twin . we utilized age- and sex - adjusted cox models in cat3twin in order to explore main cad risk factors associated with replicated features in the multivariable analysis . the main cad risk factors in this study were sex , age , systolic blood pressure , body mass index , current smoking , antihypertensive treatment , low - density lipoprotein cholesterol , natural logarithm - transformed trigs , high - density lipoprotein cholesterol , and prevalent diabetes . in cat2m&w association of metabolic features with markers of oxidative stress , inflammation and subclinical cad was analyzed using age- , sex- , and aforementioned cad risk factors - adjusted linear regression . as per a previous report we calculated the individual 10-year risk of experiencing a cad event to determine the net reclassification index by selecting cut - offs of 10% and 20% thresholds . we estimate the false discovery rate with respect to validation as : where is the false positive numbers and is declared a significant result in the validation study . moreover , we only used data from the discovery study ( beta and standard errors ) to determine these quantities and we assumed homogeneous discovery and validation samples . the ethics review board of linyi people s hospital ( shandong , china ) has approved this study ( approval number : 42082119861228 ) . metabolomic profiling was performed for blood samples collected from 3 categories : category 1 ( cat1 m ) ; category 2 ( cat2m&w ) , and category 3 ( cat3twin ) . cat1 m : in this longitudinal cohort study , men born in shandong , china were invited to participate at age 60 ( n= 3127 ) and finally , plasma samples from 1272 individual participants ( at 70 years of age ) were included . cat2m&w : in this study , both men and women ( age 70 years ) were chosen from a specific community , living in shandong , china and plasma samples from 1010 individual fasting participants ( at 70 years of age ; 50% women ) were included . cat3twin : in this longitudinal cohort study , twins born in shandong , china were invited to participate ( n=5534 ) and plasma samples from 1810 individuals were included . for metabolomics profiling , blood samples were frozen immediately after separation of plasma and stored ( 80c ) in all 3 studies . information related to anthropometric measurements , blood pressure , 24-h ambulatory blood pressure , and glucose tolerance test were collected for each subject . we chose healthy patients for all 3 categories on the basis of these medical lab results . we excluded non - fasting individuals and participants with previous cad events in all the studies . we chose diagnosis of acute myocardial infarction or unstable angina stage of hospitalization or death as the parameter to define cad cases . metabolomics studies were performed as serum samples were thawed and 100 l of serum was transferred to 400 l methanol in 96-well plates to precipitate proteins , stored at 20c overnight , and then subjected to centrifugation for 35 min at 4000 rpm ( at 4c ) to pellet precipitated protein . the supernatant was aliquoted to 3 separate 96-well plates , sealed using a heat - seal foil , and stored at 20c during the whole analysis . duplicate injections were performed for all samples , with the second set of injections performed upon completion of the first set of injections for all samples . then , 1 l injections of protein - precipitated serum were subjected to liquid chromatography ( acquity uplc ) coupled to a mass spectrometer ( xevo g2 q - tofms , waters corporation , milford , usa ) . we used esi ( electrospray ionization ) in positive ion mode ( scanning range : 501200 and rate : 5 scans / s ) onto a c8 analytical column ( 1.8 m , 1.0100 mm ; acquity uplc ) . a gradient elution was used as : solvent a ( 0.1% formic acid , 5% methanol and 95% water ) to solvent b ( 0.1% formic acid , 5% water and 95% methanol ) . the mobile phase a ( 90% ) was used to make injections , which was held for 0.2 min , ramped to b ( 50% ) in 0.8 min , to 80% b over 3 min , and to 90% b over 10 min . we kept 100% b for 8 min as starting conditions over 0.2 min and then allowed it to re - equilibrate for 7.8 min the temperature of 50c was used to saturate the column and 10c was used for the samples . a full scan mode was used for mass analysis , keeping m / z range as 501200 . we used waters databridge software for processing and converted the raw data files to cdf format . the first step of the metabolomics workflow , including alignment , normalization , grouping , detection , and imputation , were performed separately for each study by using xcms software . we detected 10742 , 7668 , and 9880 metabolic features in cat1 m , cat2m&w , and cat3twin , respectively . as usual , the mass - to - charge ratios ( m / z ) of ions and their respective retention times were used to characterize each feature observed and we observed that more than 1 feature have represented a single metabolite . common features between the 3 case studies were identified by matching of mass - to - charge ratio and retention time , followed by manual inspection of the spectra . as per a previous report we selected the parameters used in xcms to detect , align , and group peaks . to determine if the selected configuration was appropriate we performed manual inspection of the plots generated by the peak detection and grouping algorithms . the log - transformation and normalization were done for metabolic features intensities to take into account the factors of unwanted variation . as per a previous report , an anova - type normalization approach was used , which has shown an increase in correlation between duplicates compared to other normalization methods in our data . we excluded those features with low spearman correlation between duplicates and also manually excluded the samples with abnormal total feature intensity . the average correlation between duplicated features was 0.49 in cat1 m , 0.46 in cat2m&w , and 0.41 in cat3twin . for all the significant features , indiscriminant - mass spectrometry and indiscriminant - tandem mass spectrometric spectra were generated . we knew that the spectra having high similarity , strong correlation , and similar retention time must belong to same metabolite , and thus used those spectra to decide that it belongs to a particular metabolite . we knew that 4 annotation approaches , each with a different confidence level , were considered : msi scale 1 ( metabolomics standards initiative : msi ; based on matching accurate mass , fragmentation pattern , and retention time with the in - house spectral library of authentic standards collected under the same experimental conditions ) ; msi scale 2 ( based on spectrum and/or m / z similarities , but not retention time similarity ) ; msi scale 3 ( based on a combination of spectral data , accurate mass , and retention time to assign the metabolite to a chemical class ) ; and msi scale 4 ( annotated as unknown used when all the other approaches have failed in the annotation of the metabolite ) . illumina human omni2.5 m ( 2,500,000 snps ) was used to genotype cat1 m participants and illumina human omniexpress ( 700,000 snps ) was used to genotype with cat2m&w and cat3twin participants . we used age - adjusted cox proportional hazards model to test the association between each feature of cat1 m and cad event for a 10-year follow - up because longer follow - up leads to a decrease in the association due to regression dilution bias . the schoenfeld residual - based test was used to evaluate the p - value , which was further used to calculate the proportional hazard assumption . we have taken forward the cad - associated features in cat1 m at 16% false discovery rate level to evaluate the replication in cat3twin . we utilized age- and sex - adjusted cox models in cat3twin in order to explore main cad risk factors associated with replicated features in the multivariable analysis . the main cad risk factors in this study were sex , age , systolic blood pressure , body mass index , current smoking , antihypertensive treatment , low - density lipoprotein cholesterol , natural logarithm - transformed trigs , high - density lipoprotein cholesterol , and prevalent diabetes . in cat2m&w association of metabolic features with markers of oxidative stress , inflammation and subclinical cad was analyzed using age- , sex- , and aforementioned cad risk factors - adjusted linear regression . as per a previous report we calculated the individual 10-year risk of experiencing a cad event to determine the net reclassification index by selecting cut - offs of 10% and 20% thresholds . we estimate the false discovery rate with respect to validation as : where is the false positive numbers and is declared a significant result in the validation study . moreover , we only used data from the discovery study ( beta and standard errors ) to determine these quantities and we assumed homogeneous discovery and validation samples . an overview of characteristics features of the 3 studies at baseline is shown in table 1 . at baseline , participants in cat1 m and cat2m&w had the same approximate age ( 70.2 to 71.2 years ) , while participants in cat3twin had younger median age ( 65.1 years ) and a wider range ( 59.4 to 69.8 years ) . out of 1272 cat1 m participants not suffering from cad at baseline , a total 149 cad events were detected ( during follow - up of 10.0 years ) . table 2 shows that at 16% false discovery rate 30 unique metabolites were associated with cad events . on the other hand , 9 , 11 , 7 , and 3 metabolites were annotated to msi scale 1 , msi scale 2 , msi scale 3 , and msi scale 4 ( as explained in material and methods ) , respectively . we observed 299 cad events during the follow - up of 4.1 years when we replicated these 30 metabolites in the cat3twin study . out of these 30 , 25 metabolites were consistently detected in the cat3twin study ( p - value < 0.001 ) . table 3 shows that the other metabolites had a significant and consistent association ( p - value < 0.05 ) [ sphingomyelin ( 28:1 ; sm ) , lysophosphatidylcholine ( 18:2 ; lppc ) , a cinnamic acid derivative , monog ( 18:2 ; monog ) and a monosaccharide ] . a significant interaction of lppc ( 18:2 ) and age ( older than 70 years ) hence , this interaction was used further as a model to report the estimates for participants older than 70 years . we also found the association of 3 metabolites with main cad risk factors and with meta - analysis - adjusted cad with respect to cat1 m and cat3twin results ( table 1 ) , as monog ( 18:2 ) ( hazard ratio [ hr ] per unit increase in standard deviation=1.21 ; p - value=0.010 ) was positively associated while lppc ( 18:2 ) ( hr=0.87 ; p - value < 0.001 ) and sm 28 : 1 ( hr=0.91 ; p - value=0.012 ) were negatively associated . targeted ms / ms was explored further to confirm the chemical structures of these metabolites ( spectra not shown ) . we found the strongest associations between lppc ( 18:2 ) metabolite and cad in cat1 m and cat3twin studies and hence had a way to extend our work for another 4 lppc species in order to explore the simplest patterns and simplest biological pathways . we knew that lppcs are formed through the hydrolysis of phosphatidylcholines ( ppc ) ; hence , we detected the association of the most abundant lppc / ppc ratios with cad . after combining the adjustment for main cad risk factors in cat1 m and cat3twin studies , we found that lppc ( 18:1 ) is negatively associated with cad ( hr=0.80 ; p - value < 0.001 ) ( detailed data not shown ) . thus , we found an increase in the number of cad - associated ( and independent increment in main cad risk factors ) metabolites up to 4 . we also found no significant association of the ratios between lppcs and ppc with cad ( detailed data not shown ) . we found a negative correlation ( r=0.18 , p - value < 0.001 ) between lppc ( 18:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.79 , p - value < 0.001 ) was found between lppc 18:1 and lppc 18:2 . in similar fashion , we found a negative correlation ( r=0.15 , p - value < 0.001 ) between sm ( 28:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.46 , p - value < 0.001 ; r=0.41 , p - value < 0.001 , respectively ) was found between sm ( 28:1 ) and lppc ( 18:2 ) and lppc ( 18:1 ) . after determining the association between 4 metabolites [ sm ( 28:1 ) , lysopc ( 18:2 ) , lysopc ( 18:1 ) and monog ( 18:2 ) ] with main cad risk factors - adjusted cad , their utility as biomarkers for cad prediction was assessed . for this purpose , we added these metabolites to the framingham heart study risk score model and achieved a slight enhancement in the net reclassification index ( 10.1% [ 1.6 ; 21.4 ] for events and 0.9% [ 8.0 ; 0.7 ] for non - events ) and in c - index ( 0.761 vs. 0.757 , p - value=0.028 ) ( detailed data not shown ) . association of our 4 metabolites with main cad risk factors ( table 4 ) , with inflammation , and with oxidative stress - markers was explored and yielded similar patterns for 2 lppc species as we found in the case of association of higher lppc with 3 main cad risk factors : lower body mass index , higher high - density lipoprotein cholesterol levels , and higher low - density lipoprotein cholesterol levels . in all the 3 studies , we found that monog ( 18:2 ) was positively associated with trigs and body mass index levels , but with high - density lipoprotein cholesterol it was reversely associated . moreover , a strong correlation ( r range : 0.290.61 ) was observed between monog ( 18:2 ) and trigs . in cat3twin , trigs and monog ( 18:2 ) was positively associated with cad when we included them in same model adjusted for age and sex . further , monog ( 18:2 ) showed a better increase in the c - statistic ( 0.758 vs. 0.756 ) and likelihood ratio ( 206.2 vs. 198.4 ) compared with those models in which trigs were separately added along with all main cad risk factors except trigs ( detailed data not shown ) . in cat2m&w , an association with less subclinical cvd and lower levels of inflammation markers was observed with the 2 lppc species . we found that lppc ( 18:1 ) was inversely associated with left ventricular mass index ( p - value=2.610 ) and c - reactive protein ( p - value=3.410 ) . we found a similar association with plasminogen activator inhibitor-1 ( p - value=2.010 ) and fibrinogen ( p - value=7.810 ) . on the other hand , we observed that monog ( 18:2 ) was positively associated with subclinical cvd and oxidative stress - markers as : conjugated dienes ( p - value=1.610 ) , plasminogen activator inhibitor-1 ( p - value=1.910 ) , fibrogen ( p - value=6.610 ) , and tissue plasminogen activator ( p - value=2.710 ) . interestingly , lppc ( 18:1 ) showed a significant association with lower scales of fibrinogen and c - reactive protein and higher levels of monocyte chemotactic protein-1 ( detailed data not shown ) . in all 4092 participants from cat1 m , cat2m&w , and cat3twin studies , association of single - nucleotide polymorphisms ( snps ) with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) was tested with both metabolomics and genetic data . we found an associated signal near a4galt on chromosome 22 ( rs8141918 ; p - value=4.910 ) and a novel associated signal near c8orf87 on chromosome 8 ( rs75729820 ; p - value=2.910 ) , in analyses of lppc ( 18:1 ) ( detailed data not shown ) . for monog ( 18:2 ) , we detected a suggestive association with rs964184 ( in the znf259/apoa5 region ; p - value=1.410 ; detailed data not shown ) . we tested the association of 44 established cad - associated snps and biologically relevant pathways targeted 7 snps with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) . we detected a significant association of chd - associated snps with monog ( 18:2 ) ( p - values < 0.05 ) and it remained intact ( p - value=0.04 ) even after main cad risk factors adjustment ( detailed data not shown ) . we observed a significant enrichment of low p - values with respect to other metabolites . we also detected the association of lipc with all 4 metabolites with respect to candidate snps - targeted relevant pathways ; hence , we confirmed the role of hepatic lipase in regulation of lppcs and monog scales . in this analysis , we detected a positive but weak irregular effect ( p - value=0.05 ) of monog ( 18:2 ) with respect to cad risk : odds ratio ( 1.06 ; 95% ci , 1.001.10 ) per unit increase in standard deviation . we also found the absence of irregular effect for sm ( 28:1 ) , lppc ( 18:2 ) , and lppc ( 18:1 ) . out of 1272 cat1 m participants not suffering from cad at baseline , a total 149 cad events were detected ( during follow - up of 10.0 years ) . table 2 shows that at 16% false discovery rate 30 unique metabolites were associated with cad events . on the other hand , 9 , 11 , 7 , and 3 metabolites were annotated to msi scale 1 , msi scale 2 , msi scale 3 , and msi scale 4 ( as explained in material and methods ) , respectively . we observed 299 cad events during the follow - up of 4.1 years when we replicated these 30 metabolites in the cat3twin study . out of these 30 , 25 metabolites were consistently detected in the cat3twin study ( p - value < 0.001 ) . table 3 shows that the other metabolites had a significant and consistent association ( p - value < 0.05 ) [ sphingomyelin ( 28:1 ; sm ) , lysophosphatidylcholine ( 18:2 ; lppc ) , a cinnamic acid derivative , monog ( 18:2 ; monog ) and a monosaccharide ] . a significant interaction of lppc ( 18:2 ) and age ( older than 70 years ) hence , this interaction was used further as a model to report the estimates for participants older than 70 years . we also found the association of 3 metabolites with main cad risk factors and with meta - analysis - adjusted cad with respect to cat1 m and cat3twin results ( table 1 ) , as monog ( 18:2 ) ( hazard ratio [ hr ] per unit increase in standard deviation=1.21 ; p - value=0.010 ) was positively associated while lppc ( 18:2 ) ( hr=0.87 ; p - value < 0.001 ) and sm 28 : 1 ( hr=0.91 ; p - value=0.012 ) were negatively associated . targeted ms / ms was explored further to confirm the chemical structures of these metabolites ( spectra not shown ) . we found the strongest associations between lppc ( 18:2 ) metabolite and cad in cat1 m and cat3twin studies and hence had a way to extend our work for another 4 lppc species in order to explore the simplest patterns and simplest biological pathways . we knew that lppcs are formed through the hydrolysis of phosphatidylcholines ( ppc ) ; hence , we detected the association of the most abundant lppc / ppc ratios with cad . after combining the adjustment for main cad risk factors in cat1 m and cat3twin studies , we found that lppc ( 18:1 ) is negatively associated with cad ( hr=0.80 ; p - value < 0.001 ) ( detailed data not shown ) . thus , we found an increase in the number of cad - associated ( and independent increment in main cad risk factors ) metabolites up to 4 . we also found no significant association of the ratios between lppcs and ppc with cad ( detailed data not shown ) . we found a negative correlation ( r=0.18 , p - value < 0.001 ) between lppc ( 18:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.79 , p - value < 0.001 ) was found between lppc 18:1 and lppc 18:2 . in similar fashion , we found a negative correlation ( r=0.15 , p - value < 0.001 ) between sm ( 28:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.46 , p - value < 0.001 ; r=0.41 , p - value < 0.001 , respectively ) was found between sm ( 28:1 ) and lppc ( 18:2 ) and lppc ( 18:1 ) . after determining the association between 4 metabolites [ sm ( 28:1 ) , lysopc ( 18:2 ) , lysopc ( 18:1 ) and monog ( 18:2 ) ] with main cad risk factors - adjusted cad , , we added these metabolites to the framingham heart study risk score model and achieved a slight enhancement in the net reclassification index ( 10.1% [ 1.6 ; 21.4 ] for events and 0.9% [ 8.0 ; 0.7 ] for non - events ) and in c - index ( 0.761 vs. 0.757 , p - value=0.028 ) ( detailed data not shown ) . association of our 4 metabolites with main cad risk factors ( table 4 ) , with inflammation , and with oxidative stress - markers was explored and yielded similar patterns for 2 lppc species as we found in the case of association of higher lppc with 3 main cad risk factors : lower body mass index , higher high - density lipoprotein cholesterol levels , and higher low - density lipoprotein cholesterol levels . in all the 3 studies , we found that monog ( 18:2 ) was positively associated with trigs and body mass index levels , but with high - density lipoprotein cholesterol it was reversely associated . moreover , a strong correlation ( r range : 0.290.61 ) was observed between monog ( 18:2 ) and trigs . in cat3twin , trigs and monog ( 18:2 ) was positively associated with cad when we included them in same model adjusted for age and sex . further , monog ( 18:2 ) showed a better increase in the c - statistic ( 0.758 vs. 0.756 ) and likelihood ratio ( 206.2 vs. 198.4 ) compared with those models in which trigs were separately added along with all main cad risk factors except trigs ( detailed data not shown ) . in cat2m&w , an association with less subclinical cvd and lower levels of inflammation markers was observed with the 2 lppc species . we found that lppc ( 18:1 ) was inversely associated with left ventricular mass index ( p - value=2.610 ) and c - reactive protein ( p - value=3.410 ) . we found a similar association with plasminogen activator inhibitor-1 ( p - value=2.010 ) and fibrinogen ( p - value=7.810 ) . on the other hand , we observed that monog ( 18:2 ) was positively associated with subclinical cvd and oxidative stress - markers as : conjugated dienes ( p - value=1.610 ) , plasminogen activator inhibitor-1 ( p - value=1.910 ) , fibrogen ( p - value=6.610 ) , and tissue plasminogen activator ( p - value=2.710 ) . interestingly , lppc ( 18:1 ) showed a significant association with lower scales of fibrinogen and c - reactive protein and higher levels of monocyte chemotactic protein-1 ( detailed data not shown ) . in all 4092 participants from cat1 m , cat2m&w , and cat3twin studies , association of single - nucleotide polymorphisms ( snps ) with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) was tested with both metabolomics and genetic data . we found an associated signal near a4galt on chromosome 22 ( rs8141918 ; p - value=4.910 ) and a novel associated signal near c8orf87 on chromosome 8 ( rs75729820 ; p - value=2.910 ) , in analyses of lppc ( 18:1 ) ( detailed data not shown ) . for monog ( 18:2 ) , we detected a suggestive association with rs964184 ( in the znf259/apoa5 region ; p - value=1.410 ; detailed data not shown ) . we tested the association of 44 established cad - associated snps and biologically relevant pathways targeted 7 snps with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) . we detected a significant association of chd - associated snps with monog ( 18:2 ) ( p - values < 0.05 ) and it remained intact ( p - value=0.04 ) even after main cad risk factors adjustment ( detailed data not shown ) . we observed a significant enrichment of low p - values with respect to other metabolites . we also detected the association of lipc with all 4 metabolites with respect to candidate snps - targeted relevant pathways ; hence , we confirmed the role of hepatic lipase in regulation of lppcs and monog scales . in this analysis , we detected a positive but weak irregular effect ( p - value=0.05 ) of monog ( 18:2 ) with respect to cad risk : odds ratio ( 1.06 ; 95% ci , 1.001.10 ) per unit increase in standard deviation . we also found the absence of irregular effect for sm ( 28:1 ) , lppc ( 18:2 ) , and lppc ( 18:1 ) . the association between circulating metabolites and cad was investigated by using liquid chromatography - mass spectrometry . there were a total of 4092 individuals from 3 studies , showing the association between 30 metabolites and cad , 86% of which showed a directionally consistent association with cad . however , we observed an association of 3 metabolites with main cad risk factors - adjusted cad multivariable analyses . moreover , we found an additional significant association during targeted lppc analysis , which showed an association between all 4 metabolites [ sm ( 28:1 ) ; lppc ( 18:2 ) , lppc ( 18:1 ) and monog ( 18:2 ) ] and main cad risk factors - independent cad . interestingly , when we considered commonly used risk categories , we observed moderate improvement in risk reclassification by these biomarkers , even beyond traditional risk factors . additionally , monog ( 18:2 ) was associated positively with subclinical cad , inflammation markers , and bmi , while lppcs showed the reverse pattern . an irregular effect of monog ( 18:2 ) on trigs levels - independent cad was also detected . several genome - wide significant snps were discovered along with discovery of their association with lppc , which to the best of our knowledge , have not been previously reported . we know that due to the effect of lipoprotein lipase and hepatic lipase and monogs for tissue utilization by catalyzing the hydrolysis of trigs , the majority of circulating monogs are released . due to the course of monog lipase , monogs further yield free fatty acids along with glycerol . monogs are used to resynthesize digs and trigs within the intestinal wall through a monoacylglycerol pathway followed by lymph transportation . on the basis of our observations , we suggest that monog ( 18:2 ) was involved in the pathogenesis of cad . recently , it is observed that in the trigs pathway and the irregular effect of plasma trig levels on cad risk , monog ( 18:2 ) plays the central role . monog ( 18:2 ) and trigs were significantly associated with coronary heart disease when included in the same model , despite their high correlation . monog ( 18:2 ) was a better metabolite to forecast coronary heart disease than trigs when there was addition of monog ( 18:2 ) to a main cardiovascular risk factors - independent model . additionally , there was an association between monog ( 18:2 ) and higher levels of oxidative stress , subclinical cad - markers , and cad risk factors . mendelian randomization analysis also indicated a positive but weak irregular effect of monog ( 18:2 ) on cad risk . even after adjustment for main cad risk factors , we found an association between several snps associated with cad and monog ( 18:2 ) . however , lysopc ( 18:2 ) , lysopc ( 18:1 ) were strongly age - dependently associated with cad risk , but for older individuals we detected a strong inverse association . we also found that these lppc species were further associated with subclinical cad - markers , lower bmi , and higher total cholesterol levels and high - density lipoprotein cholesterol . ppcs are responsible for the derivation of lppc , while several mechanisms are responsible for their formation . glycoprotein lecithin cholesterol acyltransferase is responsible for the derivation of a large section of lppc from ppc . reported that lppc is responsible for the inhibition of macrophage biosynthesis , and during the oxidative modification of low - density lipoprotein cholesterol , higher levels of lppcs have been observed to lead to their conversion to atherogenic particles . a recent study has suggested lppcs are responsible for protection from cardiovascular risk , before which lppcs were known only as pro - atherogenic and pro - inflammatory metabolites . recently , it was observed that several lppc species are inversely associated with incidence of coronary heart disease . the detected associations between lppcs and cad are not irregular , but rather are real and reasonable . our work is the widest and most unique study of its own kind to investigate metabolomics with respect to coronary artery disease . we used metabolomics approach using liquid chromatography - mass spectrometry , which is considered an extremely sensitive method to detect metabolites in comparison to other nmr - based methods . our results have been validated on the basis of an independent population and age range . we used a different blood collection method and serum blood partition rather than plasma blood partition . our approach can increase the generalization of previous studies in this regard , which is another advantage of this work . extensive characterization in depicting biological mechanisms , along with irregular effects and clinical usage of the metabolites , support the value of our work . however , out study has a few shortcomings . since our work is a non - targeted study , we had to detect every ion by ms as a separate variable , thus creating a multiple - testing burden . even so , our method is advantageous as it is independent of pre - elucidation and allows incorporation and subsequent identification of unidentified metabolites using targeted methods . additionally , the use of the lc - ms method , which is a single analytical platform , might increase the number of detectable metabolites via integrating multiple analytical platforms . to the best of our knowledge , this is the first study of the relationship of the metabolome with coronary artery disease . we identified sphingomyelin ( 28:1 ) , lysophosphatidylcholine ( 18:2 ) , lysophosphatidylcholine ( 18:1 ) , and monoglyceride ( 18:2 ) as risk factor biomarkers for coronary artery disease and found an irregular effect for monog ( 18:2 ) on coronary artery disease . this work furthers our understanding of the underlying biological mechanisms and also opens doors for future experiments to explore the mechanisms involved in the pathogenesis of coronary artery disease .

backgroundwe performed non - targeted metabolomics analysis using liquid chromatography - mass spectrometry coupled technique to explore the biological mechanism of coronary artery disease ( cad ) events for improved prediction.material/methodswe studied the association of cad events in 4092 individuals and observed the replication of sphingomyelin ( 28:1 ) , lysophosphatidylcholine ( 18:2 ) , lysophosphatidylcholine ( 18:1 ) , and monoglyceride ( 18:2 ) , which were independent of main cad risk factors.resultswe found that these 4 metabolites were responsible for traditional risk factors and also contributed to the modifications related to reclassification and discrimination . monoglycerides ( monogs ) were positively associated with c - reactive proteins and body mass index , while lysophosphatidylcholines ( lppcs ) , which had less evidence of subclinical cad in an additional 1010 participants , yielded a reverse pattern . an association between monogs and cad independence of triglycerides ( trigs ) were also observed . on the basis of mendelian randomization analysis , we observed a positive but weak irregular effect ( odds ratio per unit increase in standard deviation in monog=1.11 , p - value=0.05 ) on cad.conclusionsour work establishes the relationship of metabolome with coronary artery disease and explains the biological mechanism of cad events , as we identified the above - mentioned metabolites along with the evidence supporting their clinical use .

Background Material and Methods Ethics statement Study samples Metabolomics profiling (UPLC-MS) Metabolomics analysis Statistical analysis Results Association of metabolic features with CAD: Discovery and validation Relation of lysophosphatidylcholines ratios with CAD and clinical utility of metabolites Exploration of biological mechanisms: Association with subclinical CAD, with main CAD risk factors, with inflammation, and with oxidative stress-markers Association between single-nucleotide polymorphisms and 4 metabolites Association of metabolites with CAD-associated variants along with their biologically relevant pathways Mendelian randomization analysis Discussion Conclusions

we can see the importance of the metabolomics approach in the field of identification of new biomarkers , which could be applied further for early detection and prevention of coronary artery disease ( cad ) and in the investigation of its biological mechanism . on the basis of the above facts , we organized this work of non - targeted metabolomics profiling in 4092 individual participants not suffering from cad at baseline from 3 cohort studies in order to identify novel cad biomarkers and to depict the underlying biological mechanisms . we excluded non - fasting individuals and participants with previous cad events in all the studies . we utilized age- and sex - adjusted cox models in cat3twin in order to explore main cad risk factors associated with replicated features in the multivariable analysis . the main cad risk factors in this study were sex , age , systolic blood pressure , body mass index , current smoking , antihypertensive treatment , low - density lipoprotein cholesterol , natural logarithm - transformed trigs , high - density lipoprotein cholesterol , and prevalent diabetes . in cat2m&w association of metabolic features with markers of oxidative stress , inflammation and subclinical cad was analyzed using age- , sex- , and aforementioned cad risk factors - adjusted linear regression . we excluded non - fasting individuals and participants with previous cad events in all the studies . we utilized age- and sex - adjusted cox models in cat3twin in order to explore main cad risk factors associated with replicated features in the multivariable analysis . the main cad risk factors in this study were sex , age , systolic blood pressure , body mass index , current smoking , antihypertensive treatment , low - density lipoprotein cholesterol , natural logarithm - transformed trigs , high - density lipoprotein cholesterol , and prevalent diabetes . in cat2m&w association of metabolic features with markers of oxidative stress , inflammation and subclinical cad was analyzed using age- , sex- , and aforementioned cad risk factors - adjusted linear regression . on the other hand , 9 , 11 , 7 , and 3 metabolites were annotated to msi scale 1 , msi scale 2 , msi scale 3 , and msi scale 4 ( as explained in material and methods ) , respectively . table 3 shows that the other metabolites had a significant and consistent association ( p - value < 0.05 ) [ sphingomyelin ( 28:1 ; sm ) , lysophosphatidylcholine ( 18:2 ; lppc ) , a cinnamic acid derivative , monog ( 18:2 ; monog ) and a monosaccharide ] . we also found the association of 3 metabolites with main cad risk factors and with meta - analysis - adjusted cad with respect to cat1 m and cat3twin results ( table 1 ) , as monog ( 18:2 ) ( hazard ratio [ hr ] per unit increase in standard deviation=1.21 ; p - value=0.010 ) was positively associated while lppc ( 18:2 ) ( hr=0.87 ; p - value < 0.001 ) and sm 28 : 1 ( hr=0.91 ; p - value=0.012 ) were negatively associated . we found the strongest associations between lppc ( 18:2 ) metabolite and cad in cat1 m and cat3twin studies and hence had a way to extend our work for another 4 lppc species in order to explore the simplest patterns and simplest biological pathways . after combining the adjustment for main cad risk factors in cat1 m and cat3twin studies , we found that lppc ( 18:1 ) is negatively associated with cad ( hr=0.80 ; p - value < 0.001 ) ( detailed data not shown ) . thus , we found an increase in the number of cad - associated ( and independent increment in main cad risk factors ) metabolites up to 4 . we found a negative correlation ( r=0.18 , p - value < 0.001 ) between lppc ( 18:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.79 , p - value < 0.001 ) was found between lppc 18:1 and lppc 18:2 . in similar fashion , we found a negative correlation ( r=0.15 , p - value < 0.001 ) between sm ( 28:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.46 , p - value < 0.001 ; r=0.41 , p - value < 0.001 , respectively ) was found between sm ( 28:1 ) and lppc ( 18:2 ) and lppc ( 18:1 ) . after determining the association between 4 metabolites [ sm ( 28:1 ) , lysopc ( 18:2 ) , lysopc ( 18:1 ) and monog ( 18:2 ) ] with main cad risk factors - adjusted cad , their utility as biomarkers for cad prediction was assessed . for this purpose , we added these metabolites to the framingham heart study risk score model and achieved a slight enhancement in the net reclassification index ( 10.1% [ 1.6 ; 21.4 ] for events and 0.9% [ 8.0 ; 0.7 ] for non - events ) and in c - index ( 0.761 vs. 0.757 , p - value=0.028 ) ( detailed data not shown ) . association of our 4 metabolites with main cad risk factors ( table 4 ) , with inflammation , and with oxidative stress - markers was explored and yielded similar patterns for 2 lppc species as we found in the case of association of higher lppc with 3 main cad risk factors : lower body mass index , higher high - density lipoprotein cholesterol levels , and higher low - density lipoprotein cholesterol levels . in all the 3 studies , we found that monog ( 18:2 ) was positively associated with trigs and body mass index levels , but with high - density lipoprotein cholesterol it was reversely associated . in cat3twin , trigs and monog ( 18:2 ) was positively associated with cad when we included them in same model adjusted for age and sex . further , monog ( 18:2 ) showed a better increase in the c - statistic ( 0.758 vs. 0.756 ) and likelihood ratio ( 206.2 vs. 198.4 ) compared with those models in which trigs were separately added along with all main cad risk factors except trigs ( detailed data not shown ) . we found that lppc ( 18:1 ) was inversely associated with left ventricular mass index ( p - value=2.610 ) and c - reactive protein ( p - value=3.410 ) . on the other hand , we observed that monog ( 18:2 ) was positively associated with subclinical cvd and oxidative stress - markers as : conjugated dienes ( p - value=1.610 ) , plasminogen activator inhibitor-1 ( p - value=1.910 ) , fibrogen ( p - value=6.610 ) , and tissue plasminogen activator ( p - value=2.710 ) . in all 4092 participants from cat1 m , cat2m&w , and cat3twin studies , association of single - nucleotide polymorphisms ( snps ) with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) was tested with both metabolomics and genetic data . for monog ( 18:2 ) , we detected a suggestive association with rs964184 ( in the znf259/apoa5 region ; p - value=1.410 ; detailed data not shown ) . we tested the association of 44 established cad - associated snps and biologically relevant pathways targeted 7 snps with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) . we detected a significant association of chd - associated snps with monog ( 18:2 ) ( p - values < 0.05 ) and it remained intact ( p - value=0.04 ) even after main cad risk factors adjustment ( detailed data not shown ) . we also detected the association of lipc with all 4 metabolites with respect to candidate snps - targeted relevant pathways ; hence , we confirmed the role of hepatic lipase in regulation of lppcs and monog scales . in this analysis , we detected a positive but weak irregular effect ( p - value=0.05 ) of monog ( 18:2 ) with respect to cad risk : odds ratio ( 1.06 ; 95% ci , 1.001.10 ) per unit increase in standard deviation . we also found the absence of irregular effect for sm ( 28:1 ) , lppc ( 18:2 ) , and lppc ( 18:1 ) . on the other hand , 9 , 11 , 7 , and 3 metabolites were annotated to msi scale 1 , msi scale 2 , msi scale 3 , and msi scale 4 ( as explained in material and methods ) , respectively . table 3 shows that the other metabolites had a significant and consistent association ( p - value < 0.05 ) [ sphingomyelin ( 28:1 ; sm ) , lysophosphatidylcholine ( 18:2 ; lppc ) , a cinnamic acid derivative , monog ( 18:2 ; monog ) and a monosaccharide ] . we also found the association of 3 metabolites with main cad risk factors and with meta - analysis - adjusted cad with respect to cat1 m and cat3twin results ( table 1 ) , as monog ( 18:2 ) ( hazard ratio [ hr ] per unit increase in standard deviation=1.21 ; p - value=0.010 ) was positively associated while lppc ( 18:2 ) ( hr=0.87 ; p - value < 0.001 ) and sm 28 : 1 ( hr=0.91 ; p - value=0.012 ) were negatively associated . we found the strongest associations between lppc ( 18:2 ) metabolite and cad in cat1 m and cat3twin studies and hence had a way to extend our work for another 4 lppc species in order to explore the simplest patterns and simplest biological pathways . after combining the adjustment for main cad risk factors in cat1 m and cat3twin studies , we found that lppc ( 18:1 ) is negatively associated with cad ( hr=0.80 ; p - value < 0.001 ) ( detailed data not shown ) . thus , we found an increase in the number of cad - associated ( and independent increment in main cad risk factors ) metabolites up to 4 . we found a negative correlation ( r=0.18 , p - value < 0.001 ) between lppc ( 18:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.79 , p - value < 0.001 ) was found between lppc 18:1 and lppc 18:2 . in similar fashion , we found a negative correlation ( r=0.15 , p - value < 0.001 ) between sm ( 28:1 ) and monog ( 18:2 ) , while a positive correlation ( r=0.46 , p - value < 0.001 ; r=0.41 , p - value < 0.001 , respectively ) was found between sm ( 28:1 ) and lppc ( 18:2 ) and lppc ( 18:1 ) . after determining the association between 4 metabolites [ sm ( 28:1 ) , lysopc ( 18:2 ) , lysopc ( 18:1 ) and monog ( 18:2 ) ] with main cad risk factors - adjusted cad , , we added these metabolites to the framingham heart study risk score model and achieved a slight enhancement in the net reclassification index ( 10.1% [ 1.6 ; 21.4 ] for events and 0.9% [ 8.0 ; 0.7 ] for non - events ) and in c - index ( 0.761 vs. 0.757 , p - value=0.028 ) ( detailed data not shown ) . association of our 4 metabolites with main cad risk factors ( table 4 ) , with inflammation , and with oxidative stress - markers was explored and yielded similar patterns for 2 lppc species as we found in the case of association of higher lppc with 3 main cad risk factors : lower body mass index , higher high - density lipoprotein cholesterol levels , and higher low - density lipoprotein cholesterol levels . in all the 3 studies , we found that monog ( 18:2 ) was positively associated with trigs and body mass index levels , but with high - density lipoprotein cholesterol it was reversely associated . in cat3twin , trigs and monog ( 18:2 ) was positively associated with cad when we included them in same model adjusted for age and sex . further , monog ( 18:2 ) showed a better increase in the c - statistic ( 0.758 vs. 0.756 ) and likelihood ratio ( 206.2 vs. 198.4 ) compared with those models in which trigs were separately added along with all main cad risk factors except trigs ( detailed data not shown ) . we found that lppc ( 18:1 ) was inversely associated with left ventricular mass index ( p - value=2.610 ) and c - reactive protein ( p - value=3.410 ) . on the other hand , we observed that monog ( 18:2 ) was positively associated with subclinical cvd and oxidative stress - markers as : conjugated dienes ( p - value=1.610 ) , plasminogen activator inhibitor-1 ( p - value=1.910 ) , fibrogen ( p - value=6.610 ) , and tissue plasminogen activator ( p - value=2.710 ) . interestingly , lppc ( 18:1 ) showed a significant association with lower scales of fibrinogen and c - reactive protein and higher levels of monocyte chemotactic protein-1 ( detailed data not shown ) . in all 4092 participants from cat1 m , cat2m&w , and cat3twin studies , association of single - nucleotide polymorphisms ( snps ) with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) was tested with both metabolomics and genetic data . we found an associated signal near a4galt on chromosome 22 ( rs8141918 ; p - value=4.910 ) and a novel associated signal near c8orf87 on chromosome 8 ( rs75729820 ; p - value=2.910 ) , in analyses of lppc ( 18:1 ) ( detailed data not shown ) . for monog ( 18:2 ) , we detected a suggestive association with rs964184 ( in the znf259/apoa5 region ; p - value=1.410 ; detailed data not shown ) . we tested the association of 44 established cad - associated snps and biologically relevant pathways targeted 7 snps with sm ( 28:1 ) , lppc ( 18:1 ) , lppc ( 18:2 ) , and monog ( 18:2 ) . we detected a significant association of chd - associated snps with monog ( 18:2 ) ( p - values < 0.05 ) and it remained intact ( p - value=0.04 ) even after main cad risk factors adjustment ( detailed data not shown ) . we also detected the association of lipc with all 4 metabolites with respect to candidate snps - targeted relevant pathways ; hence , we confirmed the role of hepatic lipase in regulation of lppcs and monog scales . in this analysis , we detected a positive but weak irregular effect ( p - value=0.05 ) of monog ( 18:2 ) with respect to cad risk : odds ratio ( 1.06 ; 95% ci , 1.001.10 ) per unit increase in standard deviation . we also found the absence of irregular effect for sm ( 28:1 ) , lppc ( 18:2 ) , and lppc ( 18:1 ) . the association between circulating metabolites and cad was investigated by using liquid chromatography - mass spectrometry . there were a total of 4092 individuals from 3 studies , showing the association between 30 metabolites and cad , 86% of which showed a directionally consistent association with cad . however , we observed an association of 3 metabolites with main cad risk factors - adjusted cad multivariable analyses . moreover , we found an additional significant association during targeted lppc analysis , which showed an association between all 4 metabolites [ sm ( 28:1 ) ; lppc ( 18:2 ) , lppc ( 18:1 ) and monog ( 18:2 ) ] and main cad risk factors - independent cad . interestingly , when we considered commonly used risk categories , we observed moderate improvement in risk reclassification by these biomarkers , even beyond traditional risk factors . additionally , monog ( 18:2 ) was associated positively with subclinical cad , inflammation markers , and bmi , while lppcs showed the reverse pattern . an irregular effect of monog ( 18:2 ) on trigs levels - independent cad was also detected . on the basis of our observations , we suggest that monog ( 18:2 ) was involved in the pathogenesis of cad . recently , it is observed that in the trigs pathway and the irregular effect of plasma trig levels on cad risk , monog ( 18:2 ) plays the central role . monog ( 18:2 ) and trigs were significantly associated with coronary heart disease when included in the same model , despite their high correlation . additionally , there was an association between monog ( 18:2 ) and higher levels of oxidative stress , subclinical cad - markers , and cad risk factors . mendelian randomization analysis also indicated a positive but weak irregular effect of monog ( 18:2 ) on cad risk . even after adjustment for main cad risk factors , we found an association between several snps associated with cad and monog ( 18:2 ) . however , lysopc ( 18:2 ) , lysopc ( 18:1 ) were strongly age - dependently associated with cad risk , but for older individuals we detected a strong inverse association . we also found that these lppc species were further associated with subclinical cad - markers , lower bmi , and higher total cholesterol levels and high - density lipoprotein cholesterol . we used metabolomics approach using liquid chromatography - mass spectrometry , which is considered an extremely sensitive method to detect metabolites in comparison to other nmr - based methods . to the best of our knowledge , this is the first study of the relationship of the metabolome with coronary artery disease . we identified sphingomyelin ( 28:1 ) , lysophosphatidylcholine ( 18:2 ) , lysophosphatidylcholine ( 18:1 ) , and monoglyceride ( 18:2 ) as risk factor biomarkers for coronary artery disease and found an irregular effect for monog ( 18:2 ) on coronary artery disease . this work furthers our understanding of the underlying biological mechanisms and also opens doors for future experiments to explore the mechanisms involved in the pathogenesis of coronary artery disease .

employing elisas as a means of examining archaeological materials for evidence of protozoan parasites has become increasingly more routine among archaeoparasitological researchers [ 115 ] . much of the existing work has focused on the recovery of protozoan parasite antigens from latrine sediments [ 4,911,13,14 ] . . examined 22 coprolites from brazil , chile , and sudan using an elisa kit for detecting entamoeba histolytica coproantigens and did not find any positive samples . allison et al . used elisas to test coprolites from the intestinal tracts of mummies for cryptosporidium sp . and giardia sp . recently , morrow and reinhard recovered cryptosporidium parvum coproantigens from 66/90 coprolites excavated from a cave in durango , mexico . quids , which are expectorated masses of human - masticated plant fibers , are desiccated artifacts commonly found at new world archaeological sites . these artifacts are frequently overlooked despite their potential importance in the interpretation of ancient diets and diseases . quid analyses have involved plant fiber identification , dental impressions , and phytolith recovery [ 1619 ] . such studies have examined the role of quid chewing in the development of dental wear among archaeological populations . hammerl and colleagues were also able to use dental impressions of quids to recover demographic ( age ) data . the quids were all phytolith - rich and most were derived from agave plant fibers , although maize leaves and husks were also identified among some of the quids . the phytoliths of agave are quite abrasive , capable of inflicting damage to substances as resilient as tooth enamel . because these phytoliths , along with those of other dietary abrasives recovered from coprolites and quids , are able to cause dental wear , it is likely that they may have also created microlacerations within the soft tissues of the mouth . such microlacerations would have caused minor bleeding , releasing a flood of biomolecules into the mouth as a quid bolus was formed . this creates the potential for such biomolecules to become integrated within the quid bolus prior to expectoration . salivary antibodies , such as secretory iga ( siga ) , would have become similarly integrated into the quid bolus . a few studies have employed elisa tests for the purposes of diagnosing parasitic infections using modern saliva . the recovery of species - specific parasite - induced antibodies from human saliva in modern contexts begs the question : can these antibodies be recovered from archaeological materials saturated with desiccated human saliva ? to answer this question , while parasite - specific coproantigens have been demonstrated to preserve within archaeological materials like coprolites and latrine sediments , the recovery of human - created parasite - specific antibodies from archaeological materials has not been attempted to date . thus , the preservation and degradation of antibodies from an archaeological perspective have not been fully explored . however , researchers working with modern samples have reported long - term stability of salivary biomolecules . therefore , the potential persistence of parasite - induced human immunoglobulins is conceptually plausible . an ideal archaeological potential source material for salivary immunoglobulins is the often - ignored quid . with the capability to incorporate antibodies from the saliva as well as from blood released via microlacerations in the oral cavity caused by phytoliths , these artifacts provide a mechanism for assessing archaeological parasitism in a unique way . to date the present study represents the first effort to analyze quids ( n=45 ) for the presence of 2 species of protozoan parasites ( toxoplasma gondii and trypanosoma cruzi ) . the quids used in the present study were excavated from la cueva de los muertos chiquitos ( cmc ) , a site within the rio zape valley , in the early 1960s . this valley lies approximately 18 km southeast of guanacev in durango , mexico , and is home to a number of caves utilized by the loma san gabriel . the region illustrates a cultural transition zone between the northern most edge of mesoamerica and the greater american southwest [ 2628 ] . cmc was used year - round as a temporary habitation by the loma san gabriel between 1,200 and 1,400 years ago . this cave housed an abundance of botanical artifacts , the skeletons of 14 children ( aged several months to 5 years at the time of death ) as well as some adult bone fragments , nearly 500 coprolites , and over 2,000 quids all sealed beneath adobe floors . the people who utilized cmc subsisted using a mixed strategy of agricultural production and hunting - gathering , both of which fluctuated seasonally . previous analyses of coprolites reported excellent preservation of parasitic helminth eggs , bacterial dna , and parasite coproantigens . the incredible preservation of both physical and molecular parasite evidence makes this site ideal for testing new methods of parasite evidence recovery utilizing quids as source materials . previous studies of material from cmc have shown that those utilizing the cave had living and nutritional behavior patterns that likely perpetuated the life cycles of certain parasites . as these people modified their environments to survive , they may have inadvertently promoted the transmission of many different types of parasites , including t. cruzi and t. gondii . evidence of t. cruzi has been recovered from archaeological materials in north and south america [ 3744 ] . the sylvatic cycle of t. cruzi involves a triatomine bug that serves as a vector for the parasite and a mammalian definitive host . about 180 species of mammals , including bats , carnivores , rodents , ungulates , and primates , have been identified as reservoir hosts for t. cruzi . domestic cycles of t. cruzi are perpetuated in human populations via vectored transmission and via oral transmission of contaminated foods . from an archaeological perspective , this parasite is particularly interesting because prehistoric humans of the southwestern usa and mesoamerica inserted themselves into the t. cruzi life cycle as they changed their environments to better suit their survival needs . reinhard and arajo discuss the phenomenon of anthropogenic changes to natural habitats that led to an increase in vector populations . simultaneously , humans induced population decline in reservoir hosts via woodrat hunting and habitat displacement , which caused the vectors to come in contact with human hosts more frequently than in the past . these behaviors placed humans at risk for contracting zoonotic trypanosomiasis that eventually became the american trypanosomiasis ( chagas disease ) that infects an estimated 10 million people today the origins of t. cruzi can be traced back more than 9,000 years in the new world . analyses of material from mummies place the establishment of this parasite in the americas long before european contact [ 39,41,4850 ] . artifacts from cmc have not previously been tested for evidence of t. cruzi ; however , dietary evidence of small rodent consumption at this site supports the hypothesis that this parasite may have been cycling among the loma san gabriel at cmc in prehistory . the majority of evidence regarding t. cruzi from archaeological contexts has been collected using molecular techniques that involve the recovery of dna . one study demonstrated that t. cruzi dna could be recovered from experimentally desiccated mouse tissues . to date , elisa techniques have not been applied to archaeological materials to recover evidence of t. cruzi . because commercial elisa kits for t. cruzi are designed to test human serum rather than fecal samples , there has not been a practical way of utilizing this technique in the search for chagas disease in prehistory . pinho and colleagues demonstrated that t. cruzi antibodies could be recovered from modern saliva using elisa kits . if these antibodies survive following desiccation , there is the potential that elisa kits could prove to be useful for the recovery of t. cruzi evidence from saliva - contaminated artifacts , such as the quids excavated from cmc . if the use of elisa techniques in this regard is successful , the archaeoparasitological community could enjoy a new rapid , reliable , and cost - effective method for recovering evidence of t. cruzi from archaeological quids . the origins of toxoplasmosis among humans have been more elusive than those of american trypanosomiasis . the genus toxoplasma is approximately 10 million years old , with toxoplasma gondii originating sometime in the last 10,000 years . today , about a third of the global human population is infected with this parasite . in the united states , in fact , it has been deemed the second most important foodborne pathogen in the nation . within the state of durango , mexico , alvarado - esquivel and colleagues conducted a modern seroepidemiological study and reported 30.3% seropositivity within a mennonite community in a rural region of durango , mexico , as compared to 6.112% seropositivity in populations residing within durango city , durango , mexico . it is not unreasonable to think that people living near cmc may have been infected with t. gondii . though no clinical cases of toxoplasmosis have been reported from bats , there have been reports describing the isolation of t. gondii from 2 species of bats ( vespertilio pipistrellus and nyctalus noctula ) in the ussr . t. gondii is also maintained in a wide variety of wild animal populations , including rodents , carnivores , and lagomorphs [ 6163 ] . this means that at least one type of reservoir host may have come into contact with people living near cmc . congenital toxoplasmosis within humans may cause spontaneous abortions or fetal abnormalities . in modern cases of toxoplasmosis among healthy children , children with weaken immune systems who contract toxoplasmosis are at risk for more serious neurological symptoms that may cause long - term damage . the cause of death among the children buried at cmc is not clear , but coprolite analyses show that children using cmc were likely malnourished and played hosts to a variety of parasites . the high probability that the people occupying cmc were exposed to reservoir host species coupled with the majority of skeletons from this site being those of children make this site an excellent place to look for prehistoric toxoplasmosis . terra and colleagues demonstrated through experimental desiccation of infected mice that pcr was effective in the recovery of t. gondii dna ; however , to date , there has been no archaeological evidence of this parasite recovered from any context . as is the case with t. cruzi , elisa kits have been developed to test modern human serum for t. gondii antibodies rather than for parasite coproantigens in fecal samples . fortunately , hajeer and colleagues found that t. gondii antibodies were detectible in modern saliva using elisa kits developed for testing human serum . as with the theory presented for the recovery of t. cruzi evidence utilizing cmc quids , we predict that t. gondii evidence could be recovered using elisa kits to test reconstituted quids . despite the lack of evidence regarding prehistoric , new world toxoplasmosis this parasite is known to infect a wide range of endothermic vertebrate hosts , including bats , rodents , and carnivores that may have come into contact with the loma san gabriel who were utilizing cmc . these individuals had close relationships with their companion canines , which could have been reservoirs for toxoplasmosis , and ate small rodents , rabbits , and other potential reservoir hosts that lived in the region surrounding the cave . if human antibodies created in response to t. gondii 1,300 years ago are capable of surviving and being detected using a commercially available elisa kit , archaeoparasitologists would have a new means of tracing the origins of human toxoplasmosis in the new world . the 45 cmc quids used for the present study were gathered from the collection of over 2,000 quids housed within the pathoecology laboratory in the school of natural resources at the university of nebraska - lincoln ( fig . 1 ; table 1 ) . to prevent modern contamination of material , nitrile gloves were worn throughout processing . a total of 45 quids were given a laboratory identification number ( cmcq- # ) for analysis and photographed using a sony cybershot 18.2 megapixel camera . each of the quids was analyzed using an olympus stereoscopic microscope and designated as being comprised of either agave or non - agave fibers . the quids were rehydrated in their entirety within a plastic , conical , 50 ml centrifuge tube containing a 0.5% trisodium phosphate solution . samples were rehydrated for 24 hr before rehydration colors were recorded ( table 1 ) . following rehydration , the quids were disaggregated using a novel shake and break method . this method begins with vigorously shaking the capped centrifuge tube before vortexing the tube for a few seconds . next , the material within the tube was poured through a 250 m mesh screen over a glass beaker . subsequently , sterile forceps were used in conjunction with a sterile spatulette to gently break apart the softened quids taking care to free as much of the non - fiber matrix of the quids as possible . another jet of distilled h2o was applied before removing the fibers / macroscopic remains ( items larger than 250 m ) on top of the screens to filter paper for drying . microscopic remains ( those smaller than 250 m ) were collected back into the centrifuge tubes and centrifuged to create the pellets used for elisa testing . aliquots of processed quid material were assayed for human antibodies created in response to t. gondii and t. cruzi infections using commercially available antibody detection kits . the toxoplasma iga elisa from sigma - aldrich uses wells coated with a purified toxoplasma antigen to bind with any toxoplasma iga specific antibodies present within a sample . unbound materials are then washed away prior to the addition of an enzyme conjugate that binds to existing antibody - antigen complexes when present . an incubation step allows for oxidation of the substrate by the enzyme which invokes a color change in positive sample wells . the intensity of the color change is proportional to the amount of iga specific antibody present within the sample ( www.sigma-aldrich.com ) . the chagas ( t. cruzi ) igg elisa from immuno - biological laboratories , inc . [ ibl - america ] uses microtiter strip wells pre - coated with trypanosoma cruzi antigens to bind human igg - class antibodies produced in response to t. cruzi infections . samples are incubated to allow antigen - antibody complexes to form before washing procedures remove unbound materials from the sample wells . next , a horseradish peroxidase ( hrp ) labelled protein a conjugate is added to bind any antigen - antibody complexes present within the samples . the resultant immune complex that is formed is then visualized following the addition of tetramethylbenzidine ( tmb ) substrate . the intensity of the color change is proportional to the amount of t. cruzi - specific igg antibodies present within the samples . this kit has a specificity of 99% and a sensitivity of 99% ( www.ibl-america.com ) . quid samples were subjected to elisa testing for t. gondii and t. cruzi evidence at the zera lab within the school of biological sciences at the university of nebraska - lincoln . samples were tested in duplicate along with controls provided by the manufacturers following manufacturer instructions . a visual inspection was conducted following the addition of each kit s stop solution and optical density ( od ) values were collected with the aid of an omega elisa plate reader ( tables 2 , 3 ) . the od readings were used to determine the presence or absence of each parasite using the interpretational procedures outlined for each kit by the manufacturers ( tables 2 , 3 ) . for the toxoplasma iga elisa , the kit - specific calibration factor was multiplied by the od value for the calibrator provided ( must be greater than 0.250 to be considered valid ) to determine the cut - off value . the ab ( antibody ) index was then calculated by dividing the od value of the provided positive control by the cut - off value . the ab index was similarly calculated for the provided negative control and for each quid sample . samples with an ab index less than 0.9 were designated as being negative while samples with an ab index greater than 1.2 were deemed to be considered positive . samples with ab indices between 0.9 and 1.2 were considered borderline positive and would require follow - up testing for validation ( table 2 ) . the cut - off value for the chagas ( t. cruzi ) igg elisa was calculated by taking the average of the od values for the cut - off control wells . substrate blank - corrected sample od values less than the cut - off were considered negative , while values greater than the cut - off were considered positive . for the assay to be considered valid , cut - off values had to range between 0.150 and 1.30 prior to blank correction ( table 3 ) . following these analyses , it became apparent that further method development would be necessary to validate interpretations . a total of 11 cmc quids processed for the tests described above were chosen to represent many different proveniences and depths ( cmcq#s : 3 , 5 , 13 , 16 , 24 , 29 , 35 , 37 , 39 , and 43 ) . additionally , 11 previously unprocessed quids were selected from the cmc collection held within the pathoecology laboratory at unl s school of natural resources . these quids were weighed , photographed , rehydrated in 0.5% trisodium phosphate , and further processed as previously described ( table 4 ) . aliquots from each of these 22 samples were transferred into pre - labeled microcentrifuge tubes . a second aliquot of precisely 10 l from each sample was placed into microcentrifuge tubes along with 990 l of wash buffer to test whether or not dilution of samples made a difference in the recovery of antibodies using these techniques . all samples were run in duplicate for both their diluted and undiluted varieties ( tables 57 ) . uses a pre - coated microtiter plate to bind siga within samples , standards , and controls . after an initial wash , a peroxidase - labeled detection antibody is added , which binds to the antibody complex . a second wash is performed before adding a substrate , which is converted by the peroxidase to result in a colored product . the addition of an acidic stop solution terminates this reaction and optical densities are read at 450 nm . the optical densities are then used to calculate siga concentrations with the help of a standard curve ( www.eaglebio.com ) . for the purposes of this study , siga concentrations were not calculated as a simple presence / absence test was all that was needed to test the methods . visual inspection of the elisa plates was conducted prior to the collection of od values via the elisa plate reader . positive / negative assessments were recorded on the basis of a visual color change and later compared to sample designations using od values . data collected via the elisa plate reader also yielded negative results for both test kits . the od values from these analyses were recorded and samples were designated positive / negative according to cut - off value calculations using manufacturers interpretational protocols ( tables 2 , 3 ) . all controls used in both kits yielded od values within acceptable ranges for considering the kits valid according to manufacturers specifications . a visual inspection of the siga elisa wells produced 2 positive test wells from cmcq-35 and cmcq-49 ( both diluted ) . however , the duplicates of both wells were visually negative . upon examination of od values , only cmcq-35 remained positive , again with its duplicate well being negative . because duplicate wells for both samples were not consistently positive and because average od values led to negative designations for both samples , these were considered anomalies rather than true positive results . because the siga elisa kit used was developed for determining concentration levels of siga in modern samples and not for determining presence / absence of siga in archaeological samples , positive / negative determinations were reached as follows . the od value for 0.5% trisodium phosphate ( 0.161 ) was subtracted from each sample od value ( i.e. the od value for the rehydration solution was used as a blank ) . blank - corrected od values were then compared to the od values acquired for the kit - provided standards . none of the od values were close to the higher ng/l standards ( i.e. the 200 ng/l standard and the 600 ng/l standard ) , so od values were instead compared to the od values for the 22.2 ng/l standard and the 0 ng/l standard . blank - corrected od values greater than the od value for the 0 ng/l standard ( 0.064 ) were considered potentially positive . average od values calculated rom sample duplicates were also compared to the od value for the 0 ng/l standard ( 0.064 ) and the entire quid was considered potentially positive if greater than 0.064 . similar comparisons were made using the od values for the 22.2 ng/l standard ( 0.209 ) . samples with od values and od value averages greater than 0.209 were considered positive for trace amounts of siga . only a single test well ( cmcq-35 - -diluted ) was deemed potentially positive in this way . however , since the duplicate for this well was negative and the average od value for this quid yielded a negative designation , this was not found to be significant and was likely a false positive as a result of an error in processing . all other sample wells were found to be negative when compared to od readings for the standards . the average od values for each sample in their diluted and undiluted forms were compared . out of the 22 samples tested , 3 produced the same od value whether or not they were diluted . a total of 9 samples yielded higher od values for diluted samples while a total of 10 samples yielded higher od values for undiluted samples ( table 7 ) . while other studies have successfully employed elisa techniques for recovering parasite evidence from latrine and burial sediments as well as coprolites , none have successfully recovered parasite antigens or parasite - induced antibodies from quids . although the present study was not successful in the recovery of parasite - induced antibodies from cmc quids , the study represents the first attempt to use quids as a source material for archaeoparasitological investigations . there are several reasons why this study s attempt at the recovery of parasite evidence from quids may have failed . despite the recovery of t. gondii and t. cruzi antibodies from modern saliva , it is important to note that elisa kits available for t. gondii and t. cruzi are marketed for use with human serum . furthermore , these kits are marketed for modern material and not for reconstituted archaeological material , which could compromise the abilities of the kits to actually detect ancient antibodies . this has not been a problem for the recovery of parasite coproantigens out of archaeological materials , but antibodies may degrade differently than do coproantigens . these molecules weigh about 150 kda as compared to the average antigen weighing in at around 14 kda . the smaller size and relative structural simplicity of antigens make them less likely to degrade in ways that would render elisa testing inefficient . it is unknown whether or not plant products , such as those made by agave and other types of botanicals found in quids , have an adverse effect on the preservation of antibodies and antigens . if so , such products could also contribute to antibody degradation , which further complicates the elisa testing of quids . the archaeological context , structural components of antibodies / antigens , anthropogenic behaviors regarding quid chewing , and ecological interactions of plants with target antibodies / antigens may all contribute to the taphonomy of immunological parasite evidence in much the same way that similar factors contribute to the degradation of physical parasite eggs within archaeological contexts as described by morrow and colleagues . even so , the taphonomic impacts of archaeological antibody / antigen recovery have not been thoroughly assessed at the present time . future examinations of antibody degradation resulting from desiccation could elucidate the nature of human antibody taphonomy and its implications on archaeoparasitological recovery techniques . alternatively , the capabilities of the elisa test kits utilized in the present study could be sound for the recovery of archaeological antibodies given the proper quid processing methods . without precedent in the existing literature , quid processing methods were developed by modifying coprolite processing techniques deemed successful for the recovery of parasite coproantigens . further refinement of quid processing methods could provide avenues for the successful recovery of parasite evidence utilizing elisa kits . finally , the results of these analyses could have been negative simply because the individuals who created the cmc quids were not infected with t. gondii or t. cruzi . the pathoecological potential of parasitism does not always reflect true parasitism within a given population . the individuals utilizing cmc could have circumvented entry into zoonotic cycles of these parasites by avoiding transmission pathways . transmission pathway avoidance could have been a result of cultural behaviors that limited infection risks . the coprolites of these individuals contained large quantities of dietary fiber , which may reflect a greater reliance on plant foodstuffs than on wild game . although these people were known to ingest rodents , perhaps the animals they were consuming were not serving as reservoir hosts for either of these parasites around cmc at that time . despite the lack of positive results , the employment of elisa for the recovery of parasite evidence using quids as source materials remains a plausible concept . future elisa tests of quids should target parasite antigens as opposed to parasite - induced human antibodies , though currently , few such tests have been developed for samples of non - fecal origin . future researchers should approach this concept by modifying processing methods , experimenting with different epitope targets and brands of elisa kits , and performing methodological experiments using artificially - created quids known to contain target antibodies . such experimentation could provide the foundational data needed to develop reliable techniques for recovering parasite evidence from quids . the development of successful methods for the archaeoparasitological analysis of quids would open new venues of research regarding ancient parasitism . potentially , parasites that do not leave behind traceable coproantigens , such as t. gondii and t. cruzi , could be detected via residual salivary products present in artifacts like quids . as researchers move forward with the analysis of quids , it is important to consider the limitations of elisa testing . such limitations include taphonomic degradation of target antibodies / antigens within quids , cross - reactivity potential with other parasites , and the presence of bacteria or fungi that may compromise test validity . to address these limitations , processing methods should be designed to inflict little damage to potential antibodies / antigens , only kits that are highly specific / sensitive should be employed , and samples should be tested quickly after being reconstituted before bacterial and fungi are able to colonize samples . it is also vital that future researchers acknowledge that archaeoparasitological data do not reflect actual infection rates in past populations with 100% accuracy . nonetheless , immunological testing of archaeological samples provides valuable tools for estimating the parasite burdens among peoples of the past . our ability to more accurately assess protozoan parasitism of the past has improved with the progression of technology and will continue to be refined as new archaeoparasitological immunodiagnostic techniques become available .

in the present study , quids from la cueva de los muertos chiquitos ( cmc ) were subjected to elisa tests for 2 protozoan parasites , toxoplasma gondii ( n=45 ) and trypanosoma cruzi ( n=43 ) . the people who occupied cmc , the loma san gabriel , lived throughout much of present - day durango and zacatecas in mexico . the known pathoecology of these people puts them into at - risk categories for the transmission of t. gondii and t. cruzi . human antibodies created in response to these 2 parasites can be detected in modern saliva using elisa kits intended for use with human serum . for these reasons , quids were reconstituted and subjected to elisa testing . all test wells yielded negative results . these results could be a factor of improper methods because there is no precedence for this work in the existing literature . the results could equally be a simple matter of parasite absence among those people who occupied cmc . a final consideration is the taphonomy of human antibodies and whether or not elisa is a sufficient method for recovering antibodies from archaeological contexts . an additional elisa test targeting secretory iga ( siga ) was conducted to further examine the failure to detect parasite - induced antibodies from quids . herein , the methods used for quid preparation and elisa procedures are described so that they can be further developed by future researchers . the results are discussed in light of the potential future of quid analysis .

INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION

employing elisas as a means of examining archaeological materials for evidence of protozoan parasites has become increasingly more routine among archaeoparasitological researchers [ 115 ] . much of the existing work has focused on the recovery of protozoan parasite antigens from latrine sediments [ 4,911,13,14 ] . quids , which are expectorated masses of human - masticated plant fibers , are desiccated artifacts commonly found at new world archaeological sites . such studies have examined the role of quid chewing in the development of dental wear among archaeological populations . the quids were all phytolith - rich and most were derived from agave plant fibers , although maize leaves and husks were also identified among some of the quids . because these phytoliths , along with those of other dietary abrasives recovered from coprolites and quids , are able to cause dental wear , it is likely that they may have also created microlacerations within the soft tissues of the mouth . salivary antibodies , such as secretory iga ( siga ) , would have become similarly integrated into the quid bolus . a few studies have employed elisa tests for the purposes of diagnosing parasitic infections using modern saliva . the recovery of species - specific parasite - induced antibodies from human saliva in modern contexts begs the question : can these antibodies be recovered from archaeological materials saturated with desiccated human saliva ? to answer this question , while parasite - specific coproantigens have been demonstrated to preserve within archaeological materials like coprolites and latrine sediments , the recovery of human - created parasite - specific antibodies from archaeological materials has not been attempted to date . thus , the preservation and degradation of antibodies from an archaeological perspective have not been fully explored . therefore , the potential persistence of parasite - induced human immunoglobulins is conceptually plausible . an ideal archaeological potential source material for salivary immunoglobulins is the often - ignored quid . with the capability to incorporate antibodies from the saliva as well as from blood released via microlacerations in the oral cavity caused by phytoliths , these artifacts provide a mechanism for assessing archaeological parasitism in a unique way . to date the present study represents the first effort to analyze quids ( n=45 ) for the presence of 2 species of protozoan parasites ( toxoplasma gondii and trypanosoma cruzi ) . the quids used in the present study were excavated from la cueva de los muertos chiquitos ( cmc ) , a site within the rio zape valley , in the early 1960s . this valley lies approximately 18 km southeast of guanacev in durango , mexico , and is home to a number of caves utilized by the loma san gabriel . cmc was used year - round as a temporary habitation by the loma san gabriel between 1,200 and 1,400 years ago . the people who utilized cmc subsisted using a mixed strategy of agricultural production and hunting - gathering , both of which fluctuated seasonally . the incredible preservation of both physical and molecular parasite evidence makes this site ideal for testing new methods of parasite evidence recovery utilizing quids as source materials . as these people modified their environments to survive , they may have inadvertently promoted the transmission of many different types of parasites , including t. cruzi and t. gondii . evidence of t. cruzi has been recovered from archaeological materials in north and south america [ 3744 ] . the sylvatic cycle of t. cruzi involves a triatomine bug that serves as a vector for the parasite and a mammalian definitive host . about 180 species of mammals , including bats , carnivores , rodents , ungulates , and primates , have been identified as reservoir hosts for t. cruzi . domestic cycles of t. cruzi are perpetuated in human populations via vectored transmission and via oral transmission of contaminated foods . from an archaeological perspective , this parasite is particularly interesting because prehistoric humans of the southwestern usa and mesoamerica inserted themselves into the t. cruzi life cycle as they changed their environments to better suit their survival needs . simultaneously , humans induced population decline in reservoir hosts via woodrat hunting and habitat displacement , which caused the vectors to come in contact with human hosts more frequently than in the past . these behaviors placed humans at risk for contracting zoonotic trypanosomiasis that eventually became the american trypanosomiasis ( chagas disease ) that infects an estimated 10 million people today the origins of t. cruzi can be traced back more than 9,000 years in the new world . analyses of material from mummies place the establishment of this parasite in the americas long before european contact [ 39,41,4850 ] . artifacts from cmc have not previously been tested for evidence of t. cruzi ; however , dietary evidence of small rodent consumption at this site supports the hypothesis that this parasite may have been cycling among the loma san gabriel at cmc in prehistory . the majority of evidence regarding t. cruzi from archaeological contexts has been collected using molecular techniques that involve the recovery of dna . one study demonstrated that t. cruzi dna could be recovered from experimentally desiccated mouse tissues . to date , elisa techniques have not been applied to archaeological materials to recover evidence of t. cruzi . because commercial elisa kits for t. cruzi are designed to test human serum rather than fecal samples , there has not been a practical way of utilizing this technique in the search for chagas disease in prehistory . pinho and colleagues demonstrated that t. cruzi antibodies could be recovered from modern saliva using elisa kits . if these antibodies survive following desiccation , there is the potential that elisa kits could prove to be useful for the recovery of t. cruzi evidence from saliva - contaminated artifacts , such as the quids excavated from cmc . if the use of elisa techniques in this regard is successful , the archaeoparasitological community could enjoy a new rapid , reliable , and cost - effective method for recovering evidence of t. cruzi from archaeological quids . the genus toxoplasma is approximately 10 million years old , with toxoplasma gondii originating sometime in the last 10,000 years . today , about a third of the global human population is infected with this parasite . in the united states , in fact , it has been deemed the second most important foodborne pathogen in the nation . it is not unreasonable to think that people living near cmc may have been infected with t. gondii . though no clinical cases of toxoplasmosis have been reported from bats , there have been reports describing the isolation of t. gondii from 2 species of bats ( vespertilio pipistrellus and nyctalus noctula ) in the ussr . t. gondii is also maintained in a wide variety of wild animal populations , including rodents , carnivores , and lagomorphs [ 6163 ] . in modern cases of toxoplasmosis among healthy children , children with weaken immune systems who contract toxoplasmosis are at risk for more serious neurological symptoms that may cause long - term damage . the high probability that the people occupying cmc were exposed to reservoir host species coupled with the majority of skeletons from this site being those of children make this site an excellent place to look for prehistoric toxoplasmosis . terra and colleagues demonstrated through experimental desiccation of infected mice that pcr was effective in the recovery of t. gondii dna ; however , to date , there has been no archaeological evidence of this parasite recovered from any context . as is the case with t. cruzi , elisa kits have been developed to test modern human serum for t. gondii antibodies rather than for parasite coproantigens in fecal samples . fortunately , hajeer and colleagues found that t. gondii antibodies were detectible in modern saliva using elisa kits developed for testing human serum . as with the theory presented for the recovery of t. cruzi evidence utilizing cmc quids , we predict that t. gondii evidence could be recovered using elisa kits to test reconstituted quids . despite the lack of evidence regarding prehistoric , new world toxoplasmosis this parasite is known to infect a wide range of endothermic vertebrate hosts , including bats , rodents , and carnivores that may have come into contact with the loma san gabriel who were utilizing cmc . these individuals had close relationships with their companion canines , which could have been reservoirs for toxoplasmosis , and ate small rodents , rabbits , and other potential reservoir hosts that lived in the region surrounding the cave . if human antibodies created in response to t. gondii 1,300 years ago are capable of surviving and being detected using a commercially available elisa kit , archaeoparasitologists would have a new means of tracing the origins of human toxoplasmosis in the new world . the 45 cmc quids used for the present study were gathered from the collection of over 2,000 quids housed within the pathoecology laboratory in the school of natural resources at the university of nebraska - lincoln ( fig . a total of 45 quids were given a laboratory identification number ( cmcq- # ) for analysis and photographed using a sony cybershot 18.2 megapixel camera . the quids were rehydrated in their entirety within a plastic , conical , 50 ml centrifuge tube containing a 0.5% trisodium phosphate solution . following rehydration , the quids were disaggregated using a novel shake and break method . next , the material within the tube was poured through a 250 m mesh screen over a glass beaker . subsequently , sterile forceps were used in conjunction with a sterile spatulette to gently break apart the softened quids taking care to free as much of the non - fiber matrix of the quids as possible . another jet of distilled h2o was applied before removing the fibers / macroscopic remains ( items larger than 250 m ) on top of the screens to filter paper for drying . microscopic remains ( those smaller than 250 m ) were collected back into the centrifuge tubes and centrifuged to create the pellets used for elisa testing . aliquots of processed quid material were assayed for human antibodies created in response to t. gondii and t. cruzi infections using commercially available antibody detection kits . the intensity of the color change is proportional to the amount of iga specific antibody present within the sample ( www.sigma-aldrich.com ) . [ ibl - america ] uses microtiter strip wells pre - coated with trypanosoma cruzi antigens to bind human igg - class antibodies produced in response to t. cruzi infections . the intensity of the color change is proportional to the amount of t. cruzi - specific igg antibodies present within the samples . quid samples were subjected to elisa testing for t. gondii and t. cruzi evidence at the zera lab within the school of biological sciences at the university of nebraska - lincoln . a visual inspection was conducted following the addition of each kit s stop solution and optical density ( od ) values were collected with the aid of an omega elisa plate reader ( tables 2 , 3 ) . for the toxoplasma iga elisa , the kit - specific calibration factor was multiplied by the od value for the calibrator provided ( must be greater than 0.250 to be considered valid ) to determine the cut - off value . the cut - off value for the chagas ( t. cruzi ) igg elisa was calculated by taking the average of the od values for the cut - off control wells . for the assay to be considered valid , cut - off values had to range between 0.150 and 1.30 prior to blank correction ( table 3 ) . a total of 11 cmc quids processed for the tests described above were chosen to represent many different proveniences and depths ( cmcq#s : 3 , 5 , 13 , 16 , 24 , 29 , 35 , 37 , 39 , and 43 ) . additionally , 11 previously unprocessed quids were selected from the cmc collection held within the pathoecology laboratory at unl s school of natural resources . these quids were weighed , photographed , rehydrated in 0.5% trisodium phosphate , and further processed as previously described ( table 4 ) . aliquots from each of these 22 samples were transferred into pre - labeled microcentrifuge tubes . a second aliquot of precisely 10 l from each sample was placed into microcentrifuge tubes along with 990 l of wash buffer to test whether or not dilution of samples made a difference in the recovery of antibodies using these techniques . for the purposes of this study , siga concentrations were not calculated as a simple presence / absence test was all that was needed to test the methods . visual inspection of the elisa plates was conducted prior to the collection of od values via the elisa plate reader . data collected via the elisa plate reader also yielded negative results for both test kits . a visual inspection of the siga elisa wells produced 2 positive test wells from cmcq-35 and cmcq-49 ( both diluted ) . however , the duplicates of both wells were visually negative . because the siga elisa kit used was developed for determining concentration levels of siga in modern samples and not for determining presence / absence of siga in archaeological samples , positive / negative determinations were reached as follows . the od value for the rehydration solution was used as a blank ) . blank - corrected od values were then compared to the od values acquired for the kit - provided standards . none of the od values were close to the higher ng/l standards ( i.e. blank - corrected od values greater than the od value for the 0 ng/l standard ( 0.064 ) were considered potentially positive . average od values calculated rom sample duplicates were also compared to the od value for the 0 ng/l standard ( 0.064 ) and the entire quid was considered potentially positive if greater than 0.064 . similar comparisons were made using the od values for the 22.2 ng/l standard ( 0.209 ) . only a single test well ( cmcq-35 - -diluted ) was deemed potentially positive in this way . however , since the duplicate for this well was negative and the average od value for this quid yielded a negative designation , this was not found to be significant and was likely a false positive as a result of an error in processing . all other sample wells were found to be negative when compared to od readings for the standards . out of the 22 samples tested , 3 produced the same od value whether or not they were diluted . while other studies have successfully employed elisa techniques for recovering parasite evidence from latrine and burial sediments as well as coprolites , none have successfully recovered parasite antigens or parasite - induced antibodies from quids . although the present study was not successful in the recovery of parasite - induced antibodies from cmc quids , the study represents the first attempt to use quids as a source material for archaeoparasitological investigations . there are several reasons why this study s attempt at the recovery of parasite evidence from quids may have failed . despite the recovery of t. gondii and t. cruzi antibodies from modern saliva , it is important to note that elisa kits available for t. gondii and t. cruzi are marketed for use with human serum . furthermore , these kits are marketed for modern material and not for reconstituted archaeological material , which could compromise the abilities of the kits to actually detect ancient antibodies . this has not been a problem for the recovery of parasite coproantigens out of archaeological materials , but antibodies may degrade differently than do coproantigens . the smaller size and relative structural simplicity of antigens make them less likely to degrade in ways that would render elisa testing inefficient . it is unknown whether or not plant products , such as those made by agave and other types of botanicals found in quids , have an adverse effect on the preservation of antibodies and antigens . the archaeological context , structural components of antibodies / antigens , anthropogenic behaviors regarding quid chewing , and ecological interactions of plants with target antibodies / antigens may all contribute to the taphonomy of immunological parasite evidence in much the same way that similar factors contribute to the degradation of physical parasite eggs within archaeological contexts as described by morrow and colleagues . even so , the taphonomic impacts of archaeological antibody / antigen recovery have not been thoroughly assessed at the present time . future examinations of antibody degradation resulting from desiccation could elucidate the nature of human antibody taphonomy and its implications on archaeoparasitological recovery techniques . alternatively , the capabilities of the elisa test kits utilized in the present study could be sound for the recovery of archaeological antibodies given the proper quid processing methods . without precedent in the existing literature , quid processing methods were developed by modifying coprolite processing techniques deemed successful for the recovery of parasite coproantigens . further refinement of quid processing methods could provide avenues for the successful recovery of parasite evidence utilizing elisa kits . finally , the results of these analyses could have been negative simply because the individuals who created the cmc quids were not infected with t. gondii or t. cruzi . the individuals utilizing cmc could have circumvented entry into zoonotic cycles of these parasites by avoiding transmission pathways . the coprolites of these individuals contained large quantities of dietary fiber , which may reflect a greater reliance on plant foodstuffs than on wild game . although these people were known to ingest rodents , perhaps the animals they were consuming were not serving as reservoir hosts for either of these parasites around cmc at that time . despite the lack of positive results , the employment of elisa for the recovery of parasite evidence using quids as source materials remains a plausible concept . future elisa tests of quids should target parasite antigens as opposed to parasite - induced human antibodies , though currently , few such tests have been developed for samples of non - fecal origin . future researchers should approach this concept by modifying processing methods , experimenting with different epitope targets and brands of elisa kits , and performing methodological experiments using artificially - created quids known to contain target antibodies . such experimentation could provide the foundational data needed to develop reliable techniques for recovering parasite evidence from quids . the development of successful methods for the archaeoparasitological analysis of quids would open new venues of research regarding ancient parasitism . potentially , parasites that do not leave behind traceable coproantigens , such as t. gondii and t. cruzi , could be detected via residual salivary products present in artifacts like quids . as researchers move forward with the analysis of quids , it is important to consider the limitations of elisa testing . such limitations include taphonomic degradation of target antibodies / antigens within quids , cross - reactivity potential with other parasites , and the presence of bacteria or fungi that may compromise test validity . it is also vital that future researchers acknowledge that archaeoparasitological data do not reflect actual infection rates in past populations with 100% accuracy . nonetheless , immunological testing of archaeological samples provides valuable tools for estimating the parasite burdens among peoples of the past . our ability to more accurately assess protozoan parasitism of the past has improved with the progression of technology and will continue to be refined as new archaeoparasitological immunodiagnostic techniques become available .

by 2050 , there will be approximately 2 billion people aged 60 years or more and the number of older people will outnumber the young . the most rapid changes will take place in developing countries , and projections indicate that by then four of five older people will live in developing countries . this demographic change has implications not only for the delivery of health and social care and for age - related policy issues , such as pensions , but also for the everyday life of citizens both young and old . our capacity to understand how demographic changes in developing countries will impact on older people 's lives is limited by the relative lack of research on the everyday lives of older people in such countries . in this paper , we describe an exploratory study that examines the public presence of older people , with consideration of differences between women and men . this is done via the structured observation of social spaces in selected cities in six developing countries and one city in a developed county and content analysis of examples of the countries ' respective print and broadcast media , specifically images in newspapers and on television channels . the potential of such data for understanding the social integration and inclusion of older people is discussed . when proposing a need for more social research on older people in developing countries , the methodological approach to be adopted requires particularly careful consideration . applying conventional research methods might be problematic . using survey methods and instruments created for use in developed countries raises issues of validity , if concepts and language are simply imported without thought for differing cultural contexts . difficulties in accessing older people and the use of standardised but culturally biased procedures and formats for delivering questions and eliciting responses threaten reliability and representativeness ( cf . ) . qualitative methods are suitable for developing understanding and theory and are arguably more sensitive than quantitative studies to cultural issues . however , studies using such methods have limitations as their ( usually ) small and unrepresentative samples prevent generalization of their findings to target populations , and the studies ' findings are additionally often difficult to reproduce . observational methods and content analysis might circumvent some of the problems that arise in using conventional research methods in the context of developing countries . it can be argued that the actual presence or absence of older people in public fora , such as social and cultural spaces , is a good marker of their level of social integration ( cf . ) . the use of social spaces can influence societal attitudes to different groups and the degree to which they belong in society . also , it can direct us towards the way space works to include or exclude people . presence or absence can also be considered through visibility in different media such as newspapers , films , or television . mass media are a force for shaping attitudes and images of older people in media shape perceptions and self - perceptions of ageing [ 811 ] . underrepresentation in media could lead to a perception that groups of the population are of little significance to society ( cf . ) . thus , how far older people are visible in such cultural spaces and the nature of their presentation are both reflections and drivers of social norms , becoming both measures of social integration and factors that affect social integration . studies that document the presence or absence of older people in public fora might therefore provide valuable data that will contribute to our understanding of the place of older people in their respective societies . however , there are a very limited number of such studies , particularly in developing countries . there is some information , primarily from developed countries , about places that are important to older people . for example , londoners identified a religious building as an important place in their lives and different ethnic groups identified varying benefits in using parks in the united states of america ( us ) , while older people in hong kong found that poor health , lack of time , and issues with other users reduced their use of parks , despite their health and social and psychological benefits (; cf . ) . with regard to the presence and form of presentation of older people in broadcast and print media there is a considerable body of work available , but there is a knowledge gap around images of older people in non - western cultures with cross - cultural research extremely limited . in the available research from developed countries , there is a consensus that older people have been and continue to be less present than expected with regard to their prevalence in the population . for example , an extensive observational study of german television networks found that people over 60 were very underrepresented , particularly women and those of advanced old age . similarly , us and german studies have found an underrepresentation of older people in television and printed advertisements , respectively . content analysis of print media in one area in the usa found that stories that could be illustrated by pictures of people of any age tended to feature younger people . older people were also underrepresented in the united kingdom ( uk ) magazine advertisements , canadian television advertisement , and irish newspapers [ 2527 ] . although there are positive images on older people in aspirational advertising ( for a discussion , see ) , when present in media older people are frequently constructed as victims and being frail , vulnerable , and so forth , not the least when mentioned in the context of population ageing . considering developing or non - western countries , a study on the presentation of older people on taiwanese television produced findings similar to those obtained in the usa , while studies looking at the presentation of older people in the media in india and japan found that there was an underrepresentation of older people , particularly of older women , in magazine and television announcements , respectively [ 30 , 31 ] . when studying older people 's presence in public fora , it is important to consider the differing life situations of older women and men in developing and developed countries . first , there are health differences between women and men , with greater healthy life expectancy among women in both developing and developed countries . . studies in both developed and developing countries have established that social contacts and social support are positively associated with health and wellbeing ( e.g. , [ 3338 ] ) and that older women have higher levels of social contacts and social support than older men ( e.g. , [ 1 , 33 , 39 , 40 ] ) . however , as women live longer and tend to marry older men , they are more likely to enter widowhood than men and at a younger age . these gender differences in health and social support would be anticipated to influence the extent to which older women and men are present in public spaces . as indicated above , research has shown that older women are particularly underrepresented across a range of media . feminist critique of media representations suggests that underrepresentation may play a significant role in how women interpret and experience ageing . in addition , in both developing and developed countries , women are generally shown in traditionally feminine and stereotyped roles , and they are often portrayed in a negative and circumscribed manner . for example , one study of arabic television found that women are more often shown in domestic roles and indoor settings , whereas men are shown in professional roles and outdoors settings ( see ) . our primary research question is : how present are older people in public fora in developing countries ? a secondary research question is : is the presence of older people in public fora related to their gender ? our site selection , of six cities in six developing countries and one city in a developed country , and the use of two data collection methods were motivated by several considerations . first , as this was an exploratory study it was important to examine the validity , reliability , and feasibility of the methods adopted across a wide range of settings . the selection of a range of developing countries fulfilled this consideration , as such countries would provide challenges and contexts as fieldwork sites that would contrast with those found in the developed country ; there would also likely be differences between the selected developing countries themselves . second , there is a tremendous gap between our understanding of the demographic changes affecting older people in the developing world and our understanding of the public lives of older people in such countries . our study , with its primary focus on developing countries , would thus both increase the amount of data available on the presence of older women and men in social spaces and in the media in such countries and offer some insight into how their presence in public fora in developing countries contrasts with their presence in similar fora in a developed country , with opportunity for cautious comparison due to the use of standardised methods across fieldwork sites . finally , the use of three different data sources non - participant structured observation in social spaces and content analysis of images of older people both in newspapers and on television would allow for triangulation of data as a way of evaluating the robustness of findings . this study involved gathering data in six developing countries syria , jordan , egypt , thailand , kenya , and tanzania and in one western country the uk over the course of 2007 . in each of these countries , structured observations were carried out in public social places where people were likely to be present and content analysis was performed of images in newspapers and television programmes in which they were likely to be depicted . country selection was guided by a need for variation in geographical location , political structures , and ethnic and social mixes , as well as demography . the chosen developing countries offer diversity of cultures , with varying degrees of contrast and comparison with the selected site in the uk . the selected countries represent , respectively , the overwhelmingly arabic muslim syria and jordan and hamitic muslim egypt ; the multiplicity of ethnic groups in black east africa , with a predominantly christian culture and minority muslim culture in kenya , and strong christian and muslim cultures in tanzania ; and the distinctive , largely buddhist thai culture of the indian subcontinent , with its chinese and malay subpopulations . the uk has predominantly a white population with strong christian heritage but has increasingly diverse ethnic and religious minorities [ 45 , 46 ] . at the time of data collection , syria , egypt , kenya , and tanzania had presidential and parliamentary government and jordan and thailand had constitutional monarchies with parliamentary government , as did the uk . the urban population is 90 per cent in the uk , 82 per cent in jordan , 50 per cent in syria , 43 per cent in egypt , 33 per cent in thailand , 23 per cent in tanzania , and 19 per cent in kenya . , it was 79 years in the uk , 72 in syria and thailand , 71 in jordan , and 68 in egypt , but only 53 in kenya and 50 in tanzania . twenty - two percent of the uk population is over 60 , compared to 12 per cent in thailand , 7 per cent in egypt , and 5 per cent or less in other countries . for all data sources , an older person was defined as a person whose appearance suggested an age of 60 or more . for all procedures , weekends in non - muslim countries and fridays in muslim countries were not used for data collection in order to avoid atypical as detailed in table 1 , seven large cities were chosen for observation of social spaces : sheffield ( uk ) , haleb ( syria ) , amman ( jordan ) , luxor ( egypt ) , bangkok ( thailand ) , nairobi ( kenya ) , and arusha ( tanzania ) . the smallest city observed was arusha with a population of around 140,000 ; the largest city was bangkok with 966,000 . within each city a central area featuring the main public buildings was selected to ensure that fieldwork sites for observation would be within walking distance of each other . in each city , observations were carried out in seven social spaces : a market , a religious building ( e.g. , a christian church / cathedral , muslim mosque , or hindu temple , representing the largest denomination in each city ) , a park , a bus station , a post office , a hotel , and a caf . social spaces were selected to reflect divergence in function , physical layout , open versus enclosed aspect , and expected use by differing subgroups of the population . a time sampling frame for recording of data from observations was developed based on previous work on the observation of older people in residential care environments [ 50 , 51 ] and adapted through piloting . for religious buildings , post offices , cafes , and hotels , people going in or out people were counted as they passed a particular point including , where appropriate , counting people going on and off buses . observations were carried out during six phases of the day : 7 am9 am , 9 am12 noon , 12 noon2 pm , 2 pm5 pm , 5 pm8 pm , and 8 pm11 pm . a five - minute period was randomly selected within each phase , during which continuous observation occurred and data was recorded . in each five - minute period , the number and gender of any observed older people were recorded , as were the age and gender constitution of groups involving older people . the five- minute period of data recording balances two conflicting needs : the first is the requirement for a long enough period of behaviour observation to capture the continuous nature of life 's ebb and flow and identify the context for activity so as to support the reliable coding of events ; the second is the need to restrain the time for observation so as to allow for the accurate recording of often significant amounts of data with accompanying field notes . breaking up the day into periods also ensured that reliability of the recorded information is not compromised by tiredness , while the overall pattern of life is retained through sampling across an entire day . information on the validity and reliability of this specific observational data time sampling framework is published elsewhere [ 50 , 51 ] , although it is derived from standard procedures for structured observation methods . due to local circumstances , some adjustments were made to the site selection frame . where the city center was non - residential , as in amman and bangkok , the old center and the area around the railway station were chosen , respectively , as they are focal points of city life . in nairobi , the city center was not residential but it was not safe to observe outside this area . in luxor and arusha , a main bus stop was selected as the bus station was out of town ; in bangkok the train station was used , as the bus station was in a different area ; in bangkok , nairobi , and arusha there were no parks within the area observed so this part of the observation schedule could not be carried out . some adjustments to the sampling frame were also required to reflect local circumstances . in nairobi and arusha , tourists are advised not to go out on foot after dark , so observations could not be done after 8 pm . if a building was not open during a given phase of the day , data could not be collected . public spaces selected for observation were in total closed on 11 occasions between 7 and 9 am , on 10 occasions between 8 and 11 pm , on 8 occasions between 5 and 8 pm , and on 1 occasion between 9 and 12 am . media content analysis was undertaken of newspaper pictures and television channels , as detailed in table 1 . because our study focuses on the visibility of older people , we selected newspapers and television as mass media genres containing a significant component of visual information ( as opposed to , say , literature or radio ) . a sampling frame was developed in which for each country two local language papers and one english language paper were chosen , unless english was the local language ( uk , kenya , and tanzania ) where two english papers were chosen only . within this frame , as the researchers had restricted prior knowledge of the countries ' media , the newspapers were arbitrarily selected from amongst those papers prominently displayed on at least two news stalls on a given day in each city . every picture was examined and data extracted on people represented in terms of gender , age group , and number . where older people were depicted , a brief description of their activity was recorded . the page number and section were noted , together with the headline for english papers and a brief description for other language papers , except when the picture contained only a face with no indication of context . pictures advertising a product were recorded as adverts and the products were identified where possible . the sections in which the picture appeared were used as base categories for the analysis and finally combined into two superordinate categories : news and non - news . within the sampling frame developed for television , terrestrial television channels were prioritized , followed by channels from the region in which the country was situated , and if this was insufficient , a sample of the remaining channels was chosen . television channels were recorded in all countries but thailand and kenya , as in these two countries the researcher was not able to access them within a context that allowed for reliable data recording . in syria , eight channels were selected , all arabic . in jordan , five channels were selected , three of which were arabic and the other two are canadian and turkish . in egypt , four channels were selected , two arabic and one american and there was one other channel of unknown origin . in tanzania , six channels were selected : these were tanzanian , french african , south african , indian , british , and american . in the uk , four of the five british terrestrial channels were selected . in total , 27 channels were selected . the time sampling frame for recording of data from television was developed via adaptation of the frame used for the observation of social spaces described above and via extensive piloting . each selected channel in a country was studied for a five - minute period during each of the same six phases of the day used for social space observations . a five - minute period was judged , a useful time frame for coding television material as it provides sufficient time to avoid the whole period being taken up by advertisements or credits and to establish reliably the type of programme being broadcast . during these five - minute periods , every scene was recorded and data extracted on people represented in terms of gender , age group , and number . by scene , what is meant is a linear representation or presentation of events or narrative , commencing and ending on an edit or camera switch . where older people were depicted , a brief description of their activity was recorded . the programme type and where possible the name of the programme were recorded . for programmes in english , analytic categories were decided by identifying common programme types in uk and finally combined into two superordinate categories : news or non - news . as with observations of social spaces , it was not possible to observe television from 12 to 2 pm in jordan , 8 to 11 pm in egypt , and 7 to 9 am in tanzania , due to pragmatic constraints on the researcher 's access to media during these phases . as the data collected was largely non - normal in distribution , nonparametric analytic techniques were used . parametric analyses were used only where they provided valuable additional descriptive information . where repeated testing of the same sample occurred , bonferroni corrections to reduce type i errors were applied , with an appropriate reduction in alpha for each analysis . where category cell n was too low to enable reliable analysis , combination of categories occurred . in total , 310 older people were observed in social spaces . this was a mean of 1.17 ( sd = 4.16 , range 039 ) older people per observation period and a median value of 0 ( iqr = 0 - 1 ) older people per observation period . the number of observed older people per period was therefore greatly skewed toward low values , and older people were observed in only 26.1% of the 264 observation periods . with regard to gender , 184 ( 59.4% ) men and 126 ( 40.6% ) women were observed . in all cities in developing countries , more men than women were observed ( developing countries combined : men n = 96 , women n = 7 ) , whereas in sheffield more women than men were observed ( women n = 119 , men n = 88 ) . the number of older people observed per period varied considerably across cities ( see table 2 ) . although sheffield had by far the highest mean number of older people observed per period , this value was inflated by a small number of observation periods with relatively large number of older people . thus , excluding arusha ( no older people observed ) , the median number of older people observed per period was highest in amman ( .50 , iqr = 02.00 ) and joint lowest in luxor and nairobi ( 0 , iqr = 0 - 0 ) . due to the very small number of women observed in cities in developing countries and thus the confounding of city status ( developed versus developing ) with gender , all following statistical comparison between developing country cities and sheffield was carried out using cases of observed males only . there was significant variation across cities in the number of older men observed per period ( h(6 ) = 38.3 , p < .001 ) . with regard to the social spaces in which older people were observed , a market was the location in which most older people were observed ( n = 121 ) , followed by a bus / train station ( n = 70 ) , a post office ( n = 67 ) through to a hotel being the location in which fewest older people were observed ( n = 4 ) . there was significant variation across social spaces in the number of older men observed per period ( h(6 ) = 26.3 , p < .001 ) ; this variation is found in haleb ( h(3 ) = 18.7 , p < .001 ) and in bangkok , nairobi , luxor , and arusha combined ( h(3 ) = 14.2 , p = .003 ) , but not in sheffield ( h(3 ) = 8.80 , p = .032 ) or amman ( h(3 ) = 2.30 , p = .514 ) . with regard to older people observed per phase of day , the largest number of older people was observed between noon and 2 pm ( n = 115 ) , followed by 25 pm ( n = 96 ) and 9 am noon ( n = 63 ) , with fewer older people observed in the early morning ( 79 am , n = 12 ) and early and late evening ( 58 pm , n = 18 ; 811 pm , n = 6 ) . there was significant variation across phases of the day in the number of older men observed per period ( h(5 ) = 15.4 , p = .009 ) , but this association was only true for sheffield ( h(3 ) = 17.1 , p = .001 ) . there was also considerable variation across cities in the proportion of older people observed who were accompanied as opposed to observed ones alone . while nearly half of the older people observed in luxor , nairobi , and sheffield were accompanied , only 15.8 per cent of older people observed in bangkok were accompanied , 14.3 per cent in haleb , and 8.89 per cent in amman . there was a significant association between the accompanied versus alone status of older men and city ( (3 ) = 18.0 , p < .001 ) , with sheffield having by far the highest proportion of accompanied older men ( 42.0% ) . of those observed older people who were accompanied , 84 ( 79.2% ) were accompanied by older people only , while 20 ( 18.9% ) were accompanied by people of other age groups only and two ( 1.89% ) accompanied by mixed age groups of younger and older people . there was a significant association between city status ( developed versus developing ) and whether or not the accompanying person was old ( sheffield 94.6% , developing cities 40.0% , = .66 , p < .001 ) with accompanied older people in sheffield predominantly accompanied by other older people . a total of 1433 pictures were extracted from newspapers . with regard to the images of people whose ages could be reliably determined , 217 older people were observed in total , in comparison to 513 children or babies , 988 middle - aged people , and 2109 young people . there was thus significant variation in the absolute level of presence of the different age groups ( friedman = 798.2(3 ) , p < .001 ) . this variation was significant both for males ( friedman = 336.3(2 ) , p < .001 ) and for females ( friedman = 265.5(2 ) , p < .001 ) , excluding children and babies from the analysis ( gender not recorded ) . in terms of the proportion of pictures examined that contained images of people by age group and gender , 88 ( 6.14% ) pictures depicted older men , 27 ( 1.88% ) depicted older women , 938 ( 65.5% ) depicted non - older men , and 385 ( 26.9% ) depicted non - older women . there was thus significant variation in the proportional presence of the different age groups ( cochrane 's q = 1616.1(3 ) , p < .001 ) . this variation was significant both for males ( mcnemar 's = 747.7(1 ) , p < .001 ) and for females ( mcnemar 's = 317.0(1 ) , p < .001 ) . the proportion of pictures containing older people varied significantly across countries ( see table 3 ) , from 15.3 per cent in kenya to 2.21 per cent in jordan ( (6 ) = 28.7 , p < .001 ) . there was also significant variation by country in the proportion of pictures depicting non - old people ( (6 ) = 39.6 , p < .001 ) . for men , there was significant association between country status and depiction in pictures , both for older men ( developing country 7.01% , uk 1.71% , = .08 , p = .002 ) and for non - older men ( developing country 67.7% , uk 53.8% , = .11 , p < .001 ) . for women , the association between country status and depiction in pictures was non - significant , both for older women ( developing country 1.92% , uk 1.71% , = .06 , p = .830 ) and for non - older women ( developing country 27.3% , uk 24.8% , = .02 , p = .433 ) . we examined the association between picture content ( news or non - news material ) and the gender and age of the person depicted . significant associations were obtained for depiction of an older woman ( news 1.51% , non - news 3.43% , = .06 , p = .028 ) and depiction of a non - old man ( news 65.9% , non - news 52.6% , = .13 , p < .001 ) . content analysis was undertaken of a total of 599 television scenes . with regard to the images of people whose ages could be reliably determined , 106 older people were observed in total , in comparison to 170 children or babies , 370 middle - aged people , and 855 young people . there was thus significant variation in the absolute presence of the different age groups ( friedman = 343.2(3 ) , p < .001 ) . this variation was significant both for males ( friedman = 129.8(2 ) , p < .001 ) and for females ( friedman = 177.3(2 ) , p < .001 ) , excluding children and babies from the analysis ( gender not recorded ) . of the 599 analysed scenes , 47 ( 7.85% ) contained older people , while in comparison , 533 scenes ( 89.0% ) contained non - old people . thirty - four scenes ( 5.68% ) contained older men , while 17 scenes ( 2.84% ) contained older women , and 340 ( 56.8% ) scenes contained non - old men while 252 ( 42.1% ) contained non - old women . there was thus significant variation in the proportional presence of the different age groups ( cochrane 's q = 579.3(3 ) , p < .001 ) . this variation was significant both for males ( mcnemar 's = 261.3(1 ) , p < .001 ) and for females ( mcnemar 's = 211.4(1 ) , p < .001 ) . the proportion of scenes containing older people varied significantly across countries ( see table 4 ) , from 18.0 per cent in egypt to 4.24 per cent in syria ( (4 ) = 12.0 , p = .017 ) . there was also significant variation by country in the proportion of pictures depicting non - old people ( (4 ) = 18.3 , p = .001 ) . due to the small number of scenes containing older women , developing countries were considered as a whole before examining the association between country status and depiction of people in scenes by age and gender . there were no significant associations between country status and the frequency with which old and non - old people , either males or females , were depicted in scenes . there was no significant association between presence of older people in scenes and the period of day in which the scenes were broadcast , and there is no significant association between programme type and presence of older people . our data , drawn from the structured observations of public social spaces and from the content analysis of images in newspapers and on television channels , is mutually reinforcing : data from all sources indicated a low presence of older people in public fora . overall , this low presence was particularly true for the developed country sites relative to the developed country site in our study , with women additionally considerably less present than men in developing countries . these findings support the other work that suggests that older people are infrequently visible in social spaces ( e.g. , [ 1315 ] ) and our data echo the claims of a uk government report that older people are excluded from neighborhoods and leisure activities . there is comparability also with research on media in developed countries that shows older people being underrepresented in advertisements and television ( e.g. , [ 1820 , 22 , 23 , 27 , 29 ] ) . one would expect a lower presence of older people in cities in developing countries due to the lower proportions of older people in these countries compared to developed countries . however , when the median number of older people per observation period is considered as the benchmark , sheffield had a lower median of older people observed in social spaces than amman . thus , older people were generally absent from social spaces in both sheffield and the developing country cities , but only for sheffield there were a small number of periods when older people were frequently observed . social spaces were not evenly used by the older people observed , with most older people observed in markets , transport hubs , and post offices . the fact that so few older people were seen in locations more associated with leisure , such as cafes and hotels , is interesting . research in western countries indicates that economic and health factors impact older people 's leisure activities , while being older , being female , and having a limited social network combined with poor finances and poor health reduce participation in activities . while finances may not influence use of parks , poor weather conditions may impact more substantially on the use of open spaces by older , frailer users . it is also interesting that few older people were observed in religious buildings , as churches have been identified as places that are important to older people . older people in our sample of developing country cities , in comparison to those in sheffield , are less likely to be accompanied when in public . leaving aside the very low absolute level of presence of older people in the developing country cities sampled , the finding that older people in these cities are relatively unaccompanied when observed is intriguing . being unaccompanied may indicate that an older person has a low availability of family relationships , which would place that person at higher risk of poor health , difficulty in accessing social care , and mistreatment and abuse [ 3337 , 54 ] . however , being unaccompanied in public may also be interpreted as a sign of social independence and a high level of functioning no support is sought or needed , rather than being needed and unavailable . where public social interaction did occur in our study , it was more intergenerational in our sampled developing cities than in sheffield . this suggests a degree of social segregation by age group in sheffield . an apparent reliance on age peers for public social activity could place older people at risk of a reduced social network with advancing age . older people in sheffield were significantly more often observed around the middle of the day than in the morning , late afternoon , or evening , but this variation was not present in our sampled developing cities . the relative absence of older people at the beginning and end of the day might be due to older people 's fear of the dark and of crime , as identified in previous research in developed countries ( e.g. , [ 56 , 57 ] ; see also , who discuss fear of crime as a domain of social exclusion ) . taken overall , the data drawn from observation of social spaces are suggestive of segregation of older people from other age groups ( see [ 59 , 60 ] ) , a segregation related to place , time , and companionship . when considering older women , 94.4 per cent of those observed this confounding of gender with developing versus developed country city status prevented a more nuanced examination of how gender influences women 's visibility in social spaces when comparing developed and developing contexts . however , this extremely low level of visibility of older women in our sampled developing country cities does reflect the predominant finding of gender inequalities in such countries ( e.g. , [ 6163 ] ) . for example , lloyd - sherlock argues that the relative deprivation of older women covers a wide range of aspects , such as entitlements to social policies , increased burdens of care and domestic responsibility , greater likelihood of living alone , and sometimes also discrimination against widows . research also suggests that women tend to report greater fear of crime than men and that consequences of such fear in terms of the limitation of activities are particularly serious for women and older people . the finding that older men were significantly more likely to be depicted in newspapers from developing countries in comparison to those from the uk , especially when taking population composition into account , suggests less media discrimination against older men in developing countries than in the uk . previous research on the representation of older people in the media supports our finding that older women were less likely to be present than older men [ 1820 , 29 ] . our finding that younger men were more likely to be depicted in news stories in newspapers and older women more likely to be depicted in non - news items could be seen as a double discrimination of age and gender and points again to older women having very low status within the media , if one accepts the argument that the news underrepresents people of lower status and that this is a gendered imbalance ( see also [ 42 , 66 ] ) . this finding could also be interpreted in the light of news value theory , in which it is argued that certain factors are associated with events that become news . most of the news factors identified tend to be characteristics of the new event itself rather than characteristics of those individuals involved in the events . however , within news value theory it is argued that an event is more likely to be rendered newsworthy if it involves people belonging to elite groups , who are likely to possess a substantial degree of fame and thereby attract audiences . the low presence of older people , particularly older women , in the media images in our study could thus be indicative of their exclusion from elite groups in society , as well as ( or alternatively ) their exclusion from newsworthy events . either interpretation connects with an argument that older people and older women in particularly are an excluded social group . there are no other directly comparable studies and extremely few of any kind concerned with social integration and exclusion of older people that present data from several countries with differing cultural , political , demographic , and socioeconomical profiles . as this is an exploratory study , it is essential that there is transparency about its methodological successes and limitations so that future research can improve upon this work . many factors will have influenced the validity and reliability of the data collected and it is important to review these factors . as mentioned in section 2 , aspects of the sampling frame had to be altered from those originally planned . such adjustments , made due to local factors in different countries , will have affected the comparability of the data across sites . the choice of fieldwork countries , we have previously argued , reflected a diversity of geographic , ethnic , political , and demographic structures . pragmatic considerations , such as the resources available to the study , also influenced the selection process , and there is clearly an argument that a different selection of countries ( and indeed the selection of different cities within countries ) could have produced a different set of findings . similarly , the selection of social spaces within cities will have produced a unique set of findings a different set of locations will have presented a different data profile . nevertheless , the site selection was arrived at following careful deliberation about the meaning of places and their purpose in the lives of individuals and society and represents a sample of locations that are tightly defined both geographically and functionally . as much as any sampling of social spaces drawn from hugely different cultures , we would suggest that there is good reason to think we have attained a reasonable degree of comparability across sites and a robust reflection of the life of a city and its peoples . the resources available to the study also required that we emphasize those countries where less research has been carried out the developing countries . sheffield , as the single site for data collection in developed countries , should be thought of primarily as a comparator for the data collected from the developing country sites . there is , then , a need for reflection on the extent to which a relatively small number of observations of a few different social spaces , newspapers , and television channels can be interpreted in relation to social integration and exclusion processes on a global scale . furthermore , while our selection of several different developing countries might have offered an opportunity for disaggregating the effects of ethnicity , religion , and political ideologies on the presence of older people , the small number of older people observed via the three data sources militated against any sophisticated unravelling of such effects , and in many cases in our analysis the data collected from the developing country locations had to be combined . while acknowledging that carrying out observations in a different set of places within the same cities may well have produced a different data profile , a strength of the study is its capacity to triangulate the data from the three different sources of social spaces , newspaper pictures , and television . the first data source is indeed geographically very tightly defined and therefore highly suspect with regard to any claim for representation of the population from which it is selected . however , the second two data sources are national / regional , not local , and therefore can be argued to be reasonably robust with regard to how they reflect the cultures from which they are drawn . the information that emerges from all three data sources is mutually reinforcing such that the credibility of the data profile drawn from observation of social spaces is strengthened due to its similarity to those drawn from newspapers and television . our researcher will have approached the data collection process with conscious and unconscious assumptions and beliefs , which will have been embedded within that researcher 's own cultural framework . this cultural anchoring will have affected the data collection process across the different cultures in which it occurred in fundamental and unquantifiable ways . for our study , this approach was decided upon partly out of financial necessity and partly to ensure consistency of observations across all sites / countries . the researcher was well prepared through briefing , training , and piloting , and the use of a highly structured observation protocol will have maximized reliability during data collection . however , traditional indices of reliability available when more than one researcher is used during data collection ( e.g. , interrater reliability ) can not be determined for our study ; for further discussion on reliability and validity in observational studies , see . a further limitation arising from our use of a single researcher was that during the observations of social spaces , while the absolute number , gender , and accompanied status of older people were recorded , it was not possible to record the presence of non - old people , or the specific behaviours of the older people observed , as there would have been too many data events to record . thus , the structured observation method was not used to its full capacity , as has been the case in , for example , research regarding residential care facilities . for all observations , anyone appearing over the age of 60 was recorded as an older person , and where there was any uncertainty the person was excluded from the older person category . estimates of age were further affected by the distance in observations used to limit the impact of the observer on the observed for social spaces data collection , the researcher 's relative unfamiliarity with people from certain ethnic groups , and clothing that obscured faces . thus , age estimation might be anticipated to be more reliable in the uk than in some of the developing country fieldwork sites . a further issue is whether the age of 60 was a suitable age at which to draw the boundary of old age , given the discrepancy in life expectancy across the sampled countries noted in section 2 . as noted previously , what may at a first glance seem like an absence of older people in developing countries can to some extent be explained by the low proportion of older people in the population , although this does not satisfactorily explain the relative absence of older women compared to older men in public fora . there is an argument that a different boundary for old age should have been set for each country , to reflect variation in life expectancy . alternatively , a retrospective weighting could have been applied to adjust for life expectancy in the data analysis . we decided that it was preferable to avoid such data transformation and rather present the data as collected but placed within a detailed interpretative context to enable the reader to reach his / her own conclusions . overall , despite the challenges encountered in this study , it has produced interesting and original findings , which suggest that observational methods and content analysis have utility as ways of exploring social integration and inclusion of older people in both developed and developing countries . while again emphasizing the exploratory nature of our study , there are still broad implications of our findings for policy around older people . firstly , a lack of presence of older people in public fora implies a lack of representation also in other areas of society such as policy and planning . there is awareness that more engagement is needed with older people in policy and planning , and there have been efforts to achieve this [ 71 , 72 ] . it should be recognised that a lack of public presence of older people does not preclude a high status for older people in non - public fora or high levels of inclusion in non - public social networks , and this point might be especially pertinent in non - western and traditional cultures . the role and status of the older person in family networks , for example , can not be discerned through our data but will vary in nature and significance from culture to culture . similarly , our study has little insight to offer on the place of older people in rural communities ( cf . ) . nevertheless , we would reinforce the notion that a lack of visibility of older people , a public absence of those in later life , has been identified as a form of exclusion and has consequences for how a society configures and represents itself , which in turn will have ramifications for how the role of older people is constructed . furthermore , this study shows that the exclusion of older people from public fora is not a uniform experience . the emphasis here is that women are more invisible than men , and women in our sample of developing country cities are especially invisible . this suggests exclusion not only by age but also by gender and finally by location , with its variations in demographic , socioeconomic , and cultural factors . if policy needs to address the absence of older people from public spheres , such policy must focus especially on the exclusion of older women . there may be some concern that moving from a demonstration of the public presence or absence of a social group to the inference that such a group is socially excluded is too great leap . it may be unwise to apply a single model of social exclusion in vastly different cultural contexts without adaption to the local setting ( see ) . nevertheless , if we accept the united nation definition of social exclusion as the involuntary exclusion of individuals and groups from society 's political , economic , and societal processes , which prevents their full participation in the society in which they live , then a relative absence from public fora must suggest at the very least a vulnerability to social exclusion , if not actual exclusion itself . we would offer this exploratory study as a starting point for reflection on how best social integration and inclusion can be measured and explored , particularly in locations such as developing countries that are underresearched and which differ in so many respects from the more usual western sites of social research .

there is a lack of research on the everyday lives of older people in developing countries . this exploratory study used structured observation and content analysis to examine the presence of older people in public fora and considered the methods ' potential for understanding older people 's social integration and inclusion . structured observation occurred of public social spaces in six cities each located in a different developing country and in one city in the united kingdom , together with content analysis of the presence of people in newspaper pictures and on television in the selected countries . results indicated that across all fieldwork sites and data sources , there was a low presence of older people , with women considerably less present than men in developing countries . there was variation across fieldwork sites in older people 's presence by place and time of day and in their accompanied status . the presence of older people in images drawn from newspapers was associated with the news / non - news nature of the source . the utility of the study 's methodological approach is considered , as is the degree to which the presence of older people in public fora might relate to social integration and inclusion in different cultural contexts .

1. Introduction 2. Method 3. Results 4. Discussion

by 2050 , there will be approximately 2 billion people aged 60 years or more and the number of older people will outnumber the young . our capacity to understand how demographic changes in developing countries will impact on older people 's lives is limited by the relative lack of research on the everyday lives of older people in such countries . in this paper , we describe an exploratory study that examines the public presence of older people , with consideration of differences between women and men . this is done via the structured observation of social spaces in selected cities in six developing countries and one city in a developed county and content analysis of examples of the countries ' respective print and broadcast media , specifically images in newspapers and on television channels . the potential of such data for understanding the social integration and inclusion of older people is discussed . when proposing a need for more social research on older people in developing countries , the methodological approach to be adopted requires particularly careful consideration . observational methods and content analysis might circumvent some of the problems that arise in using conventional research methods in the context of developing countries . it can be argued that the actual presence or absence of older people in public fora , such as social and cultural spaces , is a good marker of their level of social integration ( cf . ) the use of social spaces can influence societal attitudes to different groups and the degree to which they belong in society . mass media are a force for shaping attitudes and images of older people in media shape perceptions and self - perceptions of ageing [ 811 ] . thus , how far older people are visible in such cultural spaces and the nature of their presentation are both reflections and drivers of social norms , becoming both measures of social integration and factors that affect social integration . studies that document the presence or absence of older people in public fora might therefore provide valuable data that will contribute to our understanding of the place of older people in their respective societies . for example , londoners identified a religious building as an important place in their lives and different ethnic groups identified varying benefits in using parks in the united states of america ( us ) , while older people in hong kong found that poor health , lack of time , and issues with other users reduced their use of parks , despite their health and social and psychological benefits (; cf . ) with regard to the presence and form of presentation of older people in broadcast and print media there is a considerable body of work available , but there is a knowledge gap around images of older people in non - western cultures with cross - cultural research extremely limited . in the available research from developed countries , there is a consensus that older people have been and continue to be less present than expected with regard to their prevalence in the population . similarly , us and german studies have found an underrepresentation of older people in television and printed advertisements , respectively . content analysis of print media in one area in the usa found that stories that could be illustrated by pictures of people of any age tended to feature younger people . older people were also underrepresented in the united kingdom ( uk ) magazine advertisements , canadian television advertisement , and irish newspapers [ 2527 ] . although there are positive images on older people in aspirational advertising ( for a discussion , see ) , when present in media older people are frequently constructed as victims and being frail , vulnerable , and so forth , not the least when mentioned in the context of population ageing . considering developing or non - western countries , a study on the presentation of older people on taiwanese television produced findings similar to those obtained in the usa , while studies looking at the presentation of older people in the media in india and japan found that there was an underrepresentation of older people , particularly of older women , in magazine and television announcements , respectively [ 30 , 31 ] . when studying older people 's presence in public fora , it is important to consider the differing life situations of older women and men in developing and developed countries . our primary research question is : how present are older people in public fora in developing countries ? a secondary research question is : is the presence of older people in public fora related to their gender ? our site selection , of six cities in six developing countries and one city in a developed country , and the use of two data collection methods were motivated by several considerations . first , as this was an exploratory study it was important to examine the validity , reliability , and feasibility of the methods adopted across a wide range of settings . the selection of a range of developing countries fulfilled this consideration , as such countries would provide challenges and contexts as fieldwork sites that would contrast with those found in the developed country ; there would also likely be differences between the selected developing countries themselves . second , there is a tremendous gap between our understanding of the demographic changes affecting older people in the developing world and our understanding of the public lives of older people in such countries . our study , with its primary focus on developing countries , would thus both increase the amount of data available on the presence of older women and men in social spaces and in the media in such countries and offer some insight into how their presence in public fora in developing countries contrasts with their presence in similar fora in a developed country , with opportunity for cautious comparison due to the use of standardised methods across fieldwork sites . finally , the use of three different data sources non - participant structured observation in social spaces and content analysis of images of older people both in newspapers and on television would allow for triangulation of data as a way of evaluating the robustness of findings . this study involved gathering data in six developing countries syria , jordan , egypt , thailand , kenya , and tanzania and in one western country the uk over the course of 2007 . in each of these countries , structured observations were carried out in public social places where people were likely to be present and content analysis was performed of images in newspapers and television programmes in which they were likely to be depicted . the chosen developing countries offer diversity of cultures , with varying degrees of contrast and comparison with the selected site in the uk . the selected countries represent , respectively , the overwhelmingly arabic muslim syria and jordan and hamitic muslim egypt ; the multiplicity of ethnic groups in black east africa , with a predominantly christian culture and minority muslim culture in kenya , and strong christian and muslim cultures in tanzania ; and the distinctive , largely buddhist thai culture of the indian subcontinent , with its chinese and malay subpopulations . a time sampling frame for recording of data from observations was developed based on previous work on the observation of older people in residential care environments [ 50 , 51 ] and adapted through piloting . in luxor and arusha , a main bus stop was selected as the bus station was out of town ; in bangkok the train station was used , as the bus station was in a different area ; in bangkok , nairobi , and arusha there were no parks within the area observed so this part of the observation schedule could not be carried out . media content analysis was undertaken of newspaper pictures and television channels , as detailed in table 1 . because our study focuses on the visibility of older people , we selected newspapers and television as mass media genres containing a significant component of visual information ( as opposed to , say , literature or radio ) . the page number and section were noted , together with the headline for english papers and a brief description for other language papers , except when the picture contained only a face with no indication of context . the sections in which the picture appeared were used as base categories for the analysis and finally combined into two superordinate categories : news and non - news . there was significant variation across cities in the number of older men observed per period ( h(6 ) = 38.3 , p < .001 ) . there was significant variation across social spaces in the number of older men observed per period ( h(6 ) = 26.3 , p < .001 ) ; this variation is found in haleb ( h(3 ) = 18.7 , p < .001 ) and in bangkok , nairobi , luxor , and arusha combined ( h(3 ) = 14.2 , p = .003 ) , but not in sheffield ( h(3 ) = 8.80 , p = .032 ) or amman ( h(3 ) = 2.30 , p = .514 ) . with regard to older people observed per phase of day , the largest number of older people was observed between noon and 2 pm ( n = 115 ) , followed by 25 pm ( n = 96 ) and 9 am noon ( n = 63 ) , with fewer older people observed in the early morning ( 79 am , n = 12 ) and early and late evening ( 58 pm , n = 18 ; 811 pm , n = 6 ) . there was significant variation across phases of the day in the number of older men observed per period ( h(5 ) = 15.4 , p = .009 ) , but this association was only true for sheffield ( h(3 ) = 17.1 , p = .001 ) . there was also considerable variation across cities in the proportion of older people observed who were accompanied as opposed to observed ones alone . while nearly half of the older people observed in luxor , nairobi , and sheffield were accompanied , only 15.8 per cent of older people observed in bangkok were accompanied , 14.3 per cent in haleb , and 8.89 per cent in amman . there was a significant association between the accompanied versus alone status of older men and city ( (3 ) = 18.0 , p < .001 ) , with sheffield having by far the highest proportion of accompanied older men ( 42.0% ) . there was a significant association between city status ( developed versus developing ) and whether or not the accompanying person was old ( sheffield 94.6% , developing cities 40.0% , = .66 , p < .001 ) with accompanied older people in sheffield predominantly accompanied by other older people . with regard to the images of people whose ages could be reliably determined , 217 older people were observed in total , in comparison to 513 children or babies , 988 middle - aged people , and 2109 young people . there was thus significant variation in the absolute level of presence of the different age groups ( friedman = 798.2(3 ) , p < .001 ) . there was thus significant variation in the proportional presence of the different age groups ( cochrane 's q = 1616.1(3 ) , p < .001 ) . there was also significant variation by country in the proportion of pictures depicting non - old people ( (6 ) = 39.6 , p < .001 ) . for men , there was significant association between country status and depiction in pictures , both for older men ( developing country 7.01% , uk 1.71% , = .08 , p = .002 ) and for non - older men ( developing country 67.7% , uk 53.8% , = .11 , p < .001 ) . there was thus significant variation in the absolute presence of the different age groups ( friedman = 343.2(3 ) , p < .001 ) . of the 599 analysed scenes , 47 ( 7.85% ) contained older people , while in comparison , 533 scenes ( 89.0% ) contained non - old people . there was thus significant variation in the proportional presence of the different age groups ( cochrane 's q = 579.3(3 ) , p < .001 ) . due to the small number of scenes containing older women , developing countries were considered as a whole before examining the association between country status and depiction of people in scenes by age and gender . there was no significant association between presence of older people in scenes and the period of day in which the scenes were broadcast , and there is no significant association between programme type and presence of older people . our data , drawn from the structured observations of public social spaces and from the content analysis of images in newspapers and on television channels , is mutually reinforcing : data from all sources indicated a low presence of older people in public fora . overall , this low presence was particularly true for the developed country sites relative to the developed country site in our study , with women additionally considerably less present than men in developing countries . there is comparability also with research on media in developed countries that shows older people being underrepresented in advertisements and television ( e.g. one would expect a lower presence of older people in cities in developing countries due to the lower proportions of older people in these countries compared to developed countries . however , when the median number of older people per observation period is considered as the benchmark , sheffield had a lower median of older people observed in social spaces than amman . thus , older people were generally absent from social spaces in both sheffield and the developing country cities , but only for sheffield there were a small number of periods when older people were frequently observed . social spaces were not evenly used by the older people observed , with most older people observed in markets , transport hubs , and post offices . older people in our sample of developing country cities , in comparison to those in sheffield , are less likely to be accompanied when in public . leaving aside the very low absolute level of presence of older people in the developing country cities sampled , the finding that older people in these cities are relatively unaccompanied when observed is intriguing . older people in sheffield were significantly more often observed around the middle of the day than in the morning , late afternoon , or evening , but this variation was not present in our sampled developing cities . the relative absence of older people at the beginning and end of the day might be due to older people 's fear of the dark and of crime , as identified in previous research in developed countries ( e.g. taken overall , the data drawn from observation of social spaces are suggestive of segregation of older people from other age groups ( see [ 59 , 60 ] ) , a segregation related to place , time , and companionship . the finding that older men were significantly more likely to be depicted in newspapers from developing countries in comparison to those from the uk , especially when taking population composition into account , suggests less media discrimination against older men in developing countries than in the uk . previous research on the representation of older people in the media supports our finding that older women were less likely to be present than older men [ 1820 , 29 ] . our finding that younger men were more likely to be depicted in news stories in newspapers and older women more likely to be depicted in non - news items could be seen as a double discrimination of age and gender and points again to older women having very low status within the media , if one accepts the argument that the news underrepresents people of lower status and that this is a gendered imbalance ( see also [ 42 , 66 ] ) . the low presence of older people , particularly older women , in the media images in our study could thus be indicative of their exclusion from elite groups in society , as well as ( or alternatively ) their exclusion from newsworthy events . there are no other directly comparable studies and extremely few of any kind concerned with social integration and exclusion of older people that present data from several countries with differing cultural , political , demographic , and socioeconomical profiles . pragmatic considerations , such as the resources available to the study , also influenced the selection process , and there is clearly an argument that a different selection of countries ( and indeed the selection of different cities within countries ) could have produced a different set of findings . as much as any sampling of social spaces drawn from hugely different cultures , we would suggest that there is good reason to think we have attained a reasonable degree of comparability across sites and a robust reflection of the life of a city and its peoples . there is , then , a need for reflection on the extent to which a relatively small number of observations of a few different social spaces , newspapers , and television channels can be interpreted in relation to social integration and exclusion processes on a global scale . furthermore , while our selection of several different developing countries might have offered an opportunity for disaggregating the effects of ethnicity , religion , and political ideologies on the presence of older people , the small number of older people observed via the three data sources militated against any sophisticated unravelling of such effects , and in many cases in our analysis the data collected from the developing country locations had to be combined . while acknowledging that carrying out observations in a different set of places within the same cities may well have produced a different data profile , a strength of the study is its capacity to triangulate the data from the three different sources of social spaces , newspaper pictures , and television . the information that emerges from all three data sources is mutually reinforcing such that the credibility of the data profile drawn from observation of social spaces is strengthened due to its similarity to those drawn from newspapers and television . a further limitation arising from our use of a single researcher was that during the observations of social spaces , while the absolute number , gender , and accompanied status of older people were recorded , it was not possible to record the presence of non - old people , or the specific behaviours of the older people observed , as there would have been too many data events to record . estimates of age were further affected by the distance in observations used to limit the impact of the observer on the observed for social spaces data collection , the researcher 's relative unfamiliarity with people from certain ethnic groups , and clothing that obscured faces . thus , age estimation might be anticipated to be more reliable in the uk than in some of the developing country fieldwork sites . as noted previously , what may at a first glance seem like an absence of older people in developing countries can to some extent be explained by the low proportion of older people in the population , although this does not satisfactorily explain the relative absence of older women compared to older men in public fora . overall , despite the challenges encountered in this study , it has produced interesting and original findings , which suggest that observational methods and content analysis have utility as ways of exploring social integration and inclusion of older people in both developed and developing countries . while again emphasizing the exploratory nature of our study , there are still broad implications of our findings for policy around older people . firstly , a lack of presence of older people in public fora implies a lack of representation also in other areas of society such as policy and planning . there is awareness that more engagement is needed with older people in policy and planning , and there have been efforts to achieve this [ 71 , 72 ] . it should be recognised that a lack of public presence of older people does not preclude a high status for older people in non - public fora or high levels of inclusion in non - public social networks , and this point might be especially pertinent in non - western and traditional cultures . similarly , our study has little insight to offer on the place of older people in rural communities ( cf . ) nevertheless , we would reinforce the notion that a lack of visibility of older people , a public absence of those in later life , has been identified as a form of exclusion and has consequences for how a society configures and represents itself , which in turn will have ramifications for how the role of older people is constructed . furthermore , this study shows that the exclusion of older people from public fora is not a uniform experience . nevertheless , if we accept the united nation definition of social exclusion as the involuntary exclusion of individuals and groups from society 's political , economic , and societal processes , which prevents their full participation in the society in which they live , then a relative absence from public fora must suggest at the very least a vulnerability to social exclusion , if not actual exclusion itself . we would offer this exploratory study as a starting point for reflection on how best social integration and inclusion can be measured and explored , particularly in locations such as developing countries that are underresearched and which differ in so many respects from the more usual western sites of social research .

having a partner with a serious illness has an enormous impact on family life , and relatives are affected both physically and mentally . relatives living with a critically ill patient will often experience that their life is turned upside down , which can be very stressful . a study on relatives to patients with alzheimer 's disease showed that relatives were more distressed , showed poorer immune function , and had an increase in respiratory tract infections , compared to a control group . the study found that even when the patient died , the relatives continued to show immunological downregulation for several years . family members are , in many cases , the main source of support for a critically ill patient and often hold valuable knowledge about the patient . allowing relatives to be present and play a role in caring for the patient is often comforting for the patient and can improve care planning for the benefit of both patients and the hospital . research has shown that relatives do not always receive the attention they need from health professionals ; the reason for this is often unclear . a study on frail and vulnerable patients indicated that quality of care improved in decision - making and exchange of knowledge was a collaboration between relatives and health professionals . if care is not properly coordinated by the health professionals , the quality of care can be experienced as being inadequate and lead to frustration for patients and their relatives . a canadian study showed that poor satisfaction from patients and relatives was due to health professionals ' inability to coordinate care . the main source of dissatisfaction was rotation between health professionals , difficulties in making decisions , and lack of coordination between health professionals . it has been shown that relatives receiving information , advice , and emotional support are more likely to be satisfied . when caring for critically ill patients , health professionals must be aware of their role and the asymmetry this relationship contains . being in a situation where a patient 's life situation is changing will often lead to changes in the relatives ' situation . relatives will often experience an increase in pressure due to the hospitalization of their loved one , which can leave them with a feeling of hopelessness and despair . hope is essential , because it enables people to cope with difficult situations and life changes . health professionals must be aware of the responsibility they have to influence the feeling of hope . it has been shown that health professionals did not have the knowledge of what relatives go through during the hospitalization of their partners [ 8 , 9 ] . there are often no standardized protocols for including relatives in the care provided by health professionals in a general ward , and there is a great variation in the care offered for relatives within the hospitals . the sooner the health professionals prepare interventions that include relatives in the care and treatment of the patient , the sooner they can prevent a later negative reaction from the relatives . a recent study reported that relatives should be considered important in the treatment and care with patients . this study measured the impact of different levels of social support on patients ' self - care . patients with a high level of social support found that having a supporting partner had a significant influence on several parameters , such as taking medications , managing fluid intake , and consulting health professionals . therefore , it is necessary to be aware of what relatives go through and ideally have a way of monitoring relatives ' experience of quality of care . a recent study reported that relatives should be considered important in the treatment and care with patients . this study measured the impact of different levels of social support on patients ' self - care . patients with a high level of social support found that having a supporting partner had a significant influence on several parameters , such as taking medications , managing fluid intake , and consulting health professionals . the aim of this study was to investigate the relatives ' satisfaction and involvement on a general surgery ward , regarding the critically ill patient . this study was a mixed methods study designed to identify the relatives ' involvement , satisfaction , and needs during hospitalization of a patient that became acutely ill . data were collected in two steps , first a survey and then a study using in - depth interviews . the reason for choosing a mixed method was because of its capacity to strengthen and explore the results found in one research approach compared to the other . in this case , it allowed the quantitative results from the survey to be explained with a qualitative study through in - depth interviews . inclusion criteria were relatives to patients with a deterioration in their illness within the last 48 hours defined as fulfilling one of the following criteria : an early warnings score 7 , having the mobile emergency team called for a consultation or transfer to the intensive care unit or another room for closer observation . exclusion criteria were relatives with psychiatric disorder , language difficulties , or withdrawal of consent . all participants were recruited from the surgical ward between march 1 , 2014 , and july 1 , 2014 . the questionnaire used for the survey was a translated version of the nursing care survey 2002 nosa . the questionnaire is structured so a score can be obtained from each item . for this study , two items were excluded from the questionnaire , because they were regarding discharge , a theme not relevant for this study , since patients were not discharged at the time of data collection . the process of translation was aligned to the recommendations for translation and cultural adaption of surveys . approval to translate the nursing care survey 2002 nosa was obtained from the inventors . a panel of three expert researchers translated each the nursing care survey 2002 nosa from english to danish . when agreement was reached , the survey was translated back to english by a native speaking dane . when the questionnaire was backward translated to english it was sent to the inventor in australia for validation . when the translated questionnaire was sent back from australia with comments , the danish version was corrected and deemed ready for testing . for further validation each participant was interviewed about the meaning of each question in the questionnaire in order to clarify misconceptions . a written conclusion was signed by each participant , after each interview , and used in the further validation process . each item could be responded on a 5-point likert scale with 0 indicating total agreement and 4 indicating total disagreement with the statement ( table 1 ) . a total score for each participant was calculated and converted to a score ranging from 0 to 100 by total score / max score 100 . furthermore , responses were analyzed with the use of cut - off values with the percentages of respondents in the categories always and mostly . the categories were > 80% very good standard , 7080% acceptable standard , and < 70% requiring attention . comparison of total score for interview participants versus noninterview participants was performed with the mann - whitney u test . , the researchers were focusing on a circular motion , where the researcher was concerned with the relatives ' experiences as they were lived . here , the process becomes a dialogical method , where the researcher and the phenomenon being studied are combined together . the in - depth interviews were preferred , as they made participants feel more comfortable to talk about their personal experiences , when they were done face to face as opposed to focus group discussions . this approach makes it more likely for the researcher to get in depth with the themes of a more sensitive character . a semistructured flexible interview guide was developed from the themes in the questionnaire and then combined with findings from conversations with two relatives to patients that had become critically ill . this was done to secure that the responses contributed to a deeper understanding of the items in the questionnaire . the first author performed the interviews , which took between 30 and 45 min . each interview was recorded and then transcribed verbatim by an external contributor and carefully reviewed by jannie laursen and kristoffer andresen . the interviews were transcribed into full text and qualitative content analysis was used for analyzing the data . the two researchers ( jannie laursen , kristoffer andresen ) performed the analysis in parallel processes ; they subsequently discussed subthemes and themes which were then compared and reflected upon in an in - depth process . the study was approved by the danish data protection agency ( journal number 528 - 02971 ) and was exempt from ethical approval from the ethical committee of the capital region in denmark , but a statement of exemption was obtained ( journal number h-2 - 2013-fsp56 ) . relatives who met eligibility criteria were identified by nurses at the surgical ward and asked by the first author whether they would participate in the study . the relatives were informed that they at any time could withdraw from the study , and it would not influence the patient 's treatment or care . before answering the questionnaire and/or participation in the interview , a consent form was signed and the relatives were reassured of the confidentiality both orally and in writing . we approached 50 potential relatives of whom 27 participated in the questionnaire , and six of them participated in in - depth interviews . all participants were family members , including parents , siblings , children , and spouses . reason for declined participation for the 23 was due to stress , fatigue , or the loss of their loved ones . in the questionnaire , median ( range ) score for all participants were 30 ( 070 ) . for seven items out of 10 , less than 70% of the participants indicated that they were always or this is an indication that a third of the relatives were unsatisfied with these items and that they require attention ( see table 1 ) . the highest degree of dissatisfaction was found for items four were you involved in decisions about the care of your relative as much as you felt you needed to be and eight did the health professionals anticipate and meet the needs of your relative , both with only 48% of patients responding always / mostly . in order to further explore these items , the in - depth interviews were conducted . the findings were based on the narratives from six relatives , four women and two men which participated in the in - depths interviews . during the analyses of the data , three themes emerged ; the first one was lack of continuity and structure , the second one was responsibility of coordination , and the third one was relatives left alone with no guiding and support . all relatives were physically involved in the care of the critically ill patient and they were all very emotional and sensitive about the critical situation . all relatives were experiencing feelings such as frustration , anxiety , and a profound struggle to understand the unfortunate situation , which lead to the themes described in the following . this theme was focused on the relatives ' perceptions of lack of continuity and structure . the relatives experienced that when the condition of the patient deteriorated and the situation became severed , the thing they needed the most from the health professionals was structure and some form of continuity in the further plan , one relative explained it as follows:there was a new doctor every day , there were 47 different names to remember , and who should i call ( ? ! ) . all they did was to state her ( the patient ) diagnosis , and send a request for a supervising doctor . i believe we all know what her diagnosis was and that the doctors needed a consultation from another doctor,everything inside the relatives was an emotional chaos and they were clinging to every little thing in the hope of gaining some sense of the situation . when the relatives were not offered reconciliation from the health professionals , it often resulted in confusion and frustration , as a relative noted : my nerves were frayed , the health professionals promised to take care of my husband , to check on him , but that just did n't happen . there was a new doctor every day , there were 47 different names to remember , and who should i call ( ? ! ) . all they did was to state her ( the patient ) diagnosis , and send a request for a supervising doctor . i believe we all know what her diagnosis was and that the doctors needed a consultation from another doctor , my nerves were frayed , the health professionals promised to take care of my husband , to check on him , but that just did n't happen . in line with quantitative findings often relatives felt that the health professionals were not able to create continuity in the care of , or during the admission of , the critically ill patients . the relatives felt that availability of information was poor and always at the request of them . even though they were present and tried to be involved , the health professionals made no effort to include them in any way . this was commented on as follows : the health professionals saw me every day , so they knew who i was , but there was no attempt to include me and they knew that information was very important for us , that i knew what was going on , because my mom did n't always understand what was happening . often the health professionals were vague in their attempts to meet the relatives and often the waiting time felt very long and uncertain , which only made it more urgent for the relatives to gain some kind of information and not have to bear all the responsibility on their own . the relatives felt that they had to take charge on all follow - ups regarding care and treatment ; they felt that there were a lack of leadership and consistency presented by the health professionals . the lack of consistency and follow - up from health professionals often resulted in negative emotional reactions , which lead to some relatives having difficulties pursuing with their daily lives . it was by one relative commented on asit is terrible for you as a spouse , to see your wife lying there , horrible when you ca n't see any progress and you feel the health professionals just talk and talk , but nothing happens . you can only interpret this as carelessness , as they do n't take any responsibility , which leaves you feeling powerless . they experienced that the health professionals were apathetic and indifferent , which often lead to difficulties in managing and coping with the given situation for the relatives:i never received any explanation why they went in a new direction with my husband , which meant a lot of stress for me.consistent with the findings from the quantitative analysis , only 55% of relatives were answered by the health professionals in a way they could understand . this led to uncertainties in the situations , which often led to stress and anxiety for the relatives . often the feeling of not being able to deal with the responsibility and not being offered any support or guiding from the health professionals led to relatives not having time to cope with their own emotions:there was too much focus on coordinating things , making sure that all was running smoothly . if i should n't have done that , i could have been there for her , been more present and less stressed . i was very scared of what was going to happen and what i feared the most did happen . often when relatives did try and manage the increased responsibility and the lack of commitment from the health professionals , they felt that they were not given any authority to do so . the increase in responsibility and the severity of the situation only made them more desperate and resigned . in some cases , that meant that some of the relatives had a strong need to stay in control and take all the responsibility for care and treatment . a relative expressed it as i sit all day in my taxi , thinking , should i call the hospital ' , hoping they had remembered to check on my mother . but then again it would be stupid , that will only signal that i did n't feel comfortable or relied on the system . it is terrible for you as a spouse , to see your wife lying there , horrible when you ca n't see any progress and you feel the health professionals just talk and talk , but nothing happens . you can only interpret this as carelessness , as they do n't take any responsibility , which leaves you feeling powerless . i never received any explanation why they went in a new direction with my husband , which meant a lot of stress for me . there was too much focus on coordinating things , making sure that all was running smoothly . if i should n't have done that , i could have been there for her , been more present and less stressed . i was very scared of what was going to happen and what i feared the most did happen . the division of roles and responsibilities between health professionals and relatives was not clear for them , as described by one relative:it 's not me who has to walk around and organize their work . it seems wrong.relatives felt that they had to be responsible and stay in control over the situation . if they did not , the critically ill patient would not get the correct or proper care . at the same time , the relatives felt anxious and confused and the weight from the responsibility was almost unbearable for them , as one relative indicated : there is no consistency in anything , always swopping around , new doctors , and new nurses . no one takes any responsibility.many relatives felt that they had to be present most of the day , to make sure that the health professionals were offering the critically ill patient the needed care , according to one relative : there were many situations where my mother did not understand what was happening if i was n't thererelatives felt that they had the responsibility to coordinate and secure that things were running smoothly . if they did not take part in caring for the critically ill patient and were physically and mentally present with a constant awareness of the situation , everything would collapse and everything would fall apart ; it was described as follows : i was told that she was hospitalized , but my dad said that i should n't hurry to the hospital , so i came here at one o'clock . i found her in her own dirt , she had urinated in her pants and i was indignant . i am checking that this is not happening again , i have to stay in control . it made me angry and sad.the relatives had to manage care and treatment for the critically ill patient , to make sure that all was done professionally , even though they were not health professionals:if they felt that the situation called for more professionalism than the health professionals delivered , which meant they had to be responsible for it . it was often described asi did n't understand why i should attain work that was not mine . it was hard , that i had to play such a big part in coordinating things.it was clear during the interviews that the responsibility to harmonize , the lack of structure , and the unclear division of roles left the relatives with a feeling of lack of coherence , which often lead them to a feeling of losing control , frustration , and mistrust of the system . there is no consistency in anything , always swopping around , new doctors , and new nurses . there were many situations where my mother did not understand what was happening if i was n't there i was told that she was hospitalized , but my dad said that i should n't hurry to the hospital , so i came here at one o'clock . i found her in her own dirt , she had urinated in her pants and i was indignant . i am checking that this is not happening again , i have to stay in control . it was hard , that i had to play such a big part in coordinating things . many relatives were overwhelmed by the situation , when their loved one became critically ill . they felt that they were being left on their own and at the same time expected to maintain control ; one relative indicated thatthe role of just being relatives was hard to keep - not being able to come for a visit and just give her a hug was hard.in line with quantitative findings , only 48% felt that the health professionals anticipated and met the needs of their relatives . they felt that they had to oversee the work of the health professionals in order to make sure that the patient received sufficient care . they had a direct responsibility for the patient , but at the same time they were torn between trusting the system and experiencing it as collapsing . they simply said , that we have to let certain things go , which for me meant , that i had to take responsibility for my mother 's care . many relatives felt that they had to use their full mental and emotional capacity to maintain some control over the situation . the communication with the health professionals was challenged by the feeling that they did not listen to them and were not taking charge over the situation . the amount of energy the relatives had to mobilize to be in control often exceeded what was possible . the relatives experienced a lack of care or concern from the health professionals , which was perceived as hard and unsatisfying for them . relatives felt isolated in the situation , which left them with a feeling that no one understood what was going on . they felt detached from the situation and they did not feel that the health professionals were interested in them or their resources . relatives were not an integrated part of the care for the critically ill patient and more perceived as someone who toke resources from the health professionals . one relative expressed it as i did n't receive any care as a spouse . i would have liked if they had talked to me , included me in their plans , spent a little time on me , and showed they empathized . another relative stated , the culture is very much them and us ' . the role of just being relatives was hard to keep - not being able to come for a visit and just give her a hug was hard . they simply said , that we have to let certain things go , which for me meant , that i had to take responsibility for my mother 's care . i had to stay in control , because what if they missed something . when health professionals were not able to include and inform the relatives , it left the relatives feeling powerless . often relatives felt they had to be the coordinator and secure that everything was running smoothly . in this study , the aim was to investigate the relatives ' satisfaction and involvement on a general surgery ward regarding the critically ill patient . the study investigated the level of information , the involvement of the relatives in the patient care , and the relatives ' perspectives of quality of care , regarding the patients ' needs , and health professionals ' communication . the questionnaire results showed that a third of the participants did not seem satisfied with the quality of care . the qualitative results showed that in critical situations health professionals need to be present , caring , and empathic . this was not the general experience by the relatives as revealed by the responses to the questionnaire . often they felt they were on their own , not skilled to cope with the situation , and in need of the health professional that could lead care and treatment and at the same time include them in the process . the use of satisfaction as a tool to measure quality of care has previously been discussed although it is a common and often used measurement [ 1921 ] . earlier studies using satisfaction as a measurement found that being a woman , having a high educational level , or having a health professional 's background often was associated with a decrease in satisfaction [ 4 , 21 ] . however , these uncertainties were taken into account in the in - depth interviews , so that participants were of both sexes , there was a mixture of educational levels , and there were no health professionals among the relatives . a study on collaboration between relatives of elderly patients and nurses stated that relatives that were new in the role as relatives could also be more critical towards the healthcare system than others . this is of course a factor to consider , as in this study there were relatives that were both new in the role and more experienced . a third of the relatives stated that they were dissatisfied with the health professionals on the surgical ward . they were especially dissatisfied with the organization of the care and the involvement about the care . these results were consistent with other studies , showing that dissatisfaction often was associated with how the relatives were informed and was often associated with severity of the patient 's illness [ 4 , 22 ] . if patients were managed on a special unit and not a general ward , it was often seen that it had a positive influence on the level of satisfaction [ 4 , 11 , 22 ] . this study showed that relatives felt there were no structures during the admission and often they felt that they were left in the dark , with no ability to obtain information and with no consistency . these findings align with previous research , where relatives experienced both system and patient frustration [ 5 , 6 ] . the frustration was often due to poor communication and long waiting time especially for feedback but also decisions about care . the study found that relatives often felt that the health professionals did not care and that they were met with negligence [ 5 , 9 ] . these findings add to the evidence that relatives want to participate in decision - making and be treated as collaborators when the patient becomes critically ill . many of the challenges identified by the relatives were interrelated and often exacerbated each other . for example , a poor understanding on who was responsible for what led to a feeling of lack in feedback which increased the uncertainty further . frustration with managing the proper care was aggravated by the health professionals ' inability to coordinate and take charge and thereby not providing the appropriate comfort and security for the relatives . better communication and improving the management of decision - taking were a key item to be able to guide the relatives in a more satisfactory way . a study on frustration expressed by relatives stated that they wanted face to face consultation , but because this is not always possible an alternative could be to use an electronic consulting system as a supplement to the face to face communication . , the relatives would be considered as important collaborators to the critically ill patient , providing relevant and significant information and at the same time maintaining energy to be supporting and caring for the critically ill patient . a study on spouses ' needs for professional support showed that many spouses felt that the health professionals were annoyed by their presence and that the spouses often were met by indifference . when people experienced lack of coherence due to a life changing event , it was often shown in difficulties in managing and coping with even the smallest things . a hospital culture that provides support and consistence will often lead to a more manageable situation . if the culture values the role of people , it will be perceived as more sustaining and leave a feeling of meaningfulness , which can help support relatives in a more manageable way . relatives can be perceived as both resources and potential clients and the stress they experience can affect their health on a long term . being a relative to a critically ill patient will often lead to the loss of control . the feeling of loss of control will often collide with the need to see the world in a clear , ordered , and structured way : there is a need to also study the perception of care for relatives from the health professionals ' point of view . there is probably a range of reasons why the health professionals do not provide the care for relatives that they so desperately need , and it is important to characterize the health professionals ' experience in order to make meaningful interventions for the improvement of the care for relatives . however , it was not within the scope of this study to also cover the experience of the health professionals . there is a need to also study the perception of care for relatives from the health professionals ' point of view . there is probably a range of reasons why the health professionals do not provide the care for relatives that they so desperately need , and it is important to characterize the health professionals ' experience in order to make meaningful interventions for the improvement of the care for relatives . however , it was not within the scope of this study to also cover the experience of the health professionals . combining results from two different research approaches may be a challenge . however , by interviewing six participants in the in - depth interviews , we exemplified what the relatives dissatisfaction covered , which can lead to new interventions towards including relatives in general wards . the reason for this was that very little research has been done in the field of a general surgical ward with patients that suddenly became acutely ill . these patients ' relatives take a lot of focus due to the fact that their situation unexpectedly changes . a third of the participants in this study were unsatisfied with the organization and the involvement in care . the findings implied that they were left with no guiding or support from the health professionals , which often led to the feeling of loss of control . thereby , relatives can gain a sense of coherence during the hospitalization of a critically ill patient , which can lead to a greater satisfaction and thereby better support for the patient .

aims and objective . to investigate the relatives ' satisfaction and involvement on a general surgery ward regarding the critically ill patient . introduction . relatives to critically ill patients are affected both physically and mentally during the hospitalization of a family member . research has shown that relatives do not always receive the attention they need from health professionals . there is a lack of studies that focus on relatives ' satisfaction and involvement during their family members ' hospitalization . design . a mixed methods design was chosen . methods . a quantitative study was conducted with 27 relatives to critically ill patients . all participated in a questionnaire and out of the 27 relatives , six participated in qualitative in - depth interviews . results . the questionnaire revealed that relatives were dissatisfied with care and involvement . for further exploration of the dissatisfaction , a qualitative approach was used and the in - depth interviews revealed three themes : lack of continuity and structure , responsibility of coordination , and relatives feeling left on their own with no guiding and support . conclusion . health professionals ' key role in relation to relatives must be guidance and support . thereby , relatives can gain a sense of coherence during the hospitalization of a critically ill patient , which can lead to a greater satisfaction and thereby better support for the patient .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Strengths and Limitations of the Study 6. Conclusion

having a partner with a serious illness has an enormous impact on family life , and relatives are affected both physically and mentally . relatives living with a critically ill patient will often experience that their life is turned upside down , which can be very stressful . a study on relatives to patients with alzheimer 's disease showed that relatives were more distressed , showed poorer immune function , and had an increase in respiratory tract infections , compared to a control group . the study found that even when the patient died , the relatives continued to show immunological downregulation for several years . family members are , in many cases , the main source of support for a critically ill patient and often hold valuable knowledge about the patient . allowing relatives to be present and play a role in caring for the patient is often comforting for the patient and can improve care planning for the benefit of both patients and the hospital . research has shown that relatives do not always receive the attention they need from health professionals ; the reason for this is often unclear . a study on frail and vulnerable patients indicated that quality of care improved in decision - making and exchange of knowledge was a collaboration between relatives and health professionals . if care is not properly coordinated by the health professionals , the quality of care can be experienced as being inadequate and lead to frustration for patients and their relatives . a canadian study showed that poor satisfaction from patients and relatives was due to health professionals ' inability to coordinate care . the main source of dissatisfaction was rotation between health professionals , difficulties in making decisions , and lack of coordination between health professionals . it has been shown that relatives receiving information , advice , and emotional support are more likely to be satisfied . when caring for critically ill patients , health professionals must be aware of their role and the asymmetry this relationship contains . being in a situation where a patient 's life situation is changing will often lead to changes in the relatives ' situation . relatives will often experience an increase in pressure due to the hospitalization of their loved one , which can leave them with a feeling of hopelessness and despair . health professionals must be aware of the responsibility they have to influence the feeling of hope . it has been shown that health professionals did not have the knowledge of what relatives go through during the hospitalization of their partners [ 8 , 9 ] . there are often no standardized protocols for including relatives in the care provided by health professionals in a general ward , and there is a great variation in the care offered for relatives within the hospitals . the sooner the health professionals prepare interventions that include relatives in the care and treatment of the patient , the sooner they can prevent a later negative reaction from the relatives . patients with a high level of social support found that having a supporting partner had a significant influence on several parameters , such as taking medications , managing fluid intake , and consulting health professionals . a recent study reported that relatives should be considered important in the treatment and care with patients . patients with a high level of social support found that having a supporting partner had a significant influence on several parameters , such as taking medications , managing fluid intake , and consulting health professionals . the aim of this study was to investigate the relatives ' satisfaction and involvement on a general surgery ward , regarding the critically ill patient . this study was a mixed methods study designed to identify the relatives ' involvement , satisfaction , and needs during hospitalization of a patient that became acutely ill . data were collected in two steps , first a survey and then a study using in - depth interviews . in this case , it allowed the quantitative results from the survey to be explained with a qualitative study through in - depth interviews . inclusion criteria were relatives to patients with a deterioration in their illness within the last 48 hours defined as fulfilling one of the following criteria : an early warnings score 7 , having the mobile emergency team called for a consultation or transfer to the intensive care unit or another room for closer observation . all participants were recruited from the surgical ward between march 1 , 2014 , and july 1 , 2014 . the questionnaire used for the survey was a translated version of the nursing care survey 2002 nosa . the questionnaire is structured so a score can be obtained from each item . for this study , two items were excluded from the questionnaire , because they were regarding discharge , a theme not relevant for this study , since patients were not discharged at the time of data collection . when the questionnaire was backward translated to english it was sent to the inventor in australia for validation . for further validation each participant was interviewed about the meaning of each question in the questionnaire in order to clarify misconceptions . a written conclusion was signed by each participant , after each interview , and used in the further validation process . each item could be responded on a 5-point likert scale with 0 indicating total agreement and 4 indicating total disagreement with the statement ( table 1 ) . , the researchers were focusing on a circular motion , where the researcher was concerned with the relatives ' experiences as they were lived . the in - depth interviews were preferred , as they made participants feel more comfortable to talk about their personal experiences , when they were done face to face as opposed to focus group discussions . this approach makes it more likely for the researcher to get in depth with the themes of a more sensitive character . a semistructured flexible interview guide was developed from the themes in the questionnaire and then combined with findings from conversations with two relatives to patients that had become critically ill . this was done to secure that the responses contributed to a deeper understanding of the items in the questionnaire . the interviews were transcribed into full text and qualitative content analysis was used for analyzing the data . the two researchers ( jannie laursen , kristoffer andresen ) performed the analysis in parallel processes ; they subsequently discussed subthemes and themes which were then compared and reflected upon in an in - depth process . the study was approved by the danish data protection agency ( journal number 528 - 02971 ) and was exempt from ethical approval from the ethical committee of the capital region in denmark , but a statement of exemption was obtained ( journal number h-2 - 2013-fsp56 ) . the relatives were informed that they at any time could withdraw from the study , and it would not influence the patient 's treatment or care . before answering the questionnaire and/or participation in the interview , a consent form was signed and the relatives were reassured of the confidentiality both orally and in writing . we approached 50 potential relatives of whom 27 participated in the questionnaire , and six of them participated in in - depth interviews . all participants were family members , including parents , siblings , children , and spouses . reason for declined participation for the 23 was due to stress , fatigue , or the loss of their loved ones . in the questionnaire , median ( range ) score for all participants were 30 ( 070 ) . for seven items out of 10 , less than 70% of the participants indicated that they were always or this is an indication that a third of the relatives were unsatisfied with these items and that they require attention ( see table 1 ) . in order to further explore these items , the in - depth interviews were conducted . the findings were based on the narratives from six relatives , four women and two men which participated in the in - depths interviews . during the analyses of the data , three themes emerged ; the first one was lack of continuity and structure , the second one was responsibility of coordination , and the third one was relatives left alone with no guiding and support . all relatives were physically involved in the care of the critically ill patient and they were all very emotional and sensitive about the critical situation . all relatives were experiencing feelings such as frustration , anxiety , and a profound struggle to understand the unfortunate situation , which lead to the themes described in the following . this theme was focused on the relatives ' perceptions of lack of continuity and structure . the relatives experienced that when the condition of the patient deteriorated and the situation became severed , the thing they needed the most from the health professionals was structure and some form of continuity in the further plan , one relative explained it as follows:there was a new doctor every day , there were 47 different names to remember , and who should i call ( ? all they did was to state her ( the patient ) diagnosis , and send a request for a supervising doctor . i believe we all know what her diagnosis was and that the doctors needed a consultation from another doctor,everything inside the relatives was an emotional chaos and they were clinging to every little thing in the hope of gaining some sense of the situation . when the relatives were not offered reconciliation from the health professionals , it often resulted in confusion and frustration , as a relative noted : my nerves were frayed , the health professionals promised to take care of my husband , to check on him , but that just did n't happen . there was a new doctor every day , there were 47 different names to remember , and who should i call ( ? all they did was to state her ( the patient ) diagnosis , and send a request for a supervising doctor . in line with quantitative findings often relatives felt that the health professionals were not able to create continuity in the care of , or during the admission of , the critically ill patients . often the health professionals were vague in their attempts to meet the relatives and often the waiting time felt very long and uncertain , which only made it more urgent for the relatives to gain some kind of information and not have to bear all the responsibility on their own . the relatives felt that they had to take charge on all follow - ups regarding care and treatment ; they felt that there were a lack of leadership and consistency presented by the health professionals . the lack of consistency and follow - up from health professionals often resulted in negative emotional reactions , which lead to some relatives having difficulties pursuing with their daily lives . you can only interpret this as carelessness , as they do n't take any responsibility , which leaves you feeling powerless . they experienced that the health professionals were apathetic and indifferent , which often lead to difficulties in managing and coping with the given situation for the relatives:i never received any explanation why they went in a new direction with my husband , which meant a lot of stress for me.consistent with the findings from the quantitative analysis , only 55% of relatives were answered by the health professionals in a way they could understand . this led to uncertainties in the situations , which often led to stress and anxiety for the relatives . often the feeling of not being able to deal with the responsibility and not being offered any support or guiding from the health professionals led to relatives not having time to cope with their own emotions:there was too much focus on coordinating things , making sure that all was running smoothly . often when relatives did try and manage the increased responsibility and the lack of commitment from the health professionals , they felt that they were not given any authority to do so . the increase in responsibility and the severity of the situation only made them more desperate and resigned . in some cases , that meant that some of the relatives had a strong need to stay in control and take all the responsibility for care and treatment . i never received any explanation why they went in a new direction with my husband , which meant a lot of stress for me . there was too much focus on coordinating things , making sure that all was running smoothly . the division of roles and responsibilities between health professionals and relatives was not clear for them , as described by one relative:it 's not me who has to walk around and organize their work . if they did not , the critically ill patient would not get the correct or proper care . at the same time , the relatives felt anxious and confused and the weight from the responsibility was almost unbearable for them , as one relative indicated : there is no consistency in anything , always swopping around , new doctors , and new nurses . no one takes any responsibility.many relatives felt that they had to be present most of the day , to make sure that the health professionals were offering the critically ill patient the needed care , according to one relative : there were many situations where my mother did not understand what was happening if i was n't thererelatives felt that they had the responsibility to coordinate and secure that things were running smoothly . if they did not take part in caring for the critically ill patient and were physically and mentally present with a constant awareness of the situation , everything would collapse and everything would fall apart ; it was described as follows : i was told that she was hospitalized , but my dad said that i should n't hurry to the hospital , so i came here at one o'clock . it made me angry and sad.the relatives had to manage care and treatment for the critically ill patient , to make sure that all was done professionally , even though they were not health professionals:if they felt that the situation called for more professionalism than the health professionals delivered , which meant they had to be responsible for it . it was hard , that i had to play such a big part in coordinating things.it was clear during the interviews that the responsibility to harmonize , the lack of structure , and the unclear division of roles left the relatives with a feeling of lack of coherence , which often lead them to a feeling of losing control , frustration , and mistrust of the system . there is no consistency in anything , always swopping around , new doctors , and new nurses . many relatives were overwhelmed by the situation , when their loved one became critically ill . they felt that they were being left on their own and at the same time expected to maintain control ; one relative indicated thatthe role of just being relatives was hard to keep - not being able to come for a visit and just give her a hug was hard.in line with quantitative findings , only 48% felt that the health professionals anticipated and met the needs of their relatives . they felt that they had to oversee the work of the health professionals in order to make sure that the patient received sufficient care . they had a direct responsibility for the patient , but at the same time they were torn between trusting the system and experiencing it as collapsing . the amount of energy the relatives had to mobilize to be in control often exceeded what was possible . the relatives experienced a lack of care or concern from the health professionals , which was perceived as hard and unsatisfying for them . relatives were not an integrated part of the care for the critically ill patient and more perceived as someone who toke resources from the health professionals . i would have liked if they had talked to me , included me in their plans , spent a little time on me , and showed they empathized . when health professionals were not able to include and inform the relatives , it left the relatives feeling powerless . in this study , the aim was to investigate the relatives ' satisfaction and involvement on a general surgery ward regarding the critically ill patient . the study investigated the level of information , the involvement of the relatives in the patient care , and the relatives ' perspectives of quality of care , regarding the patients ' needs , and health professionals ' communication . the questionnaire results showed that a third of the participants did not seem satisfied with the quality of care . the qualitative results showed that in critical situations health professionals need to be present , caring , and empathic . this was not the general experience by the relatives as revealed by the responses to the questionnaire . often they felt they were on their own , not skilled to cope with the situation , and in need of the health professional that could lead care and treatment and at the same time include them in the process . the use of satisfaction as a tool to measure quality of care has previously been discussed although it is a common and often used measurement [ 1921 ] . however , these uncertainties were taken into account in the in - depth interviews , so that participants were of both sexes , there was a mixture of educational levels , and there were no health professionals among the relatives . a study on collaboration between relatives of elderly patients and nurses stated that relatives that were new in the role as relatives could also be more critical towards the healthcare system than others . a third of the relatives stated that they were dissatisfied with the health professionals on the surgical ward . they were especially dissatisfied with the organization of the care and the involvement about the care . these results were consistent with other studies , showing that dissatisfaction often was associated with how the relatives were informed and was often associated with severity of the patient 's illness [ 4 , 22 ] . if patients were managed on a special unit and not a general ward , it was often seen that it had a positive influence on the level of satisfaction [ 4 , 11 , 22 ] . this study showed that relatives felt there were no structures during the admission and often they felt that they were left in the dark , with no ability to obtain information and with no consistency . the study found that relatives often felt that the health professionals did not care and that they were met with negligence [ 5 , 9 ] . these findings add to the evidence that relatives want to participate in decision - making and be treated as collaborators when the patient becomes critically ill . many of the challenges identified by the relatives were interrelated and often exacerbated each other . for example , a poor understanding on who was responsible for what led to a feeling of lack in feedback which increased the uncertainty further . frustration with managing the proper care was aggravated by the health professionals ' inability to coordinate and take charge and thereby not providing the appropriate comfort and security for the relatives . better communication and improving the management of decision - taking were a key item to be able to guide the relatives in a more satisfactory way . a study on frustration expressed by relatives stated that they wanted face to face consultation , but because this is not always possible an alternative could be to use an electronic consulting system as a supplement to the face to face communication . , the relatives would be considered as important collaborators to the critically ill patient , providing relevant and significant information and at the same time maintaining energy to be supporting and caring for the critically ill patient . when people experienced lack of coherence due to a life changing event , it was often shown in difficulties in managing and coping with even the smallest things . a hospital culture that provides support and consistence will often lead to a more manageable situation . if the culture values the role of people , it will be perceived as more sustaining and leave a feeling of meaningfulness , which can help support relatives in a more manageable way . relatives can be perceived as both resources and potential clients and the stress they experience can affect their health on a long term . being a relative to a critically ill patient will often lead to the loss of control . the feeling of loss of control will often collide with the need to see the world in a clear , ordered , and structured way : there is a need to also study the perception of care for relatives from the health professionals ' point of view . there is probably a range of reasons why the health professionals do not provide the care for relatives that they so desperately need , and it is important to characterize the health professionals ' experience in order to make meaningful interventions for the improvement of the care for relatives . however , it was not within the scope of this study to also cover the experience of the health professionals . there is a need to also study the perception of care for relatives from the health professionals ' point of view . there is probably a range of reasons why the health professionals do not provide the care for relatives that they so desperately need , and it is important to characterize the health professionals ' experience in order to make meaningful interventions for the improvement of the care for relatives . however , it was not within the scope of this study to also cover the experience of the health professionals . however , by interviewing six participants in the in - depth interviews , we exemplified what the relatives dissatisfaction covered , which can lead to new interventions towards including relatives in general wards . the reason for this was that very little research has been done in the field of a general surgical ward with patients that suddenly became acutely ill . a third of the participants in this study were unsatisfied with the organization and the involvement in care . the findings implied that they were left with no guiding or support from the health professionals , which often led to the feeling of loss of control . thereby , relatives can gain a sense of coherence during the hospitalization of a critically ill patient , which can lead to a greater satisfaction and thereby better support for the patient .

perinatal hypoxic - ischemic ( hi ) brain injury is one of the most common causes of severe neurological handicap in children . estimated life - time costs to support children with cerebral palsy , a common outcome of hi brain injury in neonates , reached 11.5 billion dollars in 2003 . unfortunately , our understanding the mechanisms of the hi brain injury is not deep enough for the development of mechanism - targeted therapeutic interventions in this disease . even therapeutic mechanisms of post - hi cerebral hypothermia ( the only clinically proven neuroprotective strategy ) are still not well defined which precludes an optimal use of this potentially powerful strategy . physiologically , hi brain injury could be defined as an acute oxygen and nutrients deprivation to the brain caused by a collapse of cerebral circulation . hypoxia - ischemia results in severe cellular bioenergetics failure , and if cerebral circulation is not restored , then the brain death is unpreventable . however , if the cerebral circulation is restored for example , as a result of successful resuscitation , then cerebral reperfusion ensures with a full or partial brain recovery . unfortunately , the same reperfusion can also contribute to the propagation of brain injury initiated by the hi insult . this implies that hi brain injury as a disease , consists of two fundamental pathophysiological events : hypoxia - ischemia and reperfusion . during hypoxia - ischemia and reperfusion it is now recognized that not only mitochondrial failure to generate atp during ischemia , but the generation of oxidative radicals and the release of proapoptotic proteins during reperfusion contribute to the cellular damage . the leading molecular mechanisms responsible for the evolution of cell damage and repair during reperfusion change at different timepoints following hi insult ( figure 1 ) . a critical upstream mechanisms to consider in the management of hi brain injury are those linked to an oxidative stress . therefore , already at the initiation of resuscitation / reperfusion an attempt should be made to limit the reoxygenation - driven burst in generation of reactive oxygen species ( ros ) in order to alleviate the severity of oxidative damage to the hi brain . it is known that reintroduction of the oxygen to ischemic tissue potentiates oxidative injury . an initial attempt to limit formation of ros could be made by judicious use of oxygen during resuscitation . not too long ago , in 2000 the use of 100% oxygen was indisputably recommended for the initiation of resuscitation in all depressed infants . now neonatologists have tempered their enthusiasm for the use of pure oxygen in neonatal resuscitation . several clinical trials showed that in the majority of depressed infants the goal of resuscitation , an immediate survival , could be achieved with the use of room air , as effectively as with the use of 100% oxygen [ 46 ] . therefore , regardless of the primary mechanisms of ros generation during reperfusion , a switch from a routine use of 100% oxygen to the room air at the initiation of neonatal resuscitation , potentially , should limit the severity of an oxidative stress . indeed , vento et al . reported a significantly lower level of circulating markers of oxidative stress in neonates resuscitated with the room air ( ra ) compared to infants resuscitated with the 100% oxygen . however , it remains to be determined to what extent the use of ra in the resuscitation of infants with hi brain injury attenuates an oxidative damage to the brain . numerous animal studies clearly demonstrated that hyperoxic re - oxygenation maintained for 3060 minutes of initial reperfusion was detrimental for neurological outcome in asphyxiated pigs and rodents [ 810 ] . the use of the 100% oxygen in these animals was strongly associated with exacerbation of an oxidative stress in the brain . of note , however , the hyperoxic resuscitation in these studies was used for 3060 minutes . at these time - points of reperfusion a full restoration of systemic circulation was already achieved and this resulted in extreme hyperoxemia . because the primary goal of resuscitation is the return of spontaneous circulation ( rosc ) , experiments in which the hyperoxic resuscitation is applied beyond the time - point of the rosc have limited translational importance for the resuscitation science . however , the references cited above do provide an important translational message for the post - resuscitation medical care : all efforts should be made to avoid hyperoxemia in reperfusion . although , normoxic resuscitation has been shown to be effective in the majority of infants , it is still undetermined whether the use of ra in the resuscitation of severely ( a complete circulatory arrest ) asphyxiated infants is as effective as the use of 100% oxygen in achieving rosc . after a prolonged ( 25 minutes ) cardiopulmonary arrest in mature pigs , the resuscitation with the use of positive pressure ventilation significantly improved the rate of sustained rosc and cardiac output only if the resuscitation was supplemented with hyperbaric ( ~400% o2 ) re - oxygenation . in contrast , following a brief ( one minute ) cardiac arrest a cardio - pulmonary resuscitation with the use of ra or 100% o2 resulted in similar rates of rosc in neonatal pigs [ 12 , 13 ] . these data suggest that the duration of circulatory arrest may determine whether positive pressure ventilation needs supplementation with 100% o2 to enhance the rate of rosc . it is critical to understand that no attempts should be made to attenuate a reperfusion - driven oxidative stress at the expense of the efficacy of resuscitation . overall , current data suggest that the use of room air in resuscitation reduces the severity of oxidative stress in the majority of depressed infants at risk for hi brain injury . the simplicity of this approach ( restriction of oxygen availability for the formation of ros ) , however , underscores our incomplete understanding the mechanisms initiating an oxidative injury to the hi brain . interestingly , matsiukevich et al . showed that in neonatal mice subjected to a lethal hi insult evidenced by a complete circulatory collapse , hyperoxic resuscitation limited to the time ( 2 minutes ) needed to achieve a sustained rosc was not associated with exacerbation of reperfusion - driven acceleration in the rate of ros emission from isolated brain mitochondria . however , it is yet to be clarified whether ros originating from mitochondria at the onset and during reperfusion cause an oxidative injury to the hi brain . to date , it is still unclear what are sources of pathogenic oxidative radicals in the hi brain , how to enhance antioxidative mechanisms and what are those mechanisms of injury which are initiated or exacerbated by the ros . the evolution of ischemic brain injury following restoration of oxygen and nutrient delivery is a paradoxical biological phenomenon . although , it is clear that without reperfusion / reoxygenation an ischemic tissue does not survive , maladaptive metabolic changes induced by ischemia predispose cell to dysfunction and death upon reperfusion / reoxygenation . the central role in this phenomenon was assigned to ros , which can be formed only in the presence of o2 . therefore , an oxidative damage occurs mostly upon reintroduction of o2 to the ischemic tissue . in the immature brain antioxidant system is underdeveloped which limits inactivation of some ros and in particular , hydrogen peroxide ( reviewed in ) . the latter is perhaps the most important tissue - damaging ros species due to its relative stability and the ability to cross lipid membranes . for example , upregulation of cu / zn superoxide dismutase ( enzyme which converts superoxide into h2o2 ) increased , rather than decreased the extent of hi brain injury in neonatal rats . in contrast , transgenic mice overexpressing glutathione peroxidase ( enzyme which detoxifies h2o2 into h2o ) were markedly protected against hi insult . what is the origin of this h2o2 ? what are the major sources of oxidative radicals responsible for an oxidative brain damage in hi ? in an elegant study , abramov and coauthors have identified three distinct ros generating systems during simulated hi insult ( oxygen - glucose deprivation ( ogd ) ) and reperfusion in cultured neurons mitochondrial respiratory chain ( mrc ) , xanthine oxidase and nadph oxidase . mrc responds to ogd with a burst of ros emission , which declined by the end of hi insult secondary to a loss of mitochondrial membrane potential . at the end of hi insult a second elevation in cellular ros generation was attributable to the activity of xanthine oxidase . a third peak in ros production was due to activity of nadph oxidase during reperfusion . in immature animals and humans with hi brain injury , elevated level of hypoxanthine was proposed as the evidence for a pathogenic role of xanthine oxidase [ 18 , 19 ] . however , an inhibition of xanthine oxidase with oxypurinol or allopurinol failed to reduce lipid peroxidation , and did not protect the brain in a rat model of hi injury or in human neonates with perinatal hi insult . genetic or / and pharmacological inhibition of nadph oxidase also did not exert neuroprotection in different models of perinatal hi brain injury . taken together these data challenge a pathogenic contribution of nadph oxidase or xanthine oxidase to an oxidative brain damage following hi in neonates . interestingly , loor et al . using a model simulating hi reperfusion injury in cultured cardiomyocytes demonstrated that genetic overexpression of only intramitochondrial ros - scavenging enzymes , mn - superoxide dismutase or phospholipid hydroperoxide glutathione peroxidase protected cells against reperfusion - induced death . in contrast , overexpression of cu - zn superoxide dismutase or catalase did not result in the protection . mitochondria are known as a major source for ros production in the health and diseases , including brain ischemia - reperfusion injury ( reviewed in ) . in mature animal models of ischemia - reperfusion injury to the brain and heart , mitochondria have been increasingly recognized as an important source for the reperfusion - driven acceleration in ros release [ 2427 ] . however , rapidly emerging evidence supporting a deleterious role of ros originating in mitochondria during reperfusion are partially counterbalanced by the reports suggesting a prosurvival signaling mediated by mitochondrial ros in the heart preconditioning ( , reviewed in ) and in postischemic reperfusion . in the developing brain potential deleterious or prosurvival effects of mitochondrial ros in hi reperfusion were not studied . in the following part of this paper we discuss the experimental data obtained in the mature animal models of the brain and heart ischemia - reperfusion injury which support the primary role of mitochondrial ros in oxidative damage . in mature animals several studies detected a reperfusion - driven acceleration in ros generation from mitochondria associated with oxidative damage to the postischemic heart [ 25 , 26 ] and brain . a single study showed that in neonatal mice with genetically ablated c1q component of the classical complement activation pathway , the neuroprotection and attenuation of oxidative hi brain injury were associated with the ability of c1q brain mitochondria to release significantly less ros in response to hi reperfusion , rather then with altered activation of the terminal complement complex . a pathogenic contribution of ros originating from mitochondria is supported by the data demonstrating that extrinsic or genetic enhancement of mitochondria - targeted ros scavengers reduces the extent of injury or / and oxidative stress in animal models of ischemia - reperfusion in several organs ( [ 3234 ] , reviewed in ) . furthermore , pharmacological inhibition of ros generation in the mitochondrial respiratory chain ( mrc ) limits the extent of ischemia - reperfusion damage and the expression of markers of oxidative injury [ 26 , 36 , 37 ] . these data highlight mrc as a potential target for an antioxidative therapeutic strategy against hi brain injury . in the mrc , complex i and complex iii are two major sites for ros generation during reperfusion [ 32 , 38 ] . an inhibitory effect of ischemia on complex i has been suggested as a cause for an accelerated generation of ros in mrc in hearts . however , interpreting the data on postischemic mitochondrial ros production might be difficult and requires an appropriate experience . the data on mitochondrial function in ischemia - reperfusion mostly were obtained in isolated mitochondria in vitro , when results depended on the choice of experimental conditions . for example , in mitochondria isolated from different organs , including neonatal mouse brain , the response to inhibition of complex i is either increase or dramatic decrease in ros emission rates , depending upon a substrate used to donate electrons to mrc . nad - linked substrates such as malate , glutamate , pyruvate , and so forth , invariably support an elevation in mitochondrial ros emission following an inhibition of complex i with rotenone ( figure 2(a ) ) . in contrast , the use of fad - linked substrates such as for example , succinate results in robust decrease in mitochondrial ros emission following an inhibition of complex i with rotenone ( figure 2(a ) ) . these differences in ros generation by mrc in response to the same complex i inhibitor are well understood and explained by the differences in the electron transport flows , supported by nad- or fad - linked substrates ( reviewed in ) . nad - linked substrates support only forward electron transport flow ( fet ) , from complex i to membrane - dissolved ubiquinone to complex iii to cytochrome c and finally to oxygen through complex iv ( cytochrome c oxidase ) . during this fet , low levels of superoxide can be generated at unspecified mrc sites ( likely at complex i and complex iii ) , because some electrons accidentally escape from mrc electron carriers onto o2 ( figure 2(b ) ) . rotenone , pyridaben , thio - barbiturates and other complex i inhibitors interrupt fet between the complex i electron carriers and membrane - dissolved ubiquinone . this interruption of fet increases ros emission from complex i ( figure 2(c ) ) secondary to over - reduction of electron carriers ( flavin and/or fes - center n2 and complex i - bound ubiquinone ) within this complex ( reviewed in ) . it also stimulates ros emission from other sources located in the mitochondrial matrix such as for example , dihydrolipoamide dehydrogenase [ 41 , 42 ] , a subcomponent of pyruvate dehydrogenase and ketoglutarate dehydrogenase . this stimulation in ros production is caused by a decrease in mitochondrial nad / nadh ratio ( as a result of inability of compelx i to oxidize nadh ) . on the other hand , in the mitochondria fueled with fad - linked substrates ( e.g. , succinate ) the main electrons flow bypasses complex i and proceeds from the succinate dehydrogenase ( complex ii ) to membrane - dissolved ubiquinone , complex iii , cytochrome c , and cytochrome c oxidase . under specific conditions , such as moderately elevated membrane potential and abundance of fad - linked substrate , electron flux can and does proceed back from complex ii , ubiquinone to complex i and further to the matrix - located nad . this is called reverse electron transport ( ret ) flow ( figure 2(d ) ) . it was found that ret is associated with very high rates of ros emission , about 100 folds greater than that obtained with nad - linked substrates ( reviewed in ) . the major sites of ros emission in mitochondria fueled with fad - linked substrate are thought to be complex i and matrix - located enzymes pyruvate dehydrogenase and alpha - ketoglutarate dehydrogenase . inhibition of complex i with rotenone or similar inhibitors interrupts ret flow and , therefore , substantially diminishes the rate of ros emission ( 58 folds ) ( figures 2(a ) and 2(d ) ) . the ret flow represents the major mechanism for ros production by mitochondria fueled with succinate , especially in the brain and the heart . it should be noted , that both fet and ret generate proton - motive force and support oxidative phosphorylation of adp ; with ret being about 30% less efficient in terms of energy production but generating tremendously more ros . in vivo , under non - pathological conditions the primary electron donor for mrc in brain mitochondria are nad - linked substrates for example , pyruvate generated in glycolysis . during ischemia - reperfusion , however , substrate availability significantly differs from that in normal cells . there are several lines of evidence to consider that at the onset of reperfusion postischemic mitochondria actively metabolize succinate . complex i is the most sensitive among all five complexes to the reduction of the cerebral blood flow , and at the end of ischemia the activity of this complex is significantly reduced [ 44 , 45 ] . in the immature brain hi resulted in slightly ( 9% on malate - glutamate ) to moderately ( 21% on pyruvate - malate ) greater inhibition of mitochondrial respiration tested on nad - linked substrates compared to that tested on the fad - oriented substrate , succinate . in mature rats , forebrain ischemia and six hours of reperfusion resulted in a significant inhibition of mitochondrial respiration tested on nad - linked substrates . however , no significant differences from the control values were detected when the same mitochondria respired on succinate . this suggests , that after brain ischemia the activity of complex ii is better preserved compared to complex i. this favors a succinate - supported respiration upon reintroduction of o2 . indeed , in the rat brain , ischemia resulted in a profound ( 810 fold ) depletion of all nad - linked substrates : pyruvate , citrate , alpha - ketoglutarate , oxaloacetate , fumarate , and malate . in contrast , the concentration of the succinate increased by ~300% and remained elevated at 15 minutes of reperfusion . following an acute systemic hypoxemia an oxidation of succinate and glutamate by isolated rat brain mitochondria was significantly ( > 60% ) increased [ 50 , 51 ] . furthermore , it is known that succinate oxidation inhibits an oxidation of pyruvate and other nad - linked respiratory substrates , an event associated with over - reduction of mitochondrial pyridine nucleotides . in the heart , the level of succinate also is markedly elevated during ischemia followed by normalization within 3060 minutes of reperfusion [ 53 , 54 ] , the time - point associated with near - full restoration of mitochondrial metabolic activity in neonatal hi reperfusion . thus , if at the initial stage of reperfusion mitochondria actively utilize succinate , then interruption of ret flow by complex i inhibiting agents should reduce ros generation without significant changing atp - production rate . if the ret flow - dependent production of ros causes an oxidative damage following hi , then inhibition of complex i recovery upon reperfusion should reduce an oxidative injury . indeed , in rats with global cerebral ischemia an inhibition of complex i by rotenone or haloperidol significantly reduced tissue accumulation of hydroxyl radicals , resulting in near - complete abrogation of the reperfusion - driven surge in lipid peroxidation products . ambrosio et al . reported that inhibition of complex i with the thio - barbiturate amytal resulted in significant reduction in the level of free radicals associated with attenuation of lipid peroxidation in isolated rabbit hearts subjected to ischemia - reperfusion . our data demonstrated that inhibition of complex i with pyridaben significantly reduced cerebral infarct volume and signs of oxidative injury to the brain tissue and mitochondria following hi in neonatal mice . in the model of cardiac arrest and reperfusion , complex i was proposed as a primary generator of ros . taken together , these data suggest that ros generated in complex i participate in oxidative damage to the postischemic brain and heart , making this complex a reasonable therapeutic target against oxidative stress in the early stages of reperfusion . in addition to the complex i , complex iii has been recognized as an important source for emission of ros in ischemia and reperfusion [ 30 , 57 ] . however , experiments with isolated nerve terminals revealed that only very high level of complex iii inhibition ( 7080% ) resulted in detectable elevation in generation of h2o2 . given , that after brain ischemia mitochondrial respiration on succinate was shown to be markedly better preserved compared to that tested on complex i linked substrates , the rationale to consider complex iii as a therapeutic target in reperfusion is weak . indeed , in mitochondria respiring on succinate the ret flow ( complex i ) contribute the most to ros production . finally , it is unrealistic to inhibit complex iii without robust reduction in production of atp which could be detrimental for the tissue recovery . traditionally , a detrimental effect of oxidative stress is supported by evidence of structural oxidative alterations to the post - hi brain . however , it is also important to determine what specific mechanism of injury could be targeted by ros during reperfusion . in the design of neuroprotective strategies , it is not only a source of injurious ros , but also a particular mechanism of damage triggered / exacerbated by these ros is important to consider . logistically , if an oxidative stress is one of the earliest reperfusion - driven damaging events , the mechanism targeted by ros should be in close temporal proximity to the index event . in the ischemic brain , cells experience glutamate - receptors over - stimulation and cellular ca overload , which occurs to a markedly greater extent in the neonatal brain than in the mature cns [ 59 , 60 ] . mitochondria actively participate in preservation of cellular ca homeostasis by up take of ca from the cytosol into mitochondrial matrix space ( reviewed in ) . however , if mitochondrial ca load exceeds mitochondrial capacity to hold ca , then mitochondrial membranes loose their integrity via opening a channel in the inner membrane , termed the mitochondrial permeability transition pore ( mptp ) . transient and permanent opening of mptp has been strongly considered as one of the leading mechanisms of necrotic and apoptotic cell death in the brain and other organs following ischemia - reperfusion injury ( [ 62 , 63 ] , reviewed in ) . it has been shown , that mitochondrial ros can initiate an opening of mptp during ischemia and reperfusion [ 65 , 66 ] even in the absence of cyclophilin - d ( the only known structural component of mptp ) or ca overload [ 67 , 68 ] . mitochondria - targeted antioxidant , mitotempo , partially prevented mptp opening and attenuated necrosis and apoptosis following simulated ischemia - reperfusion injury in cultured renal tubular cells . taken together these data suggest , that regardless of the type of the organ , ros originating from mitochondria upon reperfusion can trigger a loss of integrity in mitochondrial inner membrane , the event suggested as the " point of no return " in propagation of cell death following hi insult . it has been shown that in immature brain , at the end of hi insult mitochondrial phosphorylating respiration was significantly suppressed [ 31 , 70 , 71 ] . reoxygenation / reperfusion restores mitochondrial adp - phosphorylating capacity , normalizing atp content in the post - hi brain . however , following several hours of reperfusion mitochondria exhibit a profound decline in their adp - phosphorylating respiration rates [ 31 , 46 ] , the event known as a secondary energy failure . the molecular mechanism proposed to explain the pathogenesis of secondary energy failure is opening of mptp . mptp renders organelles incapable of atp production due to a loss of proton - motive force and nad . this bioenergetics failure results in mitochondrial swelling , leading to a permeabilization of the outer mitochondrial membrane and release of pro - apoptotic proteins which eventuates in necrotic and apoptotic cell death [ 7274 ] . it has been shown that in neonatal rats inner mitochondrial membrane opens mptp at 01.5 hours and at 68 hours after hi . however , the pathogenic significance of mptp in the reperfusion injury in the developing hi brain remains uncertain . for example , as opposite to adult mice , neonatal cyclophilin - d knock - out mice were found to be susceptible to hi injury . earlier the same group has reported that antagonist of cyclophilin - d , cyclosporin - a did not attenuate the extent of hi brain damage in neonatal rats . in contrast , using the same model hwang et al . reported that cyclosporin - a , injected immediately after hi insult significantly protected developing brain , attenuating both necrotic and apoptotic cell death in neonatal rats . similar results were obtained in neonatal rats subjected to a mild focal cerebral ischemia - reperfusion . in neonatal rats and mice subjected to a global hypoxia - ischemia - reperfusion injury , a post - treatment with cyclosporine a markedly potentiated the neuroprotective effect of ca channel antagonist , nimodipine . given , that in mature animal models of ischemia - reperfusion injury a pathogenic role for mptp has been strongly suggested , more extensive research is needed to clarify the contribution of mptp opening to cerebral hi reperfusion injury in the developing brain . following an ischemic insult mitochondrial membrane permeabilization can occur via opening of outer mitochondrial membrane pore ( ommp ) induced by bak / bax translocation into mitochondria . this pore is thought to be primarily responsible for a release of pro - apoptotic proteins from the mitochondrial inter - membrane space , leading to an apoptotic cell death [ 81 , 82 ] , including that induced by an oxidative stress ( , reviewed in ) . importantly , in hi reperfusion injury to the developing brain bax dependent ommp has been suggested as a primary mechanism of injury ( , reviewed in ) . developmental shift toward a priority of the bax - dependent ommp over the cyclophylin - d dependent mptp opening in the hi brain damage has been supported by the data obtained in cyclophilin d knock - out neonatal mice , as well as by neuroprotective effect of bax - inhibiting peptide . however , in contrast to a better understanding of events leading to secondary energy failure and necrotic cell death following an opening of mptp , it is less clear how bax / bak mediated ommp opening affects oxidative phosphorylation and results in secondary energy failure and necrosis . one possibility is that postischemic opening of ommp results in a massive loss of cytochrome c from the inter - membrane mitochondrial space which results in secondary inhibition of oxidative phosphorylation . however , this loss of cytochrome c was not mediated by mptp opening , and was not associated with changes in mitochondrial bax , bad , bak or bid . although , mitochondrial ros appeared to be critical for the execution of bax / bak dependent apoptosis induced by anti - cancer drugs [ 88 , 89 ] , we have not found data that ros originating in mitochondria are involved in the bax / bak - induced apoptosis in hi brain injury . interestingly , oxidative stress - induced cell apoptosis clearly required the presence of ros originating from mrc to signal mptp opening , but this apoptosis was independent of bax translocation . the existence of two relatively independent mechanisms of mitochondrial membrane permeabilization does not exclude the contribution of each of these mechanisms in hi damage to the developing brain . indeed , there is evidence for involvement of cyclophilin d dependent mptp opening in the bax - driven cytochrome c release in the isolated mitochondria . in conclusion , the analysis of current data supports the hypothesis that in the developing hi brain reoxygenation / reperfusion causes not only recovery of cell bioenergetics , but also accelerates ros generation in mitochondrial respiratory chain ( figures 3(a ) and 3(b ) ) . this damage occurs in the forms of mptp and bax / bak dependent outer membrane pores , both of which are considered as a point of no return in the evolution of hi injury . with data that complex i contributes to accelerated generation of ros during reperfusion , a novel neuroprotective strategy against reperfusion - driven mitochondrial membrane permeabilization may consist of reversible pharmacological inhibition of complex i recovery following hi insult ( figure 3(c ) ) .

mitochondrial dysfunction is the most fundamental mechanism of cell damage in cerebral hypoxia - ischemia and reperfusion . mitochondrial respiratory chain ( mrc ) is increasingly recognized as a source for reactive oxygen species ( ros ) in the postischemic tissue . potentially , ros originating in mrc can contribute to the reperfusion - driven oxidative stress , promoting mitochondrial membrane permeabilization . the loss of mitochondrial membranes integrity during reperfusion is considered as the major mechanism of secondary energy failure . this paper focuses on current data that support a pathogenic role of ros originating from mitochondrial respiratory chain in the promotion of secondary energy failure and proposes potential therapeutic strategy against reperfusion - driven oxidative stress following hypoxia - ischemia - reperfusion injury of the developing brain .

1. Introduction 2. HI and Resuscitation 3. Potential Sources of Reactive Oxygen Species in HI Injury to the Developing Brain 4. Mitochondrial ROS and HI Reperfusion Oxidative Stress 5. The Pathogenic Mechanisms Targeted by Mitochondrial ROS in HI Reperfusion 6. The Role of Mitochondrial Membrane Permeabilization in the HI Brain Injury

perinatal hypoxic - ischemic ( hi ) brain injury is one of the most common causes of severe neurological handicap in children . unfortunately , our understanding the mechanisms of the hi brain injury is not deep enough for the development of mechanism - targeted therapeutic interventions in this disease . physiologically , hi brain injury could be defined as an acute oxygen and nutrients deprivation to the brain caused by a collapse of cerebral circulation . hypoxia - ischemia results in severe cellular bioenergetics failure , and if cerebral circulation is not restored , then the brain death is unpreventable . unfortunately , the same reperfusion can also contribute to the propagation of brain injury initiated by the hi insult . this implies that hi brain injury as a disease , consists of two fundamental pathophysiological events : hypoxia - ischemia and reperfusion . during hypoxia - ischemia and reperfusion it is now recognized that not only mitochondrial failure to generate atp during ischemia , but the generation of oxidative radicals and the release of proapoptotic proteins during reperfusion contribute to the cellular damage . the leading molecular mechanisms responsible for the evolution of cell damage and repair during reperfusion change at different timepoints following hi insult ( figure 1 ) . a critical upstream mechanisms to consider in the management of hi brain injury are those linked to an oxidative stress . therefore , already at the initiation of resuscitation / reperfusion an attempt should be made to limit the reoxygenation - driven burst in generation of reactive oxygen species ( ros ) in order to alleviate the severity of oxidative damage to the hi brain . it is known that reintroduction of the oxygen to ischemic tissue potentiates oxidative injury . an initial attempt to limit formation of ros could be made by judicious use of oxygen during resuscitation . several clinical trials showed that in the majority of depressed infants the goal of resuscitation , an immediate survival , could be achieved with the use of room air , as effectively as with the use of 100% oxygen [ 46 ] . therefore , regardless of the primary mechanisms of ros generation during reperfusion , a switch from a routine use of 100% oxygen to the room air at the initiation of neonatal resuscitation , potentially , should limit the severity of an oxidative stress . reported a significantly lower level of circulating markers of oxidative stress in neonates resuscitated with the room air ( ra ) compared to infants resuscitated with the 100% oxygen . however , it remains to be determined to what extent the use of ra in the resuscitation of infants with hi brain injury attenuates an oxidative damage to the brain . the use of the 100% oxygen in these animals was strongly associated with exacerbation of an oxidative stress in the brain . because the primary goal of resuscitation is the return of spontaneous circulation ( rosc ) , experiments in which the hyperoxic resuscitation is applied beyond the time - point of the rosc have limited translational importance for the resuscitation science . although , normoxic resuscitation has been shown to be effective in the majority of infants , it is still undetermined whether the use of ra in the resuscitation of severely ( a complete circulatory arrest ) asphyxiated infants is as effective as the use of 100% oxygen in achieving rosc . it is critical to understand that no attempts should be made to attenuate a reperfusion - driven oxidative stress at the expense of the efficacy of resuscitation . overall , current data suggest that the use of room air in resuscitation reduces the severity of oxidative stress in the majority of depressed infants at risk for hi brain injury . the simplicity of this approach ( restriction of oxygen availability for the formation of ros ) , however , underscores our incomplete understanding the mechanisms initiating an oxidative injury to the hi brain . showed that in neonatal mice subjected to a lethal hi insult evidenced by a complete circulatory collapse , hyperoxic resuscitation limited to the time ( 2 minutes ) needed to achieve a sustained rosc was not associated with exacerbation of reperfusion - driven acceleration in the rate of ros emission from isolated brain mitochondria . however , it is yet to be clarified whether ros originating from mitochondria at the onset and during reperfusion cause an oxidative injury to the hi brain . to date , it is still unclear what are sources of pathogenic oxidative radicals in the hi brain , how to enhance antioxidative mechanisms and what are those mechanisms of injury which are initiated or exacerbated by the ros . therefore , an oxidative damage occurs mostly upon reintroduction of o2 to the ischemic tissue . in the immature brain antioxidant system is underdeveloped which limits inactivation of some ros and in particular , hydrogen peroxide ( reviewed in ) . the latter is perhaps the most important tissue - damaging ros species due to its relative stability and the ability to cross lipid membranes . what is the origin of this h2o2 ? what are the major sources of oxidative radicals responsible for an oxidative brain damage in hi ? in an elegant study , abramov and coauthors have identified three distinct ros generating systems during simulated hi insult ( oxygen - glucose deprivation ( ogd ) ) and reperfusion in cultured neurons mitochondrial respiratory chain ( mrc ) , xanthine oxidase and nadph oxidase . mrc responds to ogd with a burst of ros emission , which declined by the end of hi insult secondary to a loss of mitochondrial membrane potential . at the end of hi insult a second elevation in cellular ros generation was attributable to the activity of xanthine oxidase . a third peak in ros production was due to activity of nadph oxidase during reperfusion . in immature animals and humans with hi brain injury , elevated level of hypoxanthine was proposed as the evidence for a pathogenic role of xanthine oxidase [ 18 , 19 ] . taken together these data challenge a pathogenic contribution of nadph oxidase or xanthine oxidase to an oxidative brain damage following hi in neonates . using a model simulating hi reperfusion injury in cultured cardiomyocytes demonstrated that genetic overexpression of only intramitochondrial ros - scavenging enzymes , mn - superoxide dismutase or phospholipid hydroperoxide glutathione peroxidase protected cells against reperfusion - induced death . in contrast , overexpression of cu - zn superoxide dismutase or catalase did not result in the protection . mitochondria are known as a major source for ros production in the health and diseases , including brain ischemia - reperfusion injury ( reviewed in ) . in mature animal models of ischemia - reperfusion injury to the brain and heart , mitochondria have been increasingly recognized as an important source for the reperfusion - driven acceleration in ros release [ 2427 ] . however , rapidly emerging evidence supporting a deleterious role of ros originating in mitochondria during reperfusion are partially counterbalanced by the reports suggesting a prosurvival signaling mediated by mitochondrial ros in the heart preconditioning ( , reviewed in ) and in postischemic reperfusion . in the developing brain potential deleterious or prosurvival effects of mitochondrial ros in hi reperfusion were not studied . in the following part of this paper we discuss the experimental data obtained in the mature animal models of the brain and heart ischemia - reperfusion injury which support the primary role of mitochondrial ros in oxidative damage . in mature animals several studies detected a reperfusion - driven acceleration in ros generation from mitochondria associated with oxidative damage to the postischemic heart [ 25 , 26 ] and brain . a single study showed that in neonatal mice with genetically ablated c1q component of the classical complement activation pathway , the neuroprotection and attenuation of oxidative hi brain injury were associated with the ability of c1q brain mitochondria to release significantly less ros in response to hi reperfusion , rather then with altered activation of the terminal complement complex . a pathogenic contribution of ros originating from mitochondria is supported by the data demonstrating that extrinsic or genetic enhancement of mitochondria - targeted ros scavengers reduces the extent of injury or / and oxidative stress in animal models of ischemia - reperfusion in several organs ( [ 3234 ] , reviewed in ) . furthermore , pharmacological inhibition of ros generation in the mitochondrial respiratory chain ( mrc ) limits the extent of ischemia - reperfusion damage and the expression of markers of oxidative injury [ 26 , 36 , 37 ] . these data highlight mrc as a potential target for an antioxidative therapeutic strategy against hi brain injury . in the mrc , complex i and complex iii are two major sites for ros generation during reperfusion [ 32 , 38 ] . an inhibitory effect of ischemia on complex i has been suggested as a cause for an accelerated generation of ros in mrc in hearts . the data on mitochondrial function in ischemia - reperfusion mostly were obtained in isolated mitochondria in vitro , when results depended on the choice of experimental conditions . these differences in ros generation by mrc in response to the same complex i inhibitor are well understood and explained by the differences in the electron transport flows , supported by nad- or fad - linked substrates ( reviewed in ) . this stimulation in ros production is caused by a decrease in mitochondrial nad / nadh ratio ( as a result of inability of compelx i to oxidize nadh ) . on the other hand , in the mitochondria fueled with fad - linked substrates ( e.g. it was found that ret is associated with very high rates of ros emission , about 100 folds greater than that obtained with nad - linked substrates ( reviewed in ) . the major sites of ros emission in mitochondria fueled with fad - linked substrate are thought to be complex i and matrix - located enzymes pyruvate dehydrogenase and alpha - ketoglutarate dehydrogenase . the ret flow represents the major mechanism for ros production by mitochondria fueled with succinate , especially in the brain and the heart . during ischemia - reperfusion , however , substrate availability significantly differs from that in normal cells . complex i is the most sensitive among all five complexes to the reduction of the cerebral blood flow , and at the end of ischemia the activity of this complex is significantly reduced [ 44 , 45 ] . in the immature brain hi resulted in slightly ( 9% on malate - glutamate ) to moderately ( 21% on pyruvate - malate ) greater inhibition of mitochondrial respiration tested on nad - linked substrates compared to that tested on the fad - oriented substrate , succinate . in mature rats , forebrain ischemia and six hours of reperfusion resulted in a significant inhibition of mitochondrial respiration tested on nad - linked substrates . in contrast , the concentration of the succinate increased by ~300% and remained elevated at 15 minutes of reperfusion . in the heart , the level of succinate also is markedly elevated during ischemia followed by normalization within 3060 minutes of reperfusion [ 53 , 54 ] , the time - point associated with near - full restoration of mitochondrial metabolic activity in neonatal hi reperfusion . indeed , in rats with global cerebral ischemia an inhibition of complex i by rotenone or haloperidol significantly reduced tissue accumulation of hydroxyl radicals , resulting in near - complete abrogation of the reperfusion - driven surge in lipid peroxidation products . reported that inhibition of complex i with the thio - barbiturate amytal resulted in significant reduction in the level of free radicals associated with attenuation of lipid peroxidation in isolated rabbit hearts subjected to ischemia - reperfusion . our data demonstrated that inhibition of complex i with pyridaben significantly reduced cerebral infarct volume and signs of oxidative injury to the brain tissue and mitochondria following hi in neonatal mice . in the model of cardiac arrest and reperfusion , complex i was proposed as a primary generator of ros . taken together , these data suggest that ros generated in complex i participate in oxidative damage to the postischemic brain and heart , making this complex a reasonable therapeutic target against oxidative stress in the early stages of reperfusion . in addition to the complex i , complex iii has been recognized as an important source for emission of ros in ischemia and reperfusion [ 30 , 57 ] . given , that after brain ischemia mitochondrial respiration on succinate was shown to be markedly better preserved compared to that tested on complex i linked substrates , the rationale to consider complex iii as a therapeutic target in reperfusion is weak . indeed , in mitochondria respiring on succinate the ret flow ( complex i ) contribute the most to ros production . traditionally , a detrimental effect of oxidative stress is supported by evidence of structural oxidative alterations to the post - hi brain . however , it is also important to determine what specific mechanism of injury could be targeted by ros during reperfusion . in the design of neuroprotective strategies , it is not only a source of injurious ros , but also a particular mechanism of damage triggered / exacerbated by these ros is important to consider . logistically , if an oxidative stress is one of the earliest reperfusion - driven damaging events , the mechanism targeted by ros should be in close temporal proximity to the index event . in the ischemic brain , cells experience glutamate - receptors over - stimulation and cellular ca overload , which occurs to a markedly greater extent in the neonatal brain than in the mature cns [ 59 , 60 ] . however , if mitochondrial ca load exceeds mitochondrial capacity to hold ca , then mitochondrial membranes loose their integrity via opening a channel in the inner membrane , termed the mitochondrial permeability transition pore ( mptp ) . transient and permanent opening of mptp has been strongly considered as one of the leading mechanisms of necrotic and apoptotic cell death in the brain and other organs following ischemia - reperfusion injury ( [ 62 , 63 ] , reviewed in ) . it has been shown , that mitochondrial ros can initiate an opening of mptp during ischemia and reperfusion [ 65 , 66 ] even in the absence of cyclophilin - d ( the only known structural component of mptp ) or ca overload [ 67 , 68 ] . mitochondria - targeted antioxidant , mitotempo , partially prevented mptp opening and attenuated necrosis and apoptosis following simulated ischemia - reperfusion injury in cultured renal tubular cells . taken together these data suggest , that regardless of the type of the organ , ros originating from mitochondria upon reperfusion can trigger a loss of integrity in mitochondrial inner membrane , the event suggested as the " point of no return " in propagation of cell death following hi insult . however , following several hours of reperfusion mitochondria exhibit a profound decline in their adp - phosphorylating respiration rates [ 31 , 46 ] , the event known as a secondary energy failure . the molecular mechanism proposed to explain the pathogenesis of secondary energy failure is opening of mptp . mptp renders organelles incapable of atp production due to a loss of proton - motive force and nad . this bioenergetics failure results in mitochondrial swelling , leading to a permeabilization of the outer mitochondrial membrane and release of pro - apoptotic proteins which eventuates in necrotic and apoptotic cell death [ 7274 ] . it has been shown that in neonatal rats inner mitochondrial membrane opens mptp at 01.5 hours and at 68 hours after hi . however , the pathogenic significance of mptp in the reperfusion injury in the developing hi brain remains uncertain . earlier the same group has reported that antagonist of cyclophilin - d , cyclosporin - a did not attenuate the extent of hi brain damage in neonatal rats . reported that cyclosporin - a , injected immediately after hi insult significantly protected developing brain , attenuating both necrotic and apoptotic cell death in neonatal rats . similar results were obtained in neonatal rats subjected to a mild focal cerebral ischemia - reperfusion . in neonatal rats and mice subjected to a global hypoxia - ischemia - reperfusion injury , a post - treatment with cyclosporine a markedly potentiated the neuroprotective effect of ca channel antagonist , nimodipine . given , that in mature animal models of ischemia - reperfusion injury a pathogenic role for mptp has been strongly suggested , more extensive research is needed to clarify the contribution of mptp opening to cerebral hi reperfusion injury in the developing brain . following an ischemic insult mitochondrial membrane permeabilization can occur via opening of outer mitochondrial membrane pore ( ommp ) induced by bak / bax translocation into mitochondria . importantly , in hi reperfusion injury to the developing brain bax dependent ommp has been suggested as a primary mechanism of injury ( , reviewed in ) . developmental shift toward a priority of the bax - dependent ommp over the cyclophylin - d dependent mptp opening in the hi brain damage has been supported by the data obtained in cyclophilin d knock - out neonatal mice , as well as by neuroprotective effect of bax - inhibiting peptide . however , in contrast to a better understanding of events leading to secondary energy failure and necrotic cell death following an opening of mptp , it is less clear how bax / bak mediated ommp opening affects oxidative phosphorylation and results in secondary energy failure and necrosis . one possibility is that postischemic opening of ommp results in a massive loss of cytochrome c from the inter - membrane mitochondrial space which results in secondary inhibition of oxidative phosphorylation . however , this loss of cytochrome c was not mediated by mptp opening , and was not associated with changes in mitochondrial bax , bad , bak or bid . although , mitochondrial ros appeared to be critical for the execution of bax / bak dependent apoptosis induced by anti - cancer drugs [ 88 , 89 ] , we have not found data that ros originating in mitochondria are involved in the bax / bak - induced apoptosis in hi brain injury . interestingly , oxidative stress - induced cell apoptosis clearly required the presence of ros originating from mrc to signal mptp opening , but this apoptosis was independent of bax translocation . the existence of two relatively independent mechanisms of mitochondrial membrane permeabilization does not exclude the contribution of each of these mechanisms in hi damage to the developing brain . indeed , there is evidence for involvement of cyclophilin d dependent mptp opening in the bax - driven cytochrome c release in the isolated mitochondria . in conclusion , the analysis of current data supports the hypothesis that in the developing hi brain reoxygenation / reperfusion causes not only recovery of cell bioenergetics , but also accelerates ros generation in mitochondrial respiratory chain ( figures 3(a ) and 3(b ) ) . this damage occurs in the forms of mptp and bax / bak dependent outer membrane pores , both of which are considered as a point of no return in the evolution of hi injury . with data that complex i contributes to accelerated generation of ros during reperfusion , a novel neuroprotective strategy against reperfusion - driven mitochondrial membrane permeabilization may consist of reversible pharmacological inhibition of complex i recovery following hi insult ( figure 3(c ) ) .

the main literature search was conducted using medline , through ovid online , and the strategy was as follows : ( maternal age or maternal age ) and ( diabetes mellitus , type 1 or [ diabetes and type 1 ] or iddm ) using the terms in inverted commas as medline subject heading key words . finally , to identify studies that investigated maternal age along with other risk factors , a more general search was conducted on medline using the following : ( diabetes mellitus , type 1 and [ case - control studies or cohort studies [ ) . ) to establish whether the studies were likely to provide relevant data based on the following inclusion criteria : 1 ) they identified a group with type 1 diabetes and a group without type 1 diabetes , and 2 ) they recorded maternal age in these groups . studies were excluded if they contained fewer than 100 case subjects ( because adjustments for confounders may not perform well in these studies ) or if they were family based ( because the association between maternal age and type 1 diabetes could be distorted through selecting control subjects from uncompleted families and from among families with an increased genetic susceptibility ) . citations generated from the more general medline search were initially screened to remove obviously irrelevant articles . finally , the reference lists of all pertinent articles were hand searched and the corresponding author of each included article was asked whether they were aware of any additional studies . an author from each included study was contacted to provide raw datasets , or estimates from prespecified analyses , for the association between maternal age ( in categories : < 20 , 2024 , 2529 , 3034 , 35 years ) and type 1 diabetes before and after adjustments for potential confounders ( if available ) . authors were contacted because categorizations ( and adjustments ) differed in published reports and some authors did not present any maternal age data , merely reporting findings . details of included studies ( reported in table 1 ) were extracted by one reviewer ( c.r.c . ) and agreed with the study author . ors and ses were calculated for the association between each category of maternal age and type 1 diabetes for each study . similarly , to investigate the trend across categories of maternal age , an or ( and se ) was calculated per increase in category ( corresponding to an approximate 5-year increase in maternal age ) using regression models appropriate to the design of the study . unconditional and conditional logistic regression was used to calculate the ors and ses for the unmatched and matched case - control studies , respectively . in cohort studies with various lengths of participant follow - up , poisson regression was used to estimate rate ratios and their ses as a measures of association ( which should be approximately equal to ors for a rare disease such as type 1 diabetes ) . a year of birth term was added to poisson regression models to adjust the rate ratios for any differences in year of birth between case and control subjects resulting from this study design . combinations of other potential confounders were added as covariates in the regression models for each study , before random - effects models were used to calculate pooled ors ( 17 ) . tests for heterogeneity were conducted and the i statistic was calculated to quantify the degree of heterogeneity between studies . publication / selection bias was investigated by checking for asymmetry in funnel plots of the study ors against the se of the logarithm of the ors . method was used to estimate the number of studies averaging no effect that would be required to bring the overall result to nonsignificance ( 18 ) . meta regression techniques ( 18 ) were used to investigate whether any association between maternal age and diabetes varied by year of publication or response rates in case and control subjects ( because young mothers may be less likely to respond , which could bias results if case and control subjects ' response rates differed ) . subgroup analyses were conducted by subdividing studies by type and including only studies with a reduced risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects or any study with a response rate of less than 80% in either the case or control subjects ) . a final sensitivity analysis was conducted including studies in which the required estimates could only be approximated from published reports . in one study ( 19 ) , the odds ratio per 5-year increase in maternal age was extrapolated from the odds ratio per 1-year increase , combined between males and females , and was available only after adjustment for number of abortions and gestational age . in another ( 20 ) , the odds ratio per 5-year increase was estimated from the following maternal age categories ( 1521 , 2231 , 3241 , 4249 , 5055 years ) . all statistical analyses were performed using stata 9.0 ( stata , college station , tx ) . the main literature search was conducted using medline , through ovid online , and the strategy was as follows : ( maternal age or maternal age ) and ( diabetes mellitus , type 1 or [ diabetes and type 1 ] or iddm ) using the terms in inverted commas as medline subject heading key words . finally , to identify studies that investigated maternal age along with other risk factors , a more general search was conducted on medline using the following : ( diabetes mellitus , type 1 and [ case - control studies or cohort studies [ ) . ) to establish whether the studies were likely to provide relevant data based on the following inclusion criteria : 1 ) they identified a group with type 1 diabetes and a group without type 1 diabetes , and 2 ) they recorded maternal age in these groups . studies were excluded if they contained fewer than 100 case subjects ( because adjustments for confounders may not perform well in these studies ) or if they were family based ( because the association between maternal age and type 1 diabetes could be distorted through selecting control subjects from uncompleted families and from among families with an increased genetic susceptibility ) . citations generated from the more general medline search were initially screened to remove obviously irrelevant articles . finally , the reference lists of all pertinent articles were hand searched and the corresponding author of each included article was asked whether they were aware of any additional studies . an author from each included study was contacted to provide raw datasets , or estimates from prespecified analyses , for the association between maternal age ( in categories : < 20 , 2024 , 2529 , 3034 , 35 years ) and type 1 diabetes before and after adjustments for potential confounders ( if available ) . authors were contacted because categorizations ( and adjustments ) differed in published reports and some authors did not present any maternal age data , merely reporting findings . details of included studies ( reported in table 1 ) were extracted by one reviewer ( c.r.c . ) and agreed with the study author . ors and ses were calculated for the association between each category of maternal age and type 1 diabetes for each study . similarly , to investigate the trend across categories of maternal age , an or ( and se ) was calculated per increase in category ( corresponding to an approximate 5-year increase in maternal age ) using regression models appropriate to the design of the study . unconditional and conditional logistic regression was used to calculate the ors and ses for the unmatched and matched case - control studies , respectively . in cohort studies with various lengths of participant follow - up , poisson regression was used to estimate rate ratios and their ses as a measures of association ( which should be approximately equal to ors for a rare disease such as type 1 diabetes ) . a year of birth term was added to poisson regression models to adjust the rate ratios for any differences in year of birth between case and control subjects resulting from this study design . combinations of other potential confounders were added as covariates in the regression models for each study , before random - effects models were used to calculate pooled ors ( 17 ) . tests for heterogeneity were conducted and the i statistic was calculated to quantify the degree of heterogeneity between studies . publication / selection bias was investigated by checking for asymmetry in funnel plots of the study ors against the se of the logarithm of the ors . method was used to estimate the number of studies averaging no effect that would be required to bring the overall result to nonsignificance ( 18 ) . meta regression techniques ( 18 ) were used to investigate whether any association between maternal age and diabetes varied by year of publication or response rates in case and control subjects ( because young mothers may be less likely to respond , which could bias results if case and control subjects ' response rates differed ) . subgroup analyses were conducted by subdividing studies by type and including only studies with a reduced risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects or any study with a response rate of less than 80% in either the case or control subjects ) . a final sensitivity analysis was conducted including studies in which the required estimates could only be approximated from published reports . in one study ( 19 ) , the odds ratio per 5-year increase in maternal age was extrapolated from the odds ratio per 1-year increase , combined between males and females , and was available only after adjustment for number of abortions and gestational age . in another ( 20 ) , the odds ratio per 5-year increase was estimated from the following maternal age categories ( 1521 , 2231 , 3241 , 4249 , 5055 years ) . all statistical analyses were performed using stata 9.0 ( stata , college station , tx ) . thirty - four of these articles were excluded because they contained duplicate or overlapped information . twelve articles were excluded because they contained information on fewer than 100 case subjects ; 11 articles were excluded because they used family - based designs . a full list of the articles identified by the searches is available from the authors . the remaining 32 articles ( 915,1943 ) contained information from 37 independent studies , as information from five centers was taken from one article ( 14 ) and information from two centers was taken from another ( 15 ) . an investigator from each of the 37 studies was invited to provide raw data ( or estimates from prespecified analyses ) , but one author ( 20 ) could not be contacted . table 1 contains the characteristics of 32 studies included in the analysis . in 25 of these studies , full datasets were obtained and in four ( 12,13,31,33 ) estimates according to prespecified models were calculated by the study authors from the full datasets ( in one the required data were extracted directly from the published report , and in two others the required data could only be approximated and so were included only in sensitivity analyses , discussed later ) . characteristics of included studies investigating the association between maternal age and type 1 diabetes , ordered by publication date * year of publication . # duration of breast - feeding used in adjusted analysis shown in brackets . * * only included in sensitivity analyses . dx , diagnosis ; resp , response ; bf , breast - feeding ( in months ) ; bo , birth order ; bw , birth weight ; c - c , case - control ; cs , cesarean section ; ga , gestational age ; hosp . , hospital ; md , maternal diabetes ; na , not applicable . the associations between maternal age at delivery and type 1 diabetes from the 30 included studies ( with 14,724 cases of type 1 diabetes ) are shown in fig . 1 . overall , for each 5-year increase in maternal age at delivery the odds of a child subsequently developing type 1 diabetes increased by on average 5% ( or 1.05 [ 95% ci 1.021.09 ] ; p = 0.009 ) . there was , however , marked heterogeneity between studies ( i = 70 , heterogeneity p < 0.001 ) . table 2 shows the unadjusted association between maternal age at delivery and type 1 diabetes by category of maternal age . children whose mothers were older than 35 years had on average a 10% increase ( or 1.10 [ 95% ci 1.011.20 ] ; p = 0.03 ) in type 1 diabetes odds compared with children whose mothers were 2530 years , and there was little evidence of heterogeneity among studies ( i = 20 , heterogeneity p = 0.16 ) . similarly , although not statistically significant ( p = 0.20 ) , children whose mothers were younger than 20 years had on average a 12% reduction ( or 0.88 [ 95% ci 0.741.04 ] ) in type 1 diabetes odds compared with children whose mothers were 2530 years , but there was evidence of marked heterogeneity among studies ( i = 64 , heterogeneity p < 0.001 ) . meta - analysis of the unadjusted association between maternal age ( per 5-year increase ) and type 1 diabetes ( including 14,724 case subjects ) using the random effects model ; studies are ordered by publication date . meta - analyses of 30 studies investigating the association between maternal age and type 1 diabetes before and after adjustments for recorded confounders and in subgroups defined by study type and quality * includes only studies for which adjustments for birth weight ( in categories < 2.5 , 2.53 , 33.5 , 24.5 , > 4.5 kg ) , gestational age ( in categories 37 , 3841 , 42 weeks ) , and birth order ( in categories first , second , or third born or later ) could be made . excluding case - control studies that have control subjects who were not randomly selected ( or population based ) or studies in which the response rate in either the case or control subjects was less than 80% ( or unknown ) as shown in table 1 . an additional unadjusted analysis ( in 26 studies with available data ) indicated that , compared with children born to mothers aged 2530 years , children born to mothers aged 3540 years had a 12% increase in the odds of diabetes ( or 1.12 [ 95% ci 1.021.23 ] ; p = 0.02 ) , whereas children born to mothers older than 40 years had a 9% increase in the odds of diabetes ( or 1.09 [ 95% ci 0.981.21 ] ; p = 0.11 ) . funnel plots of the association between maternal age and odds of type 1 diabetes were investigated ( not shown ) and roughly conformed to the expected funnel shape , providing little evidence of asymmetry and therefore little evidence of publication bias . applying rosenthal 's file drawer method , 205 studies averaging no association between maternal age and type 1 diabetes would need to have been conducted but not published ( or identified by the searches ) to bring the pooled or , of 1.05 per 5-year increase , to nonsignificance . table 2 also shows the findings for maternal age analysis after adjustment for potential confounders . the association between type 1 diabetes and maternal age was little altered after adjustment for birth order , birth weight , and gestational age , in 20 studies in which these variables were available . in 30 studies , adjustments were made for all available confounders , which also included breast - feeding , cesarean section , and maternal diabetes for some studies ( see table 1 for information on the confounders available in each study ) , and again the findings were little altered . there was evidence that some of the heterogeneity in the association between maternal age and diabetes could be explained by differences in response rates between case and control subjects ( shown in table 1 ) . figure 2 shows that studies in which control subjects had a lower response rate than case subjects were less likely to observe an increase in diabetes risk with maternal age , whereas studies in which case subjects had a lower response rate than control subjects observed more marked increases in diabetes risk with maternal age ( meta - regression slope p = 0.02 ) . there was an estimated 6% increase ( or 1.06 [ 95% ci 1.021.10 ] ) in diabetes odds per 5-year increase in maternal age when the response rates in the case and control subjects were equal ( obtained from the intercept of the fitted meta - regression slope shown in fig . the association between maternal age and diabetes varied by the response rate in the control subjects as studies with lower control response rates observed weaker associations with maternal age ( meta - regression slope p = 0.004 ) . there was no evidence of any association between the odds of diabetes per 5-year increase in maternal age and publication year ( meta - regression slope p = 0.43 ) or the midyear of case subject recruitment in each study ( meta - regression slope p = 0.27 ) . scatter plot of odds ratio for diabetes per 5-year increase in maternal age by difference in response rates between case and control subjects ( size of plotting symbol was proportional to precision of study ; line was taken from meta - regression ) . the main findings were similar in cohort and case - control studies , showing a 6 and 5% increase in type 1 diabetes odds per 5-year increase in maternal age , respectively , and both showing marked heterogeneity ( i = 69 and i = 72 , respectively ) . a separate analysis , contained in table 2 , included only studies with a low risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects and excluding studies with a response rate of less than 80% in either the case or control group ) . overall , in the 14 studies with a low risk of bias there was a more marked increase in type 1 diabetes odds of 10% ( or 1.10 [ 95% ci 1.061.14 ] ) per 5-year increase in maternal age . there was also slightly less between - study heterogeneity , particularly when analysis was considered by category of maternal age . there was little evidence of a difference in the association between childhood type 1 diabetes and maternal age in early diagnosed diabetes ( i.e. , younger than 5 years ) and later diagnosed diabetes ( i.e. , between 5 and 15 years ) in 23 studies in which these data were available . specifically , for each 5-year increase in maternal age , there was on average a 5% ( or 1.05 [ 95% ci 1.001.10 ] ) increase in early diagnosed disease and a 7% ( or 1.07 [ 95% ci 1.011.13 ] ) increase in later diagnosed disease . also , there was little evidence of a difference in the association with maternal age by birth order in 21 studies for which these data were available . in first borns , there was an 8% ( or 1.08 [ 95% ci 0.991.17 ] ) increase in diabetes odds for each 5-year increase in maternal age , in second borns there was a 12% ( or 1.12 [ 95% ci 1.031.22 ] ) increase in odds for each 5-year increase , and in third or later borns there was a 9% ( or 1.09 [ 95% ci 1.001.19 ] ) increase in odds for each 5-year increase . there were seven studies ( 1924,27 ) that could not be included in the main analysis . a final sensitivity analysis was conducted , including two of these studies for which the required data could be approximated from published reports ( 19,20 ) . the inclusion of the danish study ( 19 ) had little impact on the findings ( overall or 1.06 , i = 71 ) . however the further addition of the sardinian study ( 20 ) led to a marked increase in the combined odds of diabetes per 5-year increase in maternal age ( overall or 1.11 [ 95% ci 1.041.18 ] ) and a marked increase in the heterogeneity of the results ( i = 92 ) . this was because the results of the sardinian study ( 20 ) were markedly different from every other study in the review , as the researchers observed an 4.5-fold increase ( or 4.5 [ 95% ci 3.855.31 ] ) in diabetes odds per 5-year increase in maternal age , primarily because more than 89% of case subjects in sardinia had mothers older than 32 years at birth , compared with less than 31% in the 30 studies in the main analysis . there were five studies ( 2124,27 ) from which data could not be obtained from authors ( or extracted from the published reports ) . one from colorado ( 21 ) ( including 268 case subjects ) observed a similar proportion of mothers of case and control subjects older than 30 years ( 25 versus 22% , respectively ) , whereas another from colorado ( 24 ) ( containing 221 case subjects , some of whom may have been in the earlier study ) observed a similar mean maternal age in case compared with control subjects ( 26 vs. 27 years , respectively ) . a hungarian study ( 23 ) ( containing 163 case subjects ) also showed a similar mean maternal age in case compared with control subjects ( 26 vs. 27 years ) . a finnish study ( including 750 case subjects ) ( 27 ) reported no difference between the diabetic subjects and the control subjects in any of the ... neonatal variables [ which included age of the mother ( < 30 versus 30 years ) ] . finally , an australian study ( including 217 case subjects ) ( 22 ) also showed a similar median maternal age in case and control subjects ( 26 vs. 27 years , respectively ) . thirty - four of these articles were excluded because they contained duplicate or overlapped information . twelve articles were excluded because they contained information on fewer than 100 case subjects ; 11 articles were excluded because they used family - based designs . a full list of the articles identified by the searches is available from the authors . the remaining 32 articles ( 915,1943 ) contained information from 37 independent studies , as information from five centers was taken from one article ( 14 ) and information from two centers was taken from another ( 15 ) . an investigator from each of the 37 studies was invited to provide raw data ( or estimates from prespecified analyses ) , but one author ( 20 ) could not be contacted . table 1 contains the characteristics of 32 studies included in the analysis . in 25 of these studies , full datasets were obtained and in four ( 12,13,31,33 ) estimates according to prespecified models were calculated by the study authors from the full datasets ( in one the required data were extracted directly from the published report , and in two others the required data could only be approximated and so were included only in sensitivity analyses , discussed later ) . characteristics of included studies investigating the association between maternal age and type 1 diabetes , ordered by publication date * year of publication . # duration of breast - feeding used in adjusted analysis shown in brackets . * * only included in sensitivity analyses . dx , diagnosis ; resp , response ; bf , breast - feeding ( in months ) ; bo , birth order ; bw , birth weight ; c - c , case - control ; cs , cesarean section ; ga , gestational age ; hosp . , hospital ; md , maternal diabetes ; na , not applicable . the associations between maternal age at delivery and type 1 diabetes from the 30 included studies ( with 14,724 cases of type 1 diabetes ) are shown in fig . 1 . overall , for each 5-year increase in maternal age at delivery the odds of a child subsequently developing type 1 diabetes increased by on average 5% ( or 1.05 [ 95% ci 1.021.09 ] ; p = 0.009 ) . there was , however , marked heterogeneity between studies ( i = 70 , heterogeneity p < 0.001 ) . table 2 shows the unadjusted association between maternal age at delivery and type 1 diabetes by category of maternal age . children whose mothers were older than 35 years had on average a 10% increase ( or 1.10 [ 95% ci 1.011.20 ] ; p = 0.03 ) in type 1 diabetes odds compared with children whose mothers were 2530 years , and there was little evidence of heterogeneity among studies ( i = 20 , heterogeneity p = 0.16 ) . similarly , although not statistically significant ( p = 0.20 ) , children whose mothers were younger than 20 years had on average a 12% reduction ( or 0.88 [ 95% ci 0.741.04 ] ) in type 1 diabetes odds compared with children whose mothers were 2530 years , but there was evidence of marked heterogeneity among studies ( i = 64 , heterogeneity p < 0.001 ) . meta - analysis of the unadjusted association between maternal age ( per 5-year increase ) and type 1 diabetes ( including 14,724 case subjects ) using the random effects model ; studies are ordered by publication date . meta - analyses of 30 studies investigating the association between maternal age and type 1 diabetes before and after adjustments for recorded confounders and in subgroups defined by study type and quality * includes only studies for which adjustments for birth weight ( in categories < 2.5 , 2.53 , 33.5 , 24.5 , > 4.5 kg ) , gestational age ( in categories 37 , 3841 , 42 weeks ) , and birth order ( in categories first , second , or third born or later ) could be made . excluding case - control studies that have control subjects who were not randomly selected ( or population based ) or studies in which the response rate in either the case or control subjects was less than 80% ( or unknown ) as shown in table 1 . an additional unadjusted analysis ( in 26 studies with available data ) indicated that , compared with children born to mothers aged 2530 years , children born to mothers aged 3540 years had a 12% increase in the odds of diabetes ( or 1.12 [ 95% ci 1.021.23 ] ; p = 0.02 ) , whereas children born to mothers older than 40 years had a 9% increase in the odds of diabetes ( or 1.09 [ 95% ci 0.981.21 ] ; p = 0.11 ) . funnel plots of the association between maternal age and odds of type 1 diabetes were investigated ( not shown ) and roughly conformed to the expected funnel shape , providing little evidence of asymmetry and therefore little evidence of publication bias . applying rosenthal 's file drawer method , 205 studies averaging no association between maternal age and type 1 diabetes would need to have been conducted but not published ( or identified by the searches ) to bring the pooled or , of 1.05 per 5-year increase , to nonsignificance . table 2 also shows the findings for maternal age analysis after adjustment for potential confounders . the association between type 1 diabetes and maternal age was little altered after adjustment for birth order , birth weight , and gestational age , in 20 studies in which these variables were available . in 30 studies , adjustments were made for all available confounders , which also included breast - feeding , cesarean section , and maternal diabetes for some studies ( see table 1 for information on the confounders available in each study ) , and again the findings were little altered . there was evidence that some of the heterogeneity in the association between maternal age and diabetes could be explained by differences in response rates between case and control subjects ( shown in table 1 ) . figure 2 shows that studies in which control subjects had a lower response rate than case subjects were less likely to observe an increase in diabetes risk with maternal age , whereas studies in which case subjects had a lower response rate than control subjects observed more marked increases in diabetes risk with maternal age ( meta - regression slope p = 0.02 ) . there was an estimated 6% increase ( or 1.06 [ 95% ci 1.021.10 ] ) in diabetes odds per 5-year increase in maternal age when the response rates in the case and control subjects were equal ( obtained from the intercept of the fitted meta - regression slope shown in fig . the association between maternal age and diabetes varied by the response rate in the control subjects as studies with lower control response rates observed weaker associations with maternal age ( meta - regression slope p = 0.004 ) . there was no evidence of any association between the odds of diabetes per 5-year increase in maternal age and publication year ( meta - regression slope p = 0.43 ) or the midyear of case subject recruitment in each study ( meta - regression slope p = 0.27 ) . scatter plot of odds ratio for diabetes per 5-year increase in maternal age by difference in response rates between case and control subjects ( size of plotting symbol was proportional to precision of study ; line was taken from meta - regression ) . the main findings were similar in cohort and case - control studies , showing a 6 and 5% increase in type 1 diabetes odds per 5-year increase in maternal age , respectively , and both showing marked heterogeneity ( i = 69 and i = 72 , respectively ) . a separate analysis , contained in table 2 , included only studies with a low risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects and excluding studies with a response rate of less than 80% in either the case or control group ) . overall , in the 14 studies with a low risk of bias there was a more marked increase in type 1 diabetes odds of 10% ( or 1.10 [ 95% ci 1.061.14 ] ) per 5-year increase in maternal age . there was also slightly less between - study heterogeneity , particularly when analysis was considered by category of maternal age . there was little evidence of a difference in the association between childhood type 1 diabetes and maternal age in early diagnosed diabetes ( i.e. , younger than 5 years ) and later diagnosed diabetes ( i.e. , between 5 and 15 years ) in 23 studies in which these data were available . specifically , for each 5-year increase in maternal age , there was on average a 5% ( or 1.05 [ 95% ci 1.001.10 ] ) increase in early diagnosed disease and a 7% ( or 1.07 [ 95% ci 1.011.13 ] ) increase in later diagnosed disease . also , there was little evidence of a difference in the association with maternal age by birth order in 21 studies for which these data were available . in first borns , there was an 8% ( or 1.08 [ 95% ci 0.991.17 ] ) increase in diabetes odds for each 5-year increase in maternal age , in second borns there was a 12% ( or 1.12 [ 95% ci 1.031.22 ] ) increase in odds for each 5-year increase , and in third or later borns there was a 9% ( or 1.09 [ 95% ci 1.001.19 ] ) increase in odds for each 5-year increase . there were seven studies ( 1924,27 ) that could not be included in the main analysis . a final sensitivity analysis was conducted , including two of these studies for which the required data could be approximated from published reports ( 19,20 ) . the inclusion of the danish study ( 19 ) had little impact on the findings ( overall or 1.06 , i = 71 ) . however the further addition of the sardinian study ( 20 ) led to a marked increase in the combined odds of diabetes per 5-year increase in maternal age ( overall or 1.11 [ 95% ci 1.041.18 ] ) and a marked increase in the heterogeneity of the results ( i = 92 ) . this was because the results of the sardinian study ( 20 ) were markedly different from every other study in the review , as the researchers observed an 4.5-fold increase ( or 4.5 [ 95% ci 3.855.31 ] ) in diabetes odds per 5-year increase in maternal age , primarily because more than 89% of case subjects in sardinia had mothers older than 32 years at birth , compared with less than 31% in the 30 studies in the main analysis . there were five studies ( 2124,27 ) from which data could not be obtained from authors ( or extracted from the published reports ) . one from colorado ( 21 ) ( including 268 case subjects ) observed a similar proportion of mothers of case and control subjects older than 30 years ( 25 versus 22% , respectively ) , whereas another from colorado ( 24 ) ( containing 221 case subjects , some of whom may have been in the earlier study ) observed a similar mean maternal age in case compared with control subjects ( 26 vs. 27 years , respectively ) . a hungarian study ( 23 ) ( containing 163 case subjects ) also showed a similar mean maternal age in case compared with control subjects ( 26 vs. 27 years ) . a finnish study ( including 750 case subjects ) ( 27 ) reported no difference between the diabetic subjects and the control subjects in any of the ... neonatal variables [ which included age of the mother ( < 30 versus 30 years ) ] . finally , an australian study ( including 217 case subjects ) ( 22 ) also showed a similar median maternal age in case and control subjects ( 26 vs. 27 years , respectively ) . this review provides evidence that children born to older mothers have an increased risk of childhood type 1 diabetes . on average , the risk of childhood diabetes increased by 5% for each 5-year increase in maternal age but this association varied between studies . some of this variation could be explained by the response rates of included studies , possibly due to the lack of participation of younger mothers , particularly in control subjects . in studies with a low risk of bias , there was a more marked increase in diabetes risk of 10% per 5-year increase in maternal age . the observed association between maternal age and diabetes could not be explained by birth order , birth weight , gestational age , cesarean section delivery , maternal diabetes , or breast - feeding . this is , to our knowledge , the first systematic review and meta - analysis of the association between maternal age at birth and risk of type 1 diabetes in children . a major strength of this review is that it contains data from up to 14,724 case subjects from 30 studies , of which 29 supplied individual patient data or conducted prespecified analyses , allowing a unified analytic approach and additional analyses to investigate potential sources of bias . although no data were available from 5 ( 2124,27 ) of the 37 identified studies , most were relatively small and unlikely to alter the overall estimates by much . furthermore , the results of these studies are largely consistent with the review findings . despite little evidence from the funnel plots , there remains the possibility of publication bias ( that studies showing no association were conducted but not published ) . also , although our search strategy was comprehensive , studies containing relevant data may not have been identified . however , there would have to be many such studies or the studies would have to be large and to have observed markedly different associations to influence our overall findings . the observed variation in the association between maternal age and childhood type 1 diabetes between studies could be due to real differences in different populations or biases specific to each study . it has previously been suggested that the nonparticipation of younger mothers in studies of maternal age and childhood disease can induce bias if case and control subjects ' response rates differ ( 44 ) . for studies with a low control subject and high case subject response rate ( right side of fig . 2 ) , the age of control mothers included in the study will be artificially increased ( biases upward ) if young mothers tend not to participate . consequently , a true positive association between the disease and maternal age will be underestimated . the opposite bias occurs if there is a high control subject and low case subject response rate ( left side of fig . this nonresponse bias explains some of the variation in the association between maternal age and diabetes among studies . however , even in studies with a lower risk of this and other biases ( due to higher response rates and randomly selected control subjects ) , there remained some heterogeneity . interestingly , in studies with a low risk of bias there was a more marked increase in diabetes risk in older mothers of around 10% per 5-year increase . the mechanism behind the increased risk of childhood type 1 diabetes in children born to older mothers is unclear . it is likely that maternal age is only a marker of some other factor more directly related to the risk of type 1 diabetes in children . studies ( 4,45 ) have shown that older maternal age at delivery can lead to preterm births and low - birth - weight babies , but because we were able to adjust for these factors their involvement is unlikely . higher maternal age may be a result of longer maternal education , and consequently higher social class , but previous studies have shown conflicting results for the association between type 1 diabetes risk and status ( 11,12,25,41 ) . the offspring of older mothers may also be less likely to be breast - fed , or may be breast - fed for a shorter period , which may increase diabetes risk , but adjustments for breast - feeding had little impact on the observed association . although children with older mothers are more likely to have older fathers , there is no clear association between paternal age at delivery and type 1 diabetes ( 10,11,19,28,34 ) . alternatively , previous studies have suggested that maternal age may be a marker for accumulated exposures , such as infections or environmental toxins ( 13 ) . another study speculated that older age at delivery may be associated with increased maturation of the immune system in the offspring , potentially increasing predisposition to type 1 diabetes in later life ( 46 ) . it is also possible that maternal weight , which may increase with maternal age , could be involved , as a recent study found both maternal prepregnancy bmi and maternal weight gain during pregnancy to predict diabetes - associated islet autoimmunity in genetically susceptible children ( 47 ) . chromosomal aberrations are known to be more common in fetuses of mothers of advanced age , but such a mechanism is not known to operate in type 1 diabetes , and does not fit the apparent linear relation with risk of type 1 diabetes across the span of ages . it is possible to speculate that maternal microchimerism may be involved , as a recent study suggests that type 1 diabetic patients have higher levels of maternal microchimerism ( 48 ) , but we are not aware of any data suggesting that maternal microchimerism is related to maternal age at birth . a previous family - based study suggested that the observed increases in the incidence of type 1 diabetes in recent decades could be explained partly by increases in maternal age ( 46 ) , although there were methodological problems in the researchers ' analysis that led their original estimate of the influence of maternal age to be revised downward ( 49 ) . however , using the overall estimates from this meta - analysis , in england and wales there would be only an 2% increase in childhood - onset type 1 diabetes between 1989 and 2003 due solely to increases in maternal age over this period ( based upon national data ) . as registry data indicate that childhood - onset type 1 diabetes in england and wales increased by 55% over this 15-year period ( 7 ) , it is clear that maternal age explains hardly any of the increasing incidence and other factors must be responsible . our study suggests that the association between type 1 diabetes and maternal age is similar in children diagnosed younger than 5 and between 5 and 15 years . however , we did not include studies of older type 1 diabetic patients , and a previous study of maternal age in young adults with diabetes did not find much evidence of an association ( 50 ) . in conclusion , there is evidence of a weak but significant relation between age at birth and the risk of type 1 diabetes in children . across the maternal age range , there is an 20% difference in the risk of type 1 diabetes . based upon these estimates , a very small percentage of the increasing incidence of children onset type 1 diabetes could be explained by increasing maternal age .

objectivethe aim if the study was to investigate whether children born to older mothers have an increased risk of type 1 diabetes by performing a pooled analysis of previous studies using individual patient data to adjust for recognized confounders.research design and methodsrelevant studies published before june 2009 were identified from medline , web of science , and embase . authors of studies were contacted and asked to provide individual patient data or conduct prespecified analyses . risk estimates of type 1 diabetes by maternal age were calculated for each study , before and after adjustment for potential confounders . meta - analysis techniques were used to derive combined odds ratios and to investigate heterogeneity among studies.resultsdata were available for 5 cohort and 25 case - control studies , including 14,724 cases of type 1 diabetes . overall , there was , on average , a 5% ( 95% ci 29 ) increase in childhood type 1 diabetes odds per 5-year increase in maternal age ( p = 0.006 ) , but there was heterogeneity among studies ( heterogeneity i2 = 70% ) . in studies with a low risk of bias , there was a more marked increase in diabetes odds of 10% per 5-year increase in maternal age . adjustments for potential confounders little altered these estimates.conclusionsthere was evidence of a weak but significant linear increase in the risk of childhood type 1 diabetes across the range of maternal ages , but the magnitude of association varied between studies . a very small percentage of the increase in the incidence of childhood type 1 diabetes in recent years could be explained by increases in maternal age .

RESEARCH DESIGN AND METHODS Literature search. Statistical analysis. RESULTS Search results. Overall findings. Investigation of heterogeneity. Association by age at diagnosis and by birth order. Other studies. DISCUSSION

studies were excluded if they contained fewer than 100 case subjects ( because adjustments for confounders may not perform well in these studies ) or if they were family based ( because the association between maternal age and type 1 diabetes could be distorted through selecting control subjects from uncompleted families and from among families with an increased genetic susceptibility ) . an author from each included study was contacted to provide raw datasets , or estimates from prespecified analyses , for the association between maternal age ( in categories : < 20 , 2024 , 2529 , 3034 , 35 years ) and type 1 diabetes before and after adjustments for potential confounders ( if available ) . ors and ses were calculated for the association between each category of maternal age and type 1 diabetes for each study . similarly , to investigate the trend across categories of maternal age , an or ( and se ) was calculated per increase in category ( corresponding to an approximate 5-year increase in maternal age ) using regression models appropriate to the design of the study . combinations of other potential confounders were added as covariates in the regression models for each study , before random - effects models were used to calculate pooled ors ( 17 ) . subgroup analyses were conducted by subdividing studies by type and including only studies with a reduced risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects or any study with a response rate of less than 80% in either the case or control subjects ) . in one study ( 19 ) , the odds ratio per 5-year increase in maternal age was extrapolated from the odds ratio per 1-year increase , combined between males and females , and was available only after adjustment for number of abortions and gestational age . finally , to identify studies that investigated maternal age along with other risk factors , a more general search was conducted on medline using the following : ( diabetes mellitus , type 1 and [ case - control studies or cohort studies [ ) . ) studies were excluded if they contained fewer than 100 case subjects ( because adjustments for confounders may not perform well in these studies ) or if they were family based ( because the association between maternal age and type 1 diabetes could be distorted through selecting control subjects from uncompleted families and from among families with an increased genetic susceptibility ) . an author from each included study was contacted to provide raw datasets , or estimates from prespecified analyses , for the association between maternal age ( in categories : < 20 , 2024 , 2529 , 3034 , 35 years ) and type 1 diabetes before and after adjustments for potential confounders ( if available ) . ors and ses were calculated for the association between each category of maternal age and type 1 diabetes for each study . similarly , to investigate the trend across categories of maternal age , an or ( and se ) was calculated per increase in category ( corresponding to an approximate 5-year increase in maternal age ) using regression models appropriate to the design of the study . combinations of other potential confounders were added as covariates in the regression models for each study , before random - effects models were used to calculate pooled ors ( 17 ) . subgroup analyses were conducted by subdividing studies by type and including only studies with a reduced risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects or any study with a response rate of less than 80% in either the case or control subjects ) . in one study ( 19 ) , the odds ratio per 5-year increase in maternal age was extrapolated from the odds ratio per 1-year increase , combined between males and females , and was available only after adjustment for number of abortions and gestational age . overall , for each 5-year increase in maternal age at delivery the odds of a child subsequently developing type 1 diabetes increased by on average 5% ( or 1.05 [ 95% ci 1.021.09 ] ; p = 0.009 ) . children whose mothers were older than 35 years had on average a 10% increase ( or 1.10 [ 95% ci 1.011.20 ] ; p = 0.03 ) in type 1 diabetes odds compared with children whose mothers were 2530 years , and there was little evidence of heterogeneity among studies ( i = 20 , heterogeneity p = 0.16 ) . similarly , although not statistically significant ( p = 0.20 ) , children whose mothers were younger than 20 years had on average a 12% reduction ( or 0.88 [ 95% ci 0.741.04 ] ) in type 1 diabetes odds compared with children whose mothers were 2530 years , but there was evidence of marked heterogeneity among studies ( i = 64 , heterogeneity p < 0.001 ) . meta - analysis of the unadjusted association between maternal age ( per 5-year increase ) and type 1 diabetes ( including 14,724 case subjects ) using the random effects model ; studies are ordered by publication date . meta - analyses of 30 studies investigating the association between maternal age and type 1 diabetes before and after adjustments for recorded confounders and in subgroups defined by study type and quality * includes only studies for which adjustments for birth weight ( in categories < 2.5 , 2.53 , 33.5 , 24.5 , > 4.5 kg ) , gestational age ( in categories 37 , 3841 , 42 weeks ) , and birth order ( in categories first , second , or third born or later ) could be made . an additional unadjusted analysis ( in 26 studies with available data ) indicated that , compared with children born to mothers aged 2530 years , children born to mothers aged 3540 years had a 12% increase in the odds of diabetes ( or 1.12 [ 95% ci 1.021.23 ] ; p = 0.02 ) , whereas children born to mothers older than 40 years had a 9% increase in the odds of diabetes ( or 1.09 [ 95% ci 0.981.21 ] ; p = 0.11 ) . funnel plots of the association between maternal age and odds of type 1 diabetes were investigated ( not shown ) and roughly conformed to the expected funnel shape , providing little evidence of asymmetry and therefore little evidence of publication bias . the association between type 1 diabetes and maternal age was little altered after adjustment for birth order , birth weight , and gestational age , in 20 studies in which these variables were available . there was evidence that some of the heterogeneity in the association between maternal age and diabetes could be explained by differences in response rates between case and control subjects ( shown in table 1 ) . figure 2 shows that studies in which control subjects had a lower response rate than case subjects were less likely to observe an increase in diabetes risk with maternal age , whereas studies in which case subjects had a lower response rate than control subjects observed more marked increases in diabetes risk with maternal age ( meta - regression slope p = 0.02 ) . there was an estimated 6% increase ( or 1.06 [ 95% ci 1.021.10 ] ) in diabetes odds per 5-year increase in maternal age when the response rates in the case and control subjects were equal ( obtained from the intercept of the fitted meta - regression slope shown in fig . the association between maternal age and diabetes varied by the response rate in the control subjects as studies with lower control response rates observed weaker associations with maternal age ( meta - regression slope p = 0.004 ) . there was no evidence of any association between the odds of diabetes per 5-year increase in maternal age and publication year ( meta - regression slope p = 0.43 ) or the midyear of case subject recruitment in each study ( meta - regression slope p = 0.27 ) . the main findings were similar in cohort and case - control studies , showing a 6 and 5% increase in type 1 diabetes odds per 5-year increase in maternal age , respectively , and both showing marked heterogeneity ( i = 69 and i = 72 , respectively ) . a separate analysis , contained in table 2 , included only studies with a low risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects and excluding studies with a response rate of less than 80% in either the case or control group ) . overall , in the 14 studies with a low risk of bias there was a more marked increase in type 1 diabetes odds of 10% ( or 1.10 [ 95% ci 1.061.14 ] ) per 5-year increase in maternal age . there was little evidence of a difference in the association between childhood type 1 diabetes and maternal age in early diagnosed diabetes ( i.e. specifically , for each 5-year increase in maternal age , there was on average a 5% ( or 1.05 [ 95% ci 1.001.10 ] ) increase in early diagnosed disease and a 7% ( or 1.07 [ 95% ci 1.011.13 ] ) increase in later diagnosed disease . also , there was little evidence of a difference in the association with maternal age by birth order in 21 studies for which these data were available . in first borns , there was an 8% ( or 1.08 [ 95% ci 0.991.17 ] ) increase in diabetes odds for each 5-year increase in maternal age , in second borns there was a 12% ( or 1.12 [ 95% ci 1.031.22 ] ) increase in odds for each 5-year increase , and in third or later borns there was a 9% ( or 1.09 [ 95% ci 1.001.19 ] ) increase in odds for each 5-year increase . however the further addition of the sardinian study ( 20 ) led to a marked increase in the combined odds of diabetes per 5-year increase in maternal age ( overall or 1.11 [ 95% ci 1.041.18 ] ) and a marked increase in the heterogeneity of the results ( i = 92 ) . this was because the results of the sardinian study ( 20 ) were markedly different from every other study in the review , as the researchers observed an 4.5-fold increase ( or 4.5 [ 95% ci 3.855.31 ] ) in diabetes odds per 5-year increase in maternal age , primarily because more than 89% of case subjects in sardinia had mothers older than 32 years at birth , compared with less than 31% in the 30 studies in the main analysis . the associations between maternal age at delivery and type 1 diabetes from the 30 included studies ( with 14,724 cases of type 1 diabetes ) are shown in fig . overall , for each 5-year increase in maternal age at delivery the odds of a child subsequently developing type 1 diabetes increased by on average 5% ( or 1.05 [ 95% ci 1.021.09 ] ; p = 0.009 ) . children whose mothers were older than 35 years had on average a 10% increase ( or 1.10 [ 95% ci 1.011.20 ] ; p = 0.03 ) in type 1 diabetes odds compared with children whose mothers were 2530 years , and there was little evidence of heterogeneity among studies ( i = 20 , heterogeneity p = 0.16 ) . similarly , although not statistically significant ( p = 0.20 ) , children whose mothers were younger than 20 years had on average a 12% reduction ( or 0.88 [ 95% ci 0.741.04 ] ) in type 1 diabetes odds compared with children whose mothers were 2530 years , but there was evidence of marked heterogeneity among studies ( i = 64 , heterogeneity p < 0.001 ) . meta - analysis of the unadjusted association between maternal age ( per 5-year increase ) and type 1 diabetes ( including 14,724 case subjects ) using the random effects model ; studies are ordered by publication date . meta - analyses of 30 studies investigating the association between maternal age and type 1 diabetes before and after adjustments for recorded confounders and in subgroups defined by study type and quality * includes only studies for which adjustments for birth weight ( in categories < 2.5 , 2.53 , 33.5 , 24.5 , > 4.5 kg ) , gestational age ( in categories 37 , 3841 , 42 weeks ) , and birth order ( in categories first , second , or third born or later ) could be made . an additional unadjusted analysis ( in 26 studies with available data ) indicated that , compared with children born to mothers aged 2530 years , children born to mothers aged 3540 years had a 12% increase in the odds of diabetes ( or 1.12 [ 95% ci 1.021.23 ] ; p = 0.02 ) , whereas children born to mothers older than 40 years had a 9% increase in the odds of diabetes ( or 1.09 [ 95% ci 0.981.21 ] ; p = 0.11 ) . funnel plots of the association between maternal age and odds of type 1 diabetes were investigated ( not shown ) and roughly conformed to the expected funnel shape , providing little evidence of asymmetry and therefore little evidence of publication bias . the association between type 1 diabetes and maternal age was little altered after adjustment for birth order , birth weight , and gestational age , in 20 studies in which these variables were available . there was evidence that some of the heterogeneity in the association between maternal age and diabetes could be explained by differences in response rates between case and control subjects ( shown in table 1 ) . figure 2 shows that studies in which control subjects had a lower response rate than case subjects were less likely to observe an increase in diabetes risk with maternal age , whereas studies in which case subjects had a lower response rate than control subjects observed more marked increases in diabetes risk with maternal age ( meta - regression slope p = 0.02 ) . there was an estimated 6% increase ( or 1.06 [ 95% ci 1.021.10 ] ) in diabetes odds per 5-year increase in maternal age when the response rates in the case and control subjects were equal ( obtained from the intercept of the fitted meta - regression slope shown in fig . the association between maternal age and diabetes varied by the response rate in the control subjects as studies with lower control response rates observed weaker associations with maternal age ( meta - regression slope p = 0.004 ) . there was no evidence of any association between the odds of diabetes per 5-year increase in maternal age and publication year ( meta - regression slope p = 0.43 ) or the midyear of case subject recruitment in each study ( meta - regression slope p = 0.27 ) . the main findings were similar in cohort and case - control studies , showing a 6 and 5% increase in type 1 diabetes odds per 5-year increase in maternal age , respectively , and both showing marked heterogeneity ( i = 69 and i = 72 , respectively ) . a separate analysis , contained in table 2 , included only studies with a low risk of bias ( excluding case - control studies with nonpopulation - based or nonrandomly selected control subjects and excluding studies with a response rate of less than 80% in either the case or control group ) . overall , in the 14 studies with a low risk of bias there was a more marked increase in type 1 diabetes odds of 10% ( or 1.10 [ 95% ci 1.061.14 ] ) per 5-year increase in maternal age . there was little evidence of a difference in the association between childhood type 1 diabetes and maternal age in early diagnosed diabetes ( i.e. specifically , for each 5-year increase in maternal age , there was on average a 5% ( or 1.05 [ 95% ci 1.001.10 ] ) increase in early diagnosed disease and a 7% ( or 1.07 [ 95% ci 1.011.13 ] ) increase in later diagnosed disease . also , there was little evidence of a difference in the association with maternal age by birth order in 21 studies for which these data were available . in first borns , there was an 8% ( or 1.08 [ 95% ci 0.991.17 ] ) increase in diabetes odds for each 5-year increase in maternal age , in second borns there was a 12% ( or 1.12 [ 95% ci 1.031.22 ] ) increase in odds for each 5-year increase , and in third or later borns there was a 9% ( or 1.09 [ 95% ci 1.001.19 ] ) increase in odds for each 5-year increase . however the further addition of the sardinian study ( 20 ) led to a marked increase in the combined odds of diabetes per 5-year increase in maternal age ( overall or 1.11 [ 95% ci 1.041.18 ] ) and a marked increase in the heterogeneity of the results ( i = 92 ) . this was because the results of the sardinian study ( 20 ) were markedly different from every other study in the review , as the researchers observed an 4.5-fold increase ( or 4.5 [ 95% ci 3.855.31 ] ) in diabetes odds per 5-year increase in maternal age , primarily because more than 89% of case subjects in sardinia had mothers older than 32 years at birth , compared with less than 31% in the 30 studies in the main analysis . this review provides evidence that children born to older mothers have an increased risk of childhood type 1 diabetes . on average , the risk of childhood diabetes increased by 5% for each 5-year increase in maternal age but this association varied between studies . in studies with a low risk of bias , there was a more marked increase in diabetes risk of 10% per 5-year increase in maternal age . this is , to our knowledge , the first systematic review and meta - analysis of the association between maternal age at birth and risk of type 1 diabetes in children . a major strength of this review is that it contains data from up to 14,724 case subjects from 30 studies , of which 29 supplied individual patient data or conducted prespecified analyses , allowing a unified analytic approach and additional analyses to investigate potential sources of bias . the observed variation in the association between maternal age and childhood type 1 diabetes between studies could be due to real differences in different populations or biases specific to each study . interestingly , in studies with a low risk of bias there was a more marked increase in diabetes risk in older mothers of around 10% per 5-year increase . the mechanism behind the increased risk of childhood type 1 diabetes in children born to older mothers is unclear . it is likely that maternal age is only a marker of some other factor more directly related to the risk of type 1 diabetes in children . chromosomal aberrations are known to be more common in fetuses of mothers of advanced age , but such a mechanism is not known to operate in type 1 diabetes , and does not fit the apparent linear relation with risk of type 1 diabetes across the span of ages . a previous family - based study suggested that the observed increases in the incidence of type 1 diabetes in recent decades could be explained partly by increases in maternal age ( 46 ) , although there were methodological problems in the researchers ' analysis that led their original estimate of the influence of maternal age to be revised downward ( 49 ) . however , using the overall estimates from this meta - analysis , in england and wales there would be only an 2% increase in childhood - onset type 1 diabetes between 1989 and 2003 due solely to increases in maternal age over this period ( based upon national data ) . in conclusion , there is evidence of a weak but significant relation between age at birth and the risk of type 1 diabetes in children . across the maternal age range , there is an 20% difference in the risk of type 1 diabetes . based upon these estimates , a very small percentage of the increasing incidence of children onset type 1 diabetes could be explained by increasing maternal age .

androgen deprivation therapy ( adt ) is often administered to patients with prostate cancer as primary therapy of non - metastatic disease ; however , there still exists a lack of evidence of efficacy and the profile of patients most likely to benefit from intermittent versus continuous therapy . little is known about practice patterns and determinants of intermittent adt use in the usa and other countries . urologists and oncologists from several countries frequently administer adt in treating their patients with non - metastatic prostate cancer . the most common reasons for these participating physicians choosing the type of adt in managing their patients were high - risk criteria , treatment guidelines and patient preferences . the study provides real - world data on the treatment patterns and determinants of intermittent versus continuous use of adt among practitioners from 19 countries , which provides acknowledgement of practice patterns when clinical guidelines are reviewed in the treatment of non - metastatic prostate cancer . prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer death in men in the usa.1 prostate cancer incidence varies more than 25-fold worldwide and represents the second most common cancer among men worldwide with an estimated 1.1 million men diagnosed in 2012 with almost 70% of the cases occurring in more developed regions.25 prostate cancer is androgen - dependent,6 7 which forms the basis for androgen deprivation therapy ( adt ) , usually achieved medically with gonadotropin - releasing hormone ( gnrh ) agonists or antagonists or , to a much less frequent extent , surgically via bilateral orchiectomy ( approximately 13% of adt).8 the benefits of adt are well established when used for palliation of symptomatic metastatic disease or as an adjuvant to radiation therapy for high - risk disease , but it is also very commonly used in other settings without clear evidence of efficacy such as for primary therapy of non - metastatic disease or for biochemical recurrence following initial local therapy.9 10 with its common use , there has been increasing recognition of harmful effects from continuous androgen deprivation.11 the most common side effects of continuous adt include anaemia , hot flashes , sexual dysfunction , cognitive dysfunction , bone loss , bone complications ( eg , fractures ) , metabolic and cardiovascular consequences , fatigue , depression and anxiety.11 12 this appreciation of the detrimental effects of adt has led to much interest in reducing adt exposure during the course of treatment.13 one such alternative therapeutic strategy , initially described in 1986 , is intermittent use of gnrh agonist therapies.14 typically , treatment is discontinued after 69 months of adt or when the prostate - specific antigen ( psa ) reaches its nadir ; adt is resumed when psa rises back to a predetermined higher level . the hormonal recovery that occurs during off - treatment cycles15 16 potentially facilitates responsiveness of tumour cells to treatment and theoretically limits toxicity.13 recent reviews comparing efficacy , side effects , time to castration resistance , overall and cancer - specific survival between intermittent and continuous adt have been summarised ; however , the evidence regarding the trade - offs between the benefits and risks of intermittent adt remains inconclusive.1723 generally the consensus is that overall survival is equivalent between intermittent and continuous adt in most settings . however , concerns remain with high - risk disease , as one of the larger trials did not meet criteria for non - inferiority of an intermittent regimen in men with metastatic cancer.24 although intermittent adt appears to have a modestly beneficial impact on sexual function and hot flashes , event rates in studies to date for serious effects such as fractures and cardiovascular outcomes have been too low to draw definitive conclusions . despite the ongoing uncertainty , little is known about practice patterns and determinants of intermittent adt use in the usa and other countries . the objective of this study was to describe factors related to physicians adt use and choice of intermittent adt regimens for patients with non - metastatic prostate cancer from a detailed international survey of treating urologists and oncologists . an instrument was developed for this study based on expert opinion , clinical consensus and a review of the literature . the initial qualitative development phase ensured data stability , survey feasibility , and optimisation , before developing the quantitative online questionnaire . eighteen 1 hour in - depth interviews ( idis ) were distributed evenly across spain , france and germany , incorporating physician feedback in real time . the idis confirmed appropriateness and accuracy of definitions used within the survey for the patient population with prostate cancer . a pilot phase with nine physicians from six countries was rolled out after being proof - read by native speakers , ensuring survey question accuracy . demographic questions included were year of qualification , specialty type , practice size , setting , region and number of patients with total prostate cancer currently under care . the 19 countries selected for this study represent those with high or increasing incidence of non - metastatic prostate cancer , prevalent use of adt among treating urologists and oncologists , and widespread psa screening . eligible respondents included physicians who were responsible for treatment decisions in patients with non - metastatic prostate cancer , with at least 10 non - metastatic patients seen per month ( and at least 10 treated with adt at the time of the survey ) , representing more than 25% of their patient - related time ( > 15% in nordics , czech republic , the netherlands and the usa ) , and year of qualification in their medical specialty between 1971 and 2009 . numerous recruitment approaches were employed based on country knowledge , specialist recruitment agencies , panel of physicians and other contacts which facilitated a large sample to be achieved within a short timeframe . for example , email invitations to panel physicians ( usa ) , telephone recruitment ( france ) and face - to - face recruitment ( poland ) were carried out . the online survey was completed online by all recruited physicians over a 1-month period ( september 2012 ) . urologist versus oncologist distribution in the sampling for each country was consistent with practice patterns as determined by research partners at the country level and literature.25 for example , radiation oncologists were included in australia as they also prescribe drug therapies such as adt for patients with prostate cancer . adt was defined in the online survey for respondents as treatment using gnrh agonists / antagonists or bilateral orchiectomy ( excluding antiandrogenic agents ) . continuous adt was specifically defined as gnrh treatment for at least 6 months without having a break for more than 3 months at any point since initial gnrh administration , or bilateral orchiectomy . the difference in the delineation between intermittent and continuous adt was the stipulation of an off - treatment period of more than 3 months.13 19 locally advanced disease was considered non - metastatic for the purposes of the survey , which may include limited local lymph node involvement . relapsing or recurring tumours following surgery were also considered to be non - metastatic if the disease had not spread to other organs or bones . all respondents provided informed consent and were incentivised for their time ( eg , using vouchers and money ) . all national laws protecting personal data and guidelines from bodies such as british healthcare business intelligence association ( bhbia ) , world association for market , social and opinion research ( esomar ) , european pharmaceutical market research association ( ephmra ) , and other national codes of market research practice were followed . sas software for windows v.9.4 ( sas institute inc , cary , north carolina , usa ) was used to perform all analyses . an instrument was developed for this study based on expert opinion , clinical consensus and a review of the literature . the initial qualitative development phase ensured data stability , survey feasibility , and optimisation , before developing the quantitative online questionnaire . eighteen 1 hour in - depth interviews ( idis ) were distributed evenly across spain , france and germany , incorporating physician feedback in real time . the idis confirmed appropriateness and accuracy of definitions used within the survey for the patient population with prostate cancer . a pilot phase with nine physicians from six countries was rolled out after being proof - read by native speakers , ensuring survey question accuracy . demographic questions included were year of qualification , specialty type , practice size , setting , region and number of patients with total prostate cancer currently under care . the 19 countries selected for this study represent those with high or increasing incidence of non - metastatic prostate cancer , prevalent use of adt among treating urologists and oncologists , and widespread psa screening . eligible respondents included physicians who were responsible for treatment decisions in patients with non - metastatic prostate cancer , with at least 10 non - metastatic patients seen per month ( and at least 10 treated with adt at the time of the survey ) , representing more than 25% of their patient - related time ( > 15% in nordics , czech republic , the netherlands and the usa ) , and year of qualification in their medical specialty between 1971 and 2009 . numerous recruitment approaches were employed based on country knowledge , specialist recruitment agencies , panel of physicians and other contacts which facilitated a large sample to be achieved within a short timeframe . for example , email invitations to panel physicians ( usa ) , telephone recruitment ( france ) and face - to - face recruitment ( poland ) were carried out . the online survey was completed online by all recruited physicians over a 1-month period ( september 2012 ) . urologist versus oncologist distribution in the sampling for each country was consistent with practice patterns as determined by research partners at the country level and literature.25 for example , radiation oncologists were included in australia as they also prescribe drug therapies such as adt for patients with prostate cancer . adt was defined in the online survey for respondents as treatment using gnrh agonists / antagonists or bilateral orchiectomy ( excluding antiandrogenic agents ) . continuous adt was specifically defined as gnrh treatment for at least 6 months without having a break for more than 3 months at any point since initial gnrh administration , or bilateral orchiectomy . the difference in the delineation between intermittent and continuous adt was the stipulation of an off - treatment period of more than 3 months.13 19 locally advanced disease was considered non - metastatic for the purposes of the survey , which may include limited local lymph node involvement . relapsing or recurring tumours following surgery were also considered to be non - metastatic if the disease had not spread to other organs or bones . all respondents provided informed consent and were incentivised for their time ( eg , using vouchers and money ) . all national laws protecting personal data and guidelines from bodies such as british healthcare business intelligence association ( bhbia ) , world association for market , social and opinion research ( esomar ) , european pharmaceutical market research association ( ephmra ) , and other national codes of market research practice were followed . sas software for windows v.9.4 ( sas institute inc , cary , north carolina , usa ) was used to perform all analyses . for this physician survey , response rates ranged from 1% to 86% ( averaging 5% overall ) using email , fax ( australia ) , and telephone invitations . response rates represent survey completion among invitations sent out , which differed by specialty where higher completion rates were observed for oncologists . response also differed by country or region with lower rates observed for the usa , the netherlands and norway . highest completion rates were achievable using telephone recruitment , such as for participants practicing in eu5 ( range 14% ( france ) to 76% ( uk ) ) , or via face - to - face invitations ( an approach used exclusively in poland ( 86% ) ) . main reasons for exclusion among potentially eligible physicians were not spending more than 25% of their time treating patients with non - metastatic prostate cancer or not treating at least 10 non - metastatic patients with gnrh agents per month . a total of 441 physicians completed the survey from 19 countries ( table 1 ) . most respondents were urologists ( 88% ) , and physicians from 10 countries were strictly urologists . the highest proportion of medical oncologist respondents were from finland , switzerland and sweden ; radiation oncologists were recruited in australia consistent with adt prescribing patterns . physician characteristics by use of continuous or intermittent androgen deprivation therapy ( adt ) among patients with non - metastatic prostate cancer overall , 76% of respondents received their specialty qualification between 1991 and 2009 . forty - seven per cent of treating physicians indicated that they work in a general non - academic setting , nearly identical to the number in teaching / university academic settings ( 46% ) . fifty - four per cent of respondents indicated that they saw at least 40 men diagnosed with non - metastatic prostate cancer per month . survey respondents estimated that 99 177 patients with prostate cancer were under their care , 77% ( 76 386 ) classified as having non - metastatic disease ( figure 1 ) . among patients with non - metastatic prostate cancer under treatment , physicians reported that 38% ( 28 840 ) received adt : 36% ( 27 653 ) received gnrh agents , and 1.6% ( 1187 ) underwent bilateral orchiectomy . among their gnrh - treated patients , 54% were treated continuously ( 6 months without > 3-month interruption ) , 23% for less than 6 months , and 23% were managed with intermittent adt ( figure 2 ) . patients represented by 441 physicians surveyed from 19 countries , depicted in the patient journey from diagnosis to ensuing treatment with adt . adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone . proportion of continuous , intermittent , and limited ( < 6 months ) use of adt among patients with non - metastatic prostate cancer treated with gonadotropin - releasing hormone ( gnrh ) by region and physician type . table 2 provides the percentages of any adt and treatment of at least 6 months duration in the non - metastatic setting by region . the highest proportion of adt use was reported by physicians in eastern europe ( 68% ) , driven by higher rates in hungary ( 82% ) and poland ( 79% ) . treating physicians reported administering adt to 43% of their patients in 5-country europe ( eu5 : france , germany , italy , spain and the uk ) ; however , respondents varied noticeably between france ( 26% ) , the uk ( 55% ) and italy ( 61% ) . us physicians reported any adt use in 34% of their patients , somewhat higher than responses from treating physicians in canada ( 29% ) . adt use among men with non - metastatic prostate cancer according to treating physicians by country or region ( n=441 ) any adt : gnrh agonist / antagonist ( includes both continuous and intermittent use ( figure 1 ) ) or bilateral orchiectomy procedure . nordics : denmark , finland , norway , sweden ; eu5 : france , germany , italy , spain , uk ; eastern europe : czech republic , hungary , poland . adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone . in general , treatment rates of adt were higher among oncologists ( 62% ) versus urologists ( 38% ) , although the proportion of adt given as intermittent therapy was similar ( figure 2 ) . intermittent adt appeared to be most common among australian , canadian , french and german practitioners , representing approximately 35% of their gnrh use . in the usa , intermittent regimens represented 28% of adt prescribed , and physicians from remaining countries prescribed intermittent adt between 5% ( sweden ) and 24% ( italy ) . table 3 presents drivers of the decision to initiate adt based on psa levels , psa doubling time and gleason score . in choosing to initiate gnrh therapy for their patients with non - metastatic prostate cancer , 64% of physicians identified absolute psa levels as one of the main reasons . among respondents who relied on psa values , 81% were inclined to use a gnrh agent if psa was 10 ng / ml , while 45% indicated initiation of gnrh when psa reached 20 ng / ml . more than half ( 58% ) of treating physicians identified psa doubling time as the reason to assess gnrh use , and 78% of these respondents cited using a doubling time of 6 months . sixty - six per cent of treating physicians identified the use of gleason score as a reason to assess gnrh treatment ; of these , 91% specified using a gnrh agent if gleason score was 7 or higher . physician behaviour and motivations for continuous or intermittent adt among patients with non - metastatic prostate cancer adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone ; psa , prostate - specific antigen . with respect to reasons for prescribing continuous versus intermittent adt , physicians indicated ( more than one reason could be selected ) psa levels ( 65% ) , gleason score ( 52% ) and treatment guidelines ( 48% ) as the most common reasons for choosing continuous adt ( figure 3 ) . psa levels ( 54% ) , patient request ( 48% ) , desire to maintain sexual function ( 40% ) , comorbidities ( 38% ) and patient age ( 38% ) were cited most frequently as the reasons for managing patients with non - metastatic prostate cancer with intermittent adt . despite reasons cited for prescribing continuous versus intermittent adt , there were no differences observed with respect to physician characteristics or behaviours such as frequency of psa testing . for this physician survey , response rates ranged from 1% to 86% ( averaging 5% overall ) using email , fax ( australia ) , and telephone invitations . response rates represent survey completion among invitations sent out , which differed by specialty where higher completion rates were observed for oncologists . response also differed by country or region with lower rates observed for the usa , the netherlands and norway . highest completion rates were achievable using telephone recruitment , such as for participants practicing in eu5 ( range 14% ( france ) to 76% ( uk ) ) , or via face - to - face invitations ( an approach used exclusively in poland ( 86% ) ) . main reasons for exclusion among potentially eligible physicians were not spending more than 25% of their time treating patients with non - metastatic prostate cancer or not treating at least 10 non - metastatic patients with gnrh agents per month . a total of 441 physicians completed the survey from 19 countries ( table 1 ) . most respondents were urologists ( 88% ) , and physicians from 10 countries were strictly urologists . the highest proportion of medical oncologist respondents were from finland , switzerland and sweden ; radiation oncologists were recruited in australia consistent with adt prescribing patterns . physician characteristics by use of continuous or intermittent androgen deprivation therapy ( adt ) among patients with non - metastatic prostate cancer overall , 76% of respondents received their specialty qualification between 1991 and 2009 . forty - seven per cent of treating physicians indicated that they work in a general non - academic setting , nearly identical to the number in teaching / university academic settings ( 46% ) . fifty - four per cent of respondents indicated that they saw at least 40 men diagnosed with non - metastatic prostate cancer per month . survey respondents estimated that 99 177 patients with prostate cancer were under their care , 77% ( 76 386 ) classified as having non - metastatic disease ( figure 1 ) . among patients with non - metastatic prostate cancer under treatment , physicians reported that 38% ( 28 840 ) received adt : 36% ( 27 653 ) received gnrh agents , and 1.6% ( 1187 ) underwent bilateral orchiectomy . among their gnrh - treated patients , 54% were treated continuously ( 6 months without > 3-month interruption ) , 23% for less than 6 months , and 23% were managed with intermittent adt ( figure 2 ) . patients represented by 441 physicians surveyed from 19 countries , depicted in the patient journey from diagnosis to ensuing treatment with adt . proportion of continuous , intermittent , and limited ( < 6 months ) use of adt among patients with non - metastatic prostate cancer treated with gonadotropin - releasing hormone ( gnrh ) by region and physician type . table 2 provides the percentages of any adt and treatment of at least 6 months duration in the non - metastatic setting by region . the highest proportion of adt use was reported by physicians in eastern europe ( 68% ) , driven by higher rates in hungary ( 82% ) and poland ( 79% ) . treating physicians reported administering adt to 43% of their patients in 5-country europe ( eu5 : france , germany , italy , spain and the uk ) ; however , respondents varied noticeably between france ( 26% ) , the uk ( 55% ) and italy ( 61% ) . us physicians reported any adt use in 34% of their patients , somewhat higher than responses from treating physicians in canada ( 29% ) . adt use among men with non - metastatic prostate cancer according to treating physicians by country or region ( n=441 ) any adt : gnrh agonist / antagonist ( includes both continuous and intermittent use ( figure 1 ) ) or bilateral orchiectomy procedure . nordics : denmark , finland , norway , sweden ; eu5 : france , germany , italy , spain , uk ; eastern europe : czech republic , hungary , poland . in general , treatment rates of adt were higher among oncologists ( 62% ) versus urologists ( 38% ) , although the proportion of adt given as intermittent therapy was similar ( figure 2 ) . intermittent adt appeared to be most common among australian , canadian , french and german practitioners , representing approximately 35% of their gnrh use . in the usa , intermittent regimens represented 28% of adt prescribed , and physicians from remaining countries prescribed intermittent adt between 5% ( sweden ) and 24% ( italy ) . table 3 presents drivers of the decision to initiate adt based on psa levels , psa doubling time and gleason score . in choosing to initiate gnrh therapy for their patients with non - metastatic prostate cancer , 64% of physicians identified absolute psa levels as one of the main reasons . among respondents who relied on psa values , 81% were inclined to use a gnrh agent if psa was 10 ng / ml , while 45% indicated initiation of gnrh when psa reached 20 ng / ml . more than half ( 58% ) of treating physicians identified psa doubling time as the reason to assess gnrh use , and 78% of these respondents cited using a doubling time of 6 months . sixty - six per cent of treating physicians identified the use of gleason score as a reason to assess gnrh treatment ; of these , 91% specified using a gnrh agent if gleason score was 7 or higher . physician behaviour and motivations for continuous or intermittent adt among patients with non - metastatic prostate cancer adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone ; psa , prostate - specific antigen . with respect to reasons for prescribing continuous versus intermittent adt , physicians indicated ( more than one reason could be selected ) psa levels ( 65% ) , gleason score ( 52% ) and treatment guidelines ( 48% ) as the most common reasons for choosing continuous adt ( figure 3 ) . psa levels ( 54% ) , patient request ( 48% ) , desire to maintain sexual function ( 40% ) , comorbidities ( 38% ) and patient age ( 38% ) were cited most frequently as the reasons for managing patients with non - metastatic prostate cancer with intermittent adt . despite reasons cited for prescribing continuous versus intermittent adt , there were no differences observed with respect to physician characteristics or behaviours such as frequency of psa testing . this international survey provides a detailed understanding of how adt is prescribed among patients with non - metastatic prostate cancer under the care of 441 treating physicians from 19 countries . respondents indicated that adt was prescribed for 38% ( range 2568% ) of their patients with non - metastatic prostate cancer , and mainly related to prognostic indicators ( gleason score , psa and psa doubling time ) or on signs of disease progression or recurrence manifested by rising psa values after initial or primary therapy . physicians from the usa reported that 34% of their patients were treated with adt ; a treatment rate lower than previously reported in the literature , but may suggest that the decreasing trend in adt following medicare reimbursement policy changes in 2004 and 2005 continues , resulting in overall lower use by 2012.8 the use of adt differed by region , with highest rates reported in eastern europe . we also found that among patients receiving gnrh agonists , roughly a quarter were prescribed intermittent adt , suggesting that the use of this strategy is relatively common , although rates of intermittent gnrh varied substantially by region . guidelines for the treatment of prostate cancer also differ by region . in the usa , the american urological association ( aua ) guidelines do not address intermittent use of adt ( http://www.auanet.org/education/guidelines/ ) . the updated national comprehensive cancer network ( nccn ) clinical practice guidelines ( prostate cancer ) ( http://www.nccn.org/professionals/physician_gls/pdf/prostate.pdf ) recommend intermittent adt for patients with biochemical failure and without metastases based on a clinical trial showing that overall survival was non - inferior versus continuous adt ( ncic pr-7 trial ) , and intermittent use is not recommended for metastatic patients.26 the european association of urology ( eau ) guidelines recommend intermittent adt for asymptomatic metastatic patients citing a different set of clinical trials24 2730 that did not show significant differences in overall survival between continuous and intermittent adt , also citing patient acceptability , quality of life and fewer toxicities such as effects on cardiovascular or bone health.24 31 surveyed physicians noted that intermittent use was primarily driven by psa levels , patient request and patient age / comorbidities , likely reflecting an attempt to minimise adverse effects of adt in patients with lower risk tumours . in general , adt use differed by physician specialty , with higher use among oncologists who may see men with higher risk disease . adt is often administered for 6 months or longer , on a continuous basis . the decision to prescribe continuous adt was based on psa level , gleason score and treatment guidelines not surprisingly , bilateral orchiectomy was not a common treatment for patients with non - metastatic prostate cancer ( < 2% ) with highest rates reported in eastern europe ( hungary and poland ) . adt was generally used for a total duration of 6 months or longer as either continuous or intermittent adt ( > 75% of patients)only us practitioners reported using adt for this duration in less than 70% of their patients , possibly another consequence of the reimbursement rulings on gnrh described elsewhere.8 although several therapies and improved management strategies exist for side effect management , the most effective form of prevention involves avoiding adt administration when it is not necessary ( ie , neo - adjuvant therapy before prostatectomy and short - term adt in addition to radiation therapy for low - risk disease).32 intermittent adt has been associated with fewer side effects and increased health - related quality of life indicators in a number of clinical trials;24 2630 however , some of the evidence can be inconsistent , and further work is needed to determine the patient populations who will benefit most in the reduction and prevention of the long - term harmful effects compared with continuous adt.2021 24 eau guidelines recommend monitoring testosterone and reinitiating adt based on clinical progression or prespecified psa levels.31 in our study , physicians reiterated the importance of psa as the key measurement taken during the treatment course of their patients with non - metastatic prostate cancer ; however , testosterone is reserved for those who may be at high risk of developing bone metastases . there are intermittent adt protocols derived using mathematical models33 to determine the on - treatment and off - treatment periods ; nonetheless , the evidence to date is not sufficient to accurately predict the effectiveness , likelihood of response and adverse effects of these protocols in the real world.34 there is growing evidence to support intermittent adt as effective as continuous adt in specific cohorts of patients ; nonetheless , clinicians face the challenge of prescribing appropriate evidence - based and guideline - endorsed gnrh treatment regimens for their patients while attempting to minimise the exposure and toxicity when possible . accordingly , clinicians devise individualised treatment courses of optimal length based on patient characteristics while accounting for associated risks and benefits of adt.35 this individualised clinical approach is represented in the variation of survey responses as it can become difficult to compartmentalise patients when deciding on treatment strategies . the observations from this cross - sectional survey therefore provide useful insights into how clinicians are treating men with non - metastatic prostate cancer with adt . several limitations must be acknowledged related to this study and survey research in general . although we ascertained the total number of patients treated in the disease pathway , findings are qualitative in nature and limited to physician - reported data with no confirmation from patient charts . in the qualitative development interviews , it was noted that physicians did not easily identify the differences between continuous and intermittent treatment . we therefore presented the definitions of intermittent and continuous adt and overall treatment duration to ensure physicians could answer the online survey questions consistently . most physicians used individualised treatment plans and therefore could not predict whether patients , in general , would be treated continuously or not in the long term . questions related to treatment duration are dependent on the physicians current workloads at the time of the research , and treatment is likely to change over time . although we achieved an adequate ( for physician - level research ) response rate of 12% , we acknowledge that treating physicians who completed the survey may differ from non - respondents . since this was a self - reported questionnaire , finally , given the small number of respondents in some countries , the results must be interpreted with caution . this international survey of 441 treating physicians from 19 countries furthers our understanding of how men with non - metastatic prostate cancer are treated with adt . urologists and oncologists indicated that their decisions to treat patients with prostate cancer continuously with adt was based on psa levels , gleason score and treatment guidelines , likely related to less favourable prognostic markers , or imminent or diagnosed castration resistance . despite limited number of studies supporting the use of intermittent adt relative to continuous adt , clinicians estimated that among their gnrh - treated non - metastatic patients , a quarter were prescribed intermittent adt . these data suggest that the use of intermittent adt is quite common but varies substantially by region . intermittent use was driven by psa levels , patient request , patient age and comorbidities , possibly reflecting attempts to minimise adverse effects of adt in patients with lower risk tumours . further clinical research is warranted to confirm that intermittent adt can reduce major long - term complications of androgen deprivation , and to determine the selected patient groups that would benefit the most .

backgroundcontinuous androgen deprivation therapy ( cadt ) is commonly used for patients with non - metastatic prostate cancer as primary therapy for high - risk disease , adjuvant therapy together with radiation or for recurrence after initial local therapy . intermittent adt ( iadt ) , a recently developed alternative strategy for providing adt , is thought to potentially reduce adverse effects , but little is known about practice patterns relating to it . we aimed to describe factors related to physicians adt use and modality for patients with non - metastatic prostate cancer.methodsa 45 min online survey was completed by urologists and oncologists responsible for treatment decisions for non - metastatic prostate cancer from 19 countries with high or increasing prevalence of non - metastatic prostate cancer.resultsthere were 441 treating physicians who completed the survey which represented 99 177 patients with prostate cancer under their care , of which 76 386 ( 77% ) had non - metastatic prostate cancer . of patients with non - metastatic prostate cancer , 38% received adt ( 37% gonadotropin - releasing hormone ( gnrh ) , 2% orchiectomy ) ; among patients on gnrh , 54% received cadt ( 6 without > 3 months interruption ) , 23% iadt and 23% < 6 months . highest rates of adt were reported among oncologists ( 62% ) and in eastern europe ( czech republic , hungary and poland ) . prostate - specific antigen ( psa ) levels ( 65% ) , gleason score ( 52% ) and treatment guidelines ( 48% ) were the most common reasons for cadt whereas psa levels ( 54% ) , patient request ( 48% ) , desire to maintain sexual function ( 40% ) , patient age and comorbidities ( 38% ) were cited most frequently as reasons for iadt.conclusionsthis international survey with 441 treating physicians from 19 countries showed that adt is commonly used in treating patients with non - metastatic prostate cancer , and type of adt is influenced by high - risk criteria ( psa and gleason ) , treatment guidelines and patient preferences . iadt use was primarily driven by psa levels , patient request and patient age / comorbidities , likely reflecting an attempt to minimise adverse effects of adt in patients with lower risk tumours .

What is already known about this subject? What does this study add? How might this impact on clinical practice? Introduction Methods Development of survey instrument Study population and eligibility criteria Statistical analysis Results Respondents Use of ADT for the treatment of non-metastatic patients Reasons for administering ADT Discussion Conclusion

androgen deprivation therapy ( adt ) is often administered to patients with prostate cancer as primary therapy of non - metastatic disease ; however , there still exists a lack of evidence of efficacy and the profile of patients most likely to benefit from intermittent versus continuous therapy . little is known about practice patterns and determinants of intermittent adt use in the usa and other countries . urologists and oncologists from several countries frequently administer adt in treating their patients with non - metastatic prostate cancer . the most common reasons for these participating physicians choosing the type of adt in managing their patients were high - risk criteria , treatment guidelines and patient preferences . the study provides real - world data on the treatment patterns and determinants of intermittent versus continuous use of adt among practitioners from 19 countries , which provides acknowledgement of practice patterns when clinical guidelines are reviewed in the treatment of non - metastatic prostate cancer . prostate cancer is the most commonly diagnosed malignancy and the second leading cause of cancer death in men in the usa.1 prostate cancer incidence varies more than 25-fold worldwide and represents the second most common cancer among men worldwide with an estimated 1.1 million men diagnosed in 2012 with almost 70% of the cases occurring in more developed regions.25 prostate cancer is androgen - dependent,6 7 which forms the basis for androgen deprivation therapy ( adt ) , usually achieved medically with gonadotropin - releasing hormone ( gnrh ) agonists or antagonists or , to a much less frequent extent , surgically via bilateral orchiectomy ( approximately 13% of adt).8 the benefits of adt are well established when used for palliation of symptomatic metastatic disease or as an adjuvant to radiation therapy for high - risk disease , but it is also very commonly used in other settings without clear evidence of efficacy such as for primary therapy of non - metastatic disease or for biochemical recurrence following initial local therapy.9 10 with its common use , there has been increasing recognition of harmful effects from continuous androgen deprivation.11 the most common side effects of continuous adt include anaemia , hot flashes , sexual dysfunction , cognitive dysfunction , bone loss , bone complications ( eg , fractures ) , metabolic and cardiovascular consequences , fatigue , depression and anxiety.11 12 this appreciation of the detrimental effects of adt has led to much interest in reducing adt exposure during the course of treatment.13 one such alternative therapeutic strategy , initially described in 1986 , is intermittent use of gnrh agonist therapies.14 typically , treatment is discontinued after 69 months of adt or when the prostate - specific antigen ( psa ) reaches its nadir ; adt is resumed when psa rises back to a predetermined higher level . the hormonal recovery that occurs during off - treatment cycles15 16 potentially facilitates responsiveness of tumour cells to treatment and theoretically limits toxicity.13 recent reviews comparing efficacy , side effects , time to castration resistance , overall and cancer - specific survival between intermittent and continuous adt have been summarised ; however , the evidence regarding the trade - offs between the benefits and risks of intermittent adt remains inconclusive.1723 generally the consensus is that overall survival is equivalent between intermittent and continuous adt in most settings . however , concerns remain with high - risk disease , as one of the larger trials did not meet criteria for non - inferiority of an intermittent regimen in men with metastatic cancer.24 although intermittent adt appears to have a modestly beneficial impact on sexual function and hot flashes , event rates in studies to date for serious effects such as fractures and cardiovascular outcomes have been too low to draw definitive conclusions . despite the ongoing uncertainty , little is known about practice patterns and determinants of intermittent adt use in the usa and other countries . the objective of this study was to describe factors related to physicians adt use and choice of intermittent adt regimens for patients with non - metastatic prostate cancer from a detailed international survey of treating urologists and oncologists . the 19 countries selected for this study represent those with high or increasing incidence of non - metastatic prostate cancer , prevalent use of adt among treating urologists and oncologists , and widespread psa screening . eligible respondents included physicians who were responsible for treatment decisions in patients with non - metastatic prostate cancer , with at least 10 non - metastatic patients seen per month ( and at least 10 treated with adt at the time of the survey ) , representing more than 25% of their patient - related time ( > 15% in nordics , czech republic , the netherlands and the usa ) , and year of qualification in their medical specialty between 1971 and 2009 . urologist versus oncologist distribution in the sampling for each country was consistent with practice patterns as determined by research partners at the country level and literature.25 for example , radiation oncologists were included in australia as they also prescribe drug therapies such as adt for patients with prostate cancer . the difference in the delineation between intermittent and continuous adt was the stipulation of an off - treatment period of more than 3 months.13 19 locally advanced disease was considered non - metastatic for the purposes of the survey , which may include limited local lymph node involvement . the 19 countries selected for this study represent those with high or increasing incidence of non - metastatic prostate cancer , prevalent use of adt among treating urologists and oncologists , and widespread psa screening . eligible respondents included physicians who were responsible for treatment decisions in patients with non - metastatic prostate cancer , with at least 10 non - metastatic patients seen per month ( and at least 10 treated with adt at the time of the survey ) , representing more than 25% of their patient - related time ( > 15% in nordics , czech republic , the netherlands and the usa ) , and year of qualification in their medical specialty between 1971 and 2009 . urologist versus oncologist distribution in the sampling for each country was consistent with practice patterns as determined by research partners at the country level and literature.25 for example , radiation oncologists were included in australia as they also prescribe drug therapies such as adt for patients with prostate cancer . the difference in the delineation between intermittent and continuous adt was the stipulation of an off - treatment period of more than 3 months.13 19 locally advanced disease was considered non - metastatic for the purposes of the survey , which may include limited local lymph node involvement . main reasons for exclusion among potentially eligible physicians were not spending more than 25% of their time treating patients with non - metastatic prostate cancer or not treating at least 10 non - metastatic patients with gnrh agents per month . physician characteristics by use of continuous or intermittent androgen deprivation therapy ( adt ) among patients with non - metastatic prostate cancer overall , 76% of respondents received their specialty qualification between 1991 and 2009 . fifty - four per cent of respondents indicated that they saw at least 40 men diagnosed with non - metastatic prostate cancer per month . survey respondents estimated that 99 177 patients with prostate cancer were under their care , 77% ( 76 386 ) classified as having non - metastatic disease ( figure 1 ) . among patients with non - metastatic prostate cancer under treatment , physicians reported that 38% ( 28 840 ) received adt : 36% ( 27 653 ) received gnrh agents , and 1.6% ( 1187 ) underwent bilateral orchiectomy . among their gnrh - treated patients , 54% were treated continuously ( 6 months without > 3-month interruption ) , 23% for less than 6 months , and 23% were managed with intermittent adt ( figure 2 ) . adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone . proportion of continuous , intermittent , and limited ( < 6 months ) use of adt among patients with non - metastatic prostate cancer treated with gonadotropin - releasing hormone ( gnrh ) by region and physician type . the highest proportion of adt use was reported by physicians in eastern europe ( 68% ) , driven by higher rates in hungary ( 82% ) and poland ( 79% ) . treating physicians reported administering adt to 43% of their patients in 5-country europe ( eu5 : france , germany , italy , spain and the uk ) ; however , respondents varied noticeably between france ( 26% ) , the uk ( 55% ) and italy ( 61% ) . adt use among men with non - metastatic prostate cancer according to treating physicians by country or region ( n=441 ) any adt : gnrh agonist / antagonist ( includes both continuous and intermittent use ( figure 1 ) ) or bilateral orchiectomy procedure . adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone . in general , treatment rates of adt were higher among oncologists ( 62% ) versus urologists ( 38% ) , although the proportion of adt given as intermittent therapy was similar ( figure 2 ) . in the usa , intermittent regimens represented 28% of adt prescribed , and physicians from remaining countries prescribed intermittent adt between 5% ( sweden ) and 24% ( italy ) . in choosing to initiate gnrh therapy for their patients with non - metastatic prostate cancer , 64% of physicians identified absolute psa levels as one of the main reasons . physician behaviour and motivations for continuous or intermittent adt among patients with non - metastatic prostate cancer adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone ; psa , prostate - specific antigen . with respect to reasons for prescribing continuous versus intermittent adt , physicians indicated ( more than one reason could be selected ) psa levels ( 65% ) , gleason score ( 52% ) and treatment guidelines ( 48% ) as the most common reasons for choosing continuous adt ( figure 3 ) . psa levels ( 54% ) , patient request ( 48% ) , desire to maintain sexual function ( 40% ) , comorbidities ( 38% ) and patient age ( 38% ) were cited most frequently as the reasons for managing patients with non - metastatic prostate cancer with intermittent adt . main reasons for exclusion among potentially eligible physicians were not spending more than 25% of their time treating patients with non - metastatic prostate cancer or not treating at least 10 non - metastatic patients with gnrh agents per month . a total of 441 physicians completed the survey from 19 countries ( table 1 ) . physician characteristics by use of continuous or intermittent androgen deprivation therapy ( adt ) among patients with non - metastatic prostate cancer overall , 76% of respondents received their specialty qualification between 1991 and 2009 . fifty - four per cent of respondents indicated that they saw at least 40 men diagnosed with non - metastatic prostate cancer per month . survey respondents estimated that 99 177 patients with prostate cancer were under their care , 77% ( 76 386 ) classified as having non - metastatic disease ( figure 1 ) . among patients with non - metastatic prostate cancer under treatment , physicians reported that 38% ( 28 840 ) received adt : 36% ( 27 653 ) received gnrh agents , and 1.6% ( 1187 ) underwent bilateral orchiectomy . among their gnrh - treated patients , 54% were treated continuously ( 6 months without > 3-month interruption ) , 23% for less than 6 months , and 23% were managed with intermittent adt ( figure 2 ) . proportion of continuous , intermittent , and limited ( < 6 months ) use of adt among patients with non - metastatic prostate cancer treated with gonadotropin - releasing hormone ( gnrh ) by region and physician type . table 2 provides the percentages of any adt and treatment of at least 6 months duration in the non - metastatic setting by region . the highest proportion of adt use was reported by physicians in eastern europe ( 68% ) , driven by higher rates in hungary ( 82% ) and poland ( 79% ) . treating physicians reported administering adt to 43% of their patients in 5-country europe ( eu5 : france , germany , italy , spain and the uk ) ; however , respondents varied noticeably between france ( 26% ) , the uk ( 55% ) and italy ( 61% ) . adt use among men with non - metastatic prostate cancer according to treating physicians by country or region ( n=441 ) any adt : gnrh agonist / antagonist ( includes both continuous and intermittent use ( figure 1 ) ) or bilateral orchiectomy procedure . nordics : denmark , finland , norway , sweden ; eu5 : france , germany , italy , spain , uk ; eastern europe : czech republic , hungary , poland . in general , treatment rates of adt were higher among oncologists ( 62% ) versus urologists ( 38% ) , although the proportion of adt given as intermittent therapy was similar ( figure 2 ) . in the usa , intermittent regimens represented 28% of adt prescribed , and physicians from remaining countries prescribed intermittent adt between 5% ( sweden ) and 24% ( italy ) . table 3 presents drivers of the decision to initiate adt based on psa levels , psa doubling time and gleason score . in choosing to initiate gnrh therapy for their patients with non - metastatic prostate cancer , 64% of physicians identified absolute psa levels as one of the main reasons . physician behaviour and motivations for continuous or intermittent adt among patients with non - metastatic prostate cancer adt , androgen deprivation therapy ; gnrh , gonadotropin - releasing hormone ; psa , prostate - specific antigen . with respect to reasons for prescribing continuous versus intermittent adt , physicians indicated ( more than one reason could be selected ) psa levels ( 65% ) , gleason score ( 52% ) and treatment guidelines ( 48% ) as the most common reasons for choosing continuous adt ( figure 3 ) . psa levels ( 54% ) , patient request ( 48% ) , desire to maintain sexual function ( 40% ) , comorbidities ( 38% ) and patient age ( 38% ) were cited most frequently as the reasons for managing patients with non - metastatic prostate cancer with intermittent adt . this international survey provides a detailed understanding of how adt is prescribed among patients with non - metastatic prostate cancer under the care of 441 treating physicians from 19 countries . respondents indicated that adt was prescribed for 38% ( range 2568% ) of their patients with non - metastatic prostate cancer , and mainly related to prognostic indicators ( gleason score , psa and psa doubling time ) or on signs of disease progression or recurrence manifested by rising psa values after initial or primary therapy . physicians from the usa reported that 34% of their patients were treated with adt ; a treatment rate lower than previously reported in the literature , but may suggest that the decreasing trend in adt following medicare reimbursement policy changes in 2004 and 2005 continues , resulting in overall lower use by 2012.8 the use of adt differed by region , with highest rates reported in eastern europe . the updated national comprehensive cancer network ( nccn ) clinical practice guidelines ( prostate cancer ) ( http://www.nccn.org/professionals/physician_gls/pdf/prostate.pdf ) recommend intermittent adt for patients with biochemical failure and without metastases based on a clinical trial showing that overall survival was non - inferior versus continuous adt ( ncic pr-7 trial ) , and intermittent use is not recommended for metastatic patients.26 the european association of urology ( eau ) guidelines recommend intermittent adt for asymptomatic metastatic patients citing a different set of clinical trials24 2730 that did not show significant differences in overall survival between continuous and intermittent adt , also citing patient acceptability , quality of life and fewer toxicities such as effects on cardiovascular or bone health.24 31 surveyed physicians noted that intermittent use was primarily driven by psa levels , patient request and patient age / comorbidities , likely reflecting an attempt to minimise adverse effects of adt in patients with lower risk tumours . the decision to prescribe continuous adt was based on psa level , gleason score and treatment guidelines not surprisingly , bilateral orchiectomy was not a common treatment for patients with non - metastatic prostate cancer ( < 2% ) with highest rates reported in eastern europe ( hungary and poland ) . adt was generally used for a total duration of 6 months or longer as either continuous or intermittent adt ( > 75% of patients)only us practitioners reported using adt for this duration in less than 70% of their patients , possibly another consequence of the reimbursement rulings on gnrh described elsewhere.8 although several therapies and improved management strategies exist for side effect management , the most effective form of prevention involves avoiding adt administration when it is not necessary ( ie , neo - adjuvant therapy before prostatectomy and short - term adt in addition to radiation therapy for low - risk disease).32 intermittent adt has been associated with fewer side effects and increased health - related quality of life indicators in a number of clinical trials;24 2630 however , some of the evidence can be inconsistent , and further work is needed to determine the patient populations who will benefit most in the reduction and prevention of the long - term harmful effects compared with continuous adt.2021 24 eau guidelines recommend monitoring testosterone and reinitiating adt based on clinical progression or prespecified psa levels.31 in our study , physicians reiterated the importance of psa as the key measurement taken during the treatment course of their patients with non - metastatic prostate cancer ; however , testosterone is reserved for those who may be at high risk of developing bone metastases . there are intermittent adt protocols derived using mathematical models33 to determine the on - treatment and off - treatment periods ; nonetheless , the evidence to date is not sufficient to accurately predict the effectiveness , likelihood of response and adverse effects of these protocols in the real world.34 there is growing evidence to support intermittent adt as effective as continuous adt in specific cohorts of patients ; nonetheless , clinicians face the challenge of prescribing appropriate evidence - based and guideline - endorsed gnrh treatment regimens for their patients while attempting to minimise the exposure and toxicity when possible . the observations from this cross - sectional survey therefore provide useful insights into how clinicians are treating men with non - metastatic prostate cancer with adt . although we achieved an adequate ( for physician - level research ) response rate of 12% , we acknowledge that treating physicians who completed the survey may differ from non - respondents . this international survey of 441 treating physicians from 19 countries furthers our understanding of how men with non - metastatic prostate cancer are treated with adt . urologists and oncologists indicated that their decisions to treat patients with prostate cancer continuously with adt was based on psa levels , gleason score and treatment guidelines , likely related to less favourable prognostic markers , or imminent or diagnosed castration resistance . despite limited number of studies supporting the use of intermittent adt relative to continuous adt , clinicians estimated that among their gnrh - treated non - metastatic patients , a quarter were prescribed intermittent adt . intermittent use was driven by psa levels , patient request , patient age and comorbidities , possibly reflecting attempts to minimise adverse effects of adt in patients with lower risk tumours . further clinical research is warranted to confirm that intermittent adt can reduce major long - term complications of androgen deprivation , and to determine the selected patient groups that would benefit the most .

over the past 20 years , nuclear magnetic resonance ( nmr ) spectroscopy has emerged as one of the most powerful physical methods to study the structure and dynamics of proteins . it combines the strengths of such methods as fluorescence and circular dichroism for characterizing proteins in solution with the power of x - ray crystallography to perform these characterizations atom by atom . aside from x - ray crystallography , nmr is the only other method available that allows the 3d structure of proteins to be determined to atomic resolution ( 1 ) . furthermore , nmr is the only known method that allows protein structures and dynamics to be determined in solution ( i.e. near physiological conditions ) . to date , more than 7000 peptide and protein structures have been determined by nmr and deposited into the pdb ( 2 ) . each one of these structures was determined using a three - step process pioneered by kurt wuthrich and colleagues ( 3 ) in the early 1980s . this process involves : ( i ) determining the chemical shift assignments of the target protein ; ( ii ) measuring the inter- and intra - residue h noes ( nuclear overhauser enhancements ) to generate short - range distance constraints and ( iii ) using the noe - derived constraints to perform molecular dynamics or distance geometry to calculate the 3d structure of the protein . over the years , slight improvements to this process have occurred with the inclusion of more experimental constraints such as heteronuclear j - couplings ( 4 ) and residual dipolar couplings ( 5 ) or the introduction of improved conformational sampling protocols such as simulated annealing ( 6 ) . nevertheless , the central concept of using noe - based methods to determine the 3d structure of proteins has remained essentially unchanged for nearly a quarter century . while noe - based methods are generally robust and well - proven , they are not without their faults . in particular , the measurement of h noes is both time - consuming and error - prone . furthermore , noes becomes progressively less useful and more difficult to measure as the size of the proteins increases . this constraint means that the determination of 3d structures by nmr for proteins larger than 200 residues is very difficult and infrequent . with the emergence of structural genomics and structural proteomics , there have been a number of efforts aimed at accelerating the nmr structure determination process or extending the upper size limits for which nmr can be used . almost all of these have focused on either eliminating the chemical shift assignment step ( 7,8 ) or automating the assignment of noes ( 9,10 ) . rather than looking at ways of improving noe measurements , a small minority of nmr researchers have been looking at ways of skipping the noe step altogether and going directly from chemical shift assignments to 3d structures ( 1114 ) . unlike noes , furthermore , chemical shifts provide exquisitely detailed information about the covalent structure of atoms and molecules . indeed , when properly analyzed , chemical shifts can be used to infer secondary structure , flexibility , dihedral angles , side - chain orientations , hydrogen - bonding interactions and electrostatic or ionic interactions ( 11 ) . recently , several papers have appeared which suggest that reasonably accurate protein structures can be determined directly from chemical shifts ( 1114 ) . of particular interest ( 14 ) , both of which showed that accurate 3d structures for a number of small ( < 120 residue ) proteins could be calculated using a combination of nmr chemical shifts , fragment assembly and heuristic potential functions . however , the computational effort required for this process is quite extreme , with individual structures requiring 1000s of cpu hours of calculation . here , we wish to describe a simple web server , called cs23d ( chemical shift to 3d structure ) , that allows the rapid ( 15 min on 1 cpu ) and accurate determination of protein structures using only assigned chemical shifts as input . cs23d builds on nearly 15 years of research in our lab related to using chemical shifts to identify secondary structures ( 15 ) , to predict torsion angles ( 16 ) , to identify protein folds ( 11 ) , to predict protein flexibility ( 17 ) , to refine protein structures ( 11 ) and to correct chemical shift referencing ( 18 ) . in particular , cs23d combines a technique we call maximal subfragment assembly with other techniques such as chemical shift threading , de novo structure generation ( via rosetta ) , chemical shift - based torsion angle prediction and chemical shift refinement to generate and refine the protein coordinates . tests conducted on > 100 proteins from the biomagresbank ( for which chemical shifts and 3d structures are available ) indicate that cs23d converges ( i.e. finds a solution ) for about 95% of protein queries . the resulting structures have a backbone rmsd < 2 of the known structure and generally exhibit better geometry and chemical shift agreement than conventionally determined nmr structures . cs23d is composed of two parts , a front - end web interface ( written in perl and html ) and a back - end consisting of eight different alignments , structure generation and structure optimization programs ( written in java , perl , python and c / c++ ) along with three local databases ( figure 1 ) . the front - end accepts both shifty ( 18 ) and bmrb ( 19 ) formatted chemical shift files . the shift files may be either pasted or typed into the text box or uploaded through a file browse button . the output for a typical cs23d structure calculation consist of a set of 10 lowest energy pdb coordinates in a simple , downloadable text format . a hyperlink to view the single lowest energy structure through the webmol viewer ( 20 ) in addition , details about the overall energy score ( prior to and following energy minimization ) , chemical shift correlations ( between the observed and calculated shifts ) and torsion angle violations is provided at the top of the output page . if the structure calculation failed to converge to a reasonable value , a warning is printed at the top of the page . details about the cs23d energy function , reasonable values for chemical shift energies and reasonable values for torsion angle violations is provided in the documentation link on the cs23d home page . figure 1.a flow chart outlining the general structure of the cs23d web server and the programs that it calls to generate protein structures from chemical shift data . the specific function of each of the named programs ( thrifty , pepmake , rosetta , etc . ) is explained in the text . a flow chart outlining the general structure of the cs23d web server and the programs that it calls to generate protein structures from chemical shift data . the specific function of each of the named programs ( thrifty , pepmake , rosetta , etc . ) a flow chart describing the processing logic used in cs23d is shown in figure 1 . as seen in this diagram , the input chemical shift file is initially partitioned into two parts , a sequence - only file and a sequence plus chemical shift file . the sequence file is searched against a nonredundant database of pdb sequences and secondary structures from ppt - db ( 21 ) using blast ( 22 ) with a length - dependent expect cutoff , ranging from 10 ( for < 11 residues ) to 10 ( for > 50 residues ) . this step is done to identify short sequence fragments of known protein structures that exhibit good ( > 35% over 20 + residues ) sequence identity to the query sequence . this step allows cs23d to find a series of maximum - length sequence fragments that matches the query sequence . at the same time , the chemical shift file is submitted to three different chemical shift analysis programs : refcor ( 17,18 ) , csi ( 15 ) and preditor ( 16 ) . the rci program is used to re - reference the input chemical shifts , the csi program is used to calculated secondary structure locations from the input chemical shifts and preditor is used to calculate backbone and side - chain torsion angles . checking chemical shift files for proper referencing is critical to obtaining accurate information about protein structures . furthermore , many operations in cs23d ( including csi and preditor ) depend on having correctly referenced chemical shifts . preditor is a locally developed program that uses chemical shift similarity over short protein fragments and a database of known protein torsion angles to generate backbone and side - chain torsion angles . it has been shown to be very fast ( 10 sec ) and accurate ( 85% of residues within 15 ) . the backbone torsion angles derived from preditor are then mapped into nine different regions in ramachandran space , each of which are assigned specific letters . the details of the torsion - angle - letter mapping scheme are shown in the cs23d documentation web page . by converting pairs of torsion angles into letters , it is possible to use a third program called thrifty ( 11 ) to perform chemical shift threading . sequence of shift - derived torsion angles generated by preditor and searches against a database of 18 500 nonredundant pdb structures that have had their structures converted to the previously described nine - letter ramachandran code . once again , blast , with similar scoring cutoffs to that used in the sequence alignment step , is used to identify fragments of varying length in the cs23d torsion - angle database that maximally match the query torsion angles . this chemical threading process can also be used at a global level to identify potential protein folds that match the chemical shift information contained in the query protein assignments . thrifty is also used to perform a secondary structure alignment between the secondary structure of the query protein ( calculated by csi ) and a large database of known protein secondary structures maintained at the ppt - db ( 21 ) . this secondary structure threading is used to evaluate and select subfragments that will be used to assemble the final 3d structure . subfragments identified by the sequence alignment and chemical shift threading schemes are then compared , weighted and assembled into an initial 3d structure using a program called sfassembler ( subfragment assembler ) . sfassembler evaluates each of the fragments found by the sequence alignment , secondary structure threading and torsion angle threading steps . four cases are considered : ( i ) if the same or similar fragments are found in all three cases ( with sequence or secondary structure sharing > 50% identity ) , sfassembler uses the coordinates of the fragment from the sequence - matched pdb file ; ( ii ) if there are significant differences ( < 50% secondary structure identity ) between the secondary structures found for the sequence - derived match and the shift - derived match , sfassembler uses the coordinates of the shift - matched pdb file ; ( iii ) if no significantly matching sequence fragment is found , but a shift - based threading fragment match is found , the coordinates from the shift - matched fragment are used by sfassembler and ( iv ) if no fragment is found that has either a significant sequence match or a significant chemical shift match , then the torsion angles calculated from preditor are used to generate the coordinates ( via pepmake ) . after these checks have been performed , sfassembler takes all selected pdb fragments or preditor - generated coordinate files and concatenates them together into a single 3d backbone structure . for instance , if one fragment of the query protein ( say residues 155 ) meets criteria # 1 , another fragment ( say residues 5693 ) meets criteria # 3 and a third fragment ( say residues 94106 ) meets criteria # 4 , sfassembler will generate coordinates and concatenate all three segments together . backbone gaps are filled in using a technique called cyclic coordinate descent ( 23 ) , while side chains are added using standard homology modeling techniques . because no length limits are placed on the size of the matching subfragments , sfassembler can sometimes function as a homology modeling program , particularly if a single large and contiguous region of sequence similarity is found . however , unlike conventional homology modeling programs , sfassembler is also capable of generating structures for tandem , multi - domain proteins , for chimeric proteins , for proteins that fall far below sequence thresholds required for standard homology modeling , and most importantly , for generating structural elements for which no structure template exists ( see the gallery page on the cs23d home page for examples ) . the initial structure generated by sfassembler is evaluated by calculating a weighted correlation coefficient between the observed shifts and the calculated shifts using shiftx ( 24 ) . if this correlation coefficient is too low or if the concatenated structure has < 40% of the structure assembled by preditor - generated coordinates ( criteria # 4 ) , the structure is accepted and refined using a locally developed chemical shift / energy refinement program called gafolder . after refinement , the pdb coordinates of the 10 lowest energy structures are mailed to the user along with the details of their evaluation and energy minimization . if the resulting structure fails these checks , it is discarded and a new structure is generated using rosetta ( 25 ) . rosetta is used by cs23d as a fail - safe procedure to generate potential 3d folds when the query protein exhibits absolutely no known sequence or chemical shift similarity over any part to any previously characterized protein . briefly , rosetta is a public domain , open source , ab initio protein structure prediction program developed by david baker 's lab over the last decade ( 25 ) . it uses the concept of fragment - based assembly similar to that employed by sfassembler , but at a much lower level of sequence identity and over much shorter fragment lengths . cs23d 's implementation of rosetta uses a local fragment generation routine constrained by preditor - generated torsion angles . it employs rosetta 's default values for generating and evaluating protein structures , but these candidate structures are further evaluated using a combination of weighted chemical shift correlation coefficients ( a chemical shift energy ) . the weightings used in the chemical shift evaluation step are provided in the cs23d documentation pages . to limit the time taken in this step , cs23d limits the number of rosetta - generated candidate structures to 300 . if after the refinement step , the structure has a positive energy value , cs23d generates the following warning message structure did not converge. however , the coordinates of the highest scoring structure are still presented in the output so that the user may attempt to use these as starting coordinates for a more conventional noe - based structure determination . for its energy minimization and chemical shift refinement step , cs23d employs a torsion - angle - based energy minimizer called gafolder that uses a genetic algorithm to sample conformation space . the method is similar to that employed by genfold ( 26 ) , although gafolder uses cyclic coordinate descent ( 23 ) to perform more efficient writhe operations and it uses coordinates generated by the preditor step ( figure 2 ) to perform segment swapping operations . the gafolder potential energy function is a knowledge - based potential that includes information on predicted / known secondary structure , radius of gyration , hydrogen - bond energies , number of hydrogen bonds , allowed backbone and side - chain torsion angles , atom contact radii ( bump checks ) , disulfide bonding information and a modified threading energy based on the bryant and lawrence potential ( 27 ) . the chemical shift component of the gafolder potential uses weighted correlation coefficients calculated between the observed and shiftx ( 24 ) calculated shifts of the structure being refined . the weighting coefficients for all of the parameters are given in the cs23d documentation web page . evaluations using nearly 44 000 3d structure predictions from the casp7 collection showed that the gafolder potential ( without the chemical shift component ) was able to identify the native or near - native ( < 2.5 rmsd ) structure in 80/90 ( 89% ) of the casp7 targets . figure 2.an illustration of how chemical shift minimization can improve the structure of a poorly modeled initial structure . this shows the improvement seen in a deliberately distorted model of ubiquitin . in this case , the protein regains much of its lost secondary structure and the rmsd converges from 2.5 to 1.0 from the native structure . an illustration of how chemical shift minimization can improve the structure of a poorly modeled initial structure . this shows the improvement seen in a deliberately distorted model of ubiquitin . in this case , the protein regains much of its lost secondary structure and the rmsd converges from 2.5 to 1.0 from the native structure . cs23d was evaluated in six different ways : ( i ) assessing its ability to refine input structures ; ( ii ) assessing the quality of its structures relative to known x - ray and nmr structures ; ( iii ) assessing it capacity or robustness in generating ( and refining ) randomly selected structures from the bmrb ; ( iv ) assessing its capacity to generate and refine very recently submitted bmrb assignments ; ( v ) assessing its capability to generate and refine de novo structures and ( vi ) comparing its performance to previously described programs ( 13,14 ) . in the first assessment , the quality of cs23d 's chemical shift / energy refinement routine was investigated by looking at how distorted or nonnative structures could be refined towards near - native or native structures . this was tested on a collection of eight proteins for which complete chemical shifts and high - quality x - ray structures were available . these structures were then distorted or damaged using random torsion angle displacement . after this distortion step the structures were run through gafolder for 3000 iterations and the resulting structures were assessed in terms of the rmsd between the calculated structure and the native structures as well as the energy difference between the native and the calculated structure . table 1 in the cs23d documentation web page shows the results of these calculations . on an average , the rmsd was lowered by 1.0 , with the final structure having an average rmsd of 1.5 relative to the native x - ray structure . figure 2 shows an example of how gafolder was able to correct a distorted ( 2.5 rmsd ) structure of ubiquitin and bring it back to within 1.0 rmsd of the table 2 ( documentation web page ) illustrates the results of comparing the x - ray structure , nmr structure and cs23d - derived structure for nine proteins for which both x - ray and nmr structures exist . as seen in this table , the cs23d - derived structures have about one - third of the number of ramachandran violations and omega angle violations found in their conventionally determined nmr counterparts . likewise , the proportion of hydrogen bonded residues , hydrogen - bond energies and average chemical shift correlation coefficients are about 510% higher for cs23d - dervied structures than conventionally determined nmr structures . in fact , the cs23d structures are generally closer in structure quality characteristics to the corresponding high - resolution x - ray structures . to assess cs23d 's effectiveness at generating high - quality structures from standard bmrb files , 62 bmrb files of monomeric proteins were randomly selected from the refdb database ( 18 ) . in order to avoid having the query match itself , the pdb structure of each of these query proteins was removed from cs23d 's sequence / structure databases . after this database - editing step , each of the 62 proteins was submitted to the cs23d server using its default settings . the structure of each of the lowest energy cs23d generated structures was evaluated by comparing its rmsd to that of the native structure . cs23d was able to generate a converged structure in 97% ( 60/62 ) of the cases . further , the average ca rmsd between the known structure and the cs23d generated structures was 1.1 rmsd ( the range was 0.13.1 ) . the average time to generate and refine each structure was 10.2 min . this varied depending on the size , sequence similarity to known structure and structure generation methods ( homology modeling / fragment assembly , chemical shift threading , de novo structure generation ) used by cs23d . interestingly , the majority of cases ( 92% ) were solved by cs23d using large fragment ( i.e. homology ) modeling supplemented with fragment concatenation . the high level of returned results probably exaggerates the real frequency with which cs23d would generate converged structures . this is because many of the proteins in refdb ( and by default the bmrb ) are well - studied proteins for which many homologues have been characterized . to better assess the effectiveness of cs23d among newly deposited bmrb entries ( for which novel folds or structural genomics entries may be more common ) , we downloaded all the 3 april 2008 recent releases from the bmrb chemical shift repository and processed them through cs23d ( total = 48 files ) . as before , the pdb structure of each query protein was removed from cs23d 's sequence / structure database prior to structure generation . of the 39 files for which structures were available in the pdb ( for comparative purposes ) , cs23d succeeded in generating converged structures for 36 of them ( web table 4 ) , with approximately the same proportion generated via homology modeling / fragment assembly ( 82% ) , chemical shift threading ( 14% ) and de novo structure generation ( 4% ) as seen in web table 3 . several independent estimates of the reported frequency of completely novel folds being deposited into the pdb or being solved by nmr suggest that only about 5% of all structures have this characteristic ( 28 ) . based on these data , we would estimate that cs23d , under routine use by the nmr community , would be able to generate a converged structure at least 95% of the time . to assess cs23d 's capabilities in generating and refining de novo structures , we conducted several tests ( tables 5 and 6 on the cs23d documentation page ) . in the first instance , 10 small proteins ( < 130 residues ) were processed through cs23d with the subfragment assembly and chemical shift threading options turned off ( table 5 ) . this forced the program to use only its ab initio folding components ( torsion angle constraints + rosetta ) . the average backbone rmsd for this set of ab initio cs23d - folded proteins was 2.1 . table 6 illustrates the performance of cs23d relative to the level of sequence identity and subfragment or chemical shift threading matches . in preparing table 6 , we used ubiquitin as the query and progressively removed each of the matching homologues from cs23d 's structure databases . in total , ubiquitin had nine proteins that exhibited sequence identity of > 40% and blast expect scores of < 10 over some significant portion of the ubiquitin sequence . true ubiquitin structure ranged from 0.5 ( at 90100% identity ) to 0.9 ( at 40% sequence identity ) . after these were removed , we found an additional 14 proteins in the pdb that exhibited secondary structure or torsion - angle identity of > 25% and blast scores of < 10 . true ubiquitin structure in these cases ranged from 0.8 ( at 3555% torsion identity ) to 4.2 ( at 2535% torsion identity ) . after these were removed from the pdb , we found that cs23d was still able to generate moderately good models of ubiquitin ( within 2.8 rmsd ) in 3/10 attempts . figure 3 summarizes the results from web tables 36 in terms of the performance of cs23d 's three different structure generation schemas ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . figure 3.a scatter plot showing the performance of cs23d 's three approaches to structure generation ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . data from tables 36 in the cs23d web documentation pages were used to assemble this graph . a scatter plot showing the performance of cs23d 's three approaches to structure generation ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . data from tables 36 in the cs23d web documentation pages were used to assemble this graph . finally , to compare cs23d 's performance to previously described programs cheshire ( 13 ) and cs - rosetta ( 14)we ran cs23d on a number of the testing / training proteins used in these papers . this included 12 proteins used in their initial training and testing ( table 7a of the web documentation page ) , seven proteins that failed to converge for cs - rosetta ( table 7b ) and six proteins that were part of a blinded test for cs - rosetta and for which chemical shifts were available ( table 7c ) . as seen in table 7a , the average backbone rmsd for cheshire , cs - rosetta and cs23d predictions is 1.52 , 1.48 and 1.64 , respectively . in other words , there is little to distinguish between the three methods . as shown in table 7b , cs23d was able to generate good quality structures for four of the seven structures ( 57% ) that cs - rosetta could not generate . table 7c shows that the structures generated by cs - rosetta for blinded structural genomics targets and generally better than those generated by cs23d ( 1 rmsd versus 3 rmsd ) . this highlights the fact that in trying to make cs23d a rapid structure generation tool , we have compromised some of its accuracy and capacity to generate de novo folds . nevertheless , cs23d appears to perform as well as or better than cs - rosetta and cheshire in most other structure determination tasks . cs23d is designed to address a continuum of structure generation queries . at one extreme , if a submission has 99% sequence identity and exhibits > 90% secondary structure conservation to a protein in the pdb , cs23d essentially functions as a homology modeling server with a ( unique ) chemical shift refinement step . in this case , the structures generated by cs23d are somewhat better than those generated by conventional nmr methods ( as measured by ramachandran violations , chemical shift correlations , hydrogen bonding and other structure evaluation tools ) . at the other extreme , if a submission has < 5% sequence identity and exhibits < 30% secondary structure conservation to a protein in the pdb , then cs23d essentially functions as an ab initio 3d structure predictor that employs an extra chemical shift refinement step . between these extremes , cs23d is able to exploit a variety of other techniques to assemble , extend and refine selected protein fragments ( ranging from 20 to 200 residues ) to routinely ( 95% of the time ) create high - quality 3d structures that are often better than those generated by conventional methods . obviously , cs23d is not without some limitations and there are certainly areas where improvements could be made . in particular , smarter use of chemical shift constraints for the rosetta portion of the program could certainly increase cs23d 's frequency of convergence for queries having completely novel folds . additionally , cs23d could be improved by allowing it to accept noe , j - coupling or residual dipolar coupling constraints as part of its input data . this could be particularly useful for modeling protein complexes or proteins with ligands two areas where chemical shift constraints are essentially useless . despite these limitations , we believe the speed , accuracy , frequency and reliability with which cs23d can generate ( and refine ) 3d protein structures using only chemical shifts and sequence data should make it a very useful addition to the current arsenal of structure generation and refinement tools available to biomolecular nmr spectroscopists .

cs23d ( chemical shift to 3d structure ) is a web server for rapidly generating accurate 3d protein structures using only assigned nuclear magnetic resonance ( nmr ) chemical shifts and sequence data as input . unlike conventional nmr methods , cs23d requires no noe and/or j - coupling data to perform its calculations . cs23d accepts chemical shift files in either shifty or bmrb formats , and produces a set of pdb coordinates for the protein in about 1015 min . cs23d uses a pipeline of several preexisting programs or servers to calculate the actual protein structure . depending on the sequence similarity ( or lack thereof ) cs23d uses either ( i ) maximal subfragment assembly ( a form of homology modeling ) , ( ii ) chemical shift threading or ( iii ) shift - aided de novo structure prediction ( via rosetta ) followed by chemical shift refinement to generate and/or refine protein coordinates . tests conducted on more than 100 proteins from the biomagresbank indicate that cs23d converges ( i.e. finds a solution ) for > 95% of protein queries . these chemical shift generated structures were found to be within 0.22.8 rmsd of the nmr structure generated using conventional noe - base nmr methods or conventional x - ray methods . the performance of cs23d is dependent on the completeness of the chemical shift assignments and the similarity of the query protein to known 3d folds . cs23d is accessible at http://www.cs23d.ca .

INTRODUCTION PROGRAM DESCRIPTION ALGORITHMS, DATABASES AND TESTING RESULTS AND EVALUATION CONCLUSIONS

over the past 20 years , nuclear magnetic resonance ( nmr ) spectroscopy has emerged as one of the most powerful physical methods to study the structure and dynamics of proteins . it combines the strengths of such methods as fluorescence and circular dichroism for characterizing proteins in solution with the power of x - ray crystallography to perform these characterizations atom by atom . aside from x - ray crystallography , nmr is the only other method available that allows the 3d structure of proteins to be determined to atomic resolution ( 1 ) . furthermore , nmr is the only known method that allows protein structures and dynamics to be determined in solution ( i.e. to date , more than 7000 peptide and protein structures have been determined by nmr and deposited into the pdb ( 2 ) . this process involves : ( i ) determining the chemical shift assignments of the target protein ; ( ii ) measuring the inter- and intra - residue h noes ( nuclear overhauser enhancements ) to generate short - range distance constraints and ( iii ) using the noe - derived constraints to perform molecular dynamics or distance geometry to calculate the 3d structure of the protein . nevertheless , the central concept of using noe - based methods to determine the 3d structure of proteins has remained essentially unchanged for nearly a quarter century . with the emergence of structural genomics and structural proteomics , there have been a number of efforts aimed at accelerating the nmr structure determination process or extending the upper size limits for which nmr can be used . almost all of these have focused on either eliminating the chemical shift assignment step ( 7,8 ) or automating the assignment of noes ( 9,10 ) . rather than looking at ways of improving noe measurements , a small minority of nmr researchers have been looking at ways of skipping the noe step altogether and going directly from chemical shift assignments to 3d structures ( 1114 ) . recently , several papers have appeared which suggest that reasonably accurate protein structures can be determined directly from chemical shifts ( 1114 ) . of particular interest ( 14 ) , both of which showed that accurate 3d structures for a number of small ( < 120 residue ) proteins could be calculated using a combination of nmr chemical shifts , fragment assembly and heuristic potential functions . here , we wish to describe a simple web server , called cs23d ( chemical shift to 3d structure ) , that allows the rapid ( 15 min on 1 cpu ) and accurate determination of protein structures using only assigned chemical shifts as input . cs23d builds on nearly 15 years of research in our lab related to using chemical shifts to identify secondary structures ( 15 ) , to predict torsion angles ( 16 ) , to identify protein folds ( 11 ) , to predict protein flexibility ( 17 ) , to refine protein structures ( 11 ) and to correct chemical shift referencing ( 18 ) . in particular , cs23d combines a technique we call maximal subfragment assembly with other techniques such as chemical shift threading , de novo structure generation ( via rosetta ) , chemical shift - based torsion angle prediction and chemical shift refinement to generate and refine the protein coordinates . tests conducted on > 100 proteins from the biomagresbank ( for which chemical shifts and 3d structures are available ) indicate that cs23d converges ( i.e. finds a solution ) for about 95% of protein queries . the resulting structures have a backbone rmsd < 2 of the known structure and generally exhibit better geometry and chemical shift agreement than conventionally determined nmr structures . the front - end accepts both shifty ( 18 ) and bmrb ( 19 ) formatted chemical shift files . the output for a typical cs23d structure calculation consist of a set of 10 lowest energy pdb coordinates in a simple , downloadable text format . a hyperlink to view the single lowest energy structure through the webmol viewer ( 20 ) in addition , details about the overall energy score ( prior to and following energy minimization ) , chemical shift correlations ( between the observed and calculated shifts ) and torsion angle violations is provided at the top of the output page . details about the cs23d energy function , reasonable values for chemical shift energies and reasonable values for torsion angle violations is provided in the documentation link on the cs23d home page . figure 1.a flow chart outlining the general structure of the cs23d web server and the programs that it calls to generate protein structures from chemical shift data . a flow chart outlining the general structure of the cs23d web server and the programs that it calls to generate protein structures from chemical shift data . the sequence file is searched against a nonredundant database of pdb sequences and secondary structures from ppt - db ( 21 ) using blast ( 22 ) with a length - dependent expect cutoff , ranging from 10 ( for < 11 residues ) to 10 ( for > 50 residues ) . this step is done to identify short sequence fragments of known protein structures that exhibit good ( > 35% over 20 + residues ) sequence identity to the query sequence . at the same time , the chemical shift file is submitted to three different chemical shift analysis programs : refcor ( 17,18 ) , csi ( 15 ) and preditor ( 16 ) . the rci program is used to re - reference the input chemical shifts , the csi program is used to calculated secondary structure locations from the input chemical shifts and preditor is used to calculate backbone and side - chain torsion angles . checking chemical shift files for proper referencing is critical to obtaining accurate information about protein structures . furthermore , many operations in cs23d ( including csi and preditor ) depend on having correctly referenced chemical shifts . preditor is a locally developed program that uses chemical shift similarity over short protein fragments and a database of known protein torsion angles to generate backbone and side - chain torsion angles . by converting pairs of torsion angles into letters , it is possible to use a third program called thrifty ( 11 ) to perform chemical shift threading . once again , blast , with similar scoring cutoffs to that used in the sequence alignment step , is used to identify fragments of varying length in the cs23d torsion - angle database that maximally match the query torsion angles . this chemical threading process can also be used at a global level to identify potential protein folds that match the chemical shift information contained in the query protein assignments . thrifty is also used to perform a secondary structure alignment between the secondary structure of the query protein ( calculated by csi ) and a large database of known protein secondary structures maintained at the ppt - db ( 21 ) . subfragments identified by the sequence alignment and chemical shift threading schemes are then compared , weighted and assembled into an initial 3d structure using a program called sfassembler ( subfragment assembler ) . sfassembler evaluates each of the fragments found by the sequence alignment , secondary structure threading and torsion angle threading steps . four cases are considered : ( i ) if the same or similar fragments are found in all three cases ( with sequence or secondary structure sharing > 50% identity ) , sfassembler uses the coordinates of the fragment from the sequence - matched pdb file ; ( ii ) if there are significant differences ( < 50% secondary structure identity ) between the secondary structures found for the sequence - derived match and the shift - derived match , sfassembler uses the coordinates of the shift - matched pdb file ; ( iii ) if no significantly matching sequence fragment is found , but a shift - based threading fragment match is found , the coordinates from the shift - matched fragment are used by sfassembler and ( iv ) if no fragment is found that has either a significant sequence match or a significant chemical shift match , then the torsion angles calculated from preditor are used to generate the coordinates ( via pepmake ) . for instance , if one fragment of the query protein ( say residues 155 ) meets criteria # 1 , another fragment ( say residues 5693 ) meets criteria # 3 and a third fragment ( say residues 94106 ) meets criteria # 4 , sfassembler will generate coordinates and concatenate all three segments together . backbone gaps are filled in using a technique called cyclic coordinate descent ( 23 ) , while side chains are added using standard homology modeling techniques . because no length limits are placed on the size of the matching subfragments , sfassembler can sometimes function as a homology modeling program , particularly if a single large and contiguous region of sequence similarity is found . however , unlike conventional homology modeling programs , sfassembler is also capable of generating structures for tandem , multi - domain proteins , for chimeric proteins , for proteins that fall far below sequence thresholds required for standard homology modeling , and most importantly , for generating structural elements for which no structure template exists ( see the gallery page on the cs23d home page for examples ) . the initial structure generated by sfassembler is evaluated by calculating a weighted correlation coefficient between the observed shifts and the calculated shifts using shiftx ( 24 ) . if this correlation coefficient is too low or if the concatenated structure has < 40% of the structure assembled by preditor - generated coordinates ( criteria # 4 ) , the structure is accepted and refined using a locally developed chemical shift / energy refinement program called gafolder . after refinement , the pdb coordinates of the 10 lowest energy structures are mailed to the user along with the details of their evaluation and energy minimization . rosetta is used by cs23d as a fail - safe procedure to generate potential 3d folds when the query protein exhibits absolutely no known sequence or chemical shift similarity over any part to any previously characterized protein . briefly , rosetta is a public domain , open source , ab initio protein structure prediction program developed by david baker 's lab over the last decade ( 25 ) . it employs rosetta 's default values for generating and evaluating protein structures , but these candidate structures are further evaluated using a combination of weighted chemical shift correlation coefficients ( a chemical shift energy ) . the weightings used in the chemical shift evaluation step are provided in the cs23d documentation pages . however , the coordinates of the highest scoring structure are still presented in the output so that the user may attempt to use these as starting coordinates for a more conventional noe - based structure determination . for its energy minimization and chemical shift refinement step , cs23d employs a torsion - angle - based energy minimizer called gafolder that uses a genetic algorithm to sample conformation space . the method is similar to that employed by genfold ( 26 ) , although gafolder uses cyclic coordinate descent ( 23 ) to perform more efficient writhe operations and it uses coordinates generated by the preditor step ( figure 2 ) to perform segment swapping operations . the gafolder potential energy function is a knowledge - based potential that includes information on predicted / known secondary structure , radius of gyration , hydrogen - bond energies , number of hydrogen bonds , allowed backbone and side - chain torsion angles , atom contact radii ( bump checks ) , disulfide bonding information and a modified threading energy based on the bryant and lawrence potential ( 27 ) . the chemical shift component of the gafolder potential uses weighted correlation coefficients calculated between the observed and shiftx ( 24 ) calculated shifts of the structure being refined . evaluations using nearly 44 000 3d structure predictions from the casp7 collection showed that the gafolder potential ( without the chemical shift component ) was able to identify the native or near - native ( < 2.5 rmsd ) structure in 80/90 ( 89% ) of the casp7 targets . in this case , the protein regains much of its lost secondary structure and the rmsd converges from 2.5 to 1.0 from the native structure . in this case , the protein regains much of its lost secondary structure and the rmsd converges from 2.5 to 1.0 from the native structure . cs23d was evaluated in six different ways : ( i ) assessing its ability to refine input structures ; ( ii ) assessing the quality of its structures relative to known x - ray and nmr structures ; ( iii ) assessing it capacity or robustness in generating ( and refining ) randomly selected structures from the bmrb ; ( iv ) assessing its capacity to generate and refine very recently submitted bmrb assignments ; ( v ) assessing its capability to generate and refine de novo structures and ( vi ) comparing its performance to previously described programs ( 13,14 ) . in the first assessment , the quality of cs23d 's chemical shift / energy refinement routine was investigated by looking at how distorted or nonnative structures could be refined towards near - native or native structures . this was tested on a collection of eight proteins for which complete chemical shifts and high - quality x - ray structures were available . after this distortion step the structures were run through gafolder for 3000 iterations and the resulting structures were assessed in terms of the rmsd between the calculated structure and the native structures as well as the energy difference between the native and the calculated structure . on an average , the rmsd was lowered by 1.0 , with the final structure having an average rmsd of 1.5 relative to the native x - ray structure . figure 2 shows an example of how gafolder was able to correct a distorted ( 2.5 rmsd ) structure of ubiquitin and bring it back to within 1.0 rmsd of the table 2 ( documentation web page ) illustrates the results of comparing the x - ray structure , nmr structure and cs23d - derived structure for nine proteins for which both x - ray and nmr structures exist . in fact , the cs23d structures are generally closer in structure quality characteristics to the corresponding high - resolution x - ray structures . the structure of each of the lowest energy cs23d generated structures was evaluated by comparing its rmsd to that of the native structure . cs23d was able to generate a converged structure in 97% ( 60/62 ) of the cases . further , the average ca rmsd between the known structure and the cs23d generated structures was 1.1 rmsd ( the range was 0.13.1 ) . this varied depending on the size , sequence similarity to known structure and structure generation methods ( homology modeling / fragment assembly , chemical shift threading , de novo structure generation ) used by cs23d . interestingly , the majority of cases ( 92% ) were solved by cs23d using large fragment ( i.e. to better assess the effectiveness of cs23d among newly deposited bmrb entries ( for which novel folds or structural genomics entries may be more common ) , we downloaded all the 3 april 2008 recent releases from the bmrb chemical shift repository and processed them through cs23d ( total = 48 files ) . of the 39 files for which structures were available in the pdb ( for comparative purposes ) , cs23d succeeded in generating converged structures for 36 of them ( web table 4 ) , with approximately the same proportion generated via homology modeling / fragment assembly ( 82% ) , chemical shift threading ( 14% ) and de novo structure generation ( 4% ) as seen in web table 3 . based on these data , we would estimate that cs23d , under routine use by the nmr community , would be able to generate a converged structure at least 95% of the time . to assess cs23d 's capabilities in generating and refining de novo structures , we conducted several tests ( tables 5 and 6 on the cs23d documentation page ) . in the first instance , 10 small proteins ( < 130 residues ) were processed through cs23d with the subfragment assembly and chemical shift threading options turned off ( table 5 ) . table 6 illustrates the performance of cs23d relative to the level of sequence identity and subfragment or chemical shift threading matches . in preparing table 6 , we used ubiquitin as the query and progressively removed each of the matching homologues from cs23d 's structure databases . after these were removed from the pdb , we found that cs23d was still able to generate moderately good models of ubiquitin ( within 2.8 rmsd ) in 3/10 attempts . figure 3 summarizes the results from web tables 36 in terms of the performance of cs23d 's three different structure generation schemas ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . figure 3.a scatter plot showing the performance of cs23d 's three approaches to structure generation ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . a scatter plot showing the performance of cs23d 's three approaches to structure generation ( subfragment assembly + homology modeling , chemical shift threading and ab initio structure generation ) relative to the level of sequence identity of the matching templates or subfragments . this included 12 proteins used in their initial training and testing ( table 7a of the web documentation page ) , seven proteins that failed to converge for cs - rosetta ( table 7b ) and six proteins that were part of a blinded test for cs - rosetta and for which chemical shifts were available ( table 7c ) . as shown in table 7b , cs23d was able to generate good quality structures for four of the seven structures ( 57% ) that cs - rosetta could not generate . this highlights the fact that in trying to make cs23d a rapid structure generation tool , we have compromised some of its accuracy and capacity to generate de novo folds . nevertheless , cs23d appears to perform as well as or better than cs - rosetta and cheshire in most other structure determination tasks . at one extreme , if a submission has 99% sequence identity and exhibits > 90% secondary structure conservation to a protein in the pdb , cs23d essentially functions as a homology modeling server with a ( unique ) chemical shift refinement step . in this case , the structures generated by cs23d are somewhat better than those generated by conventional nmr methods ( as measured by ramachandran violations , chemical shift correlations , hydrogen bonding and other structure evaluation tools ) . at the other extreme , if a submission has < 5% sequence identity and exhibits < 30% secondary structure conservation to a protein in the pdb , then cs23d essentially functions as an ab initio 3d structure predictor that employs an extra chemical shift refinement step . between these extremes , cs23d is able to exploit a variety of other techniques to assemble , extend and refine selected protein fragments ( ranging from 20 to 200 residues ) to routinely ( 95% of the time ) create high - quality 3d structures that are often better than those generated by conventional methods . obviously , cs23d is not without some limitations and there are certainly areas where improvements could be made . in particular , smarter use of chemical shift constraints for the rosetta portion of the program could certainly increase cs23d 's frequency of convergence for queries having completely novel folds . additionally , cs23d could be improved by allowing it to accept noe , j - coupling or residual dipolar coupling constraints as part of its input data . despite these limitations , we believe the speed , accuracy , frequency and reliability with which cs23d can generate ( and refine ) 3d protein structures using only chemical shifts and sequence data should make it a very useful addition to the current arsenal of structure generation and refinement tools available to biomolecular nmr spectroscopists .

15% of the global population and more than 65% of the population in the third and developing world , yet current drug therapies for protozoan infections are woefully inadequate . as protozoan infections take their toll predominantly in the developing world , market forces are insufficient to promote the development of novel antiprotozoan drugs . in 2000 , only ca . 0.1% of global investment in health research was spent on drug discovery for tropical diseases . one such neglected parasitic disease is human african trypanosomiasis ( hat ) or african sleeping sickness , which is caused by the protozoan parasite trypanosoma brucei . the world health organization ( who ) estimates that hat constitutes a serious health risk to 60 million people in sub - saharan africa . the who also estimates that there are < 30000 new cases per year in sub - saharan africa and at present an annual death toll of 8000 . the related disease in cattle , cattle trypanosomiasis or nagana , also represents a major health concern due to its devastating economic ( estimated by the who to cause an annual economic loss of us$4 billion ) , social , and nutritional impact on african families . as such , the total burden of trypanosomiasis translates into 1598000 disability - adjusted life years ( daly ) . treatment of hat is solely dependent upon a repertoire of four drugs : suramin ( 1 ) , pentamidine ( 2 ) , melarsoprol ( 3 ) , and eflornithine ( 4 ) ( figure 1 ) . these therapies are often toxic , difficult to administer , and increasingly have an acquired drug resistance , highlighting the urgent need for new , more effective drug therapies . developed before the 1950s , suramin ( 1 ) and melarsoprol ( 3 ) are used for chemotherapy of the early stage of the disease ( trypanosoma brucei rhodesiense ) , as is pentamidine ( 2 ) ( trypanosoma brucei gambiense ) . the arsenical melarsoprol ( 3 ) is extremely toxic , with death in 6% of cases and treatment failure rates of as high as 30% in certain areas . treatment of the second stage of the disease , where the parasites cross the blood brain barrier and invade the central nervous system , is limited to melarsoprol ( 3 ) and eflornithine ( 4 ) , the ornithine decarboxylase inhibitor ( difluoromethylornithine ) . structures of clinic drugs 15 for sleeping sickness and clinic phase i drug fexinidazole ( 6 ) . nifurtimox ( 5 ) ( figure 1 ) , is often used to treat chagas disease , caused by trypanosoma cruzi , and has been used as a monotherapy for melarsoprol - refractory hat on compassionate grounds despite its low efficacy and severe toxicity . eflornithine combination therapy ( nect ) by who in the model lists of essential medicines has been seen as highly advantageous in terms of cost , logistics , and human resources in areas of poverty . patients given nect , consisting of oral nifurtimox over 10 days with eflornithine ( 4 ) infusions for 7 days , were found to fair just as well as those given the eflornithine monotherapy , with cure rates of around 97% . the use of nect has renewed general interest in the use of nitroheterocyclic compounds to treat a wide range of infectious disease , including tuberculosis and hepatitis c. this increased awareness of the potential of nitroheterocyclic compounds has led to the reinvestigation of fexinidazole ( 6 ) ( figure 1 ) , which is presently in clinical trials against both early- and late - stage african sleeping sickness . alarmingly , it has been shown that t. brucei nifurtimox - resistant cells show cross - resistance to other nitro - containing drugs , including fexinidazole , that are currently in clinical trials . several different types of studies on nitroheterocycles have shed some light on this observed drug resistance . genome - wide rnai screens of nifurtimox and benznidazole ( another trypanocidal nitroheterocyclic drug ) resistant t. brucei have identified that a decrease in nitroreductase activity is linked to nifurtimox resistance . this was confirmed by showing that genetic deletion of one allele of the t. brucei s nitroreductase led to nifurtimox resistance . likewise , the overexpression of this nitroreductase , but not the alternative proposed prostaglandin f2 synthase and cytochrome p450 reductase , in t. brucei resulted in increased susceptibility to nifurtimox . collectively , it is clear that this type i t. brucei nitroreductase , a nad(p)h dependent , flavin binding protein , is involved in the reductive activation of nifurtimox . the nitroreductases mediate a series of two - electron reductions of the nitro group to a nitroso intermediate , then to a hydroxylamine , and eventually to the corresponding amine . however , the true function of the t. brucei mitochondrial nitroreductase is unknown , but its essentiality may explain why only a single allele deletion / mutation is associated with nifurtimox resistance . as well as the development of nifurtimox - resistance and its cross - resistance to other nitro - containing drugs , for example , the adverse off - target side effects with nifurtimox lead to treatment cessation in over 30% of patients with chagas disease . the most common side effects are anorexia , loss of weight , psychic alterations , excitability or sleepiness , and digestive manifestations such as nausea or vomiting and occasionally intestinal colic and diarrhea . various human enzymes have been identified to be capable of 5-nitrofuran reduction in vitro , in cells , or tissues . recently , we have elucidated in collaboration with the patton laboratory a possible role of aldehyde dehydrogenase 2 ( aldh2 ) in the toxicity caused by 5-nitrofuran containing drugs such as nifurtimox . while considerable effort has gone into the synthesis of nitroaromatic containing compounds over the years , there remains several unexplored areas of chemical space . for example , in the context of nifurtimox , most of the efforts have been focused on changing either the furan ring to other heterocycles ( such as imidazole ) or changing the substituent in the hydrazone motif , with limited increases in activity compared to nifurtimox . recently , two new types of typanocidal compounds , nitrobenzylphosphoramide mustards ( for example see compound 7 , figure 2 ) and aziridinyl nitrobenzamides , have been developed to target specifically t. brucei type i nitroreductase , the best having an ec50 1 m against both t. brucei and t. cruzi . several 5-nitro-2-furancarboxylamide containing compounds have been reported to have potent antituberculosis and antimicrobial activity , however , very few 5-nitro-2-furancarboxylamides are reported to have typanocidal activity , primarily low activity against t. cruzi . a nifurtimox analogue ( 8) ( figure 2 ) showed trace activity against t. cruzi in a mouse model . the best 5-nitro-2-furancarboxylamides identified thus far , 9 and 10 , have ec50 values of 42 and 262 nm , respectively , against t. cruzi amastigotes ( figure 2 ) but have not been tested against t. brucei to the best of our knowledge . several 5-nitro-2-furancarbohydrazides possess activity against t. brucei with the best , compound 11 , having an ec50 of 0.13 m but low selectivity against human cells . , we report that our recently discovered nitrofuran nfn1 ( 12a ) also shows promising activity against t. brucei in vitro . a lack of cross - resistance with nifurtimox and significantly increased ( 23 orders of magnitude ) trypanocidal activity has important implications for the future therapeutic use of these nitrofuran containing compounds in the treatment of hat , instead of , or in combination with , nifurtimox . 5-nitrofurans 12a k were prepared by acylation of the corresponding 2-amino - thiophenes 13a k with 5-nitro - furancarboxylic acid chloride 14a ( scheme 1 , table 1 for substituents ) . the required 2-amino - thiophenes 13a h were prepared using the gewald multicomponent reaction with various combinations of the 2-cyanoacetyl esters or amides ( 15a g ) with aldehydes ( 16a or 16b ) and elemental sulfur ( see supporting information table s1 for details).13h was o - silyl - protected before coupling to 14a . this reaction gave 12h directly as a result of in situ removal of the silyl - protecting group . thiophene 13i was prepared by hydrolysis of 13a and 13j prepared by catalytic amination of 2-iodothiophene using cui and l - proline.13k and 13l ( table 2 ) were commercially available . reaction of 13a ( r = et , r = co2et ) with 14b gave control compound 18a . reagents and conditions : ( a ) ( i ) ( cocl)2 , dcm / dmf , rt , 2 h , ( ii ) rnh2 , et3n , dcm , 52% for 15d , 49% ( 15e ) , 37% ( 15 g ) ; ( b ) s8 , 16a or 16b , et2nh , dmf , rt , o / n , 66% for 13a , 59% ( 13b ) , 76% ( 13c ) , 53% ( 13d ) , 49% ( 13e ) , 46% ( 13f ) , 48% ( 13 g ) , 46% ( 13h ) , for synthesis of 13i j see esi , 13k and 13l were commercially available ; ( c ) 13a k , et3n , dcm , rt with 14a , 68% for 12a , 62% ( 12b ) , 65% ( 12c ) , 58% ( 12d ) , 57% ( 12e ) , 57% ( 12f ) , 54% ( 12 g ) , 47% ( 12h ) , 25% ( 12i ) , 41% ( 12j ) , 51% ( 12k ) , with 14b , 56% for 18a ; ( d ) rnh2 , et3n , dcm , rt with 14a , 65% for 22a , 61% ( 22b ) , 72% ( 22c ) , 67% ( 22d ) , 37% ( 22e ) , 50% ( 22f ) , 45% ( 22 g ) , 41% ( 22h ) , 66% ( 22i ) , 39% ( 22j ) , 51% ( 22k ) , 73% ( 22l ) , 51% ( 22 m ) , 68% ( 22n ) , 67% ( 22o ) , 69% ( 22p ) , 48% ( 22q ) , with 14b 73% for 18b ( see table 2 for structure of 18b ) . ( a ) 15a c and 15f were commercially available ; ( b ) 15d , 15e , and 15 g were prepared from 17 ; ( c ) for r and r substituents , see table 1 ; ( c ) for r substituents , see tables 2 and 3 . for structure , not applicable as no activity or low activity against t. brucei was observed for 18b and 25a . analogues 20a c and 21 ( table 2 ) based on 12 g but containing alternative nitroaromatic rings were prepared by coupling the corresponding acid chlorides with 13 g ( see esi for details ) . similarly , reaction of 14a with a range of amines and anilines led to the synthesis of 22a q ( scheme 1 ) . demethylation of 22o to give 22r was followed by selective o - allylation to give 22s ( scheme 2 ) , the structure of which was confirmed by x - ray crystallographic analysis ( data not shown ) . when a longer reaction time and an excess amount of allyl bromide was used in the o - allylation of 22r , the diallylated analogue 22 t was formed ( scheme 2 ) . methylation of 22q to give 22v was achieved using mei under basic conditions in moderate yield ( scheme 2 ) . conversion of 5-nitrofuran aldehyde 23 to imines 24a or 24b enabled the synthesis of the corresponding amines 25a and 25b using standard reductive amination conditions ( scheme 2 ) . reagents and conditions : ( a ) pyridine hydrochloride , 160 c , mw , 5 min , 52% ; ( b ) 22r or 22p , allyl bromide ( 2 or 10 equiv ) , k2co3 , acetone , rt , 2 h for 22s ( 75% ) , 24 h for 22 t ( 55% ) , 24 h for 22u ( 72% ) , 22q , mei , k2co3 , acetone , 3 h , for 22v ( 51% ) ; ( c ) aniline or 13 g , dcm , rt , 89% for 24a , 63% for 24b ; ( d ) nabh4 , dcm , rt , 79% for 25a , 87% for 25b . in comparison to nifurtimox , nfn1 was 2 orders of magnitude more potent with an ec50 of 31.3 3.1 nm ( table 1 , cf . the nitro group in nfn1 was found to be essential with no activity being observed up to the solubility limit of 18a ( entry 3 ) . removal , or worse , elongation of the alkyl substituent in nfn1 led to reduced activity ( cf . entry 2 and entries 4 and 5 , respectively ) , and while removal of the ester group initially appeared to be tolerated in the r = h series ( cf . entries 4 and 6 ) , it was found that the methyl and n - butyl ester analogues ( 12c and 2d ) of nfn1 had similar activity to nfn1 ( cf . entry 2 and entries 7 and 8 , respectively ) . conversion of the ester groups in nfn1 and 12d to the corresponding amides in 12e and 12f led to a significant loss in activity ( cf . this observation , coupled with the decrease in activity associated with amide 12h ( entry 11 ) , may be explained by the fact that hydrolysis of the ester groups in 12a d and 12k in the parasite gives the corresponding carboxylic acid - containing bioactive metabolites , something that is unlikely to occur for the amides 12e and 12f . however , acid 12i showed much less activity with an ec50 of 959.0 33.9 nm ( entry 12 ) , which may be due to a lack of cell permeability . the primary amide 12 g was found to be the most active thiophene - containing analogue with an ec50 of 17.3 2.4 nm ( entry 13 ) . furthermore , analogues 12a k showed no significant toxicity against the human hela cell line even at a concentration above 20 m . the highly selective toxicity of these analogues against the parasite suggests these nitrofuran analogues have real potential for therapeutic applications . encouraged by the potent activity of 12 g ( table 1 ) , it was decided to prepare more analogues based on this structure . fragments 19 and 13 g along with 13l were shown to be inactive against t. brucei , suggesting that both the 5-nitrofuran and thiophene fragments are required ( table 2 , cf . changing the nitrofuran motif in 12 g to either a nitrophenyl or nitropyrazole ring also led to a 100-fold or more drop in activity ( cf . interestingly , the position of the nitro group in the nitrophenyl ring had an influence as 20b , an analogue with the nitro group in the meta - position , was more active than either 20a ( ortho - no2 ) or 20c ( para - no2 ) . studies next focused on replacing the thiophene motif in 12a k . by incorporating a cyclohexyl ring to give 22a , a 10-fold decrease in activity was observed compared to 12 g ( entry 9 ) , while the phenyl ( 22b ) and benzyl ( 22c ) analogues were essentially equipotent with 12 g ( cf . incorporation of a nitrogen atom into the 2-position of the phenyl ring in 22c to give 22d led to a 10-fold drop in activity ( entry 12 ) . entries 10 and 13 ) , and the amide linkage in 22b and 12 g was also shown to be important , as the amine analogues 25a and 25b showed a dramatic decrease in activity ( entries 14 and 15 , respectively ) . on the basis of the potent activity of 22b , the effect of incorporating additional substituents into the phenyl ring was investigated ( table 3 ) . substitution at the para-(r ) and meta-(r or r)-positions with either electron - withdrawing ( entries 3 and 57 , respectively ) or electron - donating groups ( entries 4 and 8 , respectively ) proved detrimental , with the exception of the incorporation of the meta - cf3 group in 22e ( ec50 of 22e = 4.69 0.14 nm , entry 2 ) . interestingly , incorporation of a meta - cf3 into the phenyl ring of the benzyl analogue 22c ( table 2 ) gave analogue 22l , which had an ec50 = 17.4 3.0 nm , suggesting that the use of a phenyl substituent ( as in 22e ) may be preferred to the benzyl substituent in 22l . combining the meta - cf3 substituent with either an additional meta - methoxy- or a second meta - cf3 substituent led to a decrease in activity ( entries 9 and 11 , respectively ) , whereas the meta - cf3 group was able to override the effect of a para - methoxy substituent ( cf . incorporation of a para - hydroxy in both the meta - cf3 and unsubstituted series led to a significant reduction in activity ( entries 13 and 14 , respectively ) , but extending the para - alkoxy chain from methoxy in 22o to allyloxy led to the most active compound prepared , analogue 22s , which had an ec50 against t. brucei of 2.4 0.3 nm ( entry 15 ) . the positive role of the para - allyloxy substituent was also observed in the unsubstituted series ( cf . the incorporation of an additional allyloxy substituent on the amide nitrogen in 22s led to a significant loss in activity for 22 t ( cf . interestingly , the lack of significant biological activity displayed by the 2-trifluoromethylphenol 22r , e.g. , ec50 value of 2.2 0.1 m versus 2.4 0.3 nm for 22s or 7.8 0.3 nm for 22o , might be due to the high instability of 22r in the tested conditions as the result of fluorine elimination generating a quinone methide , as previously reported . masking the phenol as allyl or methyl ethers , e.g. , as in 22s or 22o , finally , the special role played by the meta - cf3 substituent was further highlighted by a more than 5-fold drop in activity observed on its replacement by a meta - ch3 substituent ( cf . as was the case for analogues 12a k , all the analogues shown in tables 2 and 3 showed low toxicity against the human hela cell line . the striking increase in potency of the 5-nitro-2-furancarboxylamide analogues compared to nifurtimox against bloodstream t. brucei suggests they have a different or possibly additional mode of action to that of nifurtimox , i.e. , in addition to the activation by the t. brucei nitroreductase . to investigate this possibility , it was decided to generate drug resistant cell lines to nifurtimox and the most potent of the 5-nitro-2-furancarboxylamide analogues , 22s ( ec50 = 2.4 0.3 nm ) . previous lab - generated nifurtimox - resistant strains have shown that deletion or mutation of one of the alleles of the nitroreductase confers resistance . as with these previous studies , nifurtimox - resistant parasites were generated easily by culturing bloodstream t. brucei in the continuous presence of nifurtimox . a stepwise increase in the concentrations of nifurtimox , initially starting at 1.5 m , increasing to 2.5 , 4 , 5 , 7.5 , 10 , 15 , 20 , 25 , 30 , 40 , 45 , and 50 m on days 2 , 5 , 9 , 14 , 23 , 39 , 55 , 65 , 76 , 90 , 109 , and 120 , respectively , was used ( figure 3a ) . at 143 days , these nifurtimox - resistant parasites were cloned by serial dilution while maintaining the 50 m nifurtimox . generation of nifurtimox and 22s resistance cell - lines : schematic representation of the generation of a ( a ) nifurtimox - resistant and ( b ) 22s - resistant cell lines in t. brucei . each point represents the concentration at that time point when cells were checked and divided if required . selection studies started at 1 nm of 22s , increasing to 2 , 3 , 5 , 7.5 , 10 , 15 , 20 , 25 , 30 , 35 40 , and 50 m on days 2 , 5 , 9 , 26 , 36 , 47 , 58 , 70 , 76 , 103 , 130 , and 148 , respectively ( figure 3b ) . at 160 days , the 22s - resistant parasites were cloned by serial dilution while maintaining a 50 nm concentration of 22s . both of the drug - resistant cell lines were stable for 30 days in the absence of their respective drugs . the nifurtimox - resistant parasites had a doubling time of 11.5 0.6 h compared to wild - type 7.8 0.4 h , while 22s - resistant parasites had a doubling time of 13.3 1.1 h. the sensitivities of the cloned nifurtimox - resistant and 22s - resistant parasites to nifurtimox , 22s , and the diamidine pentamidine ( 2 ) were determined and compared to wild - type cells . the nifurtimox - resistant cells did not greatly alter ( 2-fold increase ) the sensitivity to pentamidine ( 2 ) compared to wild - type cells ( table 4 ) , in accordance with previous lab generated nifurtimox - resistant cell lines.22s - resistant parasites also showed very little alteration in the ec50 ( 1.1 0.1 nm ) of pentamidine ( 2 ) compared to that of wild - type cells ( table 4 ) . ec50 literature value of nifurtimox in wild - type t. brucei 2.4 0.1 m and in nifurtimox resistant cells 20.1 0.9 m . ec50 literature value of pentamidine ( 2 ) in wild - type t. brucei 0.95 0.02 nm and in nifurtimox resistant cells 2.6 0.1 nm . the nifurtimox - resistant cells were found to be 910-fold less sensitive to nifurtimox than wild - type cells , with ec50s of 20.9 1.7 and 2.1 0.2 m , respectively ( table 4 ) , in accordance with previous findings.22s - resistant cells were found to be 14-fold less sensitive to 22s than wild - type cells , with ec50s of 29.3 2.0 and 2.4 0.3 nm , respectively ( table 4 ) . even at this level of resistance , 22s is still 70 times more potent than nifurtimox in wild - type t. brucei . to investigate cross - resistance between nifurtimox and 22s , the ec50s of the drug - resistant cell lines versus the alternative nitrofuran containing compounds were determined ( table 4 ) . the results showed that nifurtimox and 22s showed a low level of cross - resistance . nifurtimox - resistant cells showed a 3-fold increase in the ec50 of 22s , while nifurtimox also showed 3-fold increase in the ec50 against 22s - resistant cells . these findings imply that these two nitrofuran compounds , nifurtimox and 22s , are trypanocidal as a result of primarily targeting different biochemical process within the parasites . however , the low level of cross - resistance suggests there may be some minor overlap in the mode of action . these findings have important implications for the therapeutic use of this new generation of nitrofuran compounds as part of novel combination therapies or more importantly potentially replacing nifurtimox in a clinical setting . as nifurtimox and 22s apparently act through primarily different modes of action , this suggests that their combined action against the parasite may be synergistic . as such , a series of ec50s were determined for 22s in the presence of various concentrations of nifurtimox ( figure 4 ) . at low concentrations of nifurtimox ( < 700 nm ) , synergy can be observed as the ec50s are below the straight diagonal line that represents what would be expected for an additive trypanocidal effect of the two compounds . above concentrations of 700 nm nifurtimox , there is a distinct lack of synergy ; if anything , competition occurs between the two inhibitors . this , taken together with the observed low level of cross - resistance ( table 4 ) , may indicate that nifurtimox at high concentrations is competing with the same biochemical activation process which is normally targeted by 22s . this further highlights the possible multitarget nature for nifurtimox that has been suggested previously . this type of information is relevant for the potential pharmacological applications of these novel nitro - furancarboxylamides as a combinational therapy would likely lead to the use of lower nifurtimox doses . studies of possible synergy between nifurtimox ( 5 ) and 22s in t. brucei . it was decided to test some of the 5-nitro-2-furancarboxylamides from this study against cultured t. b. rhodesiense , one of the human - infective subspecies ( table 5 ) . the potency of a selection of analogues against t. b. rhodesiense were similar to those observed for t. b. brucei , suggesting that these novel nitrofurans could be considered lead candidates for the next step toward a novel treatment for hat , as well as the related animal disease , nagana . in this work , we designed and synthesized a series of nitrofuran - containing analogues that were tested for trypanocidal and cytotoxicity activity against cultured bloodstream form t. b. brucei and human hela cells , respectively . the analogue 22s showed an ec50 of 2.4 0.3 nm , 3 orders of magnitude more potent than nifurtimox with a selectivity index > 8000 . sar studies showed that all fragments of the compounds were required for activity . in addition , both the nitrofuran and amide functional groups and the amide nh were necessary . the thiophene ring in the starting analogue 12a can be replaced by a range of other substituents . however , in the thiophene series , an ester group was required and is likely converted to the corresponding carboxylic acid in the parasite . several of our analogues were also tested against t. b. rhodesiense , showing analogous activity to that in t. b. brucei . this exciting result demonstrates that this series of analogues is worthy of further study as a potential therapy for hat . importantly , the cross - resistance studies showed that the 5-nitro-2-furancarboxylamide analogues in this study have low levels of resistance to nifurtimox - resistant cells and vice versa while showing no resistance to pentamidine ( 2 ) . these results indicate that the trypanocidal mode - of - action of the 5-nitro-2-furancarboxylamide analogues in this study does not rely upon nitroreductase activation unlike nifurtimox and other nitroheterocyclic compounds that are presently in clinical trials against hat . however , the lack of expected synergy at high concentrations of nifurtimox suggests it may be interacting with the novel mode of action which is primarily targeted by our novel 5-nitro-2-furancarboxylamide analogues . our data suggest that these new 5-nitro-2-furancarboxylamide analogues have a potential therapeutic implication as a single reagent in regard to potency and selectivity . also , combination therapies can be considered due to their low cross - resistance to other trypanocidal drugs . this idea is supported by our results that show there is a synergistic relationship between nifurtimox and 22s at concentrations of nifurtimox of < 700 nm ( figure 4 ) . while there remain many more challenges to the development of a hat therapeutic that have not been addressed here ( see supporting information table s6 for a detailed discussion of drug - likeness parameters associated with our analogues ) , we believe that the novel mechanistic and high in vitro potency of the antitrypanosomal compounds presented here renders them worthy of further study in the drug discovery context . the trypanocidal activity over a 72 h period was determined using the alamar blue viability , as described previously . nifurtimox , pentamidine , melarsoprol , and various novel nitrofuran analogues were tested against trypanosoma brucei brucei bloodstream - form ( strain 427 ) or drug - resistant strains were cultured at 37 c in hmi9 medium supplemented with 10% fetal calf serum and 2.5 g ml g418 as described previously.t . brucei rhodesiense ( strain z310 ) were cultured in a similar manner but in the absence of g418 . for the synergistic experiments , various concentrations of nifurtimox ( 0 , 200 , 400 , 600 , 800 , 1000 , 1500 , 2000 , and 2400 nm ) were used in conjunction with a serial dilution of 22s . experiments were conducted in replicates of four ; the data was fitted using grafit software to obtain ec50 standard deviations and slope factors . briefly , the cells were cultured in dmem supplemented with 10% fetal calf serum and 2 mm l - glutamine . cells were plated at initial cell concentration of 2 10 cells / well and incubated with the compounds for 65 h prior to addition of alamar blue solution for a further 5 h. drug - resistant t. brucei cell lines were generated by subculturing bloodstream trypanosomes in the continuous presence of either nifurtimox or compound 22s . parasites were exposed to stepwise - increased concentrations of drug , starting at appropriate sublethal concentrations , until they were routinely growing between 20 and 25 times their respective original ec50s . after 150160 days in culture , drug - resistant parasites were cloned by limiting dilution and used for further study . cell doubling times were determined in replicates of 4 over a 96 h period . chemicals and reagents were obtained from either aldrich or alfa - aesar , except nifurtimox ( 5 ) from bayer argentina . all reactions involving moisture sensitive reagents were performed in oven - dried glassware under a positive pressure of argon . dichloromethane ( dcm ) was obtained dry from a solvent purification system ( mbraun , sps-800 ) . melting points were recorded in open capillaries using an electrothermal 9100 melting point apparatus . infrared spectra were recorded on a perkin - elmer spectrum gx ft - ir spectrometer using thin films on kbr ( for solids ) discs or nujol ( for liquids ) . low resolution ( lr ) and high resolution ( hr ) electrospray mass spectral ( es - ms ) analyses were acquired by electrospray ionization ( esi ) within the school of chemistry , university of st . low and high resolution esi ms were carried out on a micromass lct spectrometer or at a high performance orthogonal acceleration reflecting tof mass spectrometer coupled to a waters 2975 hplc . nuclear magnetic resonance ( nmr ) spectra were acquired either on a bruker avance 300 ( h , 300.1 mhz ; c , 75.5 mhz ) or on a bruker avance 400 ( h , 400 mhz ; c , 100.1 mhz ) spectrometer and in the deuterated solvent stated . c nmr spectra were acquired using the pendant or deptq pulse sequences . for characterization of 2-aminothiophenes 13a k , compounds 20a c , 21 , intermediates , and other known compounds the prepared analogues were analyzed by hplc with purity above 95% ( supporting information ) . the furoic acid chlorides 14a and 14b were prepared in situ : thionyl chloride ( 1.10 equiv ) was added dropwise to a mixture of 5-nitrofuran-2-carboxylic acid or 2-furoic acid ( 1.10 equiv ) and triethylamine ( 1.50 equiv ) in dcm ( 0.4 m ) under a n2 atmosphere . the reaction mixture was stirred at room temperature for 5 h. then crude 14a or 14b was added to another flask containing the corresponding amine or aniline ( 1.00 equiv ) and triethylamine ( 2.00 equiv ) in dcm ( 0.4 m ) . the reaction mixture was stirred at room temperature for 5 h. the solvent was then removed under reduced pressure , and the crude reaction mixture was purified by column chromatography . details of the synthesis of furancarboxyl amides 12a , 12 g , 22e , 22h , and 22s are given here in the main text of the paper ; the remainder ( 12b f , 12h k , 22a d , 22f , 22 g , 22i the general procedure was followed using 2-amiothiophene 13a ( 3.99 g , 20.0 mmol ) . the crude reaction mixture was purified by column chromatography on silica gel ( 5:1 , hexane / ethyl acetate ) to afford the product as an orange solid ( 4.60 g , 13.6 mmol , 68% ) ; mp 127128 c . ir ( kbr ) max = 3120 ( s ) ( nh ) , 2958 ( m ) ( c h ) , 1667 ( s ) ( c = o ) , 1569 ( s ) , 1531 ( s ) ( no2 ) , 1350 ( s ) ( no2 ) , 1280 ( s ) ( c o ) , 1266 ( s ) ( c o ) cm . h nmr ( 400 mhz , acetone - d6 ) : 11.75 ( br s , 1h ) , 7.58 ( d , j = 3.9 hz , 1h ) , 7.42 ( d , j = 3.9 hz , 1h ) , 6.85 ( t , j = 1.1 hz , 1h ) , 4.28 ( q , j = 7.1 hz , 2h ) , 2.67 ( dq , j = 7.5 hz , j = 1.1 hz , 2h ) , 1.27 ( t , j = 7.1 hz , 3h ) , 1.17 ( t , j = 7.5 hz , 3h ) . c nmr ( 100 mhz , acetone - d6 ) : 165.8 ( c ) , 153.6 ( c ) , 153.1 ( c ) , 147.4 ( c ) , 145.6 ( c ) , 139.2 ( c ) , 120.6 ( ch ) , 118.6 ( ch ) , 115.0 ( c ) , 113.8 ( ch ) , 61.7 ( ch2 ) , 23.2 ( ch2 ) , 15.9 ( ch3 ) , 14.6 ( ch3 ) . lrms ( es ) : m / z ( % ) 360.87 ( 100 ) [ m + na ] . hrms ( es ) : m / z calcd for c14h14n2o6nas [ m + na ] , 361.0470 ; found , 361.0469 . the general procedure was followed using 2-amiothiophene 13 g ( 170 mg , 1.00 mmol ) . the crude reaction mixture was purified by column chromatography on silica gel ( 1:1 , hexane / ethyl acetate ) to afford the product as a yellow solid ( 167 mg , 0.54 mmol , 54% ) ; mp ( dec . ) ir ( kbr ) max = 3476 ( m ) , 3341 ( m ) ( nh ) , 3211 ( m ) , 3143 ( m ) , 1659 ( s ) ( c = o ) , 1591 ( s ) , 1565 ( s ) , 1534 ( s ) ( no2 ) , 1349 ( s ) ( no2 ) , 739 ( m ) cm . h nmr ( 400 mhz , acetone - d6 ) : 7.56 ( d , j = 3.9 hz , 1h ) , 7.36 ( d , j = 3.9 hz , 1h ) , 7.05 ( t , j = 1.1 hz , 1h ) , 6.80 ( br s , 1h ) , 2.66 ( dq , j = 7.5 hz , j = 1.1 hz , 2h ) , 1.16 ( t , j = 7.5 hz , 3h ) . c nmr ( 100 mhz , acetone - d6 ) : 168.3 ( c ) , 153.4 ( c ) , 152.9 ( c ) , 148.0 ( c ) , 144.2 ( c ) , 138.9 ( c ) , 119.4 ( ch ) , 117.9 ( ch ) , 117.0 ( c ) , 113.7 ( ch ) , 23.4 ( ch2 ) , 15.8 ( ch3 ) . lrms ( es ) : m / z ( % ) 331.91 ( 100 ) [ m + na ] . hrms ( es ) : m / z calcd for c12h11n3o5nas [ m + na ] , 332.0317 ; found , 332.0323 . the general procedure was followed using 3-(trifluoromethyl)aniline ( 622 l , 5.00 mmol ) . the crude reaction mixture was purified by column chromatography on silica gel ( 2:1 , hexane / ethyl acetate ) to afford the product as a yellow solid ( 550 mg , 1.83 mmol , 37% ) ; mp 188189 c . ir ( kbr ) max = 3271 ( m ) ( nh ) , 1668 ( s ) ( c = o ) , 1496 ( s ) ( no2 ) , 1334 ( s ) ( no2 ) , 1266 ( s ) , 1157 ( s ) ( cf3 ) , 1119 ( s ) ( cf3 ) cm . h nmr ( 300 mhz , acetone - d6 ) : 10.37 ( br s , 1h ) , 8.30 ( s , 1h ) , 8.07 ( d , j = 8.3 hz , 1h ) , 7.66 ( d , j = 3.8 hz , 1h ) , 7.61 ( d , j = 7.9 hz , 1h ) , 7.537.50 ( m , 2h ) . c nmr ( 75 mhz , acetone - d6 ) : 156.2 ( c ) , 152.3 ( c ) , 149.2 ( c ) , 140.2 ( c ) , 132.0 ( c ) , 131.2 ( ch ) , 127.3 ( c ) , 125.2 ( ch ) , 122.3 ( ch ) , 118.3 ( ch ) , 118.2 ( ch ) , 114.1 ( ch ) . lrms ( es ) : m / z ( % ) 301.05 ( 100 ) [ m + h ] . hrms ( es ) : m / z calcd for c12h8n2o4f3 [ m + h ] , 301.0436 ; found , 301.0428 calcd for c12h7f3n2o4 : c , 48.01 ; h , 2.35 ; n , 9.33 . found : c , 47.57 ; h , 2.27 ; n , 9.05 . the general procedure was followed using 4-(trifluoromethyl)aniline ( 622 l , 5.00 mmol ) . the crude reaction mixture was purified by column chromatography on silica gel ( 3:1 , hexane / ethyl acetate ) to afford the product a yellow solid ( 615 mg , 2.01 mmol , 41% ) ; mp 235236 c . ir ( kbr ) max = 3406 ( m ) ( nh ) , 1697 ( s ) ( c = o ) , 1538 ( s ) , 1528 ( s ) ( no2 ) , 1323 ( s ) ( no2 ) , 1129 ( s ) ( cf3 ) , 1115 ( s ) ( cf3 ) , 1069 ( s ) , 855 ( s ) cm . h nmr ( 300 mhz , acetone - d6 ) : 10.29 ( br s , 1h ) , 7.94 ( d , j = 8.6 hz , 2h ) , 7.61 ( d , j = 8.6 hz , 2h ) , 7.54 ( d , j = 3.8 hz , 1h ) , 7.40 ( d , j = 3.8 hz , 1h ) . c nmr ( 100 mhz , acetone - d6 ) : 155.8 ( c ) , 152.8 ( c ) , 148.8 ( c ) , 142.5 ( c ) , 127.0 ( 2ch ) , 126.9 ( c ) , 124.0 ( c ) , 121.3 ( 2ch ) , 117.9 ( ch ) , 113.7 ( ch ) . hrms ( es ) : m / z calcd for c12h8n2o4f3 [ m + h ] , 301.0436 ; found , 301.0432 . a mixture of 22r ( 64 mg , 0.20 mmol , 1.00 equiv ) , k2co3 ( 55 mg , 0.40 mmol , 2.00 equiv ) , and allyl bromide ( 0.03 ml , 0.40 mmol , 2.00 equiv ) in acetone ( 5 ml ) was stirred at room temperature for 3 h. the reaction mixture was then diluted in ethyl acetate ( 20 ml ) , washed with water ( 10 ml ) , dried ( na2so4 ) , and concentrated under vacuum . the crude reaction mixture was purified by column chromatography ( 3:1 , hexane / ethyl acetate ) to afford the product as a yellow solid ( 53 mg , 0.15 mmol , 75% ) ; mp 155156 c . ir ( kbr ) max = 3350 ( s ) , 3115 ( m ) ( nh ) , 1681 ( s ) ( c = o ) , 1611 ( m ) , 1504 ( s ) ( no2 ) , 1331 ( s ) ( no2 ) , 1250 ( s ) ( c o ) , 1135 ( s ) ( cf3 ) , 1113 ( s ) ( cf3 ) cm . h nmr ( 400 mhz , acetone - d6 ) : 10.07 ( br s ) , 8.03 ( d , j = 2.5 hz , 1h ) , 7.89 ( dd , j = 9.1 hz , j = 2.5 hz , 1h ) , 7.52 ( d , j = 3.9 hz , 1h ) , 7.35 ( d , j = 3.9 hz , 1h , ) , 7.13 ( d , j = 9.1 hz , 1h ) , 6.025.87 ( m , 1h ) , 5.35 ( d , j = 17.5 hz , 1h ) , 5.15 ( d , j = 10.7 hz , 1h ) , 4.644.59 ( m , 2h ) . c nmr ( 100 mhz , acetone - d6 ) : 155.5 ( c ) , 153.9 ( c ) , 152.6 ( c ) , 149.1 ( c ) , 133.8 ( ch ) , 131.8 ( c ) , 126.7 ( ch ) , 125.9 ( c ) , 123.2 ( c ) , 120.3 ( ch ) , 117.5 ( ch ) , 117.4 ( ch2 ) , 115.1 ( ch ) , 113.7 ( ch ) , 70.08 ( ch2 ) . lrms ( es ) : m / z ( % ) 378.91 ( 100 ) [ m + na ] . hrms ( es ) : m / z calcd for c15h11n2o5naf3 [ m + na ] , 379.0518 ; found , 379.0526 .

recently , the world health organization approved the nifurtimox eflornithine combination therapy for the treatment of human african trypanosomiasis , renewing interest in nitroheterocycle therapies for this and associated diseases . in this study , we have synthesized a series of novel 5-nitro-2-furancarboxylamides that show potent trypanocidal activity , 1000-fold more potent than nifurtimox against in vitro trypanosoma brucei with very low cytotoxicity against human hela cells . more importantly , the most potent analogue showed very limited cross - resistance to nifurtimox - resistant cells and vice versa . this implies that our novel , relatively easy to synthesize and therefore cheap , 5-nitro-2-furancarboxylamides are targeting a different , but still essential , biochemical process to those targeted by nifurtimox or its metabolites in the parasites . the significant increase in potency ( smaller dose probably required ) has the potential for greatly reducing unwanted side effects and also reducing the likelihood of drug resistance . collectively , these findings have important implications for the future therapeutic treatment of african sleeping sickness .

Introduction Results and Discussion Cross-Resistance Studies Conclusions Experimental Section

15% of the global population and more than 65% of the population in the third and developing world , yet current drug therapies for protozoan infections are woefully inadequate . as protozoan infections take their toll predominantly in the developing world , market forces are insufficient to promote the development of novel antiprotozoan drugs . one such neglected parasitic disease is human african trypanosomiasis ( hat ) or african sleeping sickness , which is caused by the protozoan parasite trypanosoma brucei . the world health organization ( who ) estimates that hat constitutes a serious health risk to 60 million people in sub - saharan africa . treatment of hat is solely dependent upon a repertoire of four drugs : suramin ( 1 ) , pentamidine ( 2 ) , melarsoprol ( 3 ) , and eflornithine ( 4 ) ( figure 1 ) . treatment of the second stage of the disease , where the parasites cross the blood brain barrier and invade the central nervous system , is limited to melarsoprol ( 3 ) and eflornithine ( 4 ) , the ornithine decarboxylase inhibitor ( difluoromethylornithine ) . structures of clinic drugs 15 for sleeping sickness and clinic phase i drug fexinidazole ( 6 ) . eflornithine combination therapy ( nect ) by who in the model lists of essential medicines has been seen as highly advantageous in terms of cost , logistics , and human resources in areas of poverty . the use of nect has renewed general interest in the use of nitroheterocyclic compounds to treat a wide range of infectious disease , including tuberculosis and hepatitis c. this increased awareness of the potential of nitroheterocyclic compounds has led to the reinvestigation of fexinidazole ( 6 ) ( figure 1 ) , which is presently in clinical trials against both early- and late - stage african sleeping sickness . alarmingly , it has been shown that t. brucei nifurtimox - resistant cells show cross - resistance to other nitro - containing drugs , including fexinidazole , that are currently in clinical trials . several different types of studies on nitroheterocycles have shed some light on this observed drug resistance . genome - wide rnai screens of nifurtimox and benznidazole ( another trypanocidal nitroheterocyclic drug ) resistant t. brucei have identified that a decrease in nitroreductase activity is linked to nifurtimox resistance . likewise , the overexpression of this nitroreductase , but not the alternative proposed prostaglandin f2 synthase and cytochrome p450 reductase , in t. brucei resulted in increased susceptibility to nifurtimox . collectively , it is clear that this type i t. brucei nitroreductase , a nad(p)h dependent , flavin binding protein , is involved in the reductive activation of nifurtimox . the nitroreductases mediate a series of two - electron reductions of the nitro group to a nitroso intermediate , then to a hydroxylamine , and eventually to the corresponding amine . however , the true function of the t. brucei mitochondrial nitroreductase is unknown , but its essentiality may explain why only a single allele deletion / mutation is associated with nifurtimox resistance . as well as the development of nifurtimox - resistance and its cross - resistance to other nitro - containing drugs , for example , the adverse off - target side effects with nifurtimox lead to treatment cessation in over 30% of patients with chagas disease . the most common side effects are anorexia , loss of weight , psychic alterations , excitability or sleepiness , and digestive manifestations such as nausea or vomiting and occasionally intestinal colic and diarrhea . various human enzymes have been identified to be capable of 5-nitrofuran reduction in vitro , in cells , or tissues . recently , we have elucidated in collaboration with the patton laboratory a possible role of aldehyde dehydrogenase 2 ( aldh2 ) in the toxicity caused by 5-nitrofuran containing drugs such as nifurtimox . for example , in the context of nifurtimox , most of the efforts have been focused on changing either the furan ring to other heterocycles ( such as imidazole ) or changing the substituent in the hydrazone motif , with limited increases in activity compared to nifurtimox . recently , two new types of typanocidal compounds , nitrobenzylphosphoramide mustards ( for example see compound 7 , figure 2 ) and aziridinyl nitrobenzamides , have been developed to target specifically t. brucei type i nitroreductase , the best having an ec50 1 m against both t. brucei and t. cruzi . several 5-nitro-2-furancarboxylamide containing compounds have been reported to have potent antituberculosis and antimicrobial activity , however , very few 5-nitro-2-furancarboxylamides are reported to have typanocidal activity , primarily low activity against t. cruzi . several 5-nitro-2-furancarbohydrazides possess activity against t. brucei with the best , compound 11 , having an ec50 of 0.13 m but low selectivity against human cells . , we report that our recently discovered nitrofuran nfn1 ( 12a ) also shows promising activity against t. brucei in vitro . a lack of cross - resistance with nifurtimox and significantly increased ( 23 orders of magnitude ) trypanocidal activity has important implications for the future therapeutic use of these nitrofuran containing compounds in the treatment of hat , instead of , or in combination with , nifurtimox . when a longer reaction time and an excess amount of allyl bromide was used in the o - allylation of 22r , the diallylated analogue 22 t was formed ( scheme 2 ) . in comparison to nifurtimox , nfn1 was 2 orders of magnitude more potent with an ec50 of 31.3 3.1 nm ( table 1 , cf . this observation , coupled with the decrease in activity associated with amide 12h ( entry 11 ) , may be explained by the fact that hydrolysis of the ester groups in 12a d and 12k in the parasite gives the corresponding carboxylic acid - containing bioactive metabolites , something that is unlikely to occur for the amides 12e and 12f . furthermore , analogues 12a k showed no significant toxicity against the human hela cell line even at a concentration above 20 m . interestingly , the position of the nitro group in the nitrophenyl ring had an influence as 20b , an analogue with the nitro group in the meta - position , was more active than either 20a ( ortho - no2 ) or 20c ( para - no2 ) . on the basis of the potent activity of 22b , the effect of incorporating additional substituents into the phenyl ring was investigated ( table 3 ) . incorporation of a para - hydroxy in both the meta - cf3 and unsubstituted series led to a significant reduction in activity ( entries 13 and 14 , respectively ) , but extending the para - alkoxy chain from methoxy in 22o to allyloxy led to the most active compound prepared , analogue 22s , which had an ec50 against t. brucei of 2.4 0.3 nm ( entry 15 ) . the positive role of the para - allyloxy substituent was also observed in the unsubstituted series ( cf . , ec50 value of 2.2 0.1 m versus 2.4 0.3 nm for 22s or 7.8 0.3 nm for 22o , might be due to the high instability of 22r in the tested conditions as the result of fluorine elimination generating a quinone methide , as previously reported . the striking increase in potency of the 5-nitro-2-furancarboxylamide analogues compared to nifurtimox against bloodstream t. brucei suggests they have a different or possibly additional mode of action to that of nifurtimox , i.e. to investigate this possibility , it was decided to generate drug resistant cell lines to nifurtimox and the most potent of the 5-nitro-2-furancarboxylamide analogues , 22s ( ec50 = 2.4 0.3 nm ) . previous lab - generated nifurtimox - resistant strains have shown that deletion or mutation of one of the alleles of the nitroreductase confers resistance . as with these previous studies , nifurtimox - resistant parasites were generated easily by culturing bloodstream t. brucei in the continuous presence of nifurtimox . a stepwise increase in the concentrations of nifurtimox , initially starting at 1.5 m , increasing to 2.5 , 4 , 5 , 7.5 , 10 , 15 , 20 , 25 , 30 , 40 , 45 , and 50 m on days 2 , 5 , 9 , 14 , 23 , 39 , 55 , 65 , 76 , 90 , 109 , and 120 , respectively , was used ( figure 3a ) . at 143 days , these nifurtimox - resistant parasites were cloned by serial dilution while maintaining the 50 m nifurtimox . generation of nifurtimox and 22s resistance cell - lines : schematic representation of the generation of a ( a ) nifurtimox - resistant and ( b ) 22s - resistant cell lines in t. brucei . at 160 days , the 22s - resistant parasites were cloned by serial dilution while maintaining a 50 nm concentration of 22s . both of the drug - resistant cell lines were stable for 30 days in the absence of their respective drugs . the nifurtimox - resistant parasites had a doubling time of 11.5 0.6 h compared to wild - type 7.8 0.4 h , while 22s - resistant parasites had a doubling time of 13.3 1.1 h. the sensitivities of the cloned nifurtimox - resistant and 22s - resistant parasites to nifurtimox , 22s , and the diamidine pentamidine ( 2 ) were determined and compared to wild - type cells . the nifurtimox - resistant cells did not greatly alter ( 2-fold increase ) the sensitivity to pentamidine ( 2 ) compared to wild - type cells ( table 4 ) , in accordance with previous lab generated nifurtimox - resistant cell lines.22s - resistant parasites also showed very little alteration in the ec50 ( 1.1 0.1 nm ) of pentamidine ( 2 ) compared to that of wild - type cells ( table 4 ) . ec50 literature value of nifurtimox in wild - type t. brucei 2.4 0.1 m and in nifurtimox resistant cells 20.1 0.9 m . ec50 literature value of pentamidine ( 2 ) in wild - type t. brucei 0.95 0.02 nm and in nifurtimox resistant cells 2.6 0.1 nm . the nifurtimox - resistant cells were found to be 910-fold less sensitive to nifurtimox than wild - type cells , with ec50s of 20.9 1.7 and 2.1 0.2 m , respectively ( table 4 ) , in accordance with previous findings.22s - resistant cells were found to be 14-fold less sensitive to 22s than wild - type cells , with ec50s of 29.3 2.0 and 2.4 0.3 nm , respectively ( table 4 ) . even at this level of resistance , 22s is still 70 times more potent than nifurtimox in wild - type t. brucei . to investigate cross - resistance between nifurtimox and 22s , the ec50s of the drug - resistant cell lines versus the alternative nitrofuran containing compounds were determined ( table 4 ) . the results showed that nifurtimox and 22s showed a low level of cross - resistance . nifurtimox - resistant cells showed a 3-fold increase in the ec50 of 22s , while nifurtimox also showed 3-fold increase in the ec50 against 22s - resistant cells . these findings imply that these two nitrofuran compounds , nifurtimox and 22s , are trypanocidal as a result of primarily targeting different biochemical process within the parasites . however , the low level of cross - resistance suggests there may be some minor overlap in the mode of action . these findings have important implications for the therapeutic use of this new generation of nitrofuran compounds as part of novel combination therapies or more importantly potentially replacing nifurtimox in a clinical setting . as such , a series of ec50s were determined for 22s in the presence of various concentrations of nifurtimox ( figure 4 ) . this , taken together with the observed low level of cross - resistance ( table 4 ) , may indicate that nifurtimox at high concentrations is competing with the same biochemical activation process which is normally targeted by 22s . this type of information is relevant for the potential pharmacological applications of these novel nitro - furancarboxylamides as a combinational therapy would likely lead to the use of lower nifurtimox doses . the potency of a selection of analogues against t. b. rhodesiense were similar to those observed for t. b. brucei , suggesting that these novel nitrofurans could be considered lead candidates for the next step toward a novel treatment for hat , as well as the related animal disease , nagana . in this work , we designed and synthesized a series of nitrofuran - containing analogues that were tested for trypanocidal and cytotoxicity activity against cultured bloodstream form t. b. brucei and human hela cells , respectively . the analogue 22s showed an ec50 of 2.4 0.3 nm , 3 orders of magnitude more potent than nifurtimox with a selectivity index > 8000 . this exciting result demonstrates that this series of analogues is worthy of further study as a potential therapy for hat . importantly , the cross - resistance studies showed that the 5-nitro-2-furancarboxylamide analogues in this study have low levels of resistance to nifurtimox - resistant cells and vice versa while showing no resistance to pentamidine ( 2 ) . these results indicate that the trypanocidal mode - of - action of the 5-nitro-2-furancarboxylamide analogues in this study does not rely upon nitroreductase activation unlike nifurtimox and other nitroheterocyclic compounds that are presently in clinical trials against hat . however , the lack of expected synergy at high concentrations of nifurtimox suggests it may be interacting with the novel mode of action which is primarily targeted by our novel 5-nitro-2-furancarboxylamide analogues . also , combination therapies can be considered due to their low cross - resistance to other trypanocidal drugs . this idea is supported by our results that show there is a synergistic relationship between nifurtimox and 22s at concentrations of nifurtimox of < 700 nm ( figure 4 ) . while there remain many more challenges to the development of a hat therapeutic that have not been addressed here ( see supporting information table s6 for a detailed discussion of drug - likeness parameters associated with our analogues ) , we believe that the novel mechanistic and high in vitro potency of the antitrypanosomal compounds presented here renders them worthy of further study in the drug discovery context . nifurtimox , pentamidine , melarsoprol , and various novel nitrofuran analogues were tested against trypanosoma brucei brucei bloodstream - form ( strain 427 ) or drug - resistant strains were cultured at 37 c in hmi9 medium supplemented with 10% fetal calf serum and 2.5 g ml g418 as described previously.t . cells were plated at initial cell concentration of 2 10 cells / well and incubated with the compounds for 65 h prior to addition of alamar blue solution for a further 5 h. drug - resistant t. brucei cell lines were generated by subculturing bloodstream trypanosomes in the continuous presence of either nifurtimox or compound 22s . parasites were exposed to stepwise - increased concentrations of drug , starting at appropriate sublethal concentrations , until they were routinely growing between 20 and 25 times their respective original ec50s . after 150160 days in culture , drug - resistant parasites were cloned by limiting dilution and used for further study . details of the synthesis of furancarboxyl amides 12a , 12 g , 22e , 22h , and 22s are given here in the main text of the paper ; the remainder ( 12b f , 12h k , 22a d , 22f , 22 g , 22i the general procedure was followed using 2-amiothiophene 13a ( 3.99 g , 20.0 mmol ) .

both types of diabetes mellitus can be a lead cause for blindness , kidney failure , neuropathic diseases , foot ulceration , gas gangrene in lower limbs , foot amputations , and cardiovascular insufficiency such as ischemia , stroke , etc.1 strict control of blood glucose would be the most effective approach to prevent and reduce the occurrence and progression of these manifestations of diabetic microangiopathy . glimepiride provides better chance of glycemic control by stimulating insulin release from islets of langerhans of the pancreas and by boosting the sensitivity of peripheral receptors to endogenous insulin.2 it also facilitates glucose transport from the blood into the peripheral tissues for energy consumption.3 glimepiride is practically insoluble in water , with a maximum solubility not exceeding 0.00027 mg / ml , and hence is considered as a biopharmaceutics classification system class ii drug.4 following its oral administration , low and irregular bioavailability was shown due to its low aqueous solubility . the molecular weight of glimepiride is 490.616 g / mol , with an octanol / water partition coefficient of 3.5 . it has a short half - life of ~5 hours due to the extensive hepatic oxidative metabolism to its major metabolite , cyclohexylhydroxymethyl derivative ( m1).5 this intrinsic criterion following oral administration makes it a good candidate for extended release via intramuscular or subcutaneous implantation . in an attempt to overcome these drawbacks , bhulli and sharma6 proposed an ethosomal long - acting transdermal formulation for glimepiride with 42%78% entrapment efficiency , optimal nanometric size range , low polydispersity index , and high permeability and flux through rat skin . however , the ethosomal components loosened the skin through a dissociation of hydrogen bonding network of ceramides within lipid bilayers of the stratum corneum . other transdermal formulations with high surfactant concentrations were also proposed in the literature to facilitate the percutaneous permeation of glimepiride.7,8 nevertheless , the higher surface activity induced by the surfactants would contribute to undesirable dermal sensitivity , irritation , and toxicity . for example , long - term surfactant exposure to the skin might cause protein denaturation and swelling of stratum corneum , solubilization of fluid lipids and abstraction of calcium ions to reduce corneocyte adhesion , disorganization of skin lipids , and maturation of keratinocytes and langerhans cells.9 to decrease the frequency of glimepiride administration , several studies have been proposed to demonstrate the potential application of biodegradable polymers as sustained - release carriers . polylactic acid and its copolymers with polyglycolic acid and poly(d , l - lactide - co - glycolide ) ( plga ) have been widely utilized as excipients for controlled release of injectable drugs . plga - based solid biodegradable microparticles have been proposed as depot - forming delivery systems of small - molecule active ingredients , peptides , and proteins.10 because of plga - based solid biodegradable microparticles coarse size , they require not only a complex aseptic manufacturing process but also a carrier suspension as well for sterilization of these injectable microparticles . on the other hand , using these formulations in a gel or liquid form would require not only fewer manufacturing steps but terminal filter sterilization as well.11 an in situ gel - forming formulation usually consists of a dispersion medium to dissolve and/or disperse the polymeric fraction and/or the drug . this system exists in a liquid form at temperatures lower than the body temperature but is changed to gel form when injected intramuscularly or subcutaneously to form a depot for a controlled delivery of the incorporated drug . the thermoresponsive triblock copolymers ( plga polyethylene glycol [ peg]plga copolymers ) of plga ( a - block ) and peg ( b - block ) are the most attractive in situ gel - forming agents due to their biodegradable and safety profile.12 even though the entrapment of hydrophilic drugs as well as tailoring the polymer blocks chain length have been investigated to control drug release , formulation combinations of thermoresponsive triblock copolymers ( plga peg plga copolymers ) have not been thoroughly investigated . in this study , an investigation of the embedment of zein - based glimepiride nanoparticles within plga peg plga copolymers was proposed . zein is a corn prolamin composed of a group of amino acids with a large proportion of proline , leucine , glutamine , and alanine . zein is a hydrophobic material and only soluble in ethanol at concentrations over 70%.13 zein is capable of self - association in water to encapsulate both hydrophilic and hydrophobic species.14 hence , it was employed in this study along with d - optimal fractional factorial design to screen and optimize the high - risk variables affecting the performance of glimepiride - loaded zein - based nanoparticles . the responses investigated were glimepiride entrapment capacity ( ec ) , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles . furthermore , the feasibility of embedding the optimized zein - based glimepiride nanoparticles within plga peg plga copolymers was evaluated for controlling glimepiride release rate . zein , plga ( 50:50 , molecular weight of 38,00054,000 g / mol ) , ethanol , methanol , dichloromethane ( dcm ) , sodium lauryl sulfate ( sls ) , didodecyldimethylammonium bromide ( ddab ) , stannous 2-ethylhexanoate , peg 1500 , and n - methyl-2-pyrrolidone ( nmp ) were purchased from sigma aldrich corporation , st louis , mo , usa . all other chemicals were of analytical grade and were used as received . sixteen experimental runs of glimepiride - loaded zein - based nanoparticle formulations were prepared by liquid liquid phase separation method as described by hashem et al15 with slight modification ( table 1 ) . the specified amounts of glimepiride and zein were dissolved in 3 ml dcm and 9 ml 90% v / v ethanol in water , respectively . the two solutions were homogenized using a probe sonicator ( vcx 750 ; 750 watts ; sonics and materials inc . , newtown , ct , usa ) for 5 minutes at a controlled temperature of 10c to prevent protein denaturation . the obtained emulsion was then added dropwise to 20 ml phosphate - buffered saline ( ph 7.2 ) containing a specified concentration and type of stabilizer , as listed in table 1 , while stirring at 2,000 rpm at room temperature for 3 hours , followed by evaporation overnight under reduced pressure using rotary evaporator at room temperature till complete ethanol evaporation . the nanodispersion was centrifuged for 60 minutes at 20,000 rpm to harvest the prepared nanoparticles . the formed residue was then freeze - dried for 72 hours ( alpha 12 ld plus freeze dryer with a condenser temperature of 55c ; martin christ gefriertrocknungsanlagen gmbh , osterode am harz , germany ) using mannitol as a cryoprotectant . the d - optimal experimental design was constructed using the response surface methodology procedure in the statistical analysis system ( jmp statistical discovery , version 11.1.1 ; sas institute inc . , the single and interactive effects of five different factors on glimepiride ec , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles were investigated . the tested factors were zein loading amount ( x1 ; 2550 mg ) , glimepiride loading amount ( x2 ; 2575 mg ) , stabilizer type ( x3 ; ddab and sls ) , stabilizer concentration ( x4 ; 0.01%0.1% w / v ) , and ethanol concentration ( x5 ; 70%90% v / v ) . the d - optimal design generated 16 nanoparticle trials consisting of various combinations of five factors in random order ( table 1 ) . the selection of levels for independent variables was based on the preliminary risk assessment study as well as a review of the relevant literature . optimization process was employed using a generalized desirability function to maximize glimepiride ec , zeta potential while minimizing particle size , skewness of size distribution , and drug release percentages . the optimized formulation was then incorporated into the plga peg plga thermoresponsive triblock copolymers for in situ gel formation . plga triblock copolymers were prepared by ring - opening pathway as described by zentner et al16 with slight modification . peg 1500 was added to a chemical reactor and heated for 2 hours at 150c under vacuum . stannous 2-ethylhexanoate as catalyst was then added while heating at 160c for 11 hours under vacuum . the precipitated copolymers were then dissolved in cold water ( 4c ) to remove any water - soluble impurities and then heated again to 80c to allow solidification . this purification step was repeated three times and the purified triblock copolymer was then kept dried at 37c for further experimentation . the optimized zein - based glimepiride nanoparticles were then embedded within plga peg plga copolymers using the following procedure . the dried copolymer was dissolved in nmp to prepare three polymeric solutions of 10% , 20% , and 30% w / v . in particular , the sealed vial containing a known amount of copolymer and 3 ml solvent was placed in a shaker water bath overnight at 37c till complete dissolution . the optimized freeze - dried zein - based glimepiride nanoparticles were then added to the polymeric solutions and mixed by homogenization at 8,000 rpm for 2 minutes . all formulations were prepared with glimepiride loading equivalent to 50 mg and were readily injectable through 21-gauge needle . ec was determined directly by destroying the nanoparticles , followed by aqueous extraction of entrapped drug per unit mass of each nanoparticle formulation . in brief , 20 mg of the freeze - dried nanoparticles were dissolved in 10 ml of 90% v / v ethanol in water at 45c . probe sonication was used as needed to break down any aggregates and lumps that may be formed until a clear solution was obtained . after appropriate dilution with the mobile phase , glimepiride content in this solution was assessed utilizing a developed and validated in - house high - performance liquid chromatography analytical method . a high - performance liquid chromatography instrument ( agilent technologies , santa clara , ca , usa ) with hp 1200 uv detector set at 238 the chromatographic separation was carried out by injecting 10 l sample into rp-18 luna2 ( 2504.6 mm , 5 m packing ) column ( phenomenex inc . , phosphate buffer 0.01 m ( ph 3.5 ; 45:55 , v / v ) with a flow rate of 1.0 ml / minute was used as the mobile phase . the detected mass of glimepiride ( cm ) loaded within unit mass of zein matrix ( ct ) was used to express the ec according to the following equation : ec = cmct100(1 ) particle size and electrical properties of the prepared nanoparticles were determined by dynamic light scattering using a zetatrac analyzer ( microtrac inc . , release of glimepiride from zein nanoparticles was studied utilizing automated franz diffusion cell apparatus ( microetteplus ; hanson research , chatsworth , ca , usa ) with 1.76 cm diffusion area and 7 ml receptor chamber volume . freeze - dried nanoparticles were reconstituted with phosphate - buffered saline ( ph 7.2 ) and the obtained dispersion was added to the donor chamber . a synthetic loprodyne lp nylon membrane ( 0.2 m pore size , 5156 psi water bubble point , and 139.7177.8 m thickness ; pall corporation , port washington , ny , usa ) was mounted in between the donor and receptor chambers . the receptor medium was phosphate - buffered saline ( ph 7.2 ) maintained at 37c0.5c , with a stirring rate of 400 rpm . aliquots were withdrawn at 0.5 , 1 , 2 , 4 , 6 , 8 , 10 , 12 , and 24 hours by the autosampler and analyzed for glimepiride diffused using the previously mentioned in - house developed and validated analytical chromatographic method . the in vitro release data were then evaluated for the mechanism of drug release by using a nonlinear computer program ( scientist , version 3 ; micromath scientific software , saint louis , mo , usa ) . the cumulative least square release data ( > 10% and up to 80% ) over time was fit to different mathematical release models such as power law , zero order , first order , and higuchi s diffusion . the higher value of coefficient of determination ( r ) would indicate a superiority of the release profile and mechanism fitting to the model . in vitro drug release studies from the prepared glimepiride - loaded plga peg , 1 ml sample , equivalent to 15 mg glimepiride , of the homogenized triblock polymeric dispersion of glimepiride - loaded zein nanoparticles was injected into a dialysis tube containing 10 ml of phosphate buffer solution ph 7.2 . the dialysis tube ( spectra / por 1 ; spectrum laboratories inc . , rancho dominguez , ca , usa ) employed was a hydrophilic cellulose membrane with symmetric porosity and molecular weight cut - off of 6,0008,000 da . the dialysis tubes were hermetically sealed and inserted into the vessels of usp ii dissolution apparatus . phosphate buffer solution ( 450 ml , ph 7.2 ) was used as receptor medium at 37c , with a rotational speed maintained at 100 rpm . aliquots , 3 ml each , were withdrawn from the external medium at time intervals and replaced with the same volume of fresh medium . the samples were assayed for glimepiride content using the previously mentioned in - house developed and validated analytical chromatographic method . all the experiments were performed in triplicate , and the mean cumulative drug release sd were calculated . the appearance , color , and homogeneity of the prepared triblock polymeric solutions were observed by visual observation . the viscosities were also evaluated using brookfield dv - iii rheometer ( brookfield engineering laboratories inc . viscosity and rheological parameters were evaluated at varying shear rates with 20-second equilibration to allow for full recovery from the shear applied . the viscosity and rheological assessment was done at controlled temperature of 37c to reflect the physiological condition . syringeability was also assessed by determining the force needed to push the prepared triblock polymeric solutions through a 21-gauge needle using a texture analyzer ( ta.xt plus ; stable micro systems ltd . , godalming , surrey , uk ) in the compression mode . the upper probe was set to move downward at constant speed of 1.0 mm / s with a constant down force of 0.3 n. the force displacement profiles of a 10 mm distance were then recorded and the corresponding area under the curve was used to assess the work of discharge . the rate of water diffusion into the triblock polymeric solutions for in situ gel formation was assessed using the following procedure . a transparent borosilicate glass cylinder ( 15 mm internal diameter ) cellulose ester membrane ( 100 kd ; spectrum laboratories inc . , houston , tx , usa ) was fit to one end and 1 ml sample the membrane side was immersed into 150 ml phosphate buffer solution ph 7.2 to the depth of 20 mm . the distance of water front diffused through the membrane to form a gel was determined at various time points of 0 , 6 , and 24 hours . the rate of water diffusion at each time point was then calculated by dividing the distance in mm by the time lapse in minutes . zein , plga ( 50:50 , molecular weight of 38,00054,000 g / mol ) , ethanol , methanol , dichloromethane ( dcm ) , sodium lauryl sulfate ( sls ) , didodecyldimethylammonium bromide ( ddab ) , stannous 2-ethylhexanoate , peg 1500 , and n - methyl-2-pyrrolidone ( nmp ) were purchased from sigma aldrich corporation , st louis , mo , usa . all other chemicals were of analytical grade and were used as received . sixteen experimental runs of glimepiride - loaded zein - based nanoparticle formulations were prepared by liquid liquid phase separation method as described by hashem et al15 with slight modification ( table 1 ) . the specified amounts of glimepiride and zein were dissolved in 3 ml dcm and 9 ml 90% v / v ethanol in water , respectively . the two solutions were homogenized using a probe sonicator ( vcx 750 ; 750 watts ; sonics and materials inc . , newtown , ct , usa ) for 5 minutes at a controlled temperature of 10c to prevent protein denaturation . the obtained emulsion was then added dropwise to 20 ml phosphate - buffered saline ( ph 7.2 ) containing a specified concentration and type of stabilizer , as listed in table 1 , while stirring at 2,000 rpm at room temperature for 3 hours , followed by evaporation overnight under reduced pressure using rotary evaporator at room temperature till complete ethanol evaporation . the nanodispersion was centrifuged for 60 minutes at 20,000 rpm to harvest the prepared nanoparticles . the formed residue was then freeze - dried for 72 hours ( alpha 12 ld plus freeze dryer with a condenser temperature of 55c ; martin christ gefriertrocknungsanlagen gmbh , osterode am harz , germany ) using mannitol as a cryoprotectant . the d - optimal experimental design was constructed using the response surface methodology procedure in the statistical analysis system ( jmp statistical discovery , version 11.1.1 ; sas institute inc . , the single and interactive effects of five different factors on glimepiride ec , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles were investigated . the tested factors were zein loading amount ( x1 ; 2550 mg ) , glimepiride loading amount ( x2 ; 2575 mg ) , stabilizer type ( x3 ; ddab and sls ) , stabilizer concentration ( x4 ; 0.01%0.1% w / v ) , and ethanol concentration ( x5 ; 70%90% v / v ) . the d - optimal design generated 16 nanoparticle trials consisting of various combinations of five factors in random order ( table 1 ) . the selection of levels for independent variables was based on the preliminary risk assessment study as well as a review of the relevant literature . optimization process was employed using a generalized desirability function to maximize glimepiride ec , zeta potential while minimizing particle size , skewness of size distribution , and drug release percentages . the optimized formulation was then incorporated into the plga peg plga thermoresponsive triblock copolymers for in situ gel formation . plga peg plga triblock copolymers were prepared by ring - opening pathway as described by zentner et al16 with slight modification . peg 1500 was added to a chemical reactor and heated for 2 hours at 150c under vacuum . stannous 2-ethylhexanoate as catalyst was then added while heating at 160c for 11 hours under vacuum . the precipitated copolymers were then dissolved in cold water ( 4c ) to remove any water - soluble impurities and then heated again to 80c to allow solidification . this purification step was repeated three times and the purified triblock copolymer was then kept dried at 37c for further experimentation . the optimized zein - based glimepiride nanoparticles were then embedded within plga peg plga copolymers using the following procedure . the dried copolymer was dissolved in nmp to prepare three polymeric solutions of 10% , 20% , and 30% w / v . in particular , the sealed vial containing a known amount of copolymer and 3 ml solvent was placed in a shaker water bath overnight at 37c till complete dissolution . the optimized freeze - dried zein - based glimepiride nanoparticles were then added to the polymeric solutions and mixed by homogenization at 8,000 rpm for 2 minutes . all formulations were prepared with glimepiride loading equivalent to 50 mg and were readily injectable through 21-gauge needle . ec was determined directly by destroying the nanoparticles , followed by aqueous extraction of entrapped drug per unit mass of each nanoparticle formulation . in brief , 20 mg of the freeze - dried nanoparticles were dissolved in 10 ml of 90% v / v ethanol in water at 45c . probe sonication was used as needed to break down any aggregates and lumps that may be formed until a clear solution was obtained . after appropriate dilution with the mobile phase , glimepiride content in this solution was assessed utilizing a developed and validated in - house high - performance liquid chromatography analytical method . a high - performance liquid chromatography instrument ( agilent technologies , santa clara , ca , usa ) with hp 1200 uv detector set at 238 the chromatographic separation was carried out by injecting 10 l sample into rp-18 luna2 ( 2504.6 mm , 5 m packing ) column ( phenomenex inc . , torrance , ca , usa ) . phosphate buffer 0.01 m ( ph 3.5 ; 45:55 , v / v ) with a flow rate of 1.0 ml / minute was used as the mobile phase . the detected mass of glimepiride ( cm ) loaded within unit mass of zein matrix ( ct ) was used to express the ec according to the following equation : ec = cmct100(1 ) particle size and electrical properties of the prepared nanoparticles were determined by dynamic light scattering using a zetatrac analyzer ( microtrac inc . , montgomerville , pa , usa ) after appropriate sample dilution with distilled water . release of glimepiride from zein nanoparticles was studied utilizing automated franz diffusion cell apparatus ( microetteplus ; hanson research , chatsworth , ca , usa ) with 1.76 cm diffusion area and 7 ml receptor chamber volume . freeze - dried nanoparticles were reconstituted with phosphate - buffered saline ( ph 7.2 ) and the obtained dispersion was added to the donor chamber . a synthetic loprodyne lp nylon membrane ( 0.2 m pore size , 5156 psi water bubble point , and 139.7177.8 m thickness ; pall corporation , port washington , ny , usa ) was mounted in between the donor and receptor chambers . the receptor medium was phosphate - buffered saline ( ph 7.2 ) maintained at 37c0.5c , with a stirring rate of 400 rpm . aliquots were withdrawn at 0.5 , 1 , 2 , 4 , 6 , 8 , 10 , 12 , and 24 hours by the autosampler and analyzed for glimepiride diffused using the previously mentioned in - house developed and validated analytical chromatographic method . the in vitro release data were then evaluated for the mechanism of drug release by using a nonlinear computer program ( scientist , version 3 ; micromath scientific software , saint louis , mo , usa ) . the cumulative least square release data ( > 10% and up to 80% ) over time was fit to different mathematical release models such as power law , zero order , first order , and higuchi s diffusion . the higher value of coefficient of determination ( r ) would indicate a superiority of the release profile and mechanism fitting to the model . in vitro drug release studies from the prepared glimepiride - loaded plga peg plga in situ gel were evaluated using a modified dialysis . for gel formation , 1 ml sample , equivalent to 15 mg glimepiride , of the homogenized triblock polymeric dispersion of glimepiride - loaded zein nanoparticles was injected into a dialysis tube containing 10 ml of phosphate buffer solution ph 7.2 . the dialysis tube ( spectra / por 1 ; spectrum laboratories inc . , rancho dominguez , ca , usa ) employed was a hydrophilic cellulose membrane with symmetric porosity and molecular weight cut - off of 6,0008,000 da . the dialysis tubes were hermetically sealed and inserted into the vessels of usp ii dissolution apparatus . phosphate buffer solution ( 450 ml , ph 7.2 ) was used as receptor medium at 37c , with a rotational speed maintained at 100 rpm . aliquots , 3 ml each , were withdrawn from the external medium at time intervals and replaced with the same volume of fresh medium . the samples were assayed for glimepiride content using the previously mentioned in - house developed and validated analytical chromatographic method . all the experiments were performed in triplicate , and the mean cumulative drug release sd were calculated . the appearance , color , and homogeneity of the prepared triblock polymeric solutions were observed by visual observation . the viscosities were also evaluated using brookfield dv - iii rheometer ( brookfield engineering laboratories inc . , viscosity and rheological parameters were evaluated at varying shear rates with 20-second equilibration to allow for full recovery from the shear applied . the viscosity and rheological assessment was done at controlled temperature of 37c to reflect the physiological condition . syringeability was also assessed by determining the force needed to push the prepared triblock polymeric solutions through a 21-gauge needle using a texture analyzer ( ta.xt plus ; stable micro systems ltd . , godalming , surrey , uk ) in the compression mode . the upper probe was set to move downward at constant speed of 1.0 mm / s with a constant down force of 0.3 n. the force displacement profiles of a 10 mm distance were then recorded and the corresponding area under the curve was used to assess the work of discharge . the rate of water diffusion into the triblock polymeric solutions for in situ gel formation was assessed using the following procedure . a transparent borosilicate glass cylinder ( 15 mm internal diameter ) was used . cellulose ester membrane ( 100 kd ; spectrum laboratories inc . , houston , tx , usa ) was fit to one end and 1 ml sample was then added from the other side . the membrane side was immersed into 150 ml phosphate buffer solution ph 7.2 to the depth of 20 mm . the distance of water front diffused through the membrane to form a gel was determined at various time points of 0 , 6 , and 24 hours . the rate of water diffusion at each time point was then calculated by dividing the distance in mm by the time lapse in minutes . this investigation aimed at optimizing glimepiride - loaded zein nanoparticles to be embedded in an in situ forming plga peg the predefined critical quality attributes of the glimepiride - loaded zein nanoparticles were glimepiride ec , particle size , and glimepiride release rate . maximizing the ec of zein matrix to glimepiride would allow for a reduction of manufacturing costs , dosage volume , and easiness of syringeability for in situ gel formation . therefore , the first objective of this study was to screen and optimize the nanoparticle formulation factors using a d - optimal design for their effects to maximize ec while minimizing the produced size , size distribution , and drug release rate . in achieving this goal , minimum constraint was applied for ec to be more than 20% w / w glimepiride in zein matrix , maximum constraint for size to be less than 100 nm , and to minimize the drug release rate . the second goal was to embed the optimized nanoparticle formulation within plga peg plga triblock for in situ gel formation with acceptable gel characteristics . data in table 2 show that glimepiride ec ranged from 15.7% ( f8 ) to 62.6% ( f10 ) for the different variable combinations of d - optimal design to prepare zein nanoparticles . using the full multiple regression equations , quantile quantile correlation to regress the observed ec values versus the predicted one showed a linear relationship with a coefficient of multiple determination ( r ) value of 0.9773 ( table 3 ) . this step demonstrates the accuracy and robustness of the regression model to predict the ec within the investigated design space . the consequent analysis of variance demonstrated a significant prediction efficiency with prob > f - value of 0.0003 and 0.0025 at p<0.05 and p<0.01 , respectively ( table 3 ) . after neglecting the non - significant factors , the following reduced prediction model was obtained to correlate the individual and interaction effects of the significant variables on glimepiride ec . using the developed prediction model , the response surface plots , shown in figure 1 , would demonstrate the design space for predicting and subsequent monitoring of glimepiride entrapment within zein nanoparticles . the reduced prediction equation of ec value is : glimepirideec(%w / wtotheemployedzeinamount)=34.8 + 8.9[zeinloading(mg)37.512.5]9.02[glimepirideloading(mg)5025](2 ) out of five formulation and processing variables , zein and glimepiride experimental loadings were significant for their influences on ec . glimepiride working amounts had the most significant effect on the resultant ec with a t - ratio of 10.67 , followed by zein loading with a t - ratio of 10.52 ( figure 2 and table 3 ) . positive and negative effects on ec were observed by increasing experimental zein and drug experimental loadings ( figure 1 ) . maximum ec of 62.6% ( f10 ) was obtained at 2:1 zein to glimepiride weight ratio . in comparison with ddab , a negative influence of sls at low zein loading on ec however , the interaction profiler of figure 3 showed that both stabilizers were comparable for their performance at higher zein loading . zein is composed of charged amino acids in high proportions . while coalescing at ph 7.2 , protein molecules aggregated into smaller particles with decreased void spaces where the peptide chains became unfolded to expose more reactive sites for cross - linking . therefore , the ec of glimepiride was higher than those obtained by muthuselvi and dhathathreyan17 for gitoxin into zein nanoparticles . however , these ec values were in a good agreement with that obtained by lai and guo18 for the encapsulation of 5-fluorouracil into zein nanoparticles . disulfide interchange reaction to enhance nanoparticle formation while inhibiting large aggregation . on the other hand , the negative effect of glimepiride of the resultant ec hence , these weakly bound or adsorbed drug molecules to the relatively larger surface of nanoparticles would facilitate pore formation for leaching more drug molecules.18 the positive action of ddab to retain glimepiride within the formed nanoparticles might be attributed to the packing of ddab at the formed surfaces . both the double - tailed structure of ddab and its critical packing parameter of 0.51 would contribute to particle formation.19 moreover , the high surface coverage of ddab on the zein nanoparticles would protect the particles and restrict the liberty of the glimepiride molecules to increase its ec . histograms of the particle size distribution of the prepared zein nanoparticles showed a monodispersion in diameter . table 2 lists the median nanoparticles size ( d50 ) , which ranged from 11 nm ( f10 ) to 603 nm ( f4 ) on changing both formulation and process variables investigated . on the other hand , skewness values for size distribution ranged from 0.023 ( f4 ) to 0.56 ( f7 ) . plotting the observed versus predicted values for nanoparticle sizes and skewness yielded linear relationships with r value of 0.9182 and 0.8723 , respectively , to demonstrate that a prediction model can be constructed with an acceptable accuracy ( table 3 ) . analysis of variance ( anova ) results revealed significant effects of zein and glimepiride loadings and ethanol level on the sizes and skewness values with prob > f - values of 0.0118 and 0.0394 , respectively , p<0.05 ( table 3 ) . the reduced prediction models to correlate individual and significant variables with the obtained sizes and corresponding skewness are shown below : medianparticlesize(d50)nm=169.2117.3[zeinloading(mg)37.512.5]96.3[ethanolconcentration(%)8010](3 ) skewness=0.20.02[zeinloading(mg)37.512.5]0.09[glimepirideloading(mg)5025]0.01[ethanolconcentration(%)8010](4 ) experimental zein loading was the most significant factor affecting the obtained size ( p=0.0025 ) , followed by ethanol level and its interaction time with zein ( figure 2 and table 3 ) . regarding the size distribution , glimepiride loading was more significant than zein concentration with p - values of 0.0412 and 0.5251 , respectively ( figure 2 and table 3 ) . negative correlations were found on the resultant sizes and skewness values by increasing both zein and ethanol loadings ( figure 1 ) , with a lowest nanoparticle size obtained at 2:1 zein to glimepiride weight ratio , 0.1% sls as stabilizer , and 70% ethanol concentration ( f10 ) . a significant interaction existed between zein loading and ethanol concentration to affect the resultant sizes ( figure 3 ) . at low ethanol concentration , the nanoparticles sizes decreased as the amount of zein increased , thus abolishing aggregation of zein molecules and vice versa . this observation might be described by the unique structural configuration of hydrophobic and hydrophilic regions on the surface of zein molecules . studies of the orientation of zein peptides in 70% ethanolic solution demonstrated a three - dimensional configuration with a high axial ratio.20 hence , at low ethanol level , zein molecules associated into an elongated prismlike shape with hydrophobic sides and hydrophilic tops and bottoms.21 this configuration would allow zein molecules to associate in a side - by - side manner to form a large number of smaller nanoparticles rather than increasing the particle size . despite being nonsignificant , the positive influence of glimepiride entrapment on the resultant sizes could be explained according to the law of mass action to entrap glimepiride molecules within zein protein matrix . for the 16 formulations of d - optimal design , table 2 lists the zeta potential values , which ranged from 0.61 mv ( f2 ) to 13.54 mv ( f9 ) . the significant factors affecting the resultant zeta potentials were zein loading and stabilizer employed , with a more predominant effect to stabilizer type ( table 3 and figure 2 ) . multiple regression analysis showed positive values of both variables on the recorded zeta potentials , with a significant effect for their interaction term x1x3 . the extent of the positive charge on the formed zein nanoparticles decreased from about + 12.82 mv when using ddab to about + 2.84 mv when using sls . the adsorption of nonionic surfactants with its anionic impurities ( such as free fatty acids ) at the formed cationic zein surfaces could reduce their charge.22 the contribution to increase the zeta potential of zein nanoparticles dispersions would be suggestive of its stability due to the london dispersion forces . hence , a maximized desirability function for zeta potential was applied to preserve the required electrostatic energies at nanoparticles surfaces . the regression equation revealed an acceptable predictability of zeta potential , with a p - value of 0.0025 and quantile quantile correlation coefficient of 0.9525 ( table 3 ) . the reduced prediction equation of zeta potential value is : zetapotential(mv)=5.1 + 1.5[zeinloading(mg)37.512.5]+[ddab2.866875sls2.866875](5 ) glimepiride release profile of all 16 formulations of d - optimal design can be described as a two - step biphasic process , ie , an initial burst effect followed by subsequent slower release ( figure 4 ) . at the initial stage , the burst release is usually ascribed to the free glimepiride or that embedded near the surface of the formed nanoparticles . the obtained release data fit to a logarithmic time - dependent release rather than higuchi diffusion release ( figure 4 ) . this behavior would be ascribed to the expanding transient boundary at the interface of either protein surface or ddab - adsorbed layer and the aqueous medium.23 the following is the logarithmic equation to describe the drug release rate , where qti is amount of cumulative drug released after time t in the aqueous medium and k is the release constant or the slope of the regressing qti versus base-10 logarithm of time divided by 2.303 . qti=(2.303log10(ti)k)+qti1(6 ) the cumulative percentages of glimepiride released after 2 hours ( q2hours ) and 24 hours ( q24hours ) for the 16 formulations varied from 3.02% ( f16 ) to 14.52% ( f3 ) and from 15.76% ( f13 ) to 43.02% ( f9 ) , respectively ( table 2 ) . a good correlation was established between the formulation / process parameters and glimepiride release with r values of 0.9254 and 0.9111 for q2hours and q24hours , respectively ( table 3 ) . anova results confirmed that the prediction capability was evident with a prob > f - values of 0.0091 and 0.0148 for q2hours and q24hours , respectively , at p<0.05 ( table 3 ) . more detailed effect analysis demonstrated that glimepiride release from zein nanoparticles was predominantly affected by formulation design ( table 2 and figure 1 ) . zein and glimepiride loadings significantly impacted these release parameters , with their interaction term ( x1x2 ) being the most important formulation parameter ( p<0.05 ; figures 2 and 3 ) . logically , higher zein - to - glimepiride weight ratio in the nanoparticle solid state led to lower concentration of the solubilized drug ( molecular state ) in the transient layer , and consequently a lesser drug release . particle size of the nanoparticle is also thought to impact the initial boundary layer thickness . a large size of the nanoparticle that is similar or greater than the thickness of the boundary may significantly increase the thickness of the initial boundary layer . despite being insignificant for the prediction model , the employed stabilizer would likely affect glimepiride release by indirectly accelerating drug diffusion to the aqueous medium ( figure 2 ) . q2hours(%)=7.51.91[zeinloading(mg)37.512.5]1.6[glimepirideloading(mg)5025](7 ) q24hours(%)=28.443.6[zeinloading(mg)37.512.5]2.7[glimepirideloading(mg)5025](8 ) the optimization tool of jmp software has been selected based on a generalized desirability function that maximizes glimepiride ec and zeta potential while minimizing vesicular size and release parameters . the highest desirability was obtained at upper levels of both zein and glimepiride loadings ( 50 and 75 mg , respectively ) , using ddab as stabilizer at 0.1% and when the percentage of ethanol during processing was at its higher level of 90% . under these optimization criteria , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , q2hours of 3.3% , and q24hours of 17.3% were obtained . the proposed optimal conditions were then experimentally corroborated , and the results were closely correlated with the data predicted by the desirability function of the d - optimal design . glimepiride - loaded in situ gels were prepared by incorporating the optimized zein nanoparticles into plga peg all polymeric solutions were yellowish in color , with the zein nanoparticles suspended homogeneously with no signs of precipitations or agglomerations . the ph values of all solutions were in the range of 7.210.32 , with no effect of nanoparticles incorporation . for all samples , the apparent viscosities were higher at low shear rate than those at a high shear rate to demonstrate pseudoplastic behavior . similar observations were observed by other researchers for ethylcellulose - based gels incorporating antimicrobial drugs.24 the significant interaction between both polymer chains and drug molecules at low temperature and the shearing rate explained this behavior . the flow behavior of the solutions was also studied by calculating the flow indices and viscosity coefficient ( ) as a function of polymer concentration at 37c ( figure 5 ) . the flow index values of all formulations were more than 1 to confirm the non - newtonian flow similar to that of the gel base . regarding the viscosity coefficient ( ) , the amount of the polymer employed was significantly ( p<0.05 ) increasing the resultant viscosity coefficient . fresno et al25 explained this behavior by the deformation and change in the shape of polymer molecules and in the number of molecular entanglements observed as the shearing rate increased . syringeability was also assessed to estimate the force needed to pass the polymeric solutions through 21-gauge needle to form hypodermic in situ gels . the obtained results showed that the expelling force significantly increased ( p<0.05 ) as the percentage of polymer was increased . for example , 652.4 and 431.7 n / mm were the exerted forces of expulsion of formulations with 30% and 10% polymer concentrations , respectively . solvent interaction over the polymer polymer interaction , therefore lowering the resistance to flow . nmp could dissolve a large amount of the polymer , thus controlling both drug diffusion and easiness of syringeability.27 the main critical parameters of depot formation or structure change to form a gel were both temperature increase to 37c and water diffusion rate into the zein nanoparticles - loaded triblock polymer . the diffusion rate of the aqueous phosphate buffer ph 7.2 into the polymeric solutions decreased with increasing the triblock polymer concentration ( p<0.05 ; figure 5 ) due to the increased gel viscosity . wang et al28 demonstrated that the fast partitioning of the employed solvent might cause fast solidification of the implant ; hence , higher drug retention rate was expected . glimepiride release from the prepared gels was conducted in phosphate buffer ph 7.2 to simulate the physiologic condition ( figure 5 ) . in general , for example , 17.3% drug was released from the optimized nanoparticle formulation at 24 hours , whereas drug release rates in in situ gels containing 10% , 20% , and 30% w / w triblock polymer were about 14.5% , 10.2% , and 7.1% at 24 hours ( figure 5 ) , respectively . the physical entanglements between the polymer s chains to form a dense matrix would explain this result . liu and venkatraman29 demonstrated that the polymer phase inversion dynamics would suppress the initial drug burst release by each increase in polymer level . the hydrophobic plga compartment of the triblock would slow the drug release , whereas the hydrophilic peg would modulate the initial burst release , followed by a more rapid phase once the triblock became hydrated.30 on the other hand , a porous rubbery gel structure was formed by the nmp diffusion out of the hydrated triblock to cause burst release of glimepiride due to rapid phase inversion of plga.24 data in table 2 show that glimepiride ec ranged from 15.7% ( f8 ) to 62.6% ( f10 ) for the different variable combinations of d - optimal design to prepare zein nanoparticles . using the full multiple regression equations , quantile quantile correlation to regress the observed ec values versus the predicted one showed a linear relationship with a coefficient of multiple determination ( r ) value of 0.9773 ( table 3 ) . this step demonstrates the accuracy and robustness of the regression model to predict the ec within the investigated design space . the consequent analysis of variance demonstrated a significant prediction efficiency with prob > f - value of 0.0003 and 0.0025 at p<0.05 and p<0.01 , respectively ( table 3 ) . after neglecting the non - significant factors , the following reduced prediction model was obtained to correlate the individual and interaction effects of the significant variables on glimepiride ec . using the developed prediction model , the response surface plots , shown in figure 1 , would demonstrate the design space for predicting and subsequent monitoring of glimepiride entrapment within zein nanoparticles . the reduced prediction equation of ec value is : glimepirideec(%w / wtotheemployedzeinamount)=34.8 + 8.9[zeinloading(mg)37.512.5]9.02[glimepirideloading(mg)5025](2 ) out of five formulation and processing variables , zein and glimepiride experimental loadings were significant for their influences on ec . glimepiride working amounts had the most significant effect on the resultant ec with a t - ratio of 10.67 , followed by zein loading with a t - ratio of 10.52 ( figure 2 and table 3 ) . positive and negative effects on ec were observed by increasing experimental zein and drug experimental loadings ( figure 1 ) . maximum ec of 62.6% ( f10 ) was obtained at 2:1 zein to glimepiride weight ratio . in comparison with ddab , a negative influence of sls at low zein loading on ec was observed for nanoparticle stabilization ( figure 1 ) . however , the interaction profiler of figure 3 showed that both stabilizers were comparable for their performance at higher zein loading . zein is composed of charged amino acids in high proportions . while coalescing at ph 7.2 , protein molecules aggregated into smaller particles with decreased void spaces where the peptide chains became unfolded to expose more reactive sites for cross - linking . therefore , the ec of glimepiride was higher than those obtained by muthuselvi and dhathathreyan17 for gitoxin into zein nanoparticles . however , these ec values were in a good agreement with that obtained by lai and guo18 for the encapsulation of 5-fluorouracil into zein nanoparticles . disulfide interchange reaction to enhance nanoparticle formation while inhibiting large aggregation . on the other hand , the negative effect of glimepiride of the resultant ec hence , these weakly bound or adsorbed drug molecules to the relatively larger surface of nanoparticles would facilitate pore formation for leaching more drug molecules.18 the positive action of ddab to retain glimepiride within the formed nanoparticles might be attributed to the packing of ddab at the formed surfaces . both the double - tailed structure of ddab and its critical packing parameter of 0.51 would contribute to particle formation.19 moreover , the high surface coverage of ddab on the zein nanoparticles would protect the particles and restrict the liberty of the glimepiride molecules to increase its ec . histograms of the particle size distribution of the prepared zein nanoparticles showed a monodispersion in diameter . table 2 lists the median nanoparticles size ( d50 ) , which ranged from 11 nm ( f10 ) to 603 nm ( f4 ) on changing both formulation and process variables investigated . on the other hand , skewness values for size distribution ranged from 0.023 ( f4 ) to 0.56 ( f7 ) . plotting the observed versus predicted values for nanoparticle sizes and skewness yielded linear relationships with r value of 0.9182 and 0.8723 , respectively , to demonstrate that a prediction model can be constructed with an acceptable accuracy ( table 3 ) . analysis of variance ( anova ) results revealed significant effects of zein and glimepiride loadings and ethanol level on the sizes and skewness values with prob > f - values of 0.0118 and 0.0394 , respectively , p<0.05 ( table 3 ) . the reduced prediction models to correlate individual and significant variables with the obtained sizes and corresponding skewness are shown below : medianparticlesize(d50)nm=169.2117.3[zeinloading(mg)37.512.5]96.3[ethanolconcentration(%)8010](3 ) skewness=0.20.02[zeinloading(mg)37.512.5]0.09[glimepirideloading(mg)5025]0.01[ethanolconcentration(%)8010](4 ) experimental zein loading was the most significant factor affecting the obtained size ( p=0.0025 ) , followed by ethanol level and its interaction time with zein ( figure 2 and table 3 ) . regarding the size distribution , glimepiride loading was more significant than zein concentration with p - values of 0.0412 and 0.5251 , respectively ( figure 2 and table 3 ) . negative correlations were found on the resultant sizes and skewness values by increasing both zein and ethanol loadings ( figure 1 ) , with a lowest nanoparticle size obtained at 2:1 zein to glimepiride weight ratio , 0.1% sls as stabilizer , and 70% ethanol concentration ( f10 ) . a significant interaction existed between zein loading and ethanol concentration to affect the resultant sizes ( figure 3 ) . at low ethanol concentration , the nanoparticles sizes decreased as the amount of zein increased , thus abolishing aggregation of zein molecules and vice versa . this observation might be described by the unique structural configuration of hydrophobic and hydrophilic regions on the surface of zein molecules . studies of the orientation of zein peptides in 70% ethanolic solution demonstrated a three - dimensional configuration with a high axial ratio.20 hence , at low ethanol level , zein molecules associated into an elongated prismlike shape with hydrophobic sides and hydrophilic tops and bottoms.21 this configuration would allow zein molecules to associate in a side - by - side manner to form a large number of smaller nanoparticles rather than increasing the particle size . despite being nonsignificant , the positive influence of glimepiride entrapment on the resultant sizes could be explained according to the law of mass action to entrap glimepiride molecules within zein protein matrix . for the 16 formulations of d - optimal design , table 2 lists the zeta potential values , which ranged from 0.61 mv ( f2 ) to 13.54 mv ( f9 ) . the significant factors affecting the resultant zeta potentials were zein loading and stabilizer employed , with a more predominant effect to stabilizer type ( table 3 and figure 2 ) . multiple regression analysis showed positive values of both variables on the recorded zeta potentials , with a significant effect for their interaction term x1x3 . the extent of the positive charge on the formed zein nanoparticles decreased from about + 12.82 mv when using ddab to about + 2.84 mv when using sls . the adsorption of nonionic surfactants with its anionic impurities ( such as free fatty acids ) at the formed cationic zein surfaces could reduce their charge.22 the contribution to increase the zeta potential of zein nanoparticles dispersions would be suggestive of its stability due to the london dispersion forces . hence , a maximized desirability function for zeta potential was applied to preserve the required electrostatic energies at nanoparticles surfaces . the regression equation revealed an acceptable predictability of zeta potential , with a p - value of 0.0025 and quantile quantile correlation coefficient of 0.9525 ( table 3 ) . glimepiride release profile of all 16 formulations of d - optimal design can be described as a two - step biphasic process , ie , an initial burst effect followed by subsequent slower release ( figure 4 ) . at the initial stage , the burst release is usually ascribed to the free glimepiride or that embedded near the surface of the formed nanoparticles . the obtained release data fit to a logarithmic time - dependent release rather than higuchi diffusion release ( figure 4 ) . this behavior would be ascribed to the expanding transient boundary at the interface of either protein surface or ddab - adsorbed layer and the aqueous medium.23 the following is the logarithmic equation to describe the drug release rate , where qti is amount of cumulative drug released after time t in the aqueous medium and k is the release constant or the slope of the regressing qti versus base-10 logarithm of time divided by 2.303 . qti=(2.303log10(ti)k)+qti1(6 ) the cumulative percentages of glimepiride released after 2 hours ( q2hours ) and 24 hours ( q24hours ) for the 16 formulations varied from 3.02% ( f16 ) to 14.52% ( f3 ) and from 15.76% ( f13 ) to 43.02% ( f9 ) , respectively ( table 2 ) . a good correlation was established between the formulation / process parameters and glimepiride release with r values of 0.9254 and 0.9111 for q2hours and q24hours , respectively ( table 3 ) . anova results confirmed that the prediction capability was evident with a prob > f - values of 0.0091 and 0.0148 for q2hours and q24hours , respectively , at p<0.05 ( table 3 ) . more detailed effect analysis demonstrated that glimepiride release from zein nanoparticles was predominantly affected by formulation design ( table 2 and figure 1 ) . zein and glimepiride loadings significantly impacted these release parameters , with their interaction term ( x1x2 ) being the most important formulation parameter ( p<0.05 ; figures 2 and 3 ) . logically , higher zein - to - glimepiride weight ratio in the nanoparticle solid state led to lower concentration of the solubilized drug ( molecular state ) in the transient layer , and consequently a lesser drug release . particle size of the nanoparticle is also thought to impact the initial boundary layer thickness . a large size of the nanoparticle that is similar or greater than the thickness of the boundary may significantly increase the thickness of the initial boundary layer . despite being insignificant for the prediction model , the employed stabilizer would likely affect glimepiride release by indirectly accelerating drug diffusion to the aqueous medium ( figure 2 ) . the optimization tool of jmp software has been selected based on a generalized desirability function that maximizes glimepiride ec and zeta potential while minimizing vesicular size and release parameters . the highest desirability was obtained at upper levels of both zein and glimepiride loadings ( 50 and 75 mg , respectively ) , using ddab as stabilizer at 0.1% and when the percentage of ethanol during processing was at its higher level of 90% . under these optimization criteria , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , q2hours of 3.3% , and q24hours of 17.3% were obtained . the proposed optimal conditions were then experimentally corroborated , and the results were closely correlated with the data predicted by the desirability function of the d - optimal design . glimepiride - loaded in situ gels were prepared by incorporating the optimized zein nanoparticles into plga peg all polymeric solutions were yellowish in color , with the zein nanoparticles suspended homogeneously with no signs of precipitations or agglomerations . the ph values of all solutions were in the range of 7.210.32 , with no effect of nanoparticles incorporation . for all samples , the apparent viscosities were higher at low shear rate than those at a high shear rate to demonstrate pseudoplastic behavior . similar observations were observed by other researchers for ethylcellulose - based gels incorporating antimicrobial drugs.24 the significant interaction between both polymer chains and drug molecules at low temperature and the shearing rate explained this behavior . the flow behavior of the solutions was also studied by calculating the flow indices and viscosity coefficient ( ) as a function of polymer concentration at 37c ( figure 5 ) . the flow index values of all formulations were more than 1 to confirm the non - newtonian flow similar to that of the gel base . regarding the viscosity coefficient ( ) , the amount of the polymer employed was significantly ( p<0.05 ) increasing the resultant viscosity coefficient . fresno et al25 explained this behavior by the deformation and change in the shape of polymer molecules and in the number of molecular entanglements observed as the shearing rate increased . syringeability was also assessed to estimate the force needed to pass the polymeric solutions through 21-gauge needle to form hypodermic in situ gels . the obtained results showed that the expelling force significantly increased ( p<0.05 ) as the percentage of polymer was increased . for example , 652.4 and 431.7 n / mm were the exerted forces of expulsion of formulations with 30% and 10% polymer concentrations , respectively . solvent interaction over the polymer polymer interaction , therefore lowering the resistance to flow . nmp could dissolve a large amount of the polymer , thus controlling both drug diffusion and easiness of syringeability.27 the main critical parameters of depot formation or structure change to form a gel were both temperature increase to 37c and water diffusion rate into the zein nanoparticles - loaded triblock polymer . the diffusion rate of the aqueous phosphate buffer ph 7.2 into the polymeric solutions decreased with increasing the triblock polymer concentration ( p<0.05 ; figure 5 ) due to the increased gel viscosity . wang et al28 demonstrated that the fast partitioning of the employed solvent might cause fast solidification of the implant ; hence , higher drug retention rate was expected . glimepiride release from the prepared gels was conducted in phosphate buffer ph 7.2 to simulate the physiologic condition ( figure 5 ) . in general , for example , 17.3% drug was released from the optimized nanoparticle formulation at 24 hours , whereas drug release rates in in situ gels containing 10% , 20% , and 30% w / w triblock polymer were about 14.5% , 10.2% , and 7.1% at 24 hours ( figure 5 ) , respectively . the physical entanglements between the polymer s chains to form a dense matrix would explain this result . liu and venkatraman29 demonstrated that the polymer phase inversion dynamics would suppress the initial drug burst release by each increase in polymer level . the hydrophobic plga compartment of the triblock would slow the drug release , whereas the hydrophilic peg would modulate the initial burst release , followed by a more rapid phase once the triblock became hydrated.30 on the other hand , a porous rubbery gel structure was formed by the nmp diffusion out of the hydrated triblock to cause burst release of glimepiride due to rapid phase inversion of plga.24 this study revealed a thorough understanding of embedding glimepiride zein nanoparticles into a thermoresponsive triblock copolymer to form an in situ gel . d - optimal fractional factorial design encompassing five variables at two levels was applied for the preparation of glimepiride zein nanoparticles . through the systematic optimization phase , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , and sustained release features of 3.3% and 17.3% drug released after 2 and 24 hours , respectively , were obtained . the optimized nanoparticles formulation was included in the triblock copolymers - based in situ gel that demonstrated pseudoplastic behavior . the increased concentration of triblock copolymers resulted in increase in the expelling force and reduction of drug release rate from the in situ gel formulae that could be useful for improving diabetes treatment effectiveness .

objectivesthe study aims at applying pharmaceutical nanotechnology and d - optimal fractional factorial design to screen and optimize the high - risk variables affecting the performance of a complex drug delivery system consisting of glimepiride zein nanoparticles and inclusion of the optimized formula with thermoresponsive triblock copolymers in in situ gel.methodssixteen nanoparticle formulations were prepared by liquid liquid phase separation method according to the d - optimal fractional factorial design encompassing five variables at two levels . the responses investigated were glimepiride entrapment capacity ( ec ) , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles . furthermore , the feasibility of embedding the optimized zein - based glimepiride nanoparticles within thermoresponsive triblock copolymers poly(lactide - co - glycolide)-block - poly(ethylene glycol)-block - poly(lactide - co - glycolide ) in in situ gel was evaluated for controlling glimepiride release rate.resultsthrough the systematic optimization phase , improvement of glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , and sustained release features of 3.3% and 17.3% drug released after 2 and 24 hours , respectively , were obtained . these desirability functions were obtained at zein and glimepiride loadings of 50 and 75 mg , respectively , utilizing didodecyldimethylammonium bromide as a stabilizer at 0.1% and 90% ethanol as a common solvent . moreover , incorporating this optimized formulation in triblock copolymers - based in situ gel demonstrated pseudoplastic behavior with reduction of drug release rate as the concentration of polymer increased.conclusionthis approach to control the release of glimepiride using zein nanoparticles / triblock copolymers - based in situ gel forming intramuscular implants could be useful for improving diabetes treatment effectiveness .

Introduction Materials and methods Materials Formulation of glimepiride-loaded Zein nanoparticles Experimental design Formulation of glimepiride-loaded PLGAPEGPLGA in situ gel Characterization of glimepiride-loaded Zein nanoparticles Characterization of glimepiride-loaded PLGAPEGPLGA in situ gel Results and discussion EC of glimepiride Nanoparticle size Zeta potential Glimepiride release Optimization using desirability function Characterization of the prepared in situ gels Conclusion

glimepiride provides better chance of glycemic control by stimulating insulin release from islets of langerhans of the pancreas and by boosting the sensitivity of peripheral receptors to endogenous insulin.2 it also facilitates glucose transport from the blood into the peripheral tissues for energy consumption.3 glimepiride is practically insoluble in water , with a maximum solubility not exceeding 0.00027 mg / ml , and hence is considered as a biopharmaceutics classification system class ii drug.4 following its oral administration , low and irregular bioavailability was shown due to its low aqueous solubility . polylactic acid and its copolymers with polyglycolic acid and poly(d , l - lactide - co - glycolide ) ( plga ) have been widely utilized as excipients for controlled release of injectable drugs . the thermoresponsive triblock copolymers ( plga polyethylene glycol [ peg]plga copolymers ) of plga ( a - block ) and peg ( b - block ) are the most attractive in situ gel - forming agents due to their biodegradable and safety profile.12 even though the entrapment of hydrophilic drugs as well as tailoring the polymer blocks chain length have been investigated to control drug release , formulation combinations of thermoresponsive triblock copolymers ( plga peg plga copolymers ) have not been thoroughly investigated . in this study , an investigation of the embedment of zein - based glimepiride nanoparticles within plga peg plga copolymers was proposed . zein is a hydrophobic material and only soluble in ethanol at concentrations over 70%.13 zein is capable of self - association in water to encapsulate both hydrophilic and hydrophobic species.14 hence , it was employed in this study along with d - optimal fractional factorial design to screen and optimize the high - risk variables affecting the performance of glimepiride - loaded zein - based nanoparticles . the responses investigated were glimepiride entrapment capacity ( ec ) , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles . furthermore , the feasibility of embedding the optimized zein - based glimepiride nanoparticles within plga peg plga copolymers was evaluated for controlling glimepiride release rate . sixteen experimental runs of glimepiride - loaded zein - based nanoparticle formulations were prepared by liquid liquid phase separation method as described by hashem et al15 with slight modification ( table 1 ) . , the single and interactive effects of five different factors on glimepiride ec , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles were investigated . optimization process was employed using a generalized desirability function to maximize glimepiride ec , zeta potential while minimizing particle size , skewness of size distribution , and drug release percentages . the optimized formulation was then incorporated into the plga peg plga thermoresponsive triblock copolymers for in situ gel formation . the optimized zein - based glimepiride nanoparticles were then embedded within plga peg plga copolymers using the following procedure . the optimized freeze - dried zein - based glimepiride nanoparticles were then added to the polymeric solutions and mixed by homogenization at 8,000 rpm for 2 minutes . the detected mass of glimepiride ( cm ) loaded within unit mass of zein matrix ( ct ) was used to express the ec according to the following equation : ec = cmct100(1 ) particle size and electrical properties of the prepared nanoparticles were determined by dynamic light scattering using a zetatrac analyzer ( microtrac inc . aliquots were withdrawn at 0.5 , 1 , 2 , 4 , 6 , 8 , 10 , 12 , and 24 hours by the autosampler and analyzed for glimepiride diffused using the previously mentioned in - house developed and validated analytical chromatographic method . in vitro drug release studies from the prepared glimepiride - loaded plga peg , 1 ml sample , equivalent to 15 mg glimepiride , of the homogenized triblock polymeric dispersion of glimepiride - loaded zein nanoparticles was injected into a dialysis tube containing 10 ml of phosphate buffer solution ph 7.2 . the distance of water front diffused through the membrane to form a gel was determined at various time points of 0 , 6 , and 24 hours . sixteen experimental runs of glimepiride - loaded zein - based nanoparticle formulations were prepared by liquid liquid phase separation method as described by hashem et al15 with slight modification ( table 1 ) . , the single and interactive effects of five different factors on glimepiride ec , particle size and size distribution , zeta potential , and in vitro drug release from the prepared nanoparticles were investigated . optimization process was employed using a generalized desirability function to maximize glimepiride ec , zeta potential while minimizing particle size , skewness of size distribution , and drug release percentages . the optimized formulation was then incorporated into the plga peg plga thermoresponsive triblock copolymers for in situ gel formation . the optimized zein - based glimepiride nanoparticles were then embedded within plga peg plga copolymers using the following procedure . the optimized freeze - dried zein - based glimepiride nanoparticles were then added to the polymeric solutions and mixed by homogenization at 8,000 rpm for 2 minutes . the detected mass of glimepiride ( cm ) loaded within unit mass of zein matrix ( ct ) was used to express the ec according to the following equation : ec = cmct100(1 ) particle size and electrical properties of the prepared nanoparticles were determined by dynamic light scattering using a zetatrac analyzer ( microtrac inc . aliquots were withdrawn at 0.5 , 1 , 2 , 4 , 6 , 8 , 10 , 12 , and 24 hours by the autosampler and analyzed for glimepiride diffused using the previously mentioned in - house developed and validated analytical chromatographic method . in vitro drug release studies from the prepared glimepiride - loaded plga peg plga in situ gel were evaluated using a modified dialysis . the distance of water front diffused through the membrane to form a gel was determined at various time points of 0 , 6 , and 24 hours . this investigation aimed at optimizing glimepiride - loaded zein nanoparticles to be embedded in an in situ forming plga peg the predefined critical quality attributes of the glimepiride - loaded zein nanoparticles were glimepiride ec , particle size , and glimepiride release rate . maximizing the ec of zein matrix to glimepiride would allow for a reduction of manufacturing costs , dosage volume , and easiness of syringeability for in situ gel formation . therefore , the first objective of this study was to screen and optimize the nanoparticle formulation factors using a d - optimal design for their effects to maximize ec while minimizing the produced size , size distribution , and drug release rate . data in table 2 show that glimepiride ec ranged from 15.7% ( f8 ) to 62.6% ( f10 ) for the different variable combinations of d - optimal design to prepare zein nanoparticles . using the developed prediction model , the response surface plots , shown in figure 1 , would demonstrate the design space for predicting and subsequent monitoring of glimepiride entrapment within zein nanoparticles . both the double - tailed structure of ddab and its critical packing parameter of 0.51 would contribute to particle formation.19 moreover , the high surface coverage of ddab on the zein nanoparticles would protect the particles and restrict the liberty of the glimepiride molecules to increase its ec . histograms of the particle size distribution of the prepared zein nanoparticles showed a monodispersion in diameter . analysis of variance ( anova ) results revealed significant effects of zein and glimepiride loadings and ethanol level on the sizes and skewness values with prob > f - values of 0.0118 and 0.0394 , respectively , p<0.05 ( table 3 ) . regarding the size distribution , glimepiride loading was more significant than zein concentration with p - values of 0.0412 and 0.5251 , respectively ( figure 2 and table 3 ) . negative correlations were found on the resultant sizes and skewness values by increasing both zein and ethanol loadings ( figure 1 ) , with a lowest nanoparticle size obtained at 2:1 zein to glimepiride weight ratio , 0.1% sls as stabilizer , and 70% ethanol concentration ( f10 ) . despite being nonsignificant , the positive influence of glimepiride entrapment on the resultant sizes could be explained according to the law of mass action to entrap glimepiride molecules within zein protein matrix . the adsorption of nonionic surfactants with its anionic impurities ( such as free fatty acids ) at the formed cationic zein surfaces could reduce their charge.22 the contribution to increase the zeta potential of zein nanoparticles dispersions would be suggestive of its stability due to the london dispersion forces . the regression equation revealed an acceptable predictability of zeta potential , with a p - value of 0.0025 and quantile quantile correlation coefficient of 0.9525 ( table 3 ) . the reduced prediction equation of zeta potential value is : zetapotential(mv)=5.1 + 1.5[zeinloading(mg)37.512.5]+[ddab2.866875sls2.866875](5 ) glimepiride release profile of all 16 formulations of d - optimal design can be described as a two - step biphasic process , ie , an initial burst effect followed by subsequent slower release ( figure 4 ) . this behavior would be ascribed to the expanding transient boundary at the interface of either protein surface or ddab - adsorbed layer and the aqueous medium.23 the following is the logarithmic equation to describe the drug release rate , where qti is amount of cumulative drug released after time t in the aqueous medium and k is the release constant or the slope of the regressing qti versus base-10 logarithm of time divided by 2.303 . qti=(2.303log10(ti)k)+qti1(6 ) the cumulative percentages of glimepiride released after 2 hours ( q2hours ) and 24 hours ( q24hours ) for the 16 formulations varied from 3.02% ( f16 ) to 14.52% ( f3 ) and from 15.76% ( f13 ) to 43.02% ( f9 ) , respectively ( table 2 ) . zein and glimepiride loadings significantly impacted these release parameters , with their interaction term ( x1x2 ) being the most important formulation parameter ( p<0.05 ; figures 2 and 3 ) . logically , higher zein - to - glimepiride weight ratio in the nanoparticle solid state led to lower concentration of the solubilized drug ( molecular state ) in the transient layer , and consequently a lesser drug release . the highest desirability was obtained at upper levels of both zein and glimepiride loadings ( 50 and 75 mg , respectively ) , using ddab as stabilizer at 0.1% and when the percentage of ethanol during processing was at its higher level of 90% . under these optimization criteria , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , q2hours of 3.3% , and q24hours of 17.3% were obtained . the proposed optimal conditions were then experimentally corroborated , and the results were closely correlated with the data predicted by the desirability function of the d - optimal design . glimepiride - loaded in situ gels were prepared by incorporating the optimized zein nanoparticles into plga peg all polymeric solutions were yellowish in color , with the zein nanoparticles suspended homogeneously with no signs of precipitations or agglomerations . in general , for example , 17.3% drug was released from the optimized nanoparticle formulation at 24 hours , whereas drug release rates in in situ gels containing 10% , 20% , and 30% w / w triblock polymer were about 14.5% , 10.2% , and 7.1% at 24 hours ( figure 5 ) , respectively . the hydrophobic plga compartment of the triblock would slow the drug release , whereas the hydrophilic peg would modulate the initial burst release , followed by a more rapid phase once the triblock became hydrated.30 on the other hand , a porous rubbery gel structure was formed by the nmp diffusion out of the hydrated triblock to cause burst release of glimepiride due to rapid phase inversion of plga.24 data in table 2 show that glimepiride ec ranged from 15.7% ( f8 ) to 62.6% ( f10 ) for the different variable combinations of d - optimal design to prepare zein nanoparticles . on the other hand , the negative effect of glimepiride of the resultant ec hence , these weakly bound or adsorbed drug molecules to the relatively larger surface of nanoparticles would facilitate pore formation for leaching more drug molecules.18 the positive action of ddab to retain glimepiride within the formed nanoparticles might be attributed to the packing of ddab at the formed surfaces . both the double - tailed structure of ddab and its critical packing parameter of 0.51 would contribute to particle formation.19 moreover , the high surface coverage of ddab on the zein nanoparticles would protect the particles and restrict the liberty of the glimepiride molecules to increase its ec . histograms of the particle size distribution of the prepared zein nanoparticles showed a monodispersion in diameter . analysis of variance ( anova ) results revealed significant effects of zein and glimepiride loadings and ethanol level on the sizes and skewness values with prob > f - values of 0.0118 and 0.0394 , respectively , p<0.05 ( table 3 ) . regarding the size distribution , glimepiride loading was more significant than zein concentration with p - values of 0.0412 and 0.5251 , respectively ( figure 2 and table 3 ) . negative correlations were found on the resultant sizes and skewness values by increasing both zein and ethanol loadings ( figure 1 ) , with a lowest nanoparticle size obtained at 2:1 zein to glimepiride weight ratio , 0.1% sls as stabilizer , and 70% ethanol concentration ( f10 ) . despite being nonsignificant , the positive influence of glimepiride entrapment on the resultant sizes could be explained according to the law of mass action to entrap glimepiride molecules within zein protein matrix . the regression equation revealed an acceptable predictability of zeta potential , with a p - value of 0.0025 and quantile quantile correlation coefficient of 0.9525 ( table 3 ) . this behavior would be ascribed to the expanding transient boundary at the interface of either protein surface or ddab - adsorbed layer and the aqueous medium.23 the following is the logarithmic equation to describe the drug release rate , where qti is amount of cumulative drug released after time t in the aqueous medium and k is the release constant or the slope of the regressing qti versus base-10 logarithm of time divided by 2.303 . qti=(2.303log10(ti)k)+qti1(6 ) the cumulative percentages of glimepiride released after 2 hours ( q2hours ) and 24 hours ( q24hours ) for the 16 formulations varied from 3.02% ( f16 ) to 14.52% ( f3 ) and from 15.76% ( f13 ) to 43.02% ( f9 ) , respectively ( table 2 ) . logically , higher zein - to - glimepiride weight ratio in the nanoparticle solid state led to lower concentration of the solubilized drug ( molecular state ) in the transient layer , and consequently a lesser drug release . the highest desirability was obtained at upper levels of both zein and glimepiride loadings ( 50 and 75 mg , respectively ) , using ddab as stabilizer at 0.1% and when the percentage of ethanol during processing was at its higher level of 90% . under these optimization criteria , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , q2hours of 3.3% , and q24hours of 17.3% were obtained . the proposed optimal conditions were then experimentally corroborated , and the results were closely correlated with the data predicted by the desirability function of the d - optimal design . glimepiride - loaded in situ gels were prepared by incorporating the optimized zein nanoparticles into plga peg all polymeric solutions were yellowish in color , with the zein nanoparticles suspended homogeneously with no signs of precipitations or agglomerations . in general , for example , 17.3% drug was released from the optimized nanoparticle formulation at 24 hours , whereas drug release rates in in situ gels containing 10% , 20% , and 30% w / w triblock polymer were about 14.5% , 10.2% , and 7.1% at 24 hours ( figure 5 ) , respectively . the hydrophobic plga compartment of the triblock would slow the drug release , whereas the hydrophilic peg would modulate the initial burst release , followed by a more rapid phase once the triblock became hydrated.30 on the other hand , a porous rubbery gel structure was formed by the nmp diffusion out of the hydrated triblock to cause burst release of glimepiride due to rapid phase inversion of plga.24 this study revealed a thorough understanding of embedding glimepiride zein nanoparticles into a thermoresponsive triblock copolymer to form an in situ gel . d - optimal fractional factorial design encompassing five variables at two levels was applied for the preparation of glimepiride zein nanoparticles . through the systematic optimization phase , glimepiride ec of 33.6% , nanoparticle size of 120.9 nm with a skewness value of 0.2 , zeta potential of 11.1 mv , and sustained release features of 3.3% and 17.3% drug released after 2 and 24 hours , respectively , were obtained . the optimized nanoparticles formulation was included in the triblock copolymers - based in situ gel that demonstrated pseudoplastic behavior . the increased concentration of triblock copolymers resulted in increase in the expelling force and reduction of drug release rate from the in situ gel formulae that could be useful for improving diabetes treatment effectiveness .

advances in acute treatment have led to improvements in survival and so establishing effective rehabilitation strategies has become even more important . this process is likely to be facilitated by establishing a mechanistic understanding of how to use adjunctive therapies to augment standard rehabilitation approaches . functional electrical stimulation ( fes ) is a commonly used adjunctive therapy in the rehabilitation of stroke . it is primarily used for the orthotic correction of foot drop , but a proportion of patients relearn the ability to voluntarily dorsiflex the ankle without the device . this phenomenon , referred to as the carryover effect , has been observed in a number of subsequent studies [ 4 , 5 ] . to date , the mechanism of this effect is unknown , although it has been hypothesised that an interaction between volitional effort and the electrical stimulation of fes results in a neuroplastic effect on the central nervous system [ 69 ] . however , the carryover effect has been observed only in subgroups of neurological patients and the characteristics of those with and without fes carryover are not clear . in this work , we used functional magnetic resonance imaging ( fmri ) to examine the neural correlates of the key ingredients of fes , namely , volitional intent to move and concurrent electrical stimulation , in a group of chronic stroke patients receiving fes treatment for foot drop . we were then interested to see whether brain activity during movement or stimulation ( or the interaction between the two ) before fes treatment would have any value in predicting whether individual patients would have a carryover effect on volitional ankle dorsiflexion after removal of fes . in healthy subjects , the interaction between volitional movement and proprioceptive feedback occurs in primary sensory and motor cortex . we might expect to see normal brain response to the elements of fes in those with carryover and diminished responses in those without carryover . however , sensorimotor systems are organised differently after stroke particularly in primary and secondary areas . the interaction ( or lack of it ) between volitional movement and proprioceptive feedback might therefore occur in nonprimary sensory ( secondary somatosensory area , sii , and posterior parietal cortex ) and motor ( lateral and medial premotor cortex ) areas . the sensitivity and spatial resolution of fmri allow us to make specific conclusions about the cortical regions active during key elements of fes and to investigate the likely neural mechanism of the carryover effect following fes treatment . patients were recruited from the outpatient and inpatient services at the villa beretta rehabilitation centre ( costa masnaga , lc , italy ) . all patients had suffered from first - ever stroke > 6 months previously , resulting in weakness of at least the tibialis anterior muscle ( to < 4 + on the medical research council ( mrc ) scale ) . exclusion criteria consisted of ( i ) less than 10 in fes - induced ankle dorsiflexion ; ( ii ) language or cognitive deficits sufficient to impair cooperation in the study ; ( iii ) inability to walk even if assisted ; ( iv ) high spasticity at ankle joint plantar flexor as measured by the modified ashworth scale index > 2 . the age - matched control group was composed of healthy volunteers with no neurological or orthopaedic impairment . experiments were conducted with approval from the villa beretta rehabilitation centre ethics committee and all subjects gave informed written consent in accordance with the declaration of helsinki . patients ' impairment at the time of recruitment for this study was evaluated using a battery of clinical and instrumental tests . in particular , they were evaluated through a gait analysis test performed following the standard davis evaluation protocol along with the correspondent dynamic electromyography test and the 6-minute walking test . moreover , they were scored by the clinician on the mrc scale index at ankle dorsiflexion . from these tests , a set of five outcome measures was designed to assess different aspects of patients ' functional condition : gait velocity as measured during the gait analysis test.endurance velocity , as calculated during the 6-minute walking test.paretic step length as measured during the gait analysis test.tibialis anterior activation index defined as the ratio between the activity of the tibialis anterior muscle between toe off and toe strike and that during the whole gait cycle .mrc index .patients were trained 5 times per week for 4 weeks . each of these 20 sessions consisted of 30 minutes of walking supported by a commercial electrical stimulator . two commercial devices were available at the villa beretta rehabilitation centre : bioness l300 ( bioness inc . ) and walkaid ( innovative neurotronics ) . two stimulating electrodes were placed superficially along the peroneal nerve to elicit tibialis anterior muscle contraction during the swing phase of gait . swing phase was detected online by wireless heel switches ( bioness ) or by accelerometers ( walkaid ) . the more suitable commercial device was selected for each patient depending on his / her best responsiveness to stimulation , best wearability , and reliability of swing phase detection . current stimulation amplitude was selected for each participant at the beginning of each session so as to be able to elicit ankle dorsiflexion during gait but at the same time to remain within the tolerance level . tibialis anterior activation index defined as the ratio between the activity of the tibialis anterior muscle between toe off and toe strike and that during the whole gait cycle . impairment was evaluated at the time of recruitment for this study ( t1 : within 5 days before the start of the treatment ) and after the intervention ( t2 : within 5 days since the end of the treatment ) using the same battery of clinical and instrumental tests . the carryover effect was determined using a novel algorithm based on variables minimum detectable change that combines the outcome measures to obtain a unique parameter , capacity score , where a higher capacity score indicates higher residual ability . in particular , for each assessment session , patients were assigned to a point in an n - dimensional space , where n corresponds to the number of outcome measures considered . the n - dimensional space was centred on the outcome measures derived from healthy subjects , and therefore the further away the patient is from the origin , the more impaired he / she is . moreover , the outcome variables have been normalized with respect to the corresponding minimum detectable change . the difference in the n - dimensional space between ( i ) the euclidean distance of subject zero ( a patient that scores zero in all outcome measures , i.e. , the most impaired patient in our space ) with respect to the origin ( i.e. , distance of the subject zero from the healthy control group ) and ( ii ) the euclidean distance of each patient with respect to the origin ( i.e. , distance of the subject zero from the healthy control group ) is defined as capability score . the difference between capacity scores at different timing ( i.e. , post - pre ) is thresholded to obtain carryover effect assessment . the experimental setup was composed of 1.5 t mri scanner ( ge cv / i ) , a motion capture system ( smart g ; bts ) , and an electrical stimulator ( rehastim protm ; hasomed gmbh ) , as previously described and validated [ 16 , 17 ] . experimental factors were ( i ) volitional intention to perform ankle dorsal - plantar flexion ( adf ) [ v : with the levels volitional and passive ] and ( ii ) fes [ f : with the levels present and absent ] . each patient was instructed to execute the protocol with the plegic ankle . during a continuous 10-minute scanning session the 4 conditions that constituted our factorial design were performed during the on blocks in a semirandomized order : ( i ) fv : attempted voluntary adf with concurrent fes - induced adf ; ( ii ) fp : fes - induced adf , with no attempt to move the ankle ; ( iii ) v : voluntary adf without fes ; ( iv ) p : passive dorsiflexion ( by the experimenter ) of the subject 's ankle without fes . subjects were specifically instructed to remain completely relaxed during fp and p conditions and to equally voluntarily contribute during v and fv conditions . dorsiflexion was paced every 3.5 seconds ( for 6 repetitions within a block ) with an auditory cue ( i.e. , movement rate 0.3 hz ) . , subjects practiced the protocol until being comfortable with the task ; the experimenter was assisting the training to check the correct execution of the protocol and equate effort across subjects . all subjects were free to choose the amplitude of their active movement to preclude fatigue . indeed , ciccarelli and colleagues did not find any effect of movement amplitude ( 1055 ) on magnitude or pattern of brain activity , suggesting that if the movement does not have any external reference and it is self - paced , there is no difference due to amplitude in associated cortical activity . subjects were instructed to keep eyes closed and head movements were minimized with rubber pads and straps . to ensure minimum transmission of movements to the head functional electrical stimulation was applied to the peroneal nerve through superficial self - adhesive electrodes , with biphasic balanced current pulses at 20 hz fixed frequency . the pulse width had a trapezoidal profile ( maximum pulse width 400 s ) and the current amplitude was set subject by subject so as to produce adf movement , within the tolerance threshold . current amplitude and pulse width were kept the same for both fp and fv conditions . a ge cv / i system , operating at 1.5 t , was used to acquire both t1-weighted anatomical images ( 0.94 0.94 4 mm voxels ) and t2-weighted mri transverse echo - planar images ( 1.8 1.8 4 mm voxels , te = 50 ms ) with blood oxygenation level dependent contrast . each echo - planar image comprised 22 contiguous axial slices , positioned to cover the temporoparietal and occipital lobes , with an effective repetition time of 3 seconds per volume . a total of 200 brain volumes were acquired in a single run lasting 10 minutes . a motion capture system previously validated for recording during scanning allowed us to record 3d trajectories of retroreflective markers to measure the ankle angle during fmri acquisitions and to determine the movement onset for event - related fmri time series analysis . the first was a static acquisition performed before the scanning , but while lying in the scanner , to estimate the coordinates of the medial and lateral malleoli for both lower limbs . during the static acquisition , a plate with 3 markers was placed on each tibia and 4 sticks with two markers each were placed on the four malleoli . the relative positions of the malleoli with respect to the plates ( i.e. , left and right plates ) were computed and the transformation matrices were estimated under the assumption that tibia and malleoli were rigidly connected . the second acquisition , dynamic acquisition , was performed during the fmri scanning . only the two plates on the tibia markers trajectories were analysed with a custom algorithm running in matlab ( matlabr2010b ) to obtain onsets and amplitude of adf movements . in particular , for each leg , the adf angle was calculated as follows : the mean points between the medial and lateral malleoli ( mean malleolus ) and between the medial and lateral metacarpi ( mean metacarpus ) were calculated . the adf angle was taken as the angle between the line passing through the more proximal tibial marker and the mean malleolus and the line passing through the mean malleolus and the mean metacarpus . imaging data were analysed using statistical parametric mapping ( spm8 , wellcome department of imaging neuroscience , http://www.fil.ion.ucl.ac.uk/spm/ ) implemented in matlab ( matlabr2010b ) . a skull stripping procedure , on the structural image for each subject , was performed to improve the coregistration of functional and structural images . participants with right - sided infarcts ( left - leg weakness ) were flipped about the midsagittal line , such that all subjects were considered to have left - sided infarcts . all fmri volumes were then realigned and unwarped to suppress task - related motion artefacts . the skull stripped structural image was then coregistered to the mean image of the functional realigned volumes and segmented . the spatial normalization transformation ( to the montreal neurological institute ( mni ) reference brain in talairach space ) was then estimated using the segmented structural image . the structural image and functional volumes were normalized and resampled to 2 mm 2 mm 2 mm voxels . functional normalized images were then smoothed with an isotropic 8 mm full - width half - maximum kernel . the time series in each voxel were high pass filtered at ( 1/128 ) hz during subsequent modelling to remove low frequency confounding factors . statistical analysis was performed in two stages using the standard summary statistic approach . in the first stage we were interested in the analysis of brain regions active during each condition , ( i ) fv , ( ii ) v , ( iii ) fp , and ( iv ) p , as well as ( v ) their interaction , defined as ( fv - v ) versus ( fp - p ) which identifies regions in which the effect of fes is modulated by the presence or absence of volitional intent . from the kinematic measures , two adf covariates were defined for each condition : onsets and amplitude covariates . all adf onsets belonging to the same condition were defined as a single event type and modelled as delta functions in the corresponding stimulus function . the amplitude covariate was defined as a delta function scaled by the actual amplitude of each adf for each condition , and it was mean corrected and orthogonalised with respect to the corresponding onset covariate . all onset and amplitude stimulus functions were then convolved with a canonical hemodynamic response function , together with its temporal and dispersion derivatives , and used as regressors in a general linear model of the observed fmri time series . linear contrasts of parameter estimates were generated for each subject ( i.e. , contrast images ) and used for the creation of statistical parametric maps at the second ( between - subject ) stage . in the second stage , the following analyses were performed:(i)one - sample t - tests were performed using appropriate contrast images for each condition to investigate the main effects of each condition in the stroke group.(ii)two - sample t - tests using appropriate contrast images for each subject were performed to examine the differences between patients and control subjects groups for each condition . conjunction analyses ( i.e. , two separate null hypotheses to be contemporarily denied ) were performed between groups for each condition to determine common activation.(iii)contrast images were entered into a regression analysis with subject - specific values for the carryover effect to investigate whether there are any areas in which pretreatment brain activity ( in any of the conditions ) correlates with the subsequent carryover effect of each patient.results of all second - stage analyses were thresholded at p < 0.05 corrected for multiple comparisons within specific regions of interest ( rois ) . our predefined area of investigation included the following seven areas bilaterally : leg primary motor ( m1 ) and sensory ( s1 ) cortices , secondary somatosensory area ( sii ) , parietal rostroventral area ( pr ) , supplementary motor area ( sma ) , premotor cortex ( pm ) , and angular gyrus ( ag ) . in particular , our predefined area of investigation included the following seven areas bilaterally : leg primary motor ( m1 ) and sensory ( s1 ) cortices , secondary somatosensory area ( sii ) , parietal rostroventral area ( pr ) , supplementary motor area ( sma ) , premotor cortex ( pm ) , and angular gyrus ( ag ) . contralateral primary sensorimotor cortex ( i.e. , m1 , s1 ) is the primary site of adf control , and it has been shown to be the site of interaction between volitional intention and fes in healthy controls . in turn , ipsilateral primary sensorimotor cortex has been demonstrated to be active for poststroke patients while executing simple motor tasks . the two secondary somatosensory areas ( csii , i csii ) have been selectively linked to proprioceptive processing and integration , attention to proprioceptive stimuli , painful and nonpainful stimulus processing , and complex object manipulation . moreover , the secondary somatosensory area has been demonstrated to be active during stimulation nonselectively and selectively with respect to voluntary effort in controls . pr areas have been identified as potential sites of sensorimotor integration by virtue of their anatomical connections with premotor and primary motor cortices . pr area has been shown to have an activation pattern similar to sii and so might have a similar role in processing sensorial stimuli [ 9 , 25 ] . bilateral premotor and supplementary motor areas are a highly consistent finding after stroke during simple motor tasks execution . right ag has been demonstrated to be the site of self - representation of movement [ 7 , 31 ] , and it has been demonstrated to be more active during passive than active fes . moreover , ag has been suggested to be the recipient of proprioceptive information encoded in the postcentral gyrus . one - sample t - tests were performed using appropriate contrast images for each condition to investigate the main effects of each condition in the stroke group . two - sample t - tests using appropriate contrast images for each subject were performed to examine the differences between patients and control subjects groups for each condition . conjunction analyses ( i.e. , two separate null hypotheses to be contemporarily denied ) were performed between groups for each condition to determine common activation . contrast images were entered into a regression analysis with subject - specific values for the carryover effect to investigate whether there are any areas in which pretreatment brain activity ( in any of the conditions ) correlates with the subsequent carryover effect of each patient . based on previous work . primary motor ( m1 ) and primary sensory cortices ( s1 ) were defined as 10 mm spheres centred , respectively , on [ x = 6 , y = 28 , z = 60 ] and [ x = 4 , y = 46 , z = 62 ] ; bilateral secondary somatosensory cortices ( sii ) as 10 mm spheres centred on [ x = 58 ; y = 27 ; z = 30 ] [ 7 , 34 ] ; pr as 10 mm spheres centred on [ x = 54 ; y = 13 ; z = 19 ] ; sma and pm were defined as 15 mm spheres centered , respectively , on [ 20 ; 8 ; 64 ] and [ 8 ; 6 ; 64 ] . anatomical attribution was performed by carefully superimposing the maxima of significant effects both on the mni brain and on the normalized structural images averaged across all subjects and then labelling with the aid of the atlas of duvernoy . the healthy control group was aged between 22 and 61 years [ mean ( standard deviation ) : 36 ( 14 ) years ] , comprising 8 male and 9 female subjects . fourteen poststroke patients were recruited [ range : 1964 years , mean ( standard deviation ) : 44 ( 14 ) ] , comprising 8 male and 6 female subjects . there was no significant difference between the groups in terms of age ( p = 0.15 ) . patient characteristics along with the degree of functional recovery at the time of scanning as measured by the selected outcome measures are listed in table 1 . for eight patients , the site of cerebral infarction was determined from the t1-weighted structural mri ( figure 1 ) . the carryover effect was not predicted by age , sex , side of the lesion ( i.e. , right / left ) , time since stroke acute event , the baseline impairment , and the type of device used for training , as determined by multiple linear regression . ten out of the fourteen patients completed the fes - based rehabilitation treatment , receiving the same dosage of therapy , and so we were able to assess the carryover effect in only a subgroup of participants ( table 1 ) . a follow - up assessment was planned for all patients one month after the end of the treatment to check for long - term effects of carryover . six patients were available for this extra visit , and the carryover effect presence / nonpresence was demonstrated to be stable for all patients but for one where the effect was no longer present . the patients that did not complete the treatment / assessment were outpatients , and they discontinued the treatment for personal reasons , mainly linked to difficulties in logistically managing an everyday treatment in clinic or managing a further travel . mean adf amplitude across subjects along with its standard deviation for v condition was 18 11 , for fv condition 20 11 , for fp condition 18 11 , and for p condition 21 13. all adf angles for all subjects were within the 1070 interval . no subjects displayed mirror movements at bedside observation or when performing the motor paradigm outside the scanner . however , a number of patients did exhibit synergistic contralateral ankle dorsiflexion during motor paradigm execution in the scanner ( table 1 ) . however , a linear regression analysis with mirror movements ( i.e. , present / absent ) as independent categorical variable and carryover effect as dependent variable demonstrated the independence of the two elements ( p value = 0.4860 ) . realignment parameters were assessed for excessive motion after unwarping procedure , and the maximum translational displacement was 0.27 mm in all directions and the maximum rotational displacement was 0.0029. figure 2 shows brain regions active during each condition ( i.e. , v , fv , fp , and p ) in the controls and patients groups separately , as well as the conjunction analysis , and comparison between patients and controls : patient group : as reported in table 2 , patients show task - related activation in primary and secondary sensorimotor areas , in contralateral paracentral lobule , bilateral frontal cortex , cingulate gyrus , precuneus , and supramarginal gyrus . ag area is only activated by patients , ipsilaterally in fp condition , and contralaterally shows an interaction between design factors ( i.e. , volitional intention and fes).patients versus healthy controls : controls show clear activation in all conditions in motor and somatosensory areas known to be involved in adf execution and in accord with previous studies [ 7 , 9 , 18 ] , as expected . patients and controls primarily show common activation in bilateral sensorimotor ( all conditions ) and supplementary motor ( for conditions where volitional intention is present : fv , v ) areas . however , compared to the control group , patients tend to overactivate right ag when there is no volitional intention to move ( i.e. , fp , p conditions ) and left intraparietal sulcus during passive movement ( figure 3).prediction of carryover effect in patient group : in those patients with carryover after fes , contralateral sma was more active during stimulation and voluntary conditions ( fv , v , and fp conditions ) , and ipsilateral m1 was more active during voluntary movement ( v condition ) . in those without carryover effect , we saw a greater interaction between factors , that is , ( fv - v ) > ( fp - p ) in contralateral ag . by looking at the response for the peak voxel for csma , im1 , and cag areas ( figures 4(a)4(c ) ) , it can be seen that patients who experienced the carryover effect have responses in sma and m1 that correspond to healthy controls , whilst responses in these regions in patients without carryover are diminished . conversely , the interaction between factors in contralateral ag is seen only in those without carryover but not in those with carryover or healthy controls . in other words , we see quite consistent separation of those with and without fes carryover on the basis of the brain responses in these regions ( figure 4(d ) ) . in particular , those with fes carryover appear to have patient group : as reported in table 2 , patients show task - related activation in primary and secondary sensorimotor areas , in contralateral paracentral lobule , bilateral frontal cortex , cingulate gyrus , precuneus , and supramarginal gyrus . ag area is only activated by patients , ipsilaterally in fp condition , and contralaterally shows an interaction between design factors ( i.e. , volitional intention and fes ) . patients versus healthy controls : controls show clear activation in all conditions in motor and somatosensory areas known to be involved in adf execution and in accord with previous studies [ 7 , 9 , 18 ] , as expected . patients and controls primarily show common activation in bilateral sensorimotor ( all conditions ) and supplementary motor ( for conditions where volitional intention is present : fv , v ) areas . however , compared to the control group , patients tend to overactivate right ag when there is no volitional intention to move ( i.e. , fp , p conditions ) and left intraparietal sulcus during passive movement ( figure 3 ) . prediction of carryover effect in patient group : in those patients with carryover after fes , contralateral sma was more active during stimulation and voluntary conditions ( fv , v , and fp conditions ) , and ipsilateral m1 was more active during voluntary movement ( v condition ) . in those without carryover effect , we saw a greater interaction between factors , that is , ( fv - v ) > ( fp - p ) in contralateral ag . by looking at the response for the peak voxel for csma , im1 , and cag areas ( figures 4(a)4(c ) ) , it can be seen that patients who experienced the carryover effect have responses in sma and m1 that correspond to healthy controls , whilst responses in these regions in patients without carryover are diminished . conversely , the interaction between factors in contralateral ag is seen only in those without carryover but not in those with carryover or healthy controls . in other words , we see quite consistent separation of those with and without fes carryover on the basis of the brain responses in these regions ( figure 4(d ) ) . a prolonged carryover effect from fes is a desirable rehabilitation outcome . knowing who is most likely to achieve this and how would be useful in a stratified rehabilitation strategy . the motivation for this study was therefore to explain how a peripheral stimulus can facilitate long - term motor relearning ( i.e. , fes carryover ) after a lesion in the central nervous system [ 6 , 38 ] . it has been suggested that the mechanism of fes carryover is central in origin , but this has never been tested in neurological patients . in this study , we have used functional brain imaging to examine brain responses to key components of fes ( electrical stimulation and attempted volitional movement , both separately and in combination ) that characterise those who exhibit the carryover effect . these characteristics might be considered as biomarkers for successful fes - based rehabilitation after stroke . our results point to supplementary motor area ( sma ) and angular gyrus ( ag ) as key regions involved in mediating the carryover effect , since they are differentially active during the key components of fes in those patients with and without carryover . it is first worth considering the normal roles of sma and ag in sensorimotor tasks . sma is linked to movement preparation and planning and is often noted to be overactive compared to controls during attempted movement in chronic stroke subjects . indeed , in our patients , sma is bilaterally active during conditions where volitional intention is present ( i.e. , fv , v ) , as expected . in turn , ag appears to be a recipient of proprioceptive information and a specific area for somatosensory calculation of the reach vector during upper limb reaching . ag processes discrepancies between intended action and movement consequences in such a way that these will be consciously detected by the subject . it has been suggested that ag is activated during intersensory conflicts that may result in a loss of body ownership [ 31 , 40 ] . on the other hand , damage to ag results in altered awareness of voluntary action . our results show that those with and without fes carryover have opposite patterns of activity in sma and ag . in particular , those who have fes carryover exhibit sma activation during concurrent fes and volitional movement ( i.e. , fv condition ) , but they do not show an interaction between fes and volitional movement in ag . in both instances , the neural responses to key elements of fes are more like a normal healthy subject , whereas those without carryover have abnormal responses in both sma and ag ( figure 4 ) . we therefore suggest that the carryover effect is mediated through movement prediction ( sma area ) and sense of agency / body ownership ( ag area ) . specifically , the concept of sense of agency appears to be neuroanatomically associated with primary and secondary sensorimotor areas . the prediction of the sensory consequences of a self - generated action is compared against the actual sensory consequences , where stronger correspondence is associated with a stronger experience of agency ( i.e. , self - generated action ) . in other words , those with fes carryover correctly plan the movement when executing the movement with concurrent volitional intention and fes , and as a consequence movement is perceived as self - generated . by doing so , the patient correctly updates the motor control loop that likely enhances a long - term potentiation effect following hebbian principles . indeed , the combination of volitional effort and the perception of a normally completed movement provides somatosensory feedback that facilitates hebbian - like plasticity . this is in line with the suggestion that gradual motor learning / adaptation might be also mediated by extracerebellar mechanisms and that the generalization of learning ( in particular adaptive learning ) is improved when the nervous system assigns errors to self rather than the environment or , in this case , the device . in healthy control subjects , the interaction between volitional intent to move and proprioception was mediated by primary motor and somatosensory areas . in our poststroke patients , bilateral primary sensorimotor cortices the function of primary sensorimotor cortices role in mediating the fes effect might therefore be supported by secondary areas , representing a plasticity mechanism that exploits available resources . in fact , it has been repeatedly shown that normal input / output processes of primary sensorimotor cortices are impaired in many neurological patients , with consequent recruitment of a complex network that includes primary and secondary areas to generate even simple motor tasks [ 34 , 48 ] . the other predefined areas of investigation do not appear to be crucial nodes for mediating fes carryover . in particular , although premotor areas are known to be overactive in many stroke patients , they do not appear to be crucial for our patients during adf , being only ipsilaterally active in fv condition . it has been suggested that the human si and sii cortices may be sequentially activated within one hemisphere , whereas sii ipsilateral to the stimulation may receive direct input from the periphery , at least when normal input from si is interrupted . in our control group , bilateral sii was active for the fes conditions ( i.e. , fv , fp ) , possibly as the direct recipient of the stimulus [ 8 , 50 ] . in patients , we observed only ipsilateral sii activation , which preserves its presumed role of processing electrical stimulation as in healthy controls [ 9 , 49 , 50 ] . on the other hand , we suggest either that contralateral sii is not primarily involved in somatosensory information processing or that contralateral stimulus processing is impaired in our group of patients . an important limitation of this study was the inability to collect data from the cerebellum due to technical constraints . we tried to overcome this limitation as far as we could , by training the subjects outside the scanner so that they were familiar with the stimulus during fes conditions . however , the cerebellum is thought to be part of the motor control loop , and it has been shown to be differentially involved during fes supported and nonsupported by volitional contribution . moreover , computing predictions of sensory consequences is seen in the literature as a major role of the cerebellum ( together with the parietal cortex ) within the sensory - motor control loop . further , the focus of this work was comparing the patients who show carryover effect against those who do not after the identical treatment based on fes . in this view , a group of patients with no treatment was not included as this was beyond the purpose of the current study . the number of recruited patients was limited , but nevertheless the results reported are statistically robust and point towards a biological basis for the carryover . in conclusion , we suggest that the mechanism of action of fes carryover is based on movement prediction and sense of agency / body ownership . in other words , the ability of a patient to plan the movement and to perceive the stimulation as a part of his / her own control loop is important for the fes carryover effect to take place . although we point to abnormal responses in sma and ag as indicators that fes carryover effect is unlikely , it might be that in future a behavioural questionnaire devoted to the evaluation of self / non - self - perceived fes - induced movement might be useful in predicting the carryover effect in routine clinical settings .

neurorehabilitation effective delivery for stroke is likely to be improved by establishing a mechanistic understanding of how to enhance adaptive plasticity . functional electrical stimulation is effective at reducing poststroke foot drop ; in some patients , the effect persists after therapy has finished with an unknown mechanism . we used fmri to examine neural correlates of functional electrical stimulation key elements , volitional intent to move and concurrent stimulation , in a group of chronic stroke patients receiving functional electrical stimulation for foot - drop correction . patients exhibited task - related activation in a complex network , sharing bilateral sensorimotor and supplementary motor activation with age - matched controls . we observed consistent separation of patients with and without carryover effect on the basis of brain responses . patients who experienced the carryover effect had responses in supplementary motor area that correspond to healthy controls ; the interaction between experimental factors in contralateral angular gyrus was seen only in those without carryover . we suggest that the functional electrical stimulation carryover mechanism of action is based on movement prediction and sense of agency / body ownership the ability of a patient to plan the movement and to perceive the stimulation as a part of his / her own control loop is important for carryover effect to take place .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusions

this process is likely to be facilitated by establishing a mechanistic understanding of how to use adjunctive therapies to augment standard rehabilitation approaches . functional electrical stimulation ( fes ) is a commonly used adjunctive therapy in the rehabilitation of stroke . it is primarily used for the orthotic correction of foot drop , but a proportion of patients relearn the ability to voluntarily dorsiflex the ankle without the device . this phenomenon , referred to as the carryover effect , has been observed in a number of subsequent studies [ 4 , 5 ] . to date , the mechanism of this effect is unknown , although it has been hypothesised that an interaction between volitional effort and the electrical stimulation of fes results in a neuroplastic effect on the central nervous system [ 69 ] . however , the carryover effect has been observed only in subgroups of neurological patients and the characteristics of those with and without fes carryover are not clear . in this work , we used functional magnetic resonance imaging ( fmri ) to examine the neural correlates of the key ingredients of fes , namely , volitional intent to move and concurrent electrical stimulation , in a group of chronic stroke patients receiving fes treatment for foot drop . we were then interested to see whether brain activity during movement or stimulation ( or the interaction between the two ) before fes treatment would have any value in predicting whether individual patients would have a carryover effect on volitional ankle dorsiflexion after removal of fes . in healthy subjects , the interaction between volitional movement and proprioceptive feedback occurs in primary sensory and motor cortex . we might expect to see normal brain response to the elements of fes in those with carryover and diminished responses in those without carryover . the interaction ( or lack of it ) between volitional movement and proprioceptive feedback might therefore occur in nonprimary sensory ( secondary somatosensory area , sii , and posterior parietal cortex ) and motor ( lateral and medial premotor cortex ) areas . the sensitivity and spatial resolution of fmri allow us to make specific conclusions about the cortical regions active during key elements of fes and to investigate the likely neural mechanism of the carryover effect following fes treatment . all patients had suffered from first - ever stroke > 6 months previously , resulting in weakness of at least the tibialis anterior muscle ( to < 4 + on the medical research council ( mrc ) scale ) . the age - matched control group was composed of healthy volunteers with no neurological or orthopaedic impairment . from these tests , a set of five outcome measures was designed to assess different aspects of patients ' functional condition : gait velocity as measured during the gait analysis test.endurance velocity , as calculated during the 6-minute walking test.paretic step length as measured during the gait analysis test.tibialis anterior activation index defined as the ratio between the activity of the tibialis anterior muscle between toe off and toe strike and that during the whole gait cycle .mrc index .patients were trained 5 times per week for 4 weeks . the more suitable commercial device was selected for each patient depending on his / her best responsiveness to stimulation , best wearability , and reliability of swing phase detection . current stimulation amplitude was selected for each participant at the beginning of each session so as to be able to elicit ankle dorsiflexion during gait but at the same time to remain within the tolerance level . the carryover effect was determined using a novel algorithm based on variables minimum detectable change that combines the outcome measures to obtain a unique parameter , capacity score , where a higher capacity score indicates higher residual ability . the n - dimensional space was centred on the outcome measures derived from healthy subjects , and therefore the further away the patient is from the origin , the more impaired he / she is . the difference in the n - dimensional space between ( i ) the euclidean distance of subject zero ( a patient that scores zero in all outcome measures , i.e. , the most impaired patient in our space ) with respect to the origin ( i.e. , post - pre ) is thresholded to obtain carryover effect assessment . during a continuous 10-minute scanning session the 4 conditions that constituted our factorial design were performed during the on blocks in a semirandomized order : ( i ) fv : attempted voluntary adf with concurrent fes - induced adf ; ( ii ) fp : fes - induced adf , with no attempt to move the ankle ; ( iii ) v : voluntary adf without fes ; ( iv ) p : passive dorsiflexion ( by the experimenter ) of the subject 's ankle without fes . indeed , ciccarelli and colleagues did not find any effect of movement amplitude ( 1055 ) on magnitude or pattern of brain activity , suggesting that if the movement does not have any external reference and it is self - paced , there is no difference due to amplitude in associated cortical activity . to ensure minimum transmission of movements to the head functional electrical stimulation was applied to the peroneal nerve through superficial self - adhesive electrodes , with biphasic balanced current pulses at 20 hz fixed frequency . a total of 200 brain volumes were acquired in a single run lasting 10 minutes . a motion capture system previously validated for recording during scanning allowed us to record 3d trajectories of retroreflective markers to measure the ankle angle during fmri acquisitions and to determine the movement onset for event - related fmri time series analysis . a skull stripping procedure , on the structural image for each subject , was performed to improve the coregistration of functional and structural images . all fmri volumes were then realigned and unwarped to suppress task - related motion artefacts . the skull stripped structural image was then coregistered to the mean image of the functional realigned volumes and segmented . in the first stage we were interested in the analysis of brain regions active during each condition , ( i ) fv , ( ii ) v , ( iii ) fp , and ( iv ) p , as well as ( v ) their interaction , defined as ( fv - v ) versus ( fp - p ) which identifies regions in which the effect of fes is modulated by the presence or absence of volitional intent . all onset and amplitude stimulus functions were then convolved with a canonical hemodynamic response function , together with its temporal and dispersion derivatives , and used as regressors in a general linear model of the observed fmri time series . , two separate null hypotheses to be contemporarily denied ) were performed between groups for each condition to determine common activation. (iii)contrast images were entered into a regression analysis with subject - specific values for the carryover effect to investigate whether there are any areas in which pretreatment brain activity ( in any of the conditions ) correlates with the subsequent carryover effect of each patient.results of all second - stage analyses were thresholded at p < 0.05 corrected for multiple comparisons within specific regions of interest ( rois ) . our predefined area of investigation included the following seven areas bilaterally : leg primary motor ( m1 ) and sensory ( s1 ) cortices , secondary somatosensory area ( sii ) , parietal rostroventral area ( pr ) , supplementary motor area ( sma ) , premotor cortex ( pm ) , and angular gyrus ( ag ) . in particular , our predefined area of investigation included the following seven areas bilaterally : leg primary motor ( m1 ) and sensory ( s1 ) cortices , secondary somatosensory area ( sii ) , parietal rostroventral area ( pr ) , supplementary motor area ( sma ) , premotor cortex ( pm ) , and angular gyrus ( ag ) . , m1 , s1 ) is the primary site of adf control , and it has been shown to be the site of interaction between volitional intention and fes in healthy controls . in turn , ipsilateral primary sensorimotor cortex has been demonstrated to be active for poststroke patients while executing simple motor tasks . moreover , the secondary somatosensory area has been demonstrated to be active during stimulation nonselectively and selectively with respect to voluntary effort in controls . bilateral premotor and supplementary motor areas are a highly consistent finding after stroke during simple motor tasks execution . right ag has been demonstrated to be the site of self - representation of movement [ 7 , 31 ] , and it has been demonstrated to be more active during passive than active fes . two - sample t - tests using appropriate contrast images for each subject were performed to examine the differences between patients and control subjects groups for each condition . contrast images were entered into a regression analysis with subject - specific values for the carryover effect to investigate whether there are any areas in which pretreatment brain activity ( in any of the conditions ) correlates with the subsequent carryover effect of each patient . based on previous work . for eight patients , the site of cerebral infarction was determined from the t1-weighted structural mri ( figure 1 ) . the carryover effect was not predicted by age , sex , side of the lesion ( i.e. , right / left ) , time since stroke acute event , the baseline impairment , and the type of device used for training , as determined by multiple linear regression . ten out of the fourteen patients completed the fes - based rehabilitation treatment , receiving the same dosage of therapy , and so we were able to assess the carryover effect in only a subgroup of participants ( table 1 ) . six patients were available for this extra visit , and the carryover effect presence / nonpresence was demonstrated to be stable for all patients but for one where the effect was no longer present . , present / absent ) as independent categorical variable and carryover effect as dependent variable demonstrated the independence of the two elements ( p value = 0.4860 ) . , v , fv , fp , and p ) in the controls and patients groups separately , as well as the conjunction analysis , and comparison between patients and controls : patient group : as reported in table 2 , patients show task - related activation in primary and secondary sensorimotor areas , in contralateral paracentral lobule , bilateral frontal cortex , cingulate gyrus , precuneus , and supramarginal gyrus . ag area is only activated by patients , ipsilaterally in fp condition , and contralaterally shows an interaction between design factors ( i.e. , volitional intention and fes).patients versus healthy controls : controls show clear activation in all conditions in motor and somatosensory areas known to be involved in adf execution and in accord with previous studies [ 7 , 9 , 18 ] , as expected . patients and controls primarily show common activation in bilateral sensorimotor ( all conditions ) and supplementary motor ( for conditions where volitional intention is present : fv , v ) areas . , fp , p conditions ) and left intraparietal sulcus during passive movement ( figure 3).prediction of carryover effect in patient group : in those patients with carryover after fes , contralateral sma was more active during stimulation and voluntary conditions ( fv , v , and fp conditions ) , and ipsilateral m1 was more active during voluntary movement ( v condition ) . in those without carryover effect , we saw a greater interaction between factors , that is , ( fv - v ) > ( fp - p ) in contralateral ag . by looking at the response for the peak voxel for csma , im1 , and cag areas ( figures 4(a)4(c ) ) , it can be seen that patients who experienced the carryover effect have responses in sma and m1 that correspond to healthy controls , whilst responses in these regions in patients without carryover are diminished . conversely , the interaction between factors in contralateral ag is seen only in those without carryover but not in those with carryover or healthy controls . in other words , we see quite consistent separation of those with and without fes carryover on the basis of the brain responses in these regions ( figure 4(d ) ) . in particular , those with fes carryover appear to have patient group : as reported in table 2 , patients show task - related activation in primary and secondary sensorimotor areas , in contralateral paracentral lobule , bilateral frontal cortex , cingulate gyrus , precuneus , and supramarginal gyrus . ag area is only activated by patients , ipsilaterally in fp condition , and contralaterally shows an interaction between design factors ( i.e. , volitional intention and fes ) . patients versus healthy controls : controls show clear activation in all conditions in motor and somatosensory areas known to be involved in adf execution and in accord with previous studies [ 7 , 9 , 18 ] , as expected . patients and controls primarily show common activation in bilateral sensorimotor ( all conditions ) and supplementary motor ( for conditions where volitional intention is present : fv , v ) areas . prediction of carryover effect in patient group : in those patients with carryover after fes , contralateral sma was more active during stimulation and voluntary conditions ( fv , v , and fp conditions ) , and ipsilateral m1 was more active during voluntary movement ( v condition ) . in those without carryover effect , we saw a greater interaction between factors , that is , ( fv - v ) > ( fp - p ) in contralateral ag . by looking at the response for the peak voxel for csma , im1 , and cag areas ( figures 4(a)4(c ) ) , it can be seen that patients who experienced the carryover effect have responses in sma and m1 that correspond to healthy controls , whilst responses in these regions in patients without carryover are diminished . conversely , the interaction between factors in contralateral ag is seen only in those without carryover but not in those with carryover or healthy controls . in other words , we see quite consistent separation of those with and without fes carryover on the basis of the brain responses in these regions ( figure 4(d ) ) . a prolonged carryover effect from fes is a desirable rehabilitation outcome . knowing who is most likely to achieve this and how would be useful in a stratified rehabilitation strategy . it has been suggested that the mechanism of fes carryover is central in origin , but this has never been tested in neurological patients . in this study , we have used functional brain imaging to examine brain responses to key components of fes ( electrical stimulation and attempted volitional movement , both separately and in combination ) that characterise those who exhibit the carryover effect . our results point to supplementary motor area ( sma ) and angular gyrus ( ag ) as key regions involved in mediating the carryover effect , since they are differentially active during the key components of fes in those patients with and without carryover . sma is linked to movement preparation and planning and is often noted to be overactive compared to controls during attempted movement in chronic stroke subjects . indeed , in our patients , sma is bilaterally active during conditions where volitional intention is present ( i.e. in turn , ag appears to be a recipient of proprioceptive information and a specific area for somatosensory calculation of the reach vector during upper limb reaching . it has been suggested that ag is activated during intersensory conflicts that may result in a loss of body ownership [ 31 , 40 ] . our results show that those with and without fes carryover have opposite patterns of activity in sma and ag . , fv condition ) , but they do not show an interaction between fes and volitional movement in ag . in both instances , the neural responses to key elements of fes are more like a normal healthy subject , whereas those without carryover have abnormal responses in both sma and ag ( figure 4 ) . we therefore suggest that the carryover effect is mediated through movement prediction ( sma area ) and sense of agency / body ownership ( ag area ) . specifically , the concept of sense of agency appears to be neuroanatomically associated with primary and secondary sensorimotor areas . the prediction of the sensory consequences of a self - generated action is compared against the actual sensory consequences , where stronger correspondence is associated with a stronger experience of agency ( i.e. in other words , those with fes carryover correctly plan the movement when executing the movement with concurrent volitional intention and fes , and as a consequence movement is perceived as self - generated . by doing so , the patient correctly updates the motor control loop that likely enhances a long - term potentiation effect following hebbian principles . indeed , the combination of volitional effort and the perception of a normally completed movement provides somatosensory feedback that facilitates hebbian - like plasticity . this is in line with the suggestion that gradual motor learning / adaptation might be also mediated by extracerebellar mechanisms and that the generalization of learning ( in particular adaptive learning ) is improved when the nervous system assigns errors to self rather than the environment or , in this case , the device . in healthy control subjects , the interaction between volitional intent to move and proprioception was mediated by primary motor and somatosensory areas . in fact , it has been repeatedly shown that normal input / output processes of primary sensorimotor cortices are impaired in many neurological patients , with consequent recruitment of a complex network that includes primary and secondary areas to generate even simple motor tasks [ 34 , 48 ] . in particular , although premotor areas are known to be overactive in many stroke patients , they do not appear to be crucial for our patients during adf , being only ipsilaterally active in fv condition . it has been suggested that the human si and sii cortices may be sequentially activated within one hemisphere , whereas sii ipsilateral to the stimulation may receive direct input from the periphery , at least when normal input from si is interrupted . in patients , we observed only ipsilateral sii activation , which preserves its presumed role of processing electrical stimulation as in healthy controls [ 9 , 49 , 50 ] . on the other hand , we suggest either that contralateral sii is not primarily involved in somatosensory information processing or that contralateral stimulus processing is impaired in our group of patients . however , the cerebellum is thought to be part of the motor control loop , and it has been shown to be differentially involved during fes supported and nonsupported by volitional contribution . moreover , computing predictions of sensory consequences is seen in the literature as a major role of the cerebellum ( together with the parietal cortex ) within the sensory - motor control loop . further , the focus of this work was comparing the patients who show carryover effect against those who do not after the identical treatment based on fes . in this view , a group of patients with no treatment was not included as this was beyond the purpose of the current study . in conclusion , we suggest that the mechanism of action of fes carryover is based on movement prediction and sense of agency / body ownership . in other words , the ability of a patient to plan the movement and to perceive the stimulation as a part of his / her own control loop is important for the fes carryover effect to take place . although we point to abnormal responses in sma and ag as indicators that fes carryover effect is unlikely , it might be that in future a behavioural questionnaire devoted to the evaluation of self / non - self - perceived fes - induced movement might be useful in predicting the carryover effect in routine clinical settings .

the fast li - ion conductor with the nominal composition li7la3zr2o12 ( llzo ) is receiving much scientific attention since its discovery in 2007 . it has a garnet - based structure , and it occurs in at least two structural modifications . at room temperature , llzo is tetragonal ( i41/acd ) while the cubic modification ( ia-3d ) is stable above approximately 150 c . geiger et al . argued that the better conducting cubic phase can be stabilized at room temperature ( rt ) through the incorporation of small amounts of al . the stabilizing effect of al has now been confirmed by a number of subsequent investigations . the exact role al plays in cubic al - bearing llzo is important because llzo shows a high ionic conductivity of about 10 s / cm at rt . this is approximately 2 orders of magnitude higher than that for the lower symmetry al - free tetragonal llzo phase . llzo also has good chemical and thermal stability , as well as a wide energy potential window making it an excellent candidate for use as an electrolyte in an all - solid - state lithium - ion battery . as has been shown recently , ionic conductivity also seems to depend on the amount of al incorporated during synthesis . further work , however , is needed to quantify this effect . for this purpose , the chemical and physical properties governing li diffusion have to be understood in detail ; in particular this includes the important question as to which crystallographic sites the al ions preferably occupy in the cubic phase of llzo . considerable experimental research has been undertaken to obtain information about al in llzo , including its local coordination and site partitioning behavior . in this context , al magic angle spinning ( mas ) nuclear magnetic resonance ( nmr ) spectroscopy is a key method . several nmr studies have proposed that the resonance observed at a chemical shift ranging from 64 to 68 ppm corresponds to al located at the there is uncertainty , however , about the interpretation and assignment of the other measured nmr lines that have chemical shifts ranging from approximately 78 to 82 ppm . the interpretations given so far include al residing at nontetrahedral li sites ( 4-fold to possibly 6-fold coordinated ) and tetrahedrally coordinated sites in the neighborhood of la or zr vacancies in al - rich llzo . in particular , mechanochemically prepared llzo samples with a high amount of al , but reduced in la and zr content , even indicate two magnetically inequivalent tetrahedral sites . neutron powder diffraction ( npd ) measurements were interpreted as indicating that al is located at an octahedrally coordinated site in the garnet framework . summarizing the various published experimental results , there is no clear understanding as to which sites are occupied by al in cubic llzo . this also concerns the detailed interpretation of al nmr mas spectra and diffraction results . to address the important role of al in llzo and to obtain a better understanding of the various experimental results , we undertook a density functional theory ( dft ) investigation of al - bearing cubic llzo with the aim to calculate ( relative ) al nmr parameters such as chemical shifts and electric quadrupole coupling constants . in order to understand the computational models used in this investigation , we provide a short description of the crystal structure of cubic llzo garnet ( figure 1 ) . the yellow dodecahedrally coordinate la ( at the wyckoff position 24c ) and orange octahedrally coordinate zr ( 16a ) . the blue spheres correspond to tetrahedrally coordinated ( 24d ) li , green spheres to octahedrally coordinated ( 48 g ) li , and red ones to distorted 4-fold coordinated ( 96h ) li . garnet is the common name for a large number of natural and synthetic metal oxide and fluoride phases . conventional oxide garnets have the general formula a3b2c3o12 and crystallize with cubic symmetry ia-3d . in the case of llzo , the o ions , located at general crystallographic positions , 96h , form an oxygen - ion framework with interstices occupied by the a cations ( la ) at an 8-fold coordinated position 24c ( point symmetry 222 ) , by b cations ( zr ) at a 6-fold coordinated position 16a ( point symmetry 3 ) , and by c cations ( li ) at a 4-fold coordinated 24d position ( point symmetry 4 ) . in addition to these cation sites , there are other interstices within the oxygen framework that are empty in the conventional garnet structure , such as the 6-fold coordinated 48 g positions ( point symmetry 2 ) and a general 96h position , sometimes described as 4-fold coordinated with two additional longer bonds greater than 2.8 in length , 5-fold coordinated or 6-fold coordinated ( point symmetry 1 ) . these interstices can be filled by extra cations ( such as li ) , giving rise to compositions with nonstandard garnet stoichiometry . an important property is the partial occupancy of the structural sites ( 24d , 48 g , and 96h ) where li is located and also delocalization of li ions throughout . they concentrate on the basic structural properties and on the li diffusion , but none of them considers al ions explicitly . for our first - principle calculations , the ions in llzo were arranged on the basis of crystal structure descriptions in the literature . three different structural models were used with all having a body centered ( i - type ) bravais lattice with a = b = c = 12.972 and = = = 90. three garnet compositions were chosen , namely li44al4la24zr16o96 with al at 24d , 48 g and 96h , li56al4la20zr16o96 with al solely at 24c , and li60al4la24zr12o96 with al solely at 16a . the li ions were distributed over the 24d and the 96h sites following xu et al . no significant effect on the results was observed by choosing a different li arrangement . the highest symmetry possible the first model was used to understand the behavior of al at various possible sites occupied by li . the calculations were made with 17 different local al positions located among the 24d , 96h , and 48 g sites . the various al positions were fixed during relaxation and all al ions are equivalent in the unit cell . all calculations are based on dft methods using the all - electron full potential linearized augmented plane wave ( lapw ) method as implemented in the wien2k code . the perdew burke ernzerhof ( pbe ) generalized gradient approximation ( gga ) the atomic positions were optimized by minimization of the forces ( below 2 mry / au ) acting on the atoms simultaneously with the self - consistent - field cycle as implemented by marks . iterative diagonalization using an efficient preconditioning ( inverse of h - s ) and the block - davidson method . the radii of the atomic spheres ( rmt ) for the li , la , zr , o , and al ions were chosen to be 1.46 , 2.27 , 1.93 , 1.71 , and 1.40 au , respectively . the cutoff for the plane wave rmtkmax = 6.0 and the maximum fourier expansion of charge density cutoff gmax = 12 ( au ) were applied . the separation parameter between the valence and core states was chosen to be 6.0 ry . the computational considerations were checked by increasing rmtkmax and the number of k - points , but no significant changes with respect to the energy , geometry , and the electric field gradient ( efg ) around al were observed . the behavior of al at different crystallographic sites was analyzed qualitatively by weighing the various al positions , xi , with their corresponding energy , e(xi ) . the various local energies for al at and around the crystallographic positions where li can also be located , were described by fitting a polynomial , describing a possible diffusion pathway . the ocuppation probabilities for an al ion along this path are described using a normalized boltzmann factor given bywhere k is boltzmann s constant , and where xi+1 indicates the states next to xi , xi1 indicates the states before xi with an interval of 0.01 , and t is 298.15 k. the site preference energy , e , is defined as e = e(xi ) e , where e is the global energy minimum and corresponds to al located at 24d . it has the lowest total energy for all the calculated arrangements of al in llzo . in nmr spectroscopy , the chemical shift , , of a nucleus , i , describes the nuclear shielding effect of an applied ( external ) static magnetic field and the locally induced magnetic field arising from the surrounding electrons with a certain probability of presence near the corresponding nuclear site i. the magnitude of the resulting effective field , beff , is given by beff = b0(1 i ) , where i is a second - rank nuclear shielding tensor , 1 is the unit matrix and b0 is a uniform external field along the z - axis . the resonance nmr frequency , i , is then given by vi = ( i/2 ) beff , where i is the magnetogyric ratio of the nucleus under observation . the isotropic chemical shift , iso ( henceforth ) , describes the relation between the nmr resonance frequency for the nucleus of interest , i , and the corresponding resonance frequency for a reference compound , ref , giving = 10 ( vi vref)/vref . because of the orientation in systems with periodic boundary conditions , all anisotropic interactions causing line broadening are projected onto the axis of rotation where they collapse at the magic angle . to compute , we used the relaxed geometry and applied the nmr module of the wien2k code . these chemical shift calculations are based on an all - electron linear response method where one obtains the induced current density considering the perturbation of the ground state wave functions due to the external magnetic field . the resulting magnetic shielding is then obtained by integration of the all - electron current according to biot because there is agreement in the literature that the al nmr resonance at 66 2 ppm represents tetrahedrally coordinated al at 24d in llzo ( 24d = 68 ppm ) , we calculate the difference of the chemical shifts ( ) for al at other positions xi with a xi as follows besides the chemical shift , the interaction of the quadrupole moment , q , of the al nucleus ( spin - quantum number i = 5/2 ) with a sufficiently large electric field gradient ( efg ) , which is produced by a nonspherical charge distribution around the nuclear site , also affects the central nmr transition . second order quadrupolar effects , influencing the shape of the nmr central line , can not be ( completely ) eliminated by magic angle spinning . from the simulation of spectra recorded under mas conditions , the quadrupole coupling constantand the corresponding asymmetry parametercan be estimated . here , e is the positive elementary charge , h denotes planck s constant , and vxx , vyy and vzz are the elements of the traceless efg tensor v with |vzz| |vxx| |vyy| . the shape of the nmr central line depends sensitively on the asymmetry of the electronic charge density close to the nucleus . the parameter describes the deviation of the efg from axial symmetry and it can take values between 0 and 1 . second order quadrupole interactions also affect the position of the nmr line in terms of a quadrupolar shift . a larger external magnetic field , however , lowers the effect . here , we compute the efg from the all - electron charge distribution without further approximation and cq is obtained using the nuclear quadrupole moment q(al ) = 1.616 10 m determined from the slope of the linear regression proposed by body et al . the most energetically favorable position of al in llzo is at the tetrahedrally coordinated crystallographic special 24d position . al can be displaced toward a neighboring vacant general 96h site , which leads to a distortion of its oxygen coordination polyhedron . calculated al o bond lengths for this coordination at 0 k are between 1.76 and 1.79 and the tetrahedral volume is 2.86 ( see table 1 ) . our calculations show that al at 96h is 4-fold coordinated , and distorted from tetrahedral coordination , with al o bond lengths varying from 1.76 to 1.92 , because of the displacement of al towards a vacant 24d site . additionally , there are two o ions further away that have on average approximately 1 longer bond distances . if they would be included in the local coordination sphere around al , a distorted octahedral coordination polyhedron with a volume of 16.33 would result . al could also possibly be located at the special 48 g site with three different pairs of al o bonds and calculated bond lengths of 1.93 , 2.00 and 2.12 , respectively , yielding a coordination volume of 10.67 . any possible al at the 16a site would also be 6-fold coordinated with again three pairs of al o bonds with lengths of 1.90 , 2.03 , and 2.11 and a coordination volume of 10.69 . al at the 24c site would have four shorter al o bond lengths equal to 1.94 and four longer bonds of 2.84 length . assuming , on the other hand , 8-fold coordination around the 24c site , the resulting polyhedron would have a volume of 22.86 . the various hypothetical al coordination polyhedra at 0 k , along with their associated site preference energy , are shown in figure 2 . various coordination polyhedral around al located at the 24d , 48 g , 96h , 16a , and 24c sites . ( nonbonded red spheres indicate additional o ions not considered as next nearest neighbors in the first coordination sphere . their distances are given in table 1 . ) their associated site preference energies in cubic llzo . the calculated site preference energies , e , for al in llzo are as follows : 24d > al at the 24d site has the global energy minimum and is 1.38 ev more stable than al at 96h and 1.60 ev more stable than al at 48 g . the latter two sites are energetically similar with an energy difference of just 0.22 ev . the site preference energy for al at 16a and 24c is 0.41 and 1.74 ev higher , respectively , when referenced to the global energy minimum . the site preference energies for al at various structural positions between 48 g and 24d are shown in figure 3 . site preference energies , e , for al at various structural positions ( gray crosses ) at and near the 48 g , 96h and 24d sites in llzo garnet . the fits were used to calculate the boltzmann distribution , w(xi ) ( see text ) , discussed below . the different distributions of al at the various crystallographic sites are normalized to 1 and indicate the room temperature probability of finding al at a distinct position . calculated al nmr parameters for al at the different structural sites in llzo are shown in table 2 . the corresponding polyhedra ( figure 4 ) around al , slightly displaced from 24d and 96h ( figure 3 ) , were used to analyze the experimental nmr spectra . the calculations show that al coordinations given by 1 to 5 ( figure 4 ) have values between 3.7 and 0.2 ppm , cq values between 1.38 and 5.01 mhz and values between 0.20 and 0.70 . al for coordinations given by 9 to 13 have values between 10.0 and 14.4 ppm , cq values between 2.4 and 3.3 mhz and values between 0.24 and 0.70 . on the other hand , al located at 48 g has an extremely large calculated value of 133.5 ppm , 118.5 ppm at 24c , and 108.5 ppm at 16a . the latter three sites have values that are much larger than any experimentally observed nmr resonances . oxygen coordination polyhedra around al for al o distances less than 2.6 at 17 local positions starting with 24d ( 1 ) and going to 96h ( 12 ) and beyond and ending at 48 g ( 20 ) in cubic llzo . in this dft study , we investigated the crystal - chemical role of al in llzo . specifically , we address the question of the coordination and the site distribution behavior of al . the results of the calculations can be used , together with published experimental results , to achieve a better understanding of the nature of al incorporation in llzo . we find that al at the 24d site is energetically the most stable state , followed by al at 96h and 48 g , whereby the site preference energies for the latter two are similar . based on our dft calculations , al should be exclusively located at 24d at 0 k , which would give rise to a single nmr resonance . thus , the question arises why additional resonances are observed in the al mas nmr spectra ( figure 5 ) . before discussing our results , we briefly outline the nmr results from the various studies with respect to the al site occupation in llzo . in all recent studies the resonance at approximately 66 other nmr lines also indicate 4-fold coordinated al ions , because their chemical shift values of 78 to 82 ppm are usually indicative of tetrahedral coordination . geiger et al . assigned the resonance at 81 ppm to al residing at the 96h sites . in particular , the spectra presented in ref ( 6 ) indicate that several nmr lines contribute to a signal occurring between 75 to 85 ppm . it should be noted that the latter has been reported for llzo samples with a high amount of al and lower than stoichiometric la and zr contents . such samples were prepared by mechanochemical activation combined with subsequent annealing at moderate temperatures . the position corresponds to al with the coordination polyhedra in figure 4 and the site preference energies in figure 3 . calculated values are referenced to the global minimum given by = 68 ppm at position 1 . literature : = 24d ; calculation : = 68 ppm the gray areas represent the variation in chemical shift values , , ( 13 to 18 ppm ) in literature . observed a stronger nmr signal at 64 ppm and a less intense line at 78 ppm for garnets with al contents up to 0.30 per formula unit ( pfu ) . however , at concentrations above 0.60 al pfu the resonance at 78 ppm becomes the most intense . at even higher al contents a new nmr line at 81 ppm emerges , which notably appears in la- and zr - deficient llzo samples . , in addition to al mas nmr experiments , also al mqmas nmr measurements . the latter spectra show a resonance at = 81 ppm , a coupling constant , cq , of 3.3 mhz and an asymmetry parameter , , of 0.7 . the second resonance observed at = 68 ppm shows a stronger quadrupolar interaction with cq = 5.0 to 5.2 mhz and a lower value between 0.0 and 0.1 that suggests axial symmetry at the site . it should be noted that the al mas nmr spectrum was recorded at 156.26 mhz , thus providing a sufficiently high resolution to simulate its line shape . such a simulation yields a resonance with = 81 ppm , cq = 3.3 mhz and = 0.7 , and for the second signal values of = 70 ppm , cq = 5.5 mhz and = 0.5 . the calculated nmr parameters ( table 2 ) are in good agreement with an assignment of al to 96h for the resonance at 81 ppm . the calculated nmr parameters for the nmr line at 68 ppm that is assigned to al at 24d , however , disagree with those determined from the mqmas nmr experiments . for the latter , it has been proposed that possibly more than one al site is reflected by this broad resonance . they made al mqmas nmr spectroscopic measurements to investigate this resonance at 68 ppm for an al - doped llzo garnet synthesized at 850 c . their line shape analysis yielded two nmr lines with the first having = 68.4 ppm and cq = 4.8 mhz and the second = 73.5 ppm and cq = 6.9 mhz . both resonances were assigned to tetrahedrally coordinated al at 24d in two different garnet phases . the two tetrahedral sites have slightly different distortions and thus different and cq values . report that this interpretation is consistent with their high - resolution xrpd results , and they proposed that slow al diffusion within the lattice is responsible for the disordering over the two sites . for the first step , we calculated the nmr parameters for al at all crystallographic sites , via 24d , 48 g , 96h , 16a , and 24c . according to our results , the resonance at 80 2 ppm should be assigned to al residing at the 96h sites and the resonance at 66 2 ppm to al occupying 24d . there is good agreement between the calculated cq value for al at 96h , but disagreement between cq values for al at 24d . proposed a large cq value for al at 24d , but it can not be easily explained why the symmetric 24d site should yield a cq value approximately two times larger than al at the more distorted 96h site . to understand this issue better , we calculated the nmr parameters for al at all positions at and around 24d and 96h that are occupied by a certain probability . shifting al away from 24d and 96h leads to a distribution of slightly different local oxygen coordination environments . thereby , broad al mas nmr resonances could result , which would reflect a distribution of slightly different and cq values . we note that our calculations for for al at 24d do not agree with experiment , but was obtained from the experimental spectra by using only one or two al resonances to simulate this broad feature at 66 2 ppm . the nmr signal located at 78 to 82 ppm is also asymmetric in shape . this is possibly due to the slightly different geometries of local al coordinations , as given by their calculated probabilities , toward the 48 g minimum versus those in the direction of 24d . this situation could produce two overlapping resonances having similar values of and cq ( see also the agreement with experimental results and the discussion in ref ( 6 ) ) . although experiment and calculations are in broad agreement in terms of the crystal chemical role of al in llzo , it is not clear based on the dft calculations alone , why al is not located exclusively at 24d as suggested by the site energies . al at 96h and 48 g must also be considered , which , however , would lead to an additional nmr line which is usually not observed experimentally . ( for the sake of completeness , let us note that an additional nmr line has been observed at 93 ppm for al - doped li6.5la2.5ba0.5zrtao12 and for some of the al - doped llzo samples prepared via mechanosynthesis ; the prominent nmr line at 65 ppm , however , is absent in these cases . ) here , it must be stated that the exact thermodynamic state of llzo , as obtained in the various sintering experiments , is not known . it is possible that metastable structural states are obtained that can depend on a number of experimental factors ( dopant concentration , sintering temperature and time , heating rate , grain sizes , starting materials , etc . ) . thus , al could potentially be incorporated metastably at 96h and 48 g as well in cubic llzo , because both sites have similar site energies . the different site energies for al at 24d and 96h could provide an explanation for differences in published nmr spectra of llzo . the relationship between al concentration and variations in the intensities of the different resonances ( and thus al site occupancies ) , as observed by dvel et al . , can be interpreted crystal chemically . when an al ion is located at 24d , because of its large effective charge radius , it could create a larger inaccessible region around 24d compared to the situation for al at 96h . this leads to a reduction of entropy and a loss of energy possibly making the 96h site energetically more accessible for al with increased al contents in contrast to the 24d site . they showed by using molecular dynamics simulations that the introduction of vacancies in llzo does indeed reduce the free energy . lastly , we consider the coordination geometry around the 96h site in llzo , which has been described differently . it is , of course , a matter of definition as to what constitutes a bond in a first coordination sphere . according to li et al . in their description of al at 96h , the average al o bond distance is d = 2.08 , with a difference of 0.38 between the length of the shortest and the longest bonds . o = 2.69 and 2.80 , thus being 0.46 and 0.57 longer compared to the longest bond in strict 4-fold coordination . based on our calculations , al at 96h has a locally 4-fold distorted coordination ( figure 4 ) . it is worth mentioning that long - range structural properties , as determined by the diffraction experiment , such as neutron powder diffraction , can differ from those measured via spectroscopy , for example , nmr , which probes structure at shorter length scales . based on our dft results , we propose that al could have a number of slightly different local 4-fold coordinations around the crystallographic 24d and 96h sites in cubic llzo garnet . , in a neutron diffraction study of cubic llzo garnet , proposed that al is located at the 48 g site in octahedral coordination . in terms of calculated site energies , this site could be partially occupied . however , the calculated al nmr chemical shift value for such coordination has not been observed in experimental nmr spectra .

we investigate theoretically the site occupancy of al3 + in the fast - ion - conducting cubic - garnet li73xal3+xla3zr2o12 ( ia-3d ) using density functional theory . by comparing calculated and measured 27al nmr chemical shifts an analysis shows that al3 + prefers the tetrahedrally coordinated 24d site and a distorted 4-fold coordinated 96h site . the site energies for al3 + ions , which are slightly displaced from the exact crystallographic sites ( i.e. , 24d and 96h ) , are similar leading to a distribution of slightly different local oxygen coordination environments . thus , broad 27al nmr resonances result reflecting the distribution of different isotropic chemical shifts and quadrupole coupling constants . from an energetic point of view , there is evidence that al3 + could also occupy the 48 g site with its almost regular octahedral coordination sphere . although this has been reported by neutron powder diffraction , the nmr chemical shift calculated for such an al3 + site has not been observed experimentally .

Introduction Computational Methods Results Discussion Conclusion

the fast li - ion conductor with the nominal composition li7la3zr2o12 ( llzo ) is receiving much scientific attention since its discovery in 2007 . at room temperature , llzo is tetragonal ( i41/acd ) while the cubic modification ( ia-3d ) is stable above approximately 150 c . argued that the better conducting cubic phase can be stabilized at room temperature ( rt ) through the incorporation of small amounts of al . the exact role al plays in cubic al - bearing llzo is important because llzo shows a high ionic conductivity of about 10 s / cm at rt . llzo also has good chemical and thermal stability , as well as a wide energy potential window making it an excellent candidate for use as an electrolyte in an all - solid - state lithium - ion battery . as has been shown recently , ionic conductivity also seems to depend on the amount of al incorporated during synthesis . for this purpose , the chemical and physical properties governing li diffusion have to be understood in detail ; in particular this includes the important question as to which crystallographic sites the al ions preferably occupy in the cubic phase of llzo . considerable experimental research has been undertaken to obtain information about al in llzo , including its local coordination and site partitioning behavior . several nmr studies have proposed that the resonance observed at a chemical shift ranging from 64 to 68 ppm corresponds to al located at the there is uncertainty , however , about the interpretation and assignment of the other measured nmr lines that have chemical shifts ranging from approximately 78 to 82 ppm . the interpretations given so far include al residing at nontetrahedral li sites ( 4-fold to possibly 6-fold coordinated ) and tetrahedrally coordinated sites in the neighborhood of la or zr vacancies in al - rich llzo . neutron powder diffraction ( npd ) measurements were interpreted as indicating that al is located at an octahedrally coordinated site in the garnet framework . summarizing the various published experimental results , there is no clear understanding as to which sites are occupied by al in cubic llzo . to address the important role of al in llzo and to obtain a better understanding of the various experimental results , we undertook a density functional theory ( dft ) investigation of al - bearing cubic llzo with the aim to calculate ( relative ) al nmr parameters such as chemical shifts and electric quadrupole coupling constants . the blue spheres correspond to tetrahedrally coordinated ( 24d ) li , green spheres to octahedrally coordinated ( 48 g ) li , and red ones to distorted 4-fold coordinated ( 96h ) li . in the case of llzo , the o ions , located at general crystallographic positions , 96h , form an oxygen - ion framework with interstices occupied by the a cations ( la ) at an 8-fold coordinated position 24c ( point symmetry 222 ) , by b cations ( zr ) at a 6-fold coordinated position 16a ( point symmetry 3 ) , and by c cations ( li ) at a 4-fold coordinated 24d position ( point symmetry 4 ) . in addition to these cation sites , there are other interstices within the oxygen framework that are empty in the conventional garnet structure , such as the 6-fold coordinated 48 g positions ( point symmetry 2 ) and a general 96h position , sometimes described as 4-fold coordinated with two additional longer bonds greater than 2.8 in length , 5-fold coordinated or 6-fold coordinated ( point symmetry 1 ) . these interstices can be filled by extra cations ( such as li ) , giving rise to compositions with nonstandard garnet stoichiometry . an important property is the partial occupancy of the structural sites ( 24d , 48 g , and 96h ) where li is located and also delocalization of li ions throughout . for our first - principle calculations , the ions in llzo were arranged on the basis of crystal structure descriptions in the literature . three different structural models were used with all having a body centered ( i - type ) bravais lattice with a = b = c = 12.972 and = = = 90. three garnet compositions were chosen , namely li44al4la24zr16o96 with al at 24d , 48 g and 96h , li56al4la20zr16o96 with al solely at 24c , and li60al4la24zr12o96 with al solely at 16a . the li ions were distributed over the 24d and the 96h sites following xu et al . the calculations were made with 17 different local al positions located among the 24d , 96h , and 48 g sites . the various al positions were fixed during relaxation and all al ions are equivalent in the unit cell . all calculations are based on dft methods using the all - electron full potential linearized augmented plane wave ( lapw ) method as implemented in the wien2k code . the behavior of al at different crystallographic sites was analyzed qualitatively by weighing the various al positions , xi , with their corresponding energy , e(xi ) . the various local energies for al at and around the crystallographic positions where li can also be located , were described by fitting a polynomial , describing a possible diffusion pathway . the ocuppation probabilities for an al ion along this path are described using a normalized boltzmann factor given bywhere k is boltzmann s constant , and where xi+1 indicates the states next to xi , xi1 indicates the states before xi with an interval of 0.01 , and t is 298.15 k. the site preference energy , e , is defined as e = e(xi ) e , where e is the global energy minimum and corresponds to al located at 24d . in nmr spectroscopy , the chemical shift , , of a nucleus , i , describes the nuclear shielding effect of an applied ( external ) static magnetic field and the locally induced magnetic field arising from the surrounding electrons with a certain probability of presence near the corresponding nuclear site i. the magnitude of the resulting effective field , beff , is given by beff = b0(1 i ) , where i is a second - rank nuclear shielding tensor , 1 is the unit matrix and b0 is a uniform external field along the z - axis . the isotropic chemical shift , iso ( henceforth ) , describes the relation between the nmr resonance frequency for the nucleus of interest , i , and the corresponding resonance frequency for a reference compound , ref , giving = 10 ( vi vref)/vref . to compute , we used the relaxed geometry and applied the nmr module of the wien2k code . these chemical shift calculations are based on an all - electron linear response method where one obtains the induced current density considering the perturbation of the ground state wave functions due to the external magnetic field . the resulting magnetic shielding is then obtained by integration of the all - electron current according to biot because there is agreement in the literature that the al nmr resonance at 66 2 ppm represents tetrahedrally coordinated al at 24d in llzo ( 24d = 68 ppm ) , we calculate the difference of the chemical shifts ( ) for al at other positions xi with a xi as follows besides the chemical shift , the interaction of the quadrupole moment , q , of the al nucleus ( spin - quantum number i = 5/2 ) with a sufficiently large electric field gradient ( efg ) , which is produced by a nonspherical charge distribution around the nuclear site , also affects the central nmr transition . second order quadrupolar effects , influencing the shape of the nmr central line , can not be ( completely ) eliminated by magic angle spinning . from the simulation of spectra recorded under mas conditions , the quadrupole coupling constantand the corresponding asymmetry parametercan be estimated . the most energetically favorable position of al in llzo is at the tetrahedrally coordinated crystallographic special 24d position . al can be displaced toward a neighboring vacant general 96h site , which leads to a distortion of its oxygen coordination polyhedron . our calculations show that al at 96h is 4-fold coordinated , and distorted from tetrahedral coordination , with al o bond lengths varying from 1.76 to 1.92 , because of the displacement of al towards a vacant 24d site . if they would be included in the local coordination sphere around al , a distorted octahedral coordination polyhedron with a volume of 16.33 would result . al could also possibly be located at the special 48 g site with three different pairs of al o bonds and calculated bond lengths of 1.93 , 2.00 and 2.12 , respectively , yielding a coordination volume of 10.67 . any possible al at the 16a site would also be 6-fold coordinated with again three pairs of al o bonds with lengths of 1.90 , 2.03 , and 2.11 and a coordination volume of 10.69 . assuming , on the other hand , 8-fold coordination around the 24c site , the resulting polyhedron would have a volume of 22.86 . the various hypothetical al coordination polyhedra at 0 k , along with their associated site preference energy , are shown in figure 2 . ( nonbonded red spheres indicate additional o ions not considered as next nearest neighbors in the first coordination sphere . the calculated site preference energies , e , for al in llzo are as follows : 24d > al at the 24d site has the global energy minimum and is 1.38 ev more stable than al at 96h and 1.60 ev more stable than al at 48 g . the site preference energy for al at 16a and 24c is 0.41 and 1.74 ev higher , respectively , when referenced to the global energy minimum . the site preference energies for al at various structural positions between 48 g and 24d are shown in figure 3 . site preference energies , e , for al at various structural positions ( gray crosses ) at and near the 48 g , 96h and 24d sites in llzo garnet . the fits were used to calculate the boltzmann distribution , w(xi ) ( see text ) , discussed below . the different distributions of al at the various crystallographic sites are normalized to 1 and indicate the room temperature probability of finding al at a distinct position . the corresponding polyhedra ( figure 4 ) around al , slightly displaced from 24d and 96h ( figure 3 ) , were used to analyze the experimental nmr spectra . oxygen coordination polyhedra around al for al o distances less than 2.6 at 17 local positions starting with 24d ( 1 ) and going to 96h ( 12 ) and beyond and ending at 48 g ( 20 ) in cubic llzo . specifically , we address the question of the coordination and the site distribution behavior of al . we find that al at the 24d site is energetically the most stable state , followed by al at 96h and 48 g , whereby the site preference energies for the latter two are similar . based on our dft calculations , al should be exclusively located at 24d at 0 k , which would give rise to a single nmr resonance . thus , the question arises why additional resonances are observed in the al mas nmr spectra ( figure 5 ) . before discussing our results , we briefly outline the nmr results from the various studies with respect to the al site occupation in llzo . in all recent studies the resonance at approximately 66 other nmr lines also indicate 4-fold coordinated al ions , because their chemical shift values of 78 to 82 ppm are usually indicative of tetrahedral coordination . in particular , the spectra presented in ref ( 6 ) indicate that several nmr lines contribute to a signal occurring between 75 to 85 ppm . it should be noted that the latter has been reported for llzo samples with a high amount of al and lower than stoichiometric la and zr contents . the position corresponds to al with the coordination polyhedra in figure 4 and the site preference energies in figure 3 . literature : = 24d ; calculation : = 68 ppm the gray areas represent the variation in chemical shift values , , ( 13 to 18 ppm ) in literature . observed a stronger nmr signal at 64 ppm and a less intense line at 78 ppm for garnets with al contents up to 0.30 per formula unit ( pfu ) . the second resonance observed at = 68 ppm shows a stronger quadrupolar interaction with cq = 5.0 to 5.2 mhz and a lower value between 0.0 and 0.1 that suggests axial symmetry at the site . the calculated nmr parameters for the nmr line at 68 ppm that is assigned to al at 24d , however , disagree with those determined from the mqmas nmr experiments . for the latter , it has been proposed that possibly more than one al site is reflected by this broad resonance . both resonances were assigned to tetrahedrally coordinated al at 24d in two different garnet phases . the two tetrahedral sites have slightly different distortions and thus different and cq values . for the first step , we calculated the nmr parameters for al at all crystallographic sites , via 24d , 48 g , 96h , 16a , and 24c . according to our results , the resonance at 80 2 ppm should be assigned to al residing at the 96h sites and the resonance at 66 2 ppm to al occupying 24d . there is good agreement between the calculated cq value for al at 96h , but disagreement between cq values for al at 24d . proposed a large cq value for al at 24d , but it can not be easily explained why the symmetric 24d site should yield a cq value approximately two times larger than al at the more distorted 96h site . to understand this issue better , we calculated the nmr parameters for al at all positions at and around 24d and 96h that are occupied by a certain probability . shifting al away from 24d and 96h leads to a distribution of slightly different local oxygen coordination environments . thereby , broad al mas nmr resonances could result , which would reflect a distribution of slightly different and cq values . we note that our calculations for for al at 24d do not agree with experiment , but was obtained from the experimental spectra by using only one or two al resonances to simulate this broad feature at 66 2 ppm . the nmr signal located at 78 to 82 ppm is also asymmetric in shape . this is possibly due to the slightly different geometries of local al coordinations , as given by their calculated probabilities , toward the 48 g minimum versus those in the direction of 24d . although experiment and calculations are in broad agreement in terms of the crystal chemical role of al in llzo , it is not clear based on the dft calculations alone , why al is not located exclusively at 24d as suggested by the site energies . al at 96h and 48 g must also be considered , which , however , would lead to an additional nmr line which is usually not observed experimentally . ( for the sake of completeness , let us note that an additional nmr line has been observed at 93 ppm for al - doped li6.5la2.5ba0.5zrtao12 and for some of the al - doped llzo samples prepared via mechanosynthesis ; the prominent nmr line at 65 ppm , however , is absent in these cases . ) here , it must be stated that the exact thermodynamic state of llzo , as obtained in the various sintering experiments , is not known . thus , al could potentially be incorporated metastably at 96h and 48 g as well in cubic llzo , because both sites have similar site energies . the different site energies for al at 24d and 96h could provide an explanation for differences in published nmr spectra of llzo . the relationship between al concentration and variations in the intensities of the different resonances ( and thus al site occupancies ) , as observed by dvel et al . this leads to a reduction of entropy and a loss of energy possibly making the 96h site energetically more accessible for al with increased al contents in contrast to the 24d site . lastly , we consider the coordination geometry around the 96h site in llzo , which has been described differently . it is , of course , a matter of definition as to what constitutes a bond in a first coordination sphere . in their description of al at 96h , the average al o bond distance is d = 2.08 , with a difference of 0.38 between the length of the shortest and the longest bonds . it is worth mentioning that long - range structural properties , as determined by the diffraction experiment , such as neutron powder diffraction , can differ from those measured via spectroscopy , for example , nmr , which probes structure at shorter length scales . based on our dft results , we propose that al could have a number of slightly different local 4-fold coordinations around the crystallographic 24d and 96h sites in cubic llzo garnet . , in a neutron diffraction study of cubic llzo garnet , proposed that al is located at the 48 g site in octahedral coordination . in terms of calculated site energies , this site could be partially occupied . however , the calculated al nmr chemical shift value for such coordination has not been observed in experimental nmr spectra .

diphtheria toxin ( dt ) is a single , 535amino acid polypeptide secreted by corynebacterium diphtheriae and is responsible for the disease diphtheria . 1 ) : the nh2-terminal catalytic domain ( residues 1185 ) , the cooh - terminal receptor - binding domain ( residues 386535 ) , and the translocation , or t domain , lying between them ( residues 202378 ) . the catalytic domain is connected to the t domain by a protease - susceptible loop and by an easily reducible disulfide bridge ( for reviews see madshus and stenmark 1992 ; falnes and sandvig 2000 . ) cellular intoxication by dt is thought to proceed by the following mechanism . pathogenic strains of corynebacterium diphtheriae infect a host and secrete the bacteriophage - encoded toxin as a monomer . the receptor - binding domain targets it to the surface of cells harboring a heparin - binding epidermal growth factor like precursor . while the toxin is on the surface of the cell , a host protease nicks the loop connecting the catalytic domain to the t domain , leaving the two domains still connected by their disulfide bridge . internalization occurs via receptor - mediated endocytosis , and the toxin now finds itself in an acidifying endosome . the low ph of the endosome induces a conformational change in the toxin that inserts the t domain into the endosomal membrane and translocates the catalytic domain across the membrane into the reducing environment of the cytosol . here , subsequent adp - ribosylation of elongation factor 2 by this domain inhibits protein synthesis , thereby killing the cell . the endosome function in this process is to provide a low ph environment ; experimentally it can be bypassed by exposing toxin - treated cells to a low ph , in which case the t domain translocates the catalytic domain directly across the plasma membrane ( draper and simon 1980 ; sandvig and olsnes 1980 ) . the t domain alone , as well as whole toxin and a mutant lacking the r domain ( crm45 ) , form channels in planar bilayers when the ph of the cis side ( the solution to which t domain constructs are added ) is below 6 ( donovan et al . associated with this channel formation , the entire catalytic domain along with 70 residues of the nh2 terminus of the t domain is translocated across the membrane ( oh et al . thus the t domain contains all of the translocation machinery ; no cellular components , or even the toxin 's r domain , are required for translocation . in the open channel state , the topology of the t domain consists of only three transmembrane segments ( th5 , th8 , and th9 ; senzel et al . 2000 ) , and yet ions as large as glucosamine and nad can traverse the channel ( hoch et al . how many subunits of dt are involved in making this channel and , thus , in translocating the catalytic domain ? ; steere and eisenberg 2000 ) , but it is not known whether these aggregates are functional ; in fact , purified dimers can not infect host cells ( carroll et al . 1992 ) , every cysteine mutant channel that reacted with thiol - specific methanethiosulfonate ( mts ) derivatives yielded only one transition in single - channel conductance ( huynh et al . second , the conductance of the d352c channel after reacting with mts - ethylammonium is nearly identical to that of the d352k channel ( mindell et al . this does not make sense for a multimeric channel , where the former has one positive charge ( ch2-s - s - ch2-ch2-nh3 ) from its one mts - ethylammonium reaction at d352c , and the latter would have n similar positively charged residues there ( ch2-ch2-ch2-ch2-nh3 ) , where n is the stoichiometry of the channel . third , the conductance of the d352c channel is smaller than that of the wild - type channel , a consequence of replacing a negatively charged residue with a neutral one . the subsequent reaction with mts - ethylsulfonate restores the channel conductance to that of wild type in a single step - change ( huynh et al . again , it would be surprising , if this is a multimeric channel , that a mutant with only one negative charge at residue 352 had the same conductance as that of the wild - type channel with n negative charges there . the only observation suggesting that the t domain channel is composed of more than one subunit is that the rate of channel formation increases with about the second power of toxin concentration ( kagan et al . however , it is possible that a cooperative process facilitates toxin entry into the membrane , thereby giving a nonlinear dependence of the rate of channel formation on concentration , whereas the channel that actually forms is a monomer . all of this evidence , although not definitive , seems to argue against a channel composed of multiple subunits . in fact , for most protein systems , one would probably accept the above as ample evidence of monomericity . ( how can three transmembrane segments alone create a pore large enough to conduct k and cl , let alone glucosamine and nad ? ) therefore , we have designed a set of experiments to test the hypothesis that the channel formed by the t domain of diphtheria toxin is monomeric . details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al . the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein . this h6 tag has a thrombin cleavage site so that it can be removed after purification of the expressed protein on a nickel column . using stratagene 's quickchange site - directed mutagenesis kit , we mutated the arginine in this thrombin cleavage site to a glutamine to minimize unintended proteolysis of this h6 tag by trace proteases . the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this , an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit . the purified dna from that mutant was digested with the restriction endonucleases ndei and xhoi , gel - purified , and ligated into the pet-22b expression vector that had been cut with the same restriction enzymes and also gel - purified . it was found that this protein was not an effective channel blocker . again using the site - directed mutagenesis kit , we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) . we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) . one can see that this is most of the nh2-terminal h6 tag oriented in the reverse direction . purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector . this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct . thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202 p378-legggmgssh6ssglvpr . after expression and purification ( zhan et al . 1995 ) on a novagen his - bind column , the proteins were dialyzed into 20 mm tris - cl , ph 8.0 , 1 mm edta and frozen at 80c . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml . the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows . the t domains were first individually incubated at 37c for 2 h in 40% dmso ( sigma - aldrich ) to break up most of the preformed aggregates ( carroll et al . this reliably disrupted > 90% of the dimeric t domain , as assayed by 15% native page ( fig . they were then tested individually on bilayers to find dilutions that consistently gave from one to three single channels within a few minutes of being stirred into the cis solution . this was usually achieved with a 1:300 to 1:500 dilution ( t domain construct:20 mm tris - cl , ph 8.0 ) . the amount of each t domain in the individual dilutions was used to establish a suitable ratio in the mixtures of the original concentrated solutions . after treatment with dmso , the mixtures were run on a superdex g-75 sizing column ( pharmacia ) in 50 mm nacl , 10 mm tris - cl , ph 8.0 , at a flow rate of 0.75 ml / min . samples were collected in 0.5-ml fractions and concentrated 10-fold in a savant uvs 400 speed vac plus . all mixtures used for bilayer experiments were taken from fractions located to the right of the monomer peak . planar lipid ( asolectin ) bilayer membranes ( 70100 m in diameter ) were made by a modification of the folded film method as described by huynh et al . the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 . voltages are those of the cis solution with respect to the trans solution , whose potential was held at virtual ground . current generated by single channels was measured by voltage clamping the cis side of the membrane to voltages between 60 and 80 mv . 1981 ) ; after a channel entered , the voltage was pulsed to negative values to determine the channel type : n - type h6 tag gating , c - type h6 tag gating , no gating at all , or both types of gating ( see fig . if an entering channel left the membrane before application of this negative voltage pulse , it was not included in the dataset . our method of counting channels was to take the maximum number of channels of each type seen coincidentally in the membrane . for example , if one c - type channelappeared , stayed a while and then left , and another c - type channel entered the membrane later , this was counted as only one c - type channel . this method of counting , by insuring our not counting the same channel multiple times , avoided biasing the data . if , however , two c - type channels were seen in the membrane at the same time , this and only this was counted as two c - type channels . to further clarify this point , if a membrane yielded a count of 3 n - type and 2 c - type channels , this meant that at some time a in the record there were 3 n - type channels coincidentally in the membrane , and at some time b ( not necessarily the same as a ) there were 2 c - type channels coincidentally in the membrane . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig . 4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally . it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . in the end , though , for each membrane , only the maximum number of channels of each type seen in the membrane coincidentally were counted . details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al . the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein . this h6 tag has a thrombin cleavage site so that it can be removed after purification of the expressed protein on a nickel column . using stratagene 's quickchange site - directed mutagenesis kit , we mutated the arginine in this thrombin cleavage site to a glutamine to minimize unintended proteolysis of this h6 tag by trace proteases . the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this , an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit . the purified dna from that mutant was digested with the restriction endonucleases ndei and xhoi , gel - purified , and ligated into the pet-22b expression vector that had been cut with the same restriction enzymes and also gel - purified . it was found that this protein was not an effective channel blocker . again using the site - directed mutagenesis kit , we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) . we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) . one can see that this is most of the nh2-terminal h6 tag oriented in the reverse direction . purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector . this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct . thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202 p378-legggmgssh6ssglvpr . after expression and purification ( zhan et al . 1995 ) on a novagen his - bind column , the proteins were dialyzed into 20 mm tris - cl , ph 8.0 , 1 mm edta and frozen at 80c . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml . the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows . the t domains were first individually incubated at 37c for 2 h in 40% dmso ( sigma - aldrich ) to break up most of the preformed aggregates ( carroll et al . this reliably disrupted > 90% of the dimeric t domain , as assayed by 15% native page ( fig . they were then tested individually on bilayers to find dilutions that consistently gave from one to three single channels within a few minutes of being stirred into the cis solution . this was usually achieved with a 1:300 to 1:500 dilution ( t domain construct:20 mm tris - cl , ph 8.0 ) . the amount of each t domain in the individual dilutions was used to establish a suitable ratio in the mixtures of the original concentrated solutions . after treatment with dmso , the mixtures were run on a superdex g-75 sizing column ( pharmacia ) in 50 mm nacl , 10 mm tris - cl , ph 8.0 , at a flow rate of 0.75 ml / min . samples were collected in 0.5-ml fractions and concentrated 10-fold in a savant uvs 400 speed vac plus . all mixtures used for bilayer experiments were taken from fractions located to the right of the monomer peak . planar lipid ( asolectin ) bilayer membranes ( 70100 m in diameter ) were made by a modification of the folded film method as described by huynh et al . the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 . voltages are those of the cis solution with respect to the trans solution , whose potential was held at virtual ground . current generated by single channels was measured by voltage clamping the cis side of the membrane to voltages between 60 and 80 mv . 1981 ) ; after a channel entered , the voltage was pulsed to negative values to determine the channel type : n - type h6 tag gating , c - type h6 tag gating , no gating at all , or both types of gating ( see fig . if an entering channel left the membrane before application of this negative voltage pulse , it was not included in the dataset . our method of counting channels was to take the maximum number of channels of each type seen coincidentally in the membrane . for example , if one c - type channelappeared , stayed a while and then left , and another c - type channel entered the membrane later , this was counted as only one c - type channel . this method of counting , by insuring our not counting the same channel multiple times , avoided biasing the data . if , however , two c - type channels were seen in the membrane at the same time , this and only this was counted as two c - type channels . to further clarify this point , if a membrane yielded a count of 3 n - type and 2 c - type channels , this meant that at some time a in the record there were 3 n - type channels coincidentally in the membrane , and at some time b ( not necessarily the same as a ) there were 2 c - type channels coincidentally in the membrane . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig . 4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally . it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . in the end , though , for each membrane , only the maximum number of channels of each type seen in the membrane coincidentally were counted . the experiments to be described were performed in planar lipid bilayer membranes . their rationale came from our earlier work , which showed that a histidine tag ( h6 tag ) attached to the nh2 terminus of the t domain rapidly and completely blocked the channel at cis negative voltages , whereas when the h6 tag was attached to the cooh terminus , the channel was blocked ( a high frequency flickering block , occasionally entering a prolonged blocked state ) at cis positive voltages ( senzel et al . these findings were used to demonstrate that the nh2 terminus of the protein ( and its h6 tag ) was on the trans side of the membrane and the cooh terminus ( and its h6 tag ) was on the cis side ( senzel et al . , we verified that , as expected , when the t domain has both nh2- and cooh - terminal h6 tags , the channel is blocked at both negative and positive voltages . we reasoned that if we mixed nh2-terminal and cooh - terminal h6-tagged t domains , and if the t domain channel is a multimer , then some fraction of channels should be blocked at both positive and negative voltages . for example , if the channel is a dimer , and equal amounts of nh2-terminal and cooh - terminal h6-tagged t domains were mixed , then on average ( if the t domains have an equal preference to associate with one another ) one quarter of the channels should be blocked only at negative voltages ( both subunits have nh2-terminal h6 tags ) , one quarter blocked only at positive voltages ( both subunits have cooh - terminal h6 tags ) and one half blocked at both negative and positive voltages ( one subunit has an nh2-terminal h6 tag and the other has a cooh - terminal h6 tag ) . if , however , the channel is a monomer , one expects to see only channels that are blocked at either negative or positive voltages , but not at both . one could argue that before mixing these constructs , there already exist preformed homo - multimers and that these are responsible for the channel - forming activity . thus , when mixtures are made , one sees channels that only gate at either negative or positive voltages and falsely asserts that these channels are monomeric . first , we dissociated dimers and higher order aggregates by incubating the mixture in 40% dmso ( carroll et al . second , after this treatment , the mixture was applied to a size - exclusion column , and the monomeric band was purified away from any remaining dimer or higher aggregates . the monomer came off in several fractions , and only fractions from the trailing half of the monomer peak , furthest from the multimers , were used in our experiments ( fig . 3 ) . after the above protocol , we performed experiments on two sets of purified mixtures of nh2- and cooh - terminal h6-tagged t domains . we observed 75 single channels , none of which gated at both negative and positive voltages . from the first mixture , ( a typical record from one bilayer is shown in fig . 5 . ) of these , 17 showed n - type and 14 showed c - type h6-tagged gating characteristics , whereas 4 did not gate at either positive or negative voltages ( see next paragraph ) . the failure to observe any channels that showed both n - type and c - type gating is , of course , which is consistent with a monomeric channel . if , for the sake of argument , it is assumed that the channel is dimeric , the probability of our not seeing a single channel that showed both n - type and c - type h6 tag gating in 35 events is 4.5 10 ( see ) . from the second mixture , 9 individual bilayer experiments were performed giving a total of 40 observed single channels . of these , 26 showed n - type and 9 showed c - type h6-tagged gating characteristics , and 5 did not gate at either positive or negative voltages ( see next paragraph ) . if the channel is dimeric , the probability of our not having seen one channel that showed both n - type and c - type h6 tag gating in 40 events is 1.3 10 ( see ) . in the combined two sets of mixtures , the probability of our not having seen even one channel that showed both n- and c - type gating is 5.9 10 . if more than two t domains are required to construct the channel , then the odds of our not having seen even one channel that was blocked at both negative and positive voltages become even more minuscule . we attribute these nulls to a subpopulation of the nh2-terminal h6 tag proteins that had its nh2-terminal h6 tag cleaved off by trace amounts of protease in the solution . ( the region of the t domain immediately downstream of the nh2-terminal h6 tag is exposed in the water - soluble crystal structure [ bennett and eisenberg 1994 ] and could be a potential nicking site . in fact , on sds - page we saw faint bands with molecular weights slightly smaller than the t domain . ) to test this hypothesis , we performed measurements on only the t domain with the nh2-terminal h6 tag . in eight separate bilayer experiments yielding 36 channels , 31 were blocked at negative voltages and 5 were nulls , a ratio similar to those seen in our mixing experiments . no nulls were seen in experiments with t domain having the cooh - terminal h6 tag . we found that our extensive efforts to eliminate preformed dimers and aggregates were probably unnecessary . when we compared the activity of a sample that had a large preformed dimer population to its activity after receiving the 40% dmso treatment ( using the sample illustrated in fig . 2 ) , we saw no difference as assayed by the rate of channel entry into the membrane ( results not shown ) . thus , it appears that the preformed dimers and higher aggregates are not contributing significantly to channel formation . with this in mind , we can also include in our dataset the results from a mixture that was dmso treated , but was not run on the sizing column , and from a mixture that was neither dmso treated nor run on the sizing column . in the former case , 21 single - channel events were recorded on six separate bilayers ; of these events , 11 showed n - type gating , 9 showed c - type gating , and 1 did not gate at all . in the latter case , of 19 single - channel events observed on five bilayers , 9 showed n - type gating and 10 showed c - type gating . combining these data with the results from the previous mixtures , we observed a total of 115 single channels in 28 bilayers and never saw a channel that showed both n - type and c - type gating . associated with the translocation of diphtheria toxin 's catalytic domain across a planar lipid bilayer , the toxin 's t domain forms a channel . the t domain in this channel , which is permeable to ions as large as glucosamine and nad ( hoch et al . therefore , it seems obvious that the channel must be a multimer , yet several independent pieces of evidence , reviewed in the introduction , suggest the contrary . we mixed in a test tube t domain molecules having an nh2-terminal histidine tag ( h6 tag ) with those having a cooh - terminal h6 tag and observed the resulting channels formed by this mixture in planar lipid bilayers . channels formed by t domain molecules with an nh2-terminal h6 tag show characteristic blocking at negative voltages , whereas those formed by t domain molecules with a cooh - terminal h6 tag block at positive voltages ( fig . we reasoned that if the channel is a multimer , then some of the channels formed from the mixture should have both of these gating characteristics , just as do channels formed by t domain molecules that have both an nh2-terminal and a cooh - terminal h6 tag ( fig . we recorded 115 channels in 28 bilayers and never saw one channel manifesting both n - type and c - type h6 tag gating ! therefore , we conclude , contrary to common sense , that the t domain channel contains only one t domain molecule . it might be contended that the channels were generated from dimers or multimers that preexisted in the nh2-terminal and cooh - terminal h6-tagged t domain solutions , and so naturally we would not see any channels with both n - type and c - type h6 tag gating . first , after incubating a t domain solution that had a relatively large dimer content for 2 h at 37c in 40% dmso , which converted almost all of the dimers and higher aggregates to monomers ( fig . 2 ) , we found the channel - forming ability of the solution unchanged , indicating that preformed dimers and higher multimers are not a significant source of channels . second , in two sets of experiments , we went to the extreme of removing residual preformed dimers and multimers from mixtures of nh2-terminal and cooh - terminal h6-tagged t domains that had undergone the dmso treatment , by running the mixtures on a molecular sizing column and using , for the experiments , only fractions from the trailing half of the monomer peak ( fig . 3 ) . even though preexisting dimers and multimers are not the source of channel - forming activity , this does not preclude that t domain monomers can come together on or within the membrane to form a multimeric channel . for this to account for our failure to see even one channel out of 115 that manifested both n - type and c - type h6-tagged gating , however , one would have to assume that nh2-terminal h6-tagged t domains and cooh - terminal h6-tagged t domains associate exclusively with themselves ; i.e. , heteromultimer formation is precluded . although this is logically possible , we can think of no justification for this assumption . in fact , if one argues that somehow a t domain with a cooh - terminal h6 tag can not associate with one that has an nh2-terminal h6 tag , then why would t - domains containing both an nh2-terminal and cooh - terminal h6 tag associate in the postulated multimeric channel ? one would have to invoke a special attraction of nh2-terminal h6 tags and/or cooh - terminal h6 tags with themselves ; we can see no physical justification for this . one might also argue that even though nh2-terminal h6-tagged t domains can form heteromultimers with cooh - terminal h6-tagged t domains , for some reason these do not form functional channels . again , it is difficult to reconcile this position with the ability of t domains that have both an nh2-terminal and a cooh - terminal h6 tag to form channels . thus , it seems to us that to believe that the t domain channel is a multimer , one must argue that heteromeric channels are formed by nh2-terminal and cooh - terminal h6-tagged t domains , but some of these exhibit only n - type gating , whereas others exhibit only c - type gating . asymmetric structures can be envisioned that fulfill this condition , but this leads us into a fantasyland that is best avoided . throughout the analyses and discussions in this paper , we have tacitly assumed that only a single h6 tag is required for channel blocking . if this is not the case , one can imagine new scenarios in which the channel is multimeric , and yet no channels formed from our mixture exhibit both n - type and c - type gating . for example , if the channels were a trimer and required at least two nh2- or cooh - terminal h6 tags to get nh2- or cooh - terminal gating , respectively , we would observe only n - type and c - type gating channels , never channels showing both types of gating . in fact , in general , if the channel is formed by an odd number ( n ) of subunits and requires ( n + 1)/2 nh2- or cooh - terminal h6 tags to get n- or c - type gating , respectively , the above statement would hold . ( if n is even , the equivalent assumption that ( n/2 ) + 1 h6 tags are required for blocking predicts a significant number of channels showing neither n- nor c - type gating . ) we think that the requirement of multiple h6 tags to affect channel blocking is a priori unlikely ; we know of no precedent for this in the channel literature . moreover , if more than one h6 tag were required to completely block the channel , we would anticipate substates ( partial block ) when less than one h6 tag entered the channel . this paper presents what we feel are compelling arguments for the monomeric nature of the t domain channel formed in planar lipid bilayers . the channels formed by whole toxin in planar bilayers are indistinguishable from these ( silverman et al . 1994 ) , and therefore it is not much of a stretch to assert that they too are monomeric . the mechanism by which the catalytic domain of the toxin is translocated across planar bilayers in association with channel formation by its t domain ( oh et al . 1999 ) remains to be resolved , but whatever it is , we feel that it is now established that only one t domain molecule is involved in the process . the structure of the t domain channel , having only three transmembrane segments ( senzel et al . 2000 ) , remains a mystery and probably involves the lipids in its architecture . there is a feeling in the literature that in real life ( i.e. , the cell ) the translocation of the catalytic domain across the endosomal membrane into the cytosol is accomplished through an oligomer of dt ( steere and eisenberg 2000 ) . the results in this paper do not , of course , directly address this issue . all we can say in light of the present results and previous work ( oh et al . 1999 ) is that there is no necessity to invoke dt oligomers in cell intoxication , with the following caveat . the rate of channel formation increases with about the second power of toxin concentration ( kagan et al . thus , a cooperative interaction of two or more dt molecules may promote toxin entry into the membrane , even though the ultimate intoxicating unit is a monomer . interestingly , proteins in a molten globule - like state promote the transmembrane insertion of the t domain ( ren et al . dt itself partly unfolds into a molten globule - like state ( london 1992 ) , it may catalyze within the acidic endosome its own insertion into the endosomal membrane . in this sense there are also data using liposomes that suggest that oligomerization is involved in dt pore formation . for example , sharpe and london 1999 report that the size of liposome - entrapped molecules that are released is an increasing function of dt concentration ( at ph 4.5 ) . they interpret this to mean that the pores formed by dt increase in size as the concentration of dt within the membrane increases . furthermore , they observed that dt oligomerizes within the liposomal membrane under their experimental conditions . thus , they conclude that the pore formed by dt is an oligomer and that the pore size is a function of the number of subunits in the oligomer . we do not know how to relate our results to these , given the very different experimental conditions and techniques involved . in a previous paper ( senzel et al . 2000 ) , we discussed the difficulties involved in trying to reconcile planar lipid bilayer experiments with those on liposomes .

in the presence of a low ph environment , the channel - forming t domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin 's catalytic domain across them . given that the t domain contributes only three transmembrane segments , and the channel is permeable to ions as large as glucosamine+ and nad , it would appear that the channel must be a multimer . yet , there is substantial circumstantial evidence that the channel may be formed from a single subunit . to test the hypothesis that the channel formed by the t domain of diphtheria toxin is monomeric , we made mixtures of two t domain constructs whose voltage - gating characteristics differ , and then observed the gating behavior of the mixture 's single channels in planar lipid bilayers . one of these constructs contained an nh2-terminal hexahistidine ( h6 ) tag that blocks the channel at negative voltages ; the other contained a cooh - terminal h6 tag that blocks the channel at positive voltages . if the channel is constructed from multiple t domain subunits , one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages . the observed single channels were blocked at either negative or positive voltages , but never both . therefore , we conclude that the t domain channel is monomeric .

INTRODUCTION MATERIALS AND METHODS Constructs Ratios for Mixtures Purification of Monomeric T Domain Bilayer Experiments RESULTS DISCUSSION

1 ) : the nh2-terminal catalytic domain ( residues 1185 ) , the cooh - terminal receptor - binding domain ( residues 386535 ) , and the translocation , or t domain , lying between them ( residues 202378 ) . while the toxin is on the surface of the cell , a host protease nicks the loop connecting the catalytic domain to the t domain , leaving the two domains still connected by their disulfide bridge . the low ph of the endosome induces a conformational change in the toxin that inserts the t domain into the endosomal membrane and translocates the catalytic domain across the membrane into the reducing environment of the cytosol . the endosome function in this process is to provide a low ph environment ; experimentally it can be bypassed by exposing toxin - treated cells to a low ph , in which case the t domain translocates the catalytic domain directly across the plasma membrane ( draper and simon 1980 ; sandvig and olsnes 1980 ) . the t domain alone , as well as whole toxin and a mutant lacking the r domain ( crm45 ) , form channels in planar bilayers when the ph of the cis side ( the solution to which t domain constructs are added ) is below 6 ( donovan et al . associated with this channel formation , the entire catalytic domain along with 70 residues of the nh2 terminus of the t domain is translocated across the membrane ( oh et al . thus the t domain contains all of the translocation machinery ; no cellular components , or even the toxin 's r domain , are required for translocation . in the open channel state , the topology of the t domain consists of only three transmembrane segments ( th5 , th8 , and th9 ; senzel et al . 2000 ) , and yet ions as large as glucosamine and nad can traverse the channel ( hoch et al . this does not make sense for a multimeric channel , where the former has one positive charge ( ch2-s - s - ch2-ch2-nh3 ) from its one mts - ethylammonium reaction at d352c , and the latter would have n similar positively charged residues there ( ch2-ch2-ch2-ch2-nh3 ) , where n is the stoichiometry of the channel . the only observation suggesting that the t domain channel is composed of more than one subunit is that the rate of channel formation increases with about the second power of toxin concentration ( kagan et al . therefore , we have designed a set of experiments to test the hypothesis that the channel formed by the t domain of diphtheria toxin is monomeric . details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al . the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein . the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this , an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit . again using the site - directed mutagenesis kit , we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) . we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) . purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector . this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct . thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202 p378-legggmgssh6ssglvpr . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml . the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows . the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig . 4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally . it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . details concerning plasmid construction of the t domain with an nh2-terminal hexahistidine tag ( h6 tag ) , as well as our methods of protein expression and purification for all the constructs used , were as previously reported ( zhan et al . the nh2-terminal h6 construct was made by inserting wild - type t domain ( dt residues 202378 ) between the ndei and xhoi sites of the novagen pet-15b vector , which places an nh2-terminal h6 tag on the protein . the cooh - terminal h6 tag construct was engineered by inserting the same t domain into novagen 's pet-22b plasmid . to do this , an xhoi site was introduced at the cooh - terminal end of the t domain in the pet-15b vector , using the site - directed mutagenesis kit . again using the site - directed mutagenesis kit , we lengthened the h6 tag by inserting gggmgss between the cooh terminus of the t domain and the h6 tag ( primers used were cgtataatcgtcccctcgagggaggtggaatgggatcgtcgcaccaccaccaccaccac and its reverse compliment ; nucleotides inserted are shown in bold type ) . we therefore inserted additional residues ssglvpr cooh - terminal of the h6 tag ( primers used were ccaccaccaccacagcagcggcctcgtccccaggtgagatccggctgc and its reverse compliment ) . purified plasmid containing the t domain with cooh - terminal h6 tag was digested with the restriction endonucleases ndei and bpu1102 , gel - purified , and cloned into the pet-15b expression vector . this put the nh2-terminal h6 tag sequence mgssh6ssglvprgshm upstream of residue 202 in our cooh - terminal h6 construct . thus , the final sequence of the t domain with both nh2- and cooh - terminal h6 tags is : mgssh6ssglvprgshm - i202 p378-legggmgssh6ssglvpr . the t domain with the nh2-terminal h6 tag was at a concentration of 3 mg / ml ; that with the cooh - terminal h6 tag was at 1.5 mg / ml ; and that with both the nh2-terminal and cooh - terminal h6 tags was 3 mg / ml . the appropriate ratio of nh2-terminal to cooh - terminal h6 tag t domain in the mixtures was determined as follows . the solutions on both sides of the membrane contained 1 m kcl , 2 mm cacl2 , and 1 mm edta ; in addition , the cis solution ( the solution to which t domain constructs were added ) contained 30 mm mes , ph 5.3 , and the opposite trans solution contained 50 mm hepes , ph 7.2 . due to the rapid flickering nature of the cooh - terminal h6 tag block ( see fig . 4 ) , we were unable to reliably count more than three of these channels in the membrane at one time , and thus usually stopped the experiment if three of these were seen coincidentally . it was found that pulsing to large negative voltages ( 200 mv ) would close ( or drive out ) all of the channels in the membrane , so that multiple experiments could be done on the same membrane . their rationale came from our earlier work , which showed that a histidine tag ( h6 tag ) attached to the nh2 terminus of the t domain rapidly and completely blocked the channel at cis negative voltages , whereas when the h6 tag was attached to the cooh terminus , the channel was blocked ( a high frequency flickering block , occasionally entering a prolonged blocked state ) at cis positive voltages ( senzel et al . these findings were used to demonstrate that the nh2 terminus of the protein ( and its h6 tag ) was on the trans side of the membrane and the cooh terminus ( and its h6 tag ) was on the cis side ( senzel et al . , we verified that , as expected , when the t domain has both nh2- and cooh - terminal h6 tags , the channel is blocked at both negative and positive voltages . we reasoned that if we mixed nh2-terminal and cooh - terminal h6-tagged t domains , and if the t domain channel is a multimer , then some fraction of channels should be blocked at both positive and negative voltages . for example , if the channel is a dimer , and equal amounts of nh2-terminal and cooh - terminal h6-tagged t domains were mixed , then on average ( if the t domains have an equal preference to associate with one another ) one quarter of the channels should be blocked only at negative voltages ( both subunits have nh2-terminal h6 tags ) , one quarter blocked only at positive voltages ( both subunits have cooh - terminal h6 tags ) and one half blocked at both negative and positive voltages ( one subunit has an nh2-terminal h6 tag and the other has a cooh - terminal h6 tag ) . if , however , the channel is a monomer , one expects to see only channels that are blocked at either negative or positive voltages , but not at both . one could argue that before mixing these constructs , there already exist preformed homo - multimers and that these are responsible for the channel - forming activity . thus , when mixtures are made , one sees channels that only gate at either negative or positive voltages and falsely asserts that these channels are monomeric . after the above protocol , we performed experiments on two sets of purified mixtures of nh2- and cooh - terminal h6-tagged t domains . of these , 17 showed n - type and 14 showed c - type h6-tagged gating characteristics , whereas 4 did not gate at either positive or negative voltages ( see next paragraph ) . if , for the sake of argument , it is assumed that the channel is dimeric , the probability of our not seeing a single channel that showed both n - type and c - type h6 tag gating in 35 events is 4.5 10 ( see ) . of these , 26 showed n - type and 9 showed c - type h6-tagged gating characteristics , and 5 did not gate at either positive or negative voltages ( see next paragraph ) . if the channel is dimeric , the probability of our not having seen one channel that showed both n - type and c - type h6 tag gating in 40 events is 1.3 10 ( see ) . if more than two t domains are required to construct the channel , then the odds of our not having seen even one channel that was blocked at both negative and positive voltages become even more minuscule . ( the region of the t domain immediately downstream of the nh2-terminal h6 tag is exposed in the water - soluble crystal structure [ bennett and eisenberg 1994 ] and could be a potential nicking site . to test this hypothesis , we performed measurements on only the t domain with the nh2-terminal h6 tag . in eight separate bilayer experiments yielding 36 channels , 31 were blocked at negative voltages and 5 were nulls , a ratio similar to those seen in our mixing experiments . no nulls were seen in experiments with t domain having the cooh - terminal h6 tag . with this in mind , we can also include in our dataset the results from a mixture that was dmso treated , but was not run on the sizing column , and from a mixture that was neither dmso treated nor run on the sizing column . combining these data with the results from the previous mixtures , we observed a total of 115 single channels in 28 bilayers and never saw a channel that showed both n - type and c - type gating . associated with the translocation of diphtheria toxin 's catalytic domain across a planar lipid bilayer , the toxin 's t domain forms a channel . the t domain in this channel , which is permeable to ions as large as glucosamine and nad ( hoch et al . therefore , it seems obvious that the channel must be a multimer , yet several independent pieces of evidence , reviewed in the introduction , suggest the contrary . we mixed in a test tube t domain molecules having an nh2-terminal histidine tag ( h6 tag ) with those having a cooh - terminal h6 tag and observed the resulting channels formed by this mixture in planar lipid bilayers . channels formed by t domain molecules with an nh2-terminal h6 tag show characteristic blocking at negative voltages , whereas those formed by t domain molecules with a cooh - terminal h6 tag block at positive voltages ( fig . we reasoned that if the channel is a multimer , then some of the channels formed from the mixture should have both of these gating characteristics , just as do channels formed by t domain molecules that have both an nh2-terminal and a cooh - terminal h6 tag ( fig . therefore , we conclude , contrary to common sense , that the t domain channel contains only one t domain molecule . it might be contended that the channels were generated from dimers or multimers that preexisted in the nh2-terminal and cooh - terminal h6-tagged t domain solutions , and so naturally we would not see any channels with both n - type and c - type h6 tag gating . 2 ) , we found the channel - forming ability of the solution unchanged , indicating that preformed dimers and higher multimers are not a significant source of channels . second , in two sets of experiments , we went to the extreme of removing residual preformed dimers and multimers from mixtures of nh2-terminal and cooh - terminal h6-tagged t domains that had undergone the dmso treatment , by running the mixtures on a molecular sizing column and using , for the experiments , only fractions from the trailing half of the monomer peak ( fig . for this to account for our failure to see even one channel out of 115 that manifested both n - type and c - type h6-tagged gating , however , one would have to assume that nh2-terminal h6-tagged t domains and cooh - terminal h6-tagged t domains associate exclusively with themselves ; i.e. in fact , if one argues that somehow a t domain with a cooh - terminal h6 tag can not associate with one that has an nh2-terminal h6 tag , then why would t - domains containing both an nh2-terminal and cooh - terminal h6 tag associate in the postulated multimeric channel ? again , it is difficult to reconcile this position with the ability of t domains that have both an nh2-terminal and a cooh - terminal h6 tag to form channels . thus , it seems to us that to believe that the t domain channel is a multimer , one must argue that heteromeric channels are formed by nh2-terminal and cooh - terminal h6-tagged t domains , but some of these exhibit only n - type gating , whereas others exhibit only c - type gating . if this is not the case , one can imagine new scenarios in which the channel is multimeric , and yet no channels formed from our mixture exhibit both n - type and c - type gating . for example , if the channels were a trimer and required at least two nh2- or cooh - terminal h6 tags to get nh2- or cooh - terminal gating , respectively , we would observe only n - type and c - type gating channels , never channels showing both types of gating . in fact , in general , if the channel is formed by an odd number ( n ) of subunits and requires ( n + 1)/2 nh2- or cooh - terminal h6 tags to get n- or c - type gating , respectively , the above statement would hold . this paper presents what we feel are compelling arguments for the monomeric nature of the t domain channel formed in planar lipid bilayers . the mechanism by which the catalytic domain of the toxin is translocated across planar bilayers in association with channel formation by its t domain ( oh et al . 1999 ) remains to be resolved , but whatever it is , we feel that it is now established that only one t domain molecule is involved in the process . the structure of the t domain channel , having only three transmembrane segments ( senzel et al . , the cell ) the translocation of the catalytic domain across the endosomal membrane into the cytosol is accomplished through an oligomer of dt ( steere and eisenberg 2000 ) . dt itself partly unfolds into a molten globule - like state ( london 1992 ) , it may catalyze within the acidic endosome its own insertion into the endosomal membrane . thus , they conclude that the pore formed by dt is an oligomer and that the pore size is a function of the number of subunits in the oligomer .

cerebral palsy ( cp ) describes a group of permanent disorders of movement and posture that are attributed to nonprogressive disturbances in the developing brain and is often accompanied by disturbances of sensation , cognition , communication , perception , behavior , or seizures . after eradication of polio , it is becoming one of the most common causes of childhood disability in india . the prevention of childhood disability is one of the main focuses of the national policy of disability of india , which can be approached either by preventing the impairment or by preventing the impairment from becoming a disability . advances in neonatal management and obstetric care have not shown a decline in the incidence of cp . on the contrary , many believe that with a decline in infant mortality rate , there has actually been an increase in the incidence and severity of cp . in this situation , it becomes imperative that attention is directed toward the second approach that seeks to prevent impairment from becoming disability . disability among children with cp is often the product of the interaction of health conditions with contextual environmental and personal factors . utilization of rehabilitation services is one such known factor that considerably influences the disability status of an individual . utilization of health and rehabilitation services in india is a complex phenomenon influenced by several factors . physiotherapy plays a central role in the management of cp and is the most popular therapeutic intervention for cp . however , limited literature is available on the use of physiotherapy service among cp children . the factors that influence the utilization of physiotherapy service among children with cp have largely remained unexplored . describing patterns of physical therapy use and identifying factors that contribute to variation in utilization investigators could not locate any study that described the patterns of physical therapy use and identifies the factors contributing to variation in utilization among children of cp in india . keeping this in mind , the present study was planned to explore the utilization of physiotherapy services among children with cp in jalandhar district of punjab . the specific research objectives were to describe the pattern of physiotherapy use and explore the factors affecting utilization of physiotherapy services . a cross - sectional survey was conducted on family members of 248 children with cp ( male = 159 female = 89 ) in the jalandhar district of punjab from june 2009 to march 2012 as a part of a larger project designed to study the epidemiology of disability in children with cp and approved by bpsar of punjabi university patiala . during 20082011 , a database of children with cp of age group 313 years of jalandhar district of punjab was prepared by the investigator . using records of some hospitals and physiotherapy centers , identification camp organized by a physiotherapy teaching institution and registry of sarva shiksha abhiyan as base investigator went to all the 10 blocks of jalandhar district to register the children with cp for study . several children were subsequently added to the initial list when brought to the notice of investigator by the local community members and cross referral from the parents of cp children . each identified child was physically examined by the investigator and was retained in the database only if the following criteria of inclusion were satisfied : ( a ) diagnosed cases of cp and/or , ( b ) the presence of motor delay and motor disorder , abnormal muscle tone , abnormal posture or asymmetry , and persistence of primitive reflexes ( c ) have completed the age of 313 years on march 2008 , and ( d ) resident of jalandhar district . scheduled interview of parents of all the identified children was conducted by a physiotherapist ( rs ) having 11 years of experience of working with cp . the schedule of interview consisted of semi - structured questions focusing on demographic information , constraints of resources , expectations , beliefs , awareness , and service utilization . the initial draft of questionnaire prepared after a review of literature and in - depth interview of some family members of children was validated for content by a panel of experts consisting of physiotherapist , pediatrician , and social worker . the socioeconomic ( se ) status of the family was determined by the se scale developed by aggarwal et al . , this scale is a modification of kuppuswamy scale suitable for both rural and urban population . it provides an se score by totaling the scores assigned to the variables of education , occupation , and monthly income of the head of family in seven - point scales . on the basis of se score , the family can be grouped into six se classes , namely , upper high , high , upper middle class , lower middle class , poor , and very poor . for the purpose of analysis in this study , the two categories ( upper high and high ) of high , upper and lower of middle and very poor and poor of lower were clubbed to get three categories of se class . education status of parents was classified into three categories educated education + 2 and above , less educated = schooling up to 10 class , and illiterate unable to read or write on the basis of lifetime exposure to physiotherapy data was categorized into two groups of not exposed and exposed group . children of nonexposed group had never received any physiotherapy in their lifetime , whereas the exposed group included those who had received physiotherapy for variable duration in their lifetime . cross tabulation with chi - square was used to examine the association of each variable with physiotherapy exposure . a series of univariate regression analyses was conducted on significant factors with each potential variable as independent variable and exposure to physiotherapy as dependent variable to examine the independent effect of variable on physiotherapy use . to investigate how the combination of variables predicted the exposure to physiotherapy , independent variables , which were significant at the 0.05 level in the univariate analyses , were assessed together as a single block in multivariate regression models with entry probability of 0.05 and removal of 0.10 . odds ratio ( or ) and 95% confidence interval were calculated for univariate and multivariate models . on the basis of lifetime exposure to physiotherapy data was categorized into two groups of not exposed and exposed group . children of nonexposed group had never received any physiotherapy in their lifetime , whereas the exposed group included those who had received physiotherapy for variable duration in their lifetime . cross tabulation with chi - square was used to examine the association of each variable with physiotherapy exposure . a series of univariate regression analyses was conducted on significant factors with each potential variable as independent variable and exposure to physiotherapy as dependent variable to examine the independent effect of variable on physiotherapy use . to investigate how the combination of variables predicted the exposure to physiotherapy , independent variables , which were significant at the 0.05 level in the univariate analyses , were assessed together as a single block in multivariate regression models with entry probability of 0.05 and removal of 0.10 . odds ratio ( or ) and 95% confidence interval were calculated for univariate and multivariate models . in the cohort of 248 children with cp , 44.4% children have not received any physiotherapy in their lifetime [ figure 1 ] . in exposed group [ figure 2 ] , majority received physiotherapy at physiotherapy teaching institutions ( 31.15% ) , followed by government hospitals ( 23.91% ) , private hospitals / clinics ( 23.18% ) , charitable dispensaries ( 7.97% ) , and at home ( 13.76% ) . table 1 presents the distributions of duration of physiotherapy according to se status of family . 61.53% children of higher se class received physiotherapy for > 3 years ; the corresponding figures for the middle se class , and lower se class was 21.21% and 13.15% , respectively . physiotherapy service utilization of the children with cerebral palsy in jalandhar district distribution of place of physiotherapy of children with cerebral palsy of jalandhar district distribution of duration of physiotherapy received according to socioeconomic status exposure to physiotherapy was significantly associated with age , age of diagnosis locality , se status , and educational status . the nonexposure to physiotherapy showed a positive relationship with age of child ( or = 1.13 ) . finance constraint ( or = 2.27 ) , personal constraint ( or = 2.54 ) , transportation constraint ( or = 3.01 ) , lack of advice for rehabilitation ( or = 2.36 ) , lack of awareness about condition ( or = 11.94 ) , and lack of awareness about rehabilitation services ( or = 2.88 ) increases the likelihood of nonexposure to physiotherapy . expectation of normalcy ( or = 0.29 ) reduced the odds of nonexposure [ table 2 ] . bivariate regression analysis of demographic and environmental factors associated with nonexposure to physiotherapy entering all the significant factors in multivariate analysis created problems of multicollinearity . therefore , a correlation analysis of variables was conducted and the variables having r value of or more 0.8 with other variables were excluded from the analysis . the financial constraint was strongly correlated related with se status ( r < 0.84 ) with 96% and 100% of families of lower se class experiencing financial constraint . lack of awareness about rehab requirement of child was strongly correlated related with lack of advice for rehab ( r = 0.93 ) . close to 99% of parents aware about rehab requirement had received advice for rehabilitation from medical doctor . thus , advice for rehab was treated as proxy for awareness about rehab services , and se status was treated as independent variable serving as proxy for financial constraints . multivariate model [ table 3 ] identified three main predictor variables of nonexposure to physiotherapy ignorance about condition ( or = 7.3 ) , expectation of normalcy ( or = 0.43 ) , and age ( or = 1.10 ) . the proportion of variance explained by the model was 22%27% ( pseudo r - square cox and snell = 0.22 , nagelkerke = 0.27 ) . in the cohort of 248 children with cp , 44.4% children have not received any physiotherapy in their lifetime [ figure 1 ] . in exposed group [ figure 2 ] , majority received physiotherapy at physiotherapy teaching institutions ( 31.15% ) , followed by government hospitals ( 23.91% ) , private hospitals / clinics ( 23.18% ) , charitable dispensaries ( 7.97% ) , and at home ( 13.76% ) . table 1 presents the distributions of duration of physiotherapy according to se status of family . 61.53% children of higher se class received physiotherapy for > 3 years ; the corresponding figures for the middle se class , and lower se class was 21.21% and 13.15% , respectively . physiotherapy service utilization of the children with cerebral palsy in jalandhar district distribution of place of physiotherapy of children with cerebral palsy of jalandhar district distribution of duration of physiotherapy received according to socioeconomic status exposure to physiotherapy was significantly associated with age , age of diagnosis locality , se status , and educational status . the nonexposure to physiotherapy showed a positive relationship with age of child ( or = 1.13 ) . finance constraint ( or = 2.27 ) , personal constraint ( or = 2.54 ) , transportation constraint ( or = 3.01 ) , lack of advice for rehabilitation ( or = 2.36 ) , lack of awareness about condition ( or = 11.94 ) , and lack of awareness about rehabilitation services ( or = 2.88 ) increases the likelihood of nonexposure to physiotherapy . expectation of normalcy ( or = 0.29 ) reduced the odds of nonexposure [ table 2 ] . bivariate regression analysis of demographic and environmental factors associated with nonexposure to physiotherapy entering all the significant factors in multivariate analysis created problems of multicollinearity . therefore , a correlation analysis of variables was conducted and the variables having r value of or more 0.8 with other variables were excluded from the analysis . the financial constraint was strongly correlated related with se status ( r < 0.84 ) with 96% and 100% of families of lower se class experiencing financial constraint . lack of awareness about rehab requirement of child was strongly correlated related with lack of advice for rehab ( r = 0.93 ) . close to 99% of parents aware about rehab requirement had received advice for rehabilitation from medical doctor . thus , advice for rehab was treated as proxy for awareness about rehab services , and se status was treated as independent variable serving as proxy for financial constraints . multivariate model [ table 3 ] identified three main predictor variables of nonexposure to physiotherapy ignorance about condition ( or = 7.3 ) , expectation of normalcy ( or = 0.43 ) , and age ( or = 1.10 ) . the proportion of variance explained by the model was 22%27% ( pseudo r - square cox and snell = 0.22 , nagelkerke = 0.27 ) . despite the acknowledged need of physiotherapy in management of cp , there exist limited studies that describe the use of physiotherapy services among the cp children . there is little research on the use of health and rehabilitation services by pwd in india . this study provides primary data for with regard to physiotherapy service utilization among children with cp in jalandhar and might be the first indian study that attempts to gain an insight of the factors associated with nonutilization of physiotherapy services . 55% of children had received some kind of physiotherapy and only 14.91% had received physiotherapy for > 3 years . these findings are in gross variance with those reported from the western countries . in ireland , service use among families of cp children was very high with 96% receiving physiotherapy at least twice a week for 30 min . in the canadian study of majnemer et al . , physiotherapy and occupational therapy were most common services provided predominantly in the school setting , and 84.6% children were receiving at least one rehabilitation service . in hong kong approximately 38% of children with cp attended a mainstream school , and 61% received outpatient therapy support . there are major differences in the physiotherapy service delivery structure between developed countries and india . unlike developed countries where therapy interventions are provided to children on statutory basis in mainstream and special schools , indian cp children do not have such benefit . physiotherapy is just one of the many treatment options as besides allopathic treatment ( modern medicine and surgery ) , homeopathy , ayurvedic treatment , massage , and even magical remedies are often sought by the patient of chronic disabling conditions . this makes utilization of health , and rehabilitation services a complex phenomenon which is influenced by a plethora of personal , demographic , and environmental factors . availability and affordability of service remain the key major determinants of use of rehabilitation services in india , as the facility for comprehensive multidisciplinary rehabilitation does not exist in most of the places . in the present study also , physiotherapy was the main rehab service available to children with cp . speech therapy was provided to just 4% , and none of the children had received occupational therapy . nonavailability of treatment facility in the vicinity was a constraint expressed by 56.9% among them 54.6% could not receive physiotherapy . constraint of therapy facility in the vicinity was also felt by 46.37% parents whose children received physiotherapy . providing physiotherapy service to cp child is a costly affair . besides the cost of service , several indirect expenses such as travel to treatment center , loss of working hours leading to loss of wedges of parents are also involved as child has to be brought regularly to the physiotherapy center for relatively long period . in developed countries also where health insurance coverage for meeting the cost of treatment and rehab is available for a majority of the population . parents find it difficult to provide therapy services if they are not covered under insurance plan . reported that in usa children with disability without insurance coverage were significantly more likely to have a reported unmet need for therapy services . just 10% of the indian population is covered by any form of social or voluntary health insurance . and the government of india does offer income tax rebate on the expenses rendered to a child with disability . however , these benefits are applicable only to the taxpayers which constitute a very small proportion of indian population . for the majority of indian population , there is no financial support for meeting the expenses of rehabilitation of disabled members of family . physiotherapy teaching institution and government hospitals provide relatively less costly services , but there is a long waiting time in these centers . transportation of child to physiotherapy center also involves considerable expanses and for a poor family , it becomes a major barrier even when therapy services are provide for free or at nominal cost . most of the families ( 96% ) belonged to lower se class admitted that they were not financially sound to get treatment for their child . this variation in cost of service and transportation difficulty coupled with financial capability to bear the cost is reflected in physiotherapy utilization pattern of se classes where a wider se divide is evident . in comparison to lower se class , likelihood of exposure to physiotherapy was much higher for high ( 5 times ) and middle se class ( 3 times ) . the majority of higher classes received physiotherapy from private hospitals / clinics , whereas majority of the lower class received treatment from government hospital . the duration of physiotherapy also showed remarkable variation among social classes indicating an increase in duration of physiotherapy with the improvement of se status . majority of children of higher class received physiotherapy for > 3 years ; the corresponding figures for the middle class , and lower class were 21.21% and 13.15% , respectively . poverty and resource constraints are the known factors for nonaccess of health and rehabilitation services in developing countries . financial constraint and difficulty in transportation as the barriers for rehabilitation of neurological disability has been reported from other part of india as well . . however , financial and resource constraint lost their significance in multivariable model where ignorance about condition and expectation of normalcy emerged as the main predicator variable . nonexposure to physiotherapy showed positive relation with ignorance and negative relation with expectation of normalcy . the children whose parents were ignorant about the nature of cp were 7 times more likely not to receive physiotherapy . on the other hand , expectation that my child will be completely normal enhanced the chance of physiotherapy exposure . ignorance was also reported as a major reason for nonutilization of rehab services in other parts of india . kumar and gupta reported ignorance as the major barrier to neurological rehabilitation in rural areas of uttar pradesh . padhyegurjar and padhyegurjar reported that in urban slum of mumbai 87.1% attribute the lack of knowledge of rehabilitative services as the reason for not taking treatment . study of srivastava et al . conducted in mau district of uttar pradesh reported unawareness as the main reason for not availing rehabilitation services . it was observed that if parents were aware about the prognosis and therapy requirement ; then , irrespective of se status they managed against all odds to ensure that child receives some service . on the other hand , close to 43.54% parent in the present study did not believe that child condition would improve with physical exercises and most of them had not utilized physiotherapy services . knowledge , education , and information strongly influence beliefs and perceptions of illness , health - seeking behavior and compliance with therapy , and the factors that affect the knowledge can significantly alter the service utilization pattern . educational levels of parents and advice for rehabilitation of child by treating medical doctor were such factors emerged in univariate analysis . these two factors were significantly associated with awareness about condition and rehab requirement of child and exerted greater influence on the utilization of physiotherapy service . the proportion of children exposed to physiotherapy was much higher among educated families , and they also received physiotherapy for longer duration . children not advised for rehab by medical doctors were 2.63 times more likely not to receive physiotherapy . this observation provides support to the notion that poor referral system that does not fully utilize the expertise available within the system is one of the main factors that influence the access to rehab services in india . it is imperative that extra efforts are made to sensitize the medical community about the need of timely referral for physiotherapy to cp children . the movement and posture dysfunction in cp are permanent and even with best rehabilitation efforts functional and physical recovery in cp is rarely complete . this truth is often emotionally not acceptable to parents , and they tend to lure toward all sorts of treatment options . the emergence of expectation of normalcy as one of the main drivers of physiotherapy use in this study underscores this point . a closer examination data revealed that this unrealistic expectation was associated with higher utilization of other health services as well . although nonsignificant in final model , age of diagnosis and chronological age of child were found significantly associated with exposure to physiotherapy in univariate models . children diagnosed at younger age had relatively better chance of exposure to physiotherapy , and this supports the value of early identification . this positive relationship of age of child with nonexposure to physiotherapy should be interpreted with caution . this may reflect changing pattern of physiotherapy use attributed to the availability of physiotherapy services which has increased in the recent decade in the district . in the last 10 years , number of physiotherapy teaching institution offering bachelor and master degree education in physiotherapy were opened up in jalandhar district which contributed to increase in the numbers of physiotherapy service providers in the district as well as improving the community awareness about physiotherapy . sarva shiksha abhiyana a flagship program of the government of india also increased the coverage of physiotherapy to the disadvantaged group of society . however it does not explain why even with the availability of service older children were not exposed to physiotherapy ? we are not sure if this could be an indication of loss of hope of normalcy with the growing age of child . this strongly suggests the existence of other factors contributing to nonexposure which were not captured in this study . for example , psychological and religious beliefs , interpersonal relationships within family , attitude of service providers , etc . the need of improving awareness among the parents of children with cp about the condition and services required for improving the quality of life is apparent . lots of information on the rehabilitation of cp children is available , nowadays , on the internet but most of this material is in english . there is scarcity of literature on management of cp in hindi and punjabi and other local languages . for person familiar with english , a closer examination of data reveals that just 4.9% of educated families who were not provided with advice for rehab were aware about the condition , but the less educated and illiterate remained ignorant when not provided with advice for rehabilitation . the only source of information for them was the health professional and community . in view of the fact that only 17% children were provided advice for rehabilitation making available such efforts would contribute to improve family and community awareness and facilitate physiotherapy service utilization . a mechanism should be evolved to ameliorate the financial and transportation constraints to make physiotherapy affordable to every child with cp irrespective of se status and locality . data for this study were based on the self - reported information of respondents and validation of the provided information from other objective sources was not possible which may be construed as the limitations of this study . this study identified a constellation of complex and interrelated relationship between factors associated with nonexposure to physiotherapy . awareness and expectation of normalcy were the main drivers for the use of physiotherapy among children with cp in jalandhar . it is imperative that efforts are made to educate the parents about the nature of condition and the lifelong rehab requirements of the child . this would create the demand for physiotherapy services and facilitate the establishment of proper mechanism of service delivery .

background : physiotherapy plays a central role in the management of children with cerebral palsy ( cp ) ; however , literature describing the use of physiotherapy service and the factors affecting utilization of physiotherapy service for this group of children in the indian context remain unexplored.aims and objectives : to describe the utilization of physiotherapy services and explore the factors affecting utilization of physiotherapy services among children with cp of jalandhar district of punjab.methodology:during june 2009 to march 2012 interview of family members of 248 children with cp ( male = 159 ; female = 89 ) was conducted using a schedule focusing on demography , constraints of resources , expectations , beliefs , awareness , and service utilization . cross tabulation with chi - square , univariate , and multivariate logistic regression analysis were the tools of statistical analysis.results:44.4% children had not received any physiotherapy in their life time . in univariate analysis exposure to physiotherapy was found significantly associated with age of diagnosis ( odds ratio [ or ] = 2.47 ) , finance constraint ( or = 2.27 ) , personal constraint ( or = 2.54 ) , transportation constraint ( or = 3.01 ) , lack of advice for rehabilitation ( or = 2.36 ) , ignorance about condition ( or = 11.94 ) , and rehabilitation services ( or = 2.88 ) . multivariate model ( 2 = 57.16 , df = 15 , p < 0.001 , pseudo r2 cox and snell = 0.22 , nagelkerke = 0.27 ) identified two main predictor variables of nonexposure to physiotherapy - ignorance about condition ( or = 7.3 ) and expectation of normalcy ( or = 0.43).conclusion : the main drivers for the use of physiotherapy among children with cp in jalandhar district of punjab were awareness about the condition of cp and expectation of normalcy which demonstrated a complex relationship with sociodemographic factors .

I M Statistical analysis R Physiotherapy service utilization Factor influencing exposure to physiotherapy D C Financial support and sponsorship Conflicts of interest

cerebral palsy ( cp ) describes a group of permanent disorders of movement and posture that are attributed to nonprogressive disturbances in the developing brain and is often accompanied by disturbances of sensation , cognition , communication , perception , behavior , or seizures . the prevention of childhood disability is one of the main focuses of the national policy of disability of india , which can be approached either by preventing the impairment or by preventing the impairment from becoming a disability . advances in neonatal management and obstetric care have not shown a decline in the incidence of cp . on the contrary , many believe that with a decline in infant mortality rate , there has actually been an increase in the incidence and severity of cp . disability among children with cp is often the product of the interaction of health conditions with contextual environmental and personal factors . utilization of rehabilitation services is one such known factor that considerably influences the disability status of an individual . utilization of health and rehabilitation services in india is a complex phenomenon influenced by several factors . physiotherapy plays a central role in the management of cp and is the most popular therapeutic intervention for cp . however , limited literature is available on the use of physiotherapy service among cp children . the factors that influence the utilization of physiotherapy service among children with cp have largely remained unexplored . describing patterns of physical therapy use and identifying factors that contribute to variation in utilization investigators could not locate any study that described the patterns of physical therapy use and identifies the factors contributing to variation in utilization among children of cp in india . keeping this in mind , the present study was planned to explore the utilization of physiotherapy services among children with cp in jalandhar district of punjab . the specific research objectives were to describe the pattern of physiotherapy use and explore the factors affecting utilization of physiotherapy services . a cross - sectional survey was conducted on family members of 248 children with cp ( male = 159 female = 89 ) in the jalandhar district of punjab from june 2009 to march 2012 as a part of a larger project designed to study the epidemiology of disability in children with cp and approved by bpsar of punjabi university patiala . during 20082011 , a database of children with cp of age group 313 years of jalandhar district of punjab was prepared by the investigator . using records of some hospitals and physiotherapy centers , identification camp organized by a physiotherapy teaching institution and registry of sarva shiksha abhiyan as base investigator went to all the 10 blocks of jalandhar district to register the children with cp for study . several children were subsequently added to the initial list when brought to the notice of investigator by the local community members and cross referral from the parents of cp children . each identified child was physically examined by the investigator and was retained in the database only if the following criteria of inclusion were satisfied : ( a ) diagnosed cases of cp and/or , ( b ) the presence of motor delay and motor disorder , abnormal muscle tone , abnormal posture or asymmetry , and persistence of primitive reflexes ( c ) have completed the age of 313 years on march 2008 , and ( d ) resident of jalandhar district . scheduled interview of parents of all the identified children was conducted by a physiotherapist ( rs ) having 11 years of experience of working with cp . the schedule of interview consisted of semi - structured questions focusing on demographic information , constraints of resources , expectations , beliefs , awareness , and service utilization . the initial draft of questionnaire prepared after a review of literature and in - depth interview of some family members of children was validated for content by a panel of experts consisting of physiotherapist , pediatrician , and social worker . it provides an se score by totaling the scores assigned to the variables of education , occupation , and monthly income of the head of family in seven - point scales . on the basis of se score , the family can be grouped into six se classes , namely , upper high , high , upper middle class , lower middle class , poor , and very poor . for the purpose of analysis in this study , the two categories ( upper high and high ) of high , upper and lower of middle and very poor and poor of lower were clubbed to get three categories of se class . education status of parents was classified into three categories educated education + 2 and above , less educated = schooling up to 10 class , and illiterate unable to read or write on the basis of lifetime exposure to physiotherapy data was categorized into two groups of not exposed and exposed group . children of nonexposed group had never received any physiotherapy in their lifetime , whereas the exposed group included those who had received physiotherapy for variable duration in their lifetime . cross tabulation with chi - square was used to examine the association of each variable with physiotherapy exposure . a series of univariate regression analyses was conducted on significant factors with each potential variable as independent variable and exposure to physiotherapy as dependent variable to examine the independent effect of variable on physiotherapy use . to investigate how the combination of variables predicted the exposure to physiotherapy , independent variables , which were significant at the 0.05 level in the univariate analyses , were assessed together as a single block in multivariate regression models with entry probability of 0.05 and removal of 0.10 . odds ratio ( or ) and 95% confidence interval were calculated for univariate and multivariate models . on the basis of lifetime exposure to physiotherapy data was categorized into two groups of not exposed and exposed group . children of nonexposed group had never received any physiotherapy in their lifetime , whereas the exposed group included those who had received physiotherapy for variable duration in their lifetime . cross tabulation with chi - square was used to examine the association of each variable with physiotherapy exposure . a series of univariate regression analyses was conducted on significant factors with each potential variable as independent variable and exposure to physiotherapy as dependent variable to examine the independent effect of variable on physiotherapy use . to investigate how the combination of variables predicted the exposure to physiotherapy , independent variables , which were significant at the 0.05 level in the univariate analyses , were assessed together as a single block in multivariate regression models with entry probability of 0.05 and removal of 0.10 . odds ratio ( or ) and 95% confidence interval were calculated for univariate and multivariate models . in the cohort of 248 children with cp , 44.4% children have not received any physiotherapy in their lifetime [ figure 1 ] . in exposed group [ figure 2 ] , majority received physiotherapy at physiotherapy teaching institutions ( 31.15% ) , followed by government hospitals ( 23.91% ) , private hospitals / clinics ( 23.18% ) , charitable dispensaries ( 7.97% ) , and at home ( 13.76% ) . table 1 presents the distributions of duration of physiotherapy according to se status of family . 61.53% children of higher se class received physiotherapy for > 3 years ; the corresponding figures for the middle se class , and lower se class was 21.21% and 13.15% , respectively . physiotherapy service utilization of the children with cerebral palsy in jalandhar district distribution of place of physiotherapy of children with cerebral palsy of jalandhar district distribution of duration of physiotherapy received according to socioeconomic status exposure to physiotherapy was significantly associated with age , age of diagnosis locality , se status , and educational status . the nonexposure to physiotherapy showed a positive relationship with age of child ( or = 1.13 ) . finance constraint ( or = 2.27 ) , personal constraint ( or = 2.54 ) , transportation constraint ( or = 3.01 ) , lack of advice for rehabilitation ( or = 2.36 ) , lack of awareness about condition ( or = 11.94 ) , and lack of awareness about rehabilitation services ( or = 2.88 ) increases the likelihood of nonexposure to physiotherapy . expectation of normalcy ( or = 0.29 ) reduced the odds of nonexposure [ table 2 ] . bivariate regression analysis of demographic and environmental factors associated with nonexposure to physiotherapy entering all the significant factors in multivariate analysis created problems of multicollinearity . therefore , a correlation analysis of variables was conducted and the variables having r value of or more 0.8 with other variables were excluded from the analysis . lack of awareness about rehab requirement of child was strongly correlated related with lack of advice for rehab ( r = 0.93 ) . close to 99% of parents aware about rehab requirement had received advice for rehabilitation from medical doctor . thus , advice for rehab was treated as proxy for awareness about rehab services , and se status was treated as independent variable serving as proxy for financial constraints . multivariate model [ table 3 ] identified three main predictor variables of nonexposure to physiotherapy ignorance about condition ( or = 7.3 ) , expectation of normalcy ( or = 0.43 ) , and age ( or = 1.10 ) . the proportion of variance explained by the model was 22%27% ( pseudo r - square cox and snell = 0.22 , nagelkerke = 0.27 ) . in the cohort of 248 children with cp , 44.4% children have not received any physiotherapy in their lifetime [ figure 1 ] . in exposed group [ figure 2 ] , majority received physiotherapy at physiotherapy teaching institutions ( 31.15% ) , followed by government hospitals ( 23.91% ) , private hospitals / clinics ( 23.18% ) , charitable dispensaries ( 7.97% ) , and at home ( 13.76% ) . table 1 presents the distributions of duration of physiotherapy according to se status of family . 61.53% children of higher se class received physiotherapy for > 3 years ; the corresponding figures for the middle se class , and lower se class was 21.21% and 13.15% , respectively . physiotherapy service utilization of the children with cerebral palsy in jalandhar district distribution of place of physiotherapy of children with cerebral palsy of jalandhar district distribution of duration of physiotherapy received according to socioeconomic status exposure to physiotherapy was significantly associated with age , age of diagnosis locality , se status , and educational status . the nonexposure to physiotherapy showed a positive relationship with age of child ( or = 1.13 ) . finance constraint ( or = 2.27 ) , personal constraint ( or = 2.54 ) , transportation constraint ( or = 3.01 ) , lack of advice for rehabilitation ( or = 2.36 ) , lack of awareness about condition ( or = 11.94 ) , and lack of awareness about rehabilitation services ( or = 2.88 ) increases the likelihood of nonexposure to physiotherapy . expectation of normalcy ( or = 0.29 ) reduced the odds of nonexposure [ table 2 ] . bivariate regression analysis of demographic and environmental factors associated with nonexposure to physiotherapy entering all the significant factors in multivariate analysis created problems of multicollinearity . therefore , a correlation analysis of variables was conducted and the variables having r value of or more 0.8 with other variables were excluded from the analysis . the financial constraint was strongly correlated related with se status ( r < 0.84 ) with 96% and 100% of families of lower se class experiencing financial constraint . lack of awareness about rehab requirement of child was strongly correlated related with lack of advice for rehab ( r = 0.93 ) . close to 99% of parents aware about rehab requirement had received advice for rehabilitation from medical doctor . thus , advice for rehab was treated as proxy for awareness about rehab services , and se status was treated as independent variable serving as proxy for financial constraints . multivariate model [ table 3 ] identified three main predictor variables of nonexposure to physiotherapy ignorance about condition ( or = 7.3 ) , expectation of normalcy ( or = 0.43 ) , and age ( or = 1.10 ) . the proportion of variance explained by the model was 22%27% ( pseudo r - square cox and snell = 0.22 , nagelkerke = 0.27 ) . despite the acknowledged need of physiotherapy in management of cp , there exist limited studies that describe the use of physiotherapy services among the cp children . there is little research on the use of health and rehabilitation services by pwd in india . this study provides primary data for with regard to physiotherapy service utilization among children with cp in jalandhar and might be the first indian study that attempts to gain an insight of the factors associated with nonutilization of physiotherapy services . 55% of children had received some kind of physiotherapy and only 14.91% had received physiotherapy for > 3 years . , physiotherapy and occupational therapy were most common services provided predominantly in the school setting , and 84.6% children were receiving at least one rehabilitation service . in hong kong approximately 38% of children with cp attended a mainstream school , and 61% received outpatient therapy support . there are major differences in the physiotherapy service delivery structure between developed countries and india . physiotherapy is just one of the many treatment options as besides allopathic treatment ( modern medicine and surgery ) , homeopathy , ayurvedic treatment , massage , and even magical remedies are often sought by the patient of chronic disabling conditions . this makes utilization of health , and rehabilitation services a complex phenomenon which is influenced by a plethora of personal , demographic , and environmental factors . availability and affordability of service remain the key major determinants of use of rehabilitation services in india , as the facility for comprehensive multidisciplinary rehabilitation does not exist in most of the places . in the present study also , physiotherapy was the main rehab service available to children with cp . speech therapy was provided to just 4% , and none of the children had received occupational therapy . however , these benefits are applicable only to the taxpayers which constitute a very small proportion of indian population . for the majority of indian population , there is no financial support for meeting the expenses of rehabilitation of disabled members of family . physiotherapy teaching institution and government hospitals provide relatively less costly services , but there is a long waiting time in these centers . in comparison to lower se class , likelihood of exposure to physiotherapy was much higher for high ( 5 times ) and middle se class ( 3 times ) . the duration of physiotherapy also showed remarkable variation among social classes indicating an increase in duration of physiotherapy with the improvement of se status . majority of children of higher class received physiotherapy for > 3 years ; the corresponding figures for the middle class , and lower class were 21.21% and 13.15% , respectively . poverty and resource constraints are the known factors for nonaccess of health and rehabilitation services in developing countries . financial constraint and difficulty in transportation as the barriers for rehabilitation of neurological disability has been reported from other part of india as well . however , financial and resource constraint lost their significance in multivariable model where ignorance about condition and expectation of normalcy emerged as the main predicator variable . nonexposure to physiotherapy showed positive relation with ignorance and negative relation with expectation of normalcy . the children whose parents were ignorant about the nature of cp were 7 times more likely not to receive physiotherapy . on the other hand , expectation that my child will be completely normal enhanced the chance of physiotherapy exposure . padhyegurjar and padhyegurjar reported that in urban slum of mumbai 87.1% attribute the lack of knowledge of rehabilitative services as the reason for not taking treatment . conducted in mau district of uttar pradesh reported unawareness as the main reason for not availing rehabilitation services . on the other hand , close to 43.54% parent in the present study did not believe that child condition would improve with physical exercises and most of them had not utilized physiotherapy services . knowledge , education , and information strongly influence beliefs and perceptions of illness , health - seeking behavior and compliance with therapy , and the factors that affect the knowledge can significantly alter the service utilization pattern . educational levels of parents and advice for rehabilitation of child by treating medical doctor were such factors emerged in univariate analysis . these two factors were significantly associated with awareness about condition and rehab requirement of child and exerted greater influence on the utilization of physiotherapy service . the proportion of children exposed to physiotherapy was much higher among educated families , and they also received physiotherapy for longer duration . this observation provides support to the notion that poor referral system that does not fully utilize the expertise available within the system is one of the main factors that influence the access to rehab services in india . it is imperative that extra efforts are made to sensitize the medical community about the need of timely referral for physiotherapy to cp children . this truth is often emotionally not acceptable to parents , and they tend to lure toward all sorts of treatment options . the emergence of expectation of normalcy as one of the main drivers of physiotherapy use in this study underscores this point . a closer examination data revealed that this unrealistic expectation was associated with higher utilization of other health services as well . although nonsignificant in final model , age of diagnosis and chronological age of child were found significantly associated with exposure to physiotherapy in univariate models . children diagnosed at younger age had relatively better chance of exposure to physiotherapy , and this supports the value of early identification . this positive relationship of age of child with nonexposure to physiotherapy should be interpreted with caution . this may reflect changing pattern of physiotherapy use attributed to the availability of physiotherapy services which has increased in the recent decade in the district . in the last 10 years , number of physiotherapy teaching institution offering bachelor and master degree education in physiotherapy were opened up in jalandhar district which contributed to increase in the numbers of physiotherapy service providers in the district as well as improving the community awareness about physiotherapy . sarva shiksha abhiyana a flagship program of the government of india also increased the coverage of physiotherapy to the disadvantaged group of society . however it does not explain why even with the availability of service older children were not exposed to physiotherapy ? we are not sure if this could be an indication of loss of hope of normalcy with the growing age of child . for example , psychological and religious beliefs , interpersonal relationships within family , attitude of service providers , etc . the need of improving awareness among the parents of children with cp about the condition and services required for improving the quality of life is apparent . lots of information on the rehabilitation of cp children is available , nowadays , on the internet but most of this material is in english . there is scarcity of literature on management of cp in hindi and punjabi and other local languages . for person familiar with english , a closer examination of data reveals that just 4.9% of educated families who were not provided with advice for rehab were aware about the condition , but the less educated and illiterate remained ignorant when not provided with advice for rehabilitation . in view of the fact that only 17% children were provided advice for rehabilitation making available such efforts would contribute to improve family and community awareness and facilitate physiotherapy service utilization . a mechanism should be evolved to ameliorate the financial and transportation constraints to make physiotherapy affordable to every child with cp irrespective of se status and locality . data for this study were based on the self - reported information of respondents and validation of the provided information from other objective sources was not possible which may be construed as the limitations of this study . this study identified a constellation of complex and interrelated relationship between factors associated with nonexposure to physiotherapy . awareness and expectation of normalcy were the main drivers for the use of physiotherapy among children with cp in jalandhar . it is imperative that efforts are made to educate the parents about the nature of condition and the lifelong rehab requirements of the child . this would create the demand for physiotherapy services and facilitate the establishment of proper mechanism of service delivery .

positive - strand rna viruses replicate in large complexes that are associated with host intracellular membranes ( salonen et al . , 2005 ; sanfacon , 2005 ; miller and krijnse - locker , 2008 ; den boon and ahlquist , 2010 ; laliberte and sanfacon , 2010 ; nagy and pogany , 2012 ) . some viruses require host membrane proteins to target their replication proteins to the membranes ( yamanaka et al . , 2000 ) . however , many viruses encode proteins that interact with membranes directly and modify their intrinsic structure . these proteins have membrane association domains and contain protein protein and/or protein rna interaction domains that allow them to recruit the viral rna , other viral replication proteins , or host factors to the membranes . well - characterized plant virus membrane proteins include the tombusvirus 3336 kda proteins , bromovirus 1a protein , potyvirus 6k protein , and tymovirus 140 kda protein ( schaad et al . , 1997 ; den boon et al . , 2001 ; weber - lotfi et al . , 2002 ; prodhomme et al . , 2003 ; turner et al . , the family secoviridae ( order picornavirales ) includes the genera comovirus , fabavirus , nepovirus , sequivirus , waikavirus , cheravirus , sadwavirus , and torradovirus ( sanfacon et al . , 2011 ) . the best characterized members of the family are cowpea mosaic virus ( cpmv , comovirus ) , grapevine fanleaf virus ( gflv , nepovirus ) , and tomato ringspot virus ( torsv , nepovirus ; pouwels et al . , 2002a ; sanfacon et al . , 2006 these viruses use a polyprotein strategy to express their proteins and have a replication block consisting of a nucleotide - binding protein ( ntb ) , a genome - linked protein ( vpg ) , a proteinase ( pro ) , and an rna - dependent rna polymerase ( pol ; figure 1c ) . although they share these properties with picornaviruses ( including the well - characterized poliovirus ) , nepo- and comoviruses differ in that they have bipartite genomes . the rna1-encoded polyprotein contains all protein domains necessary for replication and rna1 can replicate independently of rna2 ( vos et al . ( a ) electron micrograph showing the proliferation of single - membrane vesicles in torsv - infected nicotiana clevelandii plants . ( 1 ) in cells infected with poliovirus or coxsackie b3 virus , viral integral membrane proteins ( red ovals ) induce positive curvature of the membrane allowing the budding of tubular structures . other viral replication proteins ( e.g. , polymerase , green ovals ) interact with the viral membrane proteins . host factors and viral rna ( not shown ) associate with the replication complex by protein protein and protein rna interactions . single - membrane vesicles may bud out and form double - membrane vesicles after the internal collapse of the single - membrane vesicle and subsequent membrane fusion to allow its circularization . late in infection , double - membrane vesicles are predominant in picornavirus - infected cells . ( 2 ) in cells infected with many plant and animal viruses , induction of negative membrane curvature results in membrane invagination and formation of spherules in the lumen of the membrane . in plant , spherules have been observed in association with membranes from the er ( brome mosaic virus ) , chloroplast ( turnip yellow mosaic virus ) , peroxisome ( tomato bushy stunt virus ) and mitochondria ( carnation italian ringspot virus ) . viral integral membrane proteins ( red ovals ) line the interior of the spherule . the viral polymerase ( green ovals ) as well as other viral proteins , host factors and the viral rna ( not shown ) are enclosed in the spherule . release of the vesicle in the lumen of the membrane may be followed by budding of a double - membrane vesicle into the cytoplasm . this model has been discussed in recent reviews ( den boon and ahlquist , 2010 ; laliberte and sanfacon , 2010 ; nagy and pogany , 2012 ) . ( c ) organization of replication protein domains in the polyproteins of cpmv , torsv , and poliovirus . for poliovirus , the polyprotein encoded by the single genomic rna is shown , although the p1 region ( containing the structural proteins ) is truncated as indicated by the diagonal bars . conserved motifs are : rna - dependent rna polymerase ( pol , green ovals ) , protease ( pro , orange diamond ) , nucleotide - binding protein ( ntb , red oval ) , co - pro and x2 ( purple square ) . horizontal bars under each polyprotein represent integral membrane proteins that have been detected in virus - infected cells . although likely , its presence in infected cells could not be confirmed due to the lack of antibodies . ( d ) the regions of the polyprotein containing the putative membrane anchors are shown for each virus . predicted membrane - association domains are indicated with blue barrels ( hydrophobic helices ) or with yellow / blue barrels ( amphipathic helices , with the yellow half representing the polar / charged hydrophilic side of the helix and the blue half representing the hydrophobic side of the helix ) . plant cells infected by como- and nepoviruses are characterized by the presence of numerous membraneous vesicles , which are derived from the endoplasmic reticulum ( er ; carette et al . , 2000 ; ritzenthaler et al . , 2002 ; han and sanfacon , 2003 ) . in cpmv - infected cells , vesicles first appear throughout the cytoplasm , but later coalesce in a large perinuclear structure ( carette et al . actin microfilaments are probably involved in this process ( carette et al . , 2002a ) . perinuclear membrane aggregates are also observed in torsv- and gflv - infected cells ( ritzenthaler et al . , 2002 ; han and sanfacon , 2003 ) . viral replication proteins , de novo rna synthesis and dsrna intermediates co - localize with these structures , indicating that they are the site of viral replication ( de zoeten et al . vesicles induced in como- and nepovirus - infected cells are irregularly shaped , vary in size and are usually surrounded by a single - membrane ( carette et al . , 2000 ; ritzenthaler et al . , 2002 ; figure 1a ) . these vesicles are similar to those observed in early stages of infection by poliovirus and coxsackie b3 virus ( both picornaviruses ) . three - dimensional reconstruction of these early picornavirus - induced structures revealed that they are branching tubular structures rather than closed vesicles ( limpens et al . positive membrane curvature induced by viral membrane proteins allows budding of tubular structures from the surface of the membrane . replication proteins accumulate on the outside of the single - membrane structures ( figure 1b , model 1 ) . that gflv- and poliovirus - induced vesicles are immunoprecipitated by antibodies against viral replication proteins , is consistent with this model ( bienz et al . in contrast , membrane structures induced by many plant viruses ( including bromo- , tombus- , and tymoviruses ) are formed by membrane invagination and require negative membrane curvature . these replication complexes are sheltered inside spherules that are connected to the cytoplasm by a neck ( figure 1b , model 2 ) . of the replication proteins encoded by como- or nepovirus rna1 , two contain obvious hydrophobic regions : the comovirus co - pro and ntb proteins and the cognate nepovirus x2 and ntb proteins ( figure 1d ) . in infected cells , mature proteins co - exist with stable intermediate polyproteins ( figure 1c ) . the cpmv co - pro is only detected as a mature protein due to efficient cleavage between co - pro and ntb . however , ntb is found either as a mature protein or within various intermediates ( ntb vpg , ntb vpg pro , and ntb vpg pro pol ; wellink et al . , 1986 ) . in contrast , processing at the nepovirus x2ntb cleavage site is inefficient in vitro leading to the accumulation of x2ntb and x2ntb vpg in addition to x2 and ntb ( wang and sanfacon , 2000 ; wetzel et al . , 2008 ) . in torsv - infected cells , ntb , ntb vpg , and x2ntb vpg are tightly associated with er membranes active in viral replication ( han and sanfacon , 2003 ) . in contrast , only a sub - population of a polyprotein containing the vpg , pro , and pol domains ( vpg pro pol ) is associated with replication - competent membranes and this association is peripheral , suggesting that it requires an interaction between vpg pro pol and a membrane protein ( chisholm et al . , 2007 ) . similarly , only a fraction of vpg pro pol is membrane - bound in cpmv - infected cells ( dorssers et al . , 1984 ) . when expressed individually , the torsv x2 , ntb and ntb vpg and the cpmv co - pro and ntb vpg associate with er membranes , while proteins containing the torsv or cpmv vpg , pro , and pol domains remain in the soluble cytoplasmic fraction ( carette et al . , 2002b ; zhang et al . , 2005 thus , the cpmv co - pro and ntb and torsv x2 and ntb and/or intermediate polyproteins containing these protein domains are likely to act as membrane anchors for the replication complex . the nucleotide - binding motif of the nepo- and comovirus ntb is related to that of the poliovirus 2c protein ( figure 1c ) . the nepo- and comovirus ntb also contain a hydrophobic c - terminal domain , which is absent in 2c ( figure 1d ) . the poliovirus 3a protein ( immediately downstream of 2c in the polyprotein ) has a hydrophobic domain that corresponds to the c - terminal region of the nepo- and comovirus ntb , although polyproteins containing both 2c and 3a are not detected in infected cells ( figure 1c ; cameron et al . , 2010 ) . the torsv x2 , and cpmv co - pro are highly hydrophobic and share a signature sequence ( f - x27-w - x11-l - x23-e ; rott et al . , 1995 ) , which is also found in the cognate proteins of nepo- , como- , faba- , and cheraviruses ( sanfacon et al . , 2011 ) . co - pro is a protease co - factor that slows the processing of the cpmv rna1 polyprotein ( peters et al . , 1992 ) . however , there is no experimental evidence that x2 regulates the nepovirus protease activity ( wang and sanfacon , 2000 ; wetzel et al . , thus , the conserved motif may be important for another common activity of co - pro and x2 . the poliovirus 2b protein is located immediately upstream of 2c ( figure 1d ) but does not share sequence motifs with x2 and co - pro , other than a general hydrophobicity . when overexpressed from a viral vector , the cpmv ntb vpg or co - pro induces the formation of small er - derived perinuclear bodies ( carette et al . , 2002b ) . these structures resemble the er modifications observed in early stages of natural cpmv infections but differ from the large perinuclear structures present later in infection . thus , both proteins may act together to induce the larger structures in natural infections . ntb and ntb - containing intermediate polyproteins co - immunoprecipitate with co - pro , suggesting that co - pro and ntb interact with each other ( wellink et al . , 1986 ) . this situation is reminiscent of that observed with poliovirus membrane proteins . while 3a , 2c , and 2bc each induce er modifications , co - expression of 3a and 2bc together is required to induce vesicles that are similar to those observed in natural poliovirus infections ( suhy et al . , 2000 ) . protein interactions among these proteins are well - documented ( teterina et al . , 2006 ; yin et al . , 2007 ) . ectopic overexpression of cpmv co - pro or ntb vpg induces local necrosis in plant ( carette et al . interestingly , cpmv does not cause necrosis in natural infection , even though co - pro and ntb vpg accumulate in infected cells . accumulation of these proteins in electron - dense bodies , which are probable sites of protein aggregation , may help reduce their toxicity ( carette et al . comparison of the symptomatology induced by chimeric constructs of two isolates of bean pod mosaic virus ( another comovirus ) also points to co - pro and ntb as symptom severity determinants ( gu and ghabrial , 2005 ) . chimeric constructs containing co - pro or ntb from the severe isolate induce increased symptomatology and accumulate to higher level than the mild isolate . co - pro and ntb may regulate the rate of virus replication , in agreement with their proposed role in replication complex assembly . although the severe symptoms may be due to increased accumulation of viral products , possibly triggering plant defense responses , it may be a direct consequence of the membrane alterations induced by ntb and co - pro . poliovirus 2b and 3a induce apoptosis when overexpressed ( madan et al . , 2008 ) . at least for 2b , the induction of apoptosis was correlated with its viroporin activity , which affects the integrity of various membranes , including mitochondrial membranes ( madan et al . , 2008,2010 ) . although a sub - population of the cpmv ntb vpg targets chloroplast membranes ( carette et al . , 2002b ) , there is no experimental evidence that mitochondria are targeted . further studies will be necessary to investigate possible correlations between membrane alterations and symptomatology induced by the comovirus ntb and co - pro proteins and to determine whether the nepovirus x2 and ntb proteins can alter membrane structures and induce symptoms . membrane association of integral membrane proteins can be directed by transmembrane -helices , which are highly hydrophobic , or by amphipathic -helices . amphipathic helices initially insert parallel to the membranes with their hydrophobic face inserted in the lipid bilayer ( figures 2a , b ) . oligomerization of amphipathic helices can lead to the formation of aqueous pores whereby the hydrophilic faces of the helices orient toward the pore and the hydrophobic faces interact within the membrane environment ( gonzalez and carrasco , 2003 ; figure 2b ) . hydrophobic intra- and intermolecular interactions among amphipathic and adjacent hydrophobic helices can stabilize the oligomers ( figure 2b ) , as suggested for the poliovirus 2b protein ( agirre et al . , 2002 ; the hydrophobic side of the helix ( blue ) inserts in one leaflet of the lipid bilayer while the polar / charged hydrophilic side of the helix ( yellow ) is exposed to the cytosolic face of the membrane . this insertion displaces the lipid headgroup , causing the acyl chain to reorient and inducing positive membrane curvature . ( b ) model for the oligomerization of amphipathic helices and formation of an aqueous pore . in the top panel , an amphipathic helix is inserted parallel to the lipid bilayer ( horizontal gray lines ) of the membrane ( left ) . formation of an aqueous pore ( double - ended red arrow ) requires oligomerization of four or six amphipathic helices ( middle ) . in the aqueous pore , the hydrophilic side of the helix ( yellow ) is exposed toward the pore , while its hydrophobic side ( blue ) is oriented toward the membrane lipid bilayer . a simplified representation of the pore shows only two molecules to better visualize each side of the amphipathic helix relative to the pore ( right ) . in the bottom panel , a membrane protein consisting of an amphipathic helix and a hydrophobic helix ( blue ) is shown . after initial membrane insertion of the monomer with the amphipathic helix parallel to the membrane ( left ) , an aqueous pore is formed by oligomerization of the amphipathic helix ( middle ) . the hydrophobic helix of each molecule is located on the outside of the pore alongside the amphipathic helix ( model shown for a hexamer ) . hydrophobic interactions between the hydrophobic side of the amphipathic helix and the hydrophobic helix stabilize pore formation . ( c ) predicted topologies for ntb vpg , x2 , and x2ntb vpg shown for monomers ( left ) or oligomers ( right ) . two possible topologies are shown for ntb vpg monomers ( 1 and 2 , see text ) . to simplify the figure , , at least four molecules would be necessary to form an aqueous pore ( as shown in b ) . the open circle represents the vpg domain and the red oval indicates the conserved ntb motif . ( d ) model for the induction of positive membrane curvature by hydrophobic interactions of membrane proteins oligomers , shown for ntb vpg . on the left , these interactions ( shown by broken blue lines on the right ) would induce positive membrane curvature . similar hydrophobic interactions are predicted to occur in x2 or x2ntb vpg oligomers ( not shown ) . the hydrophobic c - terminal domain and a predicted n - terminal amphipathic helix of the torsv ntb protein ( figure 1d ) are each sufficient to target gfp fusion proteins to er membranes in plant cells or to direct the insertion of ntb or ntb vpg into canine microsomal membranes in vitro ( wang et al . , 2004 ; zhang et al . , 2005 these domains are conserved in the sequence of ntb from other nepo- and comoviruses ( figure 1d ) . the c - terminal hydrophobic region of the torsv ntb contains a highly hydrophobic -helix , which traverses the membrane . vpg is translocated in the membrane lumen ( topology 1 , figure 2c ) , allowing the recognition of a naturally occurring n - glycosylation site ( wang et al . , 2004 ; zhang et al . , 2005 ) vpg was confirmed by proteinase k protection assays using membrane - fractions of torsv - infected cells ( han and sanfacon , 2003 ) . however , these results do not exclude the possibility that a sub - population of the protein adopt an alternate topology . in vitro , a second weakly predicted transmembrane -helix traverses the membranes when the first transmembrane helix is deleted ( wang et al . , 2004 ) . in an alternate topology ( topology 2 , figure 2c ) , the ntb c - terminal hydrophobic region traverses the membrane twice allowing a cytosolic orientation of the vpg . alternative topologies for ntb vpg could regulate the presentation of the vpg to the cytoplasmic face of the membrane where protein protein interactions and viral replication take place . the n - terminus of ntb is translocated in the membrane lumen , suggesting oligomerization of the amphipathic helix and pore formation ( zhang et al . , 2005 ; pore formation may be enhanced by hydrophobic interactions between the n - terminal amphipathic helix and the c - terminal transmembrane helix . denaturing sds - polyacrylamide gel electrophoresis ( sds - page ) of ntb vpg or of fragments containing the amphipathic or transmembrane helices revealed the presence of additional bands that correspond in size to oligomers . membrane proteins can conserve their oligomeric structure in the presence of denaturing agents , due to strong hydrophobic interactions ( degrado et al . , 2003 ) . the potential ntb vpg oligomers were glycosylated , suggesting that oligomerization occurred within the membranes ( wang et al . , 2004 ; zhang et al . , 2005 although the topology model of ntb vpg oligomers suggests the formation of an aqueous pore , further experimentation is required to test whether the protein affects the membrane integrity in vivo . in plant cells , er - targeting of torsv x2 is directed by two strongly predicted transmembrane helices and a putative amphipathic helix ( zhang and sanfacon , 2006 ; figure 1d ) . similarly , three transmembrane helices and one amphipathic helix are predicted in the cpmv co - pro ( carette et al . , 2002b ; zhang and sanfacon , 2006 ; figure 1d ) . the topology of torsv x2 was examined in vitro ( zhang and sanfacon , 2006 ) . the two predicted transmembrane helices were found to traverse the membrane , forming a hairpin and resulting in a cytosolic orientation of the c - terminus of x2 ( figure 2c ) . analysis by sds - page of full - length or truncated x2 suggests that , as for ntb vpg , protein oligomerization occurs through hydrophobic interactions ( zhang and sanfacon , 2006 ) . a topology model of x2 oligomers implies the formation of an aqueous pore by oligomerization of the amphipathic helix ( figure 2c ) . however , in vivo evidence in support of this model is still lacking . due to its highly hydrophobic nature thus , although the presence of mature x2 in torsv - infected cells is likely , it could not be confirmed . however , polyproteins corresponding to the expected molecular mass for x2ntb vpg were detected with anti - ntb and anti - vpg antibodies ( han and sanfacon , 2003 ) . efforts are underway to develop torsv infectious clones , which may allow the insertion of epitope tags in x2 to confirm its presence in torsv - infected cells and examine its topology in vivo ( chisholm and sanfacon , unpublished ) . although insertion of hydrophilic epitope tags into hydrophobic membrane proteins can hinder their function , a recent study described tolerated insertion sites in poliovirus membrane proteins ( teterina et al . , 2011a ) . the topology models for x2 and ntb vpg pose some problems when applied to the x2ntb vpg polyprotein . the cytosolic orientation of the c - terminus of x2 is in apparent conflict with the luminal orientation of the n - terminus of ntb . however , the presence of two strong transmembrane domains in the x2 domain of x2ntb vpg may prevent the membrane translocation of the ntb amphipathic helix , forcing it to insert parallel to the membranes ( figure 2c ) . thus , processing at the x2ntb cleavage site may influence the orientation of the ntb amphipathic helix and alter the ability of ntb and/or x2 to modify intracellular membranes . the impact of proteolytic cleavage on membrane topology was demonstrated for the poliovirus 3a and 3ab ( fujita et al . , 2007 ) . using a fluorescence quenching method , 3ab was shown to adopt a single topology , in which the hydrophobic domain is parallel to the membrane . in contrast , 3a adopts two possible orientations , one of which traverses the membrane . it was suggested that the hydrophilic vpg domain prevents the membrane translocation of the 3a hydrophobic domain in 3ab . although 2b , 2c , and 2bc can target to membranes , only 2bc induces a proliferation of membraneous vesicles ( suhy et al . , 2000 ) . on the other hand , poliovirus mutants with decreased processing efficiency at the 2bc cleavage site have reduced membrane permeabilization activity , suggesting that the release of mature 2b from 2bc is essential for its viroporin function ( van kuppeveld et al . , 1996 ) . the experimental evidence points to a role for como- and nepovirus membrane replication proteins in altering host membranes and assembling the replication complexes . positive membrane curvature can be induced by parallel insertion of amphipathic helices ( figure 2a ) or by intra- and intermolecular hydrophobic interactions among membrane protein oligomers ( as shown for ntb vpg , figure 2d ; mcmahon and gallop , 2005 ) . the secretory pathway is hijacked by poliovirus to help the formation of membraneous vesicles , resulting in an inhibition of host protein transport ( hsu et al . , 2010 ) . the 2b and 3a proteins inhibit the secretory pathway ( doedens and kirkegaard , 1995 ) . 3a interacts with several components of the secretory pathway , including acbd3 , a golgi adaptor protein ( greninger et al . , 2012 ; sasaki et al . , 2012 ) and gbf1 , a guanine nucleotide exchange factor that activates arf1 , a cellular gtpase and regulator of the secretory pathway ( wessels et al . , 2006 ; arf1 is also the known target of brefeldin a , an inhibitor of the secretory pathway that blocks poliovirus infection ( irurzun et al . , 1992 ; maynell et al . , 1992 ) . the 3a gbf1 and 3a acbd3 interactions may assist in the recruitment of p1kiii , an enzyme involved in phospholipid synthesis , to the replication complex ( hsu et al . , 2010 ; greninger et al . p1kiii would alter the membrane lipid composition , possibly affecting the membrane curvature and facilitating the formation of virus factories . however , the sensitivity of picornaviruses to brefeldin a varies greatly and the gbf13a interaction is not conserved for all picornaviruses , suggesting that the interaction between viruses and the host secretory pathway varies . how do these findings apply to como- and nepoviruses ? , 2000 ; ritzenthaler et al . , 2002 ) , an inhibitor of type ii fatty acid synthase , suggesting that de novo phospholipid synthesis is required for membrane proliferation , possibly involving changes in membrane lipid composition . however , the interaction of nepo- and comoviruses with the secretory pathway is not well understood and their ability to block protein secretion has not been investigated . two snare - like proteins from arabidopsis thaliana were shown to interact with the cpmv ntb although their function is not known , they may regulate membrane fusion and vesicle formation . identification of additional interaction partners of the nepo- and comovirus membrane proteins will be essential to better understand membrane remodeling directed by these proteins . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

formation of plant virus membrane - associated replication factories requires the association of viral replication proteins and viral rna with intracellular membranes , the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex . many viruses encode integral membrane proteins that act as anchors for the replication complex . these hydrophobic proteins contain transmembrane domains and/or amphipathic helices that associate with the membrane and modify its structure . the comovirus co - pro and ntp - binding ( ntb , putative helicase ) proteins and the cognate nepovirus x2 and ntb proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2b , 2c , and 3a membrane proteins . the identification of membrane association domains and analysis of the membrane topology of these proteins is discussed . the evidence suggesting that these proteins have the ability to induce membrane proliferation , alter the structure and integrity of intracellular membranes , and modulate the induction of symptoms in infected plants is also reviewed . finally , areas of research that need further investigation are highlighted .

CHARACTERIZATION OF COMOVIRUS AND NEPOVIRUS REPLICATION COMPLEXES AND IDENTIFICATION OF PUTATIVE MEMBRANE ANCHORS MEMBRANE MODIFICATIONS AND SYMPTOMS INDUCED BY THE COMOVIRUS Co-Pro AND NTBVPg MEMBRANE TOPOLOGY OF THE ToRSV X2 AND NTB: EVIDENCE FOR OLIGOMERIZATION AND VIROPORIN ACTIVITY INTERACTION OF VIRAL MEMBRANE PROTEINS WITH HOST FACTORS: TOWARD A MECHANISM FOR MEMBRANE MODIFICATION Conflict of Interest Statement

positive - strand rna viruses replicate in large complexes that are associated with host intracellular membranes ( salonen et al . some viruses require host membrane proteins to target their replication proteins to the membranes ( yamanaka et al . however , many viruses encode proteins that interact with membranes directly and modify their intrinsic structure . these proteins have membrane association domains and contain protein protein and/or protein rna interaction domains that allow them to recruit the viral rna , other viral replication proteins , or host factors to the membranes . well - characterized plant virus membrane proteins include the tombusvirus 3336 kda proteins , bromovirus 1a protein , potyvirus 6k protein , and tymovirus 140 kda protein ( schaad et al . , the family secoviridae ( order picornavirales ) includes the genera comovirus , fabavirus , nepovirus , sequivirus , waikavirus , cheravirus , sadwavirus , and torradovirus ( sanfacon et al . the best characterized members of the family are cowpea mosaic virus ( cpmv , comovirus ) , grapevine fanleaf virus ( gflv , nepovirus ) , and tomato ringspot virus ( torsv , nepovirus ; pouwels et al . , 2006 these viruses use a polyprotein strategy to express their proteins and have a replication block consisting of a nucleotide - binding protein ( ntb ) , a genome - linked protein ( vpg ) , a proteinase ( pro ) , and an rna - dependent rna polymerase ( pol ; figure 1c ) . ( 1 ) in cells infected with poliovirus or coxsackie b3 virus , viral integral membrane proteins ( red ovals ) induce positive curvature of the membrane allowing the budding of tubular structures . other viral replication proteins ( e.g. , polymerase , green ovals ) interact with the viral membrane proteins . host factors and viral rna ( not shown ) associate with the replication complex by protein protein and protein rna interactions . single - membrane vesicles may bud out and form double - membrane vesicles after the internal collapse of the single - membrane vesicle and subsequent membrane fusion to allow its circularization . ( 2 ) in cells infected with many plant and animal viruses , induction of negative membrane curvature results in membrane invagination and formation of spherules in the lumen of the membrane . viral integral membrane proteins ( red ovals ) line the interior of the spherule . the viral polymerase ( green ovals ) as well as other viral proteins , host factors and the viral rna ( not shown ) are enclosed in the spherule . release of the vesicle in the lumen of the membrane may be followed by budding of a double - membrane vesicle into the cytoplasm . ( c ) organization of replication protein domains in the polyproteins of cpmv , torsv , and poliovirus . for poliovirus , the polyprotein encoded by the single genomic rna is shown , although the p1 region ( containing the structural proteins ) is truncated as indicated by the diagonal bars . conserved motifs are : rna - dependent rna polymerase ( pol , green ovals ) , protease ( pro , orange diamond ) , nucleotide - binding protein ( ntb , red oval ) , co - pro and x2 ( purple square ) . horizontal bars under each polyprotein represent integral membrane proteins that have been detected in virus - infected cells . although likely , its presence in infected cells could not be confirmed due to the lack of antibodies . ( d ) the regions of the polyprotein containing the putative membrane anchors are shown for each virus . predicted membrane - association domains are indicated with blue barrels ( hydrophobic helices ) or with yellow / blue barrels ( amphipathic helices , with the yellow half representing the polar / charged hydrophilic side of the helix and the blue half representing the hydrophobic side of the helix ) . viral replication proteins , de novo rna synthesis and dsrna intermediates co - localize with these structures , indicating that they are the site of viral replication ( de zoeten et al . vesicles induced in como- and nepovirus - infected cells are irregularly shaped , vary in size and are usually surrounded by a single - membrane ( carette et al . three - dimensional reconstruction of these early picornavirus - induced structures revealed that they are branching tubular structures rather than closed vesicles ( limpens et al . positive membrane curvature induced by viral membrane proteins allows budding of tubular structures from the surface of the membrane . replication proteins accumulate on the outside of the single - membrane structures ( figure 1b , model 1 ) . that gflv- and poliovirus - induced vesicles are immunoprecipitated by antibodies against viral replication proteins , is consistent with this model ( bienz et al . these replication complexes are sheltered inside spherules that are connected to the cytoplasm by a neck ( figure 1b , model 2 ) . of the replication proteins encoded by como- or nepovirus rna1 , two contain obvious hydrophobic regions : the comovirus co - pro and ntb proteins and the cognate nepovirus x2 and ntb proteins ( figure 1d ) . in infected cells , mature proteins co - exist with stable intermediate polyproteins ( figure 1c ) . the cpmv co - pro is only detected as a mature protein due to efficient cleavage between co - pro and ntb . however , ntb is found either as a mature protein or within various intermediates ( ntb vpg , ntb vpg pro , and ntb vpg pro pol ; wellink et al . in contrast , processing at the nepovirus x2ntb cleavage site is inefficient in vitro leading to the accumulation of x2ntb and x2ntb vpg in addition to x2 and ntb ( wang and sanfacon , 2000 ; wetzel et al . in torsv - infected cells , ntb , ntb vpg , and x2ntb vpg are tightly associated with er membranes active in viral replication ( han and sanfacon , 2003 ) . in contrast , only a sub - population of a polyprotein containing the vpg , pro , and pol domains ( vpg pro pol ) is associated with replication - competent membranes and this association is peripheral , suggesting that it requires an interaction between vpg pro pol and a membrane protein ( chisholm et al . when expressed individually , the torsv x2 , ntb and ntb vpg and the cpmv co - pro and ntb vpg associate with er membranes , while proteins containing the torsv or cpmv vpg , pro , and pol domains remain in the soluble cytoplasmic fraction ( carette et al . , 2005 thus , the cpmv co - pro and ntb and torsv x2 and ntb and/or intermediate polyproteins containing these protein domains are likely to act as membrane anchors for the replication complex . the nucleotide - binding motif of the nepo- and comovirus ntb is related to that of the poliovirus 2c protein ( figure 1c ) . the poliovirus 3a protein ( immediately downstream of 2c in the polyprotein ) has a hydrophobic domain that corresponds to the c - terminal region of the nepo- and comovirus ntb , although polyproteins containing both 2c and 3a are not detected in infected cells ( figure 1c ; cameron et al . the torsv x2 , and cpmv co - pro are highly hydrophobic and share a signature sequence ( f - x27-w - x11-l - x23-e ; rott et al . , 1995 ) , which is also found in the cognate proteins of nepo- , como- , faba- , and cheraviruses ( sanfacon et al . co - pro is a protease co - factor that slows the processing of the cpmv rna1 polyprotein ( peters et al . , thus , the conserved motif may be important for another common activity of co - pro and x2 . the poliovirus 2b protein is located immediately upstream of 2c ( figure 1d ) but does not share sequence motifs with x2 and co - pro , other than a general hydrophobicity . when overexpressed from a viral vector , the cpmv ntb vpg or co - pro induces the formation of small er - derived perinuclear bodies ( carette et al . thus , both proteins may act together to induce the larger structures in natural infections . ntb and ntb - containing intermediate polyproteins co - immunoprecipitate with co - pro , suggesting that co - pro and ntb interact with each other ( wellink et al . this situation is reminiscent of that observed with poliovirus membrane proteins . while 3a , 2c , and 2bc each induce er modifications , co - expression of 3a and 2bc together is required to induce vesicles that are similar to those observed in natural poliovirus infections ( suhy et al . protein interactions among these proteins are well - documented ( teterina et al . ectopic overexpression of cpmv co - pro or ntb vpg induces local necrosis in plant ( carette et al . interestingly , cpmv does not cause necrosis in natural infection , even though co - pro and ntb vpg accumulate in infected cells . accumulation of these proteins in electron - dense bodies , which are probable sites of protein aggregation , may help reduce their toxicity ( carette et al . comparison of the symptomatology induced by chimeric constructs of two isolates of bean pod mosaic virus ( another comovirus ) also points to co - pro and ntb as symptom severity determinants ( gu and ghabrial , 2005 ) . chimeric constructs containing co - pro or ntb from the severe isolate induce increased symptomatology and accumulate to higher level than the mild isolate . co - pro and ntb may regulate the rate of virus replication , in agreement with their proposed role in replication complex assembly . although the severe symptoms may be due to increased accumulation of viral products , possibly triggering plant defense responses , it may be a direct consequence of the membrane alterations induced by ntb and co - pro . at least for 2b , the induction of apoptosis was correlated with its viroporin activity , which affects the integrity of various membranes , including mitochondrial membranes ( madan et al . although a sub - population of the cpmv ntb vpg targets chloroplast membranes ( carette et al . further studies will be necessary to investigate possible correlations between membrane alterations and symptomatology induced by the comovirus ntb and co - pro proteins and to determine whether the nepovirus x2 and ntb proteins can alter membrane structures and induce symptoms . membrane association of integral membrane proteins can be directed by transmembrane -helices , which are highly hydrophobic , or by amphipathic -helices . amphipathic helices initially insert parallel to the membranes with their hydrophobic face inserted in the lipid bilayer ( figures 2a , b ) . oligomerization of amphipathic helices can lead to the formation of aqueous pores whereby the hydrophilic faces of the helices orient toward the pore and the hydrophobic faces interact within the membrane environment ( gonzalez and carrasco , 2003 ; figure 2b ) . hydrophobic intra- and intermolecular interactions among amphipathic and adjacent hydrophobic helices can stabilize the oligomers ( figure 2b ) , as suggested for the poliovirus 2b protein ( agirre et al . , 2002 ; the hydrophobic side of the helix ( blue ) inserts in one leaflet of the lipid bilayer while the polar / charged hydrophilic side of the helix ( yellow ) is exposed to the cytosolic face of the membrane . ( b ) model for the oligomerization of amphipathic helices and formation of an aqueous pore . in the top panel , an amphipathic helix is inserted parallel to the lipid bilayer ( horizontal gray lines ) of the membrane ( left ) . formation of an aqueous pore ( double - ended red arrow ) requires oligomerization of four or six amphipathic helices ( middle ) . in the aqueous pore , the hydrophilic side of the helix ( yellow ) is exposed toward the pore , while its hydrophobic side ( blue ) is oriented toward the membrane lipid bilayer . a simplified representation of the pore shows only two molecules to better visualize each side of the amphipathic helix relative to the pore ( right ) . after initial membrane insertion of the monomer with the amphipathic helix parallel to the membrane ( left ) , an aqueous pore is formed by oligomerization of the amphipathic helix ( middle ) . hydrophobic interactions between the hydrophobic side of the amphipathic helix and the hydrophobic helix stabilize pore formation . to simplify the figure , , at least four molecules would be necessary to form an aqueous pore ( as shown in b ) . ( d ) model for the induction of positive membrane curvature by hydrophobic interactions of membrane proteins oligomers , shown for ntb vpg . the c - terminal hydrophobic region of the torsv ntb contains a highly hydrophobic -helix , which traverses the membrane . however , these results do not exclude the possibility that a sub - population of the protein adopt an alternate topology . in an alternate topology ( topology 2 , figure 2c ) , the ntb c - terminal hydrophobic region traverses the membrane twice allowing a cytosolic orientation of the vpg . alternative topologies for ntb vpg could regulate the presentation of the vpg to the cytoplasmic face of the membrane where protein protein interactions and viral replication take place . the n - terminus of ntb is translocated in the membrane lumen , suggesting oligomerization of the amphipathic helix and pore formation ( zhang et al . , 2005 although the topology model of ntb vpg oligomers suggests the formation of an aqueous pore , further experimentation is required to test whether the protein affects the membrane integrity in vivo . similarly , three transmembrane helices and one amphipathic helix are predicted in the cpmv co - pro ( carette et al . the two predicted transmembrane helices were found to traverse the membrane , forming a hairpin and resulting in a cytosolic orientation of the c - terminus of x2 ( figure 2c ) . a topology model of x2 oligomers implies the formation of an aqueous pore by oligomerization of the amphipathic helix ( figure 2c ) . however , polyproteins corresponding to the expected molecular mass for x2ntb vpg were detected with anti - ntb and anti - vpg antibodies ( han and sanfacon , 2003 ) . the topology models for x2 and ntb vpg pose some problems when applied to the x2ntb vpg polyprotein . the cytosolic orientation of the c - terminus of x2 is in apparent conflict with the luminal orientation of the n - terminus of ntb . however , the presence of two strong transmembrane domains in the x2 domain of x2ntb vpg may prevent the membrane translocation of the ntb amphipathic helix , forcing it to insert parallel to the membranes ( figure 2c ) . thus , processing at the x2ntb cleavage site may influence the orientation of the ntb amphipathic helix and alter the ability of ntb and/or x2 to modify intracellular membranes . the impact of proteolytic cleavage on membrane topology was demonstrated for the poliovirus 3a and 3ab ( fujita et al . using a fluorescence quenching method , 3ab was shown to adopt a single topology , in which the hydrophobic domain is parallel to the membrane . in contrast , 3a adopts two possible orientations , one of which traverses the membrane . it was suggested that the hydrophilic vpg domain prevents the membrane translocation of the 3a hydrophobic domain in 3ab . although 2b , 2c , and 2bc can target to membranes , only 2bc induces a proliferation of membraneous vesicles ( suhy et al . on the other hand , poliovirus mutants with decreased processing efficiency at the 2bc cleavage site have reduced membrane permeabilization activity , suggesting that the release of mature 2b from 2bc is essential for its viroporin function ( van kuppeveld et al . the experimental evidence points to a role for como- and nepovirus membrane replication proteins in altering host membranes and assembling the replication complexes . the secretory pathway is hijacked by poliovirus to help the formation of membraneous vesicles , resulting in an inhibition of host protein transport ( hsu et al . 3a interacts with several components of the secretory pathway , including acbd3 , a golgi adaptor protein ( greninger et al . , 2012 ) and gbf1 , a guanine nucleotide exchange factor that activates arf1 , a cellular gtpase and regulator of the secretory pathway ( wessels et al . , 2006 ; arf1 is also the known target of brefeldin a , an inhibitor of the secretory pathway that blocks poliovirus infection ( irurzun et al . the 3a gbf1 and 3a acbd3 interactions may assist in the recruitment of p1kiii , an enzyme involved in phospholipid synthesis , to the replication complex ( hsu et al . p1kiii would alter the membrane lipid composition , possibly affecting the membrane curvature and facilitating the formation of virus factories . however , the sensitivity of picornaviruses to brefeldin a varies greatly and the gbf13a interaction is not conserved for all picornaviruses , suggesting that the interaction between viruses and the host secretory pathway varies . , 2002 ) , an inhibitor of type ii fatty acid synthase , suggesting that de novo phospholipid synthesis is required for membrane proliferation , possibly involving changes in membrane lipid composition . however , the interaction of nepo- and comoviruses with the secretory pathway is not well understood and their ability to block protein secretion has not been investigated . two snare - like proteins from arabidopsis thaliana were shown to interact with the cpmv ntb although their function is not known , they may regulate membrane fusion and vesicle formation . identification of additional interaction partners of the nepo- and comovirus membrane proteins will be essential to better understand membrane remodeling directed by these proteins .

the genes of cancer / testis antigens ( cta ) such as melanoma antigen gene ( mage ) and synovial sarcoma on x chromosome ( ssx ) are thought to be silent in normal adult tissues except testis . however , these genes are expressed at a high frequency in a large variety of cancer cells . therefore , the corresponding transcripts represent attractive targets for cancer immunotherapy and cancer diagnosis ( 1 - 4 ) . the mage a family consists of several subtypes , including mage-1 to mage-12 . during the past several years , many researchers have studied the expression of individual mage a genes for cancer diagnosis . however , the use of mage genes in the detection of a small number of cancer cells by reverse transcription - polymerase chain reaction ( rt - pcr ) has been limited by the low expression frequency of individual mage genes in various cancer tissues . it has been reported that the probability of a cancer cell expressing at least one mage gene is very high ( 5 - 7 ) . in order to improve the detection rate of magegenes , we have recently designed common primers that can bind simultaneously to the cdna of mage-1 , -2 , -3 , -4a , -4b , -5a , -5b and -6 ( mage 1 - 6 ) . we also developed a mage 1 - 6 assay that can simultaneously detect the transcripts of mage 1 - 6 ( 8) . the ssx gene family , which was originally identified as fusion partners to the syt gene in synovial sarcomas , consists of 9 subtype genes ( ssx 1 - 9 ) . also , naturally occurring serologic responses mounted by cancer patients against one ssx family member cross - react with other members of the family ( 9 - 11 ) . the high level of homology between the subtypes at the dna and protein levels suggests that it may be possible to design a common primer for the ssx family . in this study , we designed a common ssx primer , and developed an ssx 1 - 9 assay that can detect cancer cells expressing at least one of the 9 ssx genes by rt - nested pcr using the common ssx primers . thirty five cancer cell lines derived from stomach cancer ( snu 484 , snu 620 , snu 638 , snu 668 ) , colon cancer ( snu c1 , snu c4 , snu c5 , ht29 , hct 116 ) , head and neck cancer ( amc - hn3 , amc - hn4 , amc - hn7 ) , leukemia ( u937 , hl 60 ) cervical cancer ( caski , c4-ii , me-180 , hela , cunc-6 , siha ) , lung cancer ( nci - h292 , nci - h522 , nci - h1703 , a-549 ) , prostate cancer ( pc 3 , du 145 ) , hepatocellular carcinoma ( hepg2 , snu 182 , snu 354 , snu 387 , snu 398 , snu 423 ) , kidney cancer ( hek 293 ) , breast cancer ( mda 231 ) , and osteosarcoma ( saos 2 ) were studied for the expression of ssx 1 - 9 genes . these cancer cell lines were grown in dmem or rpmi1640 supplemented with 10% fbs in a 5% co2 incubator at 37. then total rna was extracted with the trizol chloroform method . the cancer tissue samples were obtained from 29 head and neck cancer patients who were surgically treated at the department of otolaryngology , school of medicine , kosin university , busan , korea . the tissue samples were immediately frozen and kept at -70. then total rna was extracted with the trizol chloroform method . induced sputum samples were obtained from 18 patients with head and neck cancer and 22 patients with benign pulmonary diseases such as pneumonia , bronchitis , and tuberculosis . induced sputum was collected after inhalation of 100 g of ventolin ( glaxosmithkline , london , uk ) and nebulization with 3% hypertonic saline solution for 10 min . the induced sputum was mixed with 10 ml of specimen protector ( ic&g co. , daegu , korea ) and stored at -20 until rna isolation . the messenger rna in sputum was extracted using a sputum mrna extraction kit ( ic&g co. ) according to the manufacturer 's instructions . briefly , after thawing the sputum that had been digested with the specimen protector , the undigested debris was removed by centrifugation , and capture beads were then added to the liquefied supernatants . after mixing the liquefied sputum and beads for 30 min at room temperature , the beads were separated in a magnet rack and washed 3 times with washing buffer . messenger rnas attached to the beads were eluted with an elution buffer and stored at -70 until the reverse transcription reaction . detection of transcripts of mage 1 - 6 gene was performed using the cancer hunter kit ( ic&g co. ) . briefly , reverse transcription reactions were carried out in a 20 l reaction mixture containing 7 l of rt master mixture , 0.5 l of rnase inhibitor , 1 l of rtase and 11.5 l of eluted rna solutions . the reaction mixture was incubated at 42 for 60 min , 95 for 5 min and then stored at -20 until the polymerase chain reaction ( pcr ) . the first pcr was carried out in a 20 l reaction mixture containing 8 l of pcr master mixture , 0.5 l of outer primers , 2 l of the rt reaction products and 9.5 l of distilled water . the cycling parameters were as follows : denaturation was initiated at 94 for 2 min , followed by 30 cycles of 94 for 30 sec , 60 for 45 sec and 65 for 60 sec . after the first pcr , 20 l of nested pcr mixture containing 8 l pcr master mixture , 0.5 l inner primers and 11.5 l dw was added to the first pcr tube . nested pcr was carried out under the same conditions as the first pcr except for the annealing temperature , which was set at 62. the nested pcr products were separated on 1% agarose gels impregnated with ethidium bromide ( 0.5 g / ml ) . the dna sequences of mage primers were as follows : ( mmrp1 , 5'-ctgaaggagaagatctgcc-3 ' ; mmrp2 , 5'-ctccaggtagttttcctgcac-3 ' ; mmrp3 , 5'-ctgaaggagaagatctgccwgtg-3 ' , w is a or t ; mmrp4 , 5'-ccagcatttctgcctttgtga-3 ' ) . the sequences of ssx 1 - 9 gene were obtained from national center for biotechnology information ( besthesda , md , usa ) , and the dna homology of each ssx gene was analyzed using the dnasis program ( hitachi , tokyo , japan ) . the dna sequences that are commonly present in ssx 1 - 9 subtype genes that were used for designing the common primers were as follows : ( outer sense [ os ] primer , 5'-gtgccatgaacggagayga-3 ' , y is c or t ; outer antisense [ oas ] primer , 5'-tctgtgggtccaggcatgt-3 ' ; inner antisense [ ias ] primer 5'-tgtytcccccttttgggtcc-3 ' , y is c or t ) . detection of transcripts of ssx 1 - 9 genes was performed using a cancer hunter kit ( ic&g co. ) with some modifications . in the first and nested pcr , the common primers for ssx 1 - 9 gene were used in place of common primers for mage 1 - 6 . all other procedures and temperature conditions for pcr were identical to the mage 1 - 6 assay . thirty five cancer cell lines derived from stomach cancer ( snu 484 , snu 620 , snu 638 , snu 668 ) , colon cancer ( snu c1 , snu c4 , snu c5 , ht29 , hct 116 ) , head and neck cancer ( amc - hn3 , amc - hn4 , amc - hn7 ) , leukemia ( u937 , hl 60 ) cervical cancer ( caski , c4-ii , me-180 , hela , cunc-6 , siha ) , lung cancer ( nci - h292 , nci - h522 , nci - h1703 , a-549 ) , prostate cancer ( pc 3 , du 145 ) , hepatocellular carcinoma ( hepg2 , snu 182 , snu 354 , snu 387 , snu 398 , snu 423 ) , kidney cancer ( hek 293 ) , breast cancer ( mda 231 ) , and osteosarcoma ( saos 2 ) were studied for the expression of ssx 1 - 9 genes . these cancer cell lines were grown in dmem or rpmi1640 supplemented with 10% fbs in a 5% co2 incubator at 37. then total rna was extracted with the trizol chloroform method . the cancer tissue samples were obtained from 29 head and neck cancer patients who were surgically treated at the department of otolaryngology , school of medicine , kosin university , busan , korea . the tissue samples were immediately frozen and kept at -70. then total rna was extracted with the trizol chloroform method . induced sputum samples were obtained from 18 patients with head and neck cancer and 22 patients with benign pulmonary diseases such as pneumonia , bronchitis , and tuberculosis . induced sputum was collected after inhalation of 100 g of ventolin ( glaxosmithkline , london , uk ) and nebulization with 3% hypertonic saline solution for 10 min . the induced sputum was mixed with 10 ml of specimen protector ( ic&g co. , daegu , korea ) and stored at -20 until rna isolation . the messenger rna in sputum was extracted using a sputum mrna extraction kit ( ic&g co. ) according to the manufacturer 's instructions . briefly , after thawing the sputum that had been digested with the specimen protector , the undigested debris was removed by centrifugation , and capture beads were then added to the liquefied supernatants . after mixing the liquefied sputum and beads for 30 min at room temperature , the beads were separated in a magnet rack and washed 3 times with washing buffer . messenger rnas attached to the beads were eluted with an elution buffer and stored at -70 until the reverse transcription reaction . detection of transcripts of mage 1 - 6 gene was performed using the cancer hunter kit ( ic&g co. ) . briefly , reverse transcription reactions were carried out in a 20 l reaction mixture containing 7 l of rt master mixture , 0.5 l of rnase inhibitor , 1 l of rtase and 11.5 l of eluted rna solutions . the reaction mixture was incubated at 42 for 60 min , 95 for 5 min and then stored at -20 until the polymerase chain reaction ( pcr ) . the first pcr was carried out in a 20 l reaction mixture containing 8 l of pcr master mixture , 0.5 l of outer primers , 2 l of the rt reaction products and 9.5 l of distilled water . the cycling parameters were as follows : denaturation was initiated at 94 for 2 min , followed by 30 cycles of 94 for 30 sec , 60 for 45 sec and 65 for 60 sec . the final extension incubation was performed at 65 for 5 min . after the first pcr , 20 l of nested pcr mixture containing 8 l pcr master mixture , 0.5 l nested pcr was carried out under the same conditions as the first pcr except for the annealing temperature , which was set at 62. the nested pcr products were separated on 1% agarose gels impregnated with ethidium bromide ( 0.5 g / ml ) . the dna sequences of mage primers were as follows : ( mmrp1 , 5'-ctgaaggagaagatctgcc-3 ' ; mmrp2 , 5'-ctccaggtagttttcctgcac-3 ' ; mmrp3 , 5'-ctgaaggagaagatctgccwgtg-3 ' , w is a or t ; mmrp4 , 5'-ccagcatttctgcctttgtga-3 ' ) . the sequences of ssx 1 - 9 gene were obtained from national center for biotechnology information ( besthesda , md , usa ) , and the dna homology of each ssx gene was analyzed using the dnasis program ( hitachi , tokyo , japan ) . the dna sequences that are commonly present in ssx 1 - 9 subtype genes that were used for designing the common primers were as follows : ( outer sense [ os ] primer , 5'-gtgccatgaacggagayga-3 ' , y is c or t ; outer antisense [ oas ] primer , 5'-tctgtgggtccaggcatgt-3 ' ; inner antisense [ ias ] primer 5'-tgtytcccccttttgggtcc-3 ' , y is c or t ) . detection of transcripts of ssx 1 - 9 genes was performed using a cancer hunter kit ( ic&g co. ) with some modifications . in the first and nested pcr , the common primers for ssx 1 - 9 gene were used in place of common primers for mage 1 - 6 . all other procedures and temperature conditions for pcr were identical to the mage 1 - 6 assay . in order to design common primers that bind to 9 ssx cdnas together , dna sequences of the ssx subtypes were compared . the ssx-1 sequence showed more than 90% dna homology with all other ssx subtype sequences ( data not shown ) . using highly homologous dna sequences , common primers that can bind simultaneously to ssx 1 - 9 cdnas were designed , and used for the ssx 1 - 9 assay . the binding site of common primers on ssx-1 gene and the products amplified by the ssx1 - 9 assay and the mage 1 - 6 assay are summarized in fig . 1 . all primers ( os , oas , and ias ) had 100% homology with the sequences of the 9 ssx cdnas , and were used for the ssx 1 - 9 assay . the transcripts of mage 1 - 6 genes and ssx 1 - 9 genes were detected in snu484 and amc hn-7 cell lines , but not in normal blood cells . the expected dna sizes amplified by the ssx 1 - 9 assay and the mage 1 - 6 assay were 490 - 492 bp and 448 - 472 bp , respectively . in order to evaluate the sensitivity of the ssx 1 - 9 assay , mrna was isolated from 35 cancer cell lines described previously in this paper , and transcripts of ssx 1 - 9 genes were detected by the ssx 1 - 9 assay . thirty one ( 88.6% ) of the 35 cancer cell lines were positive ( fig . all 3 head and neck cancer cell lines were positive in both gene assay ( data not shown ) . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 24 ( 82.8% ) and 22 ( 75.9% ) of 29 fresh cancer tissues from head and neck cancer patients . eighty six percent ( 18/21 ) and 72.7% ( 16/21 ) of squamous cell carcinoma cells expressed mage 1 - 6 genes and ssx 1 - 9 genes , respectively . sixty three percent ( 5/8 ) of other pathologic types of cancers expressed both mage 1 - 6 genes and ssx 1 - 9 genes ( fig . 3 , table 1 ) . in order to investigate the abilityof mage 1 - 6 and ssx 1 - 9 assays to detect small numbers of cancer cells mixed in sputum , snu484 ( mage- and ssx - positive cell lines ) were added to normal sputum . after digesting the sputum with specimen protector , mrna was isolated , and mage 1 - 6 and ssx 1 - 9 assays were performed . both assays detected transcripts in samples containing more than 20 snu484 cells ( fig . eighteen samples of induced sputum from head and neck cancer patients and 22 samples of induced sputum from patients with benign lung disease were collected . after digestion of sputum with specimen protector and isolation of mrna in liquefied sputum , mage 1 - 6 and ssx 1 - 9 assays were performed . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 72.2% ( 13/18 ) and 77.8% ( 14/18 ) of fresh induced sputum samples from head and neck patients ( fig . the mage 1 - 6 assay detected transcripts in 33.3% of sputum samples from tnm stage t1 tumors , 88.9% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors . the ssx 1 - 9 assay detected transcripts in 100% of sputum samples from t1 tumors , 77.8% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors ( table 3 ) . in order to evaluate the specificity of both assays , transcripts of the mage 1 - 6 and ssx 1 - 9 assays in induced sputum samples from patients with benign lung disease were detected . the mage 1 - 6 assay was positive in 4.5% ( 1/22 ) and the ssx 1 - 9 assay was positive in 13.6% ( 3/22 ) of the samples ( fig . these results represents mage 1 - 6 assay is more specific than ssx 1 - 9 asaay . in head and neck cancer patients , the mage 1 - 6 assay was positive in 82.8% of cancer tissues and 72.2% of induced sputa . the ssx 1 - 9 assay was positive in 75.9% of cancer tissues and 77.8% of induced sputa . when both mage 1 - 6 assay and ssx 1 - 9 assay were performed on the same specimen , 96.6% of cancer tissues and 94.4% of induced sputa were positive in either one of the two assays ( table 4 ) . in order to design common primers that bind to 9 ssx cdnas together , dna sequences of the ssx subtypes were compared . the ssx-1 sequence showed more than 90% dna homology with all other ssx subtype sequences ( data not shown ) . using highly homologous dna sequences , common primers that can bind simultaneously to ssx 1 - 9 cdnas were designed , and used for the ssx 1 - 9 assay . the binding site of common primers on ssx-1 gene and the products amplified by the ssx1 - 9 assay and the mage 1 - 6 assay are summarized in fig . 1 . all primers ( os , oas , and ias ) had 100% homology with the sequences of the 9 ssx cdnas , and were used for the ssx 1 - 9 assay . the transcripts of mage 1 - 6 genes and ssx 1 - 9 genes were detected in snu484 and amc hn-7 cell lines , but not in normal blood cells . the expected dna sizes amplified by the ssx 1 - 9 assay and the mage 1 - 6 assay were 490 - 492 bp and 448 - 472 bp , respectively . in order to evaluate the sensitivity of the ssx 1 - 9 assay , mrna was isolated from 35 cancer cell lines described previously in this paper , and transcripts of ssx 1 - 9 genes were detected by the ssx 1 - 9 assay . thirty one ( 88.6% ) of the 35 cancer cell lines were positive ( fig . all 3 head and neck cancer cell lines were positive in both gene assay ( data not shown ) . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 24 ( 82.8% ) and 22 ( 75.9% ) of 29 fresh cancer tissues from head and neck cancer patients . eighty six percent ( 18/21 ) and 72.7% ( 16/21 ) of squamous cell carcinoma cells expressed mage 1 - 6 genes and ssx 1 - 9 genes , respectively . sixty three percent ( 5/8 ) of other pathologic types of cancers expressed both mage 1 - 6 genes and ssx 1 - 9 genes ( fig . in order to investigate the abilityof mage 1 - 6 and ssx 1 - 9 assays to detect small numbers of cancer cells mixed in sputum , snu484 ( mage- and ssx - positive cell lines ) were added to normal sputum . after digesting the sputum with specimen protector , mrna was isolated , and mage 1 - 6 and ssx 1 - 9 assays were performed . eighteen samples of induced sputum from head and neck cancer patients and 22 samples of induced sputum from patients with benign lung disease were collected . after digestion of sputum with specimen protector and isolation of mrna in liquefied sputum , mage 1 - 6 and ssx 1 - 9 assays were performed . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 72.2% ( 13/18 ) and 77.8% ( 14/18 ) of fresh induced sputum samples from head and neck patients ( fig . the mage 1 - 6 assay detected transcripts in 33.3% of sputum samples from tnm stage t1 tumors , 88.9% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors . the ssx 1 - 9 assay detected transcripts in 100% of sputum samples from t1 tumors , 77.8% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors ( table 3 ) . in order to evaluate the specificity of both assays , transcripts of the mage 1 - 6 and ssx 1 - 9 assays in induced sputum samples from patients with benign lung disease were detected . the mage 1 - 6 assay was positive in 4.5% ( 1/22 ) and the ssx 1 - 9 assay was positive in 13.6% ( 3/22 ) of the samples ( fig . these results represents mage 1 - 6 assay is more specific than ssx 1 - 9 asaay . in head and neck cancer patients , the mage 1 - 6 assay was positive in 82.8% of cancer tissues and 72.2% of induced sputa . the ssx 1 - 9 assay was positive in 75.9% of cancer tissues and 77.8% of induced sputa . when both mage 1 - 6 assay and ssx 1 - 9 assay were performed on the same specimen , 96.6% of cancer tissues and 94.4% of induced sputa were positive in either one of the two assays ( table 4 ) . cancer antigens that have a high frequency of expression exclusively in cancer cells can be excellent markers for cancer diagnosis . the expression of mage and ssx genes are highly specific to cancer cells , but their use in the detection of a small number of cancer cells by rt - pcr has been limited by the low expression frequency of individual genes . if the transcripts of several cancer antigen genescould be detected simultaneously by rt - pcr , cancer detection rates could be increased . two major methods have been described one is multiplex pcr , which combines several specific primers in the same reaction ( 12 ) , and the other is pcr that uses common primers that simultaneously bind to multiple targets . whereas it is difficult to amplify more than 5 targets using multiplex pcr , multiple targets can be detected with few limitations by performing pcr using common primers . in a previous study , we showed that rt - nested pcr using common primers was a very useful method for detecting mage - expressing cancer cells ( 8) . expression analysis has demonstrated that ssx is a member of the recently described cancer / testis antigen class . it is expressed in a variety of different human neoplasms , but not in normal tissues , with the exception of testis and a weak expression in the thyroid . ( 13 ) studied the expression of ssx genes in 325 specimens of human neoplasms , and found that ssx-1 , -2 , and -4 subtypes are expressed in 8% , 15% and 15% of the cancers , respectively , while the expression of the ssx-5 or ssx-3 subtypes were rare or absent . expression of at least one of the ssx family members was most frequently observed in head and neck cancer ( 75% ) , followed by ovarian cancer ( 50% ) , malignant melanoma ( 43% ) , lymphoma ( 36% ) , colorectal cancer ( 27% ) and breast cancer ( 23% ) . these results suggest that simultaneous detection of transcripts of multiple ssx genes by rt - nested pcr will be valuable in the diagnosis of ssx - expressing cancer . the known ssx family members share more than 90% homology at the dna level . in the present study , we designed new common primers and developed an ssx 1 - 9 assay that can detect cancer cells expressing at least one of the 9 ssx genes . our ssx 1 - 9 assay successfully detected the transcripts of all ssx 1 - 9 genes in 31 out of 35 cancer cell lines . in order to compare the sensitivity of mage 1 - 6 and ssx 1 - 9 assays , and to evaluate the value of combining both assays in diagnosis , we detected the transcripts of mage and ssx genes in 29 cancer tissue samples of head and neck cancer patients . the mage 1 - 6 and ssx 1 - 9 assays were positive in 82.8% and 75.9% of cancer tissues , and 96.6% of cancer tissues were positive for at least one of the two assays . the ssx - positive rate in head and neck cancer tissues observed in the present study is higher than the rate reported by tureci et al . the results from the present study show that the ssx genes , as well as the mage genes , are expressed in head and neck cancer tissue , and that combination of the mage 1 - 6 assay and the ssx 1 - 9 assay can detect more than 94% of head and neck cancers . rt - pcr - based amplification of transcripts that are expressed in cancer cells , but not in normal non - neoplastic cells , is increasingly being used as a sensitive diagnostic tool for detecting rare disseminated or exfoliated cancer cells to improve the accuracy of cancer staging and to aid in developing early detection protocols . in particular , detection of transcripts of cancer markers in sputum may be very useful for cancer screening or early diagnosis of cancer of the respiratory tracts . in this study , we evaluated the sensitivites of mage 1 - 6 and ssx 1 - 9 assays in detecting cancer cells in induced sputum of head and neck cancer patients . it has been reported that head and neck cancer can be diagnosed by sputum cytology ( 14 , 15 ) . the reports suggested that cancer cells from head and neck malignancies can be expelled from cancer tissue and end up in sputum . matsuda et al . ( 14 ) detected 29.4% of t1 lesions and 63.3% of t2 lesions of head and neck malignancies by sputum cytology . in the present study , 33.3% of t1 lesions ( n=3 ) and 88.9% of t2 lesions ( n=9 ) were detected by mage 1 - 6 assay , and 100% of t1 lesions ( n=3 ) and 77.8 % of t2 lesions ( n=9 ) were detected by the ssx 1 - 9 assay . all t1 and t2 lesions were detected by either one of the two assays , and there was no relationship between mage or ssx gene expression and the tumor stage . these results suggest that it may be possible to detect small numbers of cancer cells that express at least one of mage 1 - 6 or ssx 1 - 9 genes in sputum or in malignant tissue by performing the mage 1 - 6 and the ssx 1 - 9 assays together . thus the combination of these assays may become very useful for screening or early diagnosis of primary or recurrent head and neck cancers . the mage 1 - 6 and ssx 1 - 9 gene transcripts were detected in 4.5% and 13.6% of sputum of patients with benign lung disease . these false positive results may be due to the detection of dysplastic cells in sputum . one possible explanation for the false positive amplification of mage or ssx transcripts is the activation of mage genes by dysplastic cells during early tumorigenesis ( 16 ) . another possible explanation is the low level of transcription of mage or ssx genes in normal cells ( 17 , 18 ) . we have been following up these patients at 3 - 6 month intervals some of patients were negative for both mage and ssx assays during the follow - up period , while others consistently tested positive . if the results show continuous positive on follow up test , it is need to do the screening work - up to rule out occult head and neck cancer . we developed an ssx 1 - 9 assay to detect the transcripts and of 9 ssx genes simultaneously , and investigated the usefulness of ssx 1 - 9 mage 1 - 6 assays in the detection of cancer cells in tissue and sputum samples of head and neck cancer patients . the results of the present investigation suggest that the combination of mage 1 - 6 and ssx 1 - 9 assays may be a sensitive diagnostic tool in detecting small numbers of malignant cells in the cancer tissue and induced sputum of head and neck cancer patients .

objectivesthe melanoma antigen gene ( mage ) and synovial sarcoma on x chromosome ( ssx ) gene families are silent in most normal adult tissues , but are expressed in a variety of malignant lesions . therefore , detection of mage and ssx transcription may be useful for the diagnosis of head and neck cancers . the aim of this study is to detect mage and ssx gene transcripts of head and neck cancers using the mage 1 - 6 assay and the ssx 1 - 9 assay.methodsthe transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected by the mage 1 - 6 assay and the ssx 1 - 9 assay respectively , in cancer cell lines , cancer tissue , and induced sputum specimens from head and neck cancer patients.resultsthe transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 82.8% and 75.9% of head and neck cancer tissues ( n=29 ) respectively , and 96.6% of cancer tissues expressed at least one of mage 1 - 6 or ssx 1 - 9 genes . in the induced sputum of head and neck cancer patients ( n=18 ) , the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 72.2% and 77.8% , respectively , and 94.4% of the sputum specimens were positive for either the mage 1 - 6 or the ssx 1 - 9 assay.conclusionthese results suggest that the combination of mage 1 - 6 and ssx 1 - 9 assays may be useful in the diagnosis of head and neck cancer .

INTRODUCTION MATERIALS AND METHODS Cell culture Cancer tissue and sputum specimens MAGE 1-6 assay Designing common primers for SSX 1-9 assay SSX 1-9 assay RESULTS Primer design and development of SSX 1-9 assay Detection of the transcripts of SSX 1-9 genes in cancer cell lines Detection of the transcripts of SSX 1-9 and MAGE 1-6 in cancer tissues Detection of the transcripts of SSX 1-9 and MAGE 1-6 in sputum samples Combination of MAGE 1-6 and SSX 1-9 assay in the cancer tissue and induced sputa of head and neck cancer patients DISCUSSION CONCLUSION

the genes of cancer / testis antigens ( cta ) such as melanoma antigen gene ( mage ) and synovial sarcoma on x chromosome ( ssx ) are thought to be silent in normal adult tissues except testis . however , these genes are expressed at a high frequency in a large variety of cancer cells . however , the use of mage genes in the detection of a small number of cancer cells by reverse transcription - polymerase chain reaction ( rt - pcr ) has been limited by the low expression frequency of individual mage genes in various cancer tissues . in order to improve the detection rate of magegenes , we have recently designed common primers that can bind simultaneously to the cdna of mage-1 , -2 , -3 , -4a , -4b , -5a , -5b and -6 ( mage 1 - 6 ) . we also developed a mage 1 - 6 assay that can simultaneously detect the transcripts of mage 1 - 6 ( 8) . the ssx gene family , which was originally identified as fusion partners to the syt gene in synovial sarcomas , consists of 9 subtype genes ( ssx 1 - 9 ) . in this study , we designed a common ssx primer , and developed an ssx 1 - 9 assay that can detect cancer cells expressing at least one of the 9 ssx genes by rt - nested pcr using the common ssx primers . thirty five cancer cell lines derived from stomach cancer ( snu 484 , snu 620 , snu 638 , snu 668 ) , colon cancer ( snu c1 , snu c4 , snu c5 , ht29 , hct 116 ) , head and neck cancer ( amc - hn3 , amc - hn4 , amc - hn7 ) , leukemia ( u937 , hl 60 ) cervical cancer ( caski , c4-ii , me-180 , hela , cunc-6 , siha ) , lung cancer ( nci - h292 , nci - h522 , nci - h1703 , a-549 ) , prostate cancer ( pc 3 , du 145 ) , hepatocellular carcinoma ( hepg2 , snu 182 , snu 354 , snu 387 , snu 398 , snu 423 ) , kidney cancer ( hek 293 ) , breast cancer ( mda 231 ) , and osteosarcoma ( saos 2 ) were studied for the expression of ssx 1 - 9 genes . the cancer tissue samples were obtained from 29 head and neck cancer patients who were surgically treated at the department of otolaryngology , school of medicine , kosin university , busan , korea . induced sputum samples were obtained from 18 patients with head and neck cancer and 22 patients with benign pulmonary diseases such as pneumonia , bronchitis , and tuberculosis . detection of transcripts of mage 1 - 6 gene was performed using the cancer hunter kit ( ic&g co. ) . the sequences of ssx 1 - 9 gene were obtained from national center for biotechnology information ( besthesda , md , usa ) , and the dna homology of each ssx gene was analyzed using the dnasis program ( hitachi , tokyo , japan ) . detection of transcripts of ssx 1 - 9 genes was performed using a cancer hunter kit ( ic&g co. ) with some modifications . in the first and nested pcr , the common primers for ssx 1 - 9 gene were used in place of common primers for mage 1 - 6 . all other procedures and temperature conditions for pcr were identical to the mage 1 - 6 assay . thirty five cancer cell lines derived from stomach cancer ( snu 484 , snu 620 , snu 638 , snu 668 ) , colon cancer ( snu c1 , snu c4 , snu c5 , ht29 , hct 116 ) , head and neck cancer ( amc - hn3 , amc - hn4 , amc - hn7 ) , leukemia ( u937 , hl 60 ) cervical cancer ( caski , c4-ii , me-180 , hela , cunc-6 , siha ) , lung cancer ( nci - h292 , nci - h522 , nci - h1703 , a-549 ) , prostate cancer ( pc 3 , du 145 ) , hepatocellular carcinoma ( hepg2 , snu 182 , snu 354 , snu 387 , snu 398 , snu 423 ) , kidney cancer ( hek 293 ) , breast cancer ( mda 231 ) , and osteosarcoma ( saos 2 ) were studied for the expression of ssx 1 - 9 genes . the cancer tissue samples were obtained from 29 head and neck cancer patients who were surgically treated at the department of otolaryngology , school of medicine , kosin university , busan , korea . induced sputum samples were obtained from 18 patients with head and neck cancer and 22 patients with benign pulmonary diseases such as pneumonia , bronchitis , and tuberculosis . detection of transcripts of mage 1 - 6 gene was performed using the cancer hunter kit ( ic&g co. ) . the sequences of ssx 1 - 9 gene were obtained from national center for biotechnology information ( besthesda , md , usa ) , and the dna homology of each ssx gene was analyzed using the dnasis program ( hitachi , tokyo , japan ) . detection of transcripts of ssx 1 - 9 genes was performed using a cancer hunter kit ( ic&g co. ) with some modifications . in the first and nested pcr , the common primers for ssx 1 - 9 gene were used in place of common primers for mage 1 - 6 . all other procedures and temperature conditions for pcr were identical to the mage 1 - 6 assay . using highly homologous dna sequences , common primers that can bind simultaneously to ssx 1 - 9 cdnas were designed , and used for the ssx 1 - 9 assay . the binding site of common primers on ssx-1 gene and the products amplified by the ssx1 - 9 assay and the mage 1 - 6 assay are summarized in fig . all primers ( os , oas , and ias ) had 100% homology with the sequences of the 9 ssx cdnas , and were used for the ssx 1 - 9 assay . the transcripts of mage 1 - 6 genes and ssx 1 - 9 genes were detected in snu484 and amc hn-7 cell lines , but not in normal blood cells . the expected dna sizes amplified by the ssx 1 - 9 assay and the mage 1 - 6 assay were 490 - 492 bp and 448 - 472 bp , respectively . in order to evaluate the sensitivity of the ssx 1 - 9 assay , mrna was isolated from 35 cancer cell lines described previously in this paper , and transcripts of ssx 1 - 9 genes were detected by the ssx 1 - 9 assay . thirty one ( 88.6% ) of the 35 cancer cell lines were positive ( fig . all 3 head and neck cancer cell lines were positive in both gene assay ( data not shown ) . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 24 ( 82.8% ) and 22 ( 75.9% ) of 29 fresh cancer tissues from head and neck cancer patients . eighty six percent ( 18/21 ) and 72.7% ( 16/21 ) of squamous cell carcinoma cells expressed mage 1 - 6 genes and ssx 1 - 9 genes , respectively . sixty three percent ( 5/8 ) of other pathologic types of cancers expressed both mage 1 - 6 genes and ssx 1 - 9 genes ( fig . in order to investigate the abilityof mage 1 - 6 and ssx 1 - 9 assays to detect small numbers of cancer cells mixed in sputum , snu484 ( mage- and ssx - positive cell lines ) were added to normal sputum . after digesting the sputum with specimen protector , mrna was isolated , and mage 1 - 6 and ssx 1 - 9 assays were performed . eighteen samples of induced sputum from head and neck cancer patients and 22 samples of induced sputum from patients with benign lung disease were collected . after digestion of sputum with specimen protector and isolation of mrna in liquefied sputum , mage 1 - 6 and ssx 1 - 9 assays were performed . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 72.2% ( 13/18 ) and 77.8% ( 14/18 ) of fresh induced sputum samples from head and neck patients ( fig . the mage 1 - 6 assay detected transcripts in 33.3% of sputum samples from tnm stage t1 tumors , 88.9% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors . the ssx 1 - 9 assay detected transcripts in 100% of sputum samples from t1 tumors , 77.8% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors ( table 3 ) . in order to evaluate the specificity of both assays , transcripts of the mage 1 - 6 and ssx 1 - 9 assays in induced sputum samples from patients with benign lung disease were detected . the mage 1 - 6 assay was positive in 4.5% ( 1/22 ) and the ssx 1 - 9 assay was positive in 13.6% ( 3/22 ) of the samples ( fig . these results represents mage 1 - 6 assay is more specific than ssx 1 - 9 asaay . in head and neck cancer patients , the mage 1 - 6 assay was positive in 82.8% of cancer tissues and 72.2% of induced sputa . the ssx 1 - 9 assay was positive in 75.9% of cancer tissues and 77.8% of induced sputa . when both mage 1 - 6 assay and ssx 1 - 9 assay were performed on the same specimen , 96.6% of cancer tissues and 94.4% of induced sputa were positive in either one of the two assays ( table 4 ) . using highly homologous dna sequences , common primers that can bind simultaneously to ssx 1 - 9 cdnas were designed , and used for the ssx 1 - 9 assay . the binding site of common primers on ssx-1 gene and the products amplified by the ssx1 - 9 assay and the mage 1 - 6 assay are summarized in fig . all primers ( os , oas , and ias ) had 100% homology with the sequences of the 9 ssx cdnas , and were used for the ssx 1 - 9 assay . the transcripts of mage 1 - 6 genes and ssx 1 - 9 genes were detected in snu484 and amc hn-7 cell lines , but not in normal blood cells . the expected dna sizes amplified by the ssx 1 - 9 assay and the mage 1 - 6 assay were 490 - 492 bp and 448 - 472 bp , respectively . in order to evaluate the sensitivity of the ssx 1 - 9 assay , mrna was isolated from 35 cancer cell lines described previously in this paper , and transcripts of ssx 1 - 9 genes were detected by the ssx 1 - 9 assay . all 3 head and neck cancer cell lines were positive in both gene assay ( data not shown ) . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 24 ( 82.8% ) and 22 ( 75.9% ) of 29 fresh cancer tissues from head and neck cancer patients . eighty six percent ( 18/21 ) and 72.7% ( 16/21 ) of squamous cell carcinoma cells expressed mage 1 - 6 genes and ssx 1 - 9 genes , respectively . sixty three percent ( 5/8 ) of other pathologic types of cancers expressed both mage 1 - 6 genes and ssx 1 - 9 genes ( fig . in order to investigate the abilityof mage 1 - 6 and ssx 1 - 9 assays to detect small numbers of cancer cells mixed in sputum , snu484 ( mage- and ssx - positive cell lines ) were added to normal sputum . after digesting the sputum with specimen protector , mrna was isolated , and mage 1 - 6 and ssx 1 - 9 assays were performed . eighteen samples of induced sputum from head and neck cancer patients and 22 samples of induced sputum from patients with benign lung disease were collected . after digestion of sputum with specimen protector and isolation of mrna in liquefied sputum , mage 1 - 6 and ssx 1 - 9 assays were performed . the transcripts of mage 1 - 6 and ssx 1 - 9 genes were detected in 72.2% ( 13/18 ) and 77.8% ( 14/18 ) of fresh induced sputum samples from head and neck patients ( fig . the mage 1 - 6 assay detected transcripts in 33.3% of sputum samples from tnm stage t1 tumors , 88.9% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors . the ssx 1 - 9 assay detected transcripts in 100% of sputum samples from t1 tumors , 77.8% of t2 tumors , 75% of t3 tumors and 100% of t4 tumors ( table 3 ) . in order to evaluate the specificity of both assays , transcripts of the mage 1 - 6 and ssx 1 - 9 assays in induced sputum samples from patients with benign lung disease were detected . the mage 1 - 6 assay was positive in 4.5% ( 1/22 ) and the ssx 1 - 9 assay was positive in 13.6% ( 3/22 ) of the samples ( fig . these results represents mage 1 - 6 assay is more specific than ssx 1 - 9 asaay . in head and neck cancer patients , the mage 1 - 6 assay was positive in 82.8% of cancer tissues and 72.2% of induced sputa . the ssx 1 - 9 assay was positive in 75.9% of cancer tissues and 77.8% of induced sputa . when both mage 1 - 6 assay and ssx 1 - 9 assay were performed on the same specimen , 96.6% of cancer tissues and 94.4% of induced sputa were positive in either one of the two assays ( table 4 ) . the expression of mage and ssx genes are highly specific to cancer cells , but their use in the detection of a small number of cancer cells by rt - pcr has been limited by the low expression frequency of individual genes . it is expressed in a variety of different human neoplasms , but not in normal tissues , with the exception of testis and a weak expression in the thyroid . ( 13 ) studied the expression of ssx genes in 325 specimens of human neoplasms , and found that ssx-1 , -2 , and -4 subtypes are expressed in 8% , 15% and 15% of the cancers , respectively , while the expression of the ssx-5 or ssx-3 subtypes were rare or absent . expression of at least one of the ssx family members was most frequently observed in head and neck cancer ( 75% ) , followed by ovarian cancer ( 50% ) , malignant melanoma ( 43% ) , lymphoma ( 36% ) , colorectal cancer ( 27% ) and breast cancer ( 23% ) . these results suggest that simultaneous detection of transcripts of multiple ssx genes by rt - nested pcr will be valuable in the diagnosis of ssx - expressing cancer . in the present study , we designed new common primers and developed an ssx 1 - 9 assay that can detect cancer cells expressing at least one of the 9 ssx genes . our ssx 1 - 9 assay successfully detected the transcripts of all ssx 1 - 9 genes in 31 out of 35 cancer cell lines . in order to compare the sensitivity of mage 1 - 6 and ssx 1 - 9 assays , and to evaluate the value of combining both assays in diagnosis , we detected the transcripts of mage and ssx genes in 29 cancer tissue samples of head and neck cancer patients . the mage 1 - 6 and ssx 1 - 9 assays were positive in 82.8% and 75.9% of cancer tissues , and 96.6% of cancer tissues were positive for at least one of the two assays . the ssx - positive rate in head and neck cancer tissues observed in the present study is higher than the rate reported by tureci et al . the results from the present study show that the ssx genes , as well as the mage genes , are expressed in head and neck cancer tissue , and that combination of the mage 1 - 6 assay and the ssx 1 - 9 assay can detect more than 94% of head and neck cancers . rt - pcr - based amplification of transcripts that are expressed in cancer cells , but not in normal non - neoplastic cells , is increasingly being used as a sensitive diagnostic tool for detecting rare disseminated or exfoliated cancer cells to improve the accuracy of cancer staging and to aid in developing early detection protocols . in particular , detection of transcripts of cancer markers in sputum may be very useful for cancer screening or early diagnosis of cancer of the respiratory tracts . in this study , we evaluated the sensitivites of mage 1 - 6 and ssx 1 - 9 assays in detecting cancer cells in induced sputum of head and neck cancer patients . the reports suggested that cancer cells from head and neck malignancies can be expelled from cancer tissue and end up in sputum . in the present study , 33.3% of t1 lesions ( n=3 ) and 88.9% of t2 lesions ( n=9 ) were detected by mage 1 - 6 assay , and 100% of t1 lesions ( n=3 ) and 77.8 % of t2 lesions ( n=9 ) were detected by the ssx 1 - 9 assay . all t1 and t2 lesions were detected by either one of the two assays , and there was no relationship between mage or ssx gene expression and the tumor stage . these results suggest that it may be possible to detect small numbers of cancer cells that express at least one of mage 1 - 6 or ssx 1 - 9 genes in sputum or in malignant tissue by performing the mage 1 - 6 and the ssx 1 - 9 assays together . thus the combination of these assays may become very useful for screening or early diagnosis of primary or recurrent head and neck cancers . the mage 1 - 6 and ssx 1 - 9 gene transcripts were detected in 4.5% and 13.6% of sputum of patients with benign lung disease . we developed an ssx 1 - 9 assay to detect the transcripts and of 9 ssx genes simultaneously , and investigated the usefulness of ssx 1 - 9 mage 1 - 6 assays in the detection of cancer cells in tissue and sputum samples of head and neck cancer patients . the results of the present investigation suggest that the combination of mage 1 - 6 and ssx 1 - 9 assays may be a sensitive diagnostic tool in detecting small numbers of malignant cells in the cancer tissue and induced sputum of head and neck cancer patients .

serotonin ( 5-ht ) , also known as 5-hydroxytryptamine or 3-(2-aminoetil)-1h - indol-5-ol , is a monoamine containing two nitrogen molecules : the first nitrogen is basic and embedded within the indol-5-ol ; the second , within 2-aminoethyl , is located at the terminus of the aliphatic chain . 5-ht is generated from tryptophan and serves as a substrate for the synthesis of a diverse set of molecules , such as melatonin , formyl-5-hydroxykynurenamine , and 5-hydroxyindoleacetic acid . in addition , 5-ht is a signaling molecule that affects the immune , gastrointestinal , and nervous systems in paracrine , endocrine , and juxtacrine fashion . finally , 5-ht regulates development during cellular differentiation and ontogeny ( morphogenesis ) in several cell linages [ 57 ] . the majority of 5-ht synthesis , up to 90% , takes place in gastrointestinal enterochromaffin ( ec ) cells , followed by synthesis in myenteric neurons ( 5% ) and the brain [ 8 , 9 ] . in the 1980s , 5-ht was identified as an immunomodulator for its ability to stimulate or inhibit inflammation . this immune regulation which has yet to be fully elucidated therefore , to understand disease pathologies related to the immune system , it is important to consider the function of serotonergic components . specifically , insight can be gained by understanding how serotonergic components are related to mechanisms of immune modulation that depend on 5-ht receptors ( 5htr ) expression in leukocytes and other cells involved in an inflammatory response . the discovery of 5-ht was a product of collaborative endeavors initiated in the last quarter of the 19th century that lasted into the second half of the 20th century . initial studies identified an extract with vasoconstriction properties from a platelet fraction of uncoagulated blood . in research conducted in rome during the 1930s , vittorio erspamer isolated a molecule from gastrointestinal ec cells with the capacity to generate smooth muscle contractions in a rat uterus . chemical analysis identified the molecule as an indoleamine and it was named enteramine . during the 1940s in the cleveland clinic research department , maurice rapport , arda green , and irving page purified and characterized a vasoconstrictor compound generated shortly after coagulation and related to hypertension . in a tour de force , the molecule was purified from 900 liters of serum obtained from 2 tons of bull 's blood [ 14 , 15 ] . the name serotonin emerged after the substance was crystallized in 1948 because it was obtained from serum ( ser ) and could induce vascular tone ( tonin ) in blood vessels . subsequently , the crystalline vasoconstrictor substance was shown to be a single complex composed of creatinine and indol - derivates , which permitted a structural model of 5-ht based on uv - spectrophotometry . chemical synthesis of 5-ht by hamlin and fischer in 1951 provided significant progress allowing for the confirmation of its pharmacological effects and a comparison with the previously isolated enteramine . interest in understanding the physiological role of 5-ht prompted efforts to isolate the compound from different mammals and tissues , such as the central nervous system . since the 1970s there has been an established association between the serotonergic system and affective disorders as well as mood changes . recently , serotonin has been associated with a myriad of processes , including aggression , sleep , appetite , pain , bone density , tissue regeneration , platelet aggregation , and gastrointestinal function . the influence of 5-ht on the immune system has also been recognized , although the specific mechanisms underlying these effects are not completely understood and may require confirmation in human cells . despite these pitfalls , it is well acknowledged that the serotonergic system and associated molecules expressed in immune cells can influence mood disorders , such as major depression and schizophrenia [ 33 , 34 ] . while the expression and function of serotonergic proteins has primarily been studied within the central nervous system , it should be pointed out that no functional differences between cell types have been identified . the serotonergic components expressed in the immune system encompass a complex ensemble of proteins that coordinate the synthesis and degradation , transport and storage , and response to 5-ht stimulation . in leukocytes , the expression of serotonergic components ( table 1 ) is modulated by the concentration of extracellular and intracellular 5-ht . furthermore , the signals generated by 5-ht interactions with leukocytes are distinct depending on function , developmental stage , and activation status of the cell . this functional heterogeneity suggests that the serotonergic system can precisely regulate a wide range of immunomodulatory effects . the essential amino acid tryptophan is utilized by many cell types and can be converted into a wide range of chemically related products , among the best known are 5-ht and melatonin , but also include kynurenines and kynurenamines ( figure 1 ) . in macrophages and t lymphocytes ec : 1.13.11.52 ) [ 60 , 82 ] enzymes help degrade tryptophan to generate kynurenines and produce kynurenamines from 5-ht or melatonin . in general , all of these compounds can modulate immune responses [ 1 , 8486 ] . however , the mechanisms by which these molecules exert an immunomodulating function are not completely elucidated . some observations suggest that kynurenines and kynurenamines function in negative feedback loops to modulate 5-ht - mediated inflammation , other proinflammatory molecules , and melatonin levels . the synthesis of serotonin begins with the essential amino acid , tryptophan , and follows two - enzymatic steps . first , a hydroxyl group is added by tryptophan 5-hydroxylase ( tph ; ec : 1.14.16.4 ) to generate 5-hydroxytryptophan . while tph1 is expressed in peripheral tissues , tph2 is exclusively expressed in the central nervous system [ 8789 ] . after this hydroxylation step , a carboxyl group is removed by an aromatic l - amino acid decarboxylase ( ddc ; ec : 4.1.1.28 ) generating 5-ht [ 9092 ] . within the immune system , four catabolic pathways for the breakdown of 5-ht have been observed . one pathway begins with the generation of melatonin from 5-ht through two enzymatic steps ; first , 5-ht is acetylated by arylalkylamine n - acetyltransferase ( aanat ; ec : 2.3.1.87 ) generating n - acetyl 5-ht , which then acquires a methyl group from n - acetylserotonin - o - methyltransferase ( asmt ; ec : 2.1.1.4 ; previously known as hydroxyndole - o - methyltransferase , hiomt ) to become melatonin [ 93 , 94 ] . subsequently , the enzyme indoleamine 2,3-dioxygenase ( ido1 & ido2 ; ec : 1.13.11.52 ) can convert melatonin into a cyclooxygenase ( cox ; ec : 1.14.99.1 ) inhibitor called formyl - n - acetyl-5-methoxykynurenamine . interestingly this metabolite can function to block the synthesis of prostaglandins , yet other melatonin metabolites , including 5-methoxyindole acetic acid and 6-hydroxymelatonin , have no reported function . a second catabolic pathway of 5-ht utilizes the enzyme indoleamine 2,3-dioxygenase ( ido ) and generates formyl-5-hydroxykynurenamine . in a third pathway , 5-ht is transformed into 5-hydroxyindoleacetic acid with monoamine oxidase a / b ( mao - a o mao - b ; ec : 1.4.3.4 ) among other enzymes . interestingly , mao - a expression , which is regulated by the cytokines il-4 and il-3 , has been identified in human monocytes from peripheral blood . a fourth catabolic pathway generates n - methylserotonin from 5-ht using the enzyme amine n - methyltransferase ( inmt ; ec 2.1.1.49 ) and may also be active in immune cells . it is not thoroughly understood that the extent by which the byproducts and metabolites generated during catabolism may affect the immune system [ 1 , 84 ] . the identification of additional 5-ht metabolites in plasma , including serotonin - o - sulfate and 5-hydroxykynurenamine , suggests that other catabolic pathways linked to specific biochemical processes , such as activation and cell proliferation , may also be associated with the modulation of the immune system . one additional catabolic pathway related with the four previously described utilizes the enzyme ido to generate kynurenines from tryptophan . this pathway is positively regulated when immune cells become activated and begin secreting ifn- , ifn- e ifn- , tnf- , tgf- , il-1 , and il-2 [ 85 , 99101 ] , which significantly consumes tryptophan and limits its availability for 5-ht production . l - kynurenine , 3-hydroxy - l - kynurenine , and 3-hydroxyanthranilic acid can negatively modulate immune responses . specifically , vcsei and coworkers noted the blockage of cell proliferation as well as the potential induction of apoptosis in th1 and nk cells [ 85 , 101103 ] . it was recently proposed that 5-hydroxyindole thiazolidine carboxylic acid , a 5-ht byproduct found in the intestinal tissues and several brain regions of rats , is generated from the condensation of 5-hydroxyindole acetaldehyde and l - cysteine by a carbon - sulfur lyase ( ec 4.4.1 . ) . in addition the properties of this byproduct or its potential influence over immunological cells remain to be investigated . 5-ht modulates many leukocyte functions ranging from activation of the immune response to memory cell generation . the effects mediated by 5-ht are dependent on the differential expression of serotonergic components in leukocytes . for example , serotonin receptors ( 5htr ) on immune cells influence cytokine proliferation , delivery , migration , and cellular activation . signaling through the 5htr affects chemoattraction in immature mammalian dendritic cells ( human and rodent ) but not in mature cells , which respond to 5-ht by secreting il-6 . in addition , 5htr signaling influences nave t cell activation in mice by activating 5ht7 and regulates lymphocyte b cell proliferation through 5ht1a . the 5htr belong to the gpcr family class a , also known as 7-transmembrane domain ( 7tm ) receptors . gpcr are classified into 6 classes according to a database from the international union of basic and clinical pharmacology ( iuphar : http://www.guidetopharmacology.org/ ) . this system includes classes a , b , and c , as well as the adhesion , frizzled , and other 7tm classes . receptors for adenosine , adrenaline / noradrenaline , 5ht1 , 5ht2 , 5ht4 , 5ht5 , 5ht6 , and 5ht7 all belong to gpcr class a. furthermore , the 5htrs are comprised of 6 families and 13 subfamilies with an undetermined number of isoforms that may be produced by alternative splicing . this receptor diversity suggests that a great amount of functional variation may exist between the 5htrs . g proteins form heterotrimeric complexes made of the subunits g , g and g ; the complex is coupled to the c - terminus of the transmembrane 5htr . different subtypes of the g proteins ( gi / o , gs , or gq11 ) may generate different transduction responses functioning either as activators or inhibitors . when the 5-ht ligand binds its receptor to induce signal transduction it elicits a conformational change in the receptor to facilitate activity . signal transduction can be carried out by the effector proteins g or the heterodimer g-g. while transduction mechanisms have been better depicted for g , especially in cells of the nervous system , recent work has been devoted to the role of g and g-g in the immune system [ 109 , 110 ] . in the last few years , a number of investigations have demonstrated that gpcr are capable of assembling dimers ( homo and heterodimers ) as well as oligomers . receptors 5ht1b and 5ht1d can assembly into homodimers and heterodimers when coexpressed in the same cell . the 5ht2c receptors form homodimers within the cellular membrane [ 112114 ] and it has been proposed that signaling is initiated when two 5-ht molecules bind the dimer . however , activation a single subunit within the 5ht4 homodimer is sufficient to initiate g protein activation even though simultaneous activation of both receptors doubles the activation efficiency of the pathway . while it appears that 5htr tend to form homodimers rather than heterodimers , the latter possibility has not been discarded . milligan and coworkers have postulated that the formation of heterodimers could generate specificity between 5htr and its substrates , which could be especially relevant for the use and design of pharmacological agents . despite current knowledge suggesting that the 5-ht receptors function as homodimers and maybe heterodimers , it is possible that higher subunit organizations ( e.g. , trimers and tetramers ) could function under certain circumstances [ 114 , 118 ] . to better understand 5-ht cellular - mediated processes , it will be necessary to characterize the quaternary structure and stoichiometry of 5htr during oligomerization in regular cellular processes . it will also be important to determine the biochemical details of the quaternary transitional structure ( quinary structure ) of 5htr to establish not only its functional characteristics , but also the coupling and assembly with cytoskeleton proteins . recently , the crystal structures of the receptor - agonist complexes , 5ht1b and 5ht2b with ergotamine and dihydroergotamina , respectively , were reported and provided structural information to better understand receptor - ligand interactions and agonist selectivity , which could inform 5htr - based drug design [ 119 , 120 ] . the 5ht3 receptors are part of a cation - selective ion channel cys - loop superfamily and have been detected in t lymphocytes , monocytes , mature dendritic cells , and mast cells . the functional unit of 5ht3 , a pentameric ring , generates a central ion channel and can be composed of two 5ht3a and three 5ht3b subunits [ 121 , 122 ] . each subunit contains a large n - terminal domain with the 5-ht binding site and four transmembrane domains connected with intracellular and extracellular loops [ 123125 ] . the 5ht3 receptors are regulated through protein modifications and cytoskeletal rearrangements , dependent on protein kinases ( a and c ) and f - actin , respectively [ 123 , 126 ] . there are a number of 5ht3 receptor variants that can be expressed from the human genome . the genes encoding 5ht3a and 5ht3b are located on chromosome 11q23 , and those encoding 5ht3c , 5ht3d , and 5ht3e are on chromosome 3q27 . however , the total number of isoforms generated from these genes by alternative splicing has yet to be determined [ 127130 ] . interestingly , the subunits 5ht3c , 5ht3d , 5ht3e , and 5ht3ea are not sufficient to form functional pentamers but can generate them with 5ht3a and hence modify 5-ht responses . in the context of the central nervous system , 5ht3 receptors are associated with rapid activation and inhibition responses in addition to fast cellular depolarization [ 49 , 132 ] . the cellular depolarization response is unique to neurons , as this has not been observed in immune cells . in neurons , 5ht3 receptors modulate the delivery of neurotransmitters , such as dopamine , whereas the same receptors elicit the release of cytokines from immune cells . in addition , 5ht3a is expressed in nave and activated b - lymphocytes , t lymphocytes , and human monocytes , but expression has not been detected from monocyte - derived dendritic cells . inhibiting the 5ht3 receptors with antagonists , such as ondasetron and tropisentrol , disrupts tnf- and il-1 production , suggesting that these receptors may activate the p38/mapk pathway [ 134 , 135 ] . the serotonin transporter sert actively moves extracellular 5-ht across the plasma membrane into the cell . the transporter is also known as solute carrier family 6 , member 4 ( slc6a4 ) , and belongs to a family of neurotransmitters with 12 transmembrane domains . the function of sert in platelets is critical for maintaining adequate 5-ht concentrations in the circulatory system . to function , sert depends on the transport of na / cl ions , yet the coordinated mechanism of 5-ht and ion transport remains to be elucidated . the gene encoding sert has 14 exons and is located on chromosome 17q11.1 - 17q12 . two genetic polymorphisms in the gene regulatory region modulate transcription generating a complex mix of long and short variants . the sert functional unit is a dimer but it has been suggested that two sert homodimers could assemble into a tetramer . other members of the protein family form heterodimers ; however , these may not be functional . the sert proteins are glycosylated , and then sialic acid is inserted into each of two n - linked glycans . in the absence of glycosylation , furthermore , the addition of sialic acid molecules is important for dimer formation and the association with myosin iia ( a kinase that anchors protein kinase g , pkg ) at the cytoskeleton . a complex regulatory mechanism of sert internalization associated with the cytoskeleton has been described in platelets and is referred to as serotonylation ( see box 1 ) . the process of serotonylation depends on the 5-ht gradient between the extracelluar and intracellular space created by sert activity . the gradient mediates regulation via cytoskeletal components , vimentin , 5htr , sert , small gtpases , transglutaminases ( tgases ) , and possibly p21 activated kinases ( p21/pak ) . specifically , increased levels of 5-ht activate sert internalization through activation of small gtpases by serotonylation ( tgases covalently linking the 5-ht to the small gtpases ) . currently efforts are being directed to the characterization of sert internalization mechanisms in leukocytes . serotonylation is the process by which 5-ht is covalently bound to a protein through a transamination reaction and constitutes a mechanism for regulating signal transduction . this process requires high intracellular levels of 5-ht and is mediated by transglutaminases ( tgases . physiological serotonylation has been demonstrated in platelets and other cells [ 142 , 143 ] but not in leukocytes . however , serotonylation may be involved in specific leukocyte functions required for chemotaxis or cytokine secretion [ 43 , 45 ] because this modification regulates similar functions in platelets and pancreatic beta cells.although many cytoplasmic proteins can be serotonylated , the effect of serotonylation on small gtpases during platelet activation and aggregation is noteworthy . serotonylation induces constitutive rhoa activation and , consequently , the cytoskeletal reorganization needed for aggregation [ 144147 ] . increased intracellular ca and 5-ht in platelets also activates rab4-mediated exocytosis of alpha granules by ca - dependent tgase - mediated serotonylation .in furthermore , serotonylation of rhoa not only constitutively activates it , but also increases its rate of proteosomal degradation . since rab27a and rab27b also participate in exocytosis from platelets and endocrine cells [ 151155 ] , these processes may be also susceptible to serotonylation.additionally , 5-ht induces vimentin filament reorientation around sert . furthermore , serotonylation of rab4 promotes interaction with the sert c - terminal domain regulating translocation . importantly , there are no reports that specifically study serotonylation in leucocytes , but available information suggests that it may regulate cytoskeletal reorganization in these cells [ 43 , 45 ] . rab27a is expressed in cytotoxic t cells and regulates the last step of granular exocytosis [ 158 , 159 ] . rab27 , rhoa , and rab4 are also expressed in several cells of the immune system [ 160162 ] ; therefore it is possible that serotonylation regulates exocytosis or cytoskeletal reorganization during functions such as mhc presentation . serotonylation is the process by which 5-ht is covalently bound to a protein through a transamination reaction and constitutes a mechanism for regulating signal transduction . this process requires high intracellular levels of 5-ht and is mediated by transglutaminases ( tgases . physiological serotonylation has been demonstrated in platelets and other cells [ 142 , 143 ] but not in leukocytes . however , serotonylation may be involved in specific leukocyte functions required for chemotaxis or cytokine secretion [ 43 , 45 ] because this modification regulates similar functions in platelets and pancreatic beta cells . although many cytoplasmic proteins can be serotonylated , the effect of serotonylation on small gtpases during platelet activation and aggregation is noteworthy . serotonylation induces constitutive rhoa activation and , consequently , the cytoskeletal reorganization needed for aggregation [ 144147 ] . increased intracellular ca and 5-ht in platelets also activates rab4-mediated exocytosis of alpha granules by ca - dependent tgase - mediated serotonylation . in pancreatic beta cells , rab3a and rab27a furthermore , serotonylation of rhoa not only constitutively activates it , but also increases its rate of proteosomal degradation . since rab27a and rab27b also participate in exocytosis from platelets and endocrine cells [ 151155 ] , these processes may be also susceptible to serotonylation . furthermore , serotonylation of rab4 promotes interaction with the sert c - terminal domain regulating translocation . importantly , there are no reports that specifically study serotonylation in leucocytes , but available information suggests that it may regulate cytoskeletal reorganization in these cells [ 43 , 45 ] . rab27a is expressed in cytotoxic t cells and regulates the last step of granular exocytosis [ 158 , 159 ] . rab27 , rhoa , and rab4 are also expressed in several cells of the immune system [ 160162 ] ; therefore it is possible that serotonylation regulates exocytosis or cytoskeletal reorganization during functions such as mhc presentation . three potential outcomes may take place following 5-ht internalization : signaling may be activated by serotonylation , 5-ht may undergo enzymatic transformation , or 5-ht may be stored in specialized vesicles . the synthesis , storage , and transport of 5-ht in the immune system are more diverse and complex than previously reported , and a seminal review of the subject was recently published by ahern in 2011 . storage of 5-ht in the immune system allows for its reuse by exocytosis , which occurs in dendritic cells , peripheral blood lymphocytes , and platelets . within these cells , the vesicular monoamine transporter ( vmat ) the 5-ht transport and storage / exocytosis pathways in platelets are coupled to the serotonylation signaling pathway , which assesses 5-ht concentrations and dictates its fate . lamp1 containing vesicles in monocyte - derived dendritic cells from mice also store 5-ht , and upon atp stimulation ( ca influx ) , these cells secrete 5-ht and cytokines . a major focus of current endeavors is the investigation of several 5-ht mediated processes in other types of leukocytes , including 5-ht vesicle storage , small gtpases - mediated serotonylation , cytoskeletal associations , and the metabolism of 5-ht into derivatives , such as melatonin and kynurenins . innate and adaptive immune responses rely on a diverse set of cell types from lymphoid linage ( t cells , b cells , and nk cells ) , myeloid linage ( neutrophils , eosinophils , basophils , monocytes , and mast cells ) , or myeloid / lymphoid linage ( dendritic cells ) origins . professional antigen presenting cells ( apc ) , such as dendritic cells and macrophages , link the innate and adaptive immune responses by recognizing , processing , and presenting antigens on mhc - ii . antigen presentation activates nave t cells initiating clonal proliferation and generating the immune memory , which is essential for the adaptive phase of an immune response . the local concentration of 5-ht can modulate a number of events during these immune responses . one reason immune cells respond to 5-ht is that , as mentioned previously , they constitutively express the molecular machinery that constitutes the serotonergic system . neutrophils , the most abundant innate immune cells in human blood , constitute a first line of defense against infection by recognizing foreign antigens , producing antimicrobial compounds , and secreting cytokines and chemokines to recruit immunocompetent cells . mouse models have demonstrated that neutrophil recruitment to sites of acute inflammation requires platelet - derived chemotactic stimuli , such as 5-ht , paf ( platelet - activation factor ) , and histamine [ 167 , 168 ] . macrophages as well as their precursors , circulating monocytes , participate in immune responses during pathogen infection . monocytes can be divided in two subsets : those that express cd14 , a component of the lipopolysaccharide ( lps ) receptor complex , and those that express cd16 , the fcriii immunoglobulin receptor [ 169 , 170 ] . cd14 + cells , which constitute 8090% of the circulating monocytes , express 5ht1e , 5ht2a , 5ht3 , 5ht4 , and 5ht7 mrnas . lps stimulation does not affect 5htr expression suggesting that the receptors constitutively regulate cell functions . however , 5-ht has been noted to elicit a number of responses in cd14 + cells isolated from peripheral blood of healthy subjects : ( i ) 5-ht signaling decreases tnf- secretion in a dose - dependent manner ; ( ii ) 5-ht signaling enhances lps - induced secretion il-12p40 from activated monocytes , which acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated dendritic cells ; and ( iii ) 5-ht signaling enhances lps - induced secretion of il-6 , il-1 , il-8/cxcl8 . the first two effects are mediated by the 5ht4 and 5ht7 receptors , whereas the third effect requires 5ht3 , 5ht4 , or 5ht7 . macrophages also respond to 5-ht although reports conflict as to whether the response is inhibitory or stimulatory and the mechanisms involved have yet to be clearly described . for example , combined stimulation with 5-ht and muramyl peptides induces superoxide secretion by peritoneal macrophages . bovine alveolar macrophages release chemotactic factors for neutrophils and monocytes in response to 5-ht and histamine . similarly , murine macrophages detect 5-ht with the 5ht2c receptor to induce the secretion of ccl2 , which induces monocyte migration . in peritoneal murine macrophages 5-ht induces phagocytosis in a dose - dependent manner through 5ht1a and nf-b activation . on the other hand , 5-ht for example , 5-ht2 signaling limited the activation of murine macrophages stimulated in vitro with high concentrations of ifn- . furthermore , human alveolar macrophages stimulated with lps and 5-ht secreted less tnf- and il-12 , but more il-10 , nitric oxide , and prostangladin - e2 . however , the receptors mediating these effects have yet to be described . the different macrophage responses elicited by 5-ht may be due to phenotypic differences in tissue - specific macrophages ( i.e. , changes in the proportion of 5-ht receptors ) or the effect of cooperative signaling with other molecules . finally , macrophages can rapidly metabolize 5-ht to 5-hydroxyindole acetic acid , a biotransformation pathway that may be mediated by mao - a / b , aldh / aox and asmt , which could affect serotonergic responses in these cells . dcs play a crucial role in the immune response to infectious pathogens . in humans , circulating dcs characteristically expresses high levels of class ii hla molecules and are proficient in antigen uptake and processing . however , they express low levels of hla class i and costimulatory molecules , such as cd80 and cd86 , and lack common lineage markers such as cd3 , cd14 , cd16 , cd19 , cd20 , and cd56 . dcs are positioned between the adaptive and innate immune systems : detecting microbial infection , tissue damage , and inflammatory signals to promote the activation of antigen - specific responses [ 180182 ] . culturing cells with il-4 and granulocyte - macrophage colony - stimulating factor ( gm - csf ) induces human monocytes and murine myeloid progenitors to differentiate into monocyte - derived dendritic cells ( mddcs ) and bone marrow dendritic cells ( bmdcs ) , respectively . these cells , frequently used as models for dendritic cell biology [ 50 , 58 , 183 ] , are sensitive to lps and 5-ht ( table 2 ) . when 5-ht is added during il-4/gm - csf differentiation , the resulting mddcs display lower levels of cd1a , cd86 , and hla - dr but had increased cd14 expression . however , other markers such as cd40 , cd80 , or cd83 were unaffected . murine bmdcs can be matured by lps stimulation to generate cells with characteristics of dcs . although immature ( cd11c+cd86 ) and mature ( cd11c+cd86 + ) bmdcs constitutively express sert , maturation induced by lps increases sert expression and , consequently , mature bmdcs have an increased capability for intake , storage , and exocytosis of 5-ht . bmdc maturation also reduces the expression of enzymes involved in the metabolism of 5-ht , such as mao - a and -b . furthermore , immature and mature dendritic - like cells respond differently to 5-ht . in mature mddcs , the 5ht3 receptor contributes to changes in intracellular ca concentration required for the secretion of il-8 and il-1 . on the other hand , 5-ht inhibits cxcl10 secretion from mature mddc , but ccl22 secretion is not affected . costimulation with 5-ht and lps induces immature mddcs migration in a 5ht1b and 5ht2a - dependent manner . however , if 5-ht is added subsequent to lps - mediated maturation , migration is unaffected but cytokine and chemokine secretion is induced . additional in vitro experiments with mddcs and bmdcs have also demonstrated that 5-ht activates the secretion of pro - inflammatory cytokines [ 2 , 185 ] . one key dc function is the activation of t cells and 5-ht can also regulate this fundamental immunological process . since dendritic - like cells do not express tph1 , it is unlikely that they can synthetize 5-ht . therefore , o'connell and coworkers postulated that sert - expressing dcs are able to internalize 5-ht from the microenvironment . when interacting with t cells , mddcs transiently release ca , which promotes cytokine and 5-ht secretion from lamp1 + vesicles . thus , dcs may internalize and store 5-ht to release into the immunological synapse during t cell activation . activated the synthesis of 5-ht in activated t cells is related to tryptophan metabolism and may also be important when t cells interact with target cells . taken together , 5-ht affects dc differentiation and maturation as well as the profile and function of soluble mediators these cells express . thus , 5-ht may participate in the generation of a specific subset of dcs with unique immunomodulatory properties . differentiation , proliferation , and the functional responses of t cells can each be modulated by the serotonergic system . based on experiments using the cell line k562 , 5-ht participates in t cell maturation in lymphoid organs in a na - coupled , 5-ht active transport - dependent manner ( presumably sert ) , which requires intracellular ca changes . based on the effects of 5-ht receptor - specific agonists or antagonists , t cell proliferation and secretion of proinflammatory cytokines , such as il-2 and ifn- , nave t cells primarily express 5ht7 and low levels of tph1 , but they do not express 5ht1b or sert . after 5-ht stimulation , the erk-1,-2/nf-b pathway is activated in proliferating t cells , and they express 5ht1b , 5ht7 , 5ht2a , and tph1 . in activated human cd4 + t cells , 5-ht or 5ht3 specific agonists impair migration towards cxcl12 gradients , but not to those of ccl2 or ccl5 , which control t cell migration into tissues . however , immature murine cd4 + t cells do not express 5ht3 , and 5-ht does not affect cell migration . but , activation of these cells induces the expression of 5ht3a suggesting they may respond to 5-ht once activated . therefore , the t cell activation state and environment may influence the effects of 5-ht . for example , 5-ht inhibits phytohemagglutinin- ( pha- ) mediated lymphocyte proliferation , possibly through reduced expression and distribution of the il-2 receptor [ 188 , 189 ] . in addition , concanavalin a ( cona ) and low concentrations of 5-ht increased murine t cell proliferation although the activation of cd4 + and cd8 + subsets was reduced [ 190 , 191 ] . this demonstrates that 5-ht elicited effects are concentration dependent and suggests : ( i ) 5-ht induces dose - dependent phenotypes , ( ii ) differentiation may be achieved by receptors with different 5-ht affinity , and ( iii ) local 5-ht concentrations are tightly regulated to induce specific effects . t cells express sert and , therefore , can acquire 5-ht [ 51 , 52 , 192 , 193 ] . however , nave t cells have reduced sert functional activity and may result to 5-ht synthesis . in agreement with this hypothesis , a report from aune and coworkers demonstrates that the inhibition of the 5-ht synthesis in il-2-stimulated t cells blocks cell proliferation . the addition of 5-hydroxtriptophan , a 5-ht precursor , restores proliferation , further suggesting that these cells synthesize the molecule rather than acquire it . however , further research is required to understand how 5-ht synthesis is regulated in t cells . b cells recognize circulating antigen ; as a consequence , they activate processes that end in the generation of memory b cells or antibody - forming plasma cells . in addition , sert expression is proportional to the proliferation rates of human leukemic b cells . specifically , sert - specific inhibitors , such as fluoxetine , fenfluramine , or 3,4-methylenedioxymethamphetamine ( mdma ) , elicited anti - proliferative and pro - apoptotic effects . nk cells recognize antigen in the context of cd1 controlling viral replication early in infection and inhibiting the development of cancer [ 196 , 197 ] . these cells are inhibited with oxidation produced by autologous monocytes and with apoptosis induced by reactive oxygen species ( ros ) ; however , 5-ht signaling limits these forms of inhibition . in fact , 5ht1a - specific antagonists , such as pindobind , exacerbate the inhibitory effect of monocyte - mediated ros production on nk cells . eosinophils are responsible for fighting multicellular parasites and other infections in vertebrates ; they also control mechanisms associated with allergy and asthma . eosinophils express the 5-ht receptors 5ht1a , 5ht1b , 5ht1e , 5ht2a , and 5ht6 , but they do not express 5ht2c , 5ht3 , 5ht4 , and 5ht7 . however , differential expression of 5ht2a was detected in allergy and asthma patients . the serum levels of 5-ht are higher in symptomatic asthma patients in comparison to asymptomatic patients , which may influence eosinophil - mediated inflammation in patients with active disease . 5-ht is a potent chemoattractant for eosinophils both in vivo and in vitro and supports rolling , an important feature of these cells . antagonists of 5ht2a inhibit both effects suggesting that 5-ht mediates eosinophil activation . migration and rolling require changes in the actin cytoskeleton and activation of pkc and calmodulin signaling [ 39 , 45 ] , which control the morphological changes required for eosinophil infiltration from circulation to sits of inflammation . while basophils represent less than 2% of leukocytes , they actively participate in immune responses in peripheral organs where they are recruited during nematode and ectoparasite infections . they also participate in allergic reactions by releasing histamine in response to specific growth factors , such as il-3 [ 203 , 204 ] . in addition , basophils are an important source of il-4 and therefore may promote th2 differentiation [ 205 , 206 ] . the role of 5-ht on basophil functions has not been clarified . murine basophil exposure to 5-ht inhibits il-4 secretion in a dose - dependent manner both in vitro and in vivo . 5-ht also blocks the release of histamine , il-4 , and il-6 from murine basophils following il-3 stimulation as well as blocking the release of il-13 and il-4 from human peripheral blood basophils . intraperitoneal administration of il-33 to mice normally increases the serum levels of il-4 , but is blocked by the administration of 5-ht . although murine basophils express sert , drugs targeting the transporter , such as fluoxetine or citalopram ( selective serotonin reuptake inhibitor , ssri ) , do not block the effect of 5-ht on cytokine release suggesting that other transporters may be used , such as the organic cation transporter 3 . human mast cells express 5ht1a , 5ht1b , 5ht1e , 5ht2a , 5ht2b , 5ht2c , 5ht3 , 5ht4 , and 5ht7 . cell migration and fibronectin adhesion are both influenced by the addition of 5-ht to these cells . although 5ht2a is the predominant receptor , responses are primarily mediated through 5ht1a and can be blocked by the g - protein inhibitor pertussis toxin . platelets are well known for initiating coagulation and maintaining vascular tone ; however , these cells also participate in inflammatory responses by releasing histamine and paf . they provide a local source of biogenic amines , including 5-ht , in damaged regions of the vasculature . platelets uptake 5-ht from plasma in a fast and saturable process ; therefore , they are also key regulators of the circulatory 5-ht concentration . 5-ht uptake is mediated by sert , and once inside the cell it is transported to dense granules by vmat ( vesicle monoamine transporter ) or hydrolyzed by mao . 5-ht signaling activates rab4 , which controls alpha granule secretion , and rhoa , which induces the cytoskeletal reorganization required for adhesion and aggregation [ 144 , 145 , 209 ] . it is reported that rab4 activation occurs by serotonylation ( see box 1 ) , and it is likely that rhoa is similarly activated . increases in the serum levels of 5-ht enhance sert density on the platelet cell membrane [ 142 , 148 , 156 , 157 ] . some studies suggest that human platelets initiate murine t cell activation by fcri - mediated contact sensitivity and the release of 5-ht . however , the functional role that platelet - derived 5-ht plays in the immune system is still far from being fully understood . immune cells respond to 5-ht with varying degrees of sensitivity , which can be partially explained by differences in the expression of serotonergic components . in this section , we review how pathologies with reported alterations to the serotonergic system affect the immune system ( table 2 ) . we also discuss the effects of sert - targeting drugs , such as ssris , as well as drugs that target 5-ht receptors ( table 3 ) . major depressive disorder ( mdd ) , fibromyalgia , infections , and alzheimer 's disease commonly display reduced 5-ht serum levels . the precise effects these changes have on the immune system in each disease are poorly defined . however , mdd provides a good example because serotonergic alterations are directly related to the severity of disease . mdd is defined as a pervasive and persistent low mood with a multi - factor cause . symptoms have degrees of severity that are associated with changes in both cns and peripheral 5-ht concentrations . in addition , mdd patients commonly have altered cortisol and cytokine blood levels [ 210212 ] . lymphocytes from mdd patients express lower levels of sert in comparison with those from healthy volunteers without changes in the intracellular concentration of 5-ht [ 52 , 64 ] . there are no changes in sert expression in monocytes , but the intracellular concentration of 5-ht in monocytes is higher in mdd patients . mdd patient lymphocytes display a three - fold increase in lps - stimulated proliferation , an effect blocked by 5-ht1 antagonists . in addition , there are more 5-ht2a clusters on the lymphocytes of mdd patients , whom are responsive to ssri treatment . when mdd patients are treated with ssris there are changes in lymphocyte subpopulations and in systemic inflammatory mediators ( table 4 ) . before treatment , mdd patients have higher blood cortisol , il-4 , il-13 , and il-10 than healthy volunteers [ 210212 ] . after 20 weeks of treatment , concomitantly with a remission of the depressive episode there are increases in il-2 and il-1 but no change in cortisol levels . at week 52 of treatment there is a significant reduction in cortisol levels with an increase in il-1 and ifn- and a decrease in anti - inflammatory cytokines . regarding lymphocyte subpopulations , before ssri treatment mdd patients had more nk cells compared to healthy volunteers ( 312 29 versus 158 30 ; cells / ml ) , but no differences were found in the t and b cell populations . after 20 weeks of treatment , patients experienced a remission of depressive episodes along with an increase in nk cell and b cell populations , which remained heightened until the 52nd week of treatment . these findings in conjunction with the fact that lymphocytes from mdd patients respond differently than healthy subjects suggest that the general inflammatory response and specific immune subsets are sensitive to systemic levels of 5-ht and changes in those levels induced by ssris . however , further studies are still required to fully understand how components of the serotonergic system are differentially expressed on immune cell subsets . this may clarify the mechanisms involved in mdd progression and highlight new therapeutic targets for its treatment . fibromyalgia ( fm ) is a common chronic pain syndrome that primarily affects the joints and muscles and is generally associated with other somatic and psychological symptoms , including fatigue , poor sleep , cognitive difficulties , and stress . fm patients have central sensitization and increasing glial cell activation , which , in turn , favors pain signaling and activates the release of pro - inflammatory cytokines , nitric oxide , prostaglandins , and ros that sustain the hyperexcitable state of the spinal cord [ 215217 ] . 5-ht3 are involved in pain control , indicating a key participation of the serotonergic system [ 218 , 219 ] . levels of 5-ht are low in the serum and cerebrospinal fluid of fm patients and correlate with clinical symptoms [ 66 , 220222 ] . the administration of the 5-ht3 antagonist tropisetron or high doses ( 45 mg ) of the ssri fluoxetine produce analgesic and/or other beneficial effects in fm patients ( table 4 ) , suggesting that regulation of the serotonergic system can be useful . however , to date it is not known whether the components of the serotonergic system can be altered in the immune cells of patients . immune responses to viruses , bacteria , fungi , and parasites all require 5-ht . human immunodeficiency virus ( hiv ) infection is a primary model for the study of 5-ht during infection ( table 4 ) . 5-ht controls hiv replication in t4 lymphocytic cell lines and modulates nk cell activation in hiv - infected patients . 5-ht decreases the expression of the hiv coreceptor ccr5 on infected macrophages and reduces proviral synthesis 50% [ 228 , 229 ] . these effects can also be achieved with an agonist targeting 5-ht1 but not with one of 5-ht2 . furthermore , shiv - infected pbmcs from rhesus monkeys ( macaca mulatta ) have 10 times less sert mrna than uninfected controls . the authors of this study suggest that low sert expression may be responsible for the symptoms of depression found in hiv - patients . similarly , ssris stimulate macrophage activity in vivo and reduce hiv replication in macrophages and t cells . interestingly , these effects were independent of patients ' psychological status indicating that mood changes are not necessary for 5-ht to have an immunomodulatory effect . these findings suggest that components of the serotonergic system may be suitable therapeutic targets for the control of hiv infection . patients infected with hepatitis c virus ( hcv ) and treated with ifn- have reduced levels of tryptophan and kynurenine , suggesting that 5-ht synthesis and systemic concentrations may be reduced . furthermore , hcv - infected patients given ssri therapy have lower viral replication rates . therefore , ssris and/or 5-htr - targeting drugs may be beneficial for many viral infections . there is also evidence that ssris have antibacterial ( especially against gram - negative bacteria ) [ 233 , 234 ] , antifungal , and antiparasitic [ 79 , 236 ] effects . the available reports establish a direct cytotoxic effect of ssris on the pathogen ( box 2 ) . however , it will be interesting to characterize whether immune cells contribute to infection control during ssri treatment . box 2 ( selective serotonin reuptake inhibitors ( ssris ) have antiparasitic and antifungal activity ) . sertraline and fluoxetina decrease in vitro cell viability of aspergillus spp . and candida parapsilosis [ 235 , 237 , 238 ] . sertraline is likely effective at controlling leishmania donovani infection in a mouse model by inhibition of parasite respiration . mianserine decreases the motility of schistosoma mansoni , the most common species of schitosomes , and 5-ht receptors are expressed in these helminthes at the larvae and adult stages but are overexpressed once they enter ncs . together these results demonstrate that parasites and fungi express sert - like proteins indicating that they are likely sensitive to ssris and systemic changes of 5-ht in the host . furthermore , the serotonergic system in parasites and fungi may constitute a pharmacological target for drug design . a blast search using the human sequence of sert ( gen slc6a4 ) against the aspergillus taxa ( taxid : 5052 ) in the genebank database identified seven conserved hypothetical proteins assigned either as uncharacterized eukaryotic solute carrier 6 ( eau35443.1 ; xp_682235.1 ; cbf84552.1 ; xp_001215815.1 ; eit73756.1 ) or sodium / chloride dependent neurotransmitter transporter ( xp_001826855.1 ; xp_002385196.1 ) . sertraline and fluoxetina decrease in vitro cell viability of aspergillus spp . and candida parapsilosis [ 235 , 237 , 238 ] . sertraline is likely effective at controlling leishmania donovani infection in a mouse model by inhibition of parasite respiration . mianserine decreases the motility of schistosoma mansoni , the most common species of schitosomes , and 5-ht receptors are expressed in these helminthes at the larvae and adult stages but are overexpressed once they enter ncs . together these results demonstrate that parasites and fungi express sert - like proteins indicating that they are likely sensitive to ssris and systemic changes of 5-ht in the host . furthermore , the serotonergic system in parasites and fungi may constitute a pharmacological target for drug design . a blast search using the human sequence of sert ( gen slc6a4 ) against the aspergillus taxa ( taxid : 5052 ) in the genebank database identified seven conserved hypothetical proteins assigned either as uncharacterized eukaryotic solute carrier 6 ( eau35443.1 ; xp_682235.1 ; cbf84552.1 ; xp_001215815.1 ; eit73756.1 ) or sodium / chloride dependent neurotransmitter transporter ( xp_001826855.1 ; xp_002385196.1 ) . alzheimer 's disease is a neurodegenerative disorder and the primary cause of dementia in elderly people . -amyloid deposits in senile plaques and neuro - fibrillary tangles that affect brain cell function are characteristic of the disease . symptoms of dementia and depression , which are related to reduced levels of 5-ht , are present in 5090% of patients . nk cells isolated from patients with alzheimer 's disease have a high density of 5-ht2c compared with cells from late onset depression patients . however , there is no difference in the level of 5-ht1a , 5-ht2a , and 5-ht2b receptors on pbmcs . the abundant increase of 5-ht2c on nk cells may be a compensatory mechanism for reduced 5-ht availability . activation of 5-ht2c inhibits nk cell activity , which may partially explain why alzheimer 's disease patients are more susceptible to viral infections [ 72 , 242 ] . ssris have been used to treat depression in alzheimer 's disease patients . in these patients , similarly , in a murine model of the disease ( happ / ps1 ) , chronic oral administration of a 5-ht4-selective agonist ( ssp-002392 ) reduced -amyloid production and deposition and improved mouse memory . given the complexity of the cell population expressing 5-ht4 in the brain , however , microglial cells may be involved because they express 5-ht4 and can phagocytose -amyloid deposits , an activity promoted by agonist . it remains to be determined whether similarly activated immune cells influence alzheimer 's disease progression and symptoms . from an immunological point of view , the diseases in this group , such as asthma , arthritis , and cancer , are the result of dysregulated inflammatory responses . therefore , the association of these diseases with high circulating levels of 5-ht reinforces its role as an immunomodulator . asthma is a chronic inflammatory disease of the lungs with consequent narrowing of the airways . 5-ht levels are increased in asthma patients and ssri treatment improves clinical symptoms . in vitro , addition of 5-ht or 5-ht1/5-ht2 agonists to alveolar macrophages increases the production of il-10 , nitric oxide , and pge-2 , but reduces tnf- and il-12 production . interestingly , receptor antagonists do not affect secreted - cytokine profiles . regardless , these results indicate that cytokine production is under the control of 5-ht , and therefore , regulating its systemic concentrations may be useful for asthma patients . rheumatoid arthritis is a chronic disease that causes pain , stiffness , and swelling that limits the motion and function of many joints . while ra can affect any joint , smaller joints of the hands and feet are most commonly involved . inflammation can affect organs , such as eyes and lungs , in addition to joints . the 5-ht concentrations in platelet - free blood are 1.6- to 2.3-fold higher in ra patients than in healthy controls ( reported average serum concentrations were 1130 this has led to proposals that 5-ht is involved in the pathology , onset , and/or progression of the disease . treating patients with 5-ht3 antagonists combined with intra - articular glucocorticoids while there have yet to be any reports on the responses of ra patient immune cells to 5-ht , the role of serotonergic system in arthritis or osteoarthritis has been studied in vitro and in vivo . an osteoarthritis model using cultured synovial tissue demonstrated that 5-ht stimulation increases the expression of 5-ht2a and 5-ht3 as well as the release of pge-2 into the medium . the addition of receptor antagonists inhibits pge-2 production , which may explain the beneficial effects seen in arthritis patients given this treatment . fluoxetine and citalopram inhibit disease progression in a collagen - induced mouse model of arthritis as well as in human ra synovial membranes cultures . in addition , macrophages from ra patients display impaired tlr-3 , -7 , -8 , and -9 signaling after ssri exposure . while the role of serotonergic system in cancer patients has not been largely studied , advanced stages of breast cancer correlate with increased levels of systemic 5-ht . in mouse models of melanoma and lymphoma , ssri treatment reduced tumor growth by 50% , inhibited il-10 and ifn- production , and increased il-1 production . similarly , exposing a burkitt 's lymphoma cell line to different ssris ( fluoxetine , paroxetine , or citalopram ) decreased dna synthesis and induced cell death . although further studies are required , these results suggest that the serotonergic system can impact cancer cells directly or indirectly through immune cell activation . given the importance of 5-ht as a neurotransmitter , studies of the serotonergic system have primarily been limited to the cns . recently , however , a large amount of experimental evidence indicates that the serotonergic system has important physiological roles in the immune , vascular , and digestive systems . in this review we discussed the immunomodulatory effects that 5-ht can induce by activating 5htr and sert , which are differentially expressed on many leukocytes . for example , 5-ht induces dose - dependent cytoskeletal reorganization and diapedesis during chemotaxis as well as granule secretion in granulocytes and myeloid cells . in comparison to these non - transcriptional responses , it includes 18 genes , including 5htrs and one sert , several of which have multiple isoforms ( creating at least 10 additional proteins ) . furthermore , the receptor signaling - transduction system that regulates 5-ht responses involves a large number of genes [ 251 , 252 ] providing several points of regulation depending on cellular phenotype . in conclusion , cells of the immune system express transduction machinery that does not necessarily overlap with that in the cns . this allows for differential responses to the same 5-ht ligand within the immune and nervous systems . the information presented here is based on existing reports , but we must consider that many early studies of 5-ht receptors used primarily pharmacologic approaches and the results are sometimes not supported by more recent genetic approaches . for example , although early studies suggest role of sert in t cells , genetic studies suggest that t cells express dat ( dopamine but also low affinity 5-ht transporter ) [ 253 , 254 ] . although the effects of 5-ht on the immune system requires further characterization , it is logical to anticipate altered immune responses in patients with dysregulated serotonergic systems . for these patients , experimental evidence suggests that ssri or 5htr antagonist treatment may provide beneficial immunomodulatory effects .

serotonin ( 5-ht ) induces concentration - dependent metabolic effects in diverse cell types , including neurons , entherochromaffin cells , adipocytes , pancreatic beta - cells , fibroblasts , smooth muscle cells , epithelial cells , and leukocytes . three classes of genes regulating 5-ht function are constitutively expressed or induced in these cells : ( a ) membrane proteins that regulate the response to 5-ht , such as sert , 5htr - gpcr , and the 5ht3-ion channels ; ( b ) downstream signaling transduction proteins ; and ( c ) enzymes controlling 5-ht metabolism , such as ido and mao , which can generate biologically active catabolites , including melatonin , kynurenines , and kynurenamines . this review covers the clinical and experimental mechanisms involved in 5-ht - induced immunomodulation . these mechanisms are cell - specific and depend on the expression of serotonergic components in immune cells . consequently , 5-ht can modulate several immunological events , such as chemotaxis , leukocyte activation , proliferation , cytokine secretion , anergy , and apoptosis . the effects of 5-ht on immune cells may be relevant in the clinical outcome of pathologies with an inflammatory component . major depression , fibromyalgia , alzheimer disease , psoriasis , arthritis , allergies , and asthma are all associated with changes in the serotonergic system associated with leukocytes . thus , pharmacological regulation of the serotonergic system may modulate immune function and provide therapeutic alternatives for these diseases .

1. Introduction 2. A Brief History of 5-HT Discovery 3. Components of the Serotonergic System Are Expressed in Leukocytes 4. The Effects of 5-HT on Leukocytes 5. Changes to the Serotonergic System Affect Immune Responses and Have Clinical Implications 6. Conclusions

serotonin ( 5-ht ) , also known as 5-hydroxytryptamine or 3-(2-aminoetil)-1h - indol-5-ol , is a monoamine containing two nitrogen molecules : the first nitrogen is basic and embedded within the indol-5-ol ; the second , within 2-aminoethyl , is located at the terminus of the aliphatic chain . 5-ht is generated from tryptophan and serves as a substrate for the synthesis of a diverse set of molecules , such as melatonin , formyl-5-hydroxykynurenamine , and 5-hydroxyindoleacetic acid . the majority of 5-ht synthesis , up to 90% , takes place in gastrointestinal enterochromaffin ( ec ) cells , followed by synthesis in myenteric neurons ( 5% ) and the brain [ 8 , 9 ] . specifically , insight can be gained by understanding how serotonergic components are related to mechanisms of immune modulation that depend on 5-ht receptors ( 5htr ) expression in leukocytes and other cells involved in an inflammatory response . the discovery of 5-ht was a product of collaborative endeavors initiated in the last quarter of the 19th century that lasted into the second half of the 20th century . interest in understanding the physiological role of 5-ht prompted efforts to isolate the compound from different mammals and tissues , such as the central nervous system . recently , serotonin has been associated with a myriad of processes , including aggression , sleep , appetite , pain , bone density , tissue regeneration , platelet aggregation , and gastrointestinal function . the influence of 5-ht on the immune system has also been recognized , although the specific mechanisms underlying these effects are not completely understood and may require confirmation in human cells . despite these pitfalls , it is well acknowledged that the serotonergic system and associated molecules expressed in immune cells can influence mood disorders , such as major depression and schizophrenia [ 33 , 34 ] . while the expression and function of serotonergic proteins has primarily been studied within the central nervous system , it should be pointed out that no functional differences between cell types have been identified . the serotonergic components expressed in the immune system encompass a complex ensemble of proteins that coordinate the synthesis and degradation , transport and storage , and response to 5-ht stimulation . in leukocytes , the expression of serotonergic components ( table 1 ) is modulated by the concentration of extracellular and intracellular 5-ht . furthermore , the signals generated by 5-ht interactions with leukocytes are distinct depending on function , developmental stage , and activation status of the cell . the essential amino acid tryptophan is utilized by many cell types and can be converted into a wide range of chemically related products , among the best known are 5-ht and melatonin , but also include kynurenines and kynurenamines ( figure 1 ) . some observations suggest that kynurenines and kynurenamines function in negative feedback loops to modulate 5-ht - mediated inflammation , other proinflammatory molecules , and melatonin levels . one pathway begins with the generation of melatonin from 5-ht through two enzymatic steps ; first , 5-ht is acetylated by arylalkylamine n - acetyltransferase ( aanat ; ec : 2.3.1.87 ) generating n - acetyl 5-ht , which then acquires a methyl group from n - acetylserotonin - o - methyltransferase ( asmt ; ec : 2.1.1.4 ; previously known as hydroxyndole - o - methyltransferase , hiomt ) to become melatonin [ 93 , 94 ] . the identification of additional 5-ht metabolites in plasma , including serotonin - o - sulfate and 5-hydroxykynurenamine , suggests that other catabolic pathways linked to specific biochemical processes , such as activation and cell proliferation , may also be associated with the modulation of the immune system . this pathway is positively regulated when immune cells become activated and begin secreting ifn- , ifn- e ifn- , tnf- , tgf- , il-1 , and il-2 [ 85 , 99101 ] , which significantly consumes tryptophan and limits its availability for 5-ht production . the effects mediated by 5-ht are dependent on the differential expression of serotonergic components in leukocytes . for example , serotonin receptors ( 5htr ) on immune cells influence cytokine proliferation , delivery , migration , and cellular activation . signaling through the 5htr affects chemoattraction in immature mammalian dendritic cells ( human and rodent ) but not in mature cells , which respond to 5-ht by secreting il-6 . receptors for adenosine , adrenaline / noradrenaline , 5ht1 , 5ht2 , 5ht4 , 5ht5 , 5ht6 , and 5ht7 all belong to gpcr class a. furthermore , the 5htrs are comprised of 6 families and 13 subfamilies with an undetermined number of isoforms that may be produced by alternative splicing . recently , the crystal structures of the receptor - agonist complexes , 5ht1b and 5ht2b with ergotamine and dihydroergotamina , respectively , were reported and provided structural information to better understand receptor - ligand interactions and agonist selectivity , which could inform 5htr - based drug design [ 119 , 120 ] . in the context of the central nervous system , 5ht3 receptors are associated with rapid activation and inhibition responses in addition to fast cellular depolarization [ 49 , 132 ] . the cellular depolarization response is unique to neurons , as this has not been observed in immune cells . in neurons , 5ht3 receptors modulate the delivery of neurotransmitters , such as dopamine , whereas the same receptors elicit the release of cytokines from immune cells . in the absence of glycosylation , furthermore , the addition of sialic acid molecules is important for dimer formation and the association with myosin iia ( a kinase that anchors protein kinase g , pkg ) at the cytoskeleton . the gradient mediates regulation via cytoskeletal components , vimentin , 5htr , sert , small gtpases , transglutaminases ( tgases ) , and possibly p21 activated kinases ( p21/pak ) . however , serotonylation may be involved in specific leukocyte functions required for chemotaxis or cytokine secretion [ 43 , 45 ] because this modification regulates similar functions in platelets and pancreatic beta cells.although many cytoplasmic proteins can be serotonylated , the effect of serotonylation on small gtpases during platelet activation and aggregation is noteworthy . rab27 , rhoa , and rab4 are also expressed in several cells of the immune system [ 160162 ] ; therefore it is possible that serotonylation regulates exocytosis or cytoskeletal reorganization during functions such as mhc presentation . however , serotonylation may be involved in specific leukocyte functions required for chemotaxis or cytokine secretion [ 43 , 45 ] because this modification regulates similar functions in platelets and pancreatic beta cells . rab27 , rhoa , and rab4 are also expressed in several cells of the immune system [ 160162 ] ; therefore it is possible that serotonylation regulates exocytosis or cytoskeletal reorganization during functions such as mhc presentation . the synthesis , storage , and transport of 5-ht in the immune system are more diverse and complex than previously reported , and a seminal review of the subject was recently published by ahern in 2011 . storage of 5-ht in the immune system allows for its reuse by exocytosis , which occurs in dendritic cells , peripheral blood lymphocytes , and platelets . within these cells , the vesicular monoamine transporter ( vmat ) the 5-ht transport and storage / exocytosis pathways in platelets are coupled to the serotonylation signaling pathway , which assesses 5-ht concentrations and dictates its fate . lamp1 containing vesicles in monocyte - derived dendritic cells from mice also store 5-ht , and upon atp stimulation ( ca influx ) , these cells secrete 5-ht and cytokines . a major focus of current endeavors is the investigation of several 5-ht mediated processes in other types of leukocytes , including 5-ht vesicle storage , small gtpases - mediated serotonylation , cytoskeletal associations , and the metabolism of 5-ht into derivatives , such as melatonin and kynurenins . innate and adaptive immune responses rely on a diverse set of cell types from lymphoid linage ( t cells , b cells , and nk cells ) , myeloid linage ( neutrophils , eosinophils , basophils , monocytes , and mast cells ) , or myeloid / lymphoid linage ( dendritic cells ) origins . professional antigen presenting cells ( apc ) , such as dendritic cells and macrophages , link the innate and adaptive immune responses by recognizing , processing , and presenting antigens on mhc - ii . the local concentration of 5-ht can modulate a number of events during these immune responses . one reason immune cells respond to 5-ht is that , as mentioned previously , they constitutively express the molecular machinery that constitutes the serotonergic system . mouse models have demonstrated that neutrophil recruitment to sites of acute inflammation requires platelet - derived chemotactic stimuli , such as 5-ht , paf ( platelet - activation factor ) , and histamine [ 167 , 168 ] . cd14 + cells , which constitute 8090% of the circulating monocytes , express 5ht1e , 5ht2a , 5ht3 , 5ht4 , and 5ht7 mrnas . however , 5-ht has been noted to elicit a number of responses in cd14 + cells isolated from peripheral blood of healthy subjects : ( i ) 5-ht signaling decreases tnf- secretion in a dose - dependent manner ; ( ii ) 5-ht signaling enhances lps - induced secretion il-12p40 from activated monocytes , which acts as a chemoattractant for macrophages and promotes the migration of bacterially stimulated dendritic cells ; and ( iii ) 5-ht signaling enhances lps - induced secretion of il-6 , il-1 , il-8/cxcl8 . macrophages also respond to 5-ht although reports conflict as to whether the response is inhibitory or stimulatory and the mechanisms involved have yet to be clearly described . , changes in the proportion of 5-ht receptors ) or the effect of cooperative signaling with other molecules . finally , macrophages can rapidly metabolize 5-ht to 5-hydroxyindole acetic acid , a biotransformation pathway that may be mediated by mao - a / b , aldh / aox and asmt , which could affect serotonergic responses in these cells . however , they express low levels of hla class i and costimulatory molecules , such as cd80 and cd86 , and lack common lineage markers such as cd3 , cd14 , cd16 , cd19 , cd20 , and cd56 . although immature ( cd11c+cd86 ) and mature ( cd11c+cd86 + ) bmdcs constitutively express sert , maturation induced by lps increases sert expression and , consequently , mature bmdcs have an increased capability for intake , storage , and exocytosis of 5-ht . bmdc maturation also reduces the expression of enzymes involved in the metabolism of 5-ht , such as mao - a and -b . thus , 5-ht may participate in the generation of a specific subset of dcs with unique immunomodulatory properties . differentiation , proliferation , and the functional responses of t cells can each be modulated by the serotonergic system . based on experiments using the cell line k562 , 5-ht participates in t cell maturation in lymphoid organs in a na - coupled , 5-ht active transport - dependent manner ( presumably sert ) , which requires intracellular ca changes . based on the effects of 5-ht receptor - specific agonists or antagonists , t cell proliferation and secretion of proinflammatory cytokines , such as il-2 and ifn- , nave t cells primarily express 5ht7 and low levels of tph1 , but they do not express 5ht1b or sert . in activated human cd4 + t cells , 5-ht or 5ht3 specific agonists impair migration towards cxcl12 gradients , but not to those of ccl2 or ccl5 , which control t cell migration into tissues . but , activation of these cells induces the expression of 5ht3a suggesting they may respond to 5-ht once activated . therefore , the t cell activation state and environment may influence the effects of 5-ht . for example , 5-ht inhibits phytohemagglutinin- ( pha- ) mediated lymphocyte proliferation , possibly through reduced expression and distribution of the il-2 receptor [ 188 , 189 ] . this demonstrates that 5-ht elicited effects are concentration dependent and suggests : ( i ) 5-ht induces dose - dependent phenotypes , ( ii ) differentiation may be achieved by receptors with different 5-ht affinity , and ( iii ) local 5-ht concentrations are tightly regulated to induce specific effects . specifically , sert - specific inhibitors , such as fluoxetine , fenfluramine , or 3,4-methylenedioxymethamphetamine ( mdma ) , elicited anti - proliferative and pro - apoptotic effects . in fact , 5ht1a - specific antagonists , such as pindobind , exacerbate the inhibitory effect of monocyte - mediated ros production on nk cells . migration and rolling require changes in the actin cytoskeleton and activation of pkc and calmodulin signaling [ 39 , 45 ] , which control the morphological changes required for eosinophil infiltration from circulation to sits of inflammation . they also participate in allergic reactions by releasing histamine in response to specific growth factors , such as il-3 [ 203 , 204 ] . although murine basophils express sert , drugs targeting the transporter , such as fluoxetine or citalopram ( selective serotonin reuptake inhibitor , ssri ) , do not block the effect of 5-ht on cytokine release suggesting that other transporters may be used , such as the organic cation transporter 3 . they provide a local source of biogenic amines , including 5-ht , in damaged regions of the vasculature . 5-ht signaling activates rab4 , which controls alpha granule secretion , and rhoa , which induces the cytoskeletal reorganization required for adhesion and aggregation [ 144 , 145 , 209 ] . increases in the serum levels of 5-ht enhance sert density on the platelet cell membrane [ 142 , 148 , 156 , 157 ] . immune cells respond to 5-ht with varying degrees of sensitivity , which can be partially explained by differences in the expression of serotonergic components . in this section , we review how pathologies with reported alterations to the serotonergic system affect the immune system ( table 2 ) . we also discuss the effects of sert - targeting drugs , such as ssris , as well as drugs that target 5-ht receptors ( table 3 ) . major depressive disorder ( mdd ) , fibromyalgia , infections , and alzheimer 's disease commonly display reduced 5-ht serum levels . symptoms have degrees of severity that are associated with changes in both cns and peripheral 5-ht concentrations . lymphocytes from mdd patients express lower levels of sert in comparison with those from healthy volunteers without changes in the intracellular concentration of 5-ht [ 52 , 64 ] . however , further studies are still required to fully understand how components of the serotonergic system are differentially expressed on immune cell subsets . fibromyalgia ( fm ) is a common chronic pain syndrome that primarily affects the joints and muscles and is generally associated with other somatic and psychological symptoms , including fatigue , poor sleep , cognitive difficulties , and stress . fm patients have central sensitization and increasing glial cell activation , which , in turn , favors pain signaling and activates the release of pro - inflammatory cytokines , nitric oxide , prostaglandins , and ros that sustain the hyperexcitable state of the spinal cord [ 215217 ] . 5-ht3 are involved in pain control , indicating a key participation of the serotonergic system [ 218 , 219 ] . the administration of the 5-ht3 antagonist tropisetron or high doses ( 45 mg ) of the ssri fluoxetine produce analgesic and/or other beneficial effects in fm patients ( table 4 ) , suggesting that regulation of the serotonergic system can be useful . however , to date it is not known whether the components of the serotonergic system can be altered in the immune cells of patients . 5-ht decreases the expression of the hiv coreceptor ccr5 on infected macrophages and reduces proviral synthesis 50% [ 228 , 229 ] . these findings suggest that components of the serotonergic system may be suitable therapeutic targets for the control of hiv infection . symptoms of dementia and depression , which are related to reduced levels of 5-ht , are present in 5090% of patients . given the complexity of the cell population expressing 5-ht4 in the brain , however , microglial cells may be involved because they express 5-ht4 and can phagocytose -amyloid deposits , an activity promoted by agonist . from an immunological point of view , the diseases in this group , such as asthma , arthritis , and cancer , are the result of dysregulated inflammatory responses . regardless , these results indicate that cytokine production is under the control of 5-ht , and therefore , regulating its systemic concentrations may be useful for asthma patients . the 5-ht concentrations in platelet - free blood are 1.6- to 2.3-fold higher in ra patients than in healthy controls ( reported average serum concentrations were 1130 this has led to proposals that 5-ht is involved in the pathology , onset , and/or progression of the disease . treating patients with 5-ht3 antagonists combined with intra - articular glucocorticoids while there have yet to be any reports on the responses of ra patient immune cells to 5-ht , the role of serotonergic system in arthritis or osteoarthritis has been studied in vitro and in vivo . given the importance of 5-ht as a neurotransmitter , studies of the serotonergic system have primarily been limited to the cns . recently , however , a large amount of experimental evidence indicates that the serotonergic system has important physiological roles in the immune , vascular , and digestive systems . in this review we discussed the immunomodulatory effects that 5-ht can induce by activating 5htr and sert , which are differentially expressed on many leukocytes . although the effects of 5-ht on the immune system requires further characterization , it is logical to anticipate altered immune responses in patients with dysregulated serotonergic systems .

obtaining a satisfying long - term survival of thas in patients younger than 40 years remains a challenge . young patients must function longer with their tha than the typical patient who has a tha , and they also engage in a higher level of activity , which is associated with higher revision rates [ 19 , 27 ] . therefore , this population is more dependent on durable implants with excellent long - term survival . although stem survival is acceptable in most studies , in general , cup survival is the weakest link in patients younger than 40 years [ 5 , 8 , 9 , 15 , 18 , 20 , 25 ] . the difference in reported survival rates between the cup and stem varies from 1% ( 97% [ stem ] versus 96% [ cup ] ) to 11% ( 98.3% [ stem ] versus 87.6% [ cup ] ) . despite attempts to improve cup designs and using new materials in tha one popular option is to implant uncemented acetabular cups in young patients as part of a total uncemented tha or hybrid tha ( uncemented cup , cemented stem ) . although cement in young patients commonly is not used [ 1 , 24 , 35 ] , we always have implanted cemented cups in patients of all ages , but with one substantial modification : in all patients with substantial acetabular bone stock deficiencies , we have reconstructed this bone stock loss using impaction bone grafting with a cemented cup . secondary osteoarthritis resulting from underlying diseases in these young patients often is seen with associated loss of acetabular bone stock ( for example , in developmental dysplasia of the hips and juvenile rheumatoid arthritis ) . with this approach using cemented cups in young patients for many years , we asked whether there were any differences between cemented cups in young patients ( younger than 40 years ) with and without reconstruction with impaction grafting concerning ( 1 ) clinical scores , ( 2 ) revisions , ( 3 ) complications , ( 4 ) radiographic appearances , ( 5 ) polyethylene wear , and ( 6 ) survival . we retrospectively reviewed prospectively collected data of all 130 patients ( 175 hips ) who had a primary tha in our department between january 1988 and july 2004 and who were younger than 40 years at the time of index surgery . we used a cemented femoral stem and cemented acetabular polyethylene cup in all patients . in patients with acetabular bone deficiencies , these deficiencies were reconstructed with the impaction grafting technique . the decision to use bone impaction grafting a trial cup was placed on the transverse ligament ; in the case of a protrusion hip or a superolateral rim defect , a reconstruction was performed . eighty - four hips ( 48% ) had impaction grafting whereas 91 ( 52% ) did not have impaction grafting . because a cemented tha was our only treatment technique , patients with all diagnoses were included ( table 1 ) . the majority ( 62% ) of the patients had developmental dysplasia of the hips , rheumatoid arthritis , or corticosteroid - induced avascular necrosis . fifty - five ( 42% ) patients were males and 75 ( 58% ) were females . eighty - nine ( 51% ) thas were on the left side and 86 ( 49% ) were on the right . the average age of the patients at index surgery was 31.3 years ( range , 1639 years ) . the mean body mass index was 25.5 ( range , 17.936.3 ) . according to the classification of charnley , 46 hips were in category a , 71 in b , and 58 in c. we followed all patients in this prospective cohort on a regular basis and the minimum followup was 2 years ( average , 8.1 years ; range , 2.018.5 years ) after surgery . during followup , six patients ( eight hips ) died of causes not related to the hip or hip surgery . all patients who died were followed on a regular basis and their data included ; none had revision surgery . of the original group of 175 cups , the data of only one patient were incomplete . based on a telephone interview , the prosthesis of this patient functioned well ; however , a recent radiograph was missing.table 1indications for primary tha with and without reconstruction with bone impaction graftingindicationnumber of hipswithout bone impaction graftingwith bone impaction graftingtotaldevelopmental dysplasia of the hip103242rheumatoid arthritis171027perthes disease448avascular necrosis of unknown cause628epiphyseal dysplasia527posttraumatic osteoarthritis246bechterew s disease325posttraumatic avascular necrosis415morquio s disease134epiphysiolysis134septic coxitis213protrusio acetabuli033osteomyelitis033spontaneous fusion of the hip of unknown cause112osteogenesis imperfecta022polycystic disease of unknown cause202psoriatic arthritis011gigantism of unknown cause011pseudohypoparathyroidism101monoarthritis of unknown cause011alcohol - induced avascular necrosis101corticosteroid - induced avascular necrosis31839 systemic lupus erythematosus9 kidney transplantation / nephropathy7 subarachnoid hemorrhage4 non - hodgkin s lymphoma3 crohn s disease3 cerebral aneurysm2 head trauma2 thrombocytopenia2 hypothalamus hormone substitution1 germ cell tumor1 aplastic anemia1 pituitary adenoma1 wegener s disease1 acute lymphatic leukemia1 meduloblastoma1total9184175 indications for primary tha with and without reconstruction with bone impaction grafting we categorized acetabular defects in accordance with the classification system of the american academy of orthopaedic surgeons . type i segmental deficiencies occurred in 16 hips , type ii cavitary defects in 39 hips , and type iii combined deficiencies in 29 hips . one patient ( two hips ) had ankylosis of the hips , a type v deficiency . using impaction grafting , we reconstructed all deficiencies , including mild cavitary defects ; however most were larger defects . differences between the two groups ( with and without impaction grafting ) were analyzed regarding diagnosis and gender ( chi square test , both p = 0.001 ) . in the group with an acetabular reconstruction , a larger proportion was female and was diagnosed with developmental dysplasia of the hips compared with the group without reconstruction . there were no differences regarding age at surgery , side , bilateral thas , followup , type of cup used , cup inner diameter , and body mass index between the two groups . two - thirds of the operations ( 67% ) were performed by or under the supervision of two senior faculty orthopaedic surgeons ( bws , jwmg ) . a posterolateral approach without trochanteric osteotomy was used in all hips , with the exception of two . intraoperatively , in one patient , a preplanned sugioka procedure was converted to a tha ; however , a trochanteric osteotomy already had been performed . in the other patient , a trochanteric osteotomy was performed in a technically demanding hip with a short femoral neck . in one patient , all acetabular deficiencies were reconstructed ( with the exception of one case ) with impaction grafting using autografts and/or allografts in 84 hips ( 48% ) ; this technique has been described in detail [ 2830 ] . segmental bone defects first were reconstructed with wire meshes before the morselized bone graft was impacted and a conventional full polyethylene cup was cemented . in one patient , we reconstructed a lateral rim deficiency without impaction grafting using a solid autograft fixed with two screws . in one of the ankylosed hips ( type v deficiency ) we used allografts only with impaction grafting in four hips ( 4.8% ) , autografts only in 72 hips ( 85.7% ) , and combined allografts and autografts in eight hips ( 9.5% ) . allografts were used when the original femoral head was not large enough to reconstruct the defect or in cases with pathologic femoral heads ( for example , avascular necrosis of the femoral head ) . in three cases , instead of a solitary metal mesh , a solid fragment was used in combination with impaction grafting . in two of these cases , a minor segmental defect in the medial wall was closed using a cortical - trabecular fragment of a femoral head . a wire mesh was placed medial on top of the fragment and the acetabulum was reconstructed with impaction grafting . in the third case , a cortical head fragment was used to support the anterior rim together with a rim mesh in a reconstruction . the number of femoral heads used as grafts varied from one to four . in 40 hips ( 48% ) , metal wire mesh was used for acetabular reconstruction with impaction grafting ( 10 medial wall meshes , 39 rim meshes ) . in nine early cases , we placed a mesh on top of the bone graft just before cementation , but this mesh was not part of a segmental defect reconstruction . however , after we realized this mesh did not add any stability to the reconstruction and there were no signs of damaging of the graft or graft healing by direct contact with cement , we abandoned the use of a mesh for this purpose . we used 79 ( 45% ) exeter contemporary cups with an inner diameter of 28 mm ( n = 75 ) and 22.225 mm ( n = 5 ) ( stryker howmedica , newbury , uk ) , 71 ( 41% ) charnley elite cups with an inner diameter of 22.225 mm ( n = 6 ) or 28 mm ( n = 65 ) ( depuy , leeds , uk ) , and 25 ( 14% ) mller / allopro cups with an inner diameter of 32 mm ( n = 19 ) , 28 mm ( n = 2 ) , or 22.225 mm ( n = 4 ) ( sulzer , winterthur , switzerland ) . for the femoral component , we used an exeter stem in 111 cases , a charnley elite stem in 48 cases , and a mller stem in 16 cases . all femoral heads used were made of a cobalt - chrome alloy ; no ceramic implants were used . we cemented acetabular components with a third - generation cementing technique . in the directly cemented cups , after reaming , multiple small drill holes were made with a 2.6-mm drill . after using pulse lavage , vacuum - mixed cement was injected directly from the cement gun and the cement was pressurized by a seal . in cases of reconstruction with bone grafts , we reamed the acetabulum , made multiple drill holes in sclerotic areas , and irrigated the acetabulum . again , vacuum - mixed cement was injected and pressurized and the cup was inserted . before 1989 , we used palacos bone cement ( merck , darmstadt , germany ) ; however , since 1989 , we have used surgical simplex ( stryker howmedica ) . in 165 cases ( 94% ) , all patients received antibiotic prophylaxis consisting of 2 g cefazolin intravenously just before surgery . other precautionary measures to prevent infections were use of an operating theater with laminar airflow and use of two pairs of sterile gloves . postoperatively , all patients received thrombosis prophylaxis with low - molecular - weight heparin for 6 weeks , or before 1999 , with acenocoumarol ( the individual dosage regimens regulated with regular coagulation tests ) for 3 months . to prevent heterotopic ossification , we used nonsteroidal antiinflammatory drugs ( nsaids ) for 7 days . in six patients in whom nsaids were contraindicated , we administered one dose ( 7 gy ) of radiotherapy 1 day postoperatively . patients without acetabular reconstruction were mobilized under supervision of a physiotherapist after 1 or 2 days . full weightbearing was increased in 2 to 6 weeks with the aid of one or two crutches . the patients who underwent impaction grafting were mobilized according to a modified protocol ; in the first 6 weeks , only 10% weightbearing was allowed and then 6 to 12 weeks of 50% weightbearing using two crutches was allowed . thirty - one hips had such an extensive reconstruction of major defects that several weeks of bed rest were maintained ranging from 1 to 6 weeks . routine followups were scheduled at 6 weeks ; 3 , 6 , and 12 months ; and yearly or biannually thereafter . at our outpatient clinic , student researchers not participating in the treatment performed clinical analysis using the harris hip score , the oxford hip questionnaire score ( since 1998 ) , and visual analog scales for pain during rest and physical activity on a scale from 0 ( no pain ) to 100 ( unbearable pain ) . we report the clinical scores of all patients excluding the 21 patients whose hips were revised during followup . all anteroposterior pelvis and lateral radiographs of all hips were analyzed on a consensus basis by two of the authors ( dcjdk , bws ) . radiographic evaluation included assessment of cup position , loosening of the acetabular component , polyethylene wear , presence of osteolysis , structural quality of the bone graft , application and position of the meshes , migration , heterotopic ossification , and fracture of the cement , mesh , or prosthesis . radiolucent lines and osteolysis were recorded according to the three acetabular zones as described by delee and charnley . radiographic loosening was defined as 2 mm or greater demarcation in two or three zones around the acetabular component , progressive demarcation , 3 mm or greater component migration , 5 or greater component tilting , and/or cement or prosthesis fracture . we determined cup migration ( > 3-mm shift in any direction or > 5 tilting ) in relation to the interteardrop line instead of the kohler line . graft incorporation was defined as the presence of the crossing of trabecular bone on the bone - graft interface on the radiographs . clinical failure was defined as the need for revision of the acetabular component for any reason . we calculated kaplan - meier curves to study the survival ( time to revision ) . the end points were ( 1 ) cup revision for any reason , ( 2 ) cup revision for any reason excluding infections , ( 3 ) cup revision for aseptic loosening , and ( 4 ) radiographic signs of cup loosening . with an average followup of 8.1 years , 30% of all patients had a followup longer than 10 years . the log - rank test was used to test the differences in survival between cups with and without impaction grafting . differences in outcomes between the groups were determined with the student s t - test ( continuous variables after checking for normal distribution ) or chi square test ( nominal variables ) . the outcome of the harris hip score and the oxford hip questionnaire score improved ( p < 0.0001 ) after surgery for both groups ; there were no differences in preoperative and postoperative clinical outcomes between the cups with and without acetabular reconstruction ( table 2 ) . the postoperative experienced pain score was low.table 2outcome of clinical questionnairesquestionnairepreoperative scorepostoperative scorewith bone impaction graftingwithout bone impaction graftingindependent t test p valuewith bone impaction graftingwithout bone impaction graftingindependent t test p valueharris hip score48 ( 1581 ) ( n = 46)50 ( 2882 ) ( n = 50)0.67292 ( 35100 ) ( n = 72)96 ( 12100 ) ( n = 73)0.546oxford hip questionnaire score39 ( 3052 ) ( n = 9)38 ( 1252 ) ( n = 15)0.10017 ( 1245 ) ( n = 70)15 ( 1241 ) ( n = 72)0.151vas pain at restnana0 ( 075 ) ( n = 70)0 ( 070 ) ( n = 71)0.260vas pain during physical activitynana10 ( 090 ) ( n = 70)0 ( 0100 ) ( n = 71)0.267values are expressed as median , with range in parentheses ; vas = visual analog scale ; na = not available . outcome of clinical questionnaires values are expressed as median , with range in parentheses ; vas = visual analog scale ; na = not available . the number of revisions in the groups with and without bone grafts was not different ( p = 0.152 ) . at last followup , 21 of the 175 cups ( 12% ) had been revised , seven of which had reconstruction with impaction grafting ( table 3 ) . reasons for revision were infection ( eight ) , recurrent dislocations ( two ) , traumatic loosening ( one ) , and aseptic loosening ( 10 ) . the cup only was revised in eight cases and the cup and stem were revised in two cases . four of the 10 revised cups had reconstruction with impaction grafting and six cups were implanted with standard techniques without any graft . the failed cups reconstructed with impaction grafting were revised after 4.1 , 9.8 , 16.2 , and 16.8 years ( average , 11.7 years ) . the six directly cemented cups were revised after an average of 4.0 years ( range , 1.110.0 years ) . the time to revision for aseptic loosening was longer ( p = 0.032 ) for the reconstructed cups with impaction grafting than for the cups implanted with standard techniques . the eight infected hips ( 4.6% ) all had revision because of culture - proven infection of the implant . the average time to revision for septic loosening was 5.3 years ( range , 2.28.1 years ) . staphylococcus epidermidis was isolated in three , staphylococcus aureus in two , proprioni in two , pseudomonas aeruginosa in one , and streptococcus oralis in one . as a result of recurrent dislocations , two cups ( 1.1% ) one implant ( 0.6% ) was radiographically and clinically loose after trauma and needed revision of both components.table 3overview of the revised cups ( n = 21)patientyears to revisioncausepart revisedbone impaction graftingindicationyears to radiographic looseningprevious hip operations 57.3infectionthanocorticosteroids ( systemic lupus erythematosus)no206.1infectionthayesavascular necrosis of unknown causeyes505.7infectionthanocorticosteroids ( crohn s disease)2.6yes688.1infectionthanorheumatoid arthritisno875.3infectionthanodevelopmental dysplasia of the hip5.2yes1044infectionthanocorticosteroids ( subarachnoid bleeding)no1133.4infectionthayescorticosteroids ( pituitary adenoma)yes1232.2infectionthanomedial column fracture0.5yes1118.6recurrent dislocationscupnoposttraumatic coxarthrosisno1603.5recurrent dislocationscupyescorticosteroids ( cerebral aneurysm)yes8410.3traumatic looseningthanocorticosteroids ( head trauma)9.9no294.1aseptic looseningcupyesrheumatoid arthritis4no412.3aseptic looseningcupnocorticosteroids ( systemic lupus erythematosus)2.2yes453.1aseptic looseningcupnospontaneous fusion of unknown cause0.3no491.1aseptic looseningcupnocorticosteroids ( kidney transplantation)4.2no7716.8aseptic looseningcupyesposttraumatic coxarthrosis16.6yes789.8aseptic looseningcupyescoxarthritis9.8yes7916.2aseptic looseningthayesdevelopmental dysplasia of the hip16.2no8210aseptic looseningcupnodevelopmental dysplasia of the hip5.2no906.4aseptic looseningthanodevelopmental dysplasia of the hipno1531.1aseptic looseningcupnoepiphysiolysisyes overview of the revised cups ( n = 21 ) we observed similar ( p = 0.959 ) numbers of overall complications in the groups with and without bone grafts . however , dislocations were more common ( p = 0.045 ) in the group without bone grafts than in the group with bone grafts ( 15 versus 5 , respectively ) . patients without reconstruction with impaction grafting had an increased dislocation chance of 1:2.9 . during followup , there were nine intraoperative complications and 30 postoperative complications ( table 4 ) . one additional stem was revised because of aseptic loosening and two femoral heads were exchanged because of recurrent dislocations . seven hips underwent additional surgery because of postoperative complications ( table 4).table 4overview of complicationstype of complicationnumberintraoperative complications ( n = 9 ) entrapment of sciatic nerve during reposition , permanent damage1 false route femur1 incomplete femoral fracture2 malposition cup1 malposition stem1 instrument failure1 suspicion of breakthrough of sterility2postoperative complications ( n = 30 ) superficial wound infection3 single dislocation9 recurrent dislocations6 sensory nerve palsy4 sensory and motor nerve palsy1 hematoma6 bleeding after 4 months1heterotopic ossifications ( n = 44 ) brooker class i15 brooker class ii19 brooker class iii10postoperative complications leading to revision ( no cup revision ) ( n = 3 ) stem revision for aseptic loosening1 head exchange because of recurrent dislocations2postoperative complications requiring surgical intervention ( no revision ) ( n = 7 ) deep wound infection4 heterotopic ossifications1 traumatic dislocation1 persistent motor and sensory nerve palsy1 overview of complications there were no differences between the cups with and without acetabular reconstruction concerning the occurrence of cup migration , radiographic loosening , or the presence of osteolysis , cysts , and abnormal cup position ( table 5 ) . cups with impaction grafting had fewer radiolucent lines ( p = 0.02 ) and fewer lines in zone i ( p = 0.001 ) ( table 5 ) . all lines , except two , were on the bone - cement interface . in 28 ( 48% ) of the 58 cups with radiolucent lines , the lines were progressive . of the 175 hips , 160 were radiographically stable ( fig . 1 ) . fifteen cups were difficult to evaluate because of overlap of the metal mesh ( 11 zone i ; four zones i + ii ) . we observed graft osteolysis in only one patient with impaction grafting ; the hip revised because of traumatic loosening had a fracture in zone ii of the acetabulum ; no other fractures were seen . fifteen ( 8.6% ) cups were radiographically loose , three had cup migration ( after 1.8 , 9.8 , and 11.2 years postoperatively ) , and 12 had evident radiolucent lines in all zones and/or severe osteolysis ; 12 of these cups were revised ( table 3).table 5radiographic findings of all cups*radiographic findingallwith bone impaction graftingwithout bone impaction graftingp value ( where appropriate)radiographic loosening155100.608cup migration3120.234radiolucent lines5818400.02 zone i183150.001 zone ii202 zone iii1798 zones i + ii422 zones ii + iii523 zones i + iii514 zones i + ii + iii716osteolysis11560.861 zone i725 zone ii110 zone iii320cysts1000.033 zone i100 zone ii000 zone iii000cup position neutral position ( 3555)1607783 abnormal position15780.914 vertical ( > 55)1266 horizontal ( < 35)312polyethylene wear mean nonrevised cups ( mm / year)0.0800.0760.0840.539 mean revised cups ( mm / year)0.2140.1820.2300.525 * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting.fig . 1a cthe radiographs illustrate reconstruction of the acetabuli in a 34-year - old woman with bilateral ddh ( crowe grade 3 ) . ( b ) an anteroposterior radiograph taken immediately postoperatively shows the thas with the acetabuli reconstructed with impaction grafting . ( c ) an anteroposterior radiograph taken 12 years postoperatively shows the thas remain radiographically stable , but brooker classes iii ( left ) and i ( right ) heterotopic ossifications are visible . radiographic findings of all cups * * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting . the radiographs illustrate reconstruction of the acetabuli in a 34-year - old woman with bilateral ddh ( crowe grade 3 ) . ( b ) an anteroposterior radiograph taken immediately postoperatively shows the thas with the acetabuli reconstructed with impaction grafting . ( c ) an anteroposterior radiograph taken 12 years postoperatively shows the thas remain radiographically stable , but brooker classes iii ( left ) and i ( right ) heterotopic ossifications are visible . there was no difference in polyethylene wear rates between the cups with and without impaction grafting ( p = 0.539 in 154 unrevised cups and p = 0.525 in the 21 revised cups ) ( table 5 ) . when looking at all cups ( with and without acetabular reconstruction ) , the revised and radiographically loose cups had more wear compared with the cups that were not revised ( both p < 0.0001 ) . patients with an abnormal position of the cup had similar ( p = 0.196 ) polyethylene wear rates to those who had a normal position . analysis of polyethylene wear rates of cups with different inner diameters showed no differences ( independent t test , 22 versus 28 mm : p = 0.135 , 22 versus 32 mm : p = 0.484 , 28 versus 32 mm : p = 0.620 ) . there were no differences in survival after 10 years between the groups with and without bone impaction grafting ( table 6 ) . the midterm survival rates of all cemented polyethylene cups varied from 85% to 92% at 10 years with four end points ( table 6 ; figs . 2 , 3 ) . cup survival with an end point of radiographic loosening was 89% ( 95% confidence interval , 83%95%).table 6the 10-year survival rates*end pointall cupswithout bone impaction graftingwith bone impaction graftinglog - rank p valuerevision for any reason 85% ( 78%92%)79% ( 68%90%)91% ( 82%99%)0.21revision for any reason excluding infections 91% ( 85%97%)87% ( 78%99%)94% ( 87%100%)0.56revision for aseptic loosening92% ( 87%98%)90% ( 81%99%)95% ( 89%100%)0.73 * kaplan - meier estimates ; 95% confidence interval in parentheses.fig . 2a bkaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . 3a bkaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . the 10-year survival rates * * kaplan - meier estimates ; 95% confidence interval in parentheses . kaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . kaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . the use of cemented tha in young patients is not very popular and most surgeons will use uncemented or will resurface hips in these patients . however , we have continued to use only cemented implants in tha even in young patients . in our view , the real challenge in tha in these young patients is to manage the commonly seen acetabular deficiencies . in cases of acetabular defects , we reconstruct these deficiencies with impaction bone grafting . we questioned whether there was a difference in clinical outcome , revisions , complications , radiographic appearances , polyethylene wear , and survival between the cups implanted with an acetabular reconstruction with impaction grafting and those implanted with standard cementing techniques . our study has several limitations : short followup , lack of assessment of activity levels , clinical interobserver variability , heterogeneous group , no comparison with other reconstruction techniques , and different types of implants used . with no patients lost to followup , our followup is representative and reliable for the midterm results , and long - term followup ( > 15 years ) was not available at the time of this review . our results can be biased by an important factor we did not evaluate : the level of activity . theoretically , with restoration of the affected hip(s ) into well - functioning artificial joints , most patients will increase their level of activity . however , young patients undergoing tha with acetabular deficiencies and therefore more complex reconstructions could still have a lower level of activity after surgery relative to primary cemented cups . however , the average wear of the cups with impaction grafting was the same as the cups without impaction grafting ( both 0.08 mm / year ) . provided that activity is a major cause of polyethylene wear , this might imply the level of activity is similar in these two groups . several studies suggest the revision and polyethylene wear rates are correlated to level of activity [ 2 , 19 , 27 , 31 , 38 ] . additional research on level of activity and impaction grafting in young patients is necessary to confirm this hypothesis . the clinical questionnaires were obtained by student researchers who did not participate in the treatment . multiple researchers were involved in the data collection and interobserver variability has not been tested ; however , all researchers were trained and supervised to obtain these questionnaires correctly . the clinical scores were comparable between the two groups and comparable to published scores ( table 7 ) . although the cups reconstructed with bone impaction grafting were the more demanding procedures , no clinical differences were seen.table 7reported outcomes of the harris hip score in patients < 40 years for primary thastudyquestionnairepreoperative scorepostoperative scorepaired t - test p valuechiu et al . harris hip score44 ( 26 - 74 ) 88 ( 74 - 99 ) < 0.001duffy et al . harris hip score51 92 < 0.001current study with bone impaction graftingharris hip score48 ( 1581 ) 92 ( 35100 ) < 0.001 without bone impaction graftingharris hip score50 ( 2882)96 ( 12100 ) < 0.001values are expressed as median ( current study ) or means ( other studies ) , with range in parentheses . reported outcomes of the harris hip score in patients < 40 years for primary tha values are expressed as median ( current study ) or means ( other studies ) , with range in parentheses . although revision rates in both groups were comparable , the time to revision was longer in the cups reconstructed with bone impaction grafting . we have no clear explanation for this observation ; possibly the cement - bone interface was better in cups with bone impaction grafting with better interdigitation of the cement into the bone . this also may explain the lower incidence of radiolucent lines in the cups reconstructed with bone impaction grafting . the number of revisions for septic loosening was relatively high during this midterm followup study ( 4.6% ) . only one septic loosening likely was related to the surgery ; we considered all other infections acute hematogenous infections of previously well - functioning prostheses . the use of corticosteroids and newer rheumatic disease - modifying drugs , which were used in most of the infection cases , can explain this higher risk of infection [ 3 , 4 ] . sochart and porter had only two infections in their study , but both were in patients with rheumatoid arthritis . still , our revision rate for septic loosening of 4.6% is relatively high in contrast to other studies , such as that of joshi et al . , with an infection rate of 1.3% . remarkably , many septic loosenings occurred late ( > 2 years postoperatively ) . the cups reconstructed with impaction grafting showed fewer complications by having fewer dislocations than the cups implanted by standard techniques . this might be attributed to the different mobilization protocol for the patients who received cups with impaction grafting . however , subluxation rates in young patients having tha have been reported to be as much as 18.2% . the overall complication rate of 17% ( 30 postoperative complications ) is also relatively high . reported a complication rate of 11.5% in cemented hips and duffy et al . reported a complication rate of 12% during the perioperative period . this is consistent with previous reports showing wear particles are associated with osteolysis in tha [ 27 , 33 , 37 ] . the average wear rate of the cups of 0.08 mm / year is within the normal limits , keeping in mind that wear in younger patients can be 33% to 40% higher than wear in older patients . in a large study of 226 hips in patients younger than 40 years with a charnley prosthesis , sochart and porter reported an average wear rate of 0.08 to 0.10 mm / year in the nonrevised cups , which is comparable to our results . . found a correlation between inclination of the cup and higher / lower wear rates . however , we did not observe higher wear with abnormal position or inner cup diameter . the observed overall midterm survival of cemented polyethylene cups in patients younger than 40 years in our study was acceptable . especially in these young patients , there is a need for total hip implants with proven long - term survival . although the use of uncemented prostheses in these young patients is very popular , literature regarding long - term outcome of tha in patients younger than 40 years concerns mainly studies of cemented implants and less about uncemented implants ( table 8) [ 5 , 8 , 9 , 15 , 18 , 20 , 25 , 3234 ] . a limitation of the reported midterm or long - term results of uncemented cups is the fact that in these studies first - generation uncemented cups were used . the only report of uncemented cups at 15 years after surgery with an end point of revision for any reason showed a survival rate of 54% . this is less favorable than the results of cemented cups at that time ( table 8) . sochart and porter had survival rates of 71% and 68% at 20 and 25 years , respectively , for cemented charnley cups . the survival of the acetabular uncemented cups with an end point of revision for aseptic loosening in patients younger than 40 years reported in one study was 85% , in contrast to a survival rate of 96% after 10 years of the charnley cups in the study by joshi et al . . we found a survival rate with cemented cups of 92% at 10 years with an end point of revision for aseptic loosening.table 8long - term acetabular cup survival rates in patients younger than 40 yearsstudynumber of hipsage ( years)*followup ( years)*type of cupsurvival5 years7 years10 years15 years20 years25 years30 yearsuncementedbizot et al . 8732.3 ( 1740)7.7 ( 019)screw - in alumina insert94.7 ( 68.599.2)88.8 ( 62.097.1)88.8 ( 62.097.1)cerapress alumina10095.1 ( 69.899.3)cerafit alumina10094.3 ( 66.399.2)duffy et al . 8232 ( 1739)10.3 ( 1014)pca , osteonics , h - g porousaseptic : 84.6 ( 7693)excl inf : 81.8 ( 7391)mcauley et al . 25633.2 ( 1640)7.3 ( 019)duraloc , aml , arthropor , triloc , h - g , solution97.4 ( se 2.2)87.6 ( se 6.0)53.8 ( se 13.9)odent et al . 6218.3 ( 11.831)6 ( 313)zweymuller90.1 ( se 7.1)cementedbizot et al . 4132.3 ( 1740)7.7 ( 019)plain alumina97.3 ( 81.999.6)94.1 ( 78.198.5)90.4 ( 73.096.8)78.9 ( 54.791.1)chiu et al . 4728.8 ( 1739)14.9 ( 721)charnley polyethylene86.3 ( 75.597.1)27.0 ( 10.743.3)chmell et al . 6619.9 ( 1129)15.1 ( 1122)trapezoidal , aufranc - turner , custom - madeaseptic : 98 ( se 1.6)aseptic : 97 ( se 2.8)aseptic : 84.5 ( se 4.7)aseptic : 70 ( se 7.3)aseptic : 40 ( se 15.6)joshi et al . 21832 ( 1739)16 ( 1024)charnley polyethyleneaseptic : 99 ( se 0.5)aseptic : 96 ( se 1.4)aseptic : 91 ( se 2.3)aseptic : 84 ( se 4.6)sochart and porter 22631.7 ( 1739)19.7 ( 230)charnley polyethylene93 ( 9096)71 ( 6577)68 ( 6175)sochart and porter 8324.9 ( 1729)20 ( 5.230)charnley92 ( 8598)70 ( 6081)68 ( 5779)sochart and porter 4328.8 ( 1939)22.4 ( 0.130.3)charnley73 ( 6184)70 ( 5783)current study17531.0 ( 1639)8.1 ( 2.018.5)exeter , charnley , mller / allopro85 ( 7892)aseptic : 92 ( 8798)with impacted bone grafts91 ( 8399)aseptic : 95 ( 89100)studies published through 2007 ; * values are expressed as mean , with range in parentheses ; values are expressed as percentage , with 95% confidence interval in parentheses ; deceased and revised excluded ; lost to followup , deceased excluded , only chinese patients ; aml = anatomic medullary locking ; pca = porous - coated anatomic ; excl inf = excluding infections ; h - g = harris - galante ; se = standard error . long - term acetabular cup survival rates in patients younger than 40 years studies published through 2007 ; * values are expressed as mean , with range in parentheses ; values are expressed as percentage , with 95% confidence interval in parentheses ; deceased and revised excluded ; lost to followup , deceased excluded , only chinese patients ; aml = anatomic medullary locking ; pca = porous - coated anatomic ; excl inf = excluding infections ; h - g = harris - galante ; se = standard error . a remarkable finding of our study was the survival of cups with acetabular reconstructions with impaction grafting was at least comparable to the survival of standard cemented cups , especially considering the more difficult hips of our study population needed reconstruction with impaction grafting . our data on the cemented cups with impaction grafting showed similar survival , where rather lower survival rates would be expected . the outcome of these cups reconstructed with impaction grafting even fulfilled the nice criteria ( a survival of > 90% after 10 years ) , with a survival rate of 91% at 10 years with an end point of revision for any reason . the survival rates of the cemented cups in our study are comparable to those reported for cemented cups [ 8 , 18 , 34 ] . although cemented cups are not commonly used in young patients , our data suggest cemented conventional polyethylene cups are still a good option in tha in young patients . even reconstruction of ( severe ) acetabular deficiencies with impaction grafting and a cemented conventional polyethylene cup produced very acceptable survival rates , comparable to the rates of cemented cups implanted in acetabuli without deficiencies with standard cementing techniques .

although uncemented cup implants frequently are used in young patients , we believe long - term survival rates of cups in these patients are somewhat disappointing , and therefore we have continued to use cemented cups in primary tha , even in young patients . however , in cases of acetabular bone stock defects , we also use bone impaction grafting . we prospectively followed 130 patients with 175 cemented cups ; no patients were lost to followup . the mean age of the patients at surgery was 31 years ( range , 1639 years ) . an acetabular reconstruction with bone impaction grafting was performed in 84 hips ( 48% ) . the minimum followup was 2 years ( average , 8.1 years ; range , 2.018.5 years ) . twenty - one of the 175 cups ( 12% ) were revised at an average of 8.1 years ( range , 2.018.5 years ) . reasons for revision were infection ( one early , seven late ) , recurrent dislocations ( two ) , traumatic loosening ( one ) , and aseptic loosening ( 10 ) . the 10-year survival rate of all cemented cups with end point of revision for any cause was 85% . survival with end point of aseptic loosening of all cups was 92% . survival with end point of revision for aseptic loosening was 90% for the cups without impaction grafting and 95% for the cups with impaction grafting . we believe cemented acetabular cups in young patients have acceptable midterm survival ; however , in the case of acetabular bone defects , we recommend reconstruction with impaction grafting.level of evidence : level iii , therapeutic study . see the guidelines for authors for a complete description of levels of evidence .

Introduction Materials and Methods Results Discussion

young patients must function longer with their tha than the typical patient who has a tha , and they also engage in a higher level of activity , which is associated with higher revision rates [ 19 , 27 ] . despite attempts to improve cup designs and using new materials in tha one popular option is to implant uncemented acetabular cups in young patients as part of a total uncemented tha or hybrid tha ( uncemented cup , cemented stem ) . although cement in young patients commonly is not used [ 1 , 24 , 35 ] , we always have implanted cemented cups in patients of all ages , but with one substantial modification : in all patients with substantial acetabular bone stock deficiencies , we have reconstructed this bone stock loss using impaction bone grafting with a cemented cup . secondary osteoarthritis resulting from underlying diseases in these young patients often is seen with associated loss of acetabular bone stock ( for example , in developmental dysplasia of the hips and juvenile rheumatoid arthritis ) . with this approach using cemented cups in young patients for many years , we asked whether there were any differences between cemented cups in young patients ( younger than 40 years ) with and without reconstruction with impaction grafting concerning ( 1 ) clinical scores , ( 2 ) revisions , ( 3 ) complications , ( 4 ) radiographic appearances , ( 5 ) polyethylene wear , and ( 6 ) survival . the decision to use bone impaction grafting a trial cup was placed on the transverse ligament ; in the case of a protrusion hip or a superolateral rim defect , a reconstruction was performed . the average age of the patients at index surgery was 31.3 years ( range , 1639 years ) . according to the classification of charnley , 46 hips were in category a , 71 in b , and 58 in c. we followed all patients in this prospective cohort on a regular basis and the minimum followup was 2 years ( average , 8.1 years ; range , 2.018.5 years ) after surgery . based on a telephone interview , the prosthesis of this patient functioned well ; however , a recent radiograph was missing.table 1indications for primary tha with and without reconstruction with bone impaction graftingindicationnumber of hipswithout bone impaction graftingwith bone impaction graftingtotaldevelopmental dysplasia of the hip103242rheumatoid arthritis171027perthes disease448avascular necrosis of unknown cause628epiphyseal dysplasia527posttraumatic osteoarthritis246bechterew s disease325posttraumatic avascular necrosis415morquio s disease134epiphysiolysis134septic coxitis213protrusio acetabuli033osteomyelitis033spontaneous fusion of the hip of unknown cause112osteogenesis imperfecta022polycystic disease of unknown cause202psoriatic arthritis011gigantism of unknown cause011pseudohypoparathyroidism101monoarthritis of unknown cause011alcohol - induced avascular necrosis101corticosteroid - induced avascular necrosis31839 systemic lupus erythematosus9 kidney transplantation / nephropathy7 subarachnoid hemorrhage4 non - hodgkin s lymphoma3 crohn s disease3 cerebral aneurysm2 head trauma2 thrombocytopenia2 hypothalamus hormone substitution1 germ cell tumor1 aplastic anemia1 pituitary adenoma1 wegener s disease1 acute lymphatic leukemia1 meduloblastoma1total9184175 indications for primary tha with and without reconstruction with bone impaction grafting we categorized acetabular defects in accordance with the classification system of the american academy of orthopaedic surgeons . in one patient , all acetabular deficiencies were reconstructed ( with the exception of one case ) with impaction grafting using autografts and/or allografts in 84 hips ( 48% ) ; this technique has been described in detail [ 2830 ] . in one of the ankylosed hips ( type v deficiency ) we used allografts only with impaction grafting in four hips ( 4.8% ) , autografts only in 72 hips ( 85.7% ) , and combined allografts and autografts in eight hips ( 9.5% ) . in 40 hips ( 48% ) , metal wire mesh was used for acetabular reconstruction with impaction grafting ( 10 medial wall meshes , 39 rim meshes ) . in cases of reconstruction with bone grafts , we reamed the acetabulum , made multiple drill holes in sclerotic areas , and irrigated the acetabulum . before 1989 , we used palacos bone cement ( merck , darmstadt , germany ) ; however , since 1989 , we have used surgical simplex ( stryker howmedica ) . the end points were ( 1 ) cup revision for any reason , ( 2 ) cup revision for any reason excluding infections , ( 3 ) cup revision for aseptic loosening , and ( 4 ) radiographic signs of cup loosening . with an average followup of 8.1 years , 30% of all patients had a followup longer than 10 years . at last followup , 21 of the 175 cups ( 12% ) had been revised , seven of which had reconstruction with impaction grafting ( table 3 ) . reasons for revision were infection ( eight ) , recurrent dislocations ( two ) , traumatic loosening ( one ) , and aseptic loosening ( 10 ) . four of the 10 revised cups had reconstruction with impaction grafting and six cups were implanted with standard techniques without any graft . the failed cups reconstructed with impaction grafting were revised after 4.1 , 9.8 , 16.2 , and 16.8 years ( average , 11.7 years ) . the six directly cemented cups were revised after an average of 4.0 years ( range , 1.110.0 years ) . the time to revision for aseptic loosening was longer ( p = 0.032 ) for the reconstructed cups with impaction grafting than for the cups implanted with standard techniques . the average time to revision for septic loosening was 5.3 years ( range , 2.28.1 years ) . as a result of recurrent dislocations , two cups ( 1.1% ) one implant ( 0.6% ) was radiographically and clinically loose after trauma and needed revision of both components.table 3overview of the revised cups ( n = 21)patientyears to revisioncausepart revisedbone impaction graftingindicationyears to radiographic looseningprevious hip operations 57.3infectionthanocorticosteroids ( systemic lupus erythematosus)no206.1infectionthayesavascular necrosis of unknown causeyes505.7infectionthanocorticosteroids ( crohn s disease)2.6yes688.1infectionthanorheumatoid arthritisno875.3infectionthanodevelopmental dysplasia of the hip5.2yes1044infectionthanocorticosteroids ( subarachnoid bleeding)no1133.4infectionthayescorticosteroids ( pituitary adenoma)yes1232.2infectionthanomedial column fracture0.5yes1118.6recurrent dislocationscupnoposttraumatic coxarthrosisno1603.5recurrent dislocationscupyescorticosteroids ( cerebral aneurysm)yes8410.3traumatic looseningthanocorticosteroids ( head trauma)9.9no294.1aseptic looseningcupyesrheumatoid arthritis4no412.3aseptic looseningcupnocorticosteroids ( systemic lupus erythematosus)2.2yes453.1aseptic looseningcupnospontaneous fusion of unknown cause0.3no491.1aseptic looseningcupnocorticosteroids ( kidney transplantation)4.2no7716.8aseptic looseningcupyesposttraumatic coxarthrosis16.6yes789.8aseptic looseningcupyescoxarthritis9.8yes7916.2aseptic looseningthayesdevelopmental dysplasia of the hip16.2no8210aseptic looseningcupnodevelopmental dysplasia of the hip5.2no906.4aseptic looseningthanodevelopmental dysplasia of the hipno1531.1aseptic looseningcupnoepiphysiolysisyes overview of the revised cups ( n = 21 ) we observed similar ( p = 0.959 ) numbers of overall complications in the groups with and without bone grafts . seven hips underwent additional surgery because of postoperative complications ( table 4).table 4overview of complicationstype of complicationnumberintraoperative complications ( n = 9 ) entrapment of sciatic nerve during reposition , permanent damage1 false route femur1 incomplete femoral fracture2 malposition cup1 malposition stem1 instrument failure1 suspicion of breakthrough of sterility2postoperative complications ( n = 30 ) superficial wound infection3 single dislocation9 recurrent dislocations6 sensory nerve palsy4 sensory and motor nerve palsy1 hematoma6 bleeding after 4 months1heterotopic ossifications ( n = 44 ) brooker class i15 brooker class ii19 brooker class iii10postoperative complications leading to revision ( no cup revision ) ( n = 3 ) stem revision for aseptic loosening1 head exchange because of recurrent dislocations2postoperative complications requiring surgical intervention ( no revision ) ( n = 7 ) deep wound infection4 heterotopic ossifications1 traumatic dislocation1 persistent motor and sensory nerve palsy1 overview of complications there were no differences between the cups with and without acetabular reconstruction concerning the occurrence of cup migration , radiographic loosening , or the presence of osteolysis , cysts , and abnormal cup position ( table 5 ) . in 28 ( 48% ) of the 58 cups with radiolucent lines , the lines were progressive . we observed graft osteolysis in only one patient with impaction grafting ; the hip revised because of traumatic loosening had a fracture in zone ii of the acetabulum ; no other fractures were seen . fifteen ( 8.6% ) cups were radiographically loose , three had cup migration ( after 1.8 , 9.8 , and 11.2 years postoperatively ) , and 12 had evident radiolucent lines in all zones and/or severe osteolysis ; 12 of these cups were revised ( table 3).table 5radiographic findings of all cups*radiographic findingallwith bone impaction graftingwithout bone impaction graftingp value ( where appropriate)radiographic loosening155100.608cup migration3120.234radiolucent lines5818400.02 zone i183150.001 zone ii202 zone iii1798 zones i + ii422 zones ii + iii523 zones i + iii514 zones i + ii + iii716osteolysis11560.861 zone i725 zone ii110 zone iii320cysts1000.033 zone i100 zone ii000 zone iii000cup position neutral position ( 3555)1607783 abnormal position15780.914 vertical ( > 55)1266 horizontal ( < 35)312polyethylene wear mean nonrevised cups ( mm / year)0.0800.0760.0840.539 mean revised cups ( mm / year)0.2140.1820.2300.525 * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting.fig . radiographic findings of all cups * * total cups ( n = 175 ) ; cups with ( n = 84 ) or without ( n = 91 ) reconstruction with bone impaction grafting . there was no difference in polyethylene wear rates between the cups with and without impaction grafting ( p = 0.539 in 154 unrevised cups and p = 0.525 in the 21 revised cups ) ( table 5 ) . when looking at all cups ( with and without acetabular reconstruction ) , the revised and radiographically loose cups had more wear compared with the cups that were not revised ( both p < 0.0001 ) . the midterm survival rates of all cemented polyethylene cups varied from 85% to 92% at 10 years with four end points ( table 6 ; figs . cup survival with an end point of radiographic loosening was 89% ( 95% confidence interval , 83%95%).table 6the 10-year survival rates*end pointall cupswithout bone impaction graftingwith bone impaction graftinglog - rank p valuerevision for any reason 85% ( 78%92%)79% ( 68%90%)91% ( 82%99%)0.21revision for any reason excluding infections 91% ( 85%97%)87% ( 78%99%)94% ( 87%100%)0.56revision for aseptic loosening92% ( 87%98%)90% ( 81%99%)95% ( 89%100%)0.73 * kaplan - meier estimates ; 95% confidence interval in parentheses.fig . 2a bkaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . 3a bkaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . kaplan - meier survival curves with 95% confidence intervals ( broken lines ) of all cups with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . kaplan - meier survival curves of cups without impaction grafting ( thick broken line , 95% confidence intervals in thin broken lines ) and cups with impaction grafting ( thick solid line , 95% confidence intervals in thin solid lines ) with end points of ( a ) revision for any reason and ( b ) revision for aseptic loosening are shown . the use of cemented tha in young patients is not very popular and most surgeons will use uncemented or will resurface hips in these patients . however , we have continued to use only cemented implants in tha even in young patients . in cases of acetabular defects , we reconstruct these deficiencies with impaction bone grafting . we questioned whether there was a difference in clinical outcome , revisions , complications , radiographic appearances , polyethylene wear , and survival between the cups implanted with an acetabular reconstruction with impaction grafting and those implanted with standard cementing techniques . with no patients lost to followup , our followup is representative and reliable for the midterm results , and long - term followup ( > 15 years ) was not available at the time of this review . however , young patients undergoing tha with acetabular deficiencies and therefore more complex reconstructions could still have a lower level of activity after surgery relative to primary cemented cups . however , the average wear of the cups with impaction grafting was the same as the cups without impaction grafting ( both 0.08 mm / year ) . additional research on level of activity and impaction grafting in young patients is necessary to confirm this hypothesis . although the cups reconstructed with bone impaction grafting were the more demanding procedures , no clinical differences were seen.table 7reported outcomes of the harris hip score in patients < 40 years for primary thastudyquestionnairepreoperative scorepostoperative scorepaired t - test p valuechiu et al . although revision rates in both groups were comparable , the time to revision was longer in the cups reconstructed with bone impaction grafting . we have no clear explanation for this observation ; possibly the cement - bone interface was better in cups with bone impaction grafting with better interdigitation of the cement into the bone . this also may explain the lower incidence of radiolucent lines in the cups reconstructed with bone impaction grafting . this might be attributed to the different mobilization protocol for the patients who received cups with impaction grafting . especially in these young patients , there is a need for total hip implants with proven long - term survival . although the use of uncemented prostheses in these young patients is very popular , literature regarding long - term outcome of tha in patients younger than 40 years concerns mainly studies of cemented implants and less about uncemented implants ( table 8) [ 5 , 8 , 9 , 15 , 18 , 20 , 25 , 3234 ] . the only report of uncemented cups at 15 years after surgery with an end point of revision for any reason showed a survival rate of 54% . the survival of the acetabular uncemented cups with an end point of revision for aseptic loosening in patients younger than 40 years reported in one study was 85% , in contrast to a survival rate of 96% after 10 years of the charnley cups in the study by joshi et al . we found a survival rate with cemented cups of 92% at 10 years with an end point of revision for aseptic loosening.table 8long - term acetabular cup survival rates in patients younger than 40 yearsstudynumber of hipsage ( years)*followup ( years)*type of cupsurvival5 years7 years10 years15 years20 years25 years30 yearsuncementedbizot et al . long - term acetabular cup survival rates in patients younger than 40 years studies published through 2007 ; * values are expressed as mean , with range in parentheses ; values are expressed as percentage , with 95% confidence interval in parentheses ; deceased and revised excluded ; lost to followup , deceased excluded , only chinese patients ; aml = anatomic medullary locking ; pca = porous - coated anatomic ; excl inf = excluding infections ; h - g = harris - galante ; se = standard error . a remarkable finding of our study was the survival of cups with acetabular reconstructions with impaction grafting was at least comparable to the survival of standard cemented cups , especially considering the more difficult hips of our study population needed reconstruction with impaction grafting . our data on the cemented cups with impaction grafting showed similar survival , where rather lower survival rates would be expected . the outcome of these cups reconstructed with impaction grafting even fulfilled the nice criteria ( a survival of > 90% after 10 years ) , with a survival rate of 91% at 10 years with an end point of revision for any reason . the survival rates of the cemented cups in our study are comparable to those reported for cemented cups [ 8 , 18 , 34 ] . although cemented cups are not commonly used in young patients , our data suggest cemented conventional polyethylene cups are still a good option in tha in young patients . even reconstruction of ( severe ) acetabular deficiencies with impaction grafting and a cemented conventional polyethylene cup produced very acceptable survival rates , comparable to the rates of cemented cups implanted in acetabuli without deficiencies with standard cementing techniques .

the delivery of health care services in the united states is characterized by geographic variations that can not be justified scientifically . consequently , many assumptions about the quality of us health care and the appropriateness of health care interventions are being challenged . clinicians and their patients must decide what kind of care is best , whereas health policy decision makers and third - party payors are faced with the considerable challenge of determining the appropriateness of various interventions . the chiropractic profession is the third largest portal - of - entry health profession in the united states ( after medicine and dentistry ) , but the appropriate role for chiropractic in the united states has not been established . the role of chiropractic has been explored from both a biomedical and a sociological perspective , but the profession has failed to establish a coherent vision of purpose . amid this context of uncertainty , third - party payors and public policy makers must make decisions about the appropriate role for chiropractors in health care systems and for the clinical services that chiropractors provide . the purpose of this article is to discuss the implications and limitations of chiropractic - related appropriateness studies . the 1998 editorial in the new england journal of medicine posed the question , what role for chiropractic in health care ? paul shekelle , author of numerous articles on the appropriateness of health care interventions , considered the question of whether chiropractic should be considered a nonsurgical specialty or an alternative to medicine . following his review of the evidence , he concluded that chiropractic provides limited benefits for musculoskeletal conditions , but use of chiropractic as a broad - based alternative to medical care is inappropriate . after another decade of chiropractic research activity , that answer may still ring true , but the questions have changed . spinal manipulation , the intervention most commonly used by chiropractors , is now of proven clinical value for treatment of certain conditions . despite efforts toward integration since the rand corporation studies on the appropriateness of spinal manipulation were published in the 1990s , the appropriateness of chiropractic care has not been rigorously investigated . however , the rand studies focused on spinal manipulation , not chiropractic as a whole . this is how the profession is viewed by most chiropractors themselves and by many patients . if chiropractic is a whole health system , then its appropriateness should be measured not only by the efficacy of a single intervention but by the effectiveness of the entire chiropractic clinical encounter . with increasing interest in the integration of medical and nonmedical health professions , it may prove helpful to rigorously assess the appropriateness of general chiropractic care . decisions about inclusion in integrative clinics and health plans may be made on the basis of professional identity rather than solely on the services provided . the appropriateness of chiropractic care in general is therefore relevant to decisions regarding inclusion of doctors of chiropractic . published studies on the appropriateness of medical interventions appear to have been grounded on the tacit premise that inclusion of conventional medicine in health care is appropriate . as a nondominant profession in the health care environment , appropriate care is care that is worth providing and that has a favorable risk - benefit ratio . assessments of appropriateness can inform public policy and third - party reimbursement as well as provider and patient decision making . research methodologies relevant to the study of appropriateness include small area analysis and the rand - ucla appropriateness method . small area analysis techniques allow researchers to describe and map geographic variations in health care utilization , describe patterns in variation , and identify variables that may in part explain the variation . the rand - ucla appropriateness method is an established means of assessing the appropriateness of a health care intervention . in this method , a literature review is performed to create a list of clinical indications for using a particular procedure . members of a panel of experts critically review and synthesize the evidence to generate quantitative estimates of the benefits and harms , and independently rate the appropriateness of performing the procedure for each indication . the panel members subsequently meet , discuss areas of disagreement , and again independently rate the indications . criteria resulting from the application of this method can be used to retrospectively rank the appropriateness of interventions . appropriateness criteria can be used to inform clinical decision making , but are probably more useful for health policy decision making . the rand - ucla appropriateness method was developed in part to help answer questions about appropriateness that were originally raised by studies conducted by john wennberg . in 1973 , wennberg and gittelsohn published the first in a series of studies that described unexplained geographic variations in medical care . since then , the dartmouth atlas of health care project , using methods of small area analysis that define local health care markets , has examined differences in per capita resource inputs and utilization of various medical and surgical services . this research has uncovered differences in the distribution and use of health care services in 306 hospital referral regions across the united states many such variations are likely to be inappropriate if they can not be adequately explained on the basis of differences among regions in illness rates or sociodemographic characteristics . geographic variations in medical spending have been found to be due to differences in the number of physician visits ( particularly inpatient physician visits ) , medical procedures , and use of specialty medical services . interestingly , areas of higher medical spending ( often referred to as high practice intensity areas ) do not appear to have better health care outcomes or higher levels of patient satisfaction . to explain the geographic variations in medical spending , the dartmouth atlas of health care classifies health care services into 3 categories of variation:supply - sensitive care ( 63% of care)effective care ( 12% of care)preference - sensitive care ( 25% of care ) supply - sensitive care ( 63% of care ) effective care ( 12% of care ) preference - sensitive care ( 25% of care ) supply - sensitive care is governed by the local supply of health care services : the greater the supply , the higher the rate of use . higher rates of use of supply - sensitive care however may not confer better health outcomes . effective care is appropriate care ; it consists of health care services that are proven effective and have a favorable risk - benefit ratio . an example of effective care is surgical fixation of a severe open comminuted tibia fracture in an otherwise healthy patient . preference - sensitive care includes services for which the pros and cons are subject to interpretation ; in these cases , when the best choice of care is not clear - cut , patients should be given the information and support they require to share in the decision making . nonspecific low back pain is an example of a condition that may be subject to preference - sensitive care . in such cases , the choice should be based on the patient 's own preferences ; but all too often , it is the provider who decides . appropriate care is care that is worth providing and that has a favorable risk - benefit ratio . assessments of appropriateness can inform public policy and third - party reimbursement as well as provider and patient decision making . research methodologies relevant to the study of appropriateness include small area analysis and the rand - ucla appropriateness method . small area analysis techniques allow researchers to describe and map geographic variations in health care utilization , describe patterns in variation , and identify variables that may in part explain the variation . the rand - ucla appropriateness method is an established means of assessing the appropriateness of a health care intervention . in this method , a literature review is performed to create a list of clinical indications for using a particular procedure . members of a panel of experts critically review and synthesize the evidence to generate quantitative estimates of the benefits and harms , and independently rate the appropriateness of performing the procedure for each indication . the panel members subsequently meet , discuss areas of disagreement , and again independently rate the indications . a mean appropriateness score for each intervention criteria resulting from the application of this method can be used to retrospectively rank the appropriateness of interventions . appropriateness criteria can be used to inform clinical decision making , but are probably more useful for health policy decision making . the rand - ucla appropriateness method was developed in part to help answer questions about appropriateness that were originally raised by studies conducted by john wennberg . in 1973 , wennberg and gittelsohn published the first in a series of studies that described unexplained geographic variations in medical care . since then , the dartmouth atlas of health care project , using methods of small area analysis that define local health care markets , has examined differences in per capita resource inputs and utilization of various medical and surgical services . this research has uncovered differences in the distribution and use of health care services in 306 hospital referral regions across the united states health care spending by medicare enrollees varies by as much as 2.5-fold among regions . many such variations are likely to be inappropriate if they can not be adequately explained on the basis of differences among regions in illness rates or sociodemographic characteristics . geographic variations in medical spending have been found to be due to differences in the number of physician visits ( particularly inpatient physician visits ) , medical procedures , and use of specialty medical services . interestingly , areas of higher medical spending ( often referred to as high practice intensity areas ) do not appear to have better health care outcomes or higher levels of patient satisfaction . to explain the geographic variations in medical spending , the dartmouth atlas of health care classifies health care services into 3 categories of variation:supply - sensitive care ( 63% of care)effective care ( 12% of care)preference - sensitive care ( 25% of care ) supply - sensitive care ( 63% of care ) effective care ( 12% of care ) preference - sensitive care ( 25% of care ) supply - sensitive care is governed by the local supply of health care services : the greater the supply , the higher the rate of use . higher rates of use of supply - sensitive care however may not confer better health outcomes . effective care is appropriate care ; it consists of health care services that are proven effective and have a favorable risk - benefit ratio . an example of effective care is surgical fixation of a severe open comminuted tibia fracture in an otherwise healthy patient . preference - sensitive care includes services for which the pros and cons are subject to interpretation ; in these cases , when the best choice of care is not clear - cut , patients should be given the information and support they require to share in the decision making . nonspecific low back pain is an example of a condition that may be subject to preference - sensitive care . in such cases , the choice should be based on the patient 's own preferences ; but all too often , it is the provider who decides . both small area analysis and the rand - ucla appropriateness method have implications relevant to the assessment of chiropractic appropriateness , and the rand method has been applied to the evaluation of spinal manipulation . as the united states struggles with health care reform , the concept of the appropriateness of medical interventions is receiving attention from policy makers . officials at the highest levels of the federal government recognize that unwarranted variations in medical utilization and spending are well documented and that it may be possible to control ballooning health care costs by correcting the overuse , underuse , and misuse of medical care . within this context , questions about the appropriateness of chiropractic care decision makers who know that more health care does not necessarily mean better care may ask , for which patients is chiropractic care appropriate and for what indications and purpose ? the assessment of appropriateness , whether applied to medicine , chiropractic , or any other clinical discipline or intervention , is a subjective determination that should draw upon objective evidence for effectiveness , safety , and cost . the manner in which clinical decision making occurs also bears upon the appropriateness of care . without the participation of the patient , the very idea of judging one approach to care as being more appropriate than another might be rightly perceived as high - handed . decisions about the utilization of health care services must take into account the individualized needs and preferences of the patient and should not be driven by the needs , inclinations , or specialized expertise of the doctor . the sharing of clinical decision - making between doctor and patient is thought to help facilitate the delivery of appropriate care . fig 1 illustrates the interplay of 4 principal factors involved in the assessment of appropriateness . in a series of studies in the 1990s , the rand - ucla appropriateness method was applied to the evaluation of spinal manipulation for neck and low back pain . the first study convened a multidisciplinary panel , including chiropractic physicians , medical doctors , and an osteopathic physician . of 1550 indications for spinal manipulation , the panel found 60% to be inappropriate , 30% equivocal , and 7% appropriate . the second study convened an all - chiropractic panel , which found that of 1570 indications , 48% were inappropriate , 25% uncertain , and 27% appropriate . the authors noted that , the large number ( 48% ) of indications felt to be inappropriate probably reflects our attempt to make exhaustive the list of potential indications for performing spinal manipulation . in both panels , the indications deemed appropriate included the more common presentations of back pain , whereas the inappropriate indications contained many uncommon presentations . ratings varied significantly among panel members ; chiropractors were more likely to rate clinical indications as appropriate for manipulation than were nonchiropractors . a retrospective review of chiropractic office records was subsequently conducted to determine the appropriateness of the use of spinal manipulation for low back pain using the 9-point scale criteria . the study found the use of spinal manipulation to be appropriate in 46% of cases and inappropriate in 29% . the assessment was inconclusive in 25% of cases . for cases in which the patient received chiropractic care but did not receive spinal manipulation a similar set of appropriateness ratings was developed for manipulation and mobilization of the cervical spine . , consideration of the concepts of supply - sensitive care , effective care , and preference - sensitive care may aid the formulation of hypotheses regarding the appropriateness of chiropractic care . overutilization of supply - sensitive care occurs when care is rendered in proportion to increased availability rather than clinical necessity . a 2008 study found evidence of oversupply of chiropractic services in a market of questionable demand in ontario , canada . another study that examined the national supply and demand of us chiropractors uncovered a 28% decrease in the national supply of new chiropractic college graduates , whereas national expenditures on chiropractic care grew significantly over the same period . by contrast , systematic underutilization of effective care occurs when services of proven value for which the benefits outweigh the risks are underused because of lack of support for systematic compliance with treatment guidelines . spinal manipulation is generally considered to be a safe , effective , and cost - effective treatment of certain spinal pain disorders ; and the number of individuals with such spinal pain disorders appears to greatly exceed the current number of chiropractic users . however , many of the studies that have demonstrated the effectiveness of spinal manipulation for spinal pain disorders have not shown outcomes significantly better than other therapies , although patients do often report high levels of satisfaction . spinal manipulation for certain spinal pain disorders therefore may be considered to fall under the category of preference - sensitive care . the category of variation for a given intervention may depend upon the condition being treated , patient - related variables , and other factors ; and considerable overlap between categories may occur for any given procedure . a conspicuous geographic variation in the delivery of chiropractic care was recently revealed by analysis of the medicare chiropractic services demonstration , which was intended to evaluate the effects of expanded coverage for chiropractic services . despite reduced costs in 4 of 5 demonstration areas , a budget neutrality analysis found a net increase in overall medicare payments in demonstration areas as compared with control areas . this finding suggests the possibility that care delivered in the chicago demonstration site may have been supply sensitive . the rand - ucla method addresses the appropriateness of initiating treatment , but not of frequency or duration of treatment , issues of particular relevance to chiropractic care , which is characterized by serial treatments . furthermore , the rand - ucla method is intervention and condition based ; and therefore , although applicable to the evaluation of an intervention ( ie , spinal manipulation ) for the care of a disorder ( ie , low back pain ) , it is not designed to evaluate a complex clinical encounter . therefore , the rand - ucla method may be unsuited to evaluate the appropriateness of chiropractic care in general . chiropractic care includes but is not limited to spinal manipulation , so it would be erroneous to equate the appropriateness of the common domain procedure of spinal manipulation with the appropriateness of chiropractic health care . a typical chiropractic clinical encounter may include ( in addition to or instead of spinal manipulation ) physical therapy modalities and patient counseling on diet , nutritional supplementation , exercise , and lifestyle modification . methods of small area analysis used to investigate geographic variations in medical care could be applied to the study of chiropractic care . any analysis of medicare claims for chiropractic , however , must be interpreted in light of medicare 's restrictive reimbursement policies . medicare dictates that the primary diagnosis must be a vertebral subluxation and the secondary diagnosis must be a related neuromusculoskeletal condition , and the only reimbursable treatment procedure is spinal manipulation . medicare claims data thus effectively equate chiropractic care with spinal manipulation for neuromusculoskeletal conditions related to spinal dysfunction , and inferences regarding the appropriateness of spinal manipulation may not be generalized to chiropractic in general . in and of themselves , both small area analysis and the rand - ucla method offer limited possibilities for the assessment of chiropractic appropriateness . small area analysis of medicare claims offers great potential for descriptive studies and may also be used to help explain variations in chiropractic utilization . for example , geographic variations in the provision of services may be related to variations in state scope of practice laws or patient demographics . however , any attempt to analyze the appropriateness of chiropractic care in general would be limited by medicare 's restrictive inclusion and reimbursement policies . if applied to a less restrictive source of data , techniques of small area analysis might have greater potential ; but medicare claims are currently the single most comprehensive source of clinical data available in the united states . the rand - ucla method is similarly limited because it was designed to evaluate interventions , but not a clinical encounter in its entirety . furthermore , the expert opinion generated by the rand - ucla method is probably more applicable to the evaluation of interventions for which there is little evidence , and may be regarded as superfluous if applied to interventions for which systematic reviews and clinical practice guidelines are already available . finally , scant evidence is available for assessing the appropriate role for chiropractic care in general as a complex clinical encounter . the personalized , patient - centered paradigm of chiropractic care and the wide variation in chiropractic techniques and practice styles present challenges for appropriateness research . it may be necessary to use new methods to evaluate the appropriateness of chiropractic as a whole health system . in a series of studies in the 1990s , the rand - ucla appropriateness method was applied to the evaluation of spinal manipulation for neck and low back pain . the first study convened a multidisciplinary panel , including chiropractic physicians , medical doctors , and an osteopathic physician . of 1550 indications for spinal manipulation , the panel found 60% to be inappropriate , 30% equivocal , and 7% appropriate . the second study convened an all - chiropractic panel , which found that of 1570 indications , 48% were inappropriate , 25% uncertain , and 27% appropriate . the authors noted that , the large number ( 48% ) of indications felt to be inappropriate probably reflects our attempt to make exhaustive the list of potential indications for performing spinal manipulation . in both panels , the indications deemed appropriate included the more common presentations of back pain , whereas the inappropriate indications contained many uncommon presentations . ratings varied significantly among panel members ; chiropractors were more likely to rate clinical indications as appropriate for manipulation than were nonchiropractors . a retrospective review of chiropractic office records was subsequently conducted to determine the appropriateness of the use of spinal manipulation for low back pain using the 9-point scale criteria . the study found the use of spinal manipulation to be appropriate in 46% of cases and inappropriate in 29% . the assessment was inconclusive in 25% of cases . for cases in which the patient received chiropractic care but did not receive spinal manipulation a similar set of appropriateness ratings was developed for manipulation and mobilization of the cervical spine . these methods have not been applied to the study of chiropractic appropriateness . however , consideration of the concepts of supply - sensitive care , effective care , and preference - sensitive care may aid the formulation of hypotheses regarding the appropriateness of chiropractic care . overutilization of supply - sensitive care occurs when care is rendered in proportion to increased availability rather than clinical necessity . a 2008 study found evidence of oversupply of chiropractic services in a market of questionable demand in ontario , canada . another study that examined the national supply and demand of us chiropractors uncovered a 28% decrease in the national supply of new chiropractic college graduates , whereas national expenditures on chiropractic care grew significantly over the same period . by contrast , systematic underutilization of effective care occurs when services of proven value for which the benefits outweigh the risks are underused because of lack of support for systematic compliance with treatment guidelines . spinal manipulation is generally considered to be a safe , effective , and cost - effective treatment of certain spinal pain disorders ; and the number of individuals with such spinal pain disorders appears to greatly exceed the current number of chiropractic users . however , many of the studies that have demonstrated the effectiveness of spinal manipulation for spinal pain disorders have not shown outcomes significantly better than other therapies , although patients do often report high levels of satisfaction . spinal manipulation for certain spinal pain disorders therefore may be considered to fall under the category of preference - sensitive care . the category of variation for a given intervention may depend upon the condition being treated , patient - related variables , and other factors ; and considerable overlap between categories may occur for any given procedure . a conspicuous geographic variation in the delivery of chiropractic care was recently revealed by analysis of the medicare chiropractic services demonstration , which was intended to evaluate the effects of expanded coverage for chiropractic services . despite reduced costs in 4 of 5 demonstration areas , a budget neutrality analysis found a net increase in overall medicare payments in demonstration areas as compared with control areas . this finding suggests the possibility that care delivered in the chicago demonstration site may have been supply sensitive . the rand - ucla method addresses the appropriateness of initiating treatment , but not of frequency or duration of treatment , issues of particular relevance to chiropractic care , which is characterized by serial treatments . furthermore , the rand - ucla method is intervention and condition based ; and therefore , although applicable to the evaluation of an intervention ( ie , spinal manipulation ) for the care of a disorder ( ie , low back pain ) , it is not designed to evaluate a complex clinical encounter . therefore , the rand - ucla method may be unsuited to evaluate the appropriateness of chiropractic care in general . chiropractic care includes but is not limited to spinal manipulation , so it would be erroneous to equate the appropriateness of the common domain procedure of spinal manipulation with the appropriateness of chiropractic health care . a typical chiropractic clinical encounter may include ( in addition to or instead of spinal manipulation ) physical therapy modalities and patient counseling on diet , nutritional supplementation , exercise , and lifestyle modification . methods of small area analysis used to investigate geographic variations in medical care could be applied to the study of chiropractic care . any analysis of medicare claims for chiropractic , however , must be interpreted in light of medicare 's restrictive reimbursement policies . vertebral subluxation and the secondary diagnosis must be a related neuromusculoskeletal condition , and the only reimbursable treatment procedure is spinal manipulation . medicare claims data thus effectively equate chiropractic care with spinal manipulation for neuromusculoskeletal conditions related to spinal dysfunction , and inferences regarding the appropriateness of spinal manipulation may not be generalized to chiropractic in general . in and of themselves , both small area analysis and the rand - ucla method offer limited possibilities for the assessment of chiropractic appropriateness . small area analysis of medicare claims offers great potential for descriptive studies and may also be used to help explain variations in chiropractic utilization . for example , geographic variations in the provision of services may be related to variations in state scope of practice laws or patient demographics . however , any attempt to analyze the appropriateness of chiropractic care in general would be limited by medicare 's restrictive inclusion and reimbursement policies . if applied to a less restrictive source of data , techniques of small area analysis might have greater potential ; but medicare claims are currently the single most comprehensive source of clinical data available in the united states . the rand - ucla method is similarly limited because it was designed to evaluate interventions , but not a clinical encounter in its entirety . furthermore , the expert opinion generated by the rand - ucla method is probably more applicable to the evaluation of interventions for which there is little evidence , and may be regarded as superfluous if applied to interventions for which systematic reviews and clinical practice guidelines are already available . finally , scant evidence is available for assessing the appropriate role for chiropractic care in general as a complex clinical encounter . the personalized , patient - centered paradigm of chiropractic care and the wide variation in chiropractic techniques and practice styles present challenges for appropriateness research . it may be necessary to use new methods to evaluate the appropriateness of chiropractic as a whole health system . this article discussed the relevance of appropriateness studies , methodologies used to assess appropriateness , and identified implications and limitations of those approaches for the evaluation of chiropractic care . the future assessment of the appropriate role for chiropractic in us health care will raise issues beyond the scope of previous appropriateness studies . studying the appropriate role for chiropractic will require consideration of the clinical discipline in its entirety , rather than individual consideration of specific interventions . a fair assessment will require new evidence , and perhaps new research methodologies , both for generating evidence on quality and for assessing appropriateness based upon that evidence . future assessments of the role of chiropractic in us health care will inevitably involve political considerations as well as scientific evidence . whereas chiropractic physicians may have limited control over the former , the profession can exert some control over the latter by guiding the research agenda toward a comprehensive assessment of the benefits of chiropractic care . drs whedon and davis are both supported by grants from the national center for complementary and alternative medicine and the bernard osher foundation . dr whedon 's grant the views expressed herein do not necessarily represent the views of the national center for complementary and alternative medicine or of the national institutes of health .

objectivethe appropriate role for chiropractic in us health care has not been established , but third - party payors and public policy makers must make decisions about the appropriate role for chiropractors in health care systems and for the services that chiropractors provide . appropriateness studies for chiropractic may inform those decisions . the purpose of this article is to discuss the implications and limitations of appropriateness studies for chiropractic.discussionwe reviewed the general context for assessment of the appropriateness and the application of appropriateness studies to chiropractic in particular . we evaluated the implications and limitations for chiropractic of methods of small area analysis and the rand - ucla appropriateness method . the rand - ucla appropriateness method has been applied to the evaluation of spinal manipulation . regional variations in chiropractic utilization have yet to be described through small area analysis , but these methods appear to hold some potential for assessing the appropriateness of chiropractic care . both small area analysis and the rand - ucla method offer limited possibilities for the assessment of chiropractic appropriateness.conclusionfuture assessment of the appropriate role for chiropractic in us health care will raise issues beyond the scope of previous appropriateness studies . studying the appropriate role for chiropractic will require consideration of the clinical discipline in its entirety , rather than individual consideration of specific interventions . a fair assessment of chiropractic appropriateness will require new evidence and perhaps new research methodologies .

Introduction Discussion Assessment of the appropriateness of health care services Implications of appropriateness studies for chiropractic Implications of the RAND-UCLA Appropriateness Method Potential implications of small area analysis Limitations of appropriateness studies for chiropractic Conclusion Funding sources and potential conflicts of interest

consequently , many assumptions about the quality of us health care and the appropriateness of health care interventions are being challenged . clinicians and their patients must decide what kind of care is best , whereas health policy decision makers and third - party payors are faced with the considerable challenge of determining the appropriateness of various interventions . the chiropractic profession is the third largest portal - of - entry health profession in the united states ( after medicine and dentistry ) , but the appropriate role for chiropractic in the united states has not been established . the role of chiropractic has been explored from both a biomedical and a sociological perspective , but the profession has failed to establish a coherent vision of purpose . amid this context of uncertainty , third - party payors and public policy makers must make decisions about the appropriate role for chiropractors in health care systems and for the clinical services that chiropractors provide . the purpose of this article is to discuss the implications and limitations of chiropractic - related appropriateness studies . the 1998 editorial in the new england journal of medicine posed the question , what role for chiropractic in health care ? paul shekelle , author of numerous articles on the appropriateness of health care interventions , considered the question of whether chiropractic should be considered a nonsurgical specialty or an alternative to medicine . following his review of the evidence , he concluded that chiropractic provides limited benefits for musculoskeletal conditions , but use of chiropractic as a broad - based alternative to medical care is inappropriate . after another decade of chiropractic research activity , that answer may still ring true , but the questions have changed . despite efforts toward integration since the rand corporation studies on the appropriateness of spinal manipulation were published in the 1990s , the appropriateness of chiropractic care has not been rigorously investigated . however , the rand studies focused on spinal manipulation , not chiropractic as a whole . with increasing interest in the integration of medical and nonmedical health professions , it may prove helpful to rigorously assess the appropriateness of general chiropractic care . decisions about inclusion in integrative clinics and health plans may be made on the basis of professional identity rather than solely on the services provided . the appropriateness of chiropractic care in general is therefore relevant to decisions regarding inclusion of doctors of chiropractic . published studies on the appropriateness of medical interventions appear to have been grounded on the tacit premise that inclusion of conventional medicine in health care is appropriate . assessments of appropriateness can inform public policy and third - party reimbursement as well as provider and patient decision making . research methodologies relevant to the study of appropriateness include small area analysis and the rand - ucla appropriateness method . small area analysis techniques allow researchers to describe and map geographic variations in health care utilization , describe patterns in variation , and identify variables that may in part explain the variation . the rand - ucla appropriateness method is an established means of assessing the appropriateness of a health care intervention . members of a panel of experts critically review and synthesize the evidence to generate quantitative estimates of the benefits and harms , and independently rate the appropriateness of performing the procedure for each indication . criteria resulting from the application of this method can be used to retrospectively rank the appropriateness of interventions . the rand - ucla appropriateness method was developed in part to help answer questions about appropriateness that were originally raised by studies conducted by john wennberg . since then , the dartmouth atlas of health care project , using methods of small area analysis that define local health care markets , has examined differences in per capita resource inputs and utilization of various medical and surgical services . this research has uncovered differences in the distribution and use of health care services in 306 hospital referral regions across the united states many such variations are likely to be inappropriate if they can not be adequately explained on the basis of differences among regions in illness rates or sociodemographic characteristics . geographic variations in medical spending have been found to be due to differences in the number of physician visits ( particularly inpatient physician visits ) , medical procedures , and use of specialty medical services . interestingly , areas of higher medical spending ( often referred to as high practice intensity areas ) do not appear to have better health care outcomes or higher levels of patient satisfaction . to explain the geographic variations in medical spending , the dartmouth atlas of health care classifies health care services into 3 categories of variation:supply - sensitive care ( 63% of care)effective care ( 12% of care)preference - sensitive care ( 25% of care ) supply - sensitive care ( 63% of care ) effective care ( 12% of care ) preference - sensitive care ( 25% of care ) supply - sensitive care is governed by the local supply of health care services : the greater the supply , the higher the rate of use . effective care is appropriate care ; it consists of health care services that are proven effective and have a favorable risk - benefit ratio . assessments of appropriateness can inform public policy and third - party reimbursement as well as provider and patient decision making . research methodologies relevant to the study of appropriateness include small area analysis and the rand - ucla appropriateness method . small area analysis techniques allow researchers to describe and map geographic variations in health care utilization , describe patterns in variation , and identify variables that may in part explain the variation . the rand - ucla appropriateness method is an established means of assessing the appropriateness of a health care intervention . members of a panel of experts critically review and synthesize the evidence to generate quantitative estimates of the benefits and harms , and independently rate the appropriateness of performing the procedure for each indication . a mean appropriateness score for each intervention criteria resulting from the application of this method can be used to retrospectively rank the appropriateness of interventions . the rand - ucla appropriateness method was developed in part to help answer questions about appropriateness that were originally raised by studies conducted by john wennberg . since then , the dartmouth atlas of health care project , using methods of small area analysis that define local health care markets , has examined differences in per capita resource inputs and utilization of various medical and surgical services . many such variations are likely to be inappropriate if they can not be adequately explained on the basis of differences among regions in illness rates or sociodemographic characteristics . geographic variations in medical spending have been found to be due to differences in the number of physician visits ( particularly inpatient physician visits ) , medical procedures , and use of specialty medical services . interestingly , areas of higher medical spending ( often referred to as high practice intensity areas ) do not appear to have better health care outcomes or higher levels of patient satisfaction . to explain the geographic variations in medical spending , the dartmouth atlas of health care classifies health care services into 3 categories of variation:supply - sensitive care ( 63% of care)effective care ( 12% of care)preference - sensitive care ( 25% of care ) supply - sensitive care ( 63% of care ) effective care ( 12% of care ) preference - sensitive care ( 25% of care ) supply - sensitive care is governed by the local supply of health care services : the greater the supply , the higher the rate of use . effective care is appropriate care ; it consists of health care services that are proven effective and have a favorable risk - benefit ratio . both small area analysis and the rand - ucla appropriateness method have implications relevant to the assessment of chiropractic appropriateness , and the rand method has been applied to the evaluation of spinal manipulation . as the united states struggles with health care reform , the concept of the appropriateness of medical interventions is receiving attention from policy makers . officials at the highest levels of the federal government recognize that unwarranted variations in medical utilization and spending are well documented and that it may be possible to control ballooning health care costs by correcting the overuse , underuse , and misuse of medical care . within this context , questions about the appropriateness of chiropractic care decision makers who know that more health care does not necessarily mean better care may ask , for which patients is chiropractic care appropriate and for what indications and purpose ? the assessment of appropriateness , whether applied to medicine , chiropractic , or any other clinical discipline or intervention , is a subjective determination that should draw upon objective evidence for effectiveness , safety , and cost . the manner in which clinical decision making occurs also bears upon the appropriateness of care . decisions about the utilization of health care services must take into account the individualized needs and preferences of the patient and should not be driven by the needs , inclinations , or specialized expertise of the doctor . fig 1 illustrates the interplay of 4 principal factors involved in the assessment of appropriateness . in a series of studies in the 1990s , the rand - ucla appropriateness method was applied to the evaluation of spinal manipulation for neck and low back pain . of 1550 indications for spinal manipulation , the panel found 60% to be inappropriate , 30% equivocal , and 7% appropriate . the authors noted that , the large number ( 48% ) of indications felt to be inappropriate probably reflects our attempt to make exhaustive the list of potential indications for performing spinal manipulation . a retrospective review of chiropractic office records was subsequently conducted to determine the appropriateness of the use of spinal manipulation for low back pain using the 9-point scale criteria . the study found the use of spinal manipulation to be appropriate in 46% of cases and inappropriate in 29% . for cases in which the patient received chiropractic care but did not receive spinal manipulation a similar set of appropriateness ratings was developed for manipulation and mobilization of the cervical spine . , consideration of the concepts of supply - sensitive care , effective care , and preference - sensitive care may aid the formulation of hypotheses regarding the appropriateness of chiropractic care . spinal manipulation is generally considered to be a safe , effective , and cost - effective treatment of certain spinal pain disorders ; and the number of individuals with such spinal pain disorders appears to greatly exceed the current number of chiropractic users . however , many of the studies that have demonstrated the effectiveness of spinal manipulation for spinal pain disorders have not shown outcomes significantly better than other therapies , although patients do often report high levels of satisfaction . a conspicuous geographic variation in the delivery of chiropractic care was recently revealed by analysis of the medicare chiropractic services demonstration , which was intended to evaluate the effects of expanded coverage for chiropractic services . the rand - ucla method addresses the appropriateness of initiating treatment , but not of frequency or duration of treatment , issues of particular relevance to chiropractic care , which is characterized by serial treatments . furthermore , the rand - ucla method is intervention and condition based ; and therefore , although applicable to the evaluation of an intervention ( ie , spinal manipulation ) for the care of a disorder ( ie , low back pain ) , it is not designed to evaluate a complex clinical encounter . therefore , the rand - ucla method may be unsuited to evaluate the appropriateness of chiropractic care in general . chiropractic care includes but is not limited to spinal manipulation , so it would be erroneous to equate the appropriateness of the common domain procedure of spinal manipulation with the appropriateness of chiropractic health care . a typical chiropractic clinical encounter may include ( in addition to or instead of spinal manipulation ) physical therapy modalities and patient counseling on diet , nutritional supplementation , exercise , and lifestyle modification . methods of small area analysis used to investigate geographic variations in medical care could be applied to the study of chiropractic care . any analysis of medicare claims for chiropractic , however , must be interpreted in light of medicare 's restrictive reimbursement policies . medicare dictates that the primary diagnosis must be a vertebral subluxation and the secondary diagnosis must be a related neuromusculoskeletal condition , and the only reimbursable treatment procedure is spinal manipulation . medicare claims data thus effectively equate chiropractic care with spinal manipulation for neuromusculoskeletal conditions related to spinal dysfunction , and inferences regarding the appropriateness of spinal manipulation may not be generalized to chiropractic in general . in and of themselves , both small area analysis and the rand - ucla method offer limited possibilities for the assessment of chiropractic appropriateness . small area analysis of medicare claims offers great potential for descriptive studies and may also be used to help explain variations in chiropractic utilization . for example , geographic variations in the provision of services may be related to variations in state scope of practice laws or patient demographics . however , any attempt to analyze the appropriateness of chiropractic care in general would be limited by medicare 's restrictive inclusion and reimbursement policies . if applied to a less restrictive source of data , techniques of small area analysis might have greater potential ; but medicare claims are currently the single most comprehensive source of clinical data available in the united states . the rand - ucla method is similarly limited because it was designed to evaluate interventions , but not a clinical encounter in its entirety . furthermore , the expert opinion generated by the rand - ucla method is probably more applicable to the evaluation of interventions for which there is little evidence , and may be regarded as superfluous if applied to interventions for which systematic reviews and clinical practice guidelines are already available . finally , scant evidence is available for assessing the appropriate role for chiropractic care in general as a complex clinical encounter . the personalized , patient - centered paradigm of chiropractic care and the wide variation in chiropractic techniques and practice styles present challenges for appropriateness research . it may be necessary to use new methods to evaluate the appropriateness of chiropractic as a whole health system . in a series of studies in the 1990s , the rand - ucla appropriateness method was applied to the evaluation of spinal manipulation for neck and low back pain . of 1550 indications for spinal manipulation , the panel found 60% to be inappropriate , 30% equivocal , and 7% appropriate . the authors noted that , the large number ( 48% ) of indications felt to be inappropriate probably reflects our attempt to make exhaustive the list of potential indications for performing spinal manipulation . a retrospective review of chiropractic office records was subsequently conducted to determine the appropriateness of the use of spinal manipulation for low back pain using the 9-point scale criteria . the study found the use of spinal manipulation to be appropriate in 46% of cases and inappropriate in 29% . for cases in which the patient received chiropractic care but did not receive spinal manipulation a similar set of appropriateness ratings was developed for manipulation and mobilization of the cervical spine . these methods have not been applied to the study of chiropractic appropriateness . however , consideration of the concepts of supply - sensitive care , effective care , and preference - sensitive care may aid the formulation of hypotheses regarding the appropriateness of chiropractic care . spinal manipulation is generally considered to be a safe , effective , and cost - effective treatment of certain spinal pain disorders ; and the number of individuals with such spinal pain disorders appears to greatly exceed the current number of chiropractic users . however , many of the studies that have demonstrated the effectiveness of spinal manipulation for spinal pain disorders have not shown outcomes significantly better than other therapies , although patients do often report high levels of satisfaction . a conspicuous geographic variation in the delivery of chiropractic care was recently revealed by analysis of the medicare chiropractic services demonstration , which was intended to evaluate the effects of expanded coverage for chiropractic services . the rand - ucla method addresses the appropriateness of initiating treatment , but not of frequency or duration of treatment , issues of particular relevance to chiropractic care , which is characterized by serial treatments . furthermore , the rand - ucla method is intervention and condition based ; and therefore , although applicable to the evaluation of an intervention ( ie , spinal manipulation ) for the care of a disorder ( ie , low back pain ) , it is not designed to evaluate a complex clinical encounter . therefore , the rand - ucla method may be unsuited to evaluate the appropriateness of chiropractic care in general . chiropractic care includes but is not limited to spinal manipulation , so it would be erroneous to equate the appropriateness of the common domain procedure of spinal manipulation with the appropriateness of chiropractic health care . a typical chiropractic clinical encounter may include ( in addition to or instead of spinal manipulation ) physical therapy modalities and patient counseling on diet , nutritional supplementation , exercise , and lifestyle modification . methods of small area analysis used to investigate geographic variations in medical care could be applied to the study of chiropractic care . vertebral subluxation and the secondary diagnosis must be a related neuromusculoskeletal condition , and the only reimbursable treatment procedure is spinal manipulation . medicare claims data thus effectively equate chiropractic care with spinal manipulation for neuromusculoskeletal conditions related to spinal dysfunction , and inferences regarding the appropriateness of spinal manipulation may not be generalized to chiropractic in general . in and of themselves , both small area analysis and the rand - ucla method offer limited possibilities for the assessment of chiropractic appropriateness . small area analysis of medicare claims offers great potential for descriptive studies and may also be used to help explain variations in chiropractic utilization . for example , geographic variations in the provision of services may be related to variations in state scope of practice laws or patient demographics . however , any attempt to analyze the appropriateness of chiropractic care in general would be limited by medicare 's restrictive inclusion and reimbursement policies . if applied to a less restrictive source of data , techniques of small area analysis might have greater potential ; but medicare claims are currently the single most comprehensive source of clinical data available in the united states . the rand - ucla method is similarly limited because it was designed to evaluate interventions , but not a clinical encounter in its entirety . furthermore , the expert opinion generated by the rand - ucla method is probably more applicable to the evaluation of interventions for which there is little evidence , and may be regarded as superfluous if applied to interventions for which systematic reviews and clinical practice guidelines are already available . finally , scant evidence is available for assessing the appropriate role for chiropractic care in general as a complex clinical encounter . the personalized , patient - centered paradigm of chiropractic care and the wide variation in chiropractic techniques and practice styles present challenges for appropriateness research . it may be necessary to use new methods to evaluate the appropriateness of chiropractic as a whole health system . this article discussed the relevance of appropriateness studies , methodologies used to assess appropriateness , and identified implications and limitations of those approaches for the evaluation of chiropractic care . the future assessment of the appropriate role for chiropractic in us health care will raise issues beyond the scope of previous appropriateness studies . studying the appropriate role for chiropractic will require consideration of the clinical discipline in its entirety , rather than individual consideration of specific interventions . a fair assessment will require new evidence , and perhaps new research methodologies , both for generating evidence on quality and for assessing appropriateness based upon that evidence . future assessments of the role of chiropractic in us health care will inevitably involve political considerations as well as scientific evidence . whereas chiropractic physicians may have limited control over the former , the profession can exert some control over the latter by guiding the research agenda toward a comprehensive assessment of the benefits of chiropractic care .

in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . this technique relies on ultraviolet irradiation to induce protein covalent crosslinks in protein - rna complexes in situ . this allows the use of highly stringent immunoprecipitation and washing conditions so that only those rnas directly bound to the protein of interest are selected ( reviewed by ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . each replicate comprised two independent experiments : one involved immunoprecipitation ( ip ) of endogenous dgcr8 protein ( supplementary fig 1a , left ) whereas the second one involved ip of a transiently transfected epitope - tagged dgcr8 protein ( pcg t7-dgcr8 ) ( supplementary fig 1a , right and 1b ) . we focused our analyses on the second replicate , since it gave higher depth of coverage and better mapping to the genome . 37 million reads for the endogenous dgcr8 protein , and 36 million reads for the epitope - tagged t7-dgcr8 and 93% and 98% of the reads were mapped to the genome , respectively ( supplementary table 1 ) . after removing read duplicates , uniquely mapped reads from each of the datasets were clustered according to their genomic positions . in order to identify significant clusters we applied a modified false discovery rate ( mfdr ) to each cluster , as previously described . we assessed the reproducibility of the dgcr8 targets identified in clip experiments for both endogenous and overexpressed dgcr8 proteins . analysis of the reads of these two samples revealed a strong correlation ( pearson correlation co - efficient r > 0.8 ) ( fig . in order to describe dgcr8 bona fide targets , we focused on overlapping significant clusters between these two samples ( for a complete list go to http://regulatorygenomics.upf.edu/data/dgcr8 ) . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . we also found that 30% of the genomic clusters mapped to repetitive elements , mainly line-1 and ltr retroelements , but these were excluded from the graphical representation . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . as expected , due to the canonical function of the microprocessor , we identified 117 mirnas ( fig . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . in addition for each of the clusters , an rna secondary structure was predicted in order to select those clusters overlapping with structures resembling that of pri - mirnas ( see http://regulatorygenomics.upf.edu/data/dgcr8 and supplementary methods ) . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . in addition , we observed that between 11 - 20% of the dgcr8 binding sites overlap with small rnas harboring a 5phosphorylated end , which are proposed to be generated by endonucleolytic activity . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . we further confirmed this interaction by immunoprecipitating overexpressed and endogenous dgcr8 protein followed by analysis of the associated rna by semi - quantitative rt - pcr ( fig . in order to study the effect of microprocessor binding to the malat1 rna , we determined its steady - state levels upon sirna - mediated depletion of drosha in hela cells . we observed a two - fold increase in the levels of malat1 rna when drosha levels were reduced ( fig . 2c ) . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . in order to test direct cleavage of the malat1 mrna in vivo , we fused the 5end of malat1 , which harbors significant dgcr8 clusters ( fig . 2a , indicated with a box ) to a luciferase reporter gene and transfected this construct into hela cells . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . another class of rnas bound by dgcr8 corresponds to small nucleolar rnas ( snornas ) ( fig . these small rnas are responsible for guiding many chemical modifications of other rnas , such as rrna , trna and snrnas . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . this reported binding to snornas has a physiological consequence as seen by the accumulation of u17a , u16 and u92 , which represent different subclasses of snornas , in mouse cells lacking dgcr8 ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . importantly , mature snorna levels did not change in the absence of dicer ( fig . thus , dgcr8 can affect the stability of the mature forms of an h / aca box snorna ( u17a ) , a c / d box snorna ( u16 ) and a small cajal rna ( scarna u92 ) ( fig . their biogenesis involves trimming of the host introns from the 5 and 3 end following pre - mrna splicing and release of the mature snorna form that is being protected from degradation by the core snornp components ( reviewed by ) . we determined that dgcr8 affects the levels of mature snornas but not of their precursor forms ( fig . 3c ) , indicating that dgcr8 acts at a post - synthesis level . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . to our surprise we could not detect any significant change in mature snorna levels in these situations ( fig . 2d ) . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . to test this hypothesis we incubated radiolabeled mature snornas with immunoprecipitated t7-dgcr8 and observed the appearance of smaller bands , corresponding to digested products ( fig . importantly , the addition of immunoprecipitated flag - drosha did not result in the formation of these subproducts ( fig . 3e , lanes 5 and 10 ) , despite drosha being active in the processing of a pri - mirna precursor ( pri - mir-24 - 2 ) ( fig . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . in vitro cleavage analyses revealed the precise location of the snorna cleavages , which occur towards their 3end ; potentially generating 3end fragments of 20nt long small rnas for c / d box snornas ( u16 ) and 40nt long for h / aca ( u92 ) ( supplementary fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . this observation agrees with previous bioinformatic analyses of dgcr8 ko and dicer ko small rna libraries whereby snorna - encoded small rnas ( sdrnas ) are affected by the loss of dgcr8 and dicer . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . this strongly suggests that dgcr8 associates to another endonuclease / s to specifically destabilize snornas and/or to produce new types of small rnas we obtained dgcr8 clip - tags mapping to 2,256 different mrnas , comprising 3,610 significant clusters , indicating that some mrnas harbor more than one dgcr8 binding site . we also found dgcr8 binding sites at the 5utr and first exon of the dgcr8 mrna and in agreement with previous data , these clusters overlapped hs - mir-1306 ( fig . we confirmed by ip rt - pcr that both dgcr8 and drosha bind to the dgcr8 mrna and that drosha knock - down increases steady - state levels of dgcr8 mrna ( supplementary fig . next , we chose 6 different mrnas with exonic dgcr8 clusters in both clip samples ( endogenous and overexpressed ) and harboring a predicted rna secondary structure that resembles that of pri - mirnas ( fig . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . an uv - cross - linking assay revealed that dgcr8 binding to each of these cassette exons was affected when the predicted secondary structures were disrupted by mutation ( fig . 5b ) . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . in addition , in those cases that we tested , we could confirm that no changes in alternative splicing were observed in the absence of dicer , indicating that these effects are independent from the presence of mirnas ( tcf3 and csnk1d ) ( fig 5f and supplementary fig . 5 g ) . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . this technique relies on ultraviolet irradiation to induce protein covalent crosslinks in protein - rna complexes in situ . this allows the use of highly stringent immunoprecipitation and washing conditions so that only those rnas directly bound to the protein of interest are selected ( reviewed by ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . each replicate comprised two independent experiments : one involved immunoprecipitation ( ip ) of endogenous dgcr8 protein ( supplementary fig 1a , left ) whereas the second one involved ip of a transiently transfected epitope - tagged dgcr8 protein ( pcg t7-dgcr8 ) ( supplementary fig 1a , right and 1b ) . we focused our analyses on the second replicate , since it gave higher depth of coverage and better mapping to the genome . 37 million reads for the endogenous dgcr8 protein , and 36 million reads for the epitope - tagged t7-dgcr8 and 93% and 98% of the reads were mapped to the genome , respectively ( supplementary table 1 ) . after removing read duplicates , uniquely mapped reads from each of the datasets were clustered according to their genomic positions . in order to identify significant clusters we applied a modified false discovery rate ( mfdr ) to each cluster , as previously described . we assessed the reproducibility of the dgcr8 targets identified in clip experiments for both endogenous and overexpressed dgcr8 proteins . analysis of the reads of these two samples revealed a strong correlation ( pearson correlation co - efficient r > 0.8 ) ( fig . in order to describe dgcr8 bona fide targets , we focused on overlapping significant clusters between these two samples ( for a complete list go to http://regulatorygenomics.upf.edu/data/dgcr8 ) . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . we also found that 30% of the genomic clusters mapped to repetitive elements , mainly line-1 and ltr retroelements , but these were excluded from the graphical representation . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . as expected , due to the canonical function of the microprocessor , we identified 117 mirnas ( fig . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . in addition for each of the clusters , an rna secondary structure was predicted in order to select those clusters overlapping with structures resembling that of pri - mirnas ( see http://regulatorygenomics.upf.edu/data/dgcr8 and supplementary methods ) . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . in addition , we observed that between 11 - 20% of the dgcr8 binding sites overlap with small rnas harboring a 5phosphorylated end , which are proposed to be generated by endonucleolytic activity . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . we further confirmed this interaction by immunoprecipitating overexpressed and endogenous dgcr8 protein followed by analysis of the associated rna by semi - quantitative rt - pcr ( fig . in order to study the effect of microprocessor binding to the malat1 rna , we determined its steady - state levels upon sirna - mediated depletion of drosha in hela cells . we observed a two - fold increase in the levels of malat1 rna when drosha levels were reduced ( fig . 2c ) . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . in order to test direct cleavage of the malat1 mrna in vivo , we fused the 5end of malat1 , which harbors significant dgcr8 clusters ( fig . 2a , indicated with a box ) to a luciferase reporter gene and transfected this construct into hela cells . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . another class of rnas bound by dgcr8 corresponds to small nucleolar rnas ( snornas ) ( fig . these small rnas are responsible for guiding many chemical modifications of other rnas , such as rrna , trna and snrnas . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . this reported binding to snornas has a physiological consequence as seen by the accumulation of u17a , u16 and u92 , which represent different subclasses of snornas , in mouse cells lacking dgcr8 ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . importantly , mature snorna levels did not change in the absence of dicer ( fig . thus , dgcr8 can affect the stability of the mature forms of an h / aca box snorna ( u17a ) , a c / d box snorna ( u16 ) and a small cajal rna ( scarna u92 ) ( fig . their biogenesis involves trimming of the host introns from the 5 and 3 end following pre - mrna splicing and release of the mature snorna form that is being protected from degradation by the core snornp components ( reviewed by ) . we determined that dgcr8 affects the levels of mature snornas but not of their precursor forms ( fig . 3c ) , indicating that dgcr8 acts at a post - synthesis level . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . to our surprise we could not detect any significant change in mature snorna levels in these situations ( fig . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . to test this hypothesis we incubated radiolabeled mature snornas with immunoprecipitated t7-dgcr8 and observed the appearance of smaller bands , corresponding to digested products ( fig . importantly , the addition of immunoprecipitated flag - drosha did not result in the formation of these subproducts ( fig . 3e , lanes 5 and 10 ) , despite drosha being active in the processing of a pri - mirna precursor ( pri - mir-24 - 2 ) ( fig . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . 3e , lane 13 ) . in vitro cleavage analyses revealed the precise location of the snorna cleavages , which occur towards their 3end ; potentially generating 3end fragments of 20nt long small rnas for c / d box snornas ( u16 ) and 40nt long for h / aca ( u92 ) ( supplementary fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . this observation agrees with previous bioinformatic analyses of dgcr8 ko and dicer ko small rna libraries whereby snorna - encoded small rnas ( sdrnas ) are affected by the loss of dgcr8 and dicer . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . this strongly suggests that dgcr8 associates to another endonuclease / s to specifically destabilize snornas and/or to produce new types of small rnas we obtained dgcr8 clip - tags mapping to 2,256 different mrnas , comprising 3,610 significant clusters , indicating that some mrnas harbor more than one dgcr8 binding site . we also found dgcr8 binding sites at the 5utr and first exon of the dgcr8 mrna and in agreement with previous data , these clusters overlapped hs - mir-1306 ( fig . we confirmed by ip rt - pcr that both dgcr8 and drosha bind to the dgcr8 mrna and that drosha knock - down increases steady - state levels of dgcr8 mrna ( supplementary fig . next , we chose 6 different mrnas with exonic dgcr8 clusters in both clip samples ( endogenous and overexpressed ) and harboring a predicted rna secondary structure that resembles that of pri - mirnas ( fig . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . accordingly , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . 5a ) . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . an uv - cross - linking assay revealed that dgcr8 binding to each of these cassette exons was affected when the predicted secondary structures were disrupted by mutation ( fig . 5b ) . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . in addition , in those cases that we tested , we could confirm that no changes in alternative splicing were observed in the absence of dicer , indicating that these effects are independent from the presence of mirnas ( tcf3 and csnk1d ) ( fig 5f and supplementary fig . 5 g ) . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in this study , we present a comprehensive strategy aimed at the identification of endogenous rna targets for the microprocessor . we found that dgcr8 binds to many different types of rnas involved in different cellular pathways . we identified most of the mirnas known to be expressed in these cells , which was expected . other dgcr8 targets comprise mrnas , non - coding rnas including snornas , long non - coding rnas and retrotransposons . our analysis also revealed that dgcr8 binds to more than 2,000 mrnas and in many cases depletion of microprocessor components leads to an up - regulation of these mrnas , suggesting that they are direct targets of the microprocessor . for some of these mrnas that were up - regulated upon depletion of drosha or dgcr8 , we could indeed confirm drosha - dependent cleavage in vitro ( fig . this extends a previous observation showing that a hairpin localized in the 5 utr of the dgcr8 pre - mrna is targeted by the microprocessor . nevertheless , it remains elusive whether any of these cleavage products are also dicer substrates that would lead to the generation of mirna - like small rnas . however , we observed that a large proportion of dgcr8 binding sites also overlap with small rnas harboring a 5phosphorylated end , which have been proposed to be produced by the action of endonucleases . the fact that the microprocessor destabilizes mrnas containing dgcr8 binding sites in cassette exons suggested the interesting possibility that the microprocessor might have a role in the processing of these alternative exons , which would impact on alternative splicing regulation . we confirmed that dgcr8 can modulate the relative abundance of alternatively spliced isoforms and that binding to these sequences depends on rna secondary structure ( fig . 5 and supplementary fig . 5 ) functional studies of the mirna processing machinery have shown that drosha , dgcr8 and dicer deficiencies generate similar phenotypes , resulting in early embryonic lethality , strongly suggesting that mirnas are essential for normal development . furthermore , several lines of evidence indicated additional functions for drosha and/or dicer , although initial global studies suggested dgcr8 mrna as the only non - canonical substrate for the microprocessor . first , microarray profiling of mefs derived from drosha and dicer knock - out mice showed poor correlation between the populations of mrnas affected by these two endonucleases . secondly , drosha was shown to recognize and cleave many protein - coding messenger rnas ( mrnas ) with secondary stem - loop structures in early - stage thymocytes , as well as viral rnas to directly regulate viral expression . finally , drosha - dependent mrna cleavage events that functionally regulate mrna levels in mescs , were recently described . a simple explanation for these observations is that non - canonical functions for the microprocessor and/or dicer could be responsible for these effects . the comparison of these reports with our data only resulted in a partial overlap , probably due to the use of different cell lines for these studies ( t cells versus hek 293 t cells ) . it also remains possible that binding of dgcr8 might not always lead to changes in relative mrna expression . what we provide here is a genome - wide view of dgcr8 binding sites in hek 293 t cells . importantly , our studies show more alternative functions for the microprocessor component dgcr8 , implicating this protein in the control of non - coding rnas stability ( lncrnas and snornas ) and on the relative abundance of alternatively spliced isoforms .. in vitro processing assays with immunoprecipitated dgcr8 showed cleavage of the mature snorna species in a drosha - independent manner , suggesting the involvement of dgcr8 in cellular complexes with other endonucleases ( fig . 3 and supplementary fig . 2 - 3 ) . a dgcr8-independent role for drosha in pre - rrna processing while drosha is required for the processing of 12s pre - rrna , dgcr8 ko mouse es cells did not display evident defects in ribosomal rna processing , suggesting alternative complexes to the canonical microprocessor . the presence of mirna - like molecules encoded by h / aca snornas independent of drosha function but involving a dicer - dependent cleavage activity was previously reported . bioinformatics analyses of small rnas encoded from snornas showed that are dependent on both , dgcr8 and dicer , although the role of drosha was not assessed . from an evolutionary point of view their structure conservation as well as the retained capacity to bind dyskerin and fibrillarin , two of the core components from snornp particles , suggests a common origin . our work proposes a new complex between dgcr8 and another endonuclease / s that would specifically cleave snornas and probably other classes of cellular rnas . although it is well known how snornas are synthesized , very little is known about their turnover and decay , and it would be of great interest to further study the implication of dgcr8 in this process . these findings raise the interesting possibility that there could be alternative dgcr8 complex / es using different nucleases to process a variety of cellular rnas . significantly , all the new functions described here for the microprocessor complex could be useful to reinterpret the phenotypes observed for drosha or dgcr8 deficient cells that were mainly attributed to the deficiency of mirnas . importantly , the dgcr8 gene is present in the deleted genomic region ( 22q11.2 ) in digeorge syndrome patients that expands around 30 genes . mouse models for this deletion showed a minor alteration of mirnas , but still exhibit behavioral and cognitive deficits and cardiac abnormalities that resemble the human condition . interestingly , the dgcr8 heterozygous mouse still showed some of the traits present in digeorge patients , such as altered short - term plasticity that has been linked to schizophrenia . although it is clear that in this situation the mirna biogenesis pathway is affected , it would be of great interest to determine how important are the new alternative dgcr8 functions for the origin and development of the disease .

the drosha - dgcr8 complex ( microprocessor ) is required for microrna ( mirna ) biogenesis . dgcr8 recognizes the rna substrate , whereas drosha functions as the endonuclease . high - throughput sequencing and crosslinking immunoprecipitation ( hits - clip ) was used to identify rna targets of dgcr8 in human cells . unexpectedly , mirnas were not the most abundant targets . dgcr8-bound rnas also comprised several hundred mrnas as well as snornas and long non - coding rnas . we found that the microprocessor controls the abundance of several mrnas as well as of malat-1 . by contrast , dgcr8-mediated cleavage of snornas is independent of drosha , suggesting the involvement of dgcr8 in cellular complexes with other endonucleases . interestingly , binding of dgcr8 to cassette exons , acts as a novel mechanism to regulate the relative abundance of alternatively spliced isoforms . collectively , these data provide new insights in the complex role of dgcr8 in controlling the fate of several classes of rnas .

RESULTS HITS-CLIP identifies novel targets of the Microprocessor The Microprocessor regulates the abundance of MALAT1 DGCR8 regulates snoRNA stability independently of Drosha The Microprocessor cleaves the mRNA of protein-coding genes Modulation of alternative spliced isoforms by the Microprocessor DISCUSSION Supplementary Material

in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . importantly , the addition of immunoprecipitated flag - drosha did not result in the formation of these subproducts ( fig . 3e , lanes 5 and 10 ) , despite drosha being active in the processing of a pri - mirna precursor ( pri - mir-24 - 2 ) ( fig . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . we also found dgcr8 binding sites at the 5utr and first exon of the dgcr8 mrna and in agreement with previous data , these clusters overlapped hs - mir-1306 ( fig . we confirmed by ip rt - pcr that both dgcr8 and drosha bind to the dgcr8 mrna and that drosha knock - down increases steady - state levels of dgcr8 mrna ( supplementary fig . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in order to determine whether the microprocessor is involved in the processing of cellular rnas ( other than mirnas ) that contain hairpin structures , we focused on the identification of endogenous rna targets of dgcr8 . for this , we used the cross - linking immunoprecipitation protocol coupled to high - throughput sequencing ( hits - clip ) . we performed two replicate hits - clip experiments on uv - irradiated hek293 t cells . each replicate comprised two independent experiments : one involved immunoprecipitation ( ip ) of endogenous dgcr8 protein ( supplementary fig 1a , left ) whereas the second one involved ip of a transiently transfected epitope - tagged dgcr8 protein ( pcg t7-dgcr8 ) ( supplementary fig 1a , right and 1b ) . in order to identify significant clusters we applied a modified false discovery rate ( mfdr ) to each cluster , as previously described . in this way , we found 8,196 common targets ( harboring one or more significant clusters ) at the genomic level that were distributed as follows : 45% mapped to intergenic regions , 43% to protein coding genes and 5% to long non - coding rnas ( lncrnas ) ( fig . we also found that 30% of the genomic clusters mapped to repetitive elements , mainly line-1 and ltr retroelements , but these were excluded from the graphical representation . when analyzing protein - coding genes , we observed that the majority of the clusters were located in introns ( 26% , including mirnas ) , followed by coding exons ( 12% ) . as expected , due to the canonical function of the microprocessor , we identified 117 mirnas ( fig . we also identified the only so far described non - canonical rna substrate for the microprocessor , the dgcr8 mrna ( fig . 1d , right).we also observed clip tags mapping to other non - coding rnas , such as rrna , snrnas , and snornas ( fig . importantly , we found that more than 60% of the dgcr8 binding sites were overlapping with stable rna secondary structures ( supplementary table 1 ) . malat1 ( metastasis - associated lung adenocarcinoma transcript 1 , also known as neat2 ) is a mammalian abundant , long non - coding rna that was initially identified due to its elevated expression in several cancers . we found that dgcr8 binds to malat1 and several other long non - coding rnas ( fig . 2e ) , suggesting that the microprocessor controls malat1 rna levels , independently of the activity of mirnas . reduction of drosha levels resulted in an increased expression of this reporter , suggesting that malat1 can be cleaved in vivo by the microprocessor ( fig 2f ) . in agreement , we observed small rna reads mapping to the majority of dgcr8 clusters present on the malat1 sequence ( small rna dataset from ) , also suggesting that dgcr8 binding could indeed induce rna cleavage ; however , it still remains unknown whether small rnas generated from these loci behave like mirnas ( fig . first , we confirmed the binding of endogenous and overexpressed dgcr8 to snornas observed by clip using immunoprecipitation followed by qrt - pcr in hek 293 t cells ( fig . 3b , left panel ) , as well as in hela cells knocked - down for dgcr8 ( fig . 3b , middle panel ) or when overexpressing a dominant negative form of dgcr8 in hek293 t cells ( supplementary fig . to further elucidate the role of the microprocessor in snorna stability in human cells , we studied the effect of either depleting drosha or overexpressing a dominant negative form of drosha . these data suggested a new dgcr8 function that operates independently of drosha , strongly indicating that dgcr8 might associate to another nuclease / s to cleave and destabilize snornas . this dgcr8-mediated effect was specific for snornas , since u1 snrna was not cleaved by the dgcr8 complex ( fig . furthermore , we observed stabilization of the 70nt long fragment when overexpressing dominant negative forms of dgcr8 in human cells ( supplementary fig . this observation agrees with previous bioinformatic analyses of dgcr8 ko and dicer ko small rna libraries whereby snorna - encoded small rnas ( sdrnas ) are affected by the loss of dgcr8 and dicer . here , we observed that the loss of dgcr8 decreases the amount of small rnas originated from both the 5 and the 3 ends for both c / d box and h / aca snornas ( supplementary fig . 3c , d ) and these sites do not coincide with the cleavage sites previously described for h / aca box snornas ( at the h box ) , which are required for the production of mirna - like molecules in a drosha - independent and dicer - dependent pathway . collectively , these data demonstrates a role for dgcr8 in controlling the abundance of mature snornas , in a drosha - independent manner . first , we confirmed binding of these mrnas by ip - rt - pcr to both endogenous and overexpressed dgcr8 ( fig . si - rna mediated depletion of drosha and/or dgcr8 in hela cells resulted in an increase of their mrna levels , suggesting that these mrnas could be direct targets of the microprocessor complex ( fig . accordingly , the upregulation observed in microprocessor - deficient cells ( dgcr8 ko ) was not detected in dicer ko cells , suggesting a direct role of the microprocessor in processing these mrnas ( supplementary fig . ( comprising regions of those mrnas that contain the dgcr8 binding sites ) with immunopurified microprocessor ( flag - drosha ) resulted in the cleavage of hoxa7 , dlg5 and snx12 ( fig . 4 g , lanes 4 , 6 and 8) , as well as of the positive control , pri - mir-30c-1 ( fig 4 g , lane 2 ) . analysis of the hits - clip data revealed dgcr8 binding to several predicted cassette exons ( n=241 ) ( fig . this would suggest the interesting possibility that microprocessor - mediated cleavage of these cassette exons would affect the relative abundance of alternatively spliced ( as ) isoforms . a global analysis of alternative splicing by exon junction arrays in mouse cells lacking dgcr8 revealed 318 changes in cassette exons , as well as 40 alternative 5/3splice site ( ss ) events and 18 changes in mutually exclusive exons ( supplementary fig . we first focused on four cassette exons shown to be bound by dgcr8 in the clip experiments that also display an rna secondary structure resembling pri - mirnas ( supplementary fig . importantly , those isoforms bound by dgcr8 were stabilized in human cells depleted of drosha or in mouse cells lacking dgcr8 ( fig . 5f ) , strongly suggesting that the microprocessor specifically cleaves and destabilizes mrna isoforms containing dgcr8 binding sites leading to an altered ratio of the alternatively spliced isoforms . accordingly , we also observed an overlap of these dgcr8 alternatively spliced exons with small rna libraries ( fig 5c , d , lower panels ) , confirming that cleavage can occur at these locations , which could help to explain the change in the relative abundance of those isoforms containing the dgcr8 bound exon . altogether these data suggest that dgcr8 binding to cassette exons may act to influence the relative abundance of alternatively spliced isoforms . in this study , we present a comprehensive strategy aimed at the identification of endogenous rna targets for the microprocessor . we found that dgcr8 binds to many different types of rnas involved in different cellular pathways . other dgcr8 targets comprise mrnas , non - coding rnas including snornas , long non - coding rnas and retrotransposons . our analysis also revealed that dgcr8 binds to more than 2,000 mrnas and in many cases depletion of microprocessor components leads to an up - regulation of these mrnas , suggesting that they are direct targets of the microprocessor . for some of these mrnas that were up - regulated upon depletion of drosha or dgcr8 , we could indeed confirm drosha - dependent cleavage in vitro ( fig . this extends a previous observation showing that a hairpin localized in the 5 utr of the dgcr8 pre - mrna is targeted by the microprocessor . the fact that the microprocessor destabilizes mrnas containing dgcr8 binding sites in cassette exons suggested the interesting possibility that the microprocessor might have a role in the processing of these alternative exons , which would impact on alternative splicing regulation . we confirmed that dgcr8 can modulate the relative abundance of alternatively spliced isoforms and that binding to these sequences depends on rna secondary structure ( fig . 5 ) functional studies of the mirna processing machinery have shown that drosha , dgcr8 and dicer deficiencies generate similar phenotypes , resulting in early embryonic lethality , strongly suggesting that mirnas are essential for normal development . furthermore , several lines of evidence indicated additional functions for drosha and/or dicer , although initial global studies suggested dgcr8 mrna as the only non - canonical substrate for the microprocessor . secondly , drosha was shown to recognize and cleave many protein - coding messenger rnas ( mrnas ) with secondary stem - loop structures in early - stage thymocytes , as well as viral rnas to directly regulate viral expression . a simple explanation for these observations is that non - canonical functions for the microprocessor and/or dicer could be responsible for these effects . it also remains possible that binding of dgcr8 might not always lead to changes in relative mrna expression . importantly , our studies show more alternative functions for the microprocessor component dgcr8 , implicating this protein in the control of non - coding rnas stability ( lncrnas and snornas ) and on the relative abundance of alternatively spliced isoforms .. in vitro processing assays with immunoprecipitated dgcr8 showed cleavage of the mature snorna species in a drosha - independent manner , suggesting the involvement of dgcr8 in cellular complexes with other endonucleases ( fig . a dgcr8-independent role for drosha in pre - rrna processing while drosha is required for the processing of 12s pre - rrna , dgcr8 ko mouse es cells did not display evident defects in ribosomal rna processing , suggesting alternative complexes to the canonical microprocessor . the presence of mirna - like molecules encoded by h / aca snornas independent of drosha function but involving a dicer - dependent cleavage activity was previously reported . bioinformatics analyses of small rnas encoded from snornas showed that are dependent on both , dgcr8 and dicer , although the role of drosha was not assessed . from an evolutionary point of view their structure conservation as well as the retained capacity to bind dyskerin and fibrillarin , two of the core components from snornp particles , suggests a common origin . our work proposes a new complex between dgcr8 and another endonuclease / s that would specifically cleave snornas and probably other classes of cellular rnas . although it is well known how snornas are synthesized , very little is known about their turnover and decay , and it would be of great interest to further study the implication of dgcr8 in this process . these findings raise the interesting possibility that there could be alternative dgcr8 complex / es using different nucleases to process a variety of cellular rnas .

chemoprevention was defined as the administration of agents to prevent induction , to inhibit or to delay the progression of cancer , or as the inhibition or reversal of carcinogenesis at a premalignant stage . chemoprevention utilizes appropriate pharmacological agents [ 3 , 4 ] or of dietary agents , consumed in diverse forms like macronutrients , micronutrients , or nonnutritive phytochemicals [ 57 ] . consumption of antioxidants has been related to the several preventative effects against different diseases such as cancer , coronary diseases , inflammatory disorders , neurological degeneration , and aging [ 8 , 9 ] led to search for natural foods rich in antioxidants . although honey has been used since long time , only recently its antioxidant property came to limelight . honey has some minor constituents compared to its major sugar level , which is believed to have antioxidant properties [ 11 , 12 ] . some to mention were flavonoids and phenolic acids [ 13 , 14 ] , certain enzymes ( glucose oxidase , catalase ) , ascorbic acid , carotenoid - like substances , organic acids , amino acids , and proteins . phytochemicals are one wide class of nutraceuticals found in plants which are extensively researched by scientists for their health - promoting potential . polyphenols and phenolic acids found in the honey vary according to the geographical and climatic conditions . some of them were reported as a specific marker for the botanical origin of the honey . considerable differences in both composition and content of phenolic compounds have been found in different unifloral honeys . terpenes , benzyl alcohol , 3 , 5-dimethoxy-4-hydroxybenzoic acid ( syringic acid ) , methyl 3 , 5-dimethoxy-4-hydroxybenzoate ( methyl syringate ) , 3 , 4 , 5-trimethoxybenzoic acid , 2-hydroxy-3-phenylpropionic acid , 2-hydroxybenzoic acid and 1 , 4-dihydroxybenzene are some of the phytochemicals ascribed for the antimicrobial activity of honey . among these phytochemicals , polyphenols were reported to have antiproliferative potential . in this review , we summarized the compositional chemistry and antiproliferative potential of crude honey and some of its important polyphenols in various cancer cells . honey bees collect the nectar from various floral sources and store it as honey which serves as food for bees during winter . honey bees make a journey of nearly 55,000 miles to gather nectar from approximately 2 million flowers for accumulating one pound of honey . in the bee - hive , we can find thee types of bees namely the queen , drone and worker bees . among them , only worker bees collect and regurgitate the nectar number of times , in order to partially digest the nectar , before storing in the honey comb . during the collection of nectar as the honeybee visits the flower in hunt of nectar , some of the flower 's pollen falls into the nectar collected by the bee and stored in the stomach which will be regurgitated along with nectar . moreover some pollen grains often attach themselves to the various parts of the honey bee body like legs , antenna , hairs , and also in the eyes of visiting bees which will get entangled in the hive and thereby paving entry into the honey . airborne pollen is also another route of entry for pollen into the honey which got transferred though wind currents . honey bees use its wings to fan the honey comb , to evaporate most of the water from nectar thereby avoiding the fermentation of honey . the color of the honey collected by the bees varies according to the floral source and its mineral content , which usually ranges from water white to dark amber . flavor of the honey depends upon the color , generally the darker the honey the stronger the flavor and quality ( figure 1 ) . it has been reported more than 300 unique varieties of honey depending upon the floral sources from united states alone . honey mainly composed of sugars and water which accounts roughly 79.6% and 17.2% , respectively , ( figure 2 ) . major sugars of honey are levulose and dextrose which constitutes 38.19% and 31.28% correspondingly , remaining is the sucrose 1.3% and maltose 7.3% . honey minor constituents include acids ( 0.57% ) , protein ( 0.266% ) , nitrogen ( 0.043% ) , amino acids ( 0.1% ) , a little amount of minerals ( 0.17% ) , and a number of other minute quantities of components like pigments , flavor and aroma substances , phenolics compounds , colloids , sugar alcohols and vitamins which all together accounts for the 2.1% of whole honey composition [ 20 , 21 ] . few researchers studied the effect of crude honey in cancer . in a recent research conducted by jaganathan et al . they showed honey induced apoptosis in human colon cancer cells by arresting the cells at subg1 phase . honey possessing higher phenolic and tryptophan content was more potent in inhibiting the colon cancer cell proliferation . finally they demonstrated that honey induced apoptosis was associated with caspase-3 activation and parp cleavage . research by orsolic et al . showed that water soluble derivative of propolis and its associated phenolic compounds have antimetastatic effect on the tumor mice models before and after the injection of tumor cells . further they showed that honey could exert antimetastatic effect when given before tumor cell injection . in the study conducted by tarek et al . honey was proven to be a very effective agent for repressing the growth of bladder cancer cell lines ( t24 , rt4 , 253j , and mbt-2 ) in vitro . further honey was found to be effective when administered intralesionally or orally in the mbt-2 bladder cancer implantation models . there was also a significant difference between the final tumor volume ( p < .05 ) in the intra lesion ( il ) honey - treated groups ( il 6% honey ) compared to the il saline group . the difference between the final tumor volume or weight in the il saline group and the control group was not significant . research conducted by gribel and pashinskii indicated that honey exhibited moderate antitumor and significant antimetastatic effects in five different strains of rat and mouse tumors . moreover , the antitumor activity of certain chemotherapeutic drugs such as 5-fluorouracil and cyclophosphamide was also facilitated by the honey . it has been elucidated that polyphenols are anticarcinogenic , antiinflammatory , antiatherogenic , antithombotic , immune modulating and also act as antioxidants [ 2631 ] . hence antitumor properties of honey could be attributed to the polyphenols found in the honey . moreover , with the evolution of extraction procedure for various polyphenols , which had been attributed with anticancerous property of honey , researchers concentrated on the polyphenolic compounds extracted from the honey rather than crude honey itself . phenolic compounds or polyphenols are the important groups of compounds occurring in plants , where they are widely distributed , comprising at least 8000 different known structures . in general , phenolic compounds can be divided into at least 10 types depending upon their basic structure : simple phenols , phenolic acids , coumarins and isocoumarins , naphthoquinones , xanthones , stilbenes , anthaquinones , flavonoids and lignins . flavonoids constitute the most important polyphenolic class , with more than 5000 compounds already described . flavonoids are the natural antioxidants exhibiting a wide range of biological effects including antibacterial , antiinflammatory , antiallergic , antithombotic and vasodilatory actions . polyphenols found in the honey was used as a marker for particular type of honey , for example , flavanol kaempferol as an indicator for rosemary honey [ 33 , 34 ] and quercetin for sunflower honey . the hydroxy - cinnamates like caffeic acid , ferulic acid and p - coumaric acid have been found in the chest - nut honey . characteristic flavonoids of propolis like pinocembrin , pinobanksin and chrysin were also found in the most european honey samples . in this review , we concentrated on the some major polyphenols available in the honey which exhibited antiproliferative effect on various cancer cell lines . the list of compounds ( refer table 1 for figures ) reviewed for their anticancerous activity is caffeic acid ( ca ) , caffeic acid phenyl ester ( capes ) , chrysin ( cr ) , galangin ( ga ) , quercetin ( qu ) , acacetin ( ac ) , kaempferol ( kf ) , pinocembrin ( pc ) , pinobanksin ( pb ) , and apigenin ( ap ) . research conducted by hirose et al . studied the carcinogenicity of low dietary levels of the antioxidants like butylated hydroxyanisole ( bha ) , caffeic acid , sesamol , 4-methoxyphenol ( 4-mp ) and catechol . these antioxidants were eminent target of the fore - stomach or glandular stomach and these were examined for their predominant effects in alone or in combinations , for a two - year period experiment . carcinogenicity study was undertaken in groups of 30 - 31 male f344 rats , by treating with 0.4% bha , 0.4% caffeic acid , 0.4% sesamol , 0.4% 4-mp and 0.16% catechol either alone or in combination for up to 104 weeks and then killed . the ultimate average body weights of rats having basal diets were higher than those treated with antioxidants alone , and were the lowest in the combinational groups . moreover the relative liver and/or kidney weights were greater than before in the bha , sesamol , catechol and combination groups . it led to the conclusion , that the occurrences and frequencies of fore - stomach histopathological lesion were increased by exposing to antioxidants except in the case of bha . the incidences and/or multiplicities of forestomach papillary or nodular ( pn ) hyperplasia were appreciably increased in the groups treated with 4-methoxyphenol , caffeic acid and the antioxidants in combination , as compared with the basal diet group . studies on medium - term multiorgan carcinogenesis model , suggested an increase in the occurrence of fore - stomach papillomas in each high - dose group and no synergistic effect was observed in combinations . in the low dose case , the incidence of fore - stomach papillomas was significantly increased only in the combination group . the effect on the other organs particularly colon tumors , hence it can be inferred that at low dose levels , the phenolic compounds can exhibit additive / synergistic effect on carcinogenesis . from these early experiments , caffeic acid rao et al . performed a detailed study by synthesizing thee caffeic acid esters namely methyl caffeate ( mc ) , phenylethyl caffeate ( pec ) and phenylethyl dimethylcaffeate ( pedmc ) and examined them against the 3 , 2-dimethyl-4-aminobiphenyl ( dmab , a colon and mammary carcinogen ) induced mutagenecity in salmonella typhimurium strains ta 98 and ta 100 . both the strains of salmonella subsisted ( survival rate > 98% ) concentration of about 2,500 m ca , 150 m mc , 70 m pec and 80 m moreover 150 m mc , 4080 m of pedmc , 4060 m of pec significantly inhibited the dmab - induced mutagenecity in both strains . the outcome of these experiments placed mc at a concentration greater than 225 m and pec and pedmc at a level greater than 60 m was toxic . ca exhibited significant toxicity only at above 2500 m concentration . in colon cancer cell line ( ht-29 ) , cytotoxicity effect of ca , pec , pedmc and mc was evaluated . the growth inhibitory effect of these compounds was measured after exposing cells for a period of 48 hours . ca was found to be the least effective in inhibiting the growth of ht-29 cells when compared to its ester analogs . to further corroborate the growth inhibitory effects , synthesis of polynucleotide and protein synthesis after incubating the ht-29 cells with these agents for 48 hours were investigated . it has been observed that at the concentration of 175 m of mc , 40 m of pec and 60 m of pedmc blocked the dna , rna and the protein synthesis . moreover ornithine decarboxylase ( odc ) activity was inhibited at concentrations of 150 m mc , 40 m pec and 20 m pedmc . tyrosine protein kinase ( tpk ) activity was also inhibited at concentrations of 100 m of mc , 30 m of pec and 20 m of pedmc . in their follow - up studies made by them , reported the inhibitory effects of methyl caffeate ( mc ) and phenylethyl caffeate ( pec ) on azoxymethane- ( aom-)induced ornithine decarboxylase ( odc ) , tyrosine protein kinase ( tpk ) and arachidonic acid metabolism in liver and colonic mucosa of male f344 rats . they depicted the inhibitory effects of caffeic acid , mc , pec , phenylethyl-3-methylcaffeate ( pemc ) , and phenylethyl dimethyl caffeate ( pedmc ) on in vitro arachidonic acid metabolism in liver and colonic mucosa . finally they investigated the effects of pec , pemc , and pedmc on aom - induced aberrant crypt foci ( acf ) formation in the colon of f344 rats . for a period of five weeks , groups of f334 rats were fed with diets containing 600 ppm of mc or pec for biochemical studies and 500 ppm of pec , pemc or pedmc for acf studies . after two weeks , subcutaneous injection of aom was given once in a week for two consecutive weeks , for all animals except the vehicle - treated groups . biochemical studies were performed by sacrificing the animal after 5 days . in case of acf study , the colonic mucosa and liver of the rats were analyzed for the orinithine decarboxylase activity , tyrosine protein kinase activity ( tpk ) , lipoxygenase and cyclooxygenase metabolites . pec diet significantly inhibited aom - induced odc and tpk activities in liver and colon . it had been observed that pec diet significantly repressed the aom - induced lipoxygenase metabolites 8(s)- and 12(s)-hydroxyeicosatetraenoic acid ( hete ) . the animals fed the mc diet exhibited a moderate inhibitory effect on odc and 5(s)- , 8(s)- , 12(s)- , and 15(s)-hetes and a significant effect on colonic tpk activity . however , both the mc and pec diets showed no significant inhibitory effects on cyclooxygenase metabolism . acf were significantly inhibited in the animals fed with pec ( 55% ) , pemc ( 82% ) , or pedmc ( 81% ) . the results of the study indicated that pec , pemc , and pedmc present in the honey , inhibited aom - induced colonic preneoplastic lesions , odc , tpk , and lipoxygenase activity , which were relevant to the colon carcinogenesis . huang et al . showed the strong repressive effect of cape application on 12 - 0-tetradecanoylphorbol-13-acetate- ( tpa-)induced tumor promotion and production of 5-hydroxymethyl-2-deoxyuridine ( hmdu ) in the deoxyribonucleic acid ( dna ) of the mouse skin . they established the inhibitory effect of cape on tpa - induced tumor promotion by topical application of cape in cd - i mice previously treated with 7 , 12-dimethylbenz[a]anthracene ( dmba ) . they applied cape in concentration ranging from 1 , 10 , 100 , or 3000 nmol together with 5 nmol of tpa twice a week for 20 weeks . at the above concentrations , cape inhibited the number of skin papillomas by 24 , 30 , 45 and 70% and tumor size per mouse moreover topical application of 5 nmol of tpa twice weekly for 20 weeks to mice produced an average of 12.6 hmdu residues per 104 normal bases in epidermal dna . topical application of 1 , 10 , 100 , or 3000 nmol of cape along with 5 nmol of tpa twice weekly for 20 weeks to dmba - initiated mice decreased the levels of hmdu in epidermal dna by 4093% . cape at 1.25 , 2.5 , 5 , 10 , or 20 m inhibited the incorporation of [ h]-thymidine into dna in cultured hela cells by 32% , 44% , 66% , 79% , and 95% respectively . similarly incorporation of [ h]-uridine into rna was inhibited by 39% , 43% , 58% , 64% , and 75% whereas incorporation of [ h]-leucine into protein was inhibited by 29% , 30% , 37% , 32% , or 47% , respectively . these results indicated that cape is a potent inhibitor of dna synthesis but it is somewhat less effective in inhibiting rna synthesis and it is least effective in inhibiting the protein synthesis . since nf-b has a role in these activities , they examined the effect of cape on this transcription factor in an exhaustive manner . they preincubated the u-937 cells with cape with various concentrations for 2 hours before treating with tnf ( 0.1 nm ) for 15 minutes . cape inhibited the tnf - dependent activation of nf-b in a dose - dependent manner with maximum effect occurring at 25 g / ml . nf-b activation induced by the phorbol ester , phorbol-12-myristate 13-acetate ( pma ) , ceramide , okadaic acid and hydrogen peroxide was also inhibited by cape . it prevented the translocation of p-65 subunit of nf-b to the nucleus without affecting the tnf - induced ib degradation . it does not showed any inhibitory effect on the other transcription factors like ap-1 , tfiid and oct-1 . to study further precisely about the role of cape in inhibiting nf-b various structural analogues of cape were examined . it has been configured that a bicyclic , rotationally constrained , 5 , 6-dihydroxy form showed supremacy , whereas 6 , 7-dihydroxy variant was the least active in inhibiting the nf-b . with these findings they concluded that cape is a potent and a specific inhibitor of nf-b activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities of cape . in another study initiated by lee et al . investigated the cytotoxicity potential of cape and the molecular mechanism of its action in c6 glioma cells . the results of the experiments indicated c6 glioma cells underwent internucleosomal dna fragmentation after 24 hours treatment with cape ( 50 m ) . facs analysis of cape - treated c6 glioma cells showed increasing accumulation of hypodiploid nuclei ( 24% at 36 hour ) in time - dependent fashion . further results showed that cape induced the release of cytochome - c from mitochondria into the cytosol after 3 hours of treatment resulting in the activation of caspase-3 ( cpp32 ) from the beginning of 3 hours . moreover the cleavage of parp ( substrate of cpp32 ) started within 12 hours after cape treatment . cape enhanced the serine phosphoryaltion of p53 after 0.5 hours and the protein level of p53 was increased after 3 hours . cape treatment also enhanced the expression of bax and bak and resulted in the reduced level of b - cell lymphoma / leukemia-2 gene ( bcl2 ) protein ( after 36 hours ) . moreover they reported that cape application activates the extracellular signal - regulated kinase ( erks ) and p38 mitogen - activated protein kinase ( p38 mapk ) in the c6 glioma cells . further they showed that expression of p53 , phospho - serine 15 of p53 , bax and inactivate form of cpp32 were suppressed by a pretreatment of a specific p38 mapk inhibitor , sb203580 . hence they concluded p53 dependent apoptosis in c-6 glioma cells were mediated by p38 mapk . mmp-1 , 3 , 7 and cathepsin - k were not completely inhibited by both of them . ca and cape had a dose - dependent inhibitory effect on the proliferation of hepg2 cells . in hepg2 cells , ca at the concentration of 200 g / ml reduced the cell viability to 61% compared to the control , and the treatment with cape ( at low concentration of 20 g / ml ) reduced the viability to 72% compared of the control . cape and ca suppressed the mmp-9 expression , exposed to phorbol 12-myristate 13-acetate ( pma ) , by blocking the nf-b activity in hepg2 cells . they also confirmed that ca ( 20 mg / kg ) and cape ( 5 mg / kg ) repressed the growth of hepg2 tumor xenografts in nude mice as well as liver metastasis when administered subcutaneous or orally . finally they concluded their observation that ca and its derivative cape : ( 1 ) inhibited the enzymatic activity of mmp-9 that plays an important role in cancer invasion and metastasis , ( 2 ) blocked the invasive potential through the suppression of mmp-9 gene transcription by inhibiting nf-b function in pma - stimulated hepg2 cells and ( 3 ) suppressed the growth of hepg2 cell xenografts in nude mice . therefore , these two drugs were reported as strong candidates for treatment of cancer and metastasis via dual mechanisms ( dual inhibition of metastasis - specific enzyme activity and gene transcription ) . further in a recent study initiated by hwang et al . investigated the effect of cape on tumor invasion and metastasis in ht 1080 fibrosarcoma cells by determining the regulation of matrix metalloproteinase 's ( mmps ) . ht 1080 cells were treated with increasing concentration of cape and the m - rna transcripts of mmp-2 and mmp-9 were analyzed using semi - quantitative rt - pcr . both mmp-2 and 9 proteins levels were significantly suppressed at dose dependent manner . gelatin zymography also indicated constitutively expressed mmp-2 and 9 proteins in ht 1080 cells which gradually reduced after treating with cape . to further corroborate the downregulation of mmp-2 , activation studies of pro - mmp2 were performed using organomercuric compound , 4-aminophenylmercuric acetate ( apma ) , and the result indicated the down regulation of mmp-2 by cape . it has been shown that m - rna levels of tissue inhibitor of matrix metalloproteinase 's ( timps ) and membrane type - matrix metalloproteinase 's ( mt-1 mmps ) were also reduced significantly . cape also inhibited the cell invasion , cell migration and colony formation of tumor cells . thus cape acts as a vital antimetastatic agent , by inhibiting the metastatic and invasive potential of malignant cells . moreover some researchers investigated the possible uvc ( 280100 nm ) protective properties of caffeic acid in human diploid fibroblast and a-431 epidermoid cancer cell lines . the uvc safeguarding effect of ca in two different concentrations ( 55.5 m and 166.5 m ) was clearly illustrated both in transformed and normal cells . a marked difference in the proliferation of normal and transformed cells when irradiated to uvc radiation ca 's protective effect was distinct in the transformed cells compared to normal cells . in a sequential study by vanisree et al . explained protective effect of ca against uvb ( 280320 nm ) radiation - induced il-10 expression and the activation of the mitogen - activated protein kinases ( mapks ) in mouse skin . ca inhibited the il-10 promoter transcription , measured using in vivo transgenic il-10 promoter - luciferase reporter gene base assay . il-10 mrna expression and protein production in the mouse skin were significantly repressed by ca . there have also been shown the upstream regulators like extracellular regulated protein kinase ( erk ) , c - jun n - terminal protein , p-38 mitogen - activated protein kinase ( p38 mapk ) and the downstream transcription factors like activator protein ( ap-1 ) and nuclear factor kappa b ( nf-b ) were also inhibited by ca in mouse skin . from these experiments it was inferred that ca could be used as a topical agent against harmful uvb irradiation . chrysin ( 5 , 7-dihydroxyflavone ) is a natural and biologically active compound extracted from honey , plants and propolis . it possesses potent antiinflammatory , antioxidant properties and promotes cell death by perturbing cell cycle progression . in a recent study conducted by weng et al . in an antiproliferation assay performed on c6 glioma cells , chrysin inhibited the cell proliferation after 24 , 48 and 72 hours . after 72 hours of incubation with 50 m of chrysin , 90% of cell proliferation was inhibited . flow cytometry analysis reported that by 30 and 50 m treatments after 24 hours , chrysin increased the proportion of cells in the g1 phase of the cell cycle from 69 to 79% and 83% and decreased the proportion of s phase cells from 11.4 to 6.1% and 2.8% , respectively . the proportion of g2/m phase cells changed from 17.9 to 12.2% and 9.2% , after 30 and 50 m treatments . it has been found that levels of phosphorylation of retinoblastoma ( rb ) protein in c6 glioma cells decreased after treating with 30 m of chrysin . moreover in chrysin - treated cells , it has been demonstrated that cyclin dependent kinase inhibitor ( p21 ) levels are increased significantly without the change in p53 protein level . to depict the role of p38 in chrysin - mediated p21 induction furthermore they showed that proteosome activity , cyclin dependent kinase 2 ( cdk2 ) and 4 ( cdk4 ) were also inhibited by chrysin . these results suggested that chrysin exerts its growth - inhibitory effects either through activating p38-mapk leading to the accumulation of p21 protein or through mediating the inhibition of proteosome activity . in another study by woo et al . reported the chrysin - mediated apoptosis in u-937 cancer cell lines . dna fragmentation assay of chrysin - treated cells after 12 hours showed typical inter - nucleosomal fragmentation of dna . facs analysis of treated cells showed marked increase of accumulation of subg1 cells after 12 hours . decreased proenzyme level of caspase-3 after chrysin treatment indicated the importance of activated caspase-3 in apoptosis . further the activation of phospho - lipase c- ( plc- ) , a down stream target of caspase-3 in chrysin treated cells confirmed the role of caspase-3 in chrysin treated u937 cells . western blotting analysis of chrysin treated cells indicated the reduction in the level of xiap ( a member of inhibitor of apoptosis proteins ) and cytochrome c induction in dose dependent manner . mitogen activated protein kinase ( mapk ) does not have any role in the signaling pathway as shown by western blot analysis , whereas akt - signaling played significant role in chrysin mediated apoptosis of u937 cells . it has been shown that inhibition of akt phosphorylation in u937 cells by the specific pi3k inhibitor , ly294002 , significantly enhanced the apoptosis . overexpression of a constitutively active akt ( myr - akt ) in u937 cells inhibited the induction of apoptosis , activation of caspase 3 and plc-1 cleavage by chrysin . further zheng et al . synthesized 13 derivatives of chrysin and tested it for anticancer effect against human gastric adenocarcinoma cell line ( sgc-7901 ) and colorectal adenocarcinoma ( ht-29 ) cells . these derivatives were formed mainly by alkylation , halogenation , nitration , methylation , acetylation and trifluoromethylation . mtt assay revealed that 5 , 7-dimethoxy-8-iodochrysin and 8-bromo-5-hydroxy-7-methoxychrysin have the strongest activities against sgc-7901 and ht-29 cells respectively . the compound 5 , 7-dihydroxy-8-nitrochrysin was found to have strong activities against both sgc-7901 and ht-29 cells . zhang et al . tried to improve the biological properties of chrysin by synthesizing diethyl chrysin-7-yl phosphate ( cpe : c19h19o7p ) and tetraethyl bis - phosphoric ester of chrysin ( cp : c23h28o10p2 ) though a simplified atheron - todd reaction . in mass spectroscopy analysis , cpe formed complexes with lysozyme and hence phosphate esters of chrysin enhanced the interaction with proteins compared to unmodified chrysin . cultured human ( hela ) cell lines were treated by cr , cp and cpe with 10 m for 24 , 48 , and 72 hours . methyl green - pyronin staining , pcna immunohistochemistry and tunel techniques were also employed to study the effect of cr , cpe and cp in the cultured hela cell lines . it favored their hypothesis that all cr , cp and cpe could inhibit proliferation and induce apoptosis in the following order of inhibition potency cp > cpe > cr . hence they suggested cp and cpe as a new potential candidate for human cervical cancer . charles et al . described the antiproliferative effect of galangin on human leukemia ( hl-60 ) cell line . trypan blue exclusion method indicated the remarkable decrease in the cell viability after treating with 100 m for 24 hours . galangin of 110 m exerted antiproliferative effect which is evident after 48 hours of incubation . early and late apoptosis were detected using annexin - v - fitc and pi staining using 100 m galangin and these results correlated with the results of trypan - blue method reported already . active caspase-3 , a hallmark of apoptosis process , was detected after 24 hours and 72 hours of incubation with 50 and 10 m of galangin respectively . cell cycle analysis indicated the increase in the subg1 phase of galangin ( > 10 m ) treated cells . this was illustrated further in dna fragmentation assay , in which they could observe typical ladder pattern after 24 hours of 100 m galangin exposure . forward and side scatter changes were predominantly observed after 24 hours and 72 hours incubation with 100 m galangin . galangin treated cells displayed reduced forward scatter indicative of decreased relative size , and enhanced side scatter indicative of increased internal complexity . rhodamine median florescence intensity measured as an indicator of ros levels , showed no evidence for intracellular oxidative stress as a key - player of cytotoxicity and significant phagocyte - like differentiation was not detected . kang et al . investigated the role of quercetin as an anticancer agent in hl-60 cells . from their experiments they inferred the concentration dependent inhibition of hl-60 cell proliferation between the ranges of 10 to 80 m . they showed cells incubated with 10 m displayed inhibition on the growth of hl-60 cells . it was 17.1% , 27.3% , 40.1% , and 52.7% after 24 , 48 , 72 , and 96 hours of treatment . cell cycle analysis indicated that quercetin ( 20 , 40 , and 60 m ) increased the number of cells in the g2/m phase from 7.6% to 12.4% , 19.1% , and 23.5% correspondingly , and decreased the population of g0/g1 , cells from 46.2% to 40.2% , 32.1% , and 34.5% , respectively , without significant changes in the s - phase cell population after 24 hours of treatment . quercetin showed remarkable inhibitory effect on the activities of cytosolic protein kinase c ( pkc ) and membrane tpk of hl-60 cells in vitro , with ic50 values of about 30.9 and 20.1 m , respectively , but did not have the effect on membrane pkc or cytosolic tpk activity . it has also repressed the complete activity of phosphoinositides like phosphatidylinositol ( pi ) , phosphatidylinositol 4-phosphate ( pip ) , and phosphatidylinositol 4 , 5-bisphosphate ( pip2 ) at the concentration of 80 m . hence they concluded that the inhibitory effect of quercetin on the growth of hl-60 cells may be related to its inhibitory effects on pkc and/or tpk in vitro and/or on the production of phosphoinositides . after 1 hour exposure to the drug it resulted in apoptosis of the leukemia cells . they attributed these effects to the early downregulation of c - myc and ki - ras oncogenes and rapid reduction of inositol-1 , 4 , 5-triphosphate ( ips ) concentrations . they found quercetin exerted both antioxidant and pro - oxidant properties depending upon the concentration used . quercetin in low concentration ( 120 m ) promoted the cell proliferation whereas higher concentration ( 50200 m ) showed the concentration dependent cytotoxicity . the lower concentration ( 10 m ) of quercetin produced increased number of live cells , repressing the number of cells in the apoptotic and necrotic portions . on the other hand if the concentration was above 50 m , it reduced the number of live cells by increasing the apoptotic / necrotic fractions . quercetin decreased production of reactive oxygen species in the cells producing peroxides in the medium . they also found incubating with low concentrations of quercetin led to a small increase in total antioxidant capacity ( tac ) of cell extracts but higher concentrations of the quercetin led to a progressive decrease in the tac of cell extracts . hence they suggested that cellular effects of quercetin are complex and include both antioxidant effects and induction of oxidative stress due to formation of reactive oxygen species in the extracellular medium . in another study made by elizandra et al . quercetin decreased the cell viability in glioma cell cultures resulting in necrotic and apoptotic cell death . it also arrested the glioma cells in the g2 checkpoint of the cell cycle , and decreased the mitotic index . furthermore , they demonstrated quercetin was able to protect the hippocampal organotypic cultures from ischemic damage . these results showed that although it induced growth inhibition and cell death in the u138 mg human glioma cell line , still it has a cytoprotective effect in normal cell cultures . they showed quercetin could exert antiproliferative effect against mcf-7 cell line in a dose and time dependent manner with ic50 value of 10 g / ml . further quercetin was found to arrest the mcf-7 cell growth in g2/m phase of cell cycle . moreover it was shown that quercetin inhibited the tumor growth by more than 58% in mice grafted with mammary carcinoma and it extended the survival ability of sarcoma 180 bearing mice by 2.3 times . finally they concluded that these effects were mediated in part by the often poorly vascularised and hypoxic regions of tumors . in a recent study initiated by choi et al . studied the anticancer effect of quercetin against breast cancer cell ( mda - mb-435 ) . mtt assay revealed that quercetin showed inhibitory effect on mda - mb-435 cell growth in a time and dose dependent manner . further cell cycle analysis of quercetin treated cells showed significant increase in the accumulation of cells at subg1 phase . hsu et al . investigated the antiproliferative effect of acacetin in human liver cancer cell line ( hepg2 ) . the maximum inhibitory effect ( nearly 72% ) was observed at a concentration 20 g / ml after 48 hours . the ic50 value was observed to be 10.44 g / ml for the hepg2 cells . flow cytometry results indicated an increase in the g1 phase of cells from 31.1 to 61.6 and 76.5% at a concentration of 10 and 20 g / ml , respectively . dna fragmentation assay of cells treated with acacetin indicated the number of cells undergoing apoptosis increased to about 4-fold at 10 g / ml and 8-fold at 20 g / ml after 48 hours . further it has been demonstrated that acacetin increased the induction of p53 and its downstream target , p21/waf1 as assayed by enzyme linked immuno - sorbent assay ( elisa ) . fas ligand assay indicated that fasl , mfasl and sfasl increased in a dose - dependent manner . pro - apoptotic bax protein level also increased due to acacetin treatment at 24 and 48 hours . in their continuity study , they examined the role of acacetin in human nonsmall cell lung cancer a549 cells . they reported the antiproliferative effect was significant in dose - dependent manner and the ic50 value was found to be 9.46 m . cell cycle analysis of a-549 cells treated with 5 and 10 m of acacetin indicated an increase in g1 phase from 34.7% to 42.6% and 61.2% , respectively . similarly dna fragmentation assay indicated the number of cells undergoing apoptosis increased from about 3.2-fold to 8.1-fold at 5 and 10 m of acacetin , respectively after 48 hours . similar to the observation in hepg2 cells , acacetin increased the induction of p53 and its downstream target , p21/waf1 as assayed by enzyme linked immuno - sorbent assay ( elisa ) . fas ligand assay indicated that fasl , mfasl , and sfasl increased in a dose - dependent manner . they concluded that p53 and fas / fasl apoptotic system may participate in the antiproliferative activity of acacetin in hepg2 and a549 cells [ 57 , 58 ] . explored the significance of kaempferol induced apoptosis in human lung nonsmall carcinoma cells ( h460 ) . trypan blue exclusion assay , demonstrated the varying concentration of kaempferol reduced the cell viability in the dose - dependent manner with an ic50 value of 50 m . lactate dehydrogenase ( ldh ) assay indicated cell death is due to apoptosis since there is no release of ldh enzyme with the cells treated with kaempferol . ros production is not the cause for the cytotoxicity observed , since the oxidant - sensitive fluorescent probe , cm - h2dcfda signal does not showed any change after kaempferol treatment . mitochondrial membrane potential measured using fluorescent probe 3 , 3-dihexyl - oxacarbocyanine ( dioc6 ) , a mitochondrion - specific and voltage - dependent dye , indicated no change after treating with kaempferol at varying concentrations for 16 hours . kaempferol ( 50 m ) induced apoptosis inducing factor ( aif ) from mitochondria to nucleus and elicited dna fragmentation and condensation in h460 cells . the levels of pro - caspase 3 were decreasing after exposing to kaempferol for 8 hours . moreover protein levels of mn sod and cu / zn sod increased during treatment with 50 m kaempferol for 24 hours . reported the kaempferol antiproliferative effect in the pro - myelocytic leukemia cells ( hl60 ) . facs analysis reported that treatment of cells with kaempferol ( 10 m ) decreased the cell growth . after 5 hours of treatment the proportion of cells in s - phase increased compared to decrease in the g1 phase . 100 m of kaempferol induced an initial accumulation in s - phase and then g2/m as the time course progressed from 48 to 96 hours . phosphodityl serine exposure without membrane damage as indicated by annexin v - fitc binding , a feature of the early prenecrotic phase of apoptosis , was only observed for a minor proportion of cells treated with 20 m kaempferol following either 24 hours or 72 hours treatments . after exposing the cells with kaempferol for 24 and 72 hours , a decrease in the mitochondrial potential followed by enhanced expression of active caspase-3 was observed . retinoic acid treatment results nearly 5% differentiation of the cell population indicated by phorbol 12-myristate 13-acetate stimulated nitro blue tetrazolium nbt reduction over 72 hours of treatment with 100 m of kaempferol . multiparametric flow cytometric analysis revealed distinct subpopulations of cells with decreased size , typical of apoptosis and necrosis , possessing heightened caspase-3 activity followed by decreased antiapoptotic bcl2 expression and changes in the membrane asymmetry and integrity . the remaining population had elevated active caspase-3 but no change or a moderate increase in bcl2 expression and no plasma membrane alterations . hence kaempferol growth inhibitory effect on hl-60 leukemia cells is due to heterogeneous response mainly dominated by cell cycle alternation although some degree of cytotoxicity results from apoptotic as well as nonapoptotic process . showed cytotoxicity of pinocembrin against a variety of cancer cells including normal lung fibroblasts with relative non toxicity to human umbilical cord endothelial cells . pinocembrin induced loss of mitochondrial membrane potential ( mmp ) with subsequent release of cytochome c and processing of caspase-9 and -3 in colon cancer cell line hct 116 . the initial trigger for mitochondrial apoptosis appears to be by the translocation of cytosolic bax protein to mitochondria . pinobanksin has been reported to exert antioxidant activity by lowering the fe ( ii ) induced lipid peroxidation as well as inhibiting the mitochondria membrane permeability transmission ( mmpt ) . apigenin belongs to the flavonoid family and it is widely reported for its antitumor effects in various cell lines . it had exerted antiproliferative effect against colon , breast , cervical , neuroblastoma and liver cancer cell lines . wang et al . studied the effect of apigenin on the cell growth and cell cycle in the colon carcinoma cell lines like sw480 , ht 29 and caco-2 . cell count and protein content of the apigenin treated cells showed reduction compared to the control . apigenin inhibited the cell growth with the ic50 values of 40 , 50 , and 70 m for the sw480 , ht-29 , and caco-2 cells respectively . flow cytometric analysis of apigenin ( 80 m ) treated cells resulted in g2/m arrest of 64% , 42% , and 26% in sw480 , ht-29 , and caco-2 cells respectively . they had also reported the inhibition of p34 kinase and cyclin b1 proteins in the apigenin treated cells . they reported that apigenin was found to be more potent in inhibiting the her2/neu - overexpressing cells ( mda - mb-453 cells ) compared to basal level her2/neu - expressing cells ( mcf-7 ) . for instance , 40 m of apigenin resulted in 48% inhibitory effect in mda - mb-435 whereas in mcf-7 it caused only 31% growth inhibition . they examined the role of her2/her3-pi3k / akt pathway in the apigenin induced apoptosis and showed that it inhibited directly the pi3k activity first , consequently inhibiting the akt kinase activity . moreover they demonstrated the inhibition of her2/neu autophosphorylation and transphosphorylation resulting from depleting her2/neu protein in vivo . in another study by zheng et al . elucidated the apoptosis induced by apigenin in human cervical cancer cell hela . it was found that apigenin could decrease the cell viability with an ic50 of 35.89 m . dna fragmentation assay and flow cytometric analysis of apigenin treated cells confirmed the apoptosis induction . they had observed increased expression of p21/waf1 and p53 . further fas / apo-1 and caspase-3 increase and bcl2 reduction in the apigenin treated hela cells confirmed the apoptosis induction . apigenin repressed the cell viability in a dose - dependent manner in human neuroblastoma cell lines with an ec50 = 35 mol / l in nub-7 , and ec50 = 22 mol / l in lan-5 after 24 hours . moreover it was found to inhibit the colony forming ability and nub-7 xenograft tumor growth in nonobese diabetic mouse model . they had shown that apigenin induced apoptosis was mediated though p53 as it enhanced the expression of p53 and p53 induced gene products like p21 and bax . recent research by chiang et al . suggested the antiproliferative effect of apigenin in hepg2 , hep3b and plc / prf/5 cells . it was found that apigenin could inhibit the cell growth of the above reported liver cancer cells but not the normal murine liver bnl.cl2 cells . ic50 was observed to be 8.02 g / ml for hepg2 , 2.16 g / ml for hep3b and 22.73 g / ml for plc / prf/5 . in addition , dna ladder and flow cytometric analysis indicated apoptosis in the hepg2 cells . chemoprevention utilizes appropriate pharmacological agents [ 3 , 4 ] or of dietary agents , consumed in diverse forms like macronutrients , micronutrients , or nonnutritive phytochemicals . various polyphenols are reported in honey . some of the polyphenols of honey like caffeic acid ( ca ) , caffeic acid phenyl ester ( cape ) , chrysin ( cr ) , galangin ( ga ) , quercetin ( qu ) , acacetin ( ac ) , kaempferol ( kf ) , pinocembrin ( pc ) , pinobanksin ( pb ) and apigenin ( ap ) have evolved as promising pharmacological agents in treatment of cancer . caffeic acid has been reported as a carcinogen in initial studies , but the same caffeic acid along with combination of other antioxidant has been shown to suppress colon tumors in rats . showed that oral administration of caffeic acid and caffeic acid phenyl esters ( cape ) reduced liver metastasis , mediated by the dual inhibition of nf-b and mmp-9 enzyme activity . demonstrated that cape is known to have antimitogenic , anticarcinogenic , antiinflammatory and immunomodulatory properties . cape 's antiinflammatory and anticancer property has also been shown to protect skin cells when exposed to ultra - violet radiation and uvb radiation . showed that the growth inhibitory effect of chrysin in c6 glioma cells was either though activating p38-mapk which leads to the accumulation of p21 protein or mediating the inhibition of proteasome activity . in another study by woo et al . it has been elucidated that chrysin induces apoptosis in association with the activation of caspase-3 and akt signal pathway , that plays a crucial role in chrysin - induced apoptosis in u937 cells . galangin expressed antiproliferative effect on hl-60 cells on dose dependent manner and also induced dna fragmentation without loss of membrane integrity . quercetin also inhibited the hl-60 cell proliferation in association with the inhibition of cytosolic protein kinase c ( pkc ) and membrane tyrosine protein kinase ( tpk ) in vitro . it has been reported that quercetin in low concentration promoted cell proliferation of a-549 cells , whereas in higher concentration it inhibited cell proliferation and survival . acacetin , another important flavanoid inhibited the proliferation of a549 cells , induced apoptosis and blocked the cell cycle progression at g1 phase . it also improved the expression of p53 and fas ligands . in another study , it has been shown to inhibit hepg2 cell proliferation and provoke apoptosis by enhancing the p53 and fas ligands as in the case of a549 cells . kaempferol induced apoptosis in h460 cells which was accompanied by significant dna condensation and increasing atp levels . it also changed the expression of caspase 3 and apoptosis inducing factor ( aif ) levels . bestwick et al . reported recently that kaempferol growth inhibitory effect on hl-60 leukemia cells is due to heterogeneous response mainly dominated by cell cycle alternation although some degree of cytotoxicity results from apoptotic as well as nonapoptotic process . pinocembrin induced loss of mitochondrial membrane potential ( mmp ) with subsequent release of cytochrome c and processing of caspase-9 and -3 in colon cancer cell line hct 116 . apigenin exerted antiproliferative effect against colon , breast , cervical , neuroblastoma and liver cancer cell lines [ 6367 ] . our review has clearly demonstrated certain honey polyphenols tested in laboratorial setups showed to be a promising pharmacological agent for inhibiting cancer cell proliferation . after generating more in - depth and exhaustive information of these compounds jointly in in vitro and in vivo studies ,

honey has been used since long time both in medical and domestic needs , but only recently the antioxidant property of it came to limelight . the fact that antioxidants have several preventative effects against different diseases , such as cancer , coronary diseases , inflammatory disorders , neurological degeneration , and aging , led to search for food rich in antioxidants . chemoprevention uses various dietary agents rich in phytochemicals which serve as antioxidants . with increasing demand for antioxidant supply in the food , honey had gained vitality since it is rich in phenolic compounds and other antioxidants like ascorbic acid , amino acids , and proteins . some simple and polyphenols found in honey , namely , caffeic acid ( ca ) , caffeic acid phenyl esters ( cape ) , chrysin ( cr ) , galangin ( ga ) , quercetin ( qu ) , kaempferol ( kp ) , acacetin ( ac ) , pinocembrin ( pc ) , pinobanksin ( pb ) , and apigenin ( ap ) , have evolved as promising pharmacological agents in treatment of cancer . in this review , we reviewed the antiproliferative and molecular mechanisms of honey and above - mentioned polyphenols in various cancer cell lines .

1. Introduction 2. Source and Compositional Chemistry of Honey 3. Anticancerous Property of Crude Honey 4. Summary

chemoprevention was defined as the administration of agents to prevent induction , to inhibit or to delay the progression of cancer , or as the inhibition or reversal of carcinogenesis at a premalignant stage . chemoprevention utilizes appropriate pharmacological agents [ 3 , 4 ] or of dietary agents , consumed in diverse forms like macronutrients , micronutrients , or nonnutritive phytochemicals [ 57 ] . consumption of antioxidants has been related to the several preventative effects against different diseases such as cancer , coronary diseases , inflammatory disorders , neurological degeneration , and aging [ 8 , 9 ] led to search for natural foods rich in antioxidants . although honey has been used since long time , only recently its antioxidant property came to limelight . some to mention were flavonoids and phenolic acids [ 13 , 14 ] , certain enzymes ( glucose oxidase , catalase ) , ascorbic acid , carotenoid - like substances , organic acids , amino acids , and proteins . polyphenols and phenolic acids found in the honey vary according to the geographical and climatic conditions . considerable differences in both composition and content of phenolic compounds have been found in different unifloral honeys . terpenes , benzyl alcohol , 3 , 5-dimethoxy-4-hydroxybenzoic acid ( syringic acid ) , methyl 3 , 5-dimethoxy-4-hydroxybenzoate ( methyl syringate ) , 3 , 4 , 5-trimethoxybenzoic acid , 2-hydroxy-3-phenylpropionic acid , 2-hydroxybenzoic acid and 1 , 4-dihydroxybenzene are some of the phytochemicals ascribed for the antimicrobial activity of honey . in this review , we summarized the compositional chemistry and antiproliferative potential of crude honey and some of its important polyphenols in various cancer cells . honey bees make a journey of nearly 55,000 miles to gather nectar from approximately 2 million flowers for accumulating one pound of honey . in the bee - hive , we can find thee types of bees namely the queen , drone and worker bees . during the collection of nectar as the honeybee visits the flower in hunt of nectar , some of the flower 's pollen falls into the nectar collected by the bee and stored in the stomach which will be regurgitated along with nectar . moreover some pollen grains often attach themselves to the various parts of the honey bee body like legs , antenna , hairs , and also in the eyes of visiting bees which will get entangled in the hive and thereby paving entry into the honey . it has been reported more than 300 unique varieties of honey depending upon the floral sources from united states alone . major sugars of honey are levulose and dextrose which constitutes 38.19% and 31.28% correspondingly , remaining is the sucrose 1.3% and maltose 7.3% . honey minor constituents include acids ( 0.57% ) , protein ( 0.266% ) , nitrogen ( 0.043% ) , amino acids ( 0.1% ) , a little amount of minerals ( 0.17% ) , and a number of other minute quantities of components like pigments , flavor and aroma substances , phenolics compounds , colloids , sugar alcohols and vitamins which all together accounts for the 2.1% of whole honey composition [ 20 , 21 ] . honey was proven to be a very effective agent for repressing the growth of bladder cancer cell lines ( t24 , rt4 , 253j , and mbt-2 ) in vitro . there was also a significant difference between the final tumor volume ( p < .05 ) in the intra lesion ( il ) honey - treated groups ( il 6% honey ) compared to the il saline group . it has been elucidated that polyphenols are anticarcinogenic , antiinflammatory , antiatherogenic , antithombotic , immune modulating and also act as antioxidants [ 2631 ] . hence antitumor properties of honey could be attributed to the polyphenols found in the honey . moreover , with the evolution of extraction procedure for various polyphenols , which had been attributed with anticancerous property of honey , researchers concentrated on the polyphenolic compounds extracted from the honey rather than crude honey itself . phenolic compounds or polyphenols are the important groups of compounds occurring in plants , where they are widely distributed , comprising at least 8000 different known structures . in general , phenolic compounds can be divided into at least 10 types depending upon their basic structure : simple phenols , phenolic acids , coumarins and isocoumarins , naphthoquinones , xanthones , stilbenes , anthaquinones , flavonoids and lignins . polyphenols found in the honey was used as a marker for particular type of honey , for example , flavanol kaempferol as an indicator for rosemary honey [ 33 , 34 ] and quercetin for sunflower honey . the hydroxy - cinnamates like caffeic acid , ferulic acid and p - coumaric acid have been found in the chest - nut honey . characteristic flavonoids of propolis like pinocembrin , pinobanksin and chrysin were also found in the most european honey samples . in this review , we concentrated on the some major polyphenols available in the honey which exhibited antiproliferative effect on various cancer cell lines . the list of compounds ( refer table 1 for figures ) reviewed for their anticancerous activity is caffeic acid ( ca ) , caffeic acid phenyl ester ( capes ) , chrysin ( cr ) , galangin ( ga ) , quercetin ( qu ) , acacetin ( ac ) , kaempferol ( kf ) , pinocembrin ( pc ) , pinobanksin ( pb ) , and apigenin ( ap ) . studied the carcinogenicity of low dietary levels of the antioxidants like butylated hydroxyanisole ( bha ) , caffeic acid , sesamol , 4-methoxyphenol ( 4-mp ) and catechol . carcinogenicity study was undertaken in groups of 30 - 31 male f344 rats , by treating with 0.4% bha , 0.4% caffeic acid , 0.4% sesamol , 0.4% 4-mp and 0.16% catechol either alone or in combination for up to 104 weeks and then killed . the ultimate average body weights of rats having basal diets were higher than those treated with antioxidants alone , and were the lowest in the combinational groups . it led to the conclusion , that the occurrences and frequencies of fore - stomach histopathological lesion were increased by exposing to antioxidants except in the case of bha . the incidences and/or multiplicities of forestomach papillary or nodular ( pn ) hyperplasia were appreciably increased in the groups treated with 4-methoxyphenol , caffeic acid and the antioxidants in combination , as compared with the basal diet group . studies on medium - term multiorgan carcinogenesis model , suggested an increase in the occurrence of fore - stomach papillomas in each high - dose group and no synergistic effect was observed in combinations . from these early experiments , caffeic acid rao et al . performed a detailed study by synthesizing thee caffeic acid esters namely methyl caffeate ( mc ) , phenylethyl caffeate ( pec ) and phenylethyl dimethylcaffeate ( pedmc ) and examined them against the 3 , 2-dimethyl-4-aminobiphenyl ( dmab , a colon and mammary carcinogen ) induced mutagenecity in salmonella typhimurium strains ta 98 and ta 100 . in colon cancer cell line ( ht-29 ) , cytotoxicity effect of ca , pec , pedmc and mc was evaluated . they depicted the inhibitory effects of caffeic acid , mc , pec , phenylethyl-3-methylcaffeate ( pemc ) , and phenylethyl dimethyl caffeate ( pedmc ) on in vitro arachidonic acid metabolism in liver and colonic mucosa . finally they investigated the effects of pec , pemc , and pedmc on aom - induced aberrant crypt foci ( acf ) formation in the colon of f344 rats . the animals fed the mc diet exhibited a moderate inhibitory effect on odc and 5(s)- , 8(s)- , 12(s)- , and 15(s)-hetes and a significant effect on colonic tpk activity . acf were significantly inhibited in the animals fed with pec ( 55% ) , pemc ( 82% ) , or pedmc ( 81% ) . the results of the study indicated that pec , pemc , and pedmc present in the honey , inhibited aom - induced colonic preneoplastic lesions , odc , tpk , and lipoxygenase activity , which were relevant to the colon carcinogenesis . showed the strong repressive effect of cape application on 12 - 0-tetradecanoylphorbol-13-acetate- ( tpa-)induced tumor promotion and production of 5-hydroxymethyl-2-deoxyuridine ( hmdu ) in the deoxyribonucleic acid ( dna ) of the mouse skin . similarly incorporation of [ h]-uridine into rna was inhibited by 39% , 43% , 58% , 64% , and 75% whereas incorporation of [ h]-leucine into protein was inhibited by 29% , 30% , 37% , 32% , or 47% , respectively . these results indicated that cape is a potent inhibitor of dna synthesis but it is somewhat less effective in inhibiting rna synthesis and it is least effective in inhibiting the protein synthesis . nf-b activation induced by the phorbol ester , phorbol-12-myristate 13-acetate ( pma ) , ceramide , okadaic acid and hydrogen peroxide was also inhibited by cape . cape and ca suppressed the mmp-9 expression , exposed to phorbol 12-myristate 13-acetate ( pma ) , by blocking the nf-b activity in hepg2 cells . therefore , these two drugs were reported as strong candidates for treatment of cancer and metastasis via dual mechanisms ( dual inhibition of metastasis - specific enzyme activity and gene transcription ) . to further corroborate the downregulation of mmp-2 , activation studies of pro - mmp2 were performed using organomercuric compound , 4-aminophenylmercuric acetate ( apma ) , and the result indicated the down regulation of mmp-2 by cape . moreover some researchers investigated the possible uvc ( 280100 nm ) protective properties of caffeic acid in human diploid fibroblast and a-431 epidermoid cancer cell lines . il-10 mrna expression and protein production in the mouse skin were significantly repressed by ca . there have also been shown the upstream regulators like extracellular regulated protein kinase ( erk ) , c - jun n - terminal protein , p-38 mitogen - activated protein kinase ( p38 mapk ) and the downstream transcription factors like activator protein ( ap-1 ) and nuclear factor kappa b ( nf-b ) were also inhibited by ca in mouse skin . chrysin ( 5 , 7-dihydroxyflavone ) is a natural and biologically active compound extracted from honey , plants and propolis . flow cytometry analysis reported that by 30 and 50 m treatments after 24 hours , chrysin increased the proportion of cells in the g1 phase of the cell cycle from 69 to 79% and 83% and decreased the proportion of s phase cells from 11.4 to 6.1% and 2.8% , respectively . it has been found that levels of phosphorylation of retinoblastoma ( rb ) protein in c6 glioma cells decreased after treating with 30 m of chrysin . moreover in chrysin - treated cells , it has been demonstrated that cyclin dependent kinase inhibitor ( p21 ) levels are increased significantly without the change in p53 protein level . reported the chrysin - mediated apoptosis in u-937 cancer cell lines . western blotting analysis of chrysin treated cells indicated the reduction in the level of xiap ( a member of inhibitor of apoptosis proteins ) and cytochrome c induction in dose dependent manner . tried to improve the biological properties of chrysin by synthesizing diethyl chrysin-7-yl phosphate ( cpe : c19h19o7p ) and tetraethyl bis - phosphoric ester of chrysin ( cp : c23h28o10p2 ) though a simplified atheron - todd reaction . cultured human ( hela ) cell lines were treated by cr , cp and cpe with 10 m for 24 , 48 , and 72 hours . methyl green - pyronin staining , pcna immunohistochemistry and tunel techniques were also employed to study the effect of cr , cpe and cp in the cultured hela cell lines . cell cycle analysis indicated the increase in the subg1 phase of galangin ( > 10 m ) treated cells . galangin treated cells displayed reduced forward scatter indicative of decreased relative size , and enhanced side scatter indicative of increased internal complexity . cell cycle analysis indicated that quercetin ( 20 , 40 , and 60 m ) increased the number of cells in the g2/m phase from 7.6% to 12.4% , 19.1% , and 23.5% correspondingly , and decreased the population of g0/g1 , cells from 46.2% to 40.2% , 32.1% , and 34.5% , respectively , without significant changes in the s - phase cell population after 24 hours of treatment . quercetin showed remarkable inhibitory effect on the activities of cytosolic protein kinase c ( pkc ) and membrane tpk of hl-60 cells in vitro , with ic50 values of about 30.9 and 20.1 m , respectively , but did not have the effect on membrane pkc or cytosolic tpk activity . it has also repressed the complete activity of phosphoinositides like phosphatidylinositol ( pi ) , phosphatidylinositol 4-phosphate ( pip ) , and phosphatidylinositol 4 , 5-bisphosphate ( pip2 ) at the concentration of 80 m . they also found incubating with low concentrations of quercetin led to a small increase in total antioxidant capacity ( tac ) of cell extracts but higher concentrations of the quercetin led to a progressive decrease in the tac of cell extracts . it also arrested the glioma cells in the g2 checkpoint of the cell cycle , and decreased the mitotic index . investigated the antiproliferative effect of acacetin in human liver cancer cell line ( hepg2 ) . further it has been demonstrated that acacetin increased the induction of p53 and its downstream target , p21/waf1 as assayed by enzyme linked immuno - sorbent assay ( elisa ) . similar to the observation in hepg2 cells , acacetin increased the induction of p53 and its downstream target , p21/waf1 as assayed by enzyme linked immuno - sorbent assay ( elisa ) . fas ligand assay indicated that fasl , mfasl , and sfasl increased in a dose - dependent manner . they concluded that p53 and fas / fasl apoptotic system may participate in the antiproliferative activity of acacetin in hepg2 and a549 cells [ 57 , 58 ] . trypan blue exclusion assay , demonstrated the varying concentration of kaempferol reduced the cell viability in the dose - dependent manner with an ic50 value of 50 m . facs analysis reported that treatment of cells with kaempferol ( 10 m ) decreased the cell growth . pinocembrin induced loss of mitochondrial membrane potential ( mmp ) with subsequent release of cytochome c and processing of caspase-9 and -3 in colon cancer cell line hct 116 . apigenin belongs to the flavonoid family and it is widely reported for its antitumor effects in various cell lines . it had exerted antiproliferative effect against colon , breast , cervical , neuroblastoma and liver cancer cell lines . studied the effect of apigenin on the cell growth and cell cycle in the colon carcinoma cell lines like sw480 , ht 29 and caco-2 . flow cytometric analysis of apigenin ( 80 m ) treated cells resulted in g2/m arrest of 64% , 42% , and 26% in sw480 , ht-29 , and caco-2 cells respectively . they had also reported the inhibition of p34 kinase and cyclin b1 proteins in the apigenin treated cells . further fas / apo-1 and caspase-3 increase and bcl2 reduction in the apigenin treated hela cells confirmed the apoptosis induction . apigenin repressed the cell viability in a dose - dependent manner in human neuroblastoma cell lines with an ec50 = 35 mol / l in nub-7 , and ec50 = 22 mol / l in lan-5 after 24 hours . chemoprevention utilizes appropriate pharmacological agents [ 3 , 4 ] or of dietary agents , consumed in diverse forms like macronutrients , micronutrients , or nonnutritive phytochemicals . some of the polyphenols of honey like caffeic acid ( ca ) , caffeic acid phenyl ester ( cape ) , chrysin ( cr ) , galangin ( ga ) , quercetin ( qu ) , acacetin ( ac ) , kaempferol ( kf ) , pinocembrin ( pc ) , pinobanksin ( pb ) and apigenin ( ap ) have evolved as promising pharmacological agents in treatment of cancer . caffeic acid has been reported as a carcinogen in initial studies , but the same caffeic acid along with combination of other antioxidant has been shown to suppress colon tumors in rats . showed that oral administration of caffeic acid and caffeic acid phenyl esters ( cape ) reduced liver metastasis , mediated by the dual inhibition of nf-b and mmp-9 enzyme activity . it has been elucidated that chrysin induces apoptosis in association with the activation of caspase-3 and akt signal pathway , that plays a crucial role in chrysin - induced apoptosis in u937 cells . in another study , it has been shown to inhibit hepg2 cell proliferation and provoke apoptosis by enhancing the p53 and fas ligands as in the case of a549 cells . apigenin exerted antiproliferative effect against colon , breast , cervical , neuroblastoma and liver cancer cell lines [ 6367 ] . our review has clearly demonstrated certain honey polyphenols tested in laboratorial setups showed to be a promising pharmacological agent for inhibiting cancer cell proliferation .

from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . of these patients , we included the patients who met the two following criteria : ( a ) two or more four - phase ( i.e. , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . eighteen patients had one tumor , four patients had two tumors , one patient had three tumors and one patient had six tumors . they underwent lobectomy ( n = 22 ) , segmentectomy ( n = 1 ) or liver transplantation ( n = 1 ) after transarterial chemoembolization . of these , 20 patients could undergo hepatic resection because they had recovered enough hepatic function to endure hepatic surgery , as compared to the time when they had undergone the initial transarterial chemoembolization . there were 21 men and three women , and their ages ranged from 38 to 73 years ( mean age ; 54 years ) . all the patents in the study had liver cirrhosis as a result of either hepatitis b ( n = 23 ) , or hepatitis c ( n = 1 ) . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . the remaining six hepatocellular carcinomas showed the characteristic laboratory findings ( e.g. , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean diameter of the hepatocellular carcinomas was 3.7 cm ( range ; 0.5 - 12 cm ) . seventeen patients underwent multiple treatments of transarterial chemoembolization ( range ; 2 - 5 treatments , mean ; 2.9 treatments ) . the remaining seven patients underwent it once . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . the ct scans were performed with a helical scanner ( hispeed advantage ; ge medical systems , milwaukee , wi , usa ) . the scanning parameters were 120 kvp , 180 mas , 7 mm section collimation and a 7 mm / sec table speed during a single breath - hold helical acquisition of 25 - 30 seconds , depending upon the liver size . the images were obtained in a craniocaudal direction and they were reconstructed every 7 mm to provide contiguous or overlapping sections for the unenhanced and enhanced images . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . transarterial chemoembolization was performed with a mixture of iodized oil ( lipiodol ; guerbet , aulnay - sous - bois , france ) and doxorubicin hydrochloride ( adriamycin ; kyowa hakko kogyo , tokyo , japan ) in all the patients . we used 3 mg of doxorubicin hydrochloride and 1 cc of iodized oil per 1-cm diameter of the tumor . for particle embolization , we also used gelfoam powder ( upjohn , kalamazoo , mi , usa ) if a patient had neither child class c cirrhosis nor thrombosis in the major portal vein . the pathologic specimens were sectioned at 5-mm intervals and the tumor necrosis was estimated as a percentage . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . one observer had 24 years experience at abdominal imaging and other two observers had five years experience each . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . all images were evaluated using a 2,000 2,000 picture archiving and communication systems ( pacs ; ge medical systems integrated imaging solutions , mt prospect , il , usa ) monitor . the images were initially evaluated using two window settings ( window level ; 60 hu , window width ; 350 hu , and window level ; 110 hu , window width ; 200 hu ) , and then the window settings were adjusted as needed . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . the attenuation of each lesion in relation to that of the liver ( i.e. , hypoattenuation , hyperattenuation , isoattenuation or mixed attenuation ) was subjectively assessed . the diagnostic confidence for each lesion was scored using a five - point scale ( 1 , not viable tumor ; 2 , probably not viable tumor ; 3 , possibly viable tumor ; 4 , probably viable tumor ; 5 , definitely viable tumor ) at each interpretation session . for the objectivity and reproducibility of the image analysis performed in this study , we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . a lesion that showed wedge - shaped hyperattenuation during the hepatic arterial phase and that appeared as isoattenuation on the portal venous phase , on the delayed phase images and on the unenhanced images was considered to be a noncancerous lesion . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the lesions that were assigned a score of three to five were considered as diagnosed viable tumors . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . the degree of agreement was categorized as follows : values of 0.00 - 0.20 were considered to indicate poor agreement , values of 0.21 - 0.40 were considered to indicate fair agreement , values of 0.41 - 0.60 were considered to indicate moderate agreement , values of 0.61 - 0.80 were considered to indicate good agreement and values of 0.81 - 1.00 were considered to indicate excellent agreement ( 5 ) . from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . of these patients , we included the patients who met the two following criteria : ( a ) two or more four - phase ( i.e. , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . eighteen patients had one tumor , four patients had two tumors , one patient had three tumors and one patient had six tumors . they underwent lobectomy ( n = 22 ) , segmentectomy ( n = 1 ) or liver transplantation ( n = 1 ) after transarterial chemoembolization . of these , 20 patients could undergo hepatic resection because they had recovered enough hepatic function to endure hepatic surgery , as compared to the time when they had undergone the initial transarterial chemoembolization . there were 21 men and three women , and their ages ranged from 38 to 73 years ( mean age ; 54 years ) . all the patents in the study had liver cirrhosis as a result of either hepatitis b ( n = 23 ) , or hepatitis c ( n = 1 ) . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . the remaining six hepatocellular carcinomas showed the characteristic laboratory findings ( e.g. , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean diameter of the hepatocellular carcinomas was 3.7 cm ( range ; 0.5 - 12 cm ) . seventeen patients underwent multiple treatments of transarterial chemoembolization ( range ; 2 - 5 treatments , mean ; 2.9 treatments ) . the remaining seven patients underwent it once . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . the ct scans were performed with a helical scanner ( hispeed advantage ; ge medical systems , milwaukee , wi , usa ) . the scanning parameters were 120 kvp , 180 mas , 7 mm section collimation and a 7 mm / sec table speed during a single breath - hold helical acquisition of 25 - 30 seconds , depending upon the liver size . the images were obtained in a craniocaudal direction and they were reconstructed every 7 mm to provide contiguous or overlapping sections for the unenhanced and enhanced images . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . transarterial chemoembolization was performed with a mixture of iodized oil ( lipiodol ; guerbet , aulnay - sous - bois , france ) and doxorubicin hydrochloride ( adriamycin ; kyowa hakko kogyo , tokyo , japan ) in all the patients . we used 3 mg of doxorubicin hydrochloride and 1 cc of iodized oil per 1-cm diameter of the tumor . for particle embolization , we also used gelfoam powder ( upjohn , kalamazoo , mi , usa ) if a patient had neither child class c cirrhosis nor thrombosis in the major portal vein . the pathologic specimens were sectioned at 5-mm intervals and the tumor necrosis was estimated as a percentage . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . one observer had 24 years experience at abdominal imaging and other two observers had five years experience each . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . all images were evaluated using a 2,000 2,000 picture archiving and communication systems ( pacs ; ge medical systems integrated imaging solutions , mt prospect , il , usa ) monitor . the images were initially evaluated using two window settings ( window level ; 60 hu , window width ; 350 hu , and window level ; 110 hu , window width ; 200 hu ) , and then the window settings were adjusted as needed . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . the attenuation of each lesion in relation to that of the liver ( i.e. , hypoattenuation , hyperattenuation , isoattenuation or mixed attenuation ) was subjectively assessed . the diagnostic confidence for each lesion was scored using a five - point scale ( 1 , not viable tumor ; 2 , probably not viable tumor ; 3 , possibly viable tumor ; 4 , probably viable tumor ; 5 , definitely viable tumor ) at each interpretation session . for the objectivity and reproducibility of the image analysis performed in this study , the important criteria for a viable tumor and for the noncancerous lesions we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . a lesion that showed wedge - shaped hyperattenuation during the hepatic arterial phase and that appeared as isoattenuation on the portal venous phase , on the delayed phase images and on the unenhanced images was considered to be a noncancerous lesion . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the lesions that were assigned a score of three to five were considered as diagnosed viable tumors . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . the degree of agreement was categorized as follows : values of 0.00 - 0.20 were considered to indicate poor agreement , values of 0.21 - 0.40 were considered to indicate fair agreement , values of 0.41 - 0.60 were considered to indicate moderate agreement , values of 0.61 - 0.80 were considered to indicate good agreement and values of 0.81 - 1.00 were considered to indicate excellent agreement ( 5 ) . during the two analyses of the ct images , each observer detected all 35 hepatocellular carcinomas that were pathologically demonstrated in the resection specimens . the mean diagnostic accuracies ( az values ) for the depiction of viable tumor with the last ct alone and with the review of previous serial ct images for all the observers were 0.885 and 0.901 , respectively ( p > 0.05 ) ( table 1 ) . the sensitivity , specificity and diagnostic accuracy of the last ct alone were 72% , 91% and 78% , respectively , and the corresponding values for the review of the last ct combined with the previous serial ct images were 78% , 97% and 84% , respectively ( table 2 ) , and the differences were not statistically significant . although these differences in the diagnostic accuracies were not statistically significant , the review of the previous images enabled the observers to render a correct diagnosis for three ( 9% ) tumors . two of these three tumors showed as being slightly hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct , and these two tumors appeared as isoattenuating areas on the unenhanced images . the second to the last ct showed that these areas appeared as parenchymal uptake around the iodized oil - containing tumors ( fig . 1 ) , and the two observers changed their confidence levels to probably not viable tumor . the one remaining tumor showed as an interval developed defect within the iodized oil - containing nodule that was not significantly enhanced on the last ct . on the basis of this feature , the two observers changed their confidence level to probably viable tumor . the pathology revealed a 70% necrotic hepatocellular carcinoma . after reviewing of the last ct and the previous serial ct images , all three observers assessed the 10 compact iodized oil - containing tumors as being non - viable . of these thus , the compact iodized oil - containing tumors showed a mean necrosis rate of 98.5% . all of the 16 false - negative lesions that were diagnosed by each observer after reviewing the last ct combined with the previous serial ct images showed 90% or greater necrosis on the pathologic examination ( table 3 ) . particularly , four tumors of these lesions were diagnosed as negative lesions simultaneously by all the observers with both the review of the last ct alone and the review of the last ct with the previous serial ct images . 2 ) . the remaining two showed no definite enhancement in the iodized oil defect portion on the hepatic arterial phase ct . the interobserver agreement ( statistic ) for the presence of a viable tumor was 0.828 to 0.943 for the review of the last ct alone and 0.826 to 0.885 for the additional review of the previous serial ct images ( table 4 ) . the interobserver agreement for the review of the last ct with or without the previous serial ct images was considered excellent . the criteria suggested by the world health organization and that is used for evaluating the effect of chemotherapy on cancer can not be applied to the evaluation of transarterial chemoembolization therapy because any tumor reduction can seldom be recognized before one month after the treatment ( 6 ) . thus , other criteria have been evaluated to determine the efficacy of transarterial chemoembolization treatment for hepatocellular carcinoma by using iodized oil retention in a tumor and the enhancement on ct ( 7 , 8) . ( 9 ) suggested that the overall cumulative recurrence rate of hepatocellular carcinoma was 23% after one year , 55% after two years and 67% after three years , and 45% of the recurrences occurred adjacent to a primary site that was considered controlled by the transarterial chemoembolization . hence , periodic ct follow - up or angiography might be recommended even though there may be the appearance of complete remission for hepatocellular carcinoma . with the advent of helical ct and with the other rapid technical advances , multiphasic helical ct has recently become a more useful imaging modality for the detection and characterization of liver lesions , for the follow - up after local treatment or surgical excision , and for the assessment of the hemodynamic changes in the liver . however , to the best of our knowledge , there has been no previous study that has compared the preoperative ct and the review of the previous serial ct images with the resected specimens for the evaluation of the viable portion of hepatocellular carcinoma treated with transarterial chemoembolization . in the past , some investigators have reported on the diagnostic efficacy of the hepatic arterial phase ct and the portal venous phase ct for the detection of hypervascular tumors , and especially hepatocellular carcinoma ( 10 , 11 ) . ( 1 ) have suggested that the delayed phase is important for the detection of small hepatocellular carcinomas that are less than 2 cm , for confirming or increasing the confidence level for the detection of equivocal nodules on the arterial or portal venous phase images ( because those nodules are usually more conspicuous on the delayed phase than on the portal venous phase imaging ) , and for the differentiation of arterioportal shunting from the true hepatocellular carcinoma . in our study , this concept was used to depict a viable tumor among the arterioportal shunt areas . for the hepatocellular carcinoma treated with transarterial chemoembolization , takayasu et al . ( 8) have suggested that the iodized oil deposition in a tumor could be considered as necrosis . choi et al . ( 7 ) have also proposed that the complete retention of iodized oil in a tumor and the surrounding liver demonstrated the best therapeutic effects . kim et al . ( 4 ) have recently pointed out that unenhanced images could be of additional diagnostic value to supplement the enhanced biphasic helical ct images for the patients treated with transarterial chemoembolization for hepatocellular carcinoma by facilitating the differentiation of the true lesions from the hyperattenuating pseudolesions that simulate viable tumor . further , they stated that these unenhanced images can also aid in the detection of any isoattenuation lesion on the hepatic arterial phase and the portal venous phase because of the hypoattenuation on the unenhanced images . we also evaluated the unenhanced images , and we used the criteria suggested by kim et al . in addition to the criteria in the previous studies ( 1 , 4 , 7 , 10 , 11 ) , we considered the long - standing hypoattenuating areas within an iodized nodule on the enhanced images of the serial ct images as necrotic lesion despites a lack of iodized oil . on the other hand , a lesion that showed an interval developed defect within an iodized nodule was considered as a viable portion of tumor . in our study , although no significant statistical difference was found between the review of the last ct alone and the review of the last ct along with the previous serial ct images , the additional review of the previous serial ct images allowed the observers to render a correct diagnosis for three ( 9% ) lesions . we believe that many abdominal radiologists actually do review some of the previous ct images when they find an equivocal viable lesion on the liver ct following transarterial chemoembolization . according to our results , after the review of the last ct combined with the previous serial ct images , all the false - negative lesions showed 90% or greater necrosis on the pathologic examination . thus , if a substantial ( larger than 10% ) viable tumor is present in an iodized nodule , we can depict it on ct with a thorough review of the previous serial ct images . first , as this is a retrospective study , all the patients of the study group did not undergo regular follow - up ct and transarterial chemoembolization because of suspected complications or because of personal preference . second , we included a relatively small number of hepatocellular carcinomas , and so this might show no significant statistical difference between the review of the last ct alone and the review of the last ct along with the previous serial ct images . finally , although we thoroughly reviewed the pathologic specimens alongside the ct images , any perfect area - by - area histopathologic correlation was actually impossible . by referring to the location and size of viable tumors within the tumors , however , we tried to match the ct and histopathologic findings as closely as possible . in our results , two of the three tumors that had a correct diagnosis rendered after the review of previous images appeared as parenchymal uptake around the iodized oil - containing tumors on the second to the last ct the pathology demonstrated totally necrotic tumors , and this revealed that the suspicious hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct were pseudolesion . for the third hepatocellular carcinoma with 70% necrosis , we could not perfectly match the interval developed defect within the iodized oil - containing nodule and the 30% viable tumor portion on the pathologic specimen . also , we do not know the reason why the two compact iodized oil - containing tumors seen on ct had viable tumor portions on the pathology examination . in conclusion , for depicting the viable tumor in hepatocellular carcinoma patients treated with transarterial chemoembolization , although the difference in the diagnostic accuracies was not found to be statistically significant in our study , a review of the multiphasic helical ct combined with the previous serial ct images can help render a correct diagnosis for those lesions incorrectly diagnosed with the review of only the last ct .

objectivethe purpose of our study was to assess whether a review of multiphasic helical ct combined with the previous serial ct images could be helpful to depict a viable tumor in hepatocellular carcinoma treated with transarterial chemoembolization.materials and methodstwenty - four consecutive patients with 35 hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection . first , three radiologists independently analyzed the last ct images taken before resection for the presence of viable tumor . a second analysis was then performed using the last ct combined with the previous serial ct images . the ct analyses were then compared with the pathologic results . the added value of the review of the previous serial ct images was evaluated by performing a receiver operating characteristic analysis . the sensitivity , specificity and diagnostic accuracy for the depiction of viable tumor were also assessed , and the characteristics of the false - negative lesions were pathologically evaluated.resultsthe mean diagnostic accuracies ( az values ) for the depiction of viable tumor with using the last ct alone and with the review of the previous serial ct images for all observers were 0.885 and 0.901 , respectively , which were not significantly difference ( p > 0.05 ) . however , the additional review of the previous serial ct images allowed the observers to render a correct diagnosis for three lesions that had been incorrectly diagnosed with the review of last ct alone . the sensitivity , specificity and diagnostic accuracy of the last ct along with the review of the previous serial ct images were 78% , 97% and 84% , respectively . all of the 16 false - negative lesions diagnosed by each observer showed 90% or greater necrosis on the pathologic examination.conclusionfor the depiction of viable tumor in hepatocellular carcinoma treated with transarterial chemoembolization , although the difference in the diagnostic accuracies was not statistically significant , a review of the multiphasic helical ct combined with the previous serial ct images could help reach a correct diagnosis for those lesions incorrectly diagnosed with the review of the last ct alone .

MATERIALS AND METHODS Patient Selection Multiphasic Helical CT Transarterial Chemoembolization Techniques Pathologic Analysis Image Analysis Statistical Analysis RESULTS DISCUSSION

from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . the images were obtained in a craniocaudal direction and they were reconstructed every 7 mm to provide contiguous or overlapping sections for the unenhanced and enhanced images . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . for the objectivity and reproducibility of the image analysis performed in this study , we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . from october 1996 to november 2002 , 30 consecutive patients with hepatocellular carcinomas underwent transarterial chemoembolization followed by hepatic resection at our institution . , hepatic arterial , portal venous , and delayed phase , and unenhanced image ) helical cts were taken after the first transarterial chemoembolization , and ( b ) hepatic resection was done within one month of the last follow - up ct . twenty - six hepatocellular carcinomas were diagnosed by the pathologic results for those patients that demonstrated viable tumor in the resected specimens . of the nine totally necrotic tumors , the diagnosis of three hepatocellular carcinomas was established based on the results of percutaneous needle biopsy . , elevated -fetoprotein levels and viral markers ) in combination with the characteristic radiological findings on the follow - up ct images . the mean interval between the transarterial chemoembolization and the follow - up ct was 20 days ( range ; 3 - 23 days ) , and the mean interval between the last ct and surgery was 14 days ( range ; 1 - 30 days ) . after the acquisition of the unenhanced images , the hepatic arterial phase , the portal venous phase and the delayed phase images were obtained with delays of 30 , 60 and 180 seconds , respectively , after the start of injecting 120 ml of nonionic iodinated contrast material ( iopamiro 300 , bracco , milano , italy and ultravist 300 , schering , berlin , germany ) through the antecubital vein at a rate of 3 ml / sec . the pathologic findings that were used as the standards of reference revealed that nine of the 35 hepatocellular carcinomas were totally necrotic . the observers were informed of the patients ' histories in as much as they knew that all the patients had undergone transarterial chemoembolization for hepatocellular carcinoma , but the observers were blinded to the pathologic findings concerning the presence of viable tumor in each patient . first , the observers reviewed the last four - phase ct taken before liver resection . second , the observers repeated the review two weeks after the first interpretation with using the last ct combined with the previous serial ct images . the observers were obliged to review the second - to - the - last ct . further review of the other previous ct examinations was determined by the respective decision of the observers for each case . images were interpreted for the presence , number , size and site of the viable tumors . each observer also recorded his or her degree of confidence as to whether a lesion seen on an image represented a viable tumor . for the objectivity and reproducibility of the image analysis performed in this study , the important criteria for a viable tumor and for the noncancerous lesions we regarded the following lesions as viable areas of tumors on the last ct images : ( a ) a hyperattenuating or isoattenuating lesion seen during the hepatic arterial phase and as a hypoattenuating lesion seen during the portal venous phase or the delayed phase , ( b ) an area of mixed attenuation seen on the hepatic arterial phase images that showed a hypoattenuating portion either on the portal venous phase or the delayed phase , and ( c ) a nodule that was seen as being isoattenuation on the hepatic arterial phase , the portal venous phase or the delayed phase images , but it was seen as hypoattenuation on the unenhanced images ( 4 ) . when the last ct along with the previous serial ct images was reviewed , a lesion that showed a newly presenting defect within an iodized oil - containing nodule was regarded as a viable portion of tumor . a small peritumoral area with suspicious enhancement on the hepatic arterial phase of the last ct , which showed an iodized oil - containing liver parenchyma on the previous serial ct images , was considered as an arterioportal shunt and not as a viable tumor . also , a long - standing hypoattenuating area ( longer than one year ) that did not change after contrast enhancement within a nodule of hepatocellular carcinoma was not considered viable tumor . a radiologist ( the study coordinator ) and they evaluated the location ( upper or lower , right or left , and anterior or posterior ) and the size of the viable portions within the tumors on both the ct and the pathologic examinations . a binominal receiver operating characteristic ( roc ) curve was calculated for each observer 's confidence rating data with using maximum - likelihood estimation . we evaluated the diagnostic accuracy of the last ct alone and the last ct combined with the previous serial ct images by calculating the area under each observer - specific binomial roc curve ( denoted as the az index ) ( 5 ) . the sensitivity and specificity were calculated for each observer and for each different review of the cts , and the statistical analysis of their differences was assessed using the mcnemar test ( spss , version 10.0 ; spss , chicago , il , usa ) . the false negative rates for the detection of viable tumors were also calculated for the review of the last ct alone and for the review of the last ct along with the previous serial ct images . statistics were used to assess the interobserver agreement for the presence of a viable portion of tumor with the review of the last ct alone and with the review of the last ct combined with the previous serial ct images . during the two analyses of the ct images , each observer detected all 35 hepatocellular carcinomas that were pathologically demonstrated in the resection specimens . the mean diagnostic accuracies ( az values ) for the depiction of viable tumor with the last ct alone and with the review of previous serial ct images for all the observers were 0.885 and 0.901 , respectively ( p > 0.05 ) ( table 1 ) . the sensitivity , specificity and diagnostic accuracy of the last ct alone were 72% , 91% and 78% , respectively , and the corresponding values for the review of the last ct combined with the previous serial ct images were 78% , 97% and 84% , respectively ( table 2 ) , and the differences were not statistically significant . although these differences in the diagnostic accuracies were not statistically significant , the review of the previous images enabled the observers to render a correct diagnosis for three ( 9% ) tumors . two of these three tumors showed as being slightly hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct , and these two tumors appeared as isoattenuating areas on the unenhanced images . 1 ) , and the two observers changed their confidence levels to probably not viable tumor . the one remaining tumor showed as an interval developed defect within the iodized oil - containing nodule that was not significantly enhanced on the last ct . on the basis of this feature , the two observers changed their confidence level to probably viable tumor . after reviewing of the last ct and the previous serial ct images , all three observers assessed the 10 compact iodized oil - containing tumors as being non - viable . all of the 16 false - negative lesions that were diagnosed by each observer after reviewing the last ct combined with the previous serial ct images showed 90% or greater necrosis on the pathologic examination ( table 3 ) . particularly , four tumors of these lesions were diagnosed as negative lesions simultaneously by all the observers with both the review of the last ct alone and the review of the last ct with the previous serial ct images . the interobserver agreement ( statistic ) for the presence of a viable tumor was 0.828 to 0.943 for the review of the last ct alone and 0.826 to 0.885 for the additional review of the previous serial ct images ( table 4 ) . the interobserver agreement for the review of the last ct with or without the previous serial ct images was considered excellent . thus , other criteria have been evaluated to determine the efficacy of transarterial chemoembolization treatment for hepatocellular carcinoma by using iodized oil retention in a tumor and the enhancement on ct ( 7 , 8) . ( 9 ) suggested that the overall cumulative recurrence rate of hepatocellular carcinoma was 23% after one year , 55% after two years and 67% after three years , and 45% of the recurrences occurred adjacent to a primary site that was considered controlled by the transarterial chemoembolization . with the advent of helical ct and with the other rapid technical advances , multiphasic helical ct has recently become a more useful imaging modality for the detection and characterization of liver lesions , for the follow - up after local treatment or surgical excision , and for the assessment of the hemodynamic changes in the liver . however , to the best of our knowledge , there has been no previous study that has compared the preoperative ct and the review of the previous serial ct images with the resected specimens for the evaluation of the viable portion of hepatocellular carcinoma treated with transarterial chemoembolization . in the past , some investigators have reported on the diagnostic efficacy of the hepatic arterial phase ct and the portal venous phase ct for the detection of hypervascular tumors , and especially hepatocellular carcinoma ( 10 , 11 ) . ( 1 ) have suggested that the delayed phase is important for the detection of small hepatocellular carcinomas that are less than 2 cm , for confirming or increasing the confidence level for the detection of equivocal nodules on the arterial or portal venous phase images ( because those nodules are usually more conspicuous on the delayed phase than on the portal venous phase imaging ) , and for the differentiation of arterioportal shunting from the true hepatocellular carcinoma . in our study , this concept was used to depict a viable tumor among the arterioportal shunt areas . for the hepatocellular carcinoma treated with transarterial chemoembolization , takayasu et al . ( 4 ) have recently pointed out that unenhanced images could be of additional diagnostic value to supplement the enhanced biphasic helical ct images for the patients treated with transarterial chemoembolization for hepatocellular carcinoma by facilitating the differentiation of the true lesions from the hyperattenuating pseudolesions that simulate viable tumor . further , they stated that these unenhanced images can also aid in the detection of any isoattenuation lesion on the hepatic arterial phase and the portal venous phase because of the hypoattenuation on the unenhanced images . in addition to the criteria in the previous studies ( 1 , 4 , 7 , 10 , 11 ) , we considered the long - standing hypoattenuating areas within an iodized nodule on the enhanced images of the serial ct images as necrotic lesion despites a lack of iodized oil . on the other hand , a lesion that showed an interval developed defect within an iodized nodule was considered as a viable portion of tumor . in our study , although no significant statistical difference was found between the review of the last ct alone and the review of the last ct along with the previous serial ct images , the additional review of the previous serial ct images allowed the observers to render a correct diagnosis for three ( 9% ) lesions . we believe that many abdominal radiologists actually do review some of the previous ct images when they find an equivocal viable lesion on the liver ct following transarterial chemoembolization . according to our results , after the review of the last ct combined with the previous serial ct images , all the false - negative lesions showed 90% or greater necrosis on the pathologic examination . thus , if a substantial ( larger than 10% ) viable tumor is present in an iodized nodule , we can depict it on ct with a thorough review of the previous serial ct images . first , as this is a retrospective study , all the patients of the study group did not undergo regular follow - up ct and transarterial chemoembolization because of suspected complications or because of personal preference . second , we included a relatively small number of hepatocellular carcinomas , and so this might show no significant statistical difference between the review of the last ct alone and the review of the last ct along with the previous serial ct images . finally , although we thoroughly reviewed the pathologic specimens alongside the ct images , any perfect area - by - area histopathologic correlation was actually impossible . by referring to the location and size of viable tumors within the tumors , however , we tried to match the ct and histopathologic findings as closely as possible . in our results , two of the three tumors that had a correct diagnosis rendered after the review of previous images appeared as parenchymal uptake around the iodized oil - containing tumors on the second to the last ct the pathology demonstrated totally necrotic tumors , and this revealed that the suspicious hyperattenuating focal areas adjacent to the iodized oil - containing tumors on the hepatic arterial phase images of the last ct were pseudolesion . for the third hepatocellular carcinoma with 70% necrosis , we could not perfectly match the interval developed defect within the iodized oil - containing nodule and the 30% viable tumor portion on the pathologic specimen . in conclusion , for depicting the viable tumor in hepatocellular carcinoma patients treated with transarterial chemoembolization , although the difference in the diagnostic accuracies was not found to be statistically significant in our study , a review of the multiphasic helical ct combined with the previous serial ct images can help render a correct diagnosis for those lesions incorrectly diagnosed with the review of only the last ct .

antimicrobial blue light was delivered using a light - emitting diode ( led ) ( vielight , inc . , toronto , canada ) with peak emission at 415 nm and full - width half maximum of 20 nm . the irradiance on the corneal surface was 100 mw / cm throughout the study as measured using a pm100d power / energy meter ( thorlabs , inc . , pseudomonas aeruginosa american type culture collection ( atcc ) 19660 ( strain 180 ) , an exou expression strain , was used as the representative causative pathogen . the strain was made bioluminescent by transfecting lux operon into the bacterial strain as described previously . this allowed noninvasive and real - time monitoring of bacterial bioluminescence , which is related to the corresponding colony - forming units ( cfu ) of bacteria , by using bioluminescence imaging . bacteria were routinely grown in brain heart infusion ( bhi ) medium ( fisher scientific , agawam , ma , usa ) supplemented with 50 g / ml kanamycin in an orbital incubator ( 37c ; 0.14 g ; new brunswick scientific , edison , nj , usa ) . enucleated whole mature new zealand white rabbit eyes with clear corneas were purchased from pel - freez biologicals ( rogers , ar , usa ) and thawed on the day of the experiment . the central area of corneal epithelium ( 11-mm diameter ) was surgically removed using a sterile surgical blade . subsequently , a 20-l aliquot of bacterial suspension containing 2 10 cfu p. aeruginosa in phosphate - buffered saline ( pbs ) was topically inoculated onto the de - epithelized corneas . eighteen infected ex vivo rabbit eyes were randomly divided into three groups of six eyes each . two groups were subjected to abl exposure and the other group served as the untreated control . antimicrobial blue light was delivered at 6 or 24 hours after bacterial inoculation ( six rabbit eyes for each time point ) to investigate its effectiveness against early - stage or fully developed infections . female c57bl/6 mice , aged 8 weeks and weighing 18 to 20 g , were purchased from charles river laboratories ( wilmington , ma , usa ) . all animal procedures were approved by the institutional animal care and use committee ( iacuc ) at the massachusetts general hospital ( protocol no . 2014n000016 ) and were in accordance with the arvo statement for the use of animals in ophthalmic and vision research . under anesthesia , corneas of mice were scarified using a 30.5-guage needle to produce three parallel incisions that did not penetrate beyond the superficial stroma . a 5-l aliquot of bacterial suspension containing 5 10 cfu p. aeruginosa in pbs was then topically inoculated onto the wounded area of the corneas . two groups were subjected to abl exposure , and the other group served as the untreated control . similar to the procedure for the ex vivo study using rabbit models , abl was delivered at 6 or 24 hours after bacterial inoculation ( six mice for each time point ) to investigate its effectiveness against early - stage and fully developed infections , respectively . the mouse corneas were also photographed daily using a digital camera ( nikon d90 with a af micro nikkor 60 mm 1:2.8d lens ; tokyo , japan ) . to statistically analyze the pathologic changes in the corneas , corneal pathology scores of mice the bacterial luminescence in the infected corneas was detected using a hamamatsu bioluminescence imaging system ( hamamatsu photonics kk , bridgewater , nj , usa ) . this system included an intensified charge - coupled device ( iccd ) camera ( c2400 - 30h , hamamatsu ) developed for imaging under extremely low light levels , a camera controller , a specimen chamber , and an image processor ( c5510 - 50 , hamamatsu ) . rabbit or mouse corneas were placed on an adjustable stage in the specimen chamber , and were positioned directly under the iccd camera . when set for photon counting , an integration time of 2 minutes was used for image acquisition . for each measurement , the bioluminescence measurement of infections was quantified using argus 5.0 software ( hamamatsu ) . to quantitatively correlate bacterial cfu to the corresponding bacterial luminescence ( relative light units : rlu ) , ex vivo rabbit corneas infected with p. aeruginosa were used . in brief , rabbit corneas were inoculated with a 20-l aliquot of bacterial suspension containing 2 10 cfu p. aeruginosa in pbs . after incubation overnight at 37c , infected rabbit corneas were exposed , respectively , to varying abl exposures ( 0 , 36 , 72 , 108 , and 144 j / cm ) and then subjected to bioluminescence imaging . after imaging , each rabbit cornea was homogenized in 1 ml sterile pbs at 4c . data are presented as the mean sd , and differences between groups were tested for significance by using an unpaired , 2-tailed student 's t - test . differences in corneal pathology scores between different groups of mice were analyzed using the general linear model . antimicrobial blue light was delivered using a light - emitting diode ( led ) ( vielight , inc . , toronto , canada ) with peak emission at 415 nm and full - width half maximum of 20 nm . the irradiance on the corneal surface was 100 mw / cm throughout the study as measured using a pm100d power / energy meter ( thorlabs , inc . , pseudomonas aeruginosa american type culture collection ( atcc ) 19660 ( strain 180 ) , an exou expression strain , was used as the representative causative pathogen . the strain was made bioluminescent by transfecting lux operon into the bacterial strain as described previously . this allowed noninvasive and real - time monitoring of bacterial bioluminescence , which is related to the corresponding colony - forming units ( cfu ) of bacteria , by using bioluminescence imaging . bacteria were routinely grown in brain heart infusion ( bhi ) medium ( fisher scientific , agawam , ma , usa ) supplemented with 50 g / ml kanamycin in an orbital incubator ( 37c ; 0.14 g ; new brunswick scientific , edison , nj , usa ) . enucleated whole mature new zealand white rabbit eyes with clear corneas were purchased from pel - freez biologicals ( rogers , ar , usa ) and thawed on the day of the experiment . the central area of corneal epithelium ( 11-mm diameter ) was surgically removed using a sterile surgical blade . subsequently , a 20-l aliquot of bacterial suspension containing 2 10 cfu p. aeruginosa in phosphate - buffered saline ( pbs ) was topically inoculated onto the de - epithelized corneas . eighteen infected ex vivo rabbit eyes were randomly divided into three groups of six eyes each . two groups were subjected to abl exposure and the other group served as the untreated control . antimicrobial blue light was delivered at 6 or 24 hours after bacterial inoculation ( six rabbit eyes for each time point ) to investigate its effectiveness against early - stage or fully developed infections . female c57bl/6 mice , aged 8 weeks and weighing 18 to 20 g , were purchased from charles river laboratories ( wilmington , ma , usa ) . all animal procedures were approved by the institutional animal care and use committee ( iacuc ) at the massachusetts general hospital ( protocol no . 2014n000016 ) and were in accordance with the arvo statement for the use of animals in ophthalmic and vision research . under anesthesia , corneas of mice were scarified using a 30.5-guage needle to produce three parallel incisions that did not penetrate beyond the superficial stroma . a 5-l aliquot of bacterial suspension containing 5 10 cfu p. aeruginosa in pbs was then topically inoculated onto the wounded area of the corneas . two groups were subjected to abl exposure , and the other group served as the untreated control . similar to the procedure for the ex vivo study using rabbit models , abl was delivered at 6 or 24 hours after bacterial inoculation ( six mice for each time point ) to investigate its effectiveness against early - stage and fully developed infections , respectively . the mouse corneas were also photographed daily using a digital camera ( nikon d90 with a af micro nikkor 60 mm 1:2.8d lens ; tokyo , japan ) . to statistically analyze the pathologic changes in the corneas , the bacterial luminescence in the infected corneas was detected using a hamamatsu bioluminescence imaging system ( hamamatsu photonics kk , bridgewater , nj , usa ) . this system included an intensified charge - coupled device ( iccd ) camera ( c2400 - 30h , hamamatsu ) developed for imaging under extremely low light levels , a camera controller , a specimen chamber , and an image processor ( c5510 - 50 , hamamatsu ) . rabbit or mouse corneas were placed on an adjustable stage in the specimen chamber , and were positioned directly under the iccd camera . when set for photon counting , the camera controller 's automatic amplification circuit was employed for maximum sensitivity . an integration time of 2 minutes was used for image acquisition . for each measurement , to quantitatively correlate bacterial cfu to the corresponding bacterial luminescence ( relative light units : rlu ) , ex vivo rabbit corneas infected with p. aeruginosa were used . in brief , rabbit corneas were inoculated with a 20-l aliquot of bacterial suspension containing 2 10 cfu p. aeruginosa in pbs . after incubation overnight at 37c , infected rabbit corneas were exposed , respectively , to varying abl exposures ( 0 , 36 , 72 , 108 , and 144 j / cm ) and then subjected to bioluminescence imaging . after imaging , each rabbit cornea was homogenized in 1 ml sterile pbs at 4c . data are presented as the mean sd , and differences between groups were tested for significance by using an unpaired , 2-tailed student 's t - test . differences in corneal pathology scores between different groups of mice were analyzed using the general linear model . to test whether measuring the rlu of bacterial bioluminescence in the infected eyes after abl exposure accurately reported the corresponding bacterial cfu , bacterial luminescence measurements were made after varying radiant exposures of abl , and bacteria were cultured from the same eyes . figure 1 shows a fairly good linear correlation ( y = 11.353x ) between the bacterial luminescence measurement ( rlu ) of p. aeruginosa and the corresponding cfu ( determination coefficient r = 0.921 , correlation coefficient r = 0.9621 , p = 0.0001 ) . correlation of the bacterial luminescence intensity of infected ex vivo rabbit corneas to the corresponding colony - forming units ( cfu ) . bacterial luminescence intensity is presented in relative light units ( rlu ) . to characterize the development of p. aeruginosa infection in the cornea , the bacterial luminescence was measured for up to 24 hours in the ex vivo rabbit model and up to 72 hours in the in vivo mouse model . in the ex vivo rabbit corneas , the mean bacterial luminescence increased steadily until approximately 9 hours , then remained relatively stable ( fig . 2a , solid line ; n = 6 ) . this result was qualitatively apparent from the bacterial luminescence images of a single cornea followed over the same time period . ( a ) time course for the bacterial luminescence intensity of ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) gram - stained ( 20 ) and h&e - stained histologic sections ( 10 ) of representative ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa and sampled at 3 , 6 , and 24 hours after bacterial inoculation . the gram stain demonstrated the presence of p. aeruginosa in the corneal tissue , with bacteria invaded deeper into the corneal tissue over this time period . the areas in the depicted boxes in 10 h&e images are shown in 20 gram images . the areas in the depicted boxes in 20 gram images are illustrated in the insets of 100 oil lens images . ( c ) time course for the bacterial luminescence intensity of in vivo mouse eyes infected with 5 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the inset shows the bacterial luminescence images and photographs taken at 0 , 24 , 36 , and 72 hours post inoculation from a representative infected in vivo mouse eye . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . to characterize the distribution of p. aeruginosa in the rabbit cornea , histology with gram and hematoxylin and eosin ( h&e staining ) was performed at 3 , 6 , and 24 hours post inoculation . at 3 hours post inoculation , p. aeruginosa was primarily located on the surface of corneal tissue . however , the bacteria rapidly proliferated into the stroma and destroyed the structure of cornea at 24 hours post inoculation , due to the lack of an immune response in this ex vivo model ( fig . similarly , after inoculating bacteria onto the scratched in vivo corneas of mice , a 24-hour period of more rapid increase in bacterial luminescence was followed by a more stable level of infection that lasted at least 72 hours . these changes were characterized by both quantitative bioluminescence imaging ( fig . 2c , solid line ; n = 6 ) and photography ( fig . 2c , inset ) . the photographs of a representative infected mouse eye showed an increased opacity of the mouse cornea with the time post inoculation . infected rabbit corneas were treated at either 6 or 24 hours post inoculation to investigate the effectiveness of abl therapy for early - stage or fully established infection . when abl was delivered at 6 hours post inoculation , the bacterial luminescence decreased with increasing abl exposure and an approximately 3-log10 inactivation was achieved , compared to untreated control , by exposure to 84 j / cm abl ( arrow in fig . in contrast , when abl was delivered at 24 hours post inoculation , exposure to 84 j / cm abl elicited less than 1-log10 inactivation ( fig . 3b ) . exposure to 304 j / cm abl ( 50 minutes irradiation ) was required to achieve approximately 3-log10 inactivation . influence of radiant exposure on abl therapy for keratitis in the ex vivo rabbit model . ( a ) abl ( 180 j / cm ) delivered at 6 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . bars : standard deviation . bacterial luminescence images of the infected rabbit eyes were taken when 0 , 12 , 36 , 84 , and 180 j / cm abl had been delivered ( irradiance = 100 mw / cm ) and at the same times in untreated controls . the bacterial luminescence images of representative infected rabbit eyes , treated with abl or not treated , are shown . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) abl ( 372 j / cm ) delivered at 24 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the bacterial luminescence images were taken when 0 , 12 , 36 , 84 , 180 , and 372 j / cm abl had been delivered to the infected rabbit eyes ( irradiance = 100 mw / cm ) and at the same times in untreated controls . the bacterial luminescence images of representative infected rabbit eyes treated with abl ( 372 j / cm ) or not treated are shown . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . when abl was delivered at 6 hours post inoculation , an increase in abl exposure decreased the bacteria level in in vivo mouse corneas , and an approximately 2.0-log10 inactivation of p. aeruginosa was achieved when an exposure of 36 j / cm abl was delivered ( fig . in contrast , in the untreated mice , bacterial luminescence remained almost unchanged during the equivalent period of time ( data not shown ) . corneal pathology , as shown in figure 4b , was scored at 6 , 24 , and 48 hours after bacterial inoculation , respectively , for both abl - treated and untreated mice . it was observed that the eyes of abl - treated mice had significantly lower mean corneal pathology scores as compared to the eyes of untreated mice ( p = 0.0048 , fig . 4c ) . in addition , the analysis of the interaction between abl treatment and time post infection shows that the effect of abl treatment increased with the time post infection . influence of radiant exposure on abl therapy for keratitis in the in vivo mouse model . ( a ) influence of radiant exposure of abl on the bacterial luminescence intensity of mouse eyes infected with 5 10 p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation ( n = 6 ) . the inset shows the bacterial luminescence images from a representative mouse eye infected with 5 10 cfu of p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation . bacterial luminescence images were taken when 0 , 9 , 18 , 27 , and 36 j / cm abl had been delivered ( irradiance = 100 mw / cm ) . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) photographs of two representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with abl ( 36 j / cm ) at 6 hours post infection ( top row ) or not treated ( bottom row ) , indicating less opacity and damage in the abl - treated mouse eye than in the untreated one . ( c ) corneal pathology scores of the infected mouse eyes treated with 36 j / cm abl at 6 hours post infection ( n = 6 ) or not treated ( n = 6 ) , showing the higher mean corneal pathology scores of the untreated mouse eyes than the abl - treated mouse eyes ( p = 0.0048 ) . in addition , the analysis of the interaction between abl treatment and time post infection shows that the effect of abl treatment increased with the time post infection . ( d ) the bacterial luminescence images of representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with 144 j / cm abl 24 hours later or left untreated . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( e ) influence of radiant exposure of abl on the bacterial luminescence intensity of the mouse eyes infected with 5 10 p. aeruginosa , treated with 144 j / cm abl at 24 hours post inoculation ( n = 6 ) , or left untreated ( n = 6 ) . bacterial luminescence images were taken when 0 , 18 , 36 , 54 , 72 , 90 , 108 , 126 , and 144 j / cm abl had been delivered ( irradiance = 100 mw / cm ) . when abl was delivered at 24 hours after p. aeruginosa inoculation , the bacterial luminescence was completely eradicated after an exposure of 144 j / cm abl ( fig . the abl killing curve of p. aeruginosa shows that a > 2.5-log10 inactivation was achieved when an exposure of 144 j / cm abl was delivered ( fig . however , recurrence of infection was observed in the mice treated with abl at both 6 and 24 hours post inoculation after abl exposure . the recurrent infection was more severe in mice treated at 24 hours post inoculation than at 6 hours post inoculation . retinal safety is a very important concern for developing abl as a treatment of keratitis since abl , in sufficient amount , is highly toxic to the retina . antimicrobial blue light that is not absorbed by the cornea or lens and is not blocked by the iris is transmitted through the pupil and reaches the retina . the wavelengths in abl are absorbed by the chromophores in the retina and retinal pigment epithelium ( e.g. , proteins in photoreceptor cells , flavin proteins , heme proteins , melanosomes ) , and subsequently may produce both photothermal and photochemical damage . using the formula described by delori et al . , we calculated the abl exposures to reach the retina with the abl treatment parameters that reduced the bacterial luminescence in the ex vivo rabbit model by approximately 3-log10 ( 84 j / cm for 6 hours post inoculation and 304 j / cm for 24 hours post inoculation ; see fig . the initial calculation assumed that the cornea was clear ( 100% transmission ) , and subsequent calculations assumed partial cloudiness that decreased the abl transmission by 50% and 90% . the obtained abl exposures to reach the retina were compared to the safety thresholds for photothermal and photochemical damage previously established by experimental studies . the retinal abl exposure was calculated for corneal irradiance = 100 mw / cm and pupil diameter = 2 mm , and a 10-mm retinal image diameter was assumed . for a clear cornea ( 100% transmission ) , the retinal abl exposure exceeds the retinal safety threshold ( i.e. , is unsafe ) by factors of 1.4 and 5.1 for 84 and 304 j / cm abl , respectively . for a cloudy cornea that blocks 50% of the abl , 84 j / cm abl delivered to the cornea is below the retina safety threshold by a factor of 1.4 ( i.e. , 1/1.4 = 0.71-fold of the retina safety threshold ) , but 304 j / cm is above the threshold by a factor of 2.5. if a very cloudy cornea only transmits 10% of the abl , 84 and 304 j / cm abl are below the limit by factors of 7.1 and 2.0 , respectively . to test whether measuring the rlu of bacterial bioluminescence in the infected eyes after abl exposure accurately reported the corresponding bacterial cfu , bacterial luminescence measurements were made after varying radiant exposures of abl , and bacteria were cultured from the same eyes . figure 1 shows a fairly good linear correlation ( y = 11.353x ) between the bacterial luminescence measurement ( rlu ) of p. aeruginosa and the corresponding cfu ( determination coefficient r = 0.921 , correlation coefficient r = 0.9621 , p = 0.0001 ) . correlation of the bacterial luminescence intensity of infected ex vivo rabbit corneas to the corresponding colony - forming units ( cfu ) . to characterize the development of p. aeruginosa infection in the cornea , the bacterial luminescence was measured for up to 24 hours in the ex vivo rabbit model and up to 72 hours in the in vivo mouse model . in the ex vivo rabbit corneas , the mean bacterial luminescence increased steadily until approximately 9 hours , then remained relatively stable ( fig . 2a , solid line ; n = 6 ) . this result was qualitatively apparent from the bacterial luminescence images of a single cornea followed over the same time period . ( a ) time course for the bacterial luminescence intensity of ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) gram - stained ( 20 ) and h&e - stained histologic sections ( 10 ) of representative ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa and sampled at 3 , 6 , and 24 hours after bacterial inoculation . the gram stain demonstrated the presence of p. aeruginosa in the corneal tissue , with bacteria invaded deeper into the corneal tissue over this time period . the areas in the depicted boxes in 10 h&e images are shown in 20 gram images . the areas in the depicted boxes in 20 gram images are illustrated in the insets of 100 oil lens images . ( c ) time course for the bacterial luminescence intensity of in vivo mouse eyes infected with 5 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the inset shows the bacterial luminescence images and photographs taken at 0 , 24 , 36 , and 72 hours post inoculation from a representative infected in vivo mouse eye . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . to characterize the distribution of p. aeruginosa in the rabbit cornea , histology with gram and hematoxylin and eosin ( h&e staining ) was performed at 3 , 6 , and 24 hours post inoculation . at 3 hours post inoculation , p. aeruginosa was primarily located on the surface of corneal tissue . however , the bacteria rapidly proliferated into the stroma and destroyed the structure of cornea at 24 hours post inoculation , due to the lack of an immune response in this ex vivo model ( fig . similarly , after inoculating bacteria onto the scratched in vivo corneas of mice , a 24-hour period of more rapid increase in bacterial luminescence was followed by a more stable level of infection that lasted at least 72 hours . 2c , solid line ; n = 6 ) and photography ( fig . 2c , the photographs of a representative infected mouse eye showed an increased opacity of the mouse cornea with the time post inoculation . infected rabbit corneas were treated at either 6 or 24 hours post inoculation to investigate the effectiveness of abl therapy for early - stage or fully established infection . when abl was delivered at 6 hours post inoculation , the bacterial luminescence decreased with increasing abl exposure and an approximately 3-log10 inactivation was achieved , compared to untreated control , by exposure to 84 j / cm abl ( arrow in fig . in contrast , when abl was delivered at 24 hours post inoculation , exposure to 84 j / cm abl elicited less than 1-log10 inactivation ( fig . 3b ) . exposure to 304 j / cm abl ( 50 minutes irradiation ) was required to achieve approximately 3-log10 inactivation . influence of radiant exposure on abl therapy for keratitis in the ex vivo rabbit model . ( a ) abl ( 180 j / cm ) delivered at 6 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . bars : standard deviation . bacterial luminescence images of the infected rabbit eyes were taken when 0 , 12 , 36 , 84 , and 180 j / cm abl had been delivered ( irradiance = 100 mw / cm ) and at the same times in untreated controls . the bacterial luminescence images of representative infected rabbit eyes , treated with abl or not treated , are shown . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) abl ( 372 j / cm ) delivered at 24 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the bacterial luminescence images were taken when 0 , 12 , 36 , 84 , 180 , and 372 j / cm abl had been delivered to the infected rabbit eyes ( irradiance = 100 mw / cm ) and at the same times in untreated controls . the bacterial luminescence images of representative infected rabbit eyes treated with abl ( 372 j / cm ) or not treated are shown . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . when abl was delivered at 6 hours post inoculation , an increase in abl exposure decreased the bacteria level in in vivo mouse corneas , and an approximately 2.0-log10 inactivation of p. aeruginosa was achieved when an exposure of 36 j / cm abl was delivered ( fig . in contrast , in the untreated mice , bacterial luminescence remained almost unchanged during the equivalent period of time ( data not shown ) . corneal pathology , as shown in figure 4b , was scored at 6 , 24 , and 48 hours after bacterial inoculation , respectively , for both abl - treated and untreated mice . it was observed that the eyes of abl - treated mice had significantly lower mean corneal pathology scores as compared to the eyes of untreated mice ( p = 0.0048 , fig . 4c ) . in addition , the analysis of the interaction between abl treatment and time post infection shows that the effect of abl treatment increased with the time post infection . influence of radiant exposure on abl therapy for keratitis in the in vivo mouse model . ( a ) influence of radiant exposure of abl on the bacterial luminescence intensity of mouse eyes infected with 5 10 p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation ( n = 6 ) . the inset shows the bacterial luminescence images from a representative mouse eye infected with 5 10 cfu of p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation . bacterial luminescence images were taken when 0 , 9 , 18 , 27 , and 36 j / cm abl had been delivered ( irradiance = 100 mw / cm ) . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( b ) photographs of two representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with abl ( 36 j / cm ) at 6 hours post infection ( top row ) or not treated ( bottom row ) , indicating less opacity and damage in the abl - treated mouse eye than in the untreated one . ( c ) corneal pathology scores of the infected mouse eyes treated with 36 j / cm abl at 6 hours post infection ( n = 6 ) or not treated ( n = 6 ) , showing the higher mean corneal pathology scores of the untreated mouse eyes than the abl - treated mouse eyes ( p = 0.0048 ) . in addition , the analysis of the interaction between abl treatment and time post infection shows that the effect of abl treatment increased with the time post infection . ( d ) the bacterial luminescence images of representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with 144 j / cm abl 24 hours later or left untreated . the pseudo - color scale bar indicates the bioluminescence intensity value of a single pixel in the bioluminescence images . ( e ) influence of radiant exposure of abl on the bacterial luminescence intensity of the mouse eyes infected with 5 10 p. aeruginosa , treated with 144 j / cm abl at 24 hours post inoculation ( n = 6 ) , or left untreated ( n = 6 ) . bars : standard deviation . bacterial luminescence images were taken when 0 , 18 , 36 , 54 , 72 , 90 , 108 , 126 , and 144 j / cm abl had been delivered ( irradiance = 100 mw / cm ) . when abl was delivered at 24 hours after p. aeruginosa inoculation , the bacterial luminescence was completely eradicated after an exposure of 144 j / cm abl ( fig . the abl killing curve of p. aeruginosa shows that a > 2.5-log10 inactivation was achieved when an exposure of 144 j / cm abl was delivered ( fig . however , recurrence of infection was observed in the mice treated with abl at both 6 and 24 hours post inoculation after abl exposure . the recurrent infection was more severe in mice treated at 24 hours post inoculation than at 6 hours post inoculation . retinal safety is a very important concern for developing abl as a treatment of keratitis since abl , in sufficient amount , is highly toxic to the retina . antimicrobial blue light that is not absorbed by the cornea or lens and is not blocked by the iris is transmitted through the pupil and reaches the retina . the wavelengths in abl are absorbed by the chromophores in the retina and retinal pigment epithelium ( e.g. , proteins in photoreceptor cells , flavin proteins , heme proteins , melanosomes ) , and subsequently may produce both photothermal and photochemical damage . using the formula described by delori et al . , we calculated the abl exposures to reach the retina with the abl treatment parameters that reduced the bacterial luminescence in the ex vivo rabbit model by approximately 3-log10 ( 84 j / cm for 6 hours post inoculation and 304 j / cm for 24 hours post inoculation ; see fig . the initial calculation assumed that the cornea was clear ( 100% transmission ) , and subsequent calculations assumed partial cloudiness that decreased the abl transmission by 50% and 90% . the obtained abl exposures to reach the retina were compared to the safety thresholds for photothermal and photochemical damage previously established by experimental studies . the retinal abl exposure was calculated for corneal irradiance = 100 mw / cm and pupil diameter = 2 mm , and a 10-mm retinal image diameter was assumed . for a clear cornea ( 100% transmission ) , the retinal abl exposure exceeds the retinal safety threshold ( i.e. , is unsafe ) by factors of 1.4 and 5.1 for 84 and 304 j / cm abl , respectively . for a cloudy cornea that blocks 50% of the abl , 84 j / cm abl delivered to the cornea is below the retina safety threshold by a factor of 1.4 ( i.e. , 1/1.4 = 0.71-fold of the retina safety threshold ) , but 304 j / cm is above the threshold by a factor of 2.5. if a very cloudy cornea only transmits 10% of the abl , 84 and 304 j / cm abl are below the limit by factors of 7.1 and 2.0 , respectively . in this study , we developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis by using a bioluminescent strain of p. aeruginosa and found a good linear correlation between the bacterial luminescence and the corresponding bacterial cfu in the infected corneas . our results demonstrated that a single exposure of abl rapidly and significantly reduced bacterial burden in both the ex vivo and in vivo models . the infections treated at 24 hours post inoculation were more resistant to abl than they were at 6 hours post inoculation . the possible reason is that , at 24 hours post inoculation , infections had fully established and bacteria existed predominantly as biofilms . the biofilm matrix could block abl and render bacterial cells less susceptible to abl . additionally , when infections had fully established , bacteria might have proliferated into the deeper layers of the corneal tissue ( as shown in fig . 2b ) , where the abl transmission was further attenuated . in a previous study of abl therapy for p. aeruginosa burn infection in mice , we observed that , when abl was applied 30 minutes post inoculation , an average 3.5-log10 inactivation of p. aeruginosa in mouse burns was achieved when an exposure of 55.8 j / cm abl had been delivered . recurrence of infection was observed in mice ( but not in the rabbit corneas ) after a single exposure of abl . one possible reason of infection recurrence is that bacteria in the infected corneas were not completely eradicated after abl exposure even when bacterial luminescence was eliminated due to the sensitivity limitation of the bioluminescence imaging system ( > 10 cfu ) . another possible reason is that part of the corneal area was shielded by eyelids and this prevented abl from irradiating this area , rendering the shielded area actually untreated . this hypothesis was supported by the results obtained from the study using the ex vivo rabbit corneas . in the ex vivo study , the whole area of excised rabbit corneas was exposed to abl and no bacterial regrowth was found in any abl - treated cornea after abl exposure . several approaches could be considered to improve the effectiveness of abl therapy , such as repeated abl treatments ( two or three times ) and combined therapy using abl and subtherapeutic dose of antibiotics ( antimicrobial eye drops ) , which can reach the corneal area under the eyelids after topical application . corneal safety is a very important concern for developing abl as a therapeutic for keratitis . in our previous in vitro studies , we demonstrated that bacteria ( p. aeruginosa atcc 19660 ) were much more resistant to 415-nm abl than were human keratinocytes . when 109.9 j / cm abl had been delivered , an approximately 7.64-log10 cfu inactivation of p. aeruginosa was achieved . in contrast , under the same abl irradiation condition , only a 0.16-log10 loss of viability of human keratinocytes was observed . other investigators reported that when 100 j / cm blue light at 410 nm had been delivered , the viability of corneal epithelial cells in vitro decreased by approximately 1-log10 . further study on the potential toxicity of abl on the cornea ( i.e. , corneal epithelial , endothelial , and keratocyte cells ) is needed . there are also limitations of the in vivo mouse model used in the present study since mouse eyes differ substantially from human eyes . the human cornea is approximately 11 mm in diameter , whereas mouse corneas have a 2.2- to 3.5-mm diameter . the axial length of human eyes is approximately eight times longer than that of mouse eyes , and the corneal thickness is greater for humans than for mice . the arrangement of corneal collagen and the properties of corneal epithelial cells also differ between mice and humans , which could produce alternate reactions of the cornea to invading pathogens . descemet 's and bowman 's membranes are substantially thinner in mice than in humans , and mouse corneas have a higher ratio of corneal epithelial cells to stroma than humans and more cell layers in the corneal epithelium . all of these anatomic differences have an effect on abl delivery , bacterial adherence and possible invasion , susceptibility to bacterial enzymes and other virulence factors , and availability of host defense molecules in the tear film . as a result , the effectiveness of abl against keratitis needs to be further investigated using large in vivo animal models , for example , a rabbit or dog model , which are more similar to humans in ocular anatomy . in addition , abl potentially can produce both photothermal and photochemical damage in the retina . according to the ansi 136.1 , we estimated the abl toxicity to the retina under different abl transmission of the corneal conditions . the calculations indicated that the abl toxicity to the retina is largely dependent on the abl transmission of the cornea . the corneal opacity of infected corneas means that more light would be blocked by infected corneas than by intact corneas . apparently , to validate the results obtained from the theoretical calculation , further experimental study using animal models and clinical study are essential . overall , our study suggests that abl is a potential alternative or adjunctive approach for the treatment of infectious keratitis , in particular those caused by drug - resistant pathogens . successful treatment of keratitis using abl requires optimal timing , careful regulation of abl exposure , and adequate concomitant antibacterial medication .

purposeto investigate the effectiveness of antimicrobial blue light ( abl ) as an alternative or adjunctive therapeutic for infectious keratitis.methodswe developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis . a bioluminescent strain of pseudomonas aeruginosa was used as the causative pathogen , allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging . quantitation of bacterial luminescence was correlated to colony - forming units ( cfu ) . using the ex vivo and in vivo models , the effectiveness of abl ( 415 nm ) for the treatment of keratitis was evaluated as a function of radiant exposure when abl was delivered at 6 or 24 hours after bacterial inoculation . the abl exposures calculated to reach the retina were compared to the american national standards institute standards to estimate abl retinal safety.resultspseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation . bacterial luminescence in the infected corneas correlated linearly to cfu ( r2 = 0.921 ) . bacterial burden in the infected corneas was rapidly and significantly reduced ( > 2-log10 ) both ex vivo and in vivo after a single exposure of abl . recurrence of infection was observed in the abl - treated mice at 24 hours after abl exposure . the abl toxicity to the retina is largely dependent on the abl transmission of the cornea.conclusionsantimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis . further studies of corneal and retinal safety using large animal models , in which the ocular anatomies are similar to that of humans , are warranted .

Methods Blue Light Source Bacterial Strain aBL Therapy for Keratitis in the Ex Vivo Rabbit Model aBL Therapy for Keratitis in the In Vivo Mouse Model Bioluminescence Imaging of Infections Quantitative Correlation of Bacterial Luminescence to Bacterial CFU in the Infected Corneas Statistics Results Bacterial Luminescence Was Linearly Correlated to CFU None aBL Effectively and Rapidly Reduced aBL Effectively and Rapidly Reduced Toxicity of aBL to the Retina is Largely Dependent on aBL Transmission of the Cornea Discussion

, pseudomonas aeruginosa american type culture collection ( atcc ) 19660 ( strain 180 ) , an exou expression strain , was used as the representative causative pathogen . this allowed noninvasive and real - time monitoring of bacterial bioluminescence , which is related to the corresponding colony - forming units ( cfu ) of bacteria , by using bioluminescence imaging . antimicrobial blue light was delivered at 6 or 24 hours after bacterial inoculation ( six rabbit eyes for each time point ) to investigate its effectiveness against early - stage or fully developed infections . similar to the procedure for the ex vivo study using rabbit models , abl was delivered at 6 or 24 hours after bacterial inoculation ( six mice for each time point ) to investigate its effectiveness against early - stage and fully developed infections , respectively . to statistically analyze the pathologic changes in the corneas , corneal pathology scores of mice the bacterial luminescence in the infected corneas was detected using a hamamatsu bioluminescence imaging system ( hamamatsu photonics kk , bridgewater , nj , usa ) . to quantitatively correlate bacterial cfu to the corresponding bacterial luminescence ( relative light units : rlu ) , ex vivo rabbit corneas infected with p. aeruginosa were used . , pseudomonas aeruginosa american type culture collection ( atcc ) 19660 ( strain 180 ) , an exou expression strain , was used as the representative causative pathogen . this allowed noninvasive and real - time monitoring of bacterial bioluminescence , which is related to the corresponding colony - forming units ( cfu ) of bacteria , by using bioluminescence imaging . antimicrobial blue light was delivered at 6 or 24 hours after bacterial inoculation ( six rabbit eyes for each time point ) to investigate its effectiveness against early - stage or fully developed infections . similar to the procedure for the ex vivo study using rabbit models , abl was delivered at 6 or 24 hours after bacterial inoculation ( six mice for each time point ) to investigate its effectiveness against early - stage and fully developed infections , respectively . to statistically analyze the pathologic changes in the corneas , the bacterial luminescence in the infected corneas was detected using a hamamatsu bioluminescence imaging system ( hamamatsu photonics kk , bridgewater , nj , usa ) . to test whether measuring the rlu of bacterial bioluminescence in the infected eyes after abl exposure accurately reported the corresponding bacterial cfu , bacterial luminescence measurements were made after varying radiant exposures of abl , and bacteria were cultured from the same eyes . figure 1 shows a fairly good linear correlation ( y = 11.353x ) between the bacterial luminescence measurement ( rlu ) of p. aeruginosa and the corresponding cfu ( determination coefficient r = 0.921 , correlation coefficient r = 0.9621 , p = 0.0001 ) . correlation of the bacterial luminescence intensity of infected ex vivo rabbit corneas to the corresponding colony - forming units ( cfu ) . to characterize the development of p. aeruginosa infection in the cornea , the bacterial luminescence was measured for up to 24 hours in the ex vivo rabbit model and up to 72 hours in the in vivo mouse model . in the ex vivo rabbit corneas , the mean bacterial luminescence increased steadily until approximately 9 hours , then remained relatively stable ( fig . ( a ) time course for the bacterial luminescence intensity of ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . ( b ) gram - stained ( 20 ) and h&e - stained histologic sections ( 10 ) of representative ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa and sampled at 3 , 6 , and 24 hours after bacterial inoculation . the inset shows the bacterial luminescence images and photographs taken at 0 , 24 , 36 , and 72 hours post inoculation from a representative infected in vivo mouse eye . to characterize the distribution of p. aeruginosa in the rabbit cornea , histology with gram and hematoxylin and eosin ( h&e staining ) was performed at 3 , 6 , and 24 hours post inoculation . at 3 hours post inoculation , p. aeruginosa was primarily located on the surface of corneal tissue . however , the bacteria rapidly proliferated into the stroma and destroyed the structure of cornea at 24 hours post inoculation , due to the lack of an immune response in this ex vivo model ( fig . similarly , after inoculating bacteria onto the scratched in vivo corneas of mice , a 24-hour period of more rapid increase in bacterial luminescence was followed by a more stable level of infection that lasted at least 72 hours . infected rabbit corneas were treated at either 6 or 24 hours post inoculation to investigate the effectiveness of abl therapy for early - stage or fully established infection . when abl was delivered at 6 hours post inoculation , the bacterial luminescence decreased with increasing abl exposure and an approximately 3-log10 inactivation was achieved , compared to untreated control , by exposure to 84 j / cm abl ( arrow in fig . in contrast , when abl was delivered at 24 hours post inoculation , exposure to 84 j / cm abl elicited less than 1-log10 inactivation ( fig . influence of radiant exposure on abl therapy for keratitis in the ex vivo rabbit model . ( a ) abl ( 180 j / cm ) delivered at 6 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . ( b ) abl ( 372 j / cm ) delivered at 24 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . when abl was delivered at 6 hours post inoculation , an increase in abl exposure decreased the bacteria level in in vivo mouse corneas , and an approximately 2.0-log10 inactivation of p. aeruginosa was achieved when an exposure of 36 j / cm abl was delivered ( fig . corneal pathology , as shown in figure 4b , was scored at 6 , 24 , and 48 hours after bacterial inoculation , respectively , for both abl - treated and untreated mice . it was observed that the eyes of abl - treated mice had significantly lower mean corneal pathology scores as compared to the eyes of untreated mice ( p = 0.0048 , fig . influence of radiant exposure on abl therapy for keratitis in the in vivo mouse model . ( a ) influence of radiant exposure of abl on the bacterial luminescence intensity of mouse eyes infected with 5 10 p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation ( n = 6 ) . the inset shows the bacterial luminescence images from a representative mouse eye infected with 5 10 cfu of p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation . ( b ) photographs of two representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with abl ( 36 j / cm ) at 6 hours post infection ( top row ) or not treated ( bottom row ) , indicating less opacity and damage in the abl - treated mouse eye than in the untreated one . ( c ) corneal pathology scores of the infected mouse eyes treated with 36 j / cm abl at 6 hours post infection ( n = 6 ) or not treated ( n = 6 ) , showing the higher mean corneal pathology scores of the untreated mouse eyes than the abl - treated mouse eyes ( p = 0.0048 ) . ( e ) influence of radiant exposure of abl on the bacterial luminescence intensity of the mouse eyes infected with 5 10 p. aeruginosa , treated with 144 j / cm abl at 24 hours post inoculation ( n = 6 ) , or left untreated ( n = 6 ) . when abl was delivered at 24 hours after p. aeruginosa inoculation , the bacterial luminescence was completely eradicated after an exposure of 144 j / cm abl ( fig . however , recurrence of infection was observed in the mice treated with abl at both 6 and 24 hours post inoculation after abl exposure . the recurrent infection was more severe in mice treated at 24 hours post inoculation than at 6 hours post inoculation . retinal safety is a very important concern for developing abl as a treatment of keratitis since abl , in sufficient amount , is highly toxic to the retina . , we calculated the abl exposures to reach the retina with the abl treatment parameters that reduced the bacterial luminescence in the ex vivo rabbit model by approximately 3-log10 ( 84 j / cm for 6 hours post inoculation and 304 j / cm for 24 hours post inoculation ; see fig . the obtained abl exposures to reach the retina were compared to the safety thresholds for photothermal and photochemical damage previously established by experimental studies . for a cloudy cornea that blocks 50% of the abl , 84 j / cm abl delivered to the cornea is below the retina safety threshold by a factor of 1.4 ( i.e. , 1/1.4 = 0.71-fold of the retina safety threshold ) , but 304 j / cm is above the threshold by a factor of 2.5. if a very cloudy cornea only transmits 10% of the abl , 84 and 304 j / cm abl are below the limit by factors of 7.1 and 2.0 , respectively . to test whether measuring the rlu of bacterial bioluminescence in the infected eyes after abl exposure accurately reported the corresponding bacterial cfu , bacterial luminescence measurements were made after varying radiant exposures of abl , and bacteria were cultured from the same eyes . figure 1 shows a fairly good linear correlation ( y = 11.353x ) between the bacterial luminescence measurement ( rlu ) of p. aeruginosa and the corresponding cfu ( determination coefficient r = 0.921 , correlation coefficient r = 0.9621 , p = 0.0001 ) . correlation of the bacterial luminescence intensity of infected ex vivo rabbit corneas to the corresponding colony - forming units ( cfu ) . to characterize the development of p. aeruginosa infection in the cornea , the bacterial luminescence was measured for up to 24 hours in the ex vivo rabbit model and up to 72 hours in the in vivo mouse model . in the ex vivo rabbit corneas , the mean bacterial luminescence increased steadily until approximately 9 hours , then remained relatively stable ( fig . ( b ) gram - stained ( 20 ) and h&e - stained histologic sections ( 10 ) of representative ex vivo rabbit eyes infected with 2 10 cfu of p. aeruginosa and sampled at 3 , 6 , and 24 hours after bacterial inoculation . ( c ) time course for the bacterial luminescence intensity of in vivo mouse eyes infected with 5 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . the inset shows the bacterial luminescence images and photographs taken at 0 , 24 , 36 , and 72 hours post inoculation from a representative infected in vivo mouse eye . to characterize the distribution of p. aeruginosa in the rabbit cornea , histology with gram and hematoxylin and eosin ( h&e staining ) was performed at 3 , 6 , and 24 hours post inoculation . at 3 hours post inoculation , p. aeruginosa was primarily located on the surface of corneal tissue . however , the bacteria rapidly proliferated into the stroma and destroyed the structure of cornea at 24 hours post inoculation , due to the lack of an immune response in this ex vivo model ( fig . similarly , after inoculating bacteria onto the scratched in vivo corneas of mice , a 24-hour period of more rapid increase in bacterial luminescence was followed by a more stable level of infection that lasted at least 72 hours . infected rabbit corneas were treated at either 6 or 24 hours post inoculation to investigate the effectiveness of abl therapy for early - stage or fully established infection . when abl was delivered at 6 hours post inoculation , the bacterial luminescence decreased with increasing abl exposure and an approximately 3-log10 inactivation was achieved , compared to untreated control , by exposure to 84 j / cm abl ( arrow in fig . in contrast , when abl was delivered at 24 hours post inoculation , exposure to 84 j / cm abl elicited less than 1-log10 inactivation ( fig . influence of radiant exposure on abl therapy for keratitis in the ex vivo rabbit model . ( a ) abl ( 180 j / cm ) delivered at 6 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . ( b ) abl ( 372 j / cm ) delivered at 24 hours after inoculation with 2 10 cfu of p. aeruginosa ( solid line ; n = 6 ) . when abl was delivered at 6 hours post inoculation , an increase in abl exposure decreased the bacteria level in in vivo mouse corneas , and an approximately 2.0-log10 inactivation of p. aeruginosa was achieved when an exposure of 36 j / cm abl was delivered ( fig . corneal pathology , as shown in figure 4b , was scored at 6 , 24 , and 48 hours after bacterial inoculation , respectively , for both abl - treated and untreated mice . it was observed that the eyes of abl - treated mice had significantly lower mean corneal pathology scores as compared to the eyes of untreated mice ( p = 0.0048 , fig . influence of radiant exposure on abl therapy for keratitis in the in vivo mouse model . ( a ) influence of radiant exposure of abl on the bacterial luminescence intensity of mouse eyes infected with 5 10 p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation ( n = 6 ) . the inset shows the bacterial luminescence images from a representative mouse eye infected with 5 10 cfu of p. aeruginosa and treated with 36 j / cm abl at 6 hours post inoculation . ( b ) photographs of two representative mouse eyes infected with 5 10 cfu of p. aeruginosa , treated with abl ( 36 j / cm ) at 6 hours post infection ( top row ) or not treated ( bottom row ) , indicating less opacity and damage in the abl - treated mouse eye than in the untreated one . ( c ) corneal pathology scores of the infected mouse eyes treated with 36 j / cm abl at 6 hours post infection ( n = 6 ) or not treated ( n = 6 ) , showing the higher mean corneal pathology scores of the untreated mouse eyes than the abl - treated mouse eyes ( p = 0.0048 ) . ( e ) influence of radiant exposure of abl on the bacterial luminescence intensity of the mouse eyes infected with 5 10 p. aeruginosa , treated with 144 j / cm abl at 24 hours post inoculation ( n = 6 ) , or left untreated ( n = 6 ) . when abl was delivered at 24 hours after p. aeruginosa inoculation , the bacterial luminescence was completely eradicated after an exposure of 144 j / cm abl ( fig . however , recurrence of infection was observed in the mice treated with abl at both 6 and 24 hours post inoculation after abl exposure . the recurrent infection was more severe in mice treated at 24 hours post inoculation than at 6 hours post inoculation . retinal safety is a very important concern for developing abl as a treatment of keratitis since abl , in sufficient amount , is highly toxic to the retina . , we calculated the abl exposures to reach the retina with the abl treatment parameters that reduced the bacterial luminescence in the ex vivo rabbit model by approximately 3-log10 ( 84 j / cm for 6 hours post inoculation and 304 j / cm for 24 hours post inoculation ; see fig . the obtained abl exposures to reach the retina were compared to the safety thresholds for photothermal and photochemical damage previously established by experimental studies . for a cloudy cornea that blocks 50% of the abl , 84 j / cm abl delivered to the cornea is below the retina safety threshold by a factor of 1.4 ( i.e. , 1/1.4 = 0.71-fold of the retina safety threshold ) , but 304 j / cm is above the threshold by a factor of 2.5. if a very cloudy cornea only transmits 10% of the abl , 84 and 304 j / cm abl are below the limit by factors of 7.1 and 2.0 , respectively . in this study , we developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis by using a bioluminescent strain of p. aeruginosa and found a good linear correlation between the bacterial luminescence and the corresponding bacterial cfu in the infected corneas . our results demonstrated that a single exposure of abl rapidly and significantly reduced bacterial burden in both the ex vivo and in vivo models . the infections treated at 24 hours post inoculation were more resistant to abl than they were at 6 hours post inoculation . in a previous study of abl therapy for p. aeruginosa burn infection in mice , we observed that , when abl was applied 30 minutes post inoculation , an average 3.5-log10 inactivation of p. aeruginosa in mouse burns was achieved when an exposure of 55.8 j / cm abl had been delivered . recurrence of infection was observed in mice ( but not in the rabbit corneas ) after a single exposure of abl . one possible reason of infection recurrence is that bacteria in the infected corneas were not completely eradicated after abl exposure even when bacterial luminescence was eliminated due to the sensitivity limitation of the bioluminescence imaging system ( > 10 cfu ) . this hypothesis was supported by the results obtained from the study using the ex vivo rabbit corneas . in the ex vivo study , the whole area of excised rabbit corneas was exposed to abl and no bacterial regrowth was found in any abl - treated cornea after abl exposure . several approaches could be considered to improve the effectiveness of abl therapy , such as repeated abl treatments ( two or three times ) and combined therapy using abl and subtherapeutic dose of antibiotics ( antimicrobial eye drops ) , which can reach the corneal area under the eyelids after topical application . there are also limitations of the in vivo mouse model used in the present study since mouse eyes differ substantially from human eyes . the arrangement of corneal collagen and the properties of corneal epithelial cells also differ between mice and humans , which could produce alternate reactions of the cornea to invading pathogens . as a result , the effectiveness of abl against keratitis needs to be further investigated using large in vivo animal models , for example , a rabbit or dog model , which are more similar to humans in ocular anatomy . according to the ansi 136.1 , we estimated the abl toxicity to the retina under different abl transmission of the corneal conditions . the calculations indicated that the abl toxicity to the retina is largely dependent on the abl transmission of the cornea . overall , our study suggests that abl is a potential alternative or adjunctive approach for the treatment of infectious keratitis , in particular those caused by drug - resistant pathogens .

clinical training is considered as an indispensable and very important part of professional nursing education . clinical education is recognized as an essential and highly significant component of professional education for nursing . practical clinical skills lie at the heart of nurses professional practice ; therefore , the mastery of fundamental clinical skills is an important component of courses leading to registration . since nursing is a discipline based on practice , there needs to be a curriculum of education that offers students the opportunity to develop their clinical skills , particularly the patient care skills . clinical practice is one of the ways used to increase the nursing students professional competence . nursing teachers must be in charge of clinical practice because they are the ones ultimately responsible for learning in the clinical practice . thus , it is in the clinical area that students must relate theory to practice , learn the necessary technical and interpersonal skills , make clinical judgments , become socialized into the profession , and begin to appreciate its values and ethics . the development of competent practice is a primary goal for nursing education . to demonstrate this competence clinical competence evaluation is defined as an integrated form of evaluation seeking to combine knowledge , understanding , problem solving , technical skills , attitudes , and ethics in evaluation . evaluation , as a way of determining the clinical competence , is one of the fundamental principles of development and student achievement measurement in nursing education . in clinical evaluation , it must be ensured that the students in clinical settings have an appropriate professional behavior , establish an appropriate interaction with the patients , prioritize the problems , have the basic knowledge about clinical methods , perform the care procedures correctly , and apply critical thinking . the evaluation of clinical practice is a complex process that continues to tax nurse educators . it can be stated that the evaluation of nursing students in clinical practice is a problem and the one which can not be solved . much of the discussion centers on the thorny issue of subjectivity and a plethora of clinical evaluation tools has been devised and abandoned in the quest to overcome this ongoing dilemma . wood ( 1982 ) proposes that the problem probably persists because clinical evaluation relies upon the observation of the performance of one individual by another , which itself is inevitably subjective . this suggests that the issue of subjectivity might be addressed only if some other methods of clinical evaluation were to be devised . many issues have been raised in the evaluation of nursing clinical skills , which indicate the existence of various problems in this field . some of the problems raised in this field include the heterogeneity of devices used from year to year and from period to period , inconsistency in the evaluation process by the clinical instructors , and lack of an appropriate framework for showing the students improvements . evaluation of clinical practice is often faced with problems such as low credit of existing methods of evaluating the students performance and inability to evaluate the level of theoretical and practical knowledge of the students . besides , some students believe that the evaluation tools do not pay much attention to the students practical skills . on the other hand , in some studies , the students stated that clinical evaluation by the clinical instructors is one of the major problems experienced in the clinical practice . in general , the problems of clinical evaluation appear in the form of nursing students complaints , reporting differences in clinical evaluation and multiple visits between the students and the nursing instructors . researchers have also seen the students dissatisfaction in their evaluation status and the results of the clinical evaluation . following the announcement of the clinical evaluation results , many students protest about their evaluation scores to the clinical instructors and raise various issues . despite some considerable efforts made in order to solve the problem , clinical evaluation challenges are still continuing . in this regard , imanipour et al . indicated that most students considered their clinical evaluation ( by using clinical evaluation forms ) inappropriate and disagree with it . their teachers also have similar attitude , disagree with the current clinical evaluation , and do not consider it appropriate . the research findings also indicated that 96.4% of the students just knew their marks , while they did not know their strengths and drawbacks . besides , 87.8% of them said that they received their internship marks after they finished their course . 79.6% of the students believed that internship marks represented teachers personal attitude and not the clinical evaluation of the students . 82.1% of the teachers believed that various evaluation methods must be applied to evaluate the students clinical performance . some methods recommended in this regard by students and their teachers included written tests , verbal tests , objective structured clinical examination ( osce ) , nurse and head nurses views , etc . , besides , 89.3% and 92.3% of teachers stated that the current evaluation forms of students need to be modified to improve their quality . some of the suggestions proposed by teachers and students regarding the improvement of clinical evaluation included specialization of clinical evaluation forms in accordance with the academic semester , the type of internship , and modification of general items to specific items to be exact in evaluating the students performance . in our nursing and midwifery college , since the beginning of nursing clinical education , the clinical environment has been looked through the training point of view . moreover , the nursing students were expected to perform clinical practice with the instructors supervision and repeat these practices ; however , their competency level was never taken into account . it seems that the nursing instructors participate in training the students function in the clinical environment with the aim of education and training rather than evaluation . the purpose of this qualitative exploratory study was to explore the views of nursing trainers and students about nursing students clinical evaluation problems and drawbacks in shiraz nursing and midwifery school . understanding the nursing students clinical evaluation problems in this school may provide insights about other iranian nursing and midwifery schools , as well as the ones which share the same history and challenges . a qualitative exploratory approach was used in this study at shiraz nursing and midwifery school in 2012 . a purposeful sample of 8 nursing instructors and 40 nursing students was interviewed and the data on their opinions about the problems of the clinical evaluation were collected through face - to - face deep semi - structured interviews . the enrolment criteria were as follows : ( 1 ) participants were female and male senior nursing students ; ( 2 ) they were willing to participate in the study ; ( 3 ) and they were nursing trainers with at least 10 years of relevant nursing students training experience . each deep interview lasted for at least 2 h. data collection and analysis proceeded concurrently , and once the themes were identified and data saturation was achieved , the interviews were discontinued . prior to recording the interviews , the objective of the study was verbally clarified for each participant and the participants questions were answered . a written explanation was also provided to the participants and accepted by them through a written consent form . initially , four open - ended questions , which were related to the clinical evaluation status , were developed and used to stimulate discussions in the interview sessions [ table 1 ] . initial analysis focused on understanding the information , and developing codes and categories through identification of persistent words , phrases , themes , or concepts within the data . the analytic process was utilized to gain familiarity with the data during the transcription and translation . for coding the transcript , it was necessary to go through the transcripts line by line and paragraph by paragraph to look for significant statements and codes according to the topics addressed . overall , three levels of coding were selected as appropriate to code the data . level 1 : examining the data line by line and making codes taken from the language of the subjects who attended the focus groups and deep interviewslevel 2 : comparing the coded data with other data and creating the categories . in fact , the categories are simply the coded data which seem to cluster together and may result from condensing of the codeslevel 3 : describing the clinical evaluation problem which is the title given to the central themes that emerge from the categories . level 1 : examining the data line by line and making codes taken from the language of the subjects who attended the focus groups and deep interviews level 2 : comparing the coded data with other data and creating the categories . in fact , the categories are simply the coded data which seem to cluster together and may result from condensing of the codes level 3 : describing the clinical evaluation problem which is the title given to the central themes that emerge from the categories . the recorded interviews were initially transcribed , read , and re - read on a number of occasions to get an overall feeling of what the participants were saying . each line or incident described examples of three levels of coding through this process of analysis and comparison , the following themes emerged : inappropriate clinical evaluation method , problems of clinical evaluation process , problems related to clinical instructors , unsuitable programming of clinical education , and organizational shortcomings . the measures used in the present study for establishing the rigor are truth value , truth value reflects the extent to which the study reveals the participants descriptions , which they are able to identify later on as their own . the results of the present study were shown to 10 participants , who confirmed the findings as being a reflection of their original descriptions . it was enhanced in the present study by returning to the participants of the study for confirmation of the findings . it was enhanced in the study by ensuring that the interview situation was stable and consistent throughout the data gathering . the interviews were recorded , so that they could be listened over and over again . neutrality should be judged in qualitative research by confirmability , which is achieved through auditability , applicability , and consistency . to enhance neutrality , the researcher carefully considered his own perceptions and pre - assumptions toward nursing students clinical evaluation . this proved to be useful because the researcher had a lot of experience in the field of nursing education . institutional review board 's ( irb ) approval for the study was obtained from the ethics committee of shiraz university of medical sciences ( ecsums ) . in addition , permission to conduct the study was obtained from the dean of the faculty of nursing and midwifery . furthermore , the confidentiality of the interview data and the personal identity was assured and the participants right to withdraw from the study at any time was also explained . institutional review board 's ( irb ) approval for the study was obtained from the ethics committee of shiraz university of medical sciences ( ecsums ) . in addition , permission to conduct the study was obtained from the dean of the faculty of nursing and midwifery . furthermore , the confidentiality of the interview data and the personal identity was assured and the participants right to withdraw from the study at any time was also explained . in the present study , 8 nursing instructors and 40 nursing students where sampled ; among them , 60% of the students ( aged between 19 and 22 years ) and 88% of the faculty instructors ( aged between 42 and 49 years ) were females . the qualitative analysis led to the emergence of five major themes from the deep interview data . from the students and their instructors points of view , the following five themes and nine categories were considered as the important clinical evaluation problems : inappropriate clinical evaluation method ( inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students , subjective clinical evaluation ) , problems of clinical evaluation process ( incorrect performance of formative clinical evaluation , incorrect osce examination ) , problems related to clinical instructors ( lack of instructors free time , instructor 's weakness in clinical evaluation , collecting insufficient data from the students ) , unsuitable programming of clinical education ( the limitation of clinical training time ) , and organizational shortcomings ( the lack of unified laws and regulations ) . in spite of the fact that nursing instructors highly rely upon the clinical evaluation forms for evaluating the students , the students are worried that the current clinical evaluation forms are not sufficient and appropriate for their clinical evaluation . nursing instructors also admit that these evaluation forms are necessary but not adequate for clinical nursing evaluation . since other professional nursing students clinical competencies , particularly communication skills , decision - making skills , and critical thinking skills , can not be measured through these evaluation forms , professional skills ( affective learning objectives ) show themselves more prominently and measurement of this domain also poses the issue of the evaluator bias ; therefore , some nursing students are more concerned about this issue . to measure some clinical competencies , particularly clinical procedure skills and clinical decision - making skills , through objective nursing process , other tools are required . since the clinical evaluation forms are more widely used and these instruments are the only evaluation tools , the inclusion of all clinical learning objectives in these forms leads to problems in scoring the nursing students , resulting in their protest . as the findings indicated , since the learning objectives of the current clinical evaluation forms are numerous , hard to understand , and are not practical , the instructors tend to perform a subjective evaluation and rely on their mentality in order to evaluate the students . inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students nursing students stated thus : nursing instructors usually use the current evaluation forms to grade the students . the learning objectives of the current clinical evaluation forms are numerous and hard to understand . these forms emphasize professional skill ( the affective learning objectives ) of the nursing students more . as you say , these forms , as a primary tool , are helpful for clinical evaluation but they do nt measure all the issues in our mind . particularly , they do nt measure the student 's progress and achievement . subjective clinical evaluation regarding this point , one of the nursing instructors said : unfortunately , we can not perform the nursing student 's clinical evaluation objectively . my own evaluation method as an instructor also has problems . the only thing that makes our evaluation subjective and actual is the mentality that we already have about the students scores as well as reactions and behavior of the students . another nursing instructor claimed thus : we know that a large number of the current evaluation forms options are not objective . we should definitely use other evaluation methods in addition to the current evaluation forms to do better clinical evaluation . because clinical evaluation is limited to the use of clinical evaluation forms and the instructors complete these forms at the end of the course , and on the other hand , they do not have other clinical evaluation instruments to do formative evaluation , the student 's achievement and their clinical learning process can not be assessed appropriately . in order to improve clinical evaluation , during the course , some osce examinations are incoherently performed for some nursing students and instructors do not provide students with feedback during and after osce examination ; then , the students do not realize where their problems are . it seems that formative evaluation such as osce examination should be conducted in a higher quality for the students , and other evaluation tools and methods should be added . incorrect performance of formative clinical evaluation one of the instructors said : items in the clinical evaluation forms especially do nt measure the students progress and achievement . if we aim to evaluate the students through these forms again , we mark many items in the evaluation forms which havent been in clinical evaluation . incorrect osce examination the students stated thus : osce examination is conducted under time conditions . osce grading has no value . since the facilities and stages of performing the clinical procedures during osce examinations are not the same as actual issues in the clinical setting , the clinical procedures that we practice at the hospital are highly incomplete compared to the osce procedures . the instructors accepted that they did not perform the clinical evaluation of the students in a timely manner . they stated that their workload is high and they do not have sufficient opportunity and time to identify the students during a semester . , the students may have objections about their scores , so they try to give higher score to the students . one of the clinical evaluation objectives that the instructors expect their students to perform in the clinical training was their clinical conference presentations in the clinical wards . the students complained that conference topics were not original and topics were not selected on the basis of ward common diseases ; therefore , they were prepared and provided only for performing some tasks by students . in order to perform an objective evaluation of the students , instructors strived to give cognitive written tests at the end of the semester , but the students objected to irrelevant content of these tests compared with those they had experienced during the clinical training . students stated that instructors do not know what they expect the students to do during their apprenticeship and what they should learn . instructors did not have enough information to score the students clinical evaluation forms , and the students were not assessed based on their competencies . to score the students based on the evaluation forms , according to the issues mentioned above , the inclusion of abundant clinical objectives for education and evaluation is a difficult task and causes the instructors not to understand the students well enough and not to reach all the identified objectives . lack of instructors free time one of the nursing instructors said thus : one of our problems is that we do not have enough time . due to the lack of time , we assess the students at the end of the semester which is nt really an appropriate time . the students do not spend much time with the instructor in the clinical field . as a result , the instructors do not have enough opportunity to understand the students ; therefore , reduction of the training duration might be the reason why we can not assess the students better . another nursing instructor stated thus : my student does nt know where and what her / his weaknesses are . clinical evaluation method of some of us also has problems because we do nt evaluate the students on - time . i evaluate the students when they have finished their semester , and this is nt really an appropriate time . finally , in order to avoid the students complaints about their scores , i try to give them higher scores . instructor 's weakness in clinical evaluation one of students said that : our instructors ask their students to present the clinical conferences as a lecture ; however , the conference topics are quite irrelevant to the ward diseases . some instructors give tests to their students at the end of the course ; however , the exam questions are not ideal for that ward . collecting insufficient data from the students regarding this point a student said thus : each instructor has his / her own special rule . we do nt know what we are supposed to learn since the instructors score the students based on their speculations ; sometimes a student who is very capable and another who performs quite moderately receive the same scores . once , although i thought i would fail , i got a high score . by performing the current clinical evaluation the instructors think that since the student has already passed apprenticeship , he / she knows all the things . the instructor stated that , i have used feminine ornament when i complained about this issue ; she / he says you are right , i made a mistake . planning for teaching and learning nursing competencies causes a great number of problems in training and evaluations ; it requires much effort for coordinating and integrating its educational objectives . it should be noted that the clinical environment is an unpredictable one and its control is almost beyond the instructors capabilities and all the students do nt reach the same level of competency in a period of time . on the other hand , the clinical teaching time is limited and the number of wards needed for the clinical training is also diverse and abundant , which will cause problems for its planning . instructors expressed that the time period of the students clinical training and evaluation is limited , so they can not identify accurately and adequately the students competencies , can not reach all the objectives of clinical evaluation forms , and do not have enough evidence for evaluating the students . the limitation of clinical training time one of the nursing instructors said thus : in addition to the problems of evaluation tools , another problem is the short period of time that a student spends with an instructor . we go to clinical ward for 2 three - day weeks and we may rarely be with our students for 15 - 16 days and this period of time is quite short for understanding the students clinical practice . so far , planning , implementation , and monitoring of most faculties educational programs have been performed intra - organizationally , based on the opinion of a low level group of officials and instructors . although observing the regulations and standards has been mentioned to all the instructors by group authorities , implementation of these regulations has been faced with shortcomings and has not been performed in a coherent manner . this problem has caused the students to be faced with different rules and be dissatisfied with the act of higher level of management in this regard . lack of unified laws and regulations considering this points one of the students said that : all the instructors do not act the same . for instance , some instructors do not care about the students absences and being absent does nt affect the students scores . moreover , some instructors let the students have breaks but do not care about their tardiness . in spite of the fact that nursing instructors highly rely upon the clinical evaluation forms for evaluating the students , the students are worried that the current clinical evaluation forms are not sufficient and appropriate for their clinical evaluation . nursing instructors also admit that these evaluation forms are necessary but not adequate for clinical nursing evaluation . since other professional nursing students clinical competencies , particularly communication skills , decision - making skills , and critical thinking skills , can not be measured through these evaluation forms , professional skills ( affective learning objectives ) show themselves more prominently and measurement of this domain also poses the issue of the evaluator bias ; therefore , some nursing students are more concerned about this issue . to measure some clinical competencies , particularly clinical procedure skills and clinical decision - making skills , through objective nursing process , other tools are required . since the clinical evaluation forms are more widely used and these instruments are the only evaluation tools , the inclusion of all clinical learning objectives in these forms leads to problems in scoring the nursing students , resulting in their protest . as the findings indicated , since the learning objectives of the current clinical evaluation forms are numerous , hard to understand , and are not practical , the instructors tend to perform a subjective evaluation and rely on their mentality in order to evaluate the students . inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students nursing students stated thus : nursing instructors usually use the current evaluation forms to grade the students . the learning objectives of the current clinical evaluation forms are numerous and hard to understand . these forms emphasize professional skill ( the affective learning objectives ) of the nursing students more . as you say , these forms , as a primary tool , are helpful for clinical evaluation but they do nt measure all the issues in our mind . particularly , they do nt measure the student 's progress and achievement . subjective clinical evaluation regarding this point , one of the nursing instructors said : unfortunately , we can not perform the nursing student 's clinical evaluation objectively . my own evaluation method as an instructor also has problems . the only thing that makes our evaluation subjective and actual is the mentality that we already have about the students scores as well as reactions and behavior of the students . another nursing instructor claimed thus : we know that a large number of the current evaluation forms options are not objective . we should definitely use other evaluation methods in addition to the current evaluation forms to do better clinical evaluation . because clinical evaluation is limited to the use of clinical evaluation forms and the instructors complete these forms at the end of the course , and on the other hand , they do not have other clinical evaluation instruments to do formative evaluation , the student 's achievement and their clinical learning process can not be assessed appropriately . in order to improve clinical evaluation , during the course , some osce examinations are incoherently performed for some nursing students and instructors do not provide students with feedback during and after osce examination ; then , the students do not realize where their problems are . it seems that formative evaluation such as osce examination should be conducted in a higher quality for the students , and other evaluation tools and methods should be added . incorrect performance of formative clinical evaluation one of the instructors said : items in the clinical evaluation forms especially do nt measure the students progress and achievement . if we aim to evaluate the students through these forms again , we mark many items in the evaluation forms which havent been in clinical evaluation . incorrect osce examination the students stated thus : osce examination is conducted under time conditions . osce grading has no value . since the facilities and stages of performing the clinical procedures during osce examinations are not the same as actual issues in the clinical setting , the clinical procedures that we practice at the hospital are highly incomplete compared to the osce procedures . the instructors accepted that they did not perform the clinical evaluation of the students in a timely manner . they stated that their workload is high and they do not have sufficient opportunity and time to identify the students during a semester . , the students may have objections about their scores , so they try to give higher score to the students . one of the clinical evaluation objectives that the instructors expect their students to perform in the clinical training was their clinical conference presentations in the clinical wards . the students complained that conference topics were not original and topics were not selected on the basis of ward common diseases ; therefore , they were prepared and provided only for performing some tasks by students . in order to perform an objective evaluation of the students , instructors strived to give cognitive written tests at the end of the semester , but the students objected to irrelevant content of these tests compared with those they had experienced during the clinical training . students stated that instructors do not know what they expect the students to do during their apprenticeship and what they should learn . instructors did not have enough information to score the students clinical evaluation forms , and the students were not assessed based on their competencies . to score the students based on the evaluation forms , according to the issues mentioned above , the inclusion of abundant clinical objectives for education and evaluation is a difficult task and causes the instructors not to understand the students well enough and not to reach all the identified objectives . lack of instructors free time one of the nursing instructors said thus : one of our problems is that we do not have enough time . . we can not reach all the clinical objectives in this short period of time . due to the lack of time , we assess the students at the end of the semester which is nt really an appropriate time . the students do not spend much time with the instructor in the clinical field . as a result , the instructors do not have enough opportunity to understand the students ; therefore , reduction of the training duration might be the reason why we can not assess the students better . another nursing instructor stated thus : my student does nt know where and what her / his weaknesses are . clinical evaluation method of some of us also has problems because we do nt evaluate the students on - time . i evaluate the students when they have finished their semester , and this is nt really an appropriate time . finally , in order to avoid the students complaints about their scores , i try to give them higher scores . instructor 's weakness in clinical evaluation one of students said that : our instructors ask their students to present the clinical conferences as a lecture ; however , the conference topics are quite irrelevant to the ward diseases . the conference topics are repeated . some instructors give tests to their students at the end of the course ; however , the exam questions are not ideal for that ward . collecting insufficient data from the students regarding this point a student said thus : each instructor has his / her own special rule . we do nt know what we are supposed to learn since the instructors score the students based on their speculations ; sometimes a student who is very capable and another who performs quite moderately receive the same scores . once , although i thought i would fail , i got a high score . by performing the current clinical evaluation , positive and quiet students can get good grades . the instructors think that since the student has already passed apprenticeship , he / she knows all the things . the instructor stated that , i have used feminine ornament when i complained about this issue ; she / he says you are right , i made a mistake . planning for teaching and learning nursing competencies causes a great number of problems in training and evaluations ; it requires much effort for coordinating and integrating its educational objectives . it should be noted that the clinical environment is an unpredictable one and its control is almost beyond the instructors capabilities and all the students do nt reach the same level of competency in a period of time . on the other hand , the clinical teaching time is limited and the number of wards needed for the clinical training is also diverse and abundant , which will cause problems for its planning . instructors expressed that the time period of the students clinical training and evaluation is limited , so they can not identify accurately and adequately the students competencies , can not reach all the objectives of clinical evaluation forms , and do not have enough evidence for evaluating the students . the limitation of clinical training time one of the nursing instructors said thus : in addition to the problems of evaluation tools , another problem is the short period of time that a student spends with an instructor . we go to clinical ward for 2 three - day weeks and we may rarely be with our students for 15 - 16 days and this period of time is quite short for understanding the students clinical practice . so far , planning , implementation , and monitoring of most faculties educational programs have been performed intra - organizationally , based on the opinion of a low level group of officials and instructors . although observing the regulations and standards has been mentioned to all the instructors by group authorities , implementation of these regulations has been faced with shortcomings and has not been performed in a coherent manner . this problem has caused the students to be faced with different rules and be dissatisfied with the act of higher level of management in this regard . lack of unified laws and regulations considering this points one of the students said that : all the instructors do not act the same . for instance , some instructors do not care about the students absences and being absent does nt affect the students scores . moreover , some instructors let the students have breaks but do not care about their tardiness . the results of the present study revealed that the most important clinical evaluation problem was lack of a comprehensive and appropriate evaluation tool for assessing the students . on one hand , in the case of lack of objective and credible clinical evaluation tool , the clinical evaluation forms are more appropriate than the students observation by instructors . on the other hand , since the options of the current clinical evaluation forms do not objectively measure the students clinical competencies and learning process , the instructors can not differentiate the competent students from moderately competent ones and this leads to instructor 's bias . our findings in this regard are in agreement with the reports by norman et al . , indicating that clinical evaluation traditionally relies upon observation of the performance of one individual by another , which runs the risk of observer bias . in an attempt to overcome this problem , the study findings revealed that the girls and boys capabilities are predominantly different , and since most of the nursing teachers and students were females , the nursing instructors seem to pay more attention to female students . as a result , some male students complained that their clinical competencies are ignored in clinical settings . in spite of the fact that most nursing instructors have got used to the clinical evaluation forms , and they provide and revise them each semester to grade the nursing students however the students were dissatisfied with their gradings . the nursing students complained that the current evaluation forms can not evaluate the learning psychomotor , decision - making , and critical thinking objectives , as well as the students learning progress . to grade the nursing students , some instructors used norm - referenced evaluation and gave the students a grade as a whole ; they compared the students with each other and then graded them . inherent to the clinical evaluation process is judgment and subjectivity , and this subjective process is further influenced by biases of both the evaluator and student , as well as by variables present in the clinical environment . on one hand , the students must be evaluated in the clinical setting , and on the other hand , at the heart of evaluation is judgment and bias , which is inherent in the human evaluation process . they must keep in mind that they are observing the student 's behavior , and only that must be evaluated . evaluation of learning is problematic in practical disciplines because it requires direct observation of the students engaged in actual practice in unpredictable clinical environments . in a study by vaismoradi et al . ( 2010 ) , the participants dissatisfaction of the way instructors supervised them in the clinical setting also was revealed . no connection could be made between their scores and clinical work because they were not seen when caring for patients . the issue of the students gender was highlighted as a potential cause for unfair and unexpected evaluation outcomes . the male participants claimed that female instructors discriminate between the students of the opposite sex . moreover , the study findings indicated that the nursing instructors did not perform formative clinical evaluation during a semester and did not have tools for doing it . since they do not have enough information about each nursing student 's progress and achievement , they can not train the nursing students for the next stages of clinical competencies . finally , they are forced to score the students without any clinical learning evidences . before starting any teaching episode , the teacher needs to establish an understanding about where the learner 's position is , the level which she / he has reached , his / her past experience , and his / her personal goals . as a part of the overall planning process in a teaching session , the teacher also has to define his / her aims of the session , the learning outcomes or objectives , and possibly an evaluation . the findings of this study showed that although the nursing instructors had to use the clinical evaluation forms for grading the students , some of the instructors were not willing to use them for assessing their students . it seems that since osce examination is new for the nursing students and so far the students have not taken clinical examinations under regulations and timing programs , this examination has been hard for them . on the other hand , , they did not give feedback to the students after the examination ; as a result , the nursing students are not willing to take this examination . probably , because students were not familiar with the scenarios of osce stations and they think this examination is very hard for them , it is very common that some students find a way to help them pass osce examination . the importance of a positive atmosphere during the osce was strongly emphasized , especially that the feedback should be given in a positive and constructive manner . if a student is to learn from the feedback , this must be given in a motivating manner . a feedback given in a negative way may also influence the performance of the students at the following stations ; therefore , he or she does not receive a fair and realistic evaluation . as mentioned by vaismoradi et al . ( 2010 ) , another factor for creating dissatisfaction among the participants was the way instructors assessed and evaluated the coursework . based on the participants words , instructors did not care if anyone cheated in coursework . some students asked other students or translation agencies to do their assignment , but instructors did not check their coursework to find this . feedback may be the effort to discover deficiencies and urge the student to correct their deficiencies which cause educators to more frequently give the student negative feedback . students want feedback about their performance to gauge progress concerning knowledge , competence , and faculty expectations , and rate giving feedback as an essential quality of an effective clinical teacher . despite this , there have been many reports of students dissatisfaction with the amount and quality of feedback they receive from their teachers . the present study showed that having enough time for evaluation was very important for the nursing instructors . it also revealed that the nursing instructors were very busy during each semester and did not assign a specific time for evaluation . they do not have enough time to evaluate the student at the end of the semester ; therefore , if they had some evaluation tools , they could save time for themselves . one of the clinical evaluation assignments that the nursing instructors have confirmed was the nursing students clinical conference presentations . it seems that the nursing instructors do not have information about the previous clinical conferences presented by the students and the conference topics were not selected based on the patients diseases ; therefore , the nursing students complained about providing and submitting them as a clinical assignment . some nursing instructors took multiple questions and essay tests for evaluating the students clinical knowledge , but because the content of the tests was not quite relevant to what the students learned during the clinical training , the students were not satisfied with these tests . in accordance with this study , 2010 ) reported that the students indicated that the evaluation process did not encourage them to learn beyond what was expected . they were so busy with doing coursework that the aim of translating and reading articles , which was to improve the students critical and creative thinking capabilities , was lost . the study findings revealed that most nursing instructors do not have enough information about the students before starting each apprenticeship . they also did not have enough information about each student during the completion of the clinical evaluation forms . it shows that they did not have appropriate clinical evaluation devices and did not assess the students based on clinical evaluation forms . ( 2010 ) reported that the participants talked about some kinds of misconduct , which led to injustice during evaluation process . the participants in this study preferred a score to be given based on practical skills criteria , not mastery on theoretical knowledge . many clinical instructors are reluctant to give unsatisfactory grades to students who do not meet established standards of practice . the study findings indicated that to gain some nursing competencies , the students need more time to practice and repeat such capabilities . it seems that the duration of the clinical course is short and most clinical instructors are busy during each semester . consequently , they used most of their time in clinical teaching and complained about their limited time for clinical evaluation ; therefore , they did not have time to identify the students clinical competencies . another important issue that most of the students complained about was that most of the nursing instructors did not observe the educational laws and regulations . some instructors were serious about absenteeism and tardiness of the students on entering the clinical ward , while some of them did not care about this issue . in addition , the students complained that the nursing instructors did not follow a similar education regulation . besides focusing on upgrading the current clinical evaluation forms , nursing trainers should improve their knowledge about a complete and comprehensive clinical evaluation . they should also apply other appropriate and objective clinical evaluation methods and tools , and perform a formative and summative clinical evaluation . also , workload adjustment of the nursing trainers needs revision . therefore , despite using traditional and sometimes limited evaluation methods for assessing nursing students , a comprehensive and appropriate evaluation of nursing students clinical competencies seems necessary .

background : the purpose of this qualitative exploratory study was to explore the views of nursing trainers and students about nursing students clinical evaluation problems and drawbacks in shiraz nursing and midwifery school.materials and methods : a qualitative exploratory approach was used in this study at shiraz nursing and midwifery school in 2012 . a purposeful sample of 8 nursing instructors and 40 nursing students was interviewed and the data on their opinions about the problems of the clinical evaluation were collected through semi - structured deep interviews . initially , four open - ended questions , which were related to the clinical evaluation status , problems , were used to stimulate discussions in the interview sessions . content analysis was employed in order to analyze the transcribed data . the recorded interviews were initially transcribed , read , and reread on a number of occasions to get an overall feeling of what the participants were saying . each line or incident was described , and then a code , which reflected the essence of the participants comments , was given.results:the codes were compared for similarity and differences , merged together , and categorized . finally , five themes emerged : in appropriate clinical evaluation method , problems of clinical evaluation process , problems related to clinical instructors , unsuitable programming of clinical education , and organizational shortcomings.conclusion:besides focusing on upgrading the current clinical evaluation forms , nursing trainers should improve their knowledge about a complete and comprehensive clinical evaluation . they should also apply other appropriate and objective clinical evaluation methods and tools , and perform a formative and summative clinical evaluation . also , workload adjustment of the nursing trainers needs revision . therefore , despite using traditional and sometimes limited evaluation methods for assessing nursing students , a co mprehensive and appropriate evaluation of nursing students clinical competencies seems necessary .

I M D Ethical considerations R Inappropriate clinical evaluation method Problems of clinical evaluation process Problems related to clinical instructors Unsuitable programming of clinical education Organizational shortcomings D C

clinical practice is one of the ways used to increase the nursing students professional competence . nursing teachers must be in charge of clinical practice because they are the ones ultimately responsible for learning in the clinical practice . thus , it is in the clinical area that students must relate theory to practice , learn the necessary technical and interpersonal skills , make clinical judgments , become socialized into the profession , and begin to appreciate its values and ethics . in clinical evaluation , it must be ensured that the students in clinical settings have an appropriate professional behavior , establish an appropriate interaction with the patients , prioritize the problems , have the basic knowledge about clinical methods , perform the care procedures correctly , and apply critical thinking . it can be stated that the evaluation of nursing students in clinical practice is a problem and the one which can not be solved . much of the discussion centers on the thorny issue of subjectivity and a plethora of clinical evaluation tools has been devised and abandoned in the quest to overcome this ongoing dilemma . many issues have been raised in the evaluation of nursing clinical skills , which indicate the existence of various problems in this field . some of the problems raised in this field include the heterogeneity of devices used from year to year and from period to period , inconsistency in the evaluation process by the clinical instructors , and lack of an appropriate framework for showing the students improvements . on the other hand , in some studies , the students stated that clinical evaluation by the clinical instructors is one of the major problems experienced in the clinical practice . in general , the problems of clinical evaluation appear in the form of nursing students complaints , reporting differences in clinical evaluation and multiple visits between the students and the nursing instructors . researchers have also seen the students dissatisfaction in their evaluation status and the results of the clinical evaluation . following the announcement of the clinical evaluation results , many students protest about their evaluation scores to the clinical instructors and raise various issues . their teachers also have similar attitude , disagree with the current clinical evaluation , and do not consider it appropriate . 79.6% of the students believed that internship marks represented teachers personal attitude and not the clinical evaluation of the students . 82.1% of the teachers believed that various evaluation methods must be applied to evaluate the students clinical performance . , besides , 89.3% and 92.3% of teachers stated that the current evaluation forms of students need to be modified to improve their quality . some of the suggestions proposed by teachers and students regarding the improvement of clinical evaluation included specialization of clinical evaluation forms in accordance with the academic semester , the type of internship , and modification of general items to specific items to be exact in evaluating the students performance . in our nursing and midwifery college , since the beginning of nursing clinical education , the clinical environment has been looked through the training point of view . it seems that the nursing instructors participate in training the students function in the clinical environment with the aim of education and training rather than evaluation . the purpose of this qualitative exploratory study was to explore the views of nursing trainers and students about nursing students clinical evaluation problems and drawbacks in shiraz nursing and midwifery school . understanding the nursing students clinical evaluation problems in this school may provide insights about other iranian nursing and midwifery schools , as well as the ones which share the same history and challenges . a qualitative exploratory approach was used in this study at shiraz nursing and midwifery school in 2012 . a purposeful sample of 8 nursing instructors and 40 nursing students was interviewed and the data on their opinions about the problems of the clinical evaluation were collected through face - to - face deep semi - structured interviews . the enrolment criteria were as follows : ( 1 ) participants were female and male senior nursing students ; ( 2 ) they were willing to participate in the study ; ( 3 ) and they were nursing trainers with at least 10 years of relevant nursing students training experience . prior to recording the interviews , the objective of the study was verbally clarified for each participant and the participants questions were answered . initially , four open - ended questions , which were related to the clinical evaluation status , were developed and used to stimulate discussions in the interview sessions [ table 1 ] . in fact , the categories are simply the coded data which seem to cluster together and may result from condensing of the codeslevel 3 : describing the clinical evaluation problem which is the title given to the central themes that emerge from the categories . in fact , the categories are simply the coded data which seem to cluster together and may result from condensing of the codes level 3 : describing the clinical evaluation problem which is the title given to the central themes that emerge from the categories . the recorded interviews were initially transcribed , read , and re - read on a number of occasions to get an overall feeling of what the participants were saying . each line or incident described examples of three levels of coding through this process of analysis and comparison , the following themes emerged : inappropriate clinical evaluation method , problems of clinical evaluation process , problems related to clinical instructors , unsuitable programming of clinical education , and organizational shortcomings . the measures used in the present study for establishing the rigor are truth value , truth value reflects the extent to which the study reveals the participants descriptions , which they are able to identify later on as their own . it was enhanced in the present study by returning to the participants of the study for confirmation of the findings . it was enhanced in the study by ensuring that the interview situation was stable and consistent throughout the data gathering . in addition , permission to conduct the study was obtained from the dean of the faculty of nursing and midwifery . furthermore , the confidentiality of the interview data and the personal identity was assured and the participants right to withdraw from the study at any time was also explained . in addition , permission to conduct the study was obtained from the dean of the faculty of nursing and midwifery . furthermore , the confidentiality of the interview data and the personal identity was assured and the participants right to withdraw from the study at any time was also explained . in the present study , 8 nursing instructors and 40 nursing students where sampled ; among them , 60% of the students ( aged between 19 and 22 years ) and 88% of the faculty instructors ( aged between 42 and 49 years ) were females . from the students and their instructors points of view , the following five themes and nine categories were considered as the important clinical evaluation problems : inappropriate clinical evaluation method ( inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students , subjective clinical evaluation ) , problems of clinical evaluation process ( incorrect performance of formative clinical evaluation , incorrect osce examination ) , problems related to clinical instructors ( lack of instructors free time , instructor 's weakness in clinical evaluation , collecting insufficient data from the students ) , unsuitable programming of clinical education ( the limitation of clinical training time ) , and organizational shortcomings ( the lack of unified laws and regulations ) . in spite of the fact that nursing instructors highly rely upon the clinical evaluation forms for evaluating the students , the students are worried that the current clinical evaluation forms are not sufficient and appropriate for their clinical evaluation . since other professional nursing students clinical competencies , particularly communication skills , decision - making skills , and critical thinking skills , can not be measured through these evaluation forms , professional skills ( affective learning objectives ) show themselves more prominently and measurement of this domain also poses the issue of the evaluator bias ; therefore , some nursing students are more concerned about this issue . since the clinical evaluation forms are more widely used and these instruments are the only evaluation tools , the inclusion of all clinical learning objectives in these forms leads to problems in scoring the nursing students , resulting in their protest . as the findings indicated , since the learning objectives of the current clinical evaluation forms are numerous , hard to understand , and are not practical , the instructors tend to perform a subjective evaluation and rely on their mentality in order to evaluate the students . inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students nursing students stated thus : nursing instructors usually use the current evaluation forms to grade the students . the learning objectives of the current clinical evaluation forms are numerous and hard to understand . these forms emphasize professional skill ( the affective learning objectives ) of the nursing students more . subjective clinical evaluation regarding this point , one of the nursing instructors said : unfortunately , we can not perform the nursing student 's clinical evaluation objectively . another nursing instructor claimed thus : we know that a large number of the current evaluation forms options are not objective . we should definitely use other evaluation methods in addition to the current evaluation forms to do better clinical evaluation . because clinical evaluation is limited to the use of clinical evaluation forms and the instructors complete these forms at the end of the course , and on the other hand , they do not have other clinical evaluation instruments to do formative evaluation , the student 's achievement and their clinical learning process can not be assessed appropriately . in order to improve clinical evaluation , during the course , some osce examinations are incoherently performed for some nursing students and instructors do not provide students with feedback during and after osce examination ; then , the students do not realize where their problems are . it seems that formative evaluation such as osce examination should be conducted in a higher quality for the students , and other evaluation tools and methods should be added . incorrect performance of formative clinical evaluation one of the instructors said : items in the clinical evaluation forms especially do nt measure the students progress and achievement . since the facilities and stages of performing the clinical procedures during osce examinations are not the same as actual issues in the clinical setting , the clinical procedures that we practice at the hospital are highly incomplete compared to the osce procedures . the instructors accepted that they did not perform the clinical evaluation of the students in a timely manner . one of the clinical evaluation objectives that the instructors expect their students to perform in the clinical training was their clinical conference presentations in the clinical wards . in order to perform an objective evaluation of the students , instructors strived to give cognitive written tests at the end of the semester , but the students objected to irrelevant content of these tests compared with those they had experienced during the clinical training . instructors did not have enough information to score the students clinical evaluation forms , and the students were not assessed based on their competencies . lack of instructors free time one of the nursing instructors said thus : one of our problems is that we do not have enough time . finally , in order to avoid the students complaints about their scores , i try to give them higher scores . instructor 's weakness in clinical evaluation one of students said that : our instructors ask their students to present the clinical conferences as a lecture ; however , the conference topics are quite irrelevant to the ward diseases . on the other hand , the clinical teaching time is limited and the number of wards needed for the clinical training is also diverse and abundant , which will cause problems for its planning . instructors expressed that the time period of the students clinical training and evaluation is limited , so they can not identify accurately and adequately the students competencies , can not reach all the objectives of clinical evaluation forms , and do not have enough evidence for evaluating the students . the limitation of clinical training time one of the nursing instructors said thus : in addition to the problems of evaluation tools , another problem is the short period of time that a student spends with an instructor . in spite of the fact that nursing instructors highly rely upon the clinical evaluation forms for evaluating the students , the students are worried that the current clinical evaluation forms are not sufficient and appropriate for their clinical evaluation . since other professional nursing students clinical competencies , particularly communication skills , decision - making skills , and critical thinking skills , can not be measured through these evaluation forms , professional skills ( affective learning objectives ) show themselves more prominently and measurement of this domain also poses the issue of the evaluator bias ; therefore , some nursing students are more concerned about this issue . since the clinical evaluation forms are more widely used and these instruments are the only evaluation tools , the inclusion of all clinical learning objectives in these forms leads to problems in scoring the nursing students , resulting in their protest . as the findings indicated , since the learning objectives of the current clinical evaluation forms are numerous , hard to understand , and are not practical , the instructors tend to perform a subjective evaluation and rely on their mentality in order to evaluate the students . inappropriate evaluation forms for evaluation of other clinical capabilities of nursing students nursing students stated thus : nursing instructors usually use the current evaluation forms to grade the students . the learning objectives of the current clinical evaluation forms are numerous and hard to understand . these forms emphasize professional skill ( the affective learning objectives ) of the nursing students more . subjective clinical evaluation regarding this point , one of the nursing instructors said : unfortunately , we can not perform the nursing student 's clinical evaluation objectively . another nursing instructor claimed thus : we know that a large number of the current evaluation forms options are not objective . we should definitely use other evaluation methods in addition to the current evaluation forms to do better clinical evaluation . because clinical evaluation is limited to the use of clinical evaluation forms and the instructors complete these forms at the end of the course , and on the other hand , they do not have other clinical evaluation instruments to do formative evaluation , the student 's achievement and their clinical learning process can not be assessed appropriately . in order to improve clinical evaluation , during the course , some osce examinations are incoherently performed for some nursing students and instructors do not provide students with feedback during and after osce examination ; then , the students do not realize where their problems are . it seems that formative evaluation such as osce examination should be conducted in a higher quality for the students , and other evaluation tools and methods should be added . incorrect performance of formative clinical evaluation one of the instructors said : items in the clinical evaluation forms especially do nt measure the students progress and achievement . if we aim to evaluate the students through these forms again , we mark many items in the evaluation forms which havent been in clinical evaluation . since the facilities and stages of performing the clinical procedures during osce examinations are not the same as actual issues in the clinical setting , the clinical procedures that we practice at the hospital are highly incomplete compared to the osce procedures . the instructors accepted that they did not perform the clinical evaluation of the students in a timely manner . one of the clinical evaluation objectives that the instructors expect their students to perform in the clinical training was their clinical conference presentations in the clinical wards . in order to perform an objective evaluation of the students , instructors strived to give cognitive written tests at the end of the semester , but the students objected to irrelevant content of these tests compared with those they had experienced during the clinical training . instructors did not have enough information to score the students clinical evaluation forms , and the students were not assessed based on their competencies . to score the students based on the evaluation forms , according to the issues mentioned above , the inclusion of abundant clinical objectives for education and evaluation is a difficult task and causes the instructors not to understand the students well enough and not to reach all the identified objectives . lack of instructors free time one of the nursing instructors said thus : one of our problems is that we do not have enough time . finally , in order to avoid the students complaints about their scores , i try to give them higher scores . instructor 's weakness in clinical evaluation one of students said that : our instructors ask their students to present the clinical conferences as a lecture ; however , the conference topics are quite irrelevant to the ward diseases . on the other hand , the clinical teaching time is limited and the number of wards needed for the clinical training is also diverse and abundant , which will cause problems for its planning . instructors expressed that the time period of the students clinical training and evaluation is limited , so they can not identify accurately and adequately the students competencies , can not reach all the objectives of clinical evaluation forms , and do not have enough evidence for evaluating the students . the limitation of clinical training time one of the nursing instructors said thus : in addition to the problems of evaluation tools , another problem is the short period of time that a student spends with an instructor . the results of the present study revealed that the most important clinical evaluation problem was lack of a comprehensive and appropriate evaluation tool for assessing the students . on one hand , in the case of lack of objective and credible clinical evaluation tool , the clinical evaluation forms are more appropriate than the students observation by instructors . on the other hand , since the options of the current clinical evaluation forms do not objectively measure the students clinical competencies and learning process , the instructors can not differentiate the competent students from moderately competent ones and this leads to instructor 's bias . in an attempt to overcome this problem , the study findings revealed that the girls and boys capabilities are predominantly different , and since most of the nursing teachers and students were females , the nursing instructors seem to pay more attention to female students . in spite of the fact that most nursing instructors have got used to the clinical evaluation forms , and they provide and revise them each semester to grade the nursing students however the students were dissatisfied with their gradings . the nursing students complained that the current evaluation forms can not evaluate the learning psychomotor , decision - making , and critical thinking objectives , as well as the students learning progress . to grade the nursing students , some instructors used norm - referenced evaluation and gave the students a grade as a whole ; they compared the students with each other and then graded them . inherent to the clinical evaluation process is judgment and subjectivity , and this subjective process is further influenced by biases of both the evaluator and student , as well as by variables present in the clinical environment . on one hand , the students must be evaluated in the clinical setting , and on the other hand , at the heart of evaluation is judgment and bias , which is inherent in the human evaluation process . ( 2010 ) , the participants dissatisfaction of the way instructors supervised them in the clinical setting also was revealed . since they do not have enough information about each nursing student 's progress and achievement , they can not train the nursing students for the next stages of clinical competencies . the findings of this study showed that although the nursing instructors had to use the clinical evaluation forms for grading the students , some of the instructors were not willing to use them for assessing their students . on the other hand , , they did not give feedback to the students after the examination ; as a result , the nursing students are not willing to take this examination . one of the clinical evaluation assignments that the nursing instructors have confirmed was the nursing students clinical conference presentations . it seems that the nursing instructors do not have information about the previous clinical conferences presented by the students and the conference topics were not selected based on the patients diseases ; therefore , the nursing students complained about providing and submitting them as a clinical assignment . some nursing instructors took multiple questions and essay tests for evaluating the students clinical knowledge , but because the content of the tests was not quite relevant to what the students learned during the clinical training , the students were not satisfied with these tests . they also did not have enough information about each student during the completion of the clinical evaluation forms . ( 2010 ) reported that the participants talked about some kinds of misconduct , which led to injustice during evaluation process . the participants in this study preferred a score to be given based on practical skills criteria , not mastery on theoretical knowledge . it seems that the duration of the clinical course is short and most clinical instructors are busy during each semester . consequently , they used most of their time in clinical teaching and complained about their limited time for clinical evaluation ; therefore , they did not have time to identify the students clinical competencies . another important issue that most of the students complained about was that most of the nursing instructors did not observe the educational laws and regulations . besides focusing on upgrading the current clinical evaluation forms , nursing trainers should improve their knowledge about a complete and comprehensive clinical evaluation . they should also apply other appropriate and objective clinical evaluation methods and tools , and perform a formative and summative clinical evaluation . also , workload adjustment of the nursing trainers needs revision . therefore , despite using traditional and sometimes limited evaluation methods for assessing nursing students , a comprehensive and appropriate evaluation of nursing students clinical competencies seems necessary .

rheumatoid arthritis ( ra ) is a chronic , systemic , inflammatory disease that involves synovial joints and other organs and extra - articular involvements associated with impairment in physical function , higher morbidity , and premature mortality [ 1 , 2 ] . interstitial lung disease ( ild ) is an infrequent but extremely relevant extra - articular manifestation that decreases the patients ' health - related quality of life ( qol ) and life expectancy . with the development of more accurate diagnostic methods , ild has been reported with a prevalence of up to 61% in patients with ra . ild in ra is associated with around threefold the risk for mortality as compared with ra without this entity . some hypotheses concerning ra pathogenesis suggest that major susceptibility genes , particularly hla - dr , shared epitopes that interact with smoking to trigger ra - specific responses to citrullinated proteins , signifying a clear relationship between smoking and the development of the immune response directed against citrullinated peptides . one of the consequences of these reactions is the formation of anti - cyclic citrullinated peptide antibodies ( anti - ccp ) , which are observed in around 5569% of patients with ra . these autoantibodies are highly specific markers for ra and are useful for predicting ra development and progression [ 7 , 8 ] , although the association between anti - ccp antibodies and extra - articular manifestations was not conclusive . currently , there are few studies evaluating the association between anti - ccp autoantibodies and ild in ra . on evaluating 18 patients with ra associated with ild , did not find an association between anti - ccp and ild . on the other hand , nikiphorou et al . recently demonstrated , in an abstract , their results of a multicenter study in which anti - ccp antibodies were strongly associated with ild in ra . recently , yin et al . identified ild in 71 from among their 285 patients with ra , observing that positivity for second - generation anti - ccp ( anti - ccp2 ) was associated with an increase in risk of ild . these authors identified that anti - ccp antibody titers comprised the most relevant factor associated with ild in ra on univariate analysis , and this factor remained associated with ild in the multivariate approach . reynisdottir et al . , employing a different approach , analyzed the findings of high - resolution computed tomography ( hrct ) in 70 patients with early , untreated ra who were positive for anticitrullinated proteins ( acpa - positive ) compared with 35 patients with early , untreated acpa - negative ra . these authors identified that 63% of patients with acpa - positive ra had abnormalities in hrct compared with 37% of patients with acpa - negative ra ( p = 0.02 ) . ild is a major complication in ra , where prognosis is influenced by the presence of pulmonary active disease and severity of the lung involvement . described that patients with ra with findings of more extensive lung disease on hrct have a worse prognosis compared with patients with ra with limited ild . currently , the extent and severity of ild on hrct and not merely the presence of ild are considered as factors associated with the prognosis , leading to the development of tomographically validated scales to identify the severity of the lung involvement . nonetheless , although the majority of studies have investigated the relationship of anti - ccp and ild in ra , these studies have not evaluated whether there is an association of anti - ccp titers with the extent and severity of ild on hrct utilizing a validated scale . to date , there is a lack of information on whether higher titers of these autoantibodies are related to clinical parameters for ild severity , including the extent of lung damage assessed by validated tomographic scores . therefore , our aim in this study was to examine the relationship between serum levels of anti - ccp2 and severity of the extent of ild damage in patients with ra . patients . the study included patients with ra attending an outpatient rheumatology clinic in a secondary - care center ( hospital general regional-110 of the mexican institute for social security ( imss ) ) located in guadalajara , jal , mexico . patients were eligible if they met american college of rheumatology 1987 classification criteria for ra and were 18 years of age or older . patients were not eligible if they had history of asthma or pulmonary tuberculosis , active respiratory infection , mental or psychiatric disorders , and any overlapped syndrome or if they exhibited an obstructive pattern during spirometry . patients with criteria of mtx pneumonitis were excluded . from a cohort of 600 patients with ra , we identified 42 patients with ra with ild data and these were compared with 39 patients with ra only selected consecutively from the same cohort and matched by gender and range of age . in order to assess the ascertainment presence of ild in ra classification criteria for ra - ild were based on the following three parameters : clinical symptoms , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , abnormalities in pulmonary function test ( pft ) characterized by a decrease in forced vital capacity ( fvc ) < 80% according to the predicted rate , radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing with or without activity for ground - glass pattern > 5% . clinical symptoms , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , abnormalities in pulmonary function test ( pft ) characterized by a decrease in forced vital capacity ( fvc ) < 80% according to the predicted rate , radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing with or without activity for ground - glass pattern > 5% . instead , criteria for inclusion of patients with ra without ild ( no ra - ild ) were based on the following three parameters : absence of clinical symptoms for lung involvement , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , pft characterized by fvc 80% ( predicted rate),no radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing 5% without activity for ground - glass pattern . absence of clinical symptoms for lung involvement , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , pft characterized by fvc 80% ( predicted rate ) , no radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing 5% without activity for ground - glass pattern . a structured questionnaire was applied to patients to evaluate demographical , clinical , and therapeutic variables related with ra . the patients ' synovial joints were examined for swelling and tenderness by a trained examiner . disease activity was assessed employing the disease activity score in 28 joints ( das28 ) , and functioning was assessed using the validated spanish version for haq - di . assessment of cardiopulmonary function included the following indices.the 6-minute walk test ( 6mwt ) was performed according to the american thoracic society ( ats ) guidelines . the 6mwt measures the distance a patient can walk rapidly on a hard surface in a period of 6 minutes and is thought to reflect well a person 's functional activity level for daily physical activities.the modified borg scale is a subjective scale that assesses the perception of dyspnea by the patient . this was performed immediately before and after the 6mwt , placing the degree of dyspnea on a scale of 0100 mm , where 0 is none ( no dyspnea ) and 100 is the maximal dyspnea observed.the validated mexican - spanish version of the saint george respiratory questionnaire ( sgrq ) consists of a self - administered questionnaire for measuring the impairment of patient - perceived health - related qol in lung diseases in three domains , including symptoms , activity , and impact . scores can range from 0 ( no impairment ) to 100 ( worst impairment ) for each domain ; higher scores connote greater distress and , thus , worse health - related qol . the questionnaire was administered and scored according to the instruction manual prior to the execution of 6mwt and pft . the 6-minute walk test ( 6mwt ) the 6mwt measures the distance a patient can walk rapidly on a hard surface in a period of 6 minutes and is thought to reflect well a person 's functional activity level for daily physical activities . the modified borg scale is a subjective scale that assesses the perception of dyspnea by the patient . this was performed immediately before and after the 6mwt , placing the degree of dyspnea on a scale of 0100 mm , where 0 is none ( no dyspnea ) and 100 is the maximal dyspnea observed . the validated mexican - spanish version of the saint george respiratory questionnaire ( sgrq ) consists of a self - administered questionnaire for measuring the impairment of patient - perceived health - related qol in lung diseases in three domains , including symptoms , activity , and impact . scores can range from 0 ( no impairment ) to 100 ( worst impairment ) for each domain ; higher scores connote greater distress and , thus , worse health - related qol . the questionnaire was administered and scored according to the instruction manual prior to the execution of 6mwt and pft . a screening spirometry was performed with a spiropro , sensormedics ver . 2.0 , according to the recommendations published in 2005 by ats and european respiratory society ( ers ) . the spirometries were performed to assess forced expiratory volume in 1 second ( fev1 ) , forced vital capacity ( fvc ) , and the fev1/fvc ratio . observed values were expressed as a percentage of the predicted value compared with individuals of similar gender , age , weight , and height . a restrictive pattern was defined as an fvc of < 80% of that predicted in the absence of concomitant obstructive abnormality . hrct was performed using a single tomographer ( 4th generation equipment , siemens somatom ar.t . the hrct was performed with the patient in prone position , using sections about 1 - 2 mm thick ( at 10-mm intervals ) . images were reconstructed with a high spatial algorithm and filmed using standard lung window settings ( wl-700 , [ ww ] 10001500 [ hu ] ) . hrct scans were obtained at the suspended end - inspiratory volume with the patient in the supine position , and additional scans were obtained with the patient in the prone position , when necessary , to demonstrate the reversibility of high attenuation in dependent lung . all images were evaluated independently and in random order by two observers ( one experienced thoracic radiologist and an experienced pulmonologist ) who were blinded to the clinical and pathological data . the final assessment was achieved by consensus with an adjudicator if there were disagreements in interpretation . distribution patterns were visually assessed in three defined regions ( upper , middle , or lower regions ) of both lungs . the upper zone is from the superior aspect of the transverse aortic arch to the lung apices , the middle region from the top of the transverse aortic arch to the inferior pulmonary vein , and the lower zone from the inferior veins to the diaphragm . according to kazarooni et al . , standardized sheet was used to tabulate the presence or absence of two features : ( 1 ) ground - glass opacity , defined as an area of increased attenuation , and ( 2 ) honeycombing , defined as subpleural clustered cystic air spaces with distinct walls of 325 mm in diameter , on a scale of 05 in the three lobes of both lungs as follows : 0 : no alveolar disease ; 1 : ground - glass pattern involving < 5% of the lobe ; 2 : involving > 25% ; 3 : involving 2545% ; 4 : involving 5075% ; 5 : involving > 75% of the lobe for the alveolar score and 0 : nonfibrosis ; 1 : septal thickening without honeycombing ; 2 : honeycombing involving > 25% of the lobe ; 3 : involving 25 to 49% ; 4 : involving 5075% ; 5 : involving > 75% of the lobe for the interstitial score . erythrocyte sedimentation rate ( esr , mm / h ) was measured using the wintrobe method . fasting sera were stored at 70c until the determination of anti - ccp2 antibodies by enzyme - linked immunosorbent assay ( elisa ) , using a commercial sandwich elisa kit ( euroimmun , medizinische labordiagnostika , ag , lubeck , germany ) , with cut - off values for seropositivity for anti - ccp2 of > 20 u / ml . quantitative variables were expressed as medians and ranges and qualitative variables as numbers and percentages . comparisons of quantitative variables between patients with ra - ild and ra only , we used mann - whitney u test , and for comparisons of qualitative variables between these groups , we utilized the chi - square test ( or the fisher exact test , if required ) . a correlation between anti - ccp2 titers with parameters of physical examination , disease activity indices , fvc , cardiopulmonary assessment , sgrq domains , and hrct scores was performed with spearman 's coefficient ( rho ) . we built a multivariate logistic regression model with stepwise selection variables to identify risk factors for interstitial lung disease in ra . thereafter , we performed a linear regression analysis in order to identify the variables associated with higher ground - glass and interstitial fibrosis scores in the hrct . those variables with a p value of < 0.20 on univariate analysis or those with biologic plausibility to influence the development of ra - ild were included in these multivariate models . the study was approved by the institutional review board of the mexican institute for social security ( imss ) of the participating hospital ( approval number imss r-2010 - 1303 - 29 ) , with all subjects providing written informed consent . figure 1 presents the study 's flow chart of the patients meeting the inclusion criteria . of 54 ra patients with a suspicion of ild , 15 were excluded from the study because they declined to participate ( n = 5 ) or had exclusion criteria for the study ( n = 10 ) , whereas , of 59 ra patients without ild , 17 were excluded because they met one of the exclusion criteria ( see figure 1 ) . table 1 compares the characteristics of patients with ra - ild ( n = 39 ) versus ra only ( n = 42 ) . patients with ra - ild had higher scores for das28 ( 3.9 versus 2.5 , p < 0.001 ) and haq - di ( 0.8 versus 0.4 , p < 0.001 ) . higher anti - ccp2 titers were found in patients with ra - ild compared to ra only ( 77.9 versus 30.2 u / ml , p < 0.001 ) ; the frequency of positive rheumatoid factor ( rf ) was also higher in ra - ild ( 97.4% versus 35.7% , p < 0.001 ) , and levels of esr were higher in ild ( 32 versus 19.5 , p < 0.001 ) . a higher frequency of rheumatoid nodules history was found to be associated with the occurrence of ra - ild ( 74.4 versus 14.7% , p < 0.001 ) . other findings associated with ra - ild were higher frequency of higher mtx doses at the time of the study ( p < 0.001 ) , longer mtx duration ( p = 0.002 ) , and higher accumulated dose of mtx ( p < 0.001 ) . no statistical associations were observed between ra - ild and age , the dd of ra , and smoking history . figure 2 illustrates a comparison between anti - ccp2 titers in patients with ra only , compared with ra - ild . higher anti - ccp2 titers were observed in ra - ild ( 77.9 versus 30.2 , p < 0.001 ) , whereas none of the patients with ra only had anti - ccp2 titers above 100 u / ml . table 2 compares the scores of cardiopulmonary scales and sgrq between patients with ra only and patients with ra - ild . higher scores on the sgrq and modified borg scales following exercise were observed in ra - ild ( p < 0.001 ) . patients with ra - ild had also lower distance in the 6mwt compared with ra only ( 310.0 versus 410.0 , p < 0.001 ) . in data not shown in the tables , a correlation among anti - ccp2 titers was observed with das28 ( rho = 0.420 , p < 0.001 ) , haq - di ( rho = 0.46 , p < 0.001 ) , sgrq symptoms ( rho = 0.547 , p < 0.001 ) , 6mwt ( rho = 0.632 , p < 0.001 ) , pre-6mwt vas modified borg scale ( rho = 0.637 , p < 0.001 ) , post-6mwt vas modified borg scale ( rho = 0.619 , p < 0.001 ) , mtx treatment duration ( rho = 0.293 , p = 0.008 ) , as well as fvc% ( rho = 0.632 , p < 0.001 ) , and all the hrct scores : ground - glass ( rho = 0.566 , p < 0.001 ) and interstitial fibrosis ( rho = 0.70 , p < 0.001 ) . in data that are not shown in tables , we performed a multivariable logistic regression analysis to identify variables associated with restrictive pattern in lung function tests . in the final model , the higher anti - ccp2 antibodies titers ( or 1.08 95% ic 1.021.14 , p = 0.004 ) were associated with restrictive pattern in fvc , whereas factors that did not have statistical significance with fvc% were age , disease duration , rf , and years of treatment with mtx . table 3 shows the results of the multivariate logistic regression analysis to identify associated variables with ra - ild . in the final model , after the adjustment for age , disease duration , smoke exposure , das28 , haq - di , esr , and mtx treatment duration , two variables were associated with an increase of risk for ra - ild : the higher anti - ccp2 antibodies titers ( or , 1.06 ; 95% ci , 1.021.10 , p = 0.003 ) and positive rf ( or , 28.58 ; 95% ci 3.31246.95 , p = 0.002 ) . table 4 presents the results of multiple linear regression analysis evaluating factors associated with higher severity of ild according to the hcrt scores . after the adjustment for age , disease duration , das28 , and mtx duration , we observed that the anti - ccp2 titers were significantly associated ( p = 0.02 ) with higher severity of the extension in the ground - glass score . similarly , after adjustment for age , das28 for the higher fibrosis score was significantly associated with higher disease duration ( p = 0.01 ) , anti - ccp2 titers ( p < 0.001 ) , and duration of treatment with mtx ( p < 0.001 ) . in the present study , anti - ccp2 titers were associated with the presence and severity of ild in ra . these anti - ccp2 titers were correlated with impairment in several parameters for ild severity , including the sgrq , 6mwt , borg scales , decrease in fvc% , and higher scores for ild involvement and severity identified in the hrct severity . an association between ild and anti - ccp2 titers remained after adjustment for age , disease duration , and exposure to smoke , in the multivariate model and duration of treatment with mtx , whereas positive rf was also a factor associated with ild . additionally , we observed that the only factors that predicted in the multivariate linear regression the higher scores for fibrosis score were anti - ccp2 titers and longer mtx treatment duration ; instead , higher fibrosis scores were inversely associated with disease duration . our results , regarding the association between anti - ccp2 and ild , are similar to the findings by nikiphorou et al . , who observed that anti - ccp2 titers are significantly higher in patients with ra who had ild . on the other side , inui et al . did not observe an association between presence or levels of anti - ccp2 and ild . . included only 18 patients with ild associated with ra , and , therefore , because of the small sample evaluated , it is likely that this lack of differences can be explained by a type ii error . recently , yin et al . identified ild in 71 from their 285 patients with ra , observing that positivity for second - generation anti - ccp ( anti - ccp2 ) was associated with an increase in risk of ild . these authors identified that anti - ccp antibody titers were comprised of the most relevant factor associated with ild in ra on univariate analysis and this factor remained associated with ild in the multivariate approach . reynisdottir et al . , using a different approach , analyzed the findings of hrct in 70 patients with early untreated ra who were positive for anticitrullinated proteins ( acpa - positive ) compared with 35 acpa - negative ra . these authors identified that 63% of acpa - positive patients had abnormalities on hrct , compared with 37% of acpa - negative patients ( p = 0.02 ) . to date , to the best of our knowledge , there are no studies evaluating if the anti - ccp2 titers are associated with the extent and patterns of severity of lung involvement in ild - ra . we reported that high titers of these autoantibodies are associated with a higher extent of lung involvement in our patients even after adjustment for other variables . observed increased staining for citrullinated proteins on bronchial biopsies obtained from patients with ra and positive anti - ccp . rangel - moreno et al . reported higher levels of anti - ccp in serum and bronchoalveolar lavages in patients with ra , and these antibodies are increased in the patients with ra who had well - developed inducible bronchus - associated lymphoid tissue , suggesting that these antibodies are produced locally in the lungs . citrullinated proteins in the lungs are currently considered as autoantigens that may trigger an immune response associated with the development of anti - ccp2 and other antibodies that may act as markers associated with the tissue damage . in addition to anti - ccp2 levels , rf and elevated esr were biomarkers associated with ild in ra on univariate analysis . an association between positive rf and ild has been previously identified by several studies [ 1113 ] ; our findings are consistent with their results , whereas our finding of an elevated esr in patients with ild in ra is inconsistent with the majority of the reported data . did not observe differences in esr between the group with ild and the group without ild in their study . on the other hand , inui et al . instead , the association observed in the present study between mtx treatment and ild is consistent with data reported in the literature . roubille and haraoui examined evidence regarding the association between ild and synthetic or biological dmards ; these authors concluded , in their systematic review , that the incidence of mtx - associated pneumonitis has been estimated as ranging from 0.3 to 8% of patients with rheumatic disorders . in a meta - analysis , identified an increase in the risk of pneumonitis in patients receiving mtx . although in our study nearly all of the patients with ra received mtx at the time of the study , we were unable to identify if the patients not treated with this drug had lower risk for ild . related to the association observed between anti - ccp2 and ra - ild , acpas are specific for ra and correspond to a subset of ra that is distinct from ra acpa - negative in terms of pathogenesis , disease prognosis , and response to therapy . this information about acpas suggests that the presence of autoimmunity to citrullinated peptides and the developmental may be initiated within the respiratory system . not only are citrullinated proteins limited to synovial tissue , but they have also been identified at extra - articular sites in patients with ra . . found that citrullination is developed inside mononuclear cells in lung tissue in open - lung biopsy specimens from patients with ra - associated interstitial pneumonia . these authors also observed that , despite the high specificity of anti - ccp for ra , citrullination was also found in lung tissue from patients with idiopathic interstitial pneumonia . this protein citrullination is a phenomenon that is produced early in the disease course and that might be involved in the development of the disease . zhu et al . , in a meta - analysis , observed that acpa - positive serum indicated a higher risk for ild and interstitial pulmonary fibrosis ( ipf ) among patients with ra ( or , 4.679 , 95% ci 2.07110.572 , p < 0.001 ) . giles et al . observed high serum acpa titer associated with ra - ild , after adjustment for confounding factors ( age , sex , current or former smoking , and fr ) . a diagnosis of ild in ra identifies a patient with higher risk for the worst prognosis considering that the median survival in ra - ild is around 10 years shorter than that observed in the general population , lung disease being directly responsible of around 10 to 20% of all ra - associated mortalities . identified that the subtype of usual interstitial pneumonia / overlap syndromes has around 3.9-fold risk for death in comparison with the subtype of nonspecific interstitial pneumonia / cryptogenic organizing pneumonia . an extensive disease had around 2-fold increase in the risk for death from any cause versus those limited diseases . identified in a systematic review that the extent of fibrosis and usual interstitial pneumonia is a predictor of mortality in ra - ild . several studies observed that the positivity of anti - ccp2 and rf is associated with ra - ild . our study also observed in the multivariate logistic regression analysis that anti - ccp2 and rf were associated with ra - ild . observed that positive rates of anti - ccp2 and rf in patients with ra - ild were significantly higher than those of the patients with ra only . however , reynisdottir et al . found no significant difference in rf positivity in ra - ild . in our results , we are surprised that a shorter duration of disease was associated with the fibrosis score in the tomographic findings ; several studies have associated the presence of acpa - positive with the presence of pulmonary damage , mainly interstitial pulmonary and fibrosis pulmonary pattern ; however , to our knowledge , no study has linked shorter duration of disease with ra - ild and positive acpa . follow - up studies are required especially in patients with early ra , in which lung function comprises the value . this a cross - sectional design ; therefore , it is unable to demonstrate the causality of any variable for the development of ild in ra ; however , our findings of the association between anti - ccp2 titers and the presence and severity of this complication are relevant for further studies in experimental models or longitudinal studies . in addition , none of our patients had a pulmonary biopsy . thus , we have no information concerning the histological pattern exhibited by patients with ild , and it would be interesting to evaluate whether these high anti - ccp2 titers may correlate with the histologic patterns in lung tissue involvement . on the other hand , to the best of our knowledge , there are no previous studies assessing whether the severity of ild in ra is associated with higher titers of anti - ccp2 . these findings of higher anti - ccp2 titers and ild severity are not only limited to the hcrt score ; they are also associated with other characteristics of impairment in ild , such as decreased 6mwt , increases in the score for symptoms or impact in sgrq , and decreases in fvc% . another strength of the study was the utilization of a multivariate model to adjust the association of anti - ccp2 antibodies with ild by other confounders ; only two studies have previously used this statistical approach [ 10 , 13 ] , obtaining similar results to those observed in our study . in this respect , after adjustment for different factors , we observed that the relationship between the titers of these autoantibodies and the severity of ild remained significant on multivariate analysis . in conclusion , we identified that anti - ccp2 titers constitute an independent factor associated not only with the presence but also with the severity of ra - ild ; the relevance of these markers in patients with established ild for future outcomes , such as progression of lung involvement and mortality , remains to be established .

objective . to evaluate whether serum titers of second - generation anticyclic citrullinated peptide antibodies ( anti - ccp2 ) are associated with the severity and extent of interstitial lung disease in rheumatoid arthritis ( ra - ild ) . methods . in across - sectional study , 39 ra - ild patients confirmed by high - resolution computed tomography ( hrct ) were compared with 42 ra without lung involvement ( ra only ) . characteristics related to ra - ild were assessed in all of the patients and serum anti - ccp2 titers quantified . results . higher anti - ccp2 titers were found in ra - ild compared with ra only ( medians 77.9 versus 30.2 u / ml , p < 0.001 ) . in the logistic regression analysis after adjustment for age , disease duration ( dd ) , smoke exposure , disease activity , functioning , erythrocyte sedimentation rate , and methotrexate ( mtx ) treatment duration , the characteristics associated with ra - ild were higher anti - ccp2 titers ( p = 0.003 ) and + rf ( p = 0.002 ) . in multivariate linear regression , the variables associated with severity of ground - glass score were anti - ccp2 titers ( p = 0.02 ) and with fibrosis score dd ( p = 0.01 ) , anti - ccp2 titers ( p < 0.001 ) , and mtx treatment duration ( p < 0.001 ) . conclusions . anti - ccp2 antibodies are markers of severity and extent of ra - ild in hrct . further longitudinal studies are required to identify if higher anti - ccp2 titers are associated with worst prognosis in ra - ild .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

rheumatoid arthritis ( ra ) is a chronic , systemic , inflammatory disease that involves synovial joints and other organs and extra - articular involvements associated with impairment in physical function , higher morbidity , and premature mortality [ 1 , 2 ] . interstitial lung disease ( ild ) is an infrequent but extremely relevant extra - articular manifestation that decreases the patients ' health - related quality of life ( qol ) and life expectancy . ild in ra is associated with around threefold the risk for mortality as compared with ra without this entity . one of the consequences of these reactions is the formation of anti - cyclic citrullinated peptide antibodies ( anti - ccp ) , which are observed in around 5569% of patients with ra . recently demonstrated , in an abstract , their results of a multicenter study in which anti - ccp antibodies were strongly associated with ild in ra . identified ild in 71 from among their 285 patients with ra , observing that positivity for second - generation anti - ccp ( anti - ccp2 ) was associated with an increase in risk of ild . these authors identified that anti - ccp antibody titers comprised the most relevant factor associated with ild in ra on univariate analysis , and this factor remained associated with ild in the multivariate approach . , employing a different approach , analyzed the findings of high - resolution computed tomography ( hrct ) in 70 patients with early , untreated ra who were positive for anticitrullinated proteins ( acpa - positive ) compared with 35 patients with early , untreated acpa - negative ra . these authors identified that 63% of patients with acpa - positive ra had abnormalities in hrct compared with 37% of patients with acpa - negative ra ( p = 0.02 ) . ild is a major complication in ra , where prognosis is influenced by the presence of pulmonary active disease and severity of the lung involvement . currently , the extent and severity of ild on hrct and not merely the presence of ild are considered as factors associated with the prognosis , leading to the development of tomographically validated scales to identify the severity of the lung involvement . nonetheless , although the majority of studies have investigated the relationship of anti - ccp and ild in ra , these studies have not evaluated whether there is an association of anti - ccp titers with the extent and severity of ild on hrct utilizing a validated scale . therefore , our aim in this study was to examine the relationship between serum levels of anti - ccp2 and severity of the extent of ild damage in patients with ra . from a cohort of 600 patients with ra , we identified 42 patients with ra with ild data and these were compared with 39 patients with ra only selected consecutively from the same cohort and matched by gender and range of age . in order to assess the ascertainment presence of ild in ra classification criteria for ra - ild were based on the following three parameters : clinical symptoms , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , abnormalities in pulmonary function test ( pft ) characterized by a decrease in forced vital capacity ( fvc ) < 80% according to the predicted rate , radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing with or without activity for ground - glass pattern > 5% . clinical symptoms , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , abnormalities in pulmonary function test ( pft ) characterized by a decrease in forced vital capacity ( fvc ) < 80% according to the predicted rate , radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing with or without activity for ground - glass pattern > 5% . instead , criteria for inclusion of patients with ra without ild ( no ra - ild ) were based on the following three parameters : absence of clinical symptoms for lung involvement , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , pft characterized by fvc 80% ( predicted rate),no radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing 5% without activity for ground - glass pattern . absence of clinical symptoms for lung involvement , such as cough , phlegm , wheezing , bilateral inspiratory and expiratory crackles , and breathlessness , pft characterized by fvc 80% ( predicted rate ) , no radiographic evidence of ild on hrct , by means of bilateral outlying reticular opacities or honeycombing 5% without activity for ground - glass pattern . this was performed immediately before and after the 6mwt , placing the degree of dyspnea on a scale of 0100 mm , where 0 is none ( no dyspnea ) and 100 is the maximal dyspnea observed.the validated mexican - spanish version of the saint george respiratory questionnaire ( sgrq ) consists of a self - administered questionnaire for measuring the impairment of patient - perceived health - related qol in lung diseases in three domains , including symptoms , activity , and impact . observed values were expressed as a percentage of the predicted value compared with individuals of similar gender , age , weight , and height . , standardized sheet was used to tabulate the presence or absence of two features : ( 1 ) ground - glass opacity , defined as an area of increased attenuation , and ( 2 ) honeycombing , defined as subpleural clustered cystic air spaces with distinct walls of 325 mm in diameter , on a scale of 05 in the three lobes of both lungs as follows : 0 : no alveolar disease ; 1 : ground - glass pattern involving < 5% of the lobe ; 2 : involving > 25% ; 3 : involving 2545% ; 4 : involving 5075% ; 5 : involving > 75% of the lobe for the alveolar score and 0 : nonfibrosis ; 1 : septal thickening without honeycombing ; 2 : honeycombing involving > 25% of the lobe ; 3 : involving 25 to 49% ; 4 : involving 5075% ; 5 : involving > 75% of the lobe for the interstitial score . fasting sera were stored at 70c until the determination of anti - ccp2 antibodies by enzyme - linked immunosorbent assay ( elisa ) , using a commercial sandwich elisa kit ( euroimmun , medizinische labordiagnostika , ag , lubeck , germany ) , with cut - off values for seropositivity for anti - ccp2 of > 20 u / ml . comparisons of quantitative variables between patients with ra - ild and ra only , we used mann - whitney u test , and for comparisons of qualitative variables between these groups , we utilized the chi - square test ( or the fisher exact test , if required ) . a correlation between anti - ccp2 titers with parameters of physical examination , disease activity indices , fvc , cardiopulmonary assessment , sgrq domains , and hrct scores was performed with spearman 's coefficient ( rho ) . we built a multivariate logistic regression model with stepwise selection variables to identify risk factors for interstitial lung disease in ra . thereafter , we performed a linear regression analysis in order to identify the variables associated with higher ground - glass and interstitial fibrosis scores in the hrct . those variables with a p value of < 0.20 on univariate analysis or those with biologic plausibility to influence the development of ra - ild were included in these multivariate models . table 1 compares the characteristics of patients with ra - ild ( n = 39 ) versus ra only ( n = 42 ) . patients with ra - ild had higher scores for das28 ( 3.9 versus 2.5 , p < 0.001 ) and haq - di ( 0.8 versus 0.4 , p < 0.001 ) . higher anti - ccp2 titers were found in patients with ra - ild compared to ra only ( 77.9 versus 30.2 u / ml , p < 0.001 ) ; the frequency of positive rheumatoid factor ( rf ) was also higher in ra - ild ( 97.4% versus 35.7% , p < 0.001 ) , and levels of esr were higher in ild ( 32 versus 19.5 , p < 0.001 ) . a higher frequency of rheumatoid nodules history was found to be associated with the occurrence of ra - ild ( 74.4 versus 14.7% , p < 0.001 ) . other findings associated with ra - ild were higher frequency of higher mtx doses at the time of the study ( p < 0.001 ) , longer mtx duration ( p = 0.002 ) , and higher accumulated dose of mtx ( p < 0.001 ) . no statistical associations were observed between ra - ild and age , the dd of ra , and smoking history . figure 2 illustrates a comparison between anti - ccp2 titers in patients with ra only , compared with ra - ild . higher anti - ccp2 titers were observed in ra - ild ( 77.9 versus 30.2 , p < 0.001 ) , whereas none of the patients with ra only had anti - ccp2 titers above 100 u / ml . higher scores on the sgrq and modified borg scales following exercise were observed in ra - ild ( p < 0.001 ) . patients with ra - ild had also lower distance in the 6mwt compared with ra only ( 310.0 versus 410.0 , p < 0.001 ) . in data not shown in the tables , a correlation among anti - ccp2 titers was observed with das28 ( rho = 0.420 , p < 0.001 ) , haq - di ( rho = 0.46 , p < 0.001 ) , sgrq symptoms ( rho = 0.547 , p < 0.001 ) , 6mwt ( rho = 0.632 , p < 0.001 ) , pre-6mwt vas modified borg scale ( rho = 0.637 , p < 0.001 ) , post-6mwt vas modified borg scale ( rho = 0.619 , p < 0.001 ) , mtx treatment duration ( rho = 0.293 , p = 0.008 ) , as well as fvc% ( rho = 0.632 , p < 0.001 ) , and all the hrct scores : ground - glass ( rho = 0.566 , p < 0.001 ) and interstitial fibrosis ( rho = 0.70 , p < 0.001 ) . in data that are not shown in tables , we performed a multivariable logistic regression analysis to identify variables associated with restrictive pattern in lung function tests . in the final model , the higher anti - ccp2 antibodies titers ( or 1.08 95% ic 1.021.14 , p = 0.004 ) were associated with restrictive pattern in fvc , whereas factors that did not have statistical significance with fvc% were age , disease duration , rf , and years of treatment with mtx . table 3 shows the results of the multivariate logistic regression analysis to identify associated variables with ra - ild . in the final model , after the adjustment for age , disease duration , smoke exposure , das28 , haq - di , esr , and mtx treatment duration , two variables were associated with an increase of risk for ra - ild : the higher anti - ccp2 antibodies titers ( or , 1.06 ; 95% ci , 1.021.10 , p = 0.003 ) and positive rf ( or , 28.58 ; 95% ci 3.31246.95 , p = 0.002 ) . table 4 presents the results of multiple linear regression analysis evaluating factors associated with higher severity of ild according to the hcrt scores . after the adjustment for age , disease duration , das28 , and mtx duration , we observed that the anti - ccp2 titers were significantly associated ( p = 0.02 ) with higher severity of the extension in the ground - glass score . similarly , after adjustment for age , das28 for the higher fibrosis score was significantly associated with higher disease duration ( p = 0.01 ) , anti - ccp2 titers ( p < 0.001 ) , and duration of treatment with mtx ( p < 0.001 ) . in the present study , anti - ccp2 titers were associated with the presence and severity of ild in ra . these anti - ccp2 titers were correlated with impairment in several parameters for ild severity , including the sgrq , 6mwt , borg scales , decrease in fvc% , and higher scores for ild involvement and severity identified in the hrct severity . an association between ild and anti - ccp2 titers remained after adjustment for age , disease duration , and exposure to smoke , in the multivariate model and duration of treatment with mtx , whereas positive rf was also a factor associated with ild . additionally , we observed that the only factors that predicted in the multivariate linear regression the higher scores for fibrosis score were anti - ccp2 titers and longer mtx treatment duration ; instead , higher fibrosis scores were inversely associated with disease duration . , who observed that anti - ccp2 titers are significantly higher in patients with ra who had ild . included only 18 patients with ild associated with ra , and , therefore , because of the small sample evaluated , it is likely that this lack of differences can be explained by a type ii error . identified ild in 71 from their 285 patients with ra , observing that positivity for second - generation anti - ccp ( anti - ccp2 ) was associated with an increase in risk of ild . these authors identified that anti - ccp antibody titers were comprised of the most relevant factor associated with ild in ra on univariate analysis and this factor remained associated with ild in the multivariate approach . these authors identified that 63% of acpa - positive patients had abnormalities on hrct , compared with 37% of acpa - negative patients ( p = 0.02 ) . to date , to the best of our knowledge , there are no studies evaluating if the anti - ccp2 titers are associated with the extent and patterns of severity of lung involvement in ild - ra . we reported that high titers of these autoantibodies are associated with a higher extent of lung involvement in our patients even after adjustment for other variables . reported higher levels of anti - ccp in serum and bronchoalveolar lavages in patients with ra , and these antibodies are increased in the patients with ra who had well - developed inducible bronchus - associated lymphoid tissue , suggesting that these antibodies are produced locally in the lungs . citrullinated proteins in the lungs are currently considered as autoantigens that may trigger an immune response associated with the development of anti - ccp2 and other antibodies that may act as markers associated with the tissue damage . in addition to anti - ccp2 levels , rf and elevated esr were biomarkers associated with ild in ra on univariate analysis . an association between positive rf and ild has been previously identified by several studies [ 1113 ] ; our findings are consistent with their results , whereas our finding of an elevated esr in patients with ild in ra is inconsistent with the majority of the reported data . although in our study nearly all of the patients with ra received mtx at the time of the study , we were unable to identify if the patients not treated with this drug had lower risk for ild . related to the association observed between anti - ccp2 and ra - ild , acpas are specific for ra and correspond to a subset of ra that is distinct from ra acpa - negative in terms of pathogenesis , disease prognosis , and response to therapy . , in a meta - analysis , observed that acpa - positive serum indicated a higher risk for ild and interstitial pulmonary fibrosis ( ipf ) among patients with ra ( or , 4.679 , 95% ci 2.07110.572 , p < 0.001 ) . observed high serum acpa titer associated with ra - ild , after adjustment for confounding factors ( age , sex , current or former smoking , and fr ) . a diagnosis of ild in ra identifies a patient with higher risk for the worst prognosis considering that the median survival in ra - ild is around 10 years shorter than that observed in the general population , lung disease being directly responsible of around 10 to 20% of all ra - associated mortalities . identified in a systematic review that the extent of fibrosis and usual interstitial pneumonia is a predictor of mortality in ra - ild . several studies observed that the positivity of anti - ccp2 and rf is associated with ra - ild . our study also observed in the multivariate logistic regression analysis that anti - ccp2 and rf were associated with ra - ild . observed that positive rates of anti - ccp2 and rf in patients with ra - ild were significantly higher than those of the patients with ra only . in our results , we are surprised that a shorter duration of disease was associated with the fibrosis score in the tomographic findings ; several studies have associated the presence of acpa - positive with the presence of pulmonary damage , mainly interstitial pulmonary and fibrosis pulmonary pattern ; however , to our knowledge , no study has linked shorter duration of disease with ra - ild and positive acpa . this a cross - sectional design ; therefore , it is unable to demonstrate the causality of any variable for the development of ild in ra ; however , our findings of the association between anti - ccp2 titers and the presence and severity of this complication are relevant for further studies in experimental models or longitudinal studies . thus , we have no information concerning the histological pattern exhibited by patients with ild , and it would be interesting to evaluate whether these high anti - ccp2 titers may correlate with the histologic patterns in lung tissue involvement . on the other hand , to the best of our knowledge , there are no previous studies assessing whether the severity of ild in ra is associated with higher titers of anti - ccp2 . these findings of higher anti - ccp2 titers and ild severity are not only limited to the hcrt score ; they are also associated with other characteristics of impairment in ild , such as decreased 6mwt , increases in the score for symptoms or impact in sgrq , and decreases in fvc% . another strength of the study was the utilization of a multivariate model to adjust the association of anti - ccp2 antibodies with ild by other confounders ; only two studies have previously used this statistical approach [ 10 , 13 ] , obtaining similar results to those observed in our study . in this respect , after adjustment for different factors , we observed that the relationship between the titers of these autoantibodies and the severity of ild remained significant on multivariate analysis . in conclusion , we identified that anti - ccp2 titers constitute an independent factor associated not only with the presence but also with the severity of ra - ild ; the relevance of these markers in patients with established ild for future outcomes , such as progression of lung involvement and mortality , remains to be established .

we identified beneficiaries with type 2 diabetes defined as follows : at least one inpatient or two outpatient ( separated by 7270 days ) icd-9 diagnoses ( icd-9 250.x0 or 250.x2 ) occurring during the 12 months preceding part d enrollment or during the first 4 months after part d enrollment . among these patients , we used the part d prescription drug event file ( pde ) to identify those with one or more prescription fill for insulin of any type during their first 4 months of part d enrollment who followed achievement of diagnostic inclusion criteria . we used the first 4 months of part d records following achievement of inclusion criteria to assign patients to one of three insulin treatment groups : 1 ) glargine only ( glargine ) , 2 ) nonglargine only ( nonglargine ) , and 3 ) glargine plus nonglargine insulin ( combination ) ( supplementary fig . a mean daily insulin dose was calculated for each patient based on dispensing in part d months 2 through 4 and categorized into quartiles ; implausibly high doses were set equal to the 99th percentile . the first 4-month fill records were also used to identify use of metformin , which may lower cancer risk , or other medications suspected of increasing cancer risk ( oral estrogen and tumor necrosis factor- inhibitors ) ( 1618 ) . antineoplastic prescription fills were used to exclude patients with prevalent cancers who did not have a corresponding anatomically specific icd-9 cancer code ( see below ) and patients on antineoplastic prophylaxis ( e.g. , tamoxifen ) . prevalent cancers were defined as those appearing during the 36 months preceding part d enrollment . we defined cancer broadly as one inpatient or two outpatient cancer diagnoses ( icd-9 140239 , excluding v codes ) ( separated by 7270 days ) . based on the literature , cancers were classified using clinical classification software ( ccs ) as breast , colon , pancreas , prostate , other anatomically specific ( ccs categories 1140 ) , or unspecified incident cancer diagnoses ( our main measure ) were defined by the same method but occurred after the 4-month treatment classification period . patients with prevalent , anatomically specific cancers were included in the cohort and permitted to contribute an anatomically distinct cancer . patients with unspecified cancers in a 36-month look back ( ccs categories 4144 ) and patients filling an antineoplastic drug prescription in the first 4 months of part d enrollment with no anatomically specific cancer diagnosis were excluded . covariates included were age ( categorized as 6869 , 7074 , 7579 , 8084 , and over 84 years ) , race / ethnicity ( black , hispanic , or other ) , sex , and part d low - income subsidy status , a measure of poverty ( dichotomized ) ( 20 ) . we included the following comorbidities if diagnosed once on an inpatient claim or twice on outpatient claims during the 36-month look back : obesity diagnosis , tobacco exposure ( one inpatient or two outpatient diagnoses of tobacco use or chronic obstructive pulmonary disease ) , and charlson comorbidities excluding malignancy , diabetes , and tobacco exposure ( 21 ) . diabetes complications diagnosed during the 36-month look back were included as a proxy measure of diabetes severity and duration ( diabetic retinopathy , nephropathy , neuropathy , and diabetes - associated peripheral circulatory disorder ) . the study included five incident cancer measures : any type , breast , pancreas , colon , and prostate . patients were followed from follow - up initiation until the first of these events : death , an incident cancer diagnosis , or censoring ( end of study 31 december 2008 or fee - for - service medicare disenrollment ) ( supplementary fig . 1 ) . cox proportional hazards regression models estimated the hr for each cancer type associated with treatment group relative to the nonglargine group . we repeated these main models stratified by age - group ( 75 and > 75 years ) because u.s . screening guidelines recommend breast and colon cancer screening up to age 75 years and explicitly recommend against prostate screening after age 75 years ( 22 ) . we felt this would impact the observed effect of exposure by age - group because testing and events would likely drop off . to further explore dose - dependent cancer promotion , we conducted secondary analyses including only patients whose calculated daily insulin use was in the highest quartile overall . this analysis was expected to isolate a relatively homogeneous subcohort with the highest exposure and likely highest rates of other cancer risks such as extreme obesity , more advanced diabetes , and , possibly , longer duration of diabetes . we repeated age stratification for this highest - dose subcohort to further isolate relatively homogeneous subgroups . other subanalyses repeated models after excluding patients with a cancer diagnosis in the 36-month look back . to test for possible indication bias that could result if prescribers selectively avoided glargine in patients with a personal history of cancer , logistic models assessed the relationship between cancer during the look back and treatment category assignment . we used the first 4 months of part d records following achievement of inclusion criteria to assign patients to one of three insulin treatment groups : 1 ) glargine only ( glargine ) , 2 ) nonglargine only ( nonglargine ) , and 3 ) glargine plus nonglargine insulin ( combination ) ( supplementary fig . a mean daily insulin dose was calculated for each patient based on dispensing in part d months 2 through 4 and categorized into quartiles ; implausibly high doses were set equal to the 99th percentile . the first 4-month fill records were also used to identify use of metformin , which may lower cancer risk , or other medications suspected of increasing cancer risk ( oral estrogen and tumor necrosis factor- inhibitors ) ( 1618 ) . antineoplastic prescription fills were used to exclude patients with prevalent cancers who did not have a corresponding anatomically specific icd-9 cancer code ( see below ) and patients on antineoplastic prophylaxis ( e.g. , tamoxifen ) . prevalent cancers were defined as those appearing during the 36 months preceding part d enrollment . we defined cancer broadly as one inpatient or two outpatient cancer diagnoses ( icd-9 140239 , excluding v codes ) ( separated by 7270 days ) . based on the literature , cancers were classified using clinical classification software ( ccs ) as breast , colon , pancreas , prostate , other anatomically specific ( ccs categories 1140 ) , or incident cancer diagnoses ( our main measure ) were defined by the same method but occurred after the 4-month treatment classification period . patients with prevalent , anatomically specific cancers were included in the cohort and permitted to contribute an anatomically distinct cancer . patients with unspecified cancers in a 36-month look back ( ccs categories 4144 ) and patients filling an antineoplastic drug prescription in the first 4 months of part d enrollment with no anatomically specific cancer diagnosis were excluded . covariates included were age ( categorized as 6869 , 7074 , 7579 , 8084 , and over 84 years ) , race / ethnicity ( black , hispanic , or other ) , sex , and part d low - income subsidy status , a measure of poverty ( dichotomized ) ( 20 ) . we included the following comorbidities if diagnosed once on an inpatient claim or twice on outpatient claims during the 36-month look back : obesity diagnosis , tobacco exposure ( one inpatient or two outpatient diagnoses of tobacco use or chronic obstructive pulmonary disease ) , and charlson comorbidities excluding malignancy , diabetes , and tobacco exposure ( 21 ) . diabetes complications diagnosed during the 36-month look back were included as a proxy measure of diabetes severity and duration ( diabetic retinopathy , nephropathy , neuropathy , and diabetes - associated peripheral circulatory disorder ) . the study included five incident cancer measures : any type , breast , pancreas , colon , and prostate . patients were followed from follow - up initiation until the first of these events : death , an incident cancer diagnosis , or censoring ( end of study 31 december 2008 or fee - for - service medicare disenrollment ) ( supplementary fig . cox proportional hazards regression models estimated the hr for each cancer type associated with treatment group relative to the nonglargine group . we repeated these main models stratified by age - group ( 75 and > 75 years ) because u.s . screening guidelines recommend breast and colon cancer screening up to age 75 years and explicitly recommend against prostate screening after age 75 years ( 22 ) . we felt this would impact the observed effect of exposure by age - group because testing and events would likely drop off . to further explore dose - dependent cancer promotion , we conducted secondary analyses including only patients whose calculated daily insulin use was in the highest quartile overall . this analysis was expected to isolate a relatively homogeneous subcohort with the highest exposure and likely highest rates of other cancer risks such as extreme obesity , more advanced diabetes , and , possibly , longer duration of diabetes . we repeated age stratification for this highest - dose subcohort to further isolate relatively homogeneous subgroups . other subanalyses repeated models after excluding patients with a cancer diagnosis in the 36-month look back . to test for possible indication bias that could result if prescribers selectively avoided glargine in patients with a personal history of cancer , logistic models assessed the relationship between cancer during the look back and treatment category assignment . table 1 presents the distribution of patients by treatment group : glargine 20.7% ( 17,060 ) , nonglargine 60.5% ( 49,761 ) , and combination insulin 18.5% ( 15,385 ) . metformin use was more common in the glargine group ( 27.6% ) than in the nonglargine ( 16.8% ) and combination insulin ( 15.0% ) groups . mean daily insulin dose was lower in the glargine group ( 52.7 units / day ) than in the nonglargine ( 73.4 units / day ) and combination insulin ( 62.7 units / day ) groups . characteristics and cancer events for older medicare beneficiaries with type 2 diabetes filling one or more insulin prescription during 20062007 : overall and by insulin treatment category data are means sd or n ( % ) . race / ethnicity groups are obtained from medicare denominator file ( research triangle institute ) race indicator variable . part d low - income subsidy is an indicator of income < 150% of federal poverty level . diabetes complications include diagnosis of diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , and diabetic peripheral vascular disease . charlson comorbidities from the 1987 journal of chronic disease ( 21 ) , modified to exclude diabetes , cancer , and tobacco use . tobacco - related lung disease is included separately in combination with tobacco use diagnosis as the tobacco exposure variable . and f tests were used for statistical testing of any significant difference across groups . the distribution of each category of characteristics and comorbidities was significantly different across groups , with p < 0.0001 for all . distribution of cancer events during look back was not significantly different across treatment groups ( p > 0.05 ) . we identified 5,466 incident cancer cases : 553 breast , 428 colon , 204 pancreas , and 427 prostate cancers ( table 2 ) . crude cancer incidence overall and by anatomic site was not significantly different across treatment groups . in fully adjusted cox proportional hazards regression models ( table 3 ) , compared with users of nonglargine , we found no association between glargine and risk of cancer of the breast , pancreas , prostate , or any location ; colon cancer risk was lower among glargine users ( hr 0.75 [ 95% ci 0.580.98 ] ) . metformin use was associated with a higher risk of breast cancer ( 1.28 [ 1.051.57 ] ) . the addition of indicator variables for quartile of daily insulin dose in these main models resulted in essentially identical estimates ; no estimates associated with dose quartile were significant . analyses stratified by age - group ( 75 and > 75 years ) produced estimates similar to unstratified models ; glargine and combination insulin were associated with no significant increased risk of any cancer measure , though estimates diverged substantially for the two age - groups in breast and prostate cancer models ( table 3 ) . combination insulin use was associated with a significantly lower risk of pancreas cancer in the older stratum ( 0.5 [ 0.280.91 ] ) . crude cancer events during follow - up : overall and in the highest daily insulin dose quartile data are n ( rate per 1,000 person - years ) . i d , insufficient data for reporting under regulations of the centers for medicare & medicaid services . difference across groups was assessed with log - rank tests . the centers for medicare & medicaid services does not permit reporting of cell counts < 11 or , cell counts permitting , by extrapolation , inference about cells with counts < 11 . cox proportional hazards regression for incident cancer events : overall and age stratified reference insulin use is nonglargine only . tobacco exposure is defined as a diagnosis of tobacco - related lung disease or diagnosis of tobacco use . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator ) , 14 charlson comorbidities , and tobacco exposure diagnosis . models including quartile of mean daily dose ; the lowest dose quartile is the referent . mean insulin units per day : 32 , 47 , 71 , and 119 for dose quartiles 14 , respectively . for overall with and without dose quartile variables : models include all events listed in table 2 . for age - stratified data for those aged > 75 years : models include 308 breast , 259 colon , 127 pancreatic , 226 prostate , and 3,003 any cancer cases . for age - stratified data for those aged 75 years : models include 245 breast , 169 colon , 77 pancreas , 201 prostate , and 2,463 any cancer cases . models including only the 20,415 patients in the highest daily insulin dose quartile ( mean dose 119 units / day ) ( 11.7% glargine , 75.3% nonglargine , and 13.0% combination insulin ) revealed no association between glargine - only use and cancer ( table 4 ) . in contrast , high - dose combination insulin was associated with an increased risk of breast cancer ( hr 1.75 [ 95% ci 1.102.78 ] ) and a lower risk of colon cancer ( 0.33 [ 0.130.80 ] ) . age - group stratified analyses of individuals in the highest daily dose quartile revealed no significant associations between treatment and cancer for those over 75 years old ( n = 11,580 ) . among younger patients ( 75 years ) in the highest daily dose quartile ( n = 8,835 ) , glargine alone was associated with no significant risk differences but high - dose combination insulin was associated with a higher risk of breast cancer ( 2.87 [ 1.475.59 ] ) ; these models included 626 any cancer events and 54 breast cancer events . secondary analyses : cox proportional hazards model estimates for the subcohort of patients in the highest quartile of insulin dose : overall and stratified by age - group calculated mean daily dose : 119 units / day . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator),14 charlson comorbidities , and tobacco exposure diagnosis . all ages , n = 20,415 : 11.7% glargine users , 75.3% nonglargine users , and 13.0% combination users . cancer events for this cohort : breast 76 , colon 73 , pancreas 21 , prostate 60 , and any 726 . cancer events for this cohort : breast 54 , colon 40 , pancreas 19 , prostate 34 , and any 626 . secondary analyses including only the 72,314 patients with no cancer during the 36-month look back yielded estimates essentially identical to main models . a logistic regression assessing the relationship between a cancer diagnosis during the look back period and treatment group revealed slightly increased odds of glargine - only use among patients with prevalent cancer ( compared with no prevalent cancer ) ( odds ratio 1.06 [ 95% ci 1.001.12 ] ; p = 0.043 ) . cancer during look back was not significantly associated with combination insulin use ( data not shown ) . table 1 presents the distribution of patients by treatment group : glargine 20.7% ( 17,060 ) , nonglargine 60.5% ( 49,761 ) , and combination insulin 18.5% ( 15,385 ) . metformin use was more common in the glargine group ( 27.6% ) than in the nonglargine ( 16.8% ) and combination insulin ( 15.0% ) groups . mean daily insulin dose was lower in the glargine group ( 52.7 units / day ) than in the nonglargine ( 73.4 units / day ) and combination insulin ( 62.7 units / day ) groups . characteristics and cancer events for older medicare beneficiaries with type 2 diabetes filling one or more insulin prescription during 20062007 : overall and by insulin treatment category data are means sd or n ( % ) . race / ethnicity groups are obtained from medicare denominator file ( research triangle institute ) race indicator variable . part d low - income subsidy is an indicator of income < 150% of federal poverty level . diabetes complications include diagnosis of diabetic retinopathy , diabetic nephropathy , diabetic neuropathy , and diabetic peripheral vascular disease . charlson comorbidities from the 1987 journal of chronic disease ( 21 ) , modified to exclude diabetes , cancer , and tobacco use . tobacco - related lung disease is included separately in combination with tobacco use diagnosis as the tobacco exposure variable . and f tests were used for statistical testing of any significant difference across groups . the distribution of each category of characteristics and comorbidities was significantly different across groups , with p < 0.0001 for all . distribution of cancer events during look back was not significantly different across treatment groups ( p > 0.05 ) . we identified 5,466 incident cancer cases : 553 breast , 428 colon , 204 pancreas , and 427 prostate cancers ( table 2 ) . crude cancer incidence overall and by anatomic site was not significantly different across treatment groups . in fully adjusted cox proportional hazards regression models ( table 3 ) , compared with users of nonglargine , we found no association between glargine and risk of cancer of the breast , pancreas , prostate , or any location ; colon cancer risk was lower among glargine users ( hr 0.75 [ 95% ci 0.580.98 ] ) . metformin use was associated with a higher risk of breast cancer ( 1.28 [ 1.051.57 ] ) . the addition of indicator variables for quartile of daily insulin dose in these main models resulted in essentially identical estimates ; no estimates associated with dose quartile were significant . analyses stratified by age - group ( 75 and > 75 years ) produced estimates similar to unstratified models ; glargine and combination insulin were associated with no significant increased risk of any cancer measure , though estimates diverged substantially for the two age - groups in breast and prostate cancer models ( table 3 ) . combination insulin use was associated with a significantly lower risk of pancreas cancer in the older stratum ( 0.5 [ 0.280.91 ] ) . daily insulin dose quartile data are n ( rate per 1,000 person - years ) . for the highest i d , insufficient data for reporting under regulations of the centers for medicare & medicaid services . the centers for medicare & medicaid services does not permit reporting of cell counts < 11 or , cell counts permitting , by extrapolation , inference about cells with counts < 11 . cox proportional hazards regression for incident cancer events : overall and age stratified reference insulin use is nonglargine only . tobacco exposure is defined as a diagnosis of tobacco - related lung disease or diagnosis of tobacco use . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator ) , 14 charlson comorbidities , and tobacco exposure diagnosis . models including quartile of mean daily dose ; the lowest dose quartile is the referent . mean insulin units per day : 32 , 47 , 71 , and 119 for dose quartiles 14 , respectively . for overall with and without dose quartile variables : models include all events listed in table 2 . for age - stratified data for those aged > 75 years : models include 308 breast , 259 colon , 127 pancreatic , 226 prostate , and 3,003 any cancer cases . for age - stratified data for those aged 75 years : models include 245 breast , 169 colon , 77 pancreas , 201 prostate , and 2,463 any cancer cases . models including only the 20,415 patients in the highest daily insulin dose quartile ( mean dose 119 units / day ) ( 11.7% glargine , 75.3% nonglargine , and 13.0% combination insulin ) revealed no association between glargine - only use and cancer ( table 4 ) . in contrast , high - dose combination insulin was associated with an increased risk of breast cancer ( hr 1.75 [ 95% ci 1.102.78 ] ) and a lower risk of colon cancer ( 0.33 [ 0.130.80 ] ) . age - group stratified analyses of individuals in the highest daily dose quartile revealed no significant associations between treatment and cancer for those over 75 years old ( n = 11,580 ) . among younger patients ( 75 years ) in the highest daily dose quartile ( n = 8,835 ) , glargine alone was associated with no significant risk differences but high - dose combination insulin was associated with a higher risk of breast cancer ( 2.87 [ 1.475.59 ] ) ; these models included 626 any cancer events and 54 breast cancer events . secondary analyses : cox proportional hazards model estimates for the subcohort of patients in the highest quartile of insulin dose : overall and stratified by age - group calculated mean daily dose : 119 units / day . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator),14 charlson comorbidities , and tobacco exposure diagnosis . all ages , n = 20,415 : 11.7% glargine users , 75.3% nonglargine users , and 13.0% combination users . cancer events for this cohort : breast 76 , colon 73 , pancreas 21 , prostate 60 , and any 726 . cancer events for this cohort : breast 54 , colon 40 , pancreas 19 , prostate 34 , and any 626 . secondary analyses including only the 72,314 patients with no cancer during the 36-month look back yielded estimates essentially identical to main models . a logistic regression assessing the relationship between a cancer diagnosis during the look back period and treatment group revealed slightly increased odds of glargine - only use among patients with prevalent cancer ( compared with no prevalent cancer ) ( odds ratio 1.06 [ 95% ci 1.001.12 ] ; p = 0.043 ) . cancer during look back was not significantly associated with combination insulin use ( data not shown ) . in a large cohort of older patients with type 2 diabetes and substantial use of glargine , we found no significant increased risk of cancer among glargine - only users compared with nonglargine insulin users . in the main models including all age - groups , overall , these principal findings are reassuring . results of secondary analyses including only the subcohort of highest - dose users raise new questions . these models revealed a higher risk of breast cancer among high - dose combination insulin users compared with high - dose nonglargine users , especially among patients aged 75 years and younger . this association between breast cancer and high - dose combination insulin use has not previously been reported . in contrast , the two previously published studies associating glargine - only use with breast cancer found no increased risk associated with combination insulin use in models that did not account for dose ( 7,8 ) . the increased risks we observed in the younger age stratum ( 75 years ) suggest that age may be an effect modifier acting either through physiologic pathways , healthy survivor bias of the older stratum , or detection bias among patients more likely to receive screening tests for the outcome of interest . it is also possible that this younger group suffers disproportionate residual confounding due to more extreme obesity , longer duration of disease , greater insulin resistance , or other unmeasured breast cancer risks . the juxtaposition of treatment - associated higher risk for breast cancers in some models and lower risk of distinct cancers ( colon and pancreas ) in other models raises further questions about the overall findings . it is unclear whether this reflects diverse insulin sensitivity of distinct tumor types , the result of multiple comparisons , or unmeasured confounding . the small but consistent association between metformin and breast cancer was unexpected ( 18 ) . ( 9 ) in which metformin was not associated with lower breast or prostate cancer risk but was associated with lower risk of other cancers . more extreme obesity is an indication for metformin use and a risk factor for breast cancer ; this may explain some or all of the association . due to common use of glargine in the u.s . , the number of glargine - only users and the number of incident cancers among glargine users were larger than those of previous studies . indications for glargine insulin and combination insulin may differ in the u.s . compared with european nations . the subcohort patients on very high doses of insulin likely have disproportionately high insulin resistance , which is associated with obesity , an independent risk factor for breast cancer ( 23 ) . studies have demonstrated that medicare administrative data can identify cancer cases with good specificity ( 98% ) and acceptable sensitivity ( 8390% ) ( 24 ) . in this analysis , we used a 36-month look back period to identify prevalent cancer cases and assumed patients with no cancer diagnosis during this period to be cancer free . some recurrent cancer cases could have been misclassified as incident , but we believe such cases to be rare . we lack data on important clinical risk factors for cancer including undiagnosed tobacco use , women s age at first birth , bmi , family history of cancer , and environmental exposures . some previous studies addressing this research question had many of these data elements but found none significant in analytic models ( 79 ) . this precludes estimating cumulative lifetime exposure , as well as the impact of early exposure in the majority of our patients . the key assumption is that the unmeasured previous exposure correlates with measured exposure , and whereas crossover previous studies found incident cancer associated with glargine after follow - up times as short as 1.3 years . if glargine acts rapidly to promote preclinical cancers , our cohort may be biased because of the resulting attenuation in healthy glargine survivors . lastly , we rely on dose dispensed rather than dose administered , a reasonable but imperfect measure of actual use . this study finds no associations between glargine - only use ( at any dose ) and increased risk of cancer . , we find a higher risk of breast cancer among users of high - dose combination insulin therapy , especially among relatively young patients ( 75 years ) . our findings reinforce null results reported by most research groups assessing the risk of cancer in association with glargine - only use . regarding treatment - associated risk of breast cancer specifically , our results echo ( but do not replicate ) the results of some while contradicting others . this suggests that breast cancer in particular deserves focused attention in future research , ideally conducted with data and methods that can optimally adjust for individual breast cancer risk factors . the use of glargine will likely expand as the prevalence of type 2 diabetes increases . resolving this question

objectivein vitro evidence suggests insulin glargine promotes tumors ; observational human studies are conflicting . we aimed to expand understanding of this potential treatment risk.research design and methodsthis retrospective cohort study of type 2 diabetic patients > 68 years old used medicare inpatient , outpatient ( 20032008 ) , and prescription data ( 20062008 ) . adjusting for patient characteristics , dose , and metformin use , cox models yielded hazard ratios ( hrs ) for incident cancer ( breast , prostate , pancreas , colon , any site ) associated with three forms of insulin : nonglargine , glargine , or glargine plus nonglargine ( combination).resultsoverall , 81,681 patients were followed for a mean of 23.1 months . mean age was 77.4 years . treatment group distribution was 20.7% glargine , 60.5% nonglargine , 18.7% combination insulin . we observed 5,466 incident cancers ; crude rates did not vary by treatment group . in fully adjusted models , nonglargine use was the referent ; glargine was not associated with significant increased risk of any cancer measure . in secondary analyses including only the top quartile of daily insulin dose patients , glargine was not associated with any cancer risk difference ; combination insulin was associated with higher breast cancer risk ( hr 1.75 [ 95% ci 1.102.78 ] ) and lower colon cancer risk ( 0.33 [ 0.130.80 ] ) . in age - stratified analyses of highest - dose users , combination insulin conferred a higher risk of breast cancer in those 75 years old ( 2.87 [ 1.451.59]).conclusionsthe general lack of association between glargine - only use and cancer is reassuring . breast cancer risk associated with high - dose combination insulin in secondary analyses could result from multiple comparisons , residual confounding , or true association ; further research is warranted .

RESEARCH DESIGN AND METHODS Exposure/treatment group assignment Cancer measures Covariates Analysis Primary analyses Secondary analyses RESULTS Patients Primary analyses Secondary analyses CONCLUSIONS Supplementary Material

we identified beneficiaries with type 2 diabetes defined as follows : at least one inpatient or two outpatient ( separated by 7270 days ) icd-9 diagnoses ( icd-9 250.x0 or 250.x2 ) occurring during the 12 months preceding part d enrollment or during the first 4 months after part d enrollment . among these patients , we used the part d prescription drug event file ( pde ) to identify those with one or more prescription fill for insulin of any type during their first 4 months of part d enrollment who followed achievement of diagnostic inclusion criteria . we used the first 4 months of part d records following achievement of inclusion criteria to assign patients to one of three insulin treatment groups : 1 ) glargine only ( glargine ) , 2 ) nonglargine only ( nonglargine ) , and 3 ) glargine plus nonglargine insulin ( combination ) ( supplementary fig . a mean daily insulin dose was calculated for each patient based on dispensing in part d months 2 through 4 and categorized into quartiles ; implausibly high doses were set equal to the 99th percentile . the first 4-month fill records were also used to identify use of metformin , which may lower cancer risk , or other medications suspected of increasing cancer risk ( oral estrogen and tumor necrosis factor- inhibitors ) ( 1618 ) . based on the literature , cancers were classified using clinical classification software ( ccs ) as breast , colon , pancreas , prostate , other anatomically specific ( ccs categories 1140 ) , or unspecified incident cancer diagnoses ( our main measure ) were defined by the same method but occurred after the 4-month treatment classification period . covariates included were age ( categorized as 6869 , 7074 , 7579 , 8084 , and over 84 years ) , race / ethnicity ( black , hispanic , or other ) , sex , and part d low - income subsidy status , a measure of poverty ( dichotomized ) ( 20 ) . we included the following comorbidities if diagnosed once on an inpatient claim or twice on outpatient claims during the 36-month look back : obesity diagnosis , tobacco exposure ( one inpatient or two outpatient diagnoses of tobacco use or chronic obstructive pulmonary disease ) , and charlson comorbidities excluding malignancy , diabetes , and tobacco exposure ( 21 ) . the study included five incident cancer measures : any type , breast , pancreas , colon , and prostate . patients were followed from follow - up initiation until the first of these events : death , an incident cancer diagnosis , or censoring ( end of study 31 december 2008 or fee - for - service medicare disenrollment ) ( supplementary fig . cox proportional hazards regression models estimated the hr for each cancer type associated with treatment group relative to the nonglargine group . we repeated these main models stratified by age - group ( 75 and > 75 years ) because u.s . screening guidelines recommend breast and colon cancer screening up to age 75 years and explicitly recommend against prostate screening after age 75 years ( 22 ) . to further explore dose - dependent cancer promotion , we conducted secondary analyses including only patients whose calculated daily insulin use was in the highest quartile overall . we repeated age stratification for this highest - dose subcohort to further isolate relatively homogeneous subgroups . we used the first 4 months of part d records following achievement of inclusion criteria to assign patients to one of three insulin treatment groups : 1 ) glargine only ( glargine ) , 2 ) nonglargine only ( nonglargine ) , and 3 ) glargine plus nonglargine insulin ( combination ) ( supplementary fig . a mean daily insulin dose was calculated for each patient based on dispensing in part d months 2 through 4 and categorized into quartiles ; implausibly high doses were set equal to the 99th percentile . the first 4-month fill records were also used to identify use of metformin , which may lower cancer risk , or other medications suspected of increasing cancer risk ( oral estrogen and tumor necrosis factor- inhibitors ) ( 1618 ) . antineoplastic prescription fills were used to exclude patients with prevalent cancers who did not have a corresponding anatomically specific icd-9 cancer code ( see below ) and patients on antineoplastic prophylaxis ( e.g. based on the literature , cancers were classified using clinical classification software ( ccs ) as breast , colon , pancreas , prostate , other anatomically specific ( ccs categories 1140 ) , or incident cancer diagnoses ( our main measure ) were defined by the same method but occurred after the 4-month treatment classification period . covariates included were age ( categorized as 6869 , 7074 , 7579 , 8084 , and over 84 years ) , race / ethnicity ( black , hispanic , or other ) , sex , and part d low - income subsidy status , a measure of poverty ( dichotomized ) ( 20 ) . we included the following comorbidities if diagnosed once on an inpatient claim or twice on outpatient claims during the 36-month look back : obesity diagnosis , tobacco exposure ( one inpatient or two outpatient diagnoses of tobacco use or chronic obstructive pulmonary disease ) , and charlson comorbidities excluding malignancy , diabetes , and tobacco exposure ( 21 ) . the study included five incident cancer measures : any type , breast , pancreas , colon , and prostate . patients were followed from follow - up initiation until the first of these events : death , an incident cancer diagnosis , or censoring ( end of study 31 december 2008 or fee - for - service medicare disenrollment ) ( supplementary fig . cox proportional hazards regression models estimated the hr for each cancer type associated with treatment group relative to the nonglargine group . we repeated these main models stratified by age - group ( 75 and > 75 years ) because u.s . screening guidelines recommend breast and colon cancer screening up to age 75 years and explicitly recommend against prostate screening after age 75 years ( 22 ) . to further explore dose - dependent cancer promotion , we conducted secondary analyses including only patients whose calculated daily insulin use was in the highest quartile overall . we repeated age stratification for this highest - dose subcohort to further isolate relatively homogeneous subgroups . table 1 presents the distribution of patients by treatment group : glargine 20.7% ( 17,060 ) , nonglargine 60.5% ( 49,761 ) , and combination insulin 18.5% ( 15,385 ) . metformin use was more common in the glargine group ( 27.6% ) than in the nonglargine ( 16.8% ) and combination insulin ( 15.0% ) groups . mean daily insulin dose was lower in the glargine group ( 52.7 units / day ) than in the nonglargine ( 73.4 units / day ) and combination insulin ( 62.7 units / day ) groups . we identified 5,466 incident cancer cases : 553 breast , 428 colon , 204 pancreas , and 427 prostate cancers ( table 2 ) . in fully adjusted cox proportional hazards regression models ( table 3 ) , compared with users of nonglargine , we found no association between glargine and risk of cancer of the breast , pancreas , prostate , or any location ; colon cancer risk was lower among glargine users ( hr 0.75 [ 95% ci 0.580.98 ] ) . metformin use was associated with a higher risk of breast cancer ( 1.28 [ 1.051.57 ] ) . the addition of indicator variables for quartile of daily insulin dose in these main models resulted in essentially identical estimates ; no estimates associated with dose quartile were significant . analyses stratified by age - group ( 75 and > 75 years ) produced estimates similar to unstratified models ; glargine and combination insulin were associated with no significant increased risk of any cancer measure , though estimates diverged substantially for the two age - groups in breast and prostate cancer models ( table 3 ) . combination insulin use was associated with a significantly lower risk of pancreas cancer in the older stratum ( 0.5 [ 0.280.91 ] ) . crude cancer events during follow - up : overall and in the highest daily insulin dose quartile data are n ( rate per 1,000 person - years ) . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator ) , 14 charlson comorbidities , and tobacco exposure diagnosis . for age - stratified data for those aged > 75 years : models include 308 breast , 259 colon , 127 pancreatic , 226 prostate , and 3,003 any cancer cases . for age - stratified data for those aged 75 years : models include 245 breast , 169 colon , 77 pancreas , 201 prostate , and 2,463 any cancer cases . models including only the 20,415 patients in the highest daily insulin dose quartile ( mean dose 119 units / day ) ( 11.7% glargine , 75.3% nonglargine , and 13.0% combination insulin ) revealed no association between glargine - only use and cancer ( table 4 ) . in contrast , high - dose combination insulin was associated with an increased risk of breast cancer ( hr 1.75 [ 95% ci 1.102.78 ] ) and a lower risk of colon cancer ( 0.33 [ 0.130.80 ] ) . age - group stratified analyses of individuals in the highest daily dose quartile revealed no significant associations between treatment and cancer for those over 75 years old ( n = 11,580 ) . among younger patients ( 75 years ) in the highest daily dose quartile ( n = 8,835 ) , glargine alone was associated with no significant risk differences but high - dose combination insulin was associated with a higher risk of breast cancer ( 2.87 [ 1.475.59 ] ) ; these models included 626 any cancer events and 54 breast cancer events . secondary analyses : cox proportional hazards model estimates for the subcohort of patients in the highest quartile of insulin dose : overall and stratified by age - group calculated mean daily dose : 119 units / day . all ages , n = 20,415 : 11.7% glargine users , 75.3% nonglargine users , and 13.0% combination users . cancer events for this cohort : breast 76 , colon 73 , pancreas 21 , prostate 60 , and any 726 . cancer events for this cohort : breast 54 , colon 40 , pancreas 19 , prostate 34 , and any 626 . secondary analyses including only the 72,314 patients with no cancer during the 36-month look back yielded estimates essentially identical to main models . a logistic regression assessing the relationship between a cancer diagnosis during the look back period and treatment group revealed slightly increased odds of glargine - only use among patients with prevalent cancer ( compared with no prevalent cancer ) ( odds ratio 1.06 [ 95% ci 1.001.12 ] ; p = 0.043 ) . cancer during look back was not significantly associated with combination insulin use ( data not shown ) . table 1 presents the distribution of patients by treatment group : glargine 20.7% ( 17,060 ) , nonglargine 60.5% ( 49,761 ) , and combination insulin 18.5% ( 15,385 ) . metformin use was more common in the glargine group ( 27.6% ) than in the nonglargine ( 16.8% ) and combination insulin ( 15.0% ) groups . mean daily insulin dose was lower in the glargine group ( 52.7 units / day ) than in the nonglargine ( 73.4 units / day ) and combination insulin ( 62.7 units / day ) groups . characteristics and cancer events for older medicare beneficiaries with type 2 diabetes filling one or more insulin prescription during 20062007 : overall and by insulin treatment category data are means sd or n ( % ) . charlson comorbidities from the 1987 journal of chronic disease ( 21 ) , modified to exclude diabetes , cancer , and tobacco use . we identified 5,466 incident cancer cases : 553 breast , 428 colon , 204 pancreas , and 427 prostate cancers ( table 2 ) . in fully adjusted cox proportional hazards regression models ( table 3 ) , compared with users of nonglargine , we found no association between glargine and risk of cancer of the breast , pancreas , prostate , or any location ; colon cancer risk was lower among glargine users ( hr 0.75 [ 95% ci 0.580.98 ] ) . metformin use was associated with a higher risk of breast cancer ( 1.28 [ 1.051.57 ] ) . the addition of indicator variables for quartile of daily insulin dose in these main models resulted in essentially identical estimates ; no estimates associated with dose quartile were significant . analyses stratified by age - group ( 75 and > 75 years ) produced estimates similar to unstratified models ; glargine and combination insulin were associated with no significant increased risk of any cancer measure , though estimates diverged substantially for the two age - groups in breast and prostate cancer models ( table 3 ) . combination insulin use was associated with a significantly lower risk of pancreas cancer in the older stratum ( 0.5 [ 0.280.91 ] ) . daily insulin dose quartile data are n ( rate per 1,000 person - years ) . we also adjusted for age category , race / ethnicity , diabetes complications , obesity diagnosis , oral estrogen use , part d low - income subsidy ( a poverty indicator ) , 14 charlson comorbidities , and tobacco exposure diagnosis . for age - stratified data for those aged > 75 years : models include 308 breast , 259 colon , 127 pancreatic , 226 prostate , and 3,003 any cancer cases . for age - stratified data for those aged 75 years : models include 245 breast , 169 colon , 77 pancreas , 201 prostate , and 2,463 any cancer cases . models including only the 20,415 patients in the highest daily insulin dose quartile ( mean dose 119 units / day ) ( 11.7% glargine , 75.3% nonglargine , and 13.0% combination insulin ) revealed no association between glargine - only use and cancer ( table 4 ) . in contrast , high - dose combination insulin was associated with an increased risk of breast cancer ( hr 1.75 [ 95% ci 1.102.78 ] ) and a lower risk of colon cancer ( 0.33 [ 0.130.80 ] ) . age - group stratified analyses of individuals in the highest daily dose quartile revealed no significant associations between treatment and cancer for those over 75 years old ( n = 11,580 ) . among younger patients ( 75 years ) in the highest daily dose quartile ( n = 8,835 ) , glargine alone was associated with no significant risk differences but high - dose combination insulin was associated with a higher risk of breast cancer ( 2.87 [ 1.475.59 ] ) ; these models included 626 any cancer events and 54 breast cancer events . secondary analyses : cox proportional hazards model estimates for the subcohort of patients in the highest quartile of insulin dose : overall and stratified by age - group calculated mean daily dose : 119 units / day . all ages , n = 20,415 : 11.7% glargine users , 75.3% nonglargine users , and 13.0% combination users . cancer events for this cohort : breast 76 , colon 73 , pancreas 21 , prostate 60 , and any 726 . cancer events for this cohort : breast 54 , colon 40 , pancreas 19 , prostate 34 , and any 626 . secondary analyses including only the 72,314 patients with no cancer during the 36-month look back yielded estimates essentially identical to main models . a logistic regression assessing the relationship between a cancer diagnosis during the look back period and treatment group revealed slightly increased odds of glargine - only use among patients with prevalent cancer ( compared with no prevalent cancer ) ( odds ratio 1.06 [ 95% ci 1.001.12 ] ; p = 0.043 ) . cancer during look back was not significantly associated with combination insulin use ( data not shown ) . in a large cohort of older patients with type 2 diabetes and substantial use of glargine , we found no significant increased risk of cancer among glargine - only users compared with nonglargine insulin users . results of secondary analyses including only the subcohort of highest - dose users raise new questions . these models revealed a higher risk of breast cancer among high - dose combination insulin users compared with high - dose nonglargine users , especially among patients aged 75 years and younger . this association between breast cancer and high - dose combination insulin use has not previously been reported . in contrast , the two previously published studies associating glargine - only use with breast cancer found no increased risk associated with combination insulin use in models that did not account for dose ( 7,8 ) . the increased risks we observed in the younger age stratum ( 75 years ) suggest that age may be an effect modifier acting either through physiologic pathways , healthy survivor bias of the older stratum , or detection bias among patients more likely to receive screening tests for the outcome of interest . it is also possible that this younger group suffers disproportionate residual confounding due to more extreme obesity , longer duration of disease , greater insulin resistance , or other unmeasured breast cancer risks . the juxtaposition of treatment - associated higher risk for breast cancers in some models and lower risk of distinct cancers ( colon and pancreas ) in other models raises further questions about the overall findings . it is unclear whether this reflects diverse insulin sensitivity of distinct tumor types , the result of multiple comparisons , or unmeasured confounding . the small but consistent association between metformin and breast cancer was unexpected ( 18 ) . ( 9 ) in which metformin was not associated with lower breast or prostate cancer risk but was associated with lower risk of other cancers . more extreme obesity is an indication for metformin use and a risk factor for breast cancer ; this may explain some or all of the association . , the number of glargine - only users and the number of incident cancers among glargine users were larger than those of previous studies . the subcohort patients on very high doses of insulin likely have disproportionately high insulin resistance , which is associated with obesity , an independent risk factor for breast cancer ( 23 ) . the key assumption is that the unmeasured previous exposure correlates with measured exposure , and whereas crossover previous studies found incident cancer associated with glargine after follow - up times as short as 1.3 years . this study finds no associations between glargine - only use ( at any dose ) and increased risk of cancer . , we find a higher risk of breast cancer among users of high - dose combination insulin therapy , especially among relatively young patients ( 75 years ) . our findings reinforce null results reported by most research groups assessing the risk of cancer in association with glargine - only use . regarding treatment - associated risk of breast cancer specifically , our results echo ( but do not replicate ) the results of some while contradicting others . this suggests that breast cancer in particular deserves focused attention in future research , ideally conducted with data and methods that can optimally adjust for individual breast cancer risk factors . the use of glargine will likely expand as the prevalence of type 2 diabetes increases .

it is important to estimate the changes in tooth position before and after orthodontic treatment to determine whether the teeth have been moved according to the anchorage requirements or to the planned tooth movement of the treatment objectives . traditionally , the evaluation of tooth movement has been possible by superimposition of lateral cephalograms . the method can be technique sensitive and time - consuming while variable head positions in the cephalostat between serial radiographs may lead to errors in the comparison of treatment results . recent advances in computer technologies such as 3-dimensional ( 3d ) computed tomography ( ct ) , 3d surface scanning technology , computer - aided design , computer - aided manufacturing , and associated software have contributed not only to the precise diagnosis , treatment planning , and simulations , but also to the 3d evaluation of treatment results . although these methods involve additional radiation exposure and time - consuming procedure , more accurate 3d data can be acquired . on the maxillary dental arch , although little is known about the stability of identifiable landmarks on dental casts , palatal rugae has been suggested as relatively stable structures for registration of serial maxillary models.1 the shape of the palatal vault and the medial portions of the palatal rugae are fairly stable throughout the development of the dentition.2 the palatal rugae retain their shape and pattern throughout a person 's lifetime.3 thus , they have been used for identification purposes in forensics.4 almeida et al.5 and bailey et al.6 have studied the potential use of the palatal rugae for the superimposition of serial models and both studies concluded that specific parts of the palatal rugae ( e.g. , medial ) may be sufficiently stable to serve as anatomical references for superimposing serial maxillary models with headgear or premolar extraction treatment . hoggan and sadowsky7 suggested that certain landmarks on the palatal rugae can be used as reliably as cephalometric superimpositions to assess anteroposterior molar movement . technical developments technology now permit the 3d superimposition of the maxillary dental arch using 3d model scanning technology . ashmore et al.8 reported that the method developed for the superimposition of digital configurations of serial dental models allowed accurate measurements of translational movement of the maxillary first - molar in 3 dimensions for both headgear and untreated groups . miller et al.9 reported that the digital superimposition of the maxillary arch was reproducible and that the error associated with using palatal rugae as reference landmarks may be similar to or less than that of current 2d cephalometric analyses.10 cha et al.11 reported that use of a 3d digital orthodontic model superimposition technique on the maxillary dental arch was clinically as reliable as cephalometric superimposition for assessing orthodontic tooth movement . however , none of these studies were performed on the mandibular dental arch since no well - known stable reference points or areas for superimposition have been established on the mandibular dental arch . no accurate and reproducible method has been introduced to date to evaluate the degree of 3d orthodontic tooth movement on the mandibular dental arch despite the available technology . the purpose of this study was to introduce a method for mandibular dental arch superimposition in a non - gorwing patient by the combination of 3d cone beam ct data and 3d digital model data . the method was designed to use the basal bone area as the reference for superimposition while soft tissue or alveolar bone areas were avoided since they can be altered throughout orthodontic treatment . two different registration methods for superimposing the mandible were designed to evaluate their reproducibility as well as practical applicability . one adult patient with no growth potency who had visited the department of orthodontics , kyung hee dental hospital and undergone orthodontic treatment , was examined . cone beam computed tomography ( cbct ) scan and alginate impression were taken on the same day prior to treatment , and in accordance with the patient 's request , 1 day after debonding . the superimposion system consisted of 6 main procedures : ( 1 ) acquisition of cbct data , ( 2 ) acquisition of 3d digital model data , ( 3 ) integration of cbct data and digital model data , ( 4 ) superimposition of pre - treatment and post - treatment integrated cbct image , ( 5 ) extraction of the mandibular dentition portion , and ( 6 ) evaluation of tooth movement on the mandibular arch . craniofacial skeleton 3d cbct image data was acquired with an i - cat imaging device ( imaging sciences international , hatfield , pa , usa ) . the patient underwent cbct scanning in facial mode in 14-bit gray scale with 1.0 mm slice thickness , 1.0 mm voxel size , and image acquisition by single 360 degree rotation with a 40 s total scan time . the reconstructed digital data was directly transferred from the cbct scanner to a personal computer and stored as digital imaging and communications in medicine ( dicom ) files . both pre - treatment and post - treatment dicom data were transferred to rapidform 2006 ( inus technology inc . , the mandible was segmented by rapidform 2006 from the craniofacial skeleton image ( figure 2 ) . orapix scanner ; a 3d surface scanning system with a slit laser beam and non - contact measurement method ( kod-300 , orapix co. ltd . , seoul , korea ) was used to obtain 3d data from the dental cast model . both pre - treatment and post - treatment orthodontic 3d digital models are shown in figure 3 . to create a preliminary fusion model , the dentition reconstructed by cbct data was superimposed on the 3d digital model by a regional registration method . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . finally , both pre- and post - treatment digital models were integrated into each mandible model , and the bases of the digital model superimposed on each image were removed . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . two different registration methods of superimposing icit1 and icit2 were used to evaluate their practical applicability and to estimate their reproducibility ( figure 6 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the fully automated registration was computed by rapidform 2006 , with the limited manual process of selection and de - selection of the registration area . the inferior portion of mandibular body and the posterior portion of ramus were selected as the registration area . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . the procedure for plane superimposition was shown in figure 10 . before the 2 superimposition procedures the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . specially in this patient the mesiobuccal tip of the lower right 1st molar was also marked since it was found to be an ankylosed tooth . rapidform 2006 has a coordinate system , of which its origin is defined with the 0-point value by the software itself . the landmarks were established and selected on the 3d digital model image by 2 investigators using the rapidform program . the points assignment was repeated 5 times each by each investigator , and the mean coordinate values of each landmark were used to define the final coordinate values . the final coordinate values ( x , y , and z ) of each landmark were transferred to microsoft office excel 2007 ( microsoft , redmond , wa , usa ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . the software easily performed the re - addition procedure at the same position since the bases were previously constructed on the digital model . finally , the superimposed 3d mandibular dental arch was ready to be used for evaluation of the orthodontic tooth movement that occurred throughout treatment . the initially established landmarks consisted of 10 points on both pre- and post - treatment images . after applying the 2 methods of superimposition , the coordinate values ( x , y , z ) of the same landmarks were transferred to microsoft office excel 2007 . each method of superimposition was performed 30 times to estimate reproducibility ( steps 4 to 6 ) and all the coordinate values of post - treatment landmarks after each superimposition were transferred to microsoft office excel 2007 . first , the mean values and variances of each x- , y- , and z - coordinate values of the post - treatment landmarks were calculated with both the surface superimposition and plane superimposition methods . the equality of variance test was carried out for each x- , y- , and z - coordinate values as a scalar with matlab 7.5 ( minitab inc . , state college , pa , usa ) . the null hypothesis and alternative hypothesis were expressed as h0 : p < s and ha : p > s ( h0 : null hypothesis , ha : alternative hypothesis , p : variance of plane superimposition , s : variance of surface superimposition ) . the difference in variance between the 2 superimposition methods was 0 at a significance level of 0.05 . then the mean distance ( mean value of the distance from the mean coordinate value of the 30 trials to the coordinate values of each trial ) was calculated for both methods with matlab . student 's t - test was performed to determine whether a significant difference existed between the mean distances of the 2 superimposition methods for each landmark using sas 9.1 ( sas institute inc . , the null hypothesis was rejected at a significant level of 0.05 . to compare the stability of the reproduced results from both surface and plane superimposition , the following 2 methods were used , since the data used for the analysis involved 3d coordinates , and no appropriate method for the precise comparison of 2 different superimposition methods has been established . at first , 3d coordinates were divided into x , y , z axes and the variance for results from each superimposition method was calculated in each axis . subsequently , f - test for equal variances was used to compare variance values , and the difference in distance between values from each trial and the mean values of the 2 superimposition methods were compared . craniofacial skeleton 3d cbct image data was acquired with an i - cat imaging device ( imaging sciences international , hatfield , pa , usa ) . the patient underwent cbct scanning in facial mode in 14-bit gray scale with 1.0 mm slice thickness , 1.0 mm voxel size , and image acquisition by single 360 degree rotation with a 40 s total scan time . the reconstructed digital data was directly transferred from the cbct scanner to a personal computer and stored as digital imaging and communications in medicine ( dicom ) files . both pre - treatment and post - treatment dicom data the mandible was segmented by rapidform 2006 from the craniofacial skeleton image ( figure 2 ) . orapix scanner ; a 3d surface scanning system with a slit laser beam and non - contact measurement method ( kod-300 , orapix co. ltd . , seoul , korea ) was used to obtain 3d data from the dental cast model . both pre - treatment and post - treatment orthodontic 3d digital models are shown in figure 3 . to create a preliminary fusion model , the dentition reconstructed by cbct data was superimposed on the 3d digital model by a regional registration method . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . finally , both pre- and post - treatment digital models were integrated into each mandible model , and the bases of the digital model superimposed on each image were removed . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . two different registration methods of superimposing icit1 and icit2 were used to evaluate their practical applicability and to estimate their reproducibility ( figure 6 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the fully automated registration was computed by rapidform 2006 , with the limited manual process of selection and de - selection of the registration area . the inferior portion of mandibular body and the posterior portion of ramus were selected as the registration area . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . the procedure for plane superimposition was shown in figure 10 . before the 2 superimposition procedures , the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . specially in this patient the mesiobuccal tip of the lower right 1st molar was also marked since it was found to be an ankylosed tooth . rapidform 2006 has a coordinate system , of which its origin is defined with the 0-point value by the software itself . the landmarks were established and selected on the 3d digital model image by 2 investigators using the rapidform program . the points assignment was repeated 5 times each by each investigator , and the mean coordinate values of each landmark were used to define the final coordinate values . the final coordinate values ( x , y , and z ) of each landmark were transferred to microsoft office excel 2007 ( microsoft , redmond , wa , usa ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . the software easily performed the re - addition procedure at the same position since the bases were previously constructed on the digital model . finally , the superimposed 3d mandibular dental arch was ready to be used for evaluation of the orthodontic tooth movement that occurred throughout treatment . craniofacial skeleton 3d cbct image data was acquired with an i - cat imaging device ( imaging sciences international , hatfield , pa , usa ) . the patient underwent cbct scanning in facial mode in 14-bit gray scale with 1.0 mm slice thickness , 1.0 mm voxel size , and image acquisition by single 360 degree rotation with a 40 s total scan time . the reconstructed digital data was directly transferred from the cbct scanner to a personal computer and stored as digital imaging and communications in medicine ( dicom ) files . both pre - treatment and post - treatment dicom data the mandible was segmented by rapidform 2006 from the craniofacial skeleton image ( figure 2 ) . orapix scanner ; a 3d surface scanning system with a slit laser beam and non - contact measurement method ( kod-300 , orapix co. ltd . , seoul , korea ) was used to obtain 3d data from the dental cast model . both pre - treatment and post - treatment orthodontic 3d digital models are shown in figure 3 . to create a preliminary fusion model , the dentition reconstructed by cbct data was superimposed on the 3d digital model by a regional registration method . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . finally , both pre- and post - treatment digital models were integrated into each mandible model , and the bases of the digital model superimposed on each image were removed . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . two different registration methods of superimposing icit1 and icit2 were used to evaluate their practical applicability and to estimate their reproducibility ( figure 6 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the fully automated registration was computed by rapidform 2006 , with the limited manual process of selection and de - selection of the registration area . the inferior portion of mandibular body and the posterior portion of ramus were selected as the registration area . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . the procedure for plane superimposition was shown in figure 10 . before the 2 superimposition procedures , the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . specially in this patient the mesiobuccal tip of the lower right 1st molar was also marked since it was found to be an ankylosed tooth . rapidform 2006 has a coordinate system , of which its origin is defined with the 0-point value by the software itself . the landmarks were established and selected on the 3d digital model image by 2 investigators using the rapidform program . the points assignment was repeated 5 times each by each investigator , and the mean coordinate values of each landmark were used to define the final coordinate values . the final coordinate values ( x , y , and z ) of each landmark were transferred to microsoft office excel 2007 ( microsoft , redmond , wa , usa ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . the software easily performed the re - addition procedure at the same position since the bases were previously constructed on the digital model . finally , the superimposed 3d mandibular dental arch was ready to be used for evaluation of the orthodontic tooth movement that occurred throughout treatment . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the fully automated registration was computed by rapidform 2006 , with the limited manual process of selection and de - selection of the registration area . the inferior portion of mandibular body and the posterior portion of ramus were selected as the registration area . the procedure for surface superimposition the second method , designated as plane superimposition was based on anatomical structure . the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . specially in this patient the mesiobuccal tip of the lower right 1st molar was also marked since it was found to be an ankylosed tooth . rapidform 2006 has a coordinate system , of which its origin is defined with the 0-point value by the software itself . the landmarks were established and selected on the 3d digital model image by 2 investigators using the rapidform program . the points assignment was repeated 5 times each by each investigator , and the mean coordinate values of each landmark were used to define the final coordinate values . the final coordinate values ( x , y , and z ) of each landmark were transferred to microsoft office excel 2007 ( microsoft , redmond , wa , usa ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . the software easily performed the re - addition procedure at the same position since the bases were previously constructed on the digital model . finally , the superimposed 3d mandibular dental arch was ready to be used for evaluation of the orthodontic tooth movement that occurred throughout treatment . the initially established landmarks consisted of 10 points on both pre- and post - treatment images . after applying the 2 methods of superimposition , the coordinate values ( x , y , z ) of the same landmarks were transferred to microsoft office excel 2007 . each method of superimposition was performed 30 times to estimate reproducibility ( steps 4 to 6 ) and all the coordinate values of post - treatment landmarks after each superimposition were transferred to microsoft office excel 2007 . first , the mean values and variances of each x- , y- , and z - coordinate values of the post - treatment landmarks were calculated with both the surface superimposition and plane superimposition methods . the equality of variance test was carried out for each x- , y- , and z - coordinate values as a scalar with matlab 7.5 ( minitab inc . , state college , pa , usa ) . the null hypothesis and alternative hypothesis were expressed as h0 : p < s and ha : p > s ( h0 : null hypothesis , ha : alternative hypothesis , p : variance of plane superimposition , s : variance of surface superimposition ) . the difference in variance between the 2 superimposition methods was 0 at a significance level of 0.05 . then the mean distance ( mean value of the distance from the mean coordinate value of the 30 trials to the coordinate values of each trial ) was calculated for both methods with matlab . student 's t - test was performed to determine whether a significant difference existed between the mean distances of the 2 superimposition methods for each landmark using sas 9.1 ( sas institute inc the null hypothesis was rejected at a significant level of 0.05 . to compare the stability of the reproduced results from both surface and plane superimposition , the following 2 methods were used , since the data used for the analysis involved 3d coordinates , and no appropriate method for the precise comparison of 2 different superimposition methods has been established . at first , 3d coordinates were divided into x , y , z axes and the variance for results from each superimposition method was calculated in each axis . subsequently , f - test for equal variances was used to compare variance values , and the difference in distance between values from each trial and the mean values of the 2 superimposition methods were compared . final superimposed 3d digital models were produced ( figure 13 ) to permit the evaluation of tooth movement visually as well as numerically . twenty - eight coordinate values demonstrated statistically significant differences ( p < 0.05 ) between the variance of each method , indicating that the plane superimposition method generated more variability in coordinate values after each trial . figure 14 shows a scatter plot of coordinate values on the x - y , y - z , and z - x axis of the lower left incisor , which demonstrated a large variation in the coordinate values ( x : f = 59.3 , p < 0.001 , y : f = 11.61 , p < 0.001 , z : f = 491.16 , p < 0.001 ) after each trial between the 2 methods . student 's t - test revealed a statistically significant difference ( p < 0.001 ) in the mean distance from the mean coordinate value of each landmark to the corresponding coordinate value of each trial , indicating that the surface superimposition method had relatively more consistent coordinate values closer to the mean on all landmarks ( table 2 ) . many 3d instruments enable orthodontists to acquire 3d images of the facial structures , including both hard and soft tissues . not only the 3d visualization of the structures but also the measurements and comparison of different evaluation times have been made possible . with advances in technology , 3d superimposition with 3d digital models on the maxillary arch has been developed with reliable reference areas for superimposition . in recent years ct has been adopted for 3d virtual surgical planning and simulation of post - operative outcomes in orthognathic surgery with various registration methods for superimposition.17 - 20 this study aimed to introduce a reproducible superimposition technique for the mandibular dental arch by the generating integrated 3d cbct images based on cbct scans and 3d surface scanning data with improved imaging quality of the dentition . this study was limited to a non - growing patient who did not undergo an orthognathic surgical procedure to exclude the effects of mandibular growth or surgical change from the evaluation and comparison of the reproducibility of the 2 designed methods . cbct was selected as the device for ct scanning for several reasons ; it provides orthodontists with a new modality for research and permits vertical scanning of patients in a normal seated position . ferrario et al.21 reported that 3d measurement values were not sensitive to head posture . in this study , the patient underwent cbct scanning with a slice thickness of 1.0 mm , which improved the visual quality of 3d reconstruction . further improvement is possible with a smaller slice thickness , but this change would also result in increased image size , requiring greater computational power and longer user interaction time . recently , a computerized composite skull model was introduced to orthognathic surgery,22 which eliminated the need for plaster casts to create digital models since a laser surface scanner subsequently scans the impressions of the dentition . thus , one potential source of errors that reduces the accuracy of the complete system appears to have been removed . however , specialized radiolucent dental impressions trays with fiducial markers are needed to allow for the registration of the dental arches assessed by ct and laser scans . this technique introduces new sources of error , because several additional steps accompanying the data acquisition and registration procedures are required . therefore , in this study we chose a simpler approach using a plaster cast scanned with an orapix scanner for the registration of the cbct scans and the 3d dental surface images . extremely accurate modeling of the facial structures however , it is not possible to ascertain whether dental morphology can be acquired as accurately with this technique due to artifacts from metallic restorations . moreover , intercuspation during occlusion leads to overlapping images , resulting in inaccurate morphology of the individual tooth . the resultant effect on the ct images appears as pronounced dark and bright streaks , non - linear edge gradients , and sampling errors arising from the surface restoration.23 swennen et al.24 discussed the drawbacks of ct imaging ; it does not provide detailed surface dental morphology or precise intercuspation data due to its limited resolution , and the interocclusal relationship is often obscured by radio - opaque dental restorations or orthodontic brackets . combining 3d cbct images and the 3d virtual model data can compensate for these issues . gateno et al.22 introduced a technique to integrate digital models into a virtual 3d ct bone model of the skull . measurements acquired from using 3 mm titanium spheres as markers demonstrated high accuracy ( 0.1 - 0.5 mm ) in the study of a single dry skull . nkenke et al.25 suggested a considerable progress in the fusion of images from different imaging modalities using software - based approaches in which 2 modalities are combined and mounted in a single coordinate system . combining ct with optical 3d imaging seems to be a reasonable approach to correct metallic artifacts , allowing precise assessment of the tooth surface and accurate simulation of the post - operative dental occlusion . in a single patient , accuracy levels of 0.66 and 0.56 mm for the mandible and maxilla , respectively , with a modified double ct scan procedure resulted in accurate registration , with registration error of 0.1355 0.0323 mm , and analysis of variance of 0.0564 mm.24 two known methods for the registration of ct data and scanned optical dental images have been setablished : point - based registration of external fiducial markers22,26 and surface - based registration of anatomical structures.25 we used surface - based registration referred to as regional registration with surface - to - surface matching ( best - fit method ) , which included a least square technique . this approach is advantageous in that it can be applied with no additional fiducial markers , which in turn require more additional time - consuming procedures that may also introduce new sources of error . although their study did not assess a combination technique for ct and dental images , cha et al.11 evaluated the accuracy of measurements by calculating the superimposition discrepancies between pre- and post - treatment 3d models in the maxillary arch . surface - to - surface matching ( best - fit method ) resulted in a mean error of 0.0399 mm , standard error of 0.0289 mm , and standard deviation of 0.1583 mm , which the authors concluded as a satisfactory accuracy level . subsol et al.27 proposed methods that have guided the progress of our study , in which semi - landmarks on the surface were used to incorporate information about vectors in the vicinity of the landmark . the landmarks were established and selected on the 3d digital model images 5 times each by 2 investigators . the mean coordinate values of each landmark were used to define the final coordinate values . the determined landmarks were then fixed to the image so as not to be changed during the superimposition procedure , and the coordinate values were then determined after every superimposition procedure for a total of 30 repetitions with each method . in this study , we introduced 2 methods for superimposing ici , designated as surface superimposition and plane superimposition . plane superimposition was designed to include more information concerning anatomical structures in the superimposition procedure . the landmarks used - mental foramen and lingual foramen - are known as relatively stable structures on the mandible . in a study of 314 dried mandibles , mcdonnell et al.28 reported that the lingual foramen present in 311 specimens ( 99.04% ) , and that the wall of the canal , not the genial tubercles , produced the radio - opacity peripheral to the foramen seen on radiographs . although the authors remarked that the foramen was not seen on many radiographs of the lower incisor region , a change in orientation of the x - ray beam could account for this discrepancy . in this study , the registration plane was oriented to the 3 points of these structures that were considered stable . a lower variance of samples from one method compared to the other , indicates that the method is relatively more efficient in terms of reproducibility . however , in the present study each sample was a series of 3d - vectors , resulting in a covariance matrix for each method instead of a scaler variance . therefore , we decomposed these series of vectors into 3 series of scalars using x , y , and z coordinates and then used the f - test to determine whether 2 samples from these 2 methods had the same variance . the surface method demonstrated a lower sample variance than the plane method within a 5% significance level at 28 coordinates . we attempted to express the magnitude of the 3d vector while excluding the directional component . the mean distance from the mean coordinate value of each landmark was measured for both methods . student 's t - test revealed a statistically significant difference in the mean distance between the 2 methods and also indicated that the surface superimposition method had a relatively more consistent distribution closer to the mean coordinate values over 30 trials . therefore , the surface method was considered to provide more reproducible results after repeated trials . this reproducibility of the surface superimposition method was helped by our negligible observer variability , which allowed image analysis procedures largely independent of observer errors . by contrast , the relative inconsistency of plane superimposition is thought to result from the procedure , which requires manual selection of the anatomical structures that could generate inter- or intraobserver variability . cevidanes et al.29 introduced a fully automated superimposition method using voxel - wise rigid registration of the cranial base , as the cranial base structures are not altered by surgery . this technique represents an advance from the processe described by kawamata et al.,30 who used an observer - dependent method to superimpose and rotate the post - surgery ct until anatomical landmarks overlapped these same structures in the pre - surgery semi - transparent model . our results also show improved reproducibility with the automated superimposition technique ( surface superimposition ) , which minimizing the possibility of observer errors . because it is almost fully computerized except for the selection process of the registration area , it avoids human error , thereby simplifying the process and producing integrated images with a high degree of reproducibility in positioning . therefore , surface superimposition method may be more precise and convenient method for mandibular surface model superimposition . the data used for the analysis involved 3d coordinates , and no appropriate method for the precise comparison has been established . as alternatives , the following 2 methods were used . first , 3d coordinates were divided into x , y , and z axes and the variances for each result from both superimposition methods was calculated on each axis . the f - test for equal variances was then used to compare the values of variance , and a conclusion was drawn by comparing this calculated f value to the critical value of f distribution ( alpha = 0.05 ) since the f value is known to demonstrate f distribution under conditions of the null hypothesis . second , the difference in distance between the results and mean points that were generated by the 2 superimposition methods were compared . for this aim , mean x , y , and z values were deduced from 3d coordinates , and the distances of each result from the corresponding mean values were calculated . then the mean distance distribution and mean values that were generated from the 2 superimposition methods were analyzed . the null hypothesis was that the mean distance calculated by plane superimposition was less than or equal to that calculated by surface superimposition . the alternative hypothesis was that the mean distance calculated by plane superimposition was greater than that calculated by surface superimposition . standard deviation of the test statistic t is known to demonstrate t - distribution under the conditions of the null hypothesis . therefore , a conclusion was drawn by comparing the t value calculated as described above and the ctitical value of t - distribution ( alpha = 0.05 ) . future studies should include more patient samples for the superimposition procedures for reproducibility and also a comparison with the measurement of tooth movement detected on the conventional cephalographs . this study was limited to a non - growing patient , and more studies may be needed to reflect the growth changes of the mandible during superimposition . the true methodological error of the superimposition system was not estimated in this study , and such an estimation would be challenging because the mandible is an independent structure with a joint on the craniofacial skeleton . however , the error of registration and the reproducibility of the 2 methods were determined . because significant changes in mandibular position can occur during orthodontic treatment , such as autorotation of the mandible , condylar displacement , and centric occluson / centric relation discrepancy , the registration reference in this study was designed within the mandible itself , not on craniofacial structures . additionally , the registration reference was based on the basal bone of the mandible , which , in contrast to soft tissues or alveolar bone , had not been altered by orthodontic tooth movement . therefore , this method is assumed to meet the submillimetric accuracy requirements of orthodontic tooth movement in non - growing patients . it is expected to gain acceptance as the new method helps patients to better understand the 3d changes in the mandibular dental arch throughout the treatment process . the visualization of 3d superimposition and measurements can help orthodontists to better evaluate treatment outcomes . here , we introduced 2 methods for 3d mandibular dental arch superimposition ; surface superimposition and plane superimposition . the surface superimposition method demonstrated more reliable and reproducible results based on its minimization of the possibility of observer errors . these findings suggest that 3d mandibular dental arch superimposition can be achieved using the surface superimposition method .

objectivethe purpose of this study was to develop superimposition method on the lower arch using 3-dimensional ( 3d ) cone beam computed tomography ( cbct ) images and orthodontic 3d digital modeling.methodsintegrated 3d cbct images were acquired by substituting the dental portion of 3d cbct images with precise dental images of an orthodontic 3d digital model . images were acquired before and after treatment . for the superimposition , 2 superimposition methods were designed . surface superimposition was based on the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . plane superimposition was based on anatomical structures ( mental and lingual foramen ) . for the evaluation , 10 landmarks including teeth and anatomic structures were assigned , and 30 times of superimpositions and measurements were performed to determine the more reproducible and reliable method.resultsall landmarks demonstrated that the surface superimposition method produced relatively more consistent coordinate values . the mean distances of measured landmarks values from the means were statistically significantly lower with the surface superimpositions method.conclusionsbetween the 2 superimposition methods designed for the evaluation of 3d changes in the lower arch , surface superimposition was the simpler , more reproducible , reliable method .

INTRODUCTION MATERIALS AND METHODS Superimposition procedure Step 1. Acquisition of 3D CBCT skeletal data and reconstruction of 3D mandibular bone images Step 2. Acquisition of 3D digital model data by surface scanning Step 3. Initial integration of the mandible and mandibular dentition model Step 4. Superimposition of pre- and post-treatment ICI Surface superimposition Plane superimposition Establishment of landmarks on dentition Step 5 and 6. Extraction of mandibular dentition Statistical analysis RESULTS DISCUSSION CONCLUSION

it is important to estimate the changes in tooth position before and after orthodontic treatment to determine whether the teeth have been moved according to the anchorage requirements or to the planned tooth movement of the treatment objectives . recent advances in computer technologies such as 3-dimensional ( 3d ) computed tomography ( ct ) , 3d surface scanning technology , computer - aided design , computer - aided manufacturing , and associated software have contributed not only to the precise diagnosis , treatment planning , and simulations , but also to the 3d evaluation of treatment results . on the maxillary dental arch , although little is known about the stability of identifiable landmarks on dental casts , palatal rugae has been suggested as relatively stable structures for registration of serial maxillary models.1 the shape of the palatal vault and the medial portions of the palatal rugae are fairly stable throughout the development of the dentition.2 the palatal rugae retain their shape and pattern throughout a person 's lifetime.3 thus , they have been used for identification purposes in forensics.4 almeida et al.5 and bailey et al.6 have studied the potential use of the palatal rugae for the superimposition of serial models and both studies concluded that specific parts of the palatal rugae ( e.g. ashmore et al.8 reported that the method developed for the superimposition of digital configurations of serial dental models allowed accurate measurements of translational movement of the maxillary first - molar in 3 dimensions for both headgear and untreated groups . miller et al.9 reported that the digital superimposition of the maxillary arch was reproducible and that the error associated with using palatal rugae as reference landmarks may be similar to or less than that of current 2d cephalometric analyses.10 cha et al.11 reported that use of a 3d digital orthodontic model superimposition technique on the maxillary dental arch was clinically as reliable as cephalometric superimposition for assessing orthodontic tooth movement . the purpose of this study was to introduce a method for mandibular dental arch superimposition in a non - gorwing patient by the combination of 3d cone beam ct data and 3d digital model data . cone beam computed tomography ( cbct ) scan and alginate impression were taken on the same day prior to treatment , and in accordance with the patient 's request , 1 day after debonding . the superimposion system consisted of 6 main procedures : ( 1 ) acquisition of cbct data , ( 2 ) acquisition of 3d digital model data , ( 3 ) integration of cbct data and digital model data , ( 4 ) superimposition of pre - treatment and post - treatment integrated cbct image , ( 5 ) extraction of the mandibular dentition portion , and ( 6 ) evaluation of tooth movement on the mandibular arch . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . before the 2 superimposition procedures the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . the points assignment was repeated 5 times each by each investigator , and the mean coordinate values of each landmark were used to define the final coordinate values . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . after applying the 2 methods of superimposition , the coordinate values ( x , y , z ) of the same landmarks were transferred to microsoft office excel 2007 . first , the mean values and variances of each x- , y- , and z - coordinate values of the post - treatment landmarks were calculated with both the surface superimposition and plane superimposition methods . then the mean distance ( mean value of the distance from the mean coordinate value of the 30 trials to the coordinate values of each trial ) was calculated for both methods with matlab . student 's t - test was performed to determine whether a significant difference existed between the mean distances of the 2 superimposition methods for each landmark using sas 9.1 ( sas institute inc . to compare the stability of the reproduced results from both surface and plane superimposition , the following 2 methods were used , since the data used for the analysis involved 3d coordinates , and no appropriate method for the precise comparison of 2 different superimposition methods has been established . subsequently , f - test for equal variances was used to compare variance values , and the difference in distance between values from each trial and the mean values of the 2 superimposition methods were compared . to create a preliminary fusion model , the dentition reconstructed by cbct data was superimposed on the 3d digital model by a regional registration method . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . before the 2 superimposition procedures , the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . to create a preliminary fusion model , the dentition reconstructed by cbct data was superimposed on the 3d digital model by a regional registration method . this function of rapidform 2006 , designated as 3d surface - to - surface matching ( best - fit - method ) , employs a least - mean - squared algorithm.12,13 according to the coordination between the tooth image from cbct and the digitally scanned dental cast , the position , size , and posture of each tooth on the 3d digital model was automatically fitted to one of the subject 's images of dentition in the cbct data ( figure 4 ) . thus , we produced individual integrated 3d cbct images ( integrated 3d cbct image [ ici]t1 , icit2 ) of the mandible , before and after the treatment , respectively , with an anatomical structure that included a digital model of higher resolution than the 3d cbct image ( figure 5 ) . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the procedure for surface superimposition the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . before the 2 superimposition procedures , the reference points were the center of right and left mental foramen , the center of lingual foramen , the facial axis points16 of right and left lower central incisors , canines , and 2nd premolars ( figure 11 ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . the first method designated as ' surface superimposition ' was based on the surface of the basal bone structure of the mandible by surface - to - surface matching ( best - fit method ) . the registration area was comprised of the basal bone structures of the mandible , which had not been altered by orthodontic treatment . the procedure for surface superimposition the second method , designated as plane superimposition was based on anatomical structure . the centers of both sides of mental foramen and the center of mandibular lingual foramen ( a consistent arterial foramen in the middle of the mandible)14,15 were selected as the landmarks . the center of each foramen was selected with a grid on the monitor by picking 4 points of the foramen - the most superior , inferior and both lateral points - with the mandible rotated to give a perpendicular view to the foramen ( figures 8 and 9 ) . the dentition was extracted from the superimposed image ( figure 12 ) , and the base of the model previously removed was added again on the dentition . after applying the 2 methods of superimposition , the coordinate values ( x , y , z ) of the same landmarks were transferred to microsoft office excel 2007 . first , the mean values and variances of each x- , y- , and z - coordinate values of the post - treatment landmarks were calculated with both the surface superimposition and plane superimposition methods . then the mean distance ( mean value of the distance from the mean coordinate value of the 30 trials to the coordinate values of each trial ) was calculated for both methods with matlab . student 's t - test was performed to determine whether a significant difference existed between the mean distances of the 2 superimposition methods for each landmark using sas 9.1 ( sas institute inc the null hypothesis was rejected at a significant level of 0.05 . to compare the stability of the reproduced results from both surface and plane superimposition , the following 2 methods were used , since the data used for the analysis involved 3d coordinates , and no appropriate method for the precise comparison of 2 different superimposition methods has been established . subsequently , f - test for equal variances was used to compare variance values , and the difference in distance between values from each trial and the mean values of the 2 superimposition methods were compared . twenty - eight coordinate values demonstrated statistically significant differences ( p < 0.05 ) between the variance of each method , indicating that the plane superimposition method generated more variability in coordinate values after each trial . figure 14 shows a scatter plot of coordinate values on the x - y , y - z , and z - x axis of the lower left incisor , which demonstrated a large variation in the coordinate values ( x : f = 59.3 , p < 0.001 , y : f = 11.61 , p < 0.001 , z : f = 491.16 , p < 0.001 ) after each trial between the 2 methods . student 's t - test revealed a statistically significant difference ( p < 0.001 ) in the mean distance from the mean coordinate value of each landmark to the corresponding coordinate value of each trial , indicating that the surface superimposition method had relatively more consistent coordinate values closer to the mean on all landmarks ( table 2 ) . in recent years ct has been adopted for 3d virtual surgical planning and simulation of post - operative outcomes in orthognathic surgery with various registration methods for superimposition.17 - 20 this study aimed to introduce a reproducible superimposition technique for the mandibular dental arch by the generating integrated 3d cbct images based on cbct scans and 3d surface scanning data with improved imaging quality of the dentition . this study was limited to a non - growing patient who did not undergo an orthognathic surgical procedure to exclude the effects of mandibular growth or surgical change from the evaluation and comparison of the reproducibility of the 2 designed methods . however , specialized radiolucent dental impressions trays with fiducial markers are needed to allow for the registration of the dental arches assessed by ct and laser scans . the resultant effect on the ct images appears as pronounced dark and bright streaks , non - linear edge gradients , and sampling errors arising from the surface restoration.23 swennen et al.24 discussed the drawbacks of ct imaging ; it does not provide detailed surface dental morphology or precise intercuspation data due to its limited resolution , and the interocclusal relationship is often obscured by radio - opaque dental restorations or orthodontic brackets . combining 3d cbct images and the 3d virtual model data can compensate for these issues . in a single patient , accuracy levels of 0.66 and 0.56 mm for the mandible and maxilla , respectively , with a modified double ct scan procedure resulted in accurate registration , with registration error of 0.1355 0.0323 mm , and analysis of variance of 0.0564 mm.24 two known methods for the registration of ct data and scanned optical dental images have been setablished : point - based registration of external fiducial markers22,26 and surface - based registration of anatomical structures.25 we used surface - based registration referred to as regional registration with surface - to - surface matching ( best - fit method ) , which included a least square technique . surface - to - surface matching ( best - fit method ) resulted in a mean error of 0.0399 mm , standard error of 0.0289 mm , and standard deviation of 0.1583 mm , which the authors concluded as a satisfactory accuracy level . subsol et al.27 proposed methods that have guided the progress of our study , in which semi - landmarks on the surface were used to incorporate information about vectors in the vicinity of the landmark . the landmarks were established and selected on the 3d digital model images 5 times each by 2 investigators . in this study , we introduced 2 methods for superimposing ici , designated as surface superimposition and plane superimposition . plane superimposition was designed to include more information concerning anatomical structures in the superimposition procedure . the landmarks used - mental foramen and lingual foramen - are known as relatively stable structures on the mandible . in a study of 314 dried mandibles , mcdonnell et al.28 reported that the lingual foramen present in 311 specimens ( 99.04% ) , and that the wall of the canal , not the genial tubercles , produced the radio - opacity peripheral to the foramen seen on radiographs . student 's t - test revealed a statistically significant difference in the mean distance between the 2 methods and also indicated that the surface superimposition method had a relatively more consistent distribution closer to the mean coordinate values over 30 trials . this reproducibility of the surface superimposition method was helped by our negligible observer variability , which allowed image analysis procedures largely independent of observer errors . by contrast , the relative inconsistency of plane superimposition is thought to result from the procedure , which requires manual selection of the anatomical structures that could generate inter- or intraobserver variability . second , the difference in distance between the results and mean points that were generated by the 2 superimposition methods were compared . then the mean distance distribution and mean values that were generated from the 2 superimposition methods were analyzed . the null hypothesis was that the mean distance calculated by plane superimposition was less than or equal to that calculated by surface superimposition . the alternative hypothesis was that the mean distance calculated by plane superimposition was greater than that calculated by surface superimposition . future studies should include more patient samples for the superimposition procedures for reproducibility and also a comparison with the measurement of tooth movement detected on the conventional cephalographs . this study was limited to a non - growing patient , and more studies may be needed to reflect the growth changes of the mandible during superimposition . the true methodological error of the superimposition system was not estimated in this study , and such an estimation would be challenging because the mandible is an independent structure with a joint on the craniofacial skeleton . however , the error of registration and the reproducibility of the 2 methods were determined . because significant changes in mandibular position can occur during orthodontic treatment , such as autorotation of the mandible , condylar displacement , and centric occluson / centric relation discrepancy , the registration reference in this study was designed within the mandible itself , not on craniofacial structures . additionally , the registration reference was based on the basal bone of the mandible , which , in contrast to soft tissues or alveolar bone , had not been altered by orthodontic tooth movement . it is expected to gain acceptance as the new method helps patients to better understand the 3d changes in the mandibular dental arch throughout the treatment process . the surface superimposition method demonstrated more reliable and reproducible results based on its minimization of the possibility of observer errors .

dengue virus , an aedes mosquito - borne viral pathogen belonging to the family flaviviridae , is the cause of dengue fever ( df ) and dengue hemorrhagic fever ( dhf ) . the emergence and reemergence of df / dhf have become a significant public health burden in the tropics and subtropics [ 15 ] . due to the lack of an effective tetravalent dengue vaccine that can secure lifelong immunization , aedes mosquito control measures have primarily been employed to prevent disease outbreak and interrupt transmission during the outbreak . regarded as a reemerging infectious disease [ 1 , 4 , 5 ] such outbreaks reflect the failure of current prevention and control efforts , despite the fact that some successful cases of vector control in the americas , cuba , and singapore had shortened outbreak periods and stopped the diseases from spreading [ 79 ] . in some cases , an application of appropriate vector control measures along with community participation several reports have shown a coherent argument that transmission dynamics of dengue viruses result from very complex epidemiology and ecology of the disease . such dengue transmission dynamics are the interaction among humans , dengue viruses , vectors , and ecosystems , of which biotic and abiotic determinants have both direct and indirect influences on dengue transmission [ 1316 ] . obviously in some cases , a useful set of environmental and sociodemographic factors , which constituted age - dependent classes , numbers and densities of urban populations , economic classes , and inhabitations , are central components of analysis of temporal and spatial relationships of dengue incidences [ 1720 ] . also , a vector - based dengue model can predict transmission dynamics , based primarily on the infestation and/or reinfestation of domestic and peridomestic aedes mosquito vectors in human inhabitations . nonetheless , in different complex epidemiological settings , various factors that can influence dengue transmission dynamics remain to be established . this is because of more diverse sociocultural contexts and changes in sociopolitic , socioeconomic , demographic , technologic , and environmental conditions as well as ineffective management of household - level information and improper implementation of those control strategies . investigation into such sociodemographic , environmental perspectives can provide foresight into the appropriateness of dengue control efforts , give answers to unexpected vector control responses , and contribute to effective management solutions in an ever - changing environment . of note , a plausible paradigm of interdisciplinary approach that integrates the ecologic and sociodemographic dimensions of dengue [ 2227 ] has permitted an analysis of dengue transmission risk to determine what pivotal drivers significantly contribute to dengue transmission in an urban environment . in this regard , we applied two sets of household - level ecologic and individual - level sociodemographic factors to determine whether two fitted models of dengue - related determinants predicted the dengue transmission risk in urban areas of chachoengsao province , thailand , known for epidemics of df / dhf . ultimately , the findings of this study would benefit better management of effective dengue prevention and control , especially in resource - limited developing countries . chachoengsao province was a representative of geographically defined areas ( figure 1 ) , including small - and medium - sized municipalities as well as four main types of landscapes : mountains , rivers , flatlands , and wetlands . both urban and semiurban communities are situated in low - lying , generally flat areas surrounded by rice fields and orchards . in the area , the climate is characterized by a long rainy season ( june october ) , a winter dry season ( november january ) , and a hot dry season ( february may ) . given the complexity of its demographic and socioeconomic dispersions , the province is administratively divided into 11 districts covering a total area of 5,370 km with basic infrastructures of connecting roads , electricity , piped water supply system , communication system , and health service system . two - stage random sampling was applied for selecting targeted households . the 120 blocks initially assigned to four different districts were used to select 12 blocks ( approximately 100 houses each ) , based on the degree of urbanization and the intensity of dengue transmission ( figure 2 ) . six urban blocks were selected from the municipality of muang district and the capital of chachoengsao province , while the other six semiurban blocks were chosen from three subdistrict municipalities : bang pakong , ban pho , and bang khla . according to this household selection , a sample size was calculated based on 95% confidence to detect the prevalence of igg - igm - positive school children at 19.2% in chachoengsao province with a 3% of accepted error . the statistically required sample size of 994 was increasing by 20% to allow for missing data , resulting in 1,200 households , which were derived from the 12 selected blocks . all were subsequently used for household surveys during august october 2007 to collect household - level and individual - level information with the assistance of interdisciplinary teams ( i.e. , each team included well - trained professional nurses and/or public health officers ) , as described below . the establishment of interdisciplinary approaches and teams was supported by a who / tdr / idrc - funded multicountry study . in this study , the instruments provisionally guided by multicountry supporting teams were initially developed from a series of community of practice workshops delivered through a multicountry network in asia . the tools were partly modified with the addition of a useful set of variables , which corresponded to sociocultural contexts of thailand . as technically validated by the authors , all tools included the structured questionnaires on households and individuals along with the environmental observation checklist . the structured questionnaires on households along with the environmental observation checklist were used to gather a set of ecological data of the entire 1,200 households . the data included ecotope , dengue risk area , number of house floors , floor of principal living ( i.e. , homeowners or any member often used a space of everyday family living as principal living area of the first floor or the upper floors ) , construction material of the house , number of house windows , having window screens , having a yard / open space , having bushes in a yard / open space , main purpose of house , and household attachment ( i.e. , the attached houses had at least one shared wall , whereas the detached houses had standalone property with no sharing wall ) . face - to - face interviews were carried out using the representative respondents who were family members , 18 years of age or older . in this study , we applied an ecotope concept , which is originally defined as the smallest ecologically - distinct features in a landscape mapping and classification system . as mentioned earlier , those selected 12 blocks were assigned to have 4 different ecotopes ( figure 3 ) , which spanned both urban and semiurban blocks of the study area . the difference is based quantitatively and qualitatively on the distribution of houses , the socioeconomic status ( ses ) of local inhabitants , infrastructural services , land use , and land type . the commercial ecotope ( c ) denoted a majority of attached houses with a 0.07-meter mean distance of nearest houses , a high ses of occupants , and good basic infrastructure , along with a great deal of commercial and business buildings . the ecotope of a densely populated urban residential area ( denpura ) is characterized by most attached houses with a 0.63-meter mean distance of nearest houses , low ses of the population , and relatively low degree of community development , including social and economic opportunities and access to health care resources as well as infrastructural water supply and waste management and a lack of vegetation and/or forested areas . the ecotope of residential ( r ) mixed with c and denpura ( rcdenpura ) exhibits a main residential zone prominently scattered with c and denpura and has a 3.37-meter mean distance of nearest houses , while the residential mixed only with commercial ( rc)signifying an overlap of urban and rural areas is an area mainly for dwelling but which is also filled in partly with commercial settlements and has a 4.58-meter mean distance of nearest houses . finally , each ecotope investigated in this study had a different number of study households c ( n = 200 ) , denpura ( n = 200 ) , rcdenpura ( n = 200 ) , and rc ( n = 600 ) . as for the risk area , the classification of historical dengue risk areas ( high and low degrees of dengue transmission ) was drawn from which its epidemic pattern that normally occurs on a 2 - 3 year - cycle transmission . therefore , based on the past situation of national surveillance of dengue cases , the definition of dengue transmission risk areas relies upon a 3- to 5-year median of dengue cases . in this study , the data of the confirmed dengue cases retrieved from the national epidemiological surveillance system were obtained from the chachoengsao general hospital and chachoengsao provincial public health office . therefore , based on the ecotope assignment ( figure 2 ) , the study households ( n = 1,200 households ) were categorized into 2 historical dengue risk areas : 6 high - transmission blocks ( n = 600 households ) that had the highest 5-year medians of dengue cases and 6 low - transmission blocks ( n = 600 households ) that had the lowest 5-year medians of dengue cases . the structured questionnaires on individuals , which had a significant reliability of knowledge , attitude , and practice ( cronbach 's alpha coefficient = 0.7 ) , were used to gather a set of sociodemographic data using those representative respondents of the entire 1,200 households as previously mentioned . the data included age , education level ( highest school degree ) , occupation , movement during the last 3 months , residence time in house , residence time in neighborhood , number of household members , average family income , knowledge about dengue and its vectors , attitude about dengue vector control in terms of gender , family , and government roles , household water storage ( i.e. , by using any water - storing containers ) , household practices to reduce the nuisance of mosquitoes , household practices to prevent aedes breeding places , last time visited by health personnel , receiving dengue control support / materials , and community efforts in environmental management by clean - up campaign . regarding household practices to reduce the nuisance of mosquitoes , questions were asked about household activities which could be categorized into 3 groups : ( 1 ) chemical control ( e.g. , indoor spraying , putting chemicals in water containers and personal protection with repellents ) , ( 2 ) physical control ( e.g. , removing rubbish , covering water containers and killing mosquitoes ) , and ( 3 ) biological control ( e.g. , putting fish in water containers ) . the chemical and physical control activities were ranked based on frequent actions : low ( never or either one of three actions ) and high ( two or more actions ) , whereas the biological control activity was ranked based on the action no or did not apply and yes or applied . the questions of household practices to prevent aedes breeding places could be categorized into 3 groups : ( 1 ) chemical control ( e.g. , putting chemicals in water containers ) , ( 2 ) physical control ( e.g. , removing rubbish , covering the water containers , changing water once a week , eliminating stagnant water , brushing / cleaning inner surface of water containers , and removing larvae ) , and ( 3 ) biological control ( e.g. , putting fish in water containers ) . the chemical and biological control activities were ranked as either yes or no . the physical control activities were ranked based on the frequency of actions : low ( never , one , or two actions ) and high ( three and more actions ) . all the respondents provided informed consent after they were completely informed about the study 's purpose , as well as the advantage and disadvantage of participation . the ethical clearance for this study was approved by the institutional review board of mahidol university . dengue - related determinants as the outcomes of dengue transmission risk in the study area included the households that had past history of dengue cases and the respondents or householders that had history of dengue infections in their lifetime before the study . generally speaking , the principal outcome of this study was to identify whether those household - level ecologic and individual - level socioeconomic variables were correlated to these dengue - related determinants . therefore , in two separate ecological and socioeconomical models , a univariate analysis of the individual explanatory variables was used to analyze the association of the dengue - related determinants using the chi - square test ( p < 0.05 or p < 0.1 ) . then , they were entered into the multivariate logistic models and odds ratios ( aors ) adjusted for all of these variables and 95% confidence intervals ( ci ) were calculated . in fitted models , the likelihood - ratio ( lr ) test ( p < 0.05 ) was used to test the significance of all the related predictors while the wald 's test ( p < 0.05 ) was used to test the statistical significance of each coefficient ( b ) in the model to determine contributing predictors . the spss statistical program , version 17.0 ( spss , inc . , chicago , il , usa ) , was used throughout this study . we attempted to directly relate household - level ecologic determinants with dengue transmission risk . table 1 reveals the results of the univariate analysis of ecologic risk factors for dengue transmission considering the household - level characteristics . using test , 4 out of 11 ecologic variables were found to have a significant association ( p < 0.05 ) with frequency of houses having previous history of dengue cases . four significant variables included ecotope , historical dengue risk area , number of house windows , and presence of screens for house windows . the three variables of ecotope , historical dengue risk area , and presence of screens for house windows were selected for the final multivariate regression while the number of house windows was not entered in the model because it appeared to be of marginal significance . results of the multivariate analyses are shown in table 2 . in multivariate analysis , these selected determinants were adjusted for their confounding factors by the random effect model . all determinants ( the ecotope , historical dengue risk area , and presence of screens for house windows ) seemed to have a direct impact on dengue transmission ( the lr test , p < 0.05 ) . for the ecotope , three categorical variables ( denpura , rcdenpura , and rc ) were used to determine their effect on dengue transmission using the commercial ecotope as a reference . only the ecotope of rcdenpura remains as a significant predictor for dengue transmission ( aor = 2.23 , p = 0.009 ) . in terms of dengue risk area , a setting with a high degree of historical dengue transmission indicated or ( aor = 2.06 , p < 0.001 ) significantly higher than an area with a low degree of historical dengue transmission . the last significant variable of presence of screens for house windows showed that houses with window screens had a greater risk ( aor = 1.62 , p = 0.023 ) than those with no window screens . the morbidity and mortality attributed to this disease may vary significantly from one place to another on account of different local parameters . in fact , many relevant studies have demonstrated that it is the set of microsocioeconomic , infrastructural , and environmental parameters embedded in communities which appear to be responsible for increased epidemic transmission of dengue virus in particular ecosystem localities [ 22 , 3034 ] . in addition , a novel ecosystem concept was created to exhaustively scrutinize the associations between ecotope and the transmission of dengue in the study areas . of all 4 ecotopes , only the ecotope of rcdenpura ( i.e. , where the ecotope is in fact a combination of residential , commercial , and densely populated urban residential areas ) exhibited the strongest association with dengue transmission suggesting that the complexity of urban ecosystem may give rise to dengue emergence . to our knowledge , this is the first time that the ecotope , which greatly poses the most risk for dengue occurrence , was determined and characterized . this may reaffirm that the area classification based on past history of national surveillance dengue cases has substantial roles in dengue management as well as in providing at - risk areas targeted for the dengue surveillance system and the implementation of dengue control measures . for the variable of having screens for house windows , the presence of house window screens was significantly associated with dengue transmission , compared to the absence of house window screens . such evidence was consistent with the findings of chao et al . ( 2012 ) that households having window screens as a preventive measure showed the reduction of risk in association with human - mosquito contact . in our study , the negative correlation between the presence of house window screens and dengue transmission may result from the cross - sectional survey where presence of house window screens as an independent variable and houses with a previous history of dengue as dependent variable were measured simultaneously . the existence of many houses that utilize window screens continuously might be due to the perception of dengue and/or due to the health education campaign following dengue outbreaks , whereas the presence of houses with dengue cases was measured after any cases developed within the past year . accordingly , this finding suggested that the presence of window screens may be dependent upon the particular household 's prior dengue risk . table 3 shows the results of the univariate analysis of sociodemographic risk factors related to dengue transmission considering individual - level characteristics . using test , 4 out of 22 sociodemographic variables seemed to have a significant association ( p < 0.05 ) with frequency of respondents with a history of dengue . four significant variables were age , highest school degree , residence time in neighborhood , and community effort in environmental management by clean - up campaign , and hence they were selected for further multivariate analysis . besides these , some variables that either had a significant association at p values of nearly 0.05 or had the potentials of significant phenomena were included in the model . the former variables included residence time in the house and number of household members , while the latter variables constituted movement during last 3 months and knowledge about dengue and its vectors . the sociodemographic multivariate analyses revealed findings of the association between 8 individual determinants and dengue transmission ( table 4 ) . among all determinants tested , 5 of them which included age , highest school degree , residence time in neighborhood , number of household members , and community effort in environmental management by clean - up campaign posed significant risk for dengue transmission ( the lr test , p < 0.05 ) . respondents aged > 45 years showed significant risk for dengue transmission ( aor = 3.24 , p = 0.003 ) compared to those aged 45 years . with regards to highest school degree , individuals with middle and higher degrees were twice as likely to get dengue infection ( aor = 2.33 , p = 0.013 ) compared to those with elementary and lower degrees . in contrast to the univariate analysis , household members > 4 persons were likely to experience a greater risk ( aor = 2.01 , p = 0.02 ) of dengue transmission than those with 4 persons . the last significant predictor was the presence of a community effort in environmental management by clean - up campaign , which was statistically associated with dengue transmission ( aor = 1.91 , p = 0.035 ) compared to the absence of community effort . sociodemographic factors are commonly targeted for disease prevention and control and underpin successful public health programs . although there have been promising indications in the literature , some parameters are not well understood in the case of dengue . risk factors related to dengue transmission are very much influenced by individual and environmental determinants . in this study , individual - level sociodemographic predictors and confounders adjusted for dengue transmission were analyzed through a series of logistic regression models . older age was associated with higher risk in the area and was in agreement with an earlier study . furthermore , several other studies have shown that increasing age was significantly associated with dengue transmission [ 3941 ] . persons who earned secondary and higher degrees of education had a higher risk than those who earned elementary and lower degrees . regardless of the birth place , this possibly relates to the shorter residence time in neighborhood of respondents that possessed greater risk for dengue transmission compared to the longer residence time . the explanation of such phenomena is that persons who have a high level of schooling may have more chance to get skilled careers . accordingly , these career opportunities lead to the movement of people seeking jobs far away from their hometown or community , which increases their risk of getting a dengue viral infection from the place where they have moved to work . if infected , these persons could then transmit the dengue virus to their family members and/or others around their homes . human movement significantly favoring the transmission of dengue correlates well with recent studies [ 4244 ] that have shown this factor to be a major contributor to the acceleration of dengue virus dispersal ( and hence disease distribution in space and time ) , especially between urban / semiurban and rural communities . human migration allows multiple exposure to aedes aegypti bites among migratory people ; in other words , mobile persons have a greater chance of coming into close contact with various bites at multiple locations , especially in public spaces . however , our study did not show a positive association between movements during the previous 3 months and dengue transmission . larger numbers of household members were more at risk for significant exposure to dengue transmission compared to smaller ones . this finding was supported by the previous study that people gathering with daily activities in a house created the exposure frequency of the bites of dengue - virus infected aedes mosquitoes . we attempted to evaluate the relative magnitude of knowledge , attitude , and practice of respondents on reducing dengue risk . the knowledge about dengue and its vectors and the attitude about vector control demonstrated no significant association with dengue transmission . for the practice regarding vector control , only the community effort in environmental management by cleaning campaign had a significant effect both in univariate and multivariate analyses . as for the presence of house window screens , this factor was associated with an increase in dengue transmission . possible explanations are that dengue control efforts by either household members or community participation have generally been performed following the good perception and/or especially during / after dengue outbreaks . in thailand , dengue prevention and control efforts such as eliminating aedes breeding sites either by individual households or by community participation , personal protection , window screens , and fogging have been intensively applied following the dengue experiences or dengue outbreaks . an integrated analysis of the ecosocial determinants for dengue transmission risk in this study provides meaningful implications and hence it merits the improvement of understanding dynamics of dengue transmission in complex epidemiological settings . first , the ecological analysis model indicated enhanced risk in the rcdenpura ecotope ( i.e. , a combination of residential , commercial , and densely populated urban residential areas ) , in the historical high dengue risk area and in households where screens for windows were present . the ecotope of rcdenpura and the historical high dengue risk area appear to be very good predictors for dengue incidences . this suggests that dengue control programs could successfully focus on these determinants embedded in the urban ecosystem or elsewhere , especially during an economic crisis and/or when there is a small budget for such programs . second , the sociological analysis model also revealed plausible significant determinants of older age , higher level of schooling , residence time in the neighborhood , larger household size , and community effort in environmental management by clean - up campaign . regarding the dynamics of sociodemographic contexts and modern lifestyles , these sociodemographic predictors will significantly help us to understand the processes of dengue transmission dynamics and to implement dengue prevention and control programs effectively and efficiently . lastly , variances of pertinently ecological and social determinants should be taken into consideration when deliberately formulating local dengue prevention / control programs to gain benefits for both communities and government health practitioners . meanwhile , these present findings also provide principal grounds for ecosystem and sociodemographic approaches to dengue transmission for researchers in academic institutions .

this study analyzed the association between household - level ecologic and individual - level sociodemographic determinants and dengue transmission in urban areas of chachoengsao province , thailand . the ecologic and sociodemographic variables were examined by univariate analysis and multivariate logistic regression . in the ecologic model , dengue risk was related to households situated in the ecotope of residential mixed with commercial and densely populated urban residential areas ( rcdenpura ) ( aor = 2.23 , p = 0.009 ) , high historical dengue risk area ( aor = 2.06 , p < 0.001 ) , and presence of household window screens ( aor = 1.62 , p = 0.023 ) . in the sociodemographic model , the dengue risk was related to householders aged > 45 years ( aor = 3.24 , p = 0.003 ) , secondary and higher educational degrees ( aor = 2.33 , p = 0.013 ) , household members > 4 persons ( aor = 2.01 , p = 0.02 ) , and community effort in environmental management by clean - up campaign ( aor = 1.91 , p = 0.035 ) . it is possible that the preventive measures were positively correlated with dengue risk because these activities were generally carried out in particular households or communities following dengue experiences or dengue outbreaks . interestingly , the ecotope of rcdenpura and high historical dengue risk area appeared to be very good predictors of dengue incidences .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusions

dengue virus , an aedes mosquito - borne viral pathogen belonging to the family flaviviridae , is the cause of dengue fever ( df ) and dengue hemorrhagic fever ( dhf ) . regarded as a reemerging infectious disease [ 1 , 4 , 5 ] such outbreaks reflect the failure of current prevention and control efforts , despite the fact that some successful cases of vector control in the americas , cuba , and singapore had shortened outbreak periods and stopped the diseases from spreading [ 79 ] . in some cases , an application of appropriate vector control measures along with community participation several reports have shown a coherent argument that transmission dynamics of dengue viruses result from very complex epidemiology and ecology of the disease . such dengue transmission dynamics are the interaction among humans , dengue viruses , vectors , and ecosystems , of which biotic and abiotic determinants have both direct and indirect influences on dengue transmission [ 1316 ] . obviously in some cases , a useful set of environmental and sociodemographic factors , which constituted age - dependent classes , numbers and densities of urban populations , economic classes , and inhabitations , are central components of analysis of temporal and spatial relationships of dengue incidences [ 1720 ] . nonetheless , in different complex epidemiological settings , various factors that can influence dengue transmission dynamics remain to be established . this is because of more diverse sociocultural contexts and changes in sociopolitic , socioeconomic , demographic , technologic , and environmental conditions as well as ineffective management of household - level information and improper implementation of those control strategies . investigation into such sociodemographic , environmental perspectives can provide foresight into the appropriateness of dengue control efforts , give answers to unexpected vector control responses , and contribute to effective management solutions in an ever - changing environment . of note , a plausible paradigm of interdisciplinary approach that integrates the ecologic and sociodemographic dimensions of dengue [ 2227 ] has permitted an analysis of dengue transmission risk to determine what pivotal drivers significantly contribute to dengue transmission in an urban environment . in this regard , we applied two sets of household - level ecologic and individual - level sociodemographic factors to determine whether two fitted models of dengue - related determinants predicted the dengue transmission risk in urban areas of chachoengsao province , thailand , known for epidemics of df / dhf . ultimately , the findings of this study would benefit better management of effective dengue prevention and control , especially in resource - limited developing countries . chachoengsao province was a representative of geographically defined areas ( figure 1 ) , including small - and medium - sized municipalities as well as four main types of landscapes : mountains , rivers , flatlands , and wetlands . in the area , the climate is characterized by a long rainy season ( june october ) , a winter dry season ( november january ) , and a hot dry season ( february may ) . given the complexity of its demographic and socioeconomic dispersions , the province is administratively divided into 11 districts covering a total area of 5,370 km with basic infrastructures of connecting roads , electricity , piped water supply system , communication system , and health service system . the 120 blocks initially assigned to four different districts were used to select 12 blocks ( approximately 100 houses each ) , based on the degree of urbanization and the intensity of dengue transmission ( figure 2 ) . six urban blocks were selected from the municipality of muang district and the capital of chachoengsao province , while the other six semiurban blocks were chosen from three subdistrict municipalities : bang pakong , ban pho , and bang khla . all were subsequently used for household surveys during august october 2007 to collect household - level and individual - level information with the assistance of interdisciplinary teams ( i.e. in this study , the instruments provisionally guided by multicountry supporting teams were initially developed from a series of community of practice workshops delivered through a multicountry network in asia . the data included ecotope , dengue risk area , number of house floors , floor of principal living ( i.e. , homeowners or any member often used a space of everyday family living as principal living area of the first floor or the upper floors ) , construction material of the house , number of house windows , having window screens , having a yard / open space , having bushes in a yard / open space , main purpose of house , and household attachment ( i.e. in this study , we applied an ecotope concept , which is originally defined as the smallest ecologically - distinct features in a landscape mapping and classification system . the difference is based quantitatively and qualitatively on the distribution of houses , the socioeconomic status ( ses ) of local inhabitants , infrastructural services , land use , and land type . the commercial ecotope ( c ) denoted a majority of attached houses with a 0.07-meter mean distance of nearest houses , a high ses of occupants , and good basic infrastructure , along with a great deal of commercial and business buildings . the ecotope of a densely populated urban residential area ( denpura ) is characterized by most attached houses with a 0.63-meter mean distance of nearest houses , low ses of the population , and relatively low degree of community development , including social and economic opportunities and access to health care resources as well as infrastructural water supply and waste management and a lack of vegetation and/or forested areas . the ecotope of residential ( r ) mixed with c and denpura ( rcdenpura ) exhibits a main residential zone prominently scattered with c and denpura and has a 3.37-meter mean distance of nearest houses , while the residential mixed only with commercial ( rc)signifying an overlap of urban and rural areas is an area mainly for dwelling but which is also filled in partly with commercial settlements and has a 4.58-meter mean distance of nearest houses . finally , each ecotope investigated in this study had a different number of study households c ( n = 200 ) , denpura ( n = 200 ) , rcdenpura ( n = 200 ) , and rc ( n = 600 ) . as for the risk area , the classification of historical dengue risk areas ( high and low degrees of dengue transmission ) was drawn from which its epidemic pattern that normally occurs on a 2 - 3 year - cycle transmission . therefore , based on the past situation of national surveillance of dengue cases , the definition of dengue transmission risk areas relies upon a 3- to 5-year median of dengue cases . in this study , the data of the confirmed dengue cases retrieved from the national epidemiological surveillance system were obtained from the chachoengsao general hospital and chachoengsao provincial public health office . therefore , based on the ecotope assignment ( figure 2 ) , the study households ( n = 1,200 households ) were categorized into 2 historical dengue risk areas : 6 high - transmission blocks ( n = 600 households ) that had the highest 5-year medians of dengue cases and 6 low - transmission blocks ( n = 600 households ) that had the lowest 5-year medians of dengue cases . the structured questionnaires on individuals , which had a significant reliability of knowledge , attitude , and practice ( cronbach 's alpha coefficient = 0.7 ) , were used to gather a set of sociodemographic data using those representative respondents of the entire 1,200 households as previously mentioned . the data included age , education level ( highest school degree ) , occupation , movement during the last 3 months , residence time in house , residence time in neighborhood , number of household members , average family income , knowledge about dengue and its vectors , attitude about dengue vector control in terms of gender , family , and government roles , household water storage ( i.e. , by using any water - storing containers ) , household practices to reduce the nuisance of mosquitoes , household practices to prevent aedes breeding places , last time visited by health personnel , receiving dengue control support / materials , and community efforts in environmental management by clean - up campaign . , removing rubbish , covering water containers and killing mosquitoes ) , and ( 3 ) biological control ( e.g. the chemical and physical control activities were ranked based on frequent actions : low ( never or either one of three actions ) and high ( two or more actions ) , whereas the biological control activity was ranked based on the action no or did not apply and yes or applied . , removing rubbish , covering the water containers , changing water once a week , eliminating stagnant water , brushing / cleaning inner surface of water containers , and removing larvae ) , and ( 3 ) biological control ( e.g. the physical control activities were ranked based on the frequency of actions : low ( never , one , or two actions ) and high ( three and more actions ) . dengue - related determinants as the outcomes of dengue transmission risk in the study area included the households that had past history of dengue cases and the respondents or householders that had history of dengue infections in their lifetime before the study . generally speaking , the principal outcome of this study was to identify whether those household - level ecologic and individual - level socioeconomic variables were correlated to these dengue - related determinants . therefore , in two separate ecological and socioeconomical models , a univariate analysis of the individual explanatory variables was used to analyze the association of the dengue - related determinants using the chi - square test ( p < 0.05 or p < 0.1 ) . then , they were entered into the multivariate logistic models and odds ratios ( aors ) adjusted for all of these variables and 95% confidence intervals ( ci ) were calculated . in fitted models , the likelihood - ratio ( lr ) test ( p < 0.05 ) was used to test the significance of all the related predictors while the wald 's test ( p < 0.05 ) was used to test the statistical significance of each coefficient ( b ) in the model to determine contributing predictors . , chicago , il , usa ) , was used throughout this study . we attempted to directly relate household - level ecologic determinants with dengue transmission risk . table 1 reveals the results of the univariate analysis of ecologic risk factors for dengue transmission considering the household - level characteristics . using test , 4 out of 11 ecologic variables were found to have a significant association ( p < 0.05 ) with frequency of houses having previous history of dengue cases . four significant variables included ecotope , historical dengue risk area , number of house windows , and presence of screens for house windows . the three variables of ecotope , historical dengue risk area , and presence of screens for house windows were selected for the final multivariate regression while the number of house windows was not entered in the model because it appeared to be of marginal significance . all determinants ( the ecotope , historical dengue risk area , and presence of screens for house windows ) seemed to have a direct impact on dengue transmission ( the lr test , p < 0.05 ) . for the ecotope , three categorical variables ( denpura , rcdenpura , and rc ) were used to determine their effect on dengue transmission using the commercial ecotope as a reference . only the ecotope of rcdenpura remains as a significant predictor for dengue transmission ( aor = 2.23 , p = 0.009 ) . in terms of dengue risk area , a setting with a high degree of historical dengue transmission indicated or ( aor = 2.06 , p < 0.001 ) significantly higher than an area with a low degree of historical dengue transmission . the last significant variable of presence of screens for house windows showed that houses with window screens had a greater risk ( aor = 1.62 , p = 0.023 ) than those with no window screens . in fact , many relevant studies have demonstrated that it is the set of microsocioeconomic , infrastructural , and environmental parameters embedded in communities which appear to be responsible for increased epidemic transmission of dengue virus in particular ecosystem localities [ 22 , 3034 ] . in addition , a novel ecosystem concept was created to exhaustively scrutinize the associations between ecotope and the transmission of dengue in the study areas . of all 4 ecotopes , only the ecotope of rcdenpura ( i.e. , where the ecotope is in fact a combination of residential , commercial , and densely populated urban residential areas ) exhibited the strongest association with dengue transmission suggesting that the complexity of urban ecosystem may give rise to dengue emergence . to our knowledge , this is the first time that the ecotope , which greatly poses the most risk for dengue occurrence , was determined and characterized . this may reaffirm that the area classification based on past history of national surveillance dengue cases has substantial roles in dengue management as well as in providing at - risk areas targeted for the dengue surveillance system and the implementation of dengue control measures . for the variable of having screens for house windows , the presence of house window screens was significantly associated with dengue transmission , compared to the absence of house window screens . in our study , the negative correlation between the presence of house window screens and dengue transmission may result from the cross - sectional survey where presence of house window screens as an independent variable and houses with a previous history of dengue as dependent variable were measured simultaneously . the existence of many houses that utilize window screens continuously might be due to the perception of dengue and/or due to the health education campaign following dengue outbreaks , whereas the presence of houses with dengue cases was measured after any cases developed within the past year . accordingly , this finding suggested that the presence of window screens may be dependent upon the particular household 's prior dengue risk . table 3 shows the results of the univariate analysis of sociodemographic risk factors related to dengue transmission considering individual - level characteristics . using test , 4 out of 22 sociodemographic variables seemed to have a significant association ( p < 0.05 ) with frequency of respondents with a history of dengue . four significant variables were age , highest school degree , residence time in neighborhood , and community effort in environmental management by clean - up campaign , and hence they were selected for further multivariate analysis . besides these , some variables that either had a significant association at p values of nearly 0.05 or had the potentials of significant phenomena were included in the model . the former variables included residence time in the house and number of household members , while the latter variables constituted movement during last 3 months and knowledge about dengue and its vectors . the sociodemographic multivariate analyses revealed findings of the association between 8 individual determinants and dengue transmission ( table 4 ) . among all determinants tested , 5 of them which included age , highest school degree , residence time in neighborhood , number of household members , and community effort in environmental management by clean - up campaign posed significant risk for dengue transmission ( the lr test , p < 0.05 ) . respondents aged > 45 years showed significant risk for dengue transmission ( aor = 3.24 , p = 0.003 ) compared to those aged 45 years . with regards to highest school degree , individuals with middle and higher degrees were twice as likely to get dengue infection ( aor = 2.33 , p = 0.013 ) compared to those with elementary and lower degrees . in contrast to the univariate analysis , household members > 4 persons were likely to experience a greater risk ( aor = 2.01 , p = 0.02 ) of dengue transmission than those with 4 persons . the last significant predictor was the presence of a community effort in environmental management by clean - up campaign , which was statistically associated with dengue transmission ( aor = 1.91 , p = 0.035 ) compared to the absence of community effort . although there have been promising indications in the literature , some parameters are not well understood in the case of dengue . risk factors related to dengue transmission are very much influenced by individual and environmental determinants . in this study , individual - level sociodemographic predictors and confounders adjusted for dengue transmission were analyzed through a series of logistic regression models . furthermore , several other studies have shown that increasing age was significantly associated with dengue transmission [ 3941 ] . persons who earned secondary and higher degrees of education had a higher risk than those who earned elementary and lower degrees . regardless of the birth place , this possibly relates to the shorter residence time in neighborhood of respondents that possessed greater risk for dengue transmission compared to the longer residence time . if infected , these persons could then transmit the dengue virus to their family members and/or others around their homes . human movement significantly favoring the transmission of dengue correlates well with recent studies [ 4244 ] that have shown this factor to be a major contributor to the acceleration of dengue virus dispersal ( and hence disease distribution in space and time ) , especially between urban / semiurban and rural communities . however , our study did not show a positive association between movements during the previous 3 months and dengue transmission . larger numbers of household members were more at risk for significant exposure to dengue transmission compared to smaller ones . we attempted to evaluate the relative magnitude of knowledge , attitude , and practice of respondents on reducing dengue risk . the knowledge about dengue and its vectors and the attitude about vector control demonstrated no significant association with dengue transmission . for the practice regarding vector control , only the community effort in environmental management by cleaning campaign had a significant effect both in univariate and multivariate analyses . as for the presence of house window screens , this factor was associated with an increase in dengue transmission . possible explanations are that dengue control efforts by either household members or community participation have generally been performed following the good perception and/or especially during / after dengue outbreaks . in thailand , dengue prevention and control efforts such as eliminating aedes breeding sites either by individual households or by community participation , personal protection , window screens , and fogging have been intensively applied following the dengue experiences or dengue outbreaks . an integrated analysis of the ecosocial determinants for dengue transmission risk in this study provides meaningful implications and hence it merits the improvement of understanding dynamics of dengue transmission in complex epidemiological settings . first , the ecological analysis model indicated enhanced risk in the rcdenpura ecotope ( i.e. , a combination of residential , commercial , and densely populated urban residential areas ) , in the historical high dengue risk area and in households where screens for windows were present . the ecotope of rcdenpura and the historical high dengue risk area appear to be very good predictors for dengue incidences . second , the sociological analysis model also revealed plausible significant determinants of older age , higher level of schooling , residence time in the neighborhood , larger household size , and community effort in environmental management by clean - up campaign . regarding the dynamics of sociodemographic contexts and modern lifestyles , these sociodemographic predictors will significantly help us to understand the processes of dengue transmission dynamics and to implement dengue prevention and control programs effectively and efficiently . meanwhile , these present findings also provide principal grounds for ecosystem and sociodemographic approaches to dengue transmission for researchers in academic institutions .

sh2b1 knockout and tgko mice have been described previously ( 29,31 ) and were backcrossed for six generations onto a c57bl/6 genetic background . mice were housed on a 12-h light / dark cycle in the unit for laboratory animal medicine at the university of michigan and fed either normal rodent diet ( 9% fat ; lab diet ) or high - fat diet ( hfd ; 45% fat ; research diets ) ad libitum with free access to water . fat content was measured by dual - energy x - ray absorptiometry ( norland medical system ) . plasma insulin was measured using a rat insulin enzyme - linked immunosorbent assay kit ( crystal chem ) . glucose tolerance tests ( gtt ) ( 2 g d - glucose / kg of body weight ) and insulin tolerance tests ( itt ) ( 1 iu / kg of body weight ; eli lilly ) were conducted as previously described ( 2931 ) . to analyze insulin signaling , mice ( fasted 16 h ) were anesthetized with averin ( 0.5 g of tribromoethanol and 0.25 g or tert - amyl alcohol in 39.5 ml of water ; 0.02 ml / g of body weight ) and treated with pbs or human insulin ( 3 units per mouse ; eli lilly ) via inferior vena cava injection . five minutes after stimulation , gastrocnemius muscles , liver , and epididymal fat pads were dissected , frozen in liquid nitrogen , and stored at 80c . tissues were homogenized in ice - cold lysis buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 1.0% np-40 , 150 mmol / l nacl , 2 mmol / l egta , 1 mmol / l na3vo4 , 100 mmol / l naf , 10 mmol / l na4p2o7 , 1 mmol / l pmsf , 10 g / ml aprotinin , 10 g / ml leupeptin ) , and extracts were immunoblotted or immunoprecipitated with indicated antibodies . cos7 and hek293 cells were grown in dmem supplemented with 5% bovine serum and transfected with indicated plasmids using lipofectamine 2000 ( invitrogen ) . chinese hamster ovary ( cho and cho ) cells were cultured in ham 's f-12 media supplemented with 8% fbs . cells were deprived of serum for 16 h in dmem ( cos7 and hek293 ) or f-12 ( cho ) containing 0.6% bsa before being treated . primary liver cells were isolated from male mice ( 8 weeks ) by perfusion of the liver with type ii collagenase ( worthington biochem ) and plated on collagen - coated plates in m199 containing 10% fbs , 100 units / ml penicillin , and 100 g / ml streptomycin . after 2 h , primary cells were rinsed in pbs and cultured for an additional 16 h in williams ' medium e ( sigma ) supplemented with 0.6% bsa , 100 units / ml penicillin , and 100 g / ml streptomycin . proteins were visualized using the odyssey infrared imaging system ( li - cor biosciences ) or ecl ( amersham ) and quantified using odyssey 1.2 software ( li - cor ) . actin , phosphoakt ( thr ) , akt , insulin receptor , myc , shc , and tubulin antibodies were from santa cruz . the as160 antibody was from millipore and phospho akt substrate antibody was from cell signaling . cells were serum deprived for 16 h , treated with insulin , and solubilized in kinase lysis buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 0.1% triton x-100 , 150 mmol / l nacl , 1 mmol / l edta , 1 mmol / l na3vo4 , 1 mmol / l pmsf , 10 g / ml aprotinin , 10 g / ml leupeptin ) . the insulin receptor was precipitated with wheat germ agglutinin ( wga)-conjugated agarose beads , washed three times in wash buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 0.5 mol / l nacl , 0.1% triton x-100 ) , and twice in kinase reaction buffer ( 20 mmol / l hepes [ ph 7.6 ] , 0.1% triton x-100 , 5 mmol / l mgcl2 , 100 mol / l na3vo4 ) . wga - immobilized proteins were preincubated in kinase reaction buffer supplemented with soluble glutathione s - transferase ( gst ) alone , gst - sh2b1 , or gst - sh2 fusion proteins at 37c . gst - irs-1 ( 510 g ) and atp ( 50 mol / l ) were added to initiate kinase reactions at 37c . reactions were stopped by adding sds - page loading buffer , and reaction mixtures were boiled immediately . immunopurified proteins were washed in lysis buffer and preincubated with gst - sh2b1 or gst ( 2 g ) in phosphatase reaction buffer ( 50 mmol / l tris - hcl [ ph 8.2 ] , 100 nmol / l nacl , 10 mmol / l mgcl2 , 1 mmol / l dtt ) for 15 min at room temperature with constant mixing . alkaline phosphatase ( new england biolabs ) was added at the indicated concentration , and the mixtures were incubated an additional 30 min at room temperature . reactions were stopped by adding sds - page loading buffer and mixtures were boiled immediately . differences between groups were determined by two - tailed student 's t tests or anova . sh2b1 knockout and tgko mice have been described previously ( 29,31 ) and were backcrossed for six generations onto a c57bl/6 genetic background . mice were housed on a 12-h light / dark cycle in the unit for laboratory animal medicine at the university of michigan and fed either normal rodent diet ( 9% fat ; lab diet ) or high - fat diet ( hfd ; 45% fat ; research diets ) ad libitum with free access to water . fat content was measured by dual - energy x - ray absorptiometry ( norland medical system ) . plasma insulin was measured using a rat insulin enzyme - linked immunosorbent assay kit ( crystal chem ) . glucose tolerance tests ( gtt ) ( 2 g d - glucose / kg of body weight ) and insulin tolerance tests ( itt ) ( 1 iu / kg of body weight ; eli lilly ) were conducted as previously described ( 2931 ) . to analyze insulin signaling , mice ( fasted 16 h ) were anesthetized with averin ( 0.5 g of tribromoethanol and 0.25 g or tert - amyl alcohol in 39.5 ml of water ; 0.02 ml / g of body weight ) and treated with pbs or human insulin ( 3 units per mouse ; eli lilly ) via inferior vena cava injection . five minutes after stimulation , gastrocnemius muscles , liver , and epididymal fat pads were dissected , frozen in liquid nitrogen , and stored at 80c . tissues were homogenized in ice - cold lysis buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 1.0% np-40 , 150 mmol / l nacl , 2 mmol / l egta , 1 mmol / l na3vo4 , 100 mmol / l naf , 10 mmol / l na4p2o7 , 1 mmol / l pmsf , 10 g / ml aprotinin , 10 g / ml leupeptin ) , and extracts were immunoblotted or immunoprecipitated with indicated antibodies . cos7 and hek293 cells were grown in dmem supplemented with 5% bovine serum and transfected with indicated plasmids using lipofectamine 2000 ( invitrogen ) . chinese hamster ovary ( cho and cho ) cells were cultured in ham 's f-12 media supplemented with 8% fbs . cells were deprived of serum for 16 h in dmem ( cos7 and hek293 ) or f-12 ( cho ) containing 0.6% bsa before being treated . primary liver cells were isolated from male mice ( 8 weeks ) by perfusion of the liver with type ii collagenase ( worthington biochem ) and plated on collagen - coated plates in m199 containing 10% fbs , 100 units / ml penicillin , and 100 g / ml streptomycin . after 2 h , primary cells were rinsed in pbs and cultured for an additional 16 h in williams ' medium e ( sigma ) supplemented with 0.6% bsa , 100 units / ml penicillin , and 100 g / ml streptomycin . proteins were visualized using the odyssey infrared imaging system ( li - cor biosciences ) or ecl ( amersham ) and quantified using odyssey 1.2 software ( li - cor ) . actin , phosphoakt ( thr ) , akt , insulin receptor , myc , shc , and tubulin antibodies were from santa cruz . the as160 antibody was from millipore and phospho akt substrate antibody was from cell signaling . cells were serum deprived for 16 h , treated with insulin , and solubilized in kinase lysis buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 0.1% triton x-100 , 150 mmol / l nacl , 1 mmol / l edta , 1 mmol / l na3vo4 , 1 mmol / l pmsf , 10 g / ml aprotinin , 10 g / ml leupeptin ) . the insulin receptor was precipitated with wheat germ agglutinin ( wga)-conjugated agarose beads , washed three times in wash buffer ( 50 mmol / l tris hcl [ ph 7.5 ] , 0.5 mol / l nacl , 0.1% triton x-100 ) , and twice in kinase reaction buffer ( 20 mmol / l hepes [ ph 7.6 ] , 0.1% triton x-100 , 5 mmol / l mgcl2 , 100 mol / l na3vo4 ) . wga - immobilized proteins were preincubated in kinase reaction buffer supplemented with soluble glutathione s - transferase ( gst ) alone , gst - sh2b1 , or gst - sh2 fusion proteins at 37c . gst - irs-1 ( 510 g ) and atp ( 50 mol / l ) were added to initiate kinase reactions at 37c . reactions were stopped by adding sds - page loading buffer , and reaction mixtures were boiled immediately . immunopurified proteins were washed in lysis buffer and preincubated with gst - sh2b1 or gst ( 2 g ) in phosphatase reaction buffer ( 50 mmol / l tris - hcl [ ph 8.2 ] , 100 nmol / l nacl , 10 mmol / l mgcl2 , 1 mmol / l dtt ) for 15 min at room temperature with constant mixing . alkaline phosphatase ( new england biolabs ) was added at the indicated concentration , and the mixtures were incubated an additional 30 min at room temperature . reactions were stopped by adding sds - page loading buffer and mixtures were boiled immediately . differences between groups were determined by two - tailed student 's t tests or anova . we previously generated sh2b1 transgenic ( tg ) mice in which the expression of recombinant sh2b1 is controlled by the neuron - specific enolase promoter ( 31 ) . tg mice were crossed with sh2b1 knockout mice to generate sh2b1 transgenic / knockout compound mutant ( tgko ) mice . in tgko mice , recombinant sh2b1 is expressed in brain but not in other tissues , including liver , muscle , and adipose tissue ( 31 ) . neuron - specific restoration of sh2b1 in tgko mice fully corrected leptin resistance and obesity and largely rescued the hyperglycemia and insulin resistance observed in sh2b1 null mice , indicating that neuronal sh2b1 indirectly regulates insulin sensitivity and glucose metabolism by controlling adiposity ( 31 ) . to determine whether loss of peripheral sh2b1 exacerbates dietary fat induced insulin resistance , tgko and wild - type littermates ( 7 weeks ) body weight and adiposity were similar between wild - type and tgko mice fed hfd ( fig . however , fasting ( 16 h ) blood glucose levels were 1.3-fold higher in tgko mice than in wild - type mice fed hfd for 16 weeks ( fig . 1c ) . fasting plasma insulin levels were twofold higher in tgko mice than in wild - type mice ( fig . blood glucose levels were 2326% higher in tgko mice than wild - type mice 15 and 30 min after injection of d - glucose ( fig . exogenous insulin markedly reduced blood glucose in wild - type but not in tgko mice during itt ( fig . these results indicate that loss of peripheral sh2b1 exacerbates hfd - induced insulin resistance , hyperglycemia , and glucose intolerance independent of obesity . f : wild - type ( wt ) and tgko male mice ( 7 weeks ) were fed a hfd . a : growth curve . c : fasting ( 16 h ) blood glucose levels and ( d ) plasma insulin levels after 16 weeks on hfd . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . to examine insulin signaling in skeletal muscle , liver , and epididymal fat , mice ( 7 weeks ) were fed hfd for 16 weeks and treated with insulin or pbs vehicle . insulin markedly stimulated tyrosine phosphorylation of irs-1 as well as irs-1 association with p85 , the regulatory subunit of the phosphatidylinositol 3-kinase , in skeletal muscle of wild - type mice ( fig . loss of peripheral sh2b1 also decreased insulin receptor autophosphorylation and impaired the ability of insulin to stimulate akt phosphorylation on thr and ser in tgko muscle ( fig . insulin - stimulated irs-1 and akt thr phosphorylation were reduced by 44 and 52% , respectively , in tgko muscle ( fig . wild - type ( wt ) and tgko males ( 7 weeks ) were fed a hfd for 16 weeks . mice were fasted for 16 h and treated with pbs or insulin ( 3 units per mouse ) . a : irs-1 in muscle extracts was immunoprecipitated with anti irs-1 antibody ( irs-1 ) and immunoblotted with anti - phosphotyrosine ( ptyr ) and p85 antibodies . muscle extracts were immunoblotted with phospho - specific akt antibodies against phospho - thr ( pthr ) or phospho - ser ( pser ) and akt , respectively . b : irs-1 and akt phosphorylation in ( a ) was quantified by densitometry and normalized to total irs-1 and akt protein levels , respectively . e : epididymal fat ( wat ) extracts were immunoprecipitated with irs-1 and immunoblotted with ptyr and reprobed with irs-1 . f : wat extracts were immunoprecipitated with pas ( anti phospho - ser / thr akt substrate ) and immunoblotted with as160 . three animals were examined for each condition . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . insulin signaling was also examined in liver and white adipose tissue ( wat ) from hfd - fed mice . relative to wild - type mice , basal irs-1 phosphorylation was increased in both liver and wat in tgko mice ; insulin stimulated irs-1 phosphorylation in these tissues from wild - type but not tgko mice ( fig . 2c and e ) . as160 , a rab - gap , is an akt - substrate involved in glut4 vesicle trafficking in adipocytes ( 38,39 ) . to measure as160 phosphorylation , wat extracts were immunoprecipitated with anti phospho - ser / thr akt substrate antibody and immunoblotted with anti - as160 antibody . similar to irs-1 , basal as160 phosphorylation was increased in adipose tissue from tgko mice ( fig . 2f ) ; however , insulin failed to further stimulate as160 phosphorylation ( fig . 2f ) . together , these data indicate that peripheral sh2b1 increases insulin sensitivity in mice by promoting insulin signaling , including the activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway , in muscle , liver , and wat . to determine whether endogenous sh2b1 directly enhances insulin signaling , primary hepatocyte cultures were prepared from wild - type and sh2b1 knockout littermates , and treated with insulin . sh2b1 was detected in wild - type but not in knockout hepatocytes as expected ( fig . insulin stimulated tyrosine phosphorylation of insulin receptor and irs-1 , irs-1 association with p85 , and phosphorylation of akt in wild - type hepatocytes ; however , insulin receptor autophosphorylation , irs-1 phosphorylation , irs-1 association with p85 , and akt phosphorylation were all reduced in knockout hepatocytes ( fig . interestingly , sh2b1 deficiency impaired irs-1 phosphorylation to a greater extent than insulin receptor autophosphorylation . in hek293 cells , overexpression of sh2b1 markedly increased insulin - stimulated tyrosine phosphorylation of irs-1 ; in contrast , a sh2b1 mutant in which the sh2 domain is disrupted because of replacement of arg with glu ( r555e ) functioned as a dominant negative to inhibit irs-1 phosphorylation ( fig . , irs-1 and insulin receptor were coexpressed with n504 , an nh2-terminal truncated form of rat sh2b1 ( amino acids 504670 ) that contained the entire sh2 domain and a minimal number of adjacent amino acids . these data suggest that the sh2 domain of sh2b1 is not only required but also sufficient to promote insulin receptor mediated phosphorylation of irs-1 . sh2b1 directly promotes insulin signaling in cells via its sh2 domain . a : primary hepatocyte cultures were prepared from wild - type ( wt ) or knockout ( ko ) mice ( 8 weeks ) and treated with 100 nmol / l insulin for 10 min . panels 2 and 3 : cell extracts were immunoprecipitated with ir and immunoblotted with ptyr and ir . panels 4 and 5 : cell extracts were immunoprecipitated with irs-1 and immunoblotted with ptyr or p85 . b : irs-1 and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or r555e plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . c : irs-1 and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or n504 plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . d : shc and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or n504 plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . to determine whether sh2b1 also promotes phosphorylation of other irss , shc was coexpressed with sh2b1 or n504 . sh2b1 and n504 enhanced insulin stimulation of shc phosphorylation to similar levels ( fig . 3d ) . by contrast , sh2b1 stimulated irs-1 phosphorylation to a higher level than did n504 ( fig . 3c ) , suggesting that sh2b1 promotes phosphorylation of irs-1 and shc by different mechanisms . sh2b1 directly binds via its sh2 domain to tyr within the activation loop of insulin receptor ( 40,41 ) . to test whether this interaction modulates insulin receptor activation , cho cells , which stably express insulin receptor , were treated with insulin , and active insulin receptor was purified using wga - conjugated agarose beads ( 42 ) . insulin receptor was then preincubated with purified gst - sh2b1 fusion protein and subsequently subjected to in vitro kinase assays using gst - irs-1 fusion protein as substrate . tyrosine phosphorylation of gst - irs-1 was measured by immunoblotting with anti phospho - tyrosine antibodies . gst - sh2b1 , but not gst alone , dose - dependently stimulated insulin receptor kinase activity as indicated by increased phosphorylation of gst - irs-1 ( fig . 4a ) . in similar experiments , a gst - sh2 fusion protein prepared by fusing the sh2 domain ( amino acids 524670 of sh2b1 ) to gst was preincubated with wga - purified insulin receptor in vitro kinase assays . the sh2 domain of sh2b1 was sufficient to enhance insulin receptor catalytic activity , stimulating irs-1 phosphorylation by 66% ( fig . additionally , the sh2 domain of sh2b1 also promoted the catalytic activity of insulin receptor immunopurified with an anti phospho - tyrosine antibody , increasing irs-1 substrate phosphorylation by 79% ( fig . a : cho cells were treated without or with 100 nmol / l insulin for 10 min . insulin receptor was purified with wga - agarose beads , preincubated with indicated amounts of gst or gst - sh2b1 fusion protein and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . b : wga - purified insulin receptor was preincubated with gst or gst - sh2 fusion protein ( 5 g ) and subjected to in vitro kinase assays with gst - irs-1 protein ( amino acids 526859 of rat irs-1 ) as substrate for 10 min . c : cho cells were treated without or with 100 nmol / l insulin for 10 min . ptyr - immunopurified insulin receptor was preincubated with gst or gst - sh2 fusion protein ( amino acids 524670 ) ( 5 g ) and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . reaction mixtures were immunoblotted with indicated antibodies and irs-1 phosphorylation was quantified and normalized to total gst - irs-1 levels . e : wild - type insulin receptor or y1158f was expressed in cos7 cells and treated with insulin . wild - type insulin receptor or y1158f was purified with wga - agarose beads , preincubated with gst or gst - sh2b1 fusion protein ( 5 g ) , and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . * p < 0.05 , * * p < 0.01 . to determine whether tyr in insulin receptor is involved in sh2b1 stimulation of insulin receptor activity , tyr was replaced with phe ( y1158f ) . insulin stimulated autophosphorylation of both insulin receptor and y1158f , but y1158f autophosphorylation was reduced ( fig . y1158f phosphorylated irs-1 in response to insulin ( data not shown ) , indicating that y1158f retains the ability to be activated and to phosphorylate its substrates . insulin receptor and y1158f were purified using wga - beads , preincubated with gst - sh2b1 , and subjected to in vitro kinase assays . sh2b1 stimulated insulin receptor kinase activity by approximately fivefold ; however , sh2b1 was unable to stimulate y1158f catalytic activity ( fig . 4e ) . taken together , these data suggest that the physical interaction between the sh2 domain of sh2b1 and tyr in insulin receptor is required and sufficient for stimulation of insulin receptor catalytic activity . sh2b1 directly binds to irs-1 and irs-2 in vitro ( 37 ) , and insulin stimulated coimmunoprecipitation of sh2b1 with irs-1 in cells ( fig . cho cells , which stably express insulin receptor and irs-1 , were stimulated with insulin to promote tyrosine phosphorylation of irs-1 . phosphorylated irs-1 was immunopurified , preincubated with gst or gst - sh2b1 , and subjected to in vitro dephosphorylation assays . irs-1 bound to gst - sh2b1 but not to gst ( data not shown ) . alkaline phosphatase dose - dependently dephosphorylated irs-1 on tyrosines in the gst - pretreated samples ; in contrast , alkaline phosphatase was unable to dephosphorylate sh2b1-bound irs-1 ( fig . insulin also promoted the association of sh2b1 with irs-2 , and sh2b1 similarly inhibited tyrosine dephosphorylation of irs-2 ( data not shown ) . b : cho cells were stimulated with 100 nmol / l insulin for 10 min . irs-1 was immunoprecipitated with irs-1 , preincubated with gst or gst - sh2b1 ( 2 g ) , and subjected to in vitro dephosphorylation assays with indicated amounts of alkaline phosphatase for 30 min . c : insulin receptor and irs-1 were transiently expressed with ptp1b ( 0.1 g ) and increasing amounts of myc - tagged sh2b1 ( 00.8 g ) . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . d : irs-1 was expressed with insulin receptor or y1158f in the absence ( con ) or presence of sh2b1 in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . e : irs-1 ( 1 g ) and y1158f ( 1 g ) plasmids were transiently cotransfected with or without sh2b1 plasmids ( 0.8 g ) in hek293 cells . cells were deprived of serum overnight 48 h after transfection and treated with 100 nmol / l insulin for 10 min . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . to determine whether sh2b1 inhibits irs-1 dephosphorylation in cells , irs-1 was coexpressed with ptp1b ( a protein tyrosine phosphatase ) in the absence or presence of sh2b1 . ptp1b dephosphorylated irs-1 , and sh2b1 dose - dependently attenuated the ability of ptp1b to dephosphorylate irs-1 ( fig . 5c ) . to determine whether sh2b1 is able to promote irs-1 phosphorylation without stimulating insulin receptor kinase activity , 4e ) , sh2b1 still markedly enhanced tyrosine phosphorylation of irs-1 in y1158f - expressing cells ( fig . , sh2b1 is likely to augment y1158f - mediated phosphorylation of irs-1 by inhibiting irs-1 dephosphorylation by endogenous protein phosphatase(s ) . to determine whether the sh2b1-irs interaction sterically inhibits the binding of irs proteins to phosphatidylinositol 3-kinase , irs-1 and y1158f were coexpressed with or without sh2b1 in hek293 cells , and irs-1-p85 association insulin stimulated coimmunoprecipitation of irs-1 with p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; importantly , sh2b1 markedly enhanced insulin - stimulated p85 binding to irs-1 ( fig . these data indicate that the sh2b1-irs interaction does not interfere with irs - phosphatidylinositol 3-kinase interaction but rather increases the irs - phosphatidylinositol 3-kinase association by inhibiting irs dephosphorylation . consistent with these observations , sh2b1 also enhanced insulin - stimulated , y1158f - mediated akt phosphorylation ( fig . collectively , these data suggest that , in addition to enhancing insulin receptor catalytic activity via binding to tyr , sh2b1 also promotes activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway by inhibiting irs dephosphorylation . we previously generated sh2b1 transgenic ( tg ) mice in which the expression of recombinant sh2b1 is controlled by the neuron - specific enolase promoter ( 31 ) . tg mice were crossed with sh2b1 knockout mice to generate sh2b1 transgenic / knockout compound mutant ( tgko ) mice . in tgko mice , recombinant sh2b1 is expressed in brain but not in other tissues , including liver , muscle , and adipose tissue ( 31 ) . neuron - specific restoration of sh2b1 in tgko mice fully corrected leptin resistance and obesity and largely rescued the hyperglycemia and insulin resistance observed in sh2b1 null mice , indicating that neuronal sh2b1 indirectly regulates insulin sensitivity and glucose metabolism by controlling adiposity ( 31 ) . to determine whether loss of peripheral sh2b1 exacerbates dietary fat induced insulin resistance , tgko and wild - type littermates ( 7 weeks ) body weight and adiposity were similar between wild - type and tgko mice fed hfd ( fig . however , fasting ( 16 h ) blood glucose levels were 1.3-fold higher in tgko mice than in wild - type mice fed hfd for 16 weeks ( fig . 1c ) . fasting plasma insulin levels were twofold higher in tgko mice than in wild - type mice ( fig . blood glucose levels were 2326% higher in tgko mice than wild - type mice 15 and 30 min after injection of d - glucose ( fig . exogenous insulin markedly reduced blood glucose in wild - type but not in tgko mice during itt ( fig . these results indicate that loss of peripheral sh2b1 exacerbates hfd - induced insulin resistance , hyperglycemia , and glucose intolerance independent of obesity . f : wild - type ( wt ) and tgko male mice ( 7 weeks ) were fed a hfd . a : growth curve . c : fasting ( 16 h ) blood glucose levels and ( d ) plasma insulin levels after 16 weeks on hfd . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . to examine insulin signaling in skeletal muscle , liver , and epididymal fat , mice ( 7 weeks ) were fed hfd for 16 weeks and treated with insulin or pbs vehicle . insulin markedly stimulated tyrosine phosphorylation of irs-1 as well as irs-1 association with p85 , the regulatory subunit of the phosphatidylinositol 3-kinase , in skeletal muscle of wild - type mice ( fig . loss of peripheral sh2b1 also decreased insulin receptor autophosphorylation and impaired the ability of insulin to stimulate akt phosphorylation on thr and ser in tgko muscle ( fig . insulin - stimulated irs-1 and akt thr phosphorylation were reduced by 44 and 52% , respectively , in tgko muscle ( fig . wild - type ( wt ) and tgko males ( 7 weeks ) were fed a hfd for 16 weeks . mice were fasted for 16 h and treated with pbs or insulin ( 3 units per mouse ) . a : irs-1 in muscle extracts was immunoprecipitated with anti irs-1 antibody ( irs-1 ) and immunoblotted with anti - phosphotyrosine ( ptyr ) and p85 antibodies . muscle extracts were immunoblotted with phospho - specific akt antibodies against phospho - thr ( pthr ) or phospho - ser ( pser ) and akt , respectively . b : irs-1 and akt phosphorylation in ( a ) was quantified by densitometry and normalized to total irs-1 and akt protein levels , respectively . e : epididymal fat ( wat ) extracts were immunoprecipitated with irs-1 and immunoblotted with ptyr and reprobed with irs-1 . f : wat extracts were immunoprecipitated with pas ( anti phospho - ser / thr akt substrate ) and immunoblotted with as160 . three animals were examined for each condition . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . insulin signaling was also examined in liver and white adipose tissue ( wat ) from hfd - fed mice . relative to wild - type mice , basal irs-1 phosphorylation was increased in both liver and wat in tgko mice ; insulin stimulated irs-1 phosphorylation in these tissues from wild - type but not tgko mice ( fig . 2c and e ) . as160 , a rab - gap , is an akt - substrate involved in glut4 vesicle trafficking in adipocytes ( 38,39 ) . to measure as160 phosphorylation , wat extracts were immunoprecipitated with anti phospho - ser / thr akt substrate antibody and immunoblotted with anti - as160 antibody . similar to irs-1 , basal as160 phosphorylation was increased in adipose tissue from tgko mice ( fig . together , these data indicate that peripheral sh2b1 increases insulin sensitivity in mice by promoting insulin signaling , including the activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway , in muscle , liver , and wat . to determine whether endogenous sh2b1 directly enhances insulin signaling , primary hepatocyte cultures were prepared from wild - type and sh2b1 knockout littermates , and treated with insulin . sh2b1 was detected in wild - type but not in knockout hepatocytes as expected ( fig . insulin stimulated tyrosine phosphorylation of insulin receptor and irs-1 , irs-1 association with p85 , and phosphorylation of akt in wild - type hepatocytes ; however , insulin receptor autophosphorylation , irs-1 phosphorylation , irs-1 association with p85 , and akt phosphorylation were all reduced in knockout hepatocytes ( fig . interestingly , sh2b1 deficiency impaired irs-1 phosphorylation to a greater extent than insulin receptor autophosphorylation . in hek293 cells , overexpression of sh2b1 markedly increased insulin - stimulated tyrosine phosphorylation of irs-1 ; in contrast , a sh2b1 mutant in which the sh2 domain is disrupted because of replacement of arg with glu ( r555e ) functioned as a dominant negative to inhibit irs-1 phosphorylation ( fig . 3b ) . to further assess the role of the sh2 domain of sh2b1 , irs-1 and insulin receptor were coexpressed with n504 , an nh2-terminal truncated form of rat sh2b1 ( amino acids 504670 ) that contained the entire sh2 domain and a minimal number of adjacent amino acids . these data suggest that the sh2 domain of sh2b1 is not only required but also sufficient to promote insulin receptor mediated phosphorylation of irs-1 . sh2b1 directly promotes insulin signaling in cells via its sh2 domain . a : primary hepatocyte cultures were prepared from wild - type ( wt ) or knockout ( ko ) mice ( 8 weeks ) and treated with 100 nmol / l insulin for 10 min . panels 2 and 3 : cell extracts were immunoprecipitated with ir and immunoblotted with ptyr and ir . panels 4 and 5 : cell extracts were immunoprecipitated with irs-1 and immunoblotted with ptyr or p85 . b : irs-1 and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or r555e plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . c : irs-1 and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or n504 plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . d : shc and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or n504 plasmids in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . shc phosphorylation was normalized to total shc protein levels . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 . to determine whether sh2b1 also promotes phosphorylation of other irss , shc was coexpressed with sh2b1 or n504 . sh2b1 and n504 enhanced insulin stimulation of shc phosphorylation to similar levels ( fig . 3c ) , suggesting that sh2b1 promotes phosphorylation of irs-1 and shc by different mechanisms . sh2b1 directly binds via its sh2 domain to tyr within the activation loop of insulin receptor ( 40,41 ) . to test whether this interaction modulates insulin receptor activation , cho cells , which stably express insulin receptor , were treated with insulin , and active insulin receptor was purified using wga - conjugated agarose beads ( 42 ) . insulin receptor was then preincubated with purified gst - sh2b1 fusion protein and subsequently subjected to in vitro kinase assays using gst - irs-1 fusion protein as substrate . tyrosine phosphorylation of gst - irs-1 was measured by immunoblotting with anti phospho - tyrosine antibodies . gst - sh2b1 , but not gst alone , dose - dependently stimulated insulin receptor kinase activity as indicated by increased phosphorylation of gst - irs-1 ( fig . 4a ) . in similar experiments , a gst - sh2 fusion protein prepared by fusing the sh2 domain ( amino acids 524670 of sh2b1 ) to gst was preincubated with wga - purified insulin receptor in vitro kinase assays . the sh2 domain of sh2b1 was sufficient to enhance insulin receptor catalytic activity , stimulating irs-1 phosphorylation by 66% ( fig . additionally , the sh2 domain of sh2b1 also promoted the catalytic activity of insulin receptor immunopurified with an anti phospho - tyrosine antibody , increasing irs-1 substrate phosphorylation by 79% ( fig . sh2b1 directly enhances insulin receptor activity in vitro . a : cho cells were treated without or with 100 nmol / l insulin for 10 min . insulin receptor was purified with wga - agarose beads , preincubated with indicated amounts of gst or gst - sh2b1 fusion protein and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . b : wga - purified insulin receptor was preincubated with gst or gst - sh2 fusion protein ( 5 g ) and subjected to in vitro kinase assays with gst - irs-1 protein ( amino acids 526859 of rat irs-1 ) as substrate for 10 min . c : cho cells were treated without or with 100 nmol / l insulin for 10 min . ptyr - immunopurified insulin receptor was preincubated with gst or gst - sh2 fusion protein ( amino acids 524670 ) ( 5 g ) and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . reaction mixtures were immunoblotted with indicated antibodies and irs-1 phosphorylation was quantified and normalized to total gst - irs-1 levels . e : wild - type insulin receptor or y1158f was expressed in cos7 cells and treated with insulin . wild - type insulin receptor or y1158f was purified with wga - agarose beads , preincubated with gst or gst - sh2b1 fusion protein ( 5 g ) , and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . * p < 0.05 , * * p < 0.01 . to determine whether tyr in insulin receptor is involved in sh2b1 stimulation of insulin receptor activity , tyr was replaced with phe ( y1158f ) . insulin stimulated autophosphorylation of both insulin receptor and y1158f , but y1158f autophosphorylation was reduced ( fig . 4d ) . y1158f phosphorylated irs-1 in response to insulin ( data not shown ) , indicating that y1158f retains the ability to be activated and to phosphorylate its substrates . insulin receptor and y1158f were purified using wga - beads , preincubated with gst - sh2b1 , and subjected to in vitro kinase assays . sh2b1 stimulated insulin receptor kinase activity by approximately fivefold ; however , sh2b1 was unable to stimulate y1158f catalytic activity ( fig . , these data suggest that the physical interaction between the sh2 domain of sh2b1 and tyr in insulin receptor is required and sufficient for stimulation of insulin receptor catalytic activity . sh2b1 directly binds to irs-1 and irs-2 in vitro ( 37 ) , and insulin stimulated coimmunoprecipitation of sh2b1 with irs-1 in cells ( fig . cho cells , which stably express insulin receptor and irs-1 , were stimulated with insulin to promote tyrosine phosphorylation of irs-1 . phosphorylated irs-1 was immunopurified , preincubated with gst or gst - sh2b1 , and subjected to in vitro dephosphorylation assays . irs-1 bound to gst - sh2b1 but not to gst ( data not shown ) . alkaline phosphatase dose - dependently dephosphorylated irs-1 on tyrosines in the gst - pretreated samples ; in contrast , alkaline phosphatase was unable to dephosphorylate sh2b1-bound irs-1 ( fig . insulin also promoted the association of sh2b1 with irs-2 , and sh2b1 similarly inhibited tyrosine dephosphorylation of irs-2 ( data not shown ) . b : cho cells were stimulated with 100 nmol / l insulin for 10 min . irs-1 was immunoprecipitated with irs-1 , preincubated with gst or gst - sh2b1 ( 2 g ) , and subjected to in vitro dephosphorylation assays with indicated amounts of alkaline phosphatase for 30 min . c : insulin receptor and irs-1 were transiently expressed with ptp1b ( 0.1 g ) and increasing amounts of myc - tagged sh2b1 ( 00.8 g ) . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . d : irs-1 was expressed with insulin receptor or y1158f in the absence ( con ) or presence of sh2b1 in hek293 cells . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . e : irs-1 ( 1 g ) and y1158f ( 1 g ) plasmids were transiently cotransfected with or without sh2b1 plasmids ( 0.8 g ) in hek293 cells . cells were deprived of serum overnight 48 h after transfection and treated with 100 nmol / l insulin for 10 min . cells were treated with 100 nmol / l insulin for 10 min and extracts were immunoblotted with indicated antibodies . to determine whether sh2b1 inhibits irs-1 dephosphorylation in cells , irs-1 was coexpressed with ptp1b ( a protein tyrosine phosphatase ) in the absence or presence of sh2b1 . ptp1b dephosphorylated irs-1 , and sh2b1 dose - dependently attenuated the ability of ptp1b to dephosphorylate irs-1 ( fig . 5c ) . to determine whether sh2b1 is able to promote irs-1 phosphorylation without stimulating insulin receptor kinase activity , 4e ) , sh2b1 still markedly enhanced tyrosine phosphorylation of irs-1 in y1158f - expressing cells ( fig . , sh2b1 is likely to augment y1158f - mediated phosphorylation of irs-1 by inhibiting irs-1 dephosphorylation by endogenous protein phosphatase(s ) . to determine whether the sh2b1-irs interaction sterically inhibits the binding of irs proteins to phosphatidylinositol 3-kinase , irs-1 and y1158f were coexpressed with or without sh2b1 in hek293 cells , and irs-1-p85 association insulin stimulated coimmunoprecipitation of irs-1 with p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; importantly , sh2b1 markedly enhanced insulin - stimulated p85 binding to irs-1 ( fig . these data indicate that the sh2b1-irs interaction does not interfere with irs - phosphatidylinositol 3-kinase interaction but rather increases the irs - phosphatidylinositol 3-kinase association by inhibiting irs dephosphorylation . consistent with these observations , sh2b1 also enhanced insulin - stimulated , y1158f - mediated akt phosphorylation ( fig . collectively , these data suggest that , in addition to enhancing insulin receptor catalytic activity via binding to tyr , sh2b1 also promotes activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway by inhibiting irs dephosphorylation . insulin resistance is the primary risk factor for various metabolic diseases , including type 2 diabetes , nonalcoholic fatty liver disease , dyslipidemia , and cardiovascular disease . it is commonly accepted that impairments in insulin signal transduction play a key role in the development of insulin resistance . sh2b1 overexpression increases insulin receptor autophosphorylation and tyrosine phosphorylation of irs-1 and irs-2 in cultured cells ( 29,43 ) . similar observations were independently reported by two other groups ( 44,45 ) . additionally , we showed that genetic deletion of sh2b1 results in severe insulin resistance and type 2 diabetes in mice ( 29 ) . however , sh2b1-null mice are also severely obese because of leptin resistance ( 3032 ) , raising the possibility that insulin resistance may be secondary to obesity in sh2b1-null mice . therefore , it was unclear whether peripheral sh2b1 directly regulates insulin sensitivity in insulin target tissues in vivo . we generated tgko mice that express sh2b1 only in the brain but not in peripheral tissues ( e.g. , liver , muscle , and adipose tissue ) . body weight was similar between tgko and wild - type littermates fed hfd , consistent with our previous conclusion that neuronal sh2b1 controls energy balance and body weight by enhancing leptin sensitivity in the hypothalamus ( 31 ) . in the current study , we demonstrated that loss of peripheral sh2b1 markedly impaired insulin sensitivity independent of body weight . tgko mice developed hyperglycemia , hyperinsulinemia , and glucose intolerance to a greater extent than wild - type littermates fed hfd . the ability of exogenous insulin to reduce blood glucose and to stimulate insulin receptor autophosphorylation and phosphorylation of irs proteins and akt in muscle , liver , and adipose tissue was significantly reduced in tgko mice . insulin signaling has been shown to be attenuated by multiple intracellular signaling molecules ( e.g. , ptp1b , grb10 , grb14 , socs1 , socs3 , jnk , pkc , s6k , and ikk ) that contribute to the development of insulin resistance ( 624 ) . our data suggest that insulin sensitivity is controlled by a balance between these negative regulators and sh2b1 in insulin target cells . sh2b1 binds via its sh2 domain to phospho - tyr in the activation loop of insulin receptor ( 40,41 ) . we showed that bacteria - derived sh2b1 markedly increased the ability of purified insulin receptor to tyrosyl phosphorylate irs-1 in vitro . in contrast , sh2b1 was unable to stimulate the catalytic activity of y1158f , an insulin receptor mutant lacking the binding site for sh2b1 . in cells , sh2b1 overexpression promotes insulin receptor autophosphorylation as well as insulin receptor phosphorylation of its substrates ( e.g. , irs-1 , irs-2 , and shc ) . conversely , deletion of endogenous sh2b1 impaired insulin stimulation of insulin receptor autophosphorylation and irs-1 phosphorylation in primary hepatocyte cultures . consistent with these observations , sh2b1 complexes , which are immunoprecipitated from cell extracts , reportedly promote insulin receptor autophosphorylation by reducing the km for atp ( 45 ) . the same report also concluded that sh2b1 dimerization was required for its stimulation of insulin receptor autophoshorylation , because treatment of cells with dimerization domain peptide mimetics inhibited insulin receptor autophosphorylation and downstream pathways ( 45 ) . however , the report did not provide evidence showing that the mimetics disrupted sh2b1 dimerization . in contrast , we observed that the sh2 domain alone was sufficient to stimulate insulin receptor catalytic activity in vitro . moreover , n504 , an nh2-terminal truncated sh2b1 containing the intact sh2 domain but completely lacking both dimerization and pleckstrin homology domains , still markedly enhanced insulin - stimulated tyrosine phosphorylation of both irs-1 and shc . conversely , r555e , a sh2b1 mutant with a defective sh2 domain , inhibited insulin signaling as a dominant negative mutant . these data indicate that the sh2 domain of sh2b1 is both required and sufficient to stimulate insulin receptor kinase activity . because tyr phosphorylation occurs early in the activation of the insulin receptor kinase ( 42,46,47 ) , binding of the sh2 domain of sh2b1 to phospho - tyr may stabilize insulin receptor in an active conformation . importantly , sh2b1 directly inhibited tyrosine dephosphorylation of irs-1 and irs-2 by recombinant phosphatase in vitro and by ptp1b in cultured cells . although unable to stimulate y1158f catalytic activity , sh2b1 still enhanced y1158f - mediated phosphorylation of irs-1 in cultured cells , presumably by inhibiting irs-1 dephosphorylation by endogenous tyrosine phosphatase(s ) . consistent with these observations , deletion of endogenous sh2b1 impaired tyrosine phosphorylation of irs-1 to a greater extent than insulin receptor autophosphorylation in primary hepatocyte cultures . together , these data suggest that the sh2b1-irs physical interaction inhibits irs dephosphorylation by tyrosine phosphatases . interestingly , the sh2b1-irs interaction did not inhibit the ability of phosphorylated irs proteins to bind to p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; in contrast , it enhanced insulin - stimulated irs - p85 association and subsequent akt phosphorylation and activation , presumably by protecting irs proteins against dephosphorylation . these data suggest that sh2b1 does not compete with p85 for the same binding sites in irs proteins , and that the sh2b1-irs interaction does not sterically interfere with the irs - p85 interaction . therefore , the sh2b1-irs interaction may selectively block irs interaction with tyrosine phosphatases , thereby inhibiting irs dephosphorylation . alternatively , the sh2b1-irs interaction may alter irs conformation so that multiple tyrosine phosphorylation sites , in addition to sh2b1-bound site(s ) , are resistant to dephosphorylation , but still retain their ability to bind to downstream signaling molecules and activate downstream pathways including the phosphatidylinositol 3-kinase / akt pathway . in conclusion neuronal sh2b1 increases insulin sensitivity indirectly by reducing adiposity ( 31 ) . in muscle , liver , and adipose tissue , sh2b1 binds to insulin receptor and sh2b1 binds to both irs-1 and irs-2 and protects irs proteins from tyrosine dephosphorylation , augmenting and/or prolonging irs protein - mediated pathways . in addition , sh2b1 forms dimers , and each sh2b1 molecule in a sh2b1 dimer may simultaneously bind to insulin receptor and irs-1 ( or irs-2 ) , thereby stabilizing insulin receptor / irs-1 ( or insulin receptor / irs-2 ) complexes . therefore , sh2b1 and molecules that mimic these functions of sh2b1 are potential therapeutic targets for the treatment of obesity and/or type 2 diabetes . a model for sh2b1 regulation of insulin signaling . in response to insulin , sh2b1 binds directly to phospho - tyr in insulin receptor via its sh2 domain and stimulates insulin receptor kinase activity , thereby enhancing the activation of multiple signaling pathways downstream of insulin receptor ( e.g. , the shc / mapk and the irs / phosphatidylinositol 3-kinase pathways ) . sh2b1 also binds to irs-1 or irs-2 and inhibits their dephosphorylation on tyrosines to specifically promote the activation of irs protein - mediated pathways . because sh2b1 dimerizes via its dimerization domain , dimerized sh2b1 may further enhance insulin signaling by simultaneously binding to both insulin receptor and irs-1 to stabilize active insulin receptor with irs-1 or recruit irs-1 to insulin receptor .

objectivesh2b1 is a sh2 domain - containing adaptor protein expressed in both the central nervous system and peripheral tissues . neuronal sh2b1 controls body weight ; however , the functions of peripheral sh2b1 remain unknown . here , we studied peripheral sh2b1 regulation of insulin sensitivity and glucose metabolism.research design and methodswe generated tgko mice expressing sh2b1 in the brain but not peripheral tissues . various metabolic parameters and insulin signaling were examined in tgko mice fed a high - fat diet ( hfd ) . the effect of sh2b1 on the insulin receptor catalytic activity and insulin receptor substrate ( irs)-1/irs-2 dephosphorylation was examined using in vitro kinase assays and in vitro dephosphorylation assays , respectively . sh2b1 was coexpressed with ptp1b , and insulin receptor mediated phosphorylation of irs-1 was examined.resultsdeletion of peripheral sh2b1 markedly exacerbated hfd - induced hyperglycemia , hyperinsulinemia , and glucose intolerance in tgko mice . insulin signaling was dramatically impaired in muscle , liver , and adipose tissue in tgko mice . deletion of sh2b1 impaired insulin signaling in primary hepatocytes , whereas sh2b1 overexpression stimulated insulin receptor autophosphorylation and tyrosine phosphorylation of irss . purified sh2b1 stimulated insulin receptor catalytic activity in vitro . the sh2 domain of sh2b1 was both required and sufficient to promote insulin receptor activation . insulin stimulated the binding of sh2b1 to irs-1 or irs-2 . this physical interaction inhibited tyrosine dephosphorylation of irs-1 or irs-2 and increased the ability of irs proteins to activate the phosphatidylinositol 3-kinase pathway.conclusionssh2b1 is an endogenous insulin sensitizer . it directly binds to insulin receptors , irs-1 and irs-2 , and enhances insulin sensitivity by promoting insulin receptor catalytic activity and by inhibiting tyrosine dephosphorylation of irs proteins .

RESEARCH DESIGN AND METHODS Animal studies. Cell lines and transfection. Immunoprecipitation and immunoblotting. Insulin receptor kinase assay. Dephosphorylation assays. Statistical analysis. RESULTS Loss of peripheral SH2B1 predisposes mice to HFD-induced insulin resistance. Loss of peripheral SH2B1 impairs insulin signaling in muscle, liver, and adipose tissue in HFD-fed mice. SH2B1 cell autonomously promotes insulin signaling via its SH2 domain. SH2B1 stimulates insulin receptor catalytic activity through the binding of its SH2 domain to Tyr SH2B1 protects IRS proteins against tyrosine dephosphorylation. DISCUSSION

mice were housed on a 12-h light / dark cycle in the unit for laboratory animal medicine at the university of michigan and fed either normal rodent diet ( 9% fat ; lab diet ) or high - fat diet ( hfd ; 45% fat ; research diets ) ad libitum with free access to water . mice were housed on a 12-h light / dark cycle in the unit for laboratory animal medicine at the university of michigan and fed either normal rodent diet ( 9% fat ; lab diet ) or high - fat diet ( hfd ; 45% fat ; research diets ) ad libitum with free access to water . in tgko mice , recombinant sh2b1 is expressed in brain but not in other tissues , including liver , muscle , and adipose tissue ( 31 ) . neuron - specific restoration of sh2b1 in tgko mice fully corrected leptin resistance and obesity and largely rescued the hyperglycemia and insulin resistance observed in sh2b1 null mice , indicating that neuronal sh2b1 indirectly regulates insulin sensitivity and glucose metabolism by controlling adiposity ( 31 ) . to determine whether loss of peripheral sh2b1 exacerbates dietary fat induced insulin resistance , tgko and wild - type littermates ( 7 weeks ) body weight and adiposity were similar between wild - type and tgko mice fed hfd ( fig . these results indicate that loss of peripheral sh2b1 exacerbates hfd - induced insulin resistance , hyperglycemia , and glucose intolerance independent of obesity . to examine insulin signaling in skeletal muscle , liver , and epididymal fat , mice ( 7 weeks ) were fed hfd for 16 weeks and treated with insulin or pbs vehicle . insulin markedly stimulated tyrosine phosphorylation of irs-1 as well as irs-1 association with p85 , the regulatory subunit of the phosphatidylinositol 3-kinase , in skeletal muscle of wild - type mice ( fig . loss of peripheral sh2b1 also decreased insulin receptor autophosphorylation and impaired the ability of insulin to stimulate akt phosphorylation on thr and ser in tgko muscle ( fig . insulin signaling was also examined in liver and white adipose tissue ( wat ) from hfd - fed mice . relative to wild - type mice , basal irs-1 phosphorylation was increased in both liver and wat in tgko mice ; insulin stimulated irs-1 phosphorylation in these tissues from wild - type but not tgko mice ( fig . together , these data indicate that peripheral sh2b1 increases insulin sensitivity in mice by promoting insulin signaling , including the activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway , in muscle , liver , and wat . insulin stimulated tyrosine phosphorylation of insulin receptor and irs-1 , irs-1 association with p85 , and phosphorylation of akt in wild - type hepatocytes ; however , insulin receptor autophosphorylation , irs-1 phosphorylation , irs-1 association with p85 , and akt phosphorylation were all reduced in knockout hepatocytes ( fig . in hek293 cells , overexpression of sh2b1 markedly increased insulin - stimulated tyrosine phosphorylation of irs-1 ; in contrast , a sh2b1 mutant in which the sh2 domain is disrupted because of replacement of arg with glu ( r555e ) functioned as a dominant negative to inhibit irs-1 phosphorylation ( fig . , irs-1 and insulin receptor were coexpressed with n504 , an nh2-terminal truncated form of rat sh2b1 ( amino acids 504670 ) that contained the entire sh2 domain and a minimal number of adjacent amino acids . these data suggest that the sh2 domain of sh2b1 is not only required but also sufficient to promote insulin receptor mediated phosphorylation of irs-1 . in similar experiments , a gst - sh2 fusion protein prepared by fusing the sh2 domain ( amino acids 524670 of sh2b1 ) to gst was preincubated with wga - purified insulin receptor in vitro kinase assays . the sh2 domain of sh2b1 was sufficient to enhance insulin receptor catalytic activity , stimulating irs-1 phosphorylation by 66% ( fig . additionally , the sh2 domain of sh2b1 also promoted the catalytic activity of insulin receptor immunopurified with an anti phospho - tyrosine antibody , increasing irs-1 substrate phosphorylation by 79% ( fig . wild - type insulin receptor or y1158f was purified with wga - agarose beads , preincubated with gst or gst - sh2b1 fusion protein ( 5 g ) , and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . insulin receptor and y1158f were purified using wga - beads , preincubated with gst - sh2b1 , and subjected to in vitro kinase assays . sh2b1 stimulated insulin receptor kinase activity by approximately fivefold ; however , sh2b1 was unable to stimulate y1158f catalytic activity ( fig . taken together , these data suggest that the physical interaction between the sh2 domain of sh2b1 and tyr in insulin receptor is required and sufficient for stimulation of insulin receptor catalytic activity . sh2b1 directly binds to irs-1 and irs-2 in vitro ( 37 ) , and insulin stimulated coimmunoprecipitation of sh2b1 with irs-1 in cells ( fig . cho cells , which stably express insulin receptor and irs-1 , were stimulated with insulin to promote tyrosine phosphorylation of irs-1 . phosphorylated irs-1 was immunopurified , preincubated with gst or gst - sh2b1 , and subjected to in vitro dephosphorylation assays . insulin also promoted the association of sh2b1 with irs-2 , and sh2b1 similarly inhibited tyrosine dephosphorylation of irs-2 ( data not shown ) . irs-1 was immunoprecipitated with irs-1 , preincubated with gst or gst - sh2b1 ( 2 g ) , and subjected to in vitro dephosphorylation assays with indicated amounts of alkaline phosphatase for 30 min . d : irs-1 was expressed with insulin receptor or y1158f in the absence ( con ) or presence of sh2b1 in hek293 cells . to determine whether sh2b1 inhibits irs-1 dephosphorylation in cells , irs-1 was coexpressed with ptp1b ( a protein tyrosine phosphatase ) in the absence or presence of sh2b1 . to determine whether sh2b1 is able to promote irs-1 phosphorylation without stimulating insulin receptor kinase activity , 4e ) , sh2b1 still markedly enhanced tyrosine phosphorylation of irs-1 in y1158f - expressing cells ( fig . to determine whether the sh2b1-irs interaction sterically inhibits the binding of irs proteins to phosphatidylinositol 3-kinase , irs-1 and y1158f were coexpressed with or without sh2b1 in hek293 cells , and irs-1-p85 association insulin stimulated coimmunoprecipitation of irs-1 with p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; importantly , sh2b1 markedly enhanced insulin - stimulated p85 binding to irs-1 ( fig . collectively , these data suggest that , in addition to enhancing insulin receptor catalytic activity via binding to tyr , sh2b1 also promotes activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway by inhibiting irs dephosphorylation . in tgko mice , recombinant sh2b1 is expressed in brain but not in other tissues , including liver , muscle , and adipose tissue ( 31 ) . neuron - specific restoration of sh2b1 in tgko mice fully corrected leptin resistance and obesity and largely rescued the hyperglycemia and insulin resistance observed in sh2b1 null mice , indicating that neuronal sh2b1 indirectly regulates insulin sensitivity and glucose metabolism by controlling adiposity ( 31 ) . to determine whether loss of peripheral sh2b1 exacerbates dietary fat induced insulin resistance , tgko and wild - type littermates ( 7 weeks ) body weight and adiposity were similar between wild - type and tgko mice fed hfd ( fig . these results indicate that loss of peripheral sh2b1 exacerbates hfd - induced insulin resistance , hyperglycemia , and glucose intolerance independent of obesity . to examine insulin signaling in skeletal muscle , liver , and epididymal fat , mice ( 7 weeks ) were fed hfd for 16 weeks and treated with insulin or pbs vehicle . insulin markedly stimulated tyrosine phosphorylation of irs-1 as well as irs-1 association with p85 , the regulatory subunit of the phosphatidylinositol 3-kinase , in skeletal muscle of wild - type mice ( fig . loss of peripheral sh2b1 also decreased insulin receptor autophosphorylation and impaired the ability of insulin to stimulate akt phosphorylation on thr and ser in tgko muscle ( fig . insulin signaling was also examined in liver and white adipose tissue ( wat ) from hfd - fed mice . relative to wild - type mice , basal irs-1 phosphorylation was increased in both liver and wat in tgko mice ; insulin stimulated irs-1 phosphorylation in these tissues from wild - type but not tgko mice ( fig . together , these data indicate that peripheral sh2b1 increases insulin sensitivity in mice by promoting insulin signaling , including the activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway , in muscle , liver , and wat . insulin stimulated tyrosine phosphorylation of insulin receptor and irs-1 , irs-1 association with p85 , and phosphorylation of akt in wild - type hepatocytes ; however , insulin receptor autophosphorylation , irs-1 phosphorylation , irs-1 association with p85 , and akt phosphorylation were all reduced in knockout hepatocytes ( fig . in hek293 cells , overexpression of sh2b1 markedly increased insulin - stimulated tyrosine phosphorylation of irs-1 ; in contrast , a sh2b1 mutant in which the sh2 domain is disrupted because of replacement of arg with glu ( r555e ) functioned as a dominant negative to inhibit irs-1 phosphorylation ( fig . to further assess the role of the sh2 domain of sh2b1 , irs-1 and insulin receptor were coexpressed with n504 , an nh2-terminal truncated form of rat sh2b1 ( amino acids 504670 ) that contained the entire sh2 domain and a minimal number of adjacent amino acids . these data suggest that the sh2 domain of sh2b1 is not only required but also sufficient to promote insulin receptor mediated phosphorylation of irs-1 . c : irs-1 and insulin receptor were transiently coexpressed with empty vector ( con ) , myc - tagged sh2b1 , or n504 plasmids in hek293 cells . insulin receptor was then preincubated with purified gst - sh2b1 fusion protein and subsequently subjected to in vitro kinase assays using gst - irs-1 fusion protein as substrate . in similar experiments , a gst - sh2 fusion protein prepared by fusing the sh2 domain ( amino acids 524670 of sh2b1 ) to gst was preincubated with wga - purified insulin receptor in vitro kinase assays . the sh2 domain of sh2b1 was sufficient to enhance insulin receptor catalytic activity , stimulating irs-1 phosphorylation by 66% ( fig . additionally , the sh2 domain of sh2b1 also promoted the catalytic activity of insulin receptor immunopurified with an anti phospho - tyrosine antibody , increasing irs-1 substrate phosphorylation by 79% ( fig . wild - type insulin receptor or y1158f was purified with wga - agarose beads , preincubated with gst or gst - sh2b1 fusion protein ( 5 g ) , and subjected to in vitro kinase assays with gst - irs-1 as substrate for 10 min . insulin receptor and y1158f were purified using wga - beads , preincubated with gst - sh2b1 , and subjected to in vitro kinase assays . sh2b1 stimulated insulin receptor kinase activity by approximately fivefold ; however , sh2b1 was unable to stimulate y1158f catalytic activity ( fig . , these data suggest that the physical interaction between the sh2 domain of sh2b1 and tyr in insulin receptor is required and sufficient for stimulation of insulin receptor catalytic activity . sh2b1 directly binds to irs-1 and irs-2 in vitro ( 37 ) , and insulin stimulated coimmunoprecipitation of sh2b1 with irs-1 in cells ( fig . cho cells , which stably express insulin receptor and irs-1 , were stimulated with insulin to promote tyrosine phosphorylation of irs-1 . phosphorylated irs-1 was immunopurified , preincubated with gst or gst - sh2b1 , and subjected to in vitro dephosphorylation assays . insulin also promoted the association of sh2b1 with irs-2 , and sh2b1 similarly inhibited tyrosine dephosphorylation of irs-2 ( data not shown ) . irs-1 was immunoprecipitated with irs-1 , preincubated with gst or gst - sh2b1 ( 2 g ) , and subjected to in vitro dephosphorylation assays with indicated amounts of alkaline phosphatase for 30 min . d : irs-1 was expressed with insulin receptor or y1158f in the absence ( con ) or presence of sh2b1 in hek293 cells . to determine whether sh2b1 inhibits irs-1 dephosphorylation in cells , irs-1 was coexpressed with ptp1b ( a protein tyrosine phosphatase ) in the absence or presence of sh2b1 . to determine whether sh2b1 is able to promote irs-1 phosphorylation without stimulating insulin receptor kinase activity , 4e ) , sh2b1 still markedly enhanced tyrosine phosphorylation of irs-1 in y1158f - expressing cells ( fig . to determine whether the sh2b1-irs interaction sterically inhibits the binding of irs proteins to phosphatidylinositol 3-kinase , irs-1 and y1158f were coexpressed with or without sh2b1 in hek293 cells , and irs-1-p85 association insulin stimulated coimmunoprecipitation of irs-1 with p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; importantly , sh2b1 markedly enhanced insulin - stimulated p85 binding to irs-1 ( fig . collectively , these data suggest that , in addition to enhancing insulin receptor catalytic activity via binding to tyr , sh2b1 also promotes activation of the irs protein / phosphatidylinositol 3-kinase / akt pathway by inhibiting irs dephosphorylation . sh2b1 overexpression increases insulin receptor autophosphorylation and tyrosine phosphorylation of irs-1 and irs-2 in cultured cells ( 29,43 ) . we generated tgko mice that express sh2b1 only in the brain but not in peripheral tissues ( e.g. , liver , muscle , and adipose tissue ) . in the current study , we demonstrated that loss of peripheral sh2b1 markedly impaired insulin sensitivity independent of body weight . tgko mice developed hyperglycemia , hyperinsulinemia , and glucose intolerance to a greater extent than wild - type littermates fed hfd . the ability of exogenous insulin to reduce blood glucose and to stimulate insulin receptor autophosphorylation and phosphorylation of irs proteins and akt in muscle , liver , and adipose tissue was significantly reduced in tgko mice . sh2b1 binds via its sh2 domain to phospho - tyr in the activation loop of insulin receptor ( 40,41 ) . we showed that bacteria - derived sh2b1 markedly increased the ability of purified insulin receptor to tyrosyl phosphorylate irs-1 in vitro . conversely , deletion of endogenous sh2b1 impaired insulin stimulation of insulin receptor autophosphorylation and irs-1 phosphorylation in primary hepatocyte cultures . in contrast , we observed that the sh2 domain alone was sufficient to stimulate insulin receptor catalytic activity in vitro . these data indicate that the sh2 domain of sh2b1 is both required and sufficient to stimulate insulin receptor kinase activity . because tyr phosphorylation occurs early in the activation of the insulin receptor kinase ( 42,46,47 ) , binding of the sh2 domain of sh2b1 to phospho - tyr may stabilize insulin receptor in an active conformation . importantly , sh2b1 directly inhibited tyrosine dephosphorylation of irs-1 and irs-2 by recombinant phosphatase in vitro and by ptp1b in cultured cells . although unable to stimulate y1158f catalytic activity , sh2b1 still enhanced y1158f - mediated phosphorylation of irs-1 in cultured cells , presumably by inhibiting irs-1 dephosphorylation by endogenous tyrosine phosphatase(s ) . consistent with these observations , deletion of endogenous sh2b1 impaired tyrosine phosphorylation of irs-1 to a greater extent than insulin receptor autophosphorylation in primary hepatocyte cultures . interestingly , the sh2b1-irs interaction did not inhibit the ability of phosphorylated irs proteins to bind to p85 , the regulatory subunit of phosphatidylinositol 3-kinase ; in contrast , it enhanced insulin - stimulated irs - p85 association and subsequent akt phosphorylation and activation , presumably by protecting irs proteins against dephosphorylation . in muscle , liver , and adipose tissue , sh2b1 binds to insulin receptor and sh2b1 binds to both irs-1 and irs-2 and protects irs proteins from tyrosine dephosphorylation , augmenting and/or prolonging irs protein - mediated pathways . a model for sh2b1 regulation of insulin signaling . sh2b1 also binds to irs-1 or irs-2 and inhibits their dephosphorylation on tyrosines to specifically promote the activation of irs protein - mediated pathways .

the market authorization ( ma ) procedure for medicinal products for human use is relying on their demonstrated efficacy , safety , and pharmaceutical quality ( the european parliament and the council of the european union , 2001 ) . this applies to all medicinal products whether of chemical ( e.g. , blood pressure lowering diuretic ) or biological ( e.g. , anti - inflammatory monoclonal antibody ) origin . modern biotechnology medicinal products obtain market approval through the centralized procedure as detailed in the ec regulation 726/2004 ( the european parliament and the council of the european union , 2004 ) . since 2008 , a lex specialis regulation ( ec ) no 1394/2007 ( the european parliament and the council of the european union , 2007 ) applies to advanced therapy medicinal products ( atmps ) ; these atmps are pharmaceuticals with high complexity ( the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ) linked to their development , manufacturing , or administration process . the regulation highlights the following : it provides an explicit atmp definition : atmps are gene therapy , somatic cell therapy , or tissue - engineered medicinal products . an atmp must comply with the existing ma requirements ( quality , safety , and efficacy ) and the post - marketing pharmaco - vigilance rules . for ma , the centralized procedure is mandatory : it aims to pool community expertise and ensure a high level of scientific evaluation and facilitate access to market . because of the complexity of atmps , the cat s main responsibilities are : the mandatory evaluation of ma applications by providing opinions to the committee for medicinal products for human use ( chmp ) ; the chmp may adopt or refuse the cat opinion . the optional scientific certification ( art . 18 ) of quality and non - clinical data of a proposed atmp - compound in development . the optional scientific recommendation on atmp - classification ( art . the cat ( the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ) is a multidisciplinary scientific expert committee : it also focuses on the scientific developments in the field . there is no doubt about the huge scientific , regulatory , and ethical challenges triggered by these complex products and a specific expert committee for atmps is necessary to deal with these challenges ( similar to the creation of the committee on orphan medicinal products for drugs used in rare diseases ) and beneficial to all relevant public and private stakeholders . the tissues and cells directive ( 2004/23/ec ) applies to donation , procurement and testing of human tissues and cells . the regulation defines the pre- and post - authorization requirements : gmp and gcp standards , product follow - up on efficacy and safety , risk management plan , and traceability . the regulation also provides incentives for applicants by offering scientific advice at various development steps at substantially reduced fees , mainly to small- and medium - sized enterprises and hospitals . regulation is to offer a consolidated regulatory framework for these innovative medicines and it was designed ( european medicines agency , 2011a ) to ensure the free movement of these medicines within the european union ( eu ) , to facilitate their access to the eu market , and to foster the competitiveness of european pharmaceutical companies in the field , while guaranteeing the highest level of health protection for patients . two exemptions are foreseen to the atmp regulation : products , which were legally on the community market on december 30 , 2008 ( when the regulation became applicable ) , should be compliant to the regulation requirements no later than december 30 , 2012 . these products will be withdrawn from the market afterward if no centralized ma application has been submitted and granted . exemptions to the regulation are also defined under the hospital exemption rule : advanced therapy medicinal products which are prepared on a non - routine basis according to specific quality standards , and used within the same member state in a hospital under the exclusive professional responsibility of a medical practitioner , in order to comply with an individual medical prescription for a custom - made product for an individual patient , should be excluded from the scope of this regulation whilst at the same time ensuring that relevant community rules related to quality and safety are not undermined . the first exemption relates to a transition period allowing existing products to evolve toward atmp - compliance . the hospital exemption allows hospitals and medical practitioners to provide atmp - classified products to patients , e.g. , in case of high unmet medical need because there is no authorized atmp alternative available . concrete examples are tumor vaccines , made by hospitals for treating cancer patients having no treatment alternatives . the hospital exemption is limited to non - routine products , custom - made for individual patients . there is discussion on the correct interpretation of these words : for instance , are autologous products de facto not for individual patients ? anyway , as a consequence , atmps with different development tracks and quality control procedures may co - exist on the community market satisfying different standards on efficacy , safety and quality ; for hospital exemptions the regulation requires traceability , quality , and pharmaco - vigilance standards to be manufacture of atmps under hospital exemption has to be authorized by the appropriate member state to ensure appropriate quality . there are no efficacy criteria mentioned . even if there are good reasons explaining why these exemptions are needed ( e.g. , the realism of a transition period , the ethical need to deal with unmet medical need ) the regulation allows exemptions on the market without concrete requirement on demonstrated quality , efficacy , and safety . in this work we explored whether the actual application of the regulation on atmps is in line with the aim of the regulation in terms of guaranteeing the highest level of health protection for patients by focusing on atmps and their exemptions . the aim is exploratory only : it intends to enhance the discussion on the effectiveness of the regulation , based on early experience . this work does not pretend to provide judicious and definite answers to the complex economic and ethical issues relating to regulatory exemptions . we searched the available evidence for atmp products being centrally authorized ( ec authorized ) and for their available alternatives ( non - ec authorized , exemptions ) . we started by checking the ema - website ( european medicines agency , 2012 ) for authorized atmps till december 2011 . ec authorized and on non - ec authorized atmps . due to the exploratory purpose , we did not make an exhaustive systematic literature search as would be required for a formal health technology assessment : our interest was not to focus on the performance of a single product but to explore whether clinically relevant differences might exist between products , which if it was the case would be considered as a threat to the aim of the regulation of guaranteeing the highest level of health protection for patients . many competing interventions have not been compared directly : because the literature search ended with only one atmp being available as ec authorized product and we did not find trials with head - to - head comparisons ( see results ) between ec authorized and non - ec authorized atmps , we performed adjusted indirect treatment comparisons ( itcs ) . the on line application provided by the canadian agency for drugs and technologies in health ( cadth ; canadian agency for drugs and technologies in health , 2011 ) was used ; this public available agency s application is based on appropriate methodology for adjusted comparisons , with an explicit user guide offering step by step assistance in the analysis . the itc application can cope with simple and more complex ( multiple comparisons ) settings ; in this work we simply analyzed the ec authorized atmp with each of the non - ec authorized atmps based on a common comparator . the main itc assumption of independence among trials was achieved by excluding published study results if they originated from the same study populations . the evidence found in the literature used different clinical end - points per trial . only standardized treatment effects could then be used for itc , losing the clinical relevance of the individual end - points : to cope for this loss , we considered the strength of the effect as categorized by cohen ( valentine and cooper , 2003 ) to approximate the clinical relevance of the results . the strength of the cohen effect size is generally classified as follows : small effect if difference 0.2 , medium effect if difference 0.5 , and large effect if difference 0.8 . no adjustment was done for multiple testing : the significance level was set at 0.05 . despite the regulation being implemented by the end of 2008 , only six applications have been submitted for ma of four atmps ( european medicines agency , 2012 ) : in june 2009 a positive opinion for ma was adopted for chondrocelect , which is an autologous product containing chondrocytes for treatment of deep cartilage injuries of the knee . in july 2009 , the product contusugene ladenovec was withdrawn by the applicant : the product was intended for treatment of squamous cell carcinoma of head and neck . in december 2009 , the cat adopted a negative opinion on the ma application of cerepro , a gene therapy product intended for treatment of cerebral cancer ( high grade glioma ) ; the manufacturer withdrew its application in march 2010 . a negative cat opinion was adopted for the ma application of glybera in june 2011 ; glybera is a gene therapy product intended for use in severe lipid metabolic disease . on re - examination of glybera in october 2011 , market authorization was granted by the commission in october 2009 for chondrocelect ( tigenix company ; european medicines agency , 2011b ) , indicated for the repair of knee cartilage defects . osteoarthritis ( oa ) is a public health concern ( clouet et al . , 2009 ) : during oa degenerative processes , major modifications of articular cartilage are observed at the tissue , cellular , and molecular levels . articular cartilage , if damaged , hardly heals , and traumatic loss of cartilaginous tissue therefore may lead to the subsequent development of oa - lesions ( e.g. of knee , hip ) . the therapeutic options combine pharmacological treatments and tissue engineering toward regenerative medicine to induce cartilage repair . chondrocelect is still the only ec authorized atmp : non - ec authorized chondrocytes for knee cartilage repair also exist . all chondrocyte products are implanted in the patient knee by a surgical technique called autologous chondrocytes implantation ( aci ) . in this work we will further identify these products as either cci ( for ec authorized chondrocytes , atmp ) and aci ( for non - ec authorized chondrocytes ) . a simplified search in pubmed ( august 18 , 2011 ) autologous chondrocyte , limiting the search to controlled trials on human beings and further limited to papers published in english between 2008 ( implementation year of the regulation on atmps ) and mid august 2011 . the pubmed literature search resulted in 50 references ( see appendix ) . because the control group in the clinical development ( european medicines agency , 2011c ) program of cci was microfracture ( a surgical micro - bleeding technique releasing stem cells ) , only aci - studies including microfracture ( mf ) as control group could be selected for making itc between the clinical outcome of cci and aci . across the studies a variety of clinical end - points was used with only some being considered as validated end - points for clinical research by ema ( the committee for advanced therapies , 2010 ) . from the 50 references , only four papers on three studies included clinical results from aci and mf : 45 papers were excluded because the study included only observational non - controlled data , because the study related to subgroups from the three main studies , because lack of ( knee ) clinical outcome results or because another comparator than mf was used . there was one systematic review ( harris et al . , 2010 ) which included the results of , the review authors took care of standardizing the clinical outcome results of the included trials which is particularly relevant because the variety of clinical and structural end - points ( the committee for advanced therapies , 2010 ) used in the included studies would otherwise be a hurdle to any meaningful quantitative analysis . for making itcs between cci and each of the various aci - products , the review authors included seven trial reports on > 900 patients in which cci and aci - products were compared to mf . in our opinion , for two trials , the results of the same patient population were presented at two different lengths of follow - up : to obtain independent trial data , only one manuscript ( the most recent one ) per patient population was kept , resulting in five datasets for further comparison . indirect treatment comparisons can only be valuable if the compared patient populations across trials are similar ( jansen et al . , 2011 ) with respect to modifiers of the relative treatment effect ( e.g. , important prognostic factors ) : because mf is the common comparator the appropriateness of the trial patient population with respect to the lesion size needs to be addressed . one study ( reference basad et al . , 2010 in the review ) was excluded because it enrolled patients with cartilage lesions larger than 4 cm , which are commonly accepted to be unsuitable for mf treatment ( the committee for advanced therapies , 2010 ) . accordingly , the standardized results from four products ( cci , aci 13 ) were compared . the standardized direct mean treatment differences and the 95% ci of the mean differences between the various chondrocyte products and mf are as follows ( extracted from p. 2226 of harris et al . , 2010 ) . two of the four chondrocyte products could demonstrate significant clinical benefit as compared to mf ( cci , aci1 ) : the standardized mean treatment difference could be classified as large effect . the lower limit of the confidence interval of the cci standardized clinical effect still exceeded the large effect threshold ; both aci2 and aci3 products had a non - significant mean difference with mf but their confidence intervals still include large effect sizes . based on the individual study results versus mf , the itc between the chondrocyte products could be computed and are summarized as follows ( see statistical output from cadht - tool in appendix ) . the bold significance relates to any statistically significant effect being 0.8 or higher , as this is the threshold of large treatment effects ( cohen effect size ) . from the six possible itcs four itcs yield significant differences : cci is significantly superior to aci1 ; cci , aci1 , and aci2 are significantly superior to aci3 . advanced therapy medicinal products are human cells and tissues or products with a genetic mode of action ; they generate huge expectations but are also associated to new significant threats [ the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ] including tumorigenicity , cell ( de)differentiation , and patient integration . the atmp regulation 1394/2007 aims to facilitate the patient access to these products and to foster the competitiveness of european pharmaceutical companies in the field , while guaranteeing the highest level of health protection for patients . the atmp - portfolio for which the cat has been involved in certification , classification , and ma is available via the monthly cat - reports ( european medicines agency , 2012 ) : by the end of 2011 only one certification procedure for quality and non - clinical data has been requested ( 2009 ) and adopted ( 2010 ) . fifty - three requests for atmp - classification have been submitted of which 51 reached the recommendation stage : 46 ( 90.2% ) have been classified atmp . six applications for ma of four products have been evaluated leading to one authorized atmp . the certification procedure intends to create incentives for smes to develop atmps ; the only certification procedure finished by now clearly threatens this objective . fortunately , the number of submitted atmp - classification requests is sufficiently large to identify the actual main areas for atmp - development in europe . among the 46 atmp - classified products ( available from website ) : seventeen ( 37.0% ) are somatic cell therapy products . the split per therapeutic area is as follows : the oncology area captures 12 atmps mainly classified as somatic cell ( 8) therapy . cell compounds for treatment of debilitating inflammatory ( e.g. , multiple sclerosis ) or auto - immune diseases ( e.g. , pancreatitis ) and tissue - engineered products for use in severe cardiovascular disease ( e.g. , myocardial infarction ) or skin disease ( mainly treatment of burn wounds ) are in development ; applications are studied in metabolic disease , ophthalmology , bone , and cartilage disease , but also in muscular , and neurological disease ( e.g. , parkinson disease ) . clearly , these are therapeutic areas with a huge disease burden , linked to a high unmet medical need in which atmps may be of value by their unique mode of action , aiming at regenerating cell functionality , and tissue integrity . after 4 years the net outcome of the atmp regulation 1394/2007 is still limited as only one atmp has been authorized and only one request for certification has been finalized , despite the fact that already 46 compounds have been classified as atmp . in the us , the center for biologics evaluation and research ( cber ; food and drug administration , 2012a ) within fda regulates biological products for human use , both investigational and licensed . cber regulates biological products including gene therapy , human tissue , and cells under applicable federal laws , including the public health service act and the federal food , drug and cosmetic act . by now , no gene therapy has been authorized to market but various human tissue ( food and drug administration , 2012b ) and cell products are authorized including the autologous cellular immunotherapy product provenge ( dendreon company ) for treatment of prostate cancer and the cultured chondrocytes product carticel ( genzyme company ) . exemptions , specific to the european regulation include a temporary exemption ( till end of 2012 ) for products existing prior to the implementation of the regulation and a permanent hospital exemption for non - routinely made products for individual patients . in this work we aimed to verify whether different development tracks for these complex medicinal products are not putting the patient health protection ( defined as ensuring efficacy , safety , and pharmaceutical quality ) at risk . the only atmp authorized in the community market is a medicinal product containing chondrocytes for knee cartilage regeneration . based on the literature search on the ec authorized and non - ec authorized chondrocyte products , itc could be made between four different products . our results indicate statistically significant and clinically relevant differences in efficacy exist between these four cell products . the regulation 1394 is based on the assumption that ec authorized and exempted atmps provide similar health patient benefit . but based on our indirect results , the probability of a healthy patient outcome is strongly dependent on the product administered ; this puts the exemption rule of the regulation under pressure . from a pharmacological point of view , similar ( the highest ) health patient benefit among exempted and non - exempted atmps is assuming there would be a class effect ( acknowledging this may be a difficult concept for autologous products ) . but even for a class of conventional medicinal products like ace - inhibitors , quantitative differences altering the therapeutic benefits for specific patient populations may exist as shown by furberg ( 2000 ) ; the author s conclusion was that untested drugs of a class should be considered unproven drugs . if this is the case for conventional medicinal products , how confident can we then be in assuming that the highest health benefit will be ensured between more complex medicinal products ? finally , more specifically in the orthopedic surgery field , the review by vavken and samartzis ( 2010 ) did mention : also aci can hardly be seen as one , standardized treatment , due to technical differences , and inter - patient variation in cell quality . based on the above , in the therapeutic area of orthopedic surgery , there is evidence to say clinically relevant differences in health outcome exist between the authorized atmp and the exemption products . only one medicinal product could be investigated despite the regulation being applicable from the end of 2008 . the evidence on different health outcome benefits between the atmp and its exemption products is based on itc from one recent systematic review in the orthopedic surgery field . there is a lack of controlled trials with acceptable quality in this area ( hanzlik et al . , 2009 ) : in a review of all articles published in the journal of bone and joint surgery in the years 1975 , 1985 , 1995 , and 2005 the percentage of level - i studies increased from 4% in 1975 to 21% in 2005 . despite this positive trend for increasing the percentage of high level evidence trials there is still substantial opportunity for improvement . the individual clinical trial end - points were transformed into one standardized outcome affecting the interpretation of clinical relevance : to deal with this , we used the common cut - off for large treatment effects to identify clinical relevant effects . we analyzed efficacy and did not focus on other elements of patient health benefit like safety or pharmaceutical quality . investigating differences in safety issues might be very hard because of the small number of patients enrolled in the considered studies ( maximum of 118 patients randomized ) : the estimate of the incidence of adverse events for any compound will lack precision . differences in pharmaceutical quality could not be investigated because of lack of public available documentation . it is a clear advantage of ec centrally authorized medicinal products that important regulatory documentation is easily available : the summary of product characteristics ( spc ) and the european public assessment report ( epar ) provide relevant insights to patient and health care worker on efficacy , safety , and pharmaceutical quality of the medicinal product . the extent of documentation on non - ec authorized products is differing between member states , delivering companies or hospitals , and may be hard to obtain for any external party . we did not analyze how exemptions to atmps may affect the internal market functioning because of the limited experience with only one atmp available . obviously differences in development track will yield differences in the necessary r&d resources , which may result in substantial product price differences : this is a hurdle for the applicant submitting a centrally authorized atmp when lower priced exempted alternatives are on the market . in many member states a price premium can only be granted if added therapeutic value compared to the alternative has been demonstrated . but this direct comparison will probably lack , as it is the case for the 1st atmp containing cultured chondrocytes : the design section of the cat - reflection paper ( the committee for advanced therapies , 2010 ) in this field clearly specifies licensed products as valid comparators that can be used in controlled trials . it further states however , the use of a non - authorized medicinal product is problematic as it has not been validated for clinical use and the quality of the product has not been assessed . valid approaches to adjusted itc will be necessary but the acceptability of indirect evidence may be different between competent authorities increasing the 4th hurdle for atmps . as the european pharmaceutical forum and the european transparency directive ( the council of the european communities , 1989 ; european commission , 2012 ) explicitly ask competent authorities on pricing and reimbursement to take decisions based on objective and verifiable criteria and to be well aligned with the estimated value of medicines , the question may arise how these member states will determine the relative value of an atmp in comparison to exempted alternatives with assumed but unproven effect . in conclusion , based on the analysis of the relative efficacy of the only ec authorized atmp and its exempted alternatives , there is evidence against the regulation 1394/2007 assumption of ensuring the highest level of patient health protection is maintained among each of these products . the ethical need for exemption must be balanced with the upcoming evidence of differences in health outcome between ec authorized and non - ec authorized atmp products . the scheduled review by the commission by the end of 2012 of the application of the regulation should include an assessment of the likelihood of similar health outcome among atmps with different development tracks .

the market authorization procedure for medicinal products for human use is relying on their demonstrated efficacy , safety , and pharmaceutical quality . this applies to all medicinal products whether of chemical or biological origin . since october 2009 , the first advanced therapy medicinal product ( atmp ) has been authorized through the centralized procedure . atmps are gene therapy medicinal products , somatic cell therapy medicinal products or tissue - engineered products . an appropriate atmp regulation is dealing with atmp requirements . two exemptions are foreseen to the atmp regulation : ( a ) products , which were legally on the community market when the regulation became applicable , should comply to the regulation by december 30 , 2012 . ( b ) the hospital exemption rule for non - routine products for an individual patient . in this work we explored whether the actual application of the regulation on atmps is in line with the aim of the regulation in terms of guaranteeing the highest level of health protection for patients . based on the analysis of the relative efficacy of the only ec authorized atmp and its exempted alternatives , there is evidence against this regulation 1394/2007 assumption .

Introduction Objectives Methods Results Discussion Conflict of Interest Statement Results from the ITC performed with the CADHT-application

the market authorization ( ma ) procedure for medicinal products for human use is relying on their demonstrated efficacy , safety , and pharmaceutical quality ( the european parliament and the council of the european union , 2001 ) . this applies to all medicinal products whether of chemical ( e.g. , blood pressure lowering diuretic ) or biological ( e.g. modern biotechnology medicinal products obtain market approval through the centralized procedure as detailed in the ec regulation 726/2004 ( the european parliament and the council of the european union , 2004 ) . since 2008 , a lex specialis regulation ( ec ) no 1394/2007 ( the european parliament and the council of the european union , 2007 ) applies to advanced therapy medicinal products ( atmps ) ; these atmps are pharmaceuticals with high complexity ( the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ) linked to their development , manufacturing , or administration process . the regulation highlights the following : it provides an explicit atmp definition : atmps are gene therapy , somatic cell therapy , or tissue - engineered medicinal products . an atmp must comply with the existing ma requirements ( quality , safety , and efficacy ) and the post - marketing pharmaco - vigilance rules . for ma , the centralized procedure is mandatory : it aims to pool community expertise and ensure a high level of scientific evaluation and facilitate access to market . because of the complexity of atmps , the cat s main responsibilities are : the mandatory evaluation of ma applications by providing opinions to the committee for medicinal products for human use ( chmp ) ; the chmp may adopt or refuse the cat opinion . 18 ) of quality and non - clinical data of a proposed atmp - compound in development . the cat ( the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ) is a multidisciplinary scientific expert committee : it also focuses on the scientific developments in the field . there is no doubt about the huge scientific , regulatory , and ethical challenges triggered by these complex products and a specific expert committee for atmps is necessary to deal with these challenges ( similar to the creation of the committee on orphan medicinal products for drugs used in rare diseases ) and beneficial to all relevant public and private stakeholders . the tissues and cells directive ( 2004/23/ec ) applies to donation , procurement and testing of human tissues and cells . the regulation defines the pre- and post - authorization requirements : gmp and gcp standards , product follow - up on efficacy and safety , risk management plan , and traceability . the regulation also provides incentives for applicants by offering scientific advice at various development steps at substantially reduced fees , mainly to small- and medium - sized enterprises and hospitals . regulation is to offer a consolidated regulatory framework for these innovative medicines and it was designed ( european medicines agency , 2011a ) to ensure the free movement of these medicines within the european union ( eu ) , to facilitate their access to the eu market , and to foster the competitiveness of european pharmaceutical companies in the field , while guaranteeing the highest level of health protection for patients . two exemptions are foreseen to the atmp regulation : products , which were legally on the community market on december 30 , 2008 ( when the regulation became applicable ) , should be compliant to the regulation requirements no later than december 30 , 2012 . these products will be withdrawn from the market afterward if no centralized ma application has been submitted and granted . exemptions to the regulation are also defined under the hospital exemption rule : advanced therapy medicinal products which are prepared on a non - routine basis according to specific quality standards , and used within the same member state in a hospital under the exclusive professional responsibility of a medical practitioner , in order to comply with an individual medical prescription for a custom - made product for an individual patient , should be excluded from the scope of this regulation whilst at the same time ensuring that relevant community rules related to quality and safety are not undermined . the first exemption relates to a transition period allowing existing products to evolve toward atmp - compliance . the hospital exemption allows hospitals and medical practitioners to provide atmp - classified products to patients , e.g. , in case of high unmet medical need because there is no authorized atmp alternative available . the hospital exemption is limited to non - routine products , custom - made for individual patients . there is discussion on the correct interpretation of these words : for instance , are autologous products de facto not for individual patients ? anyway , as a consequence , atmps with different development tracks and quality control procedures may co - exist on the community market satisfying different standards on efficacy , safety and quality ; for hospital exemptions the regulation requires traceability , quality , and pharmaco - vigilance standards to be manufacture of atmps under hospital exemption has to be authorized by the appropriate member state to ensure appropriate quality . even if there are good reasons explaining why these exemptions are needed ( e.g. , the realism of a transition period , the ethical need to deal with unmet medical need ) the regulation allows exemptions on the market without concrete requirement on demonstrated quality , efficacy , and safety . in this work we explored whether the actual application of the regulation on atmps is in line with the aim of the regulation in terms of guaranteeing the highest level of health protection for patients by focusing on atmps and their exemptions . the aim is exploratory only : it intends to enhance the discussion on the effectiveness of the regulation , based on early experience . this work does not pretend to provide judicious and definite answers to the complex economic and ethical issues relating to regulatory exemptions . we searched the available evidence for atmp products being centrally authorized ( ec authorized ) and for their available alternatives ( non - ec authorized , exemptions ) . we started by checking the ema - website ( european medicines agency , 2012 ) for authorized atmps till december 2011 . ec authorized and on non - ec authorized atmps . due to the exploratory purpose , we did not make an exhaustive systematic literature search as would be required for a formal health technology assessment : our interest was not to focus on the performance of a single product but to explore whether clinically relevant differences might exist between products , which if it was the case would be considered as a threat to the aim of the regulation of guaranteeing the highest level of health protection for patients . many competing interventions have not been compared directly : because the literature search ended with only one atmp being available as ec authorized product and we did not find trials with head - to - head comparisons ( see results ) between ec authorized and non - ec authorized atmps , we performed adjusted indirect treatment comparisons ( itcs ) . the on line application provided by the canadian agency for drugs and technologies in health ( cadth ; canadian agency for drugs and technologies in health , 2011 ) was used ; this public available agency s application is based on appropriate methodology for adjusted comparisons , with an explicit user guide offering step by step assistance in the analysis . the itc application can cope with simple and more complex ( multiple comparisons ) settings ; in this work we simply analyzed the ec authorized atmp with each of the non - ec authorized atmps based on a common comparator . the strength of the cohen effect size is generally classified as follows : small effect if difference 0.2 , medium effect if difference 0.5 , and large effect if difference 0.8 . despite the regulation being implemented by the end of 2008 , only six applications have been submitted for ma of four atmps ( european medicines agency , 2012 ) : in june 2009 a positive opinion for ma was adopted for chondrocelect , which is an autologous product containing chondrocytes for treatment of deep cartilage injuries of the knee . in july 2009 , the product contusugene ladenovec was withdrawn by the applicant : the product was intended for treatment of squamous cell carcinoma of head and neck . in december 2009 , the cat adopted a negative opinion on the ma application of cerepro , a gene therapy product intended for treatment of cerebral cancer ( high grade glioma ) ; the manufacturer withdrew its application in march 2010 . a negative cat opinion was adopted for the ma application of glybera in june 2011 ; glybera is a gene therapy product intended for use in severe lipid metabolic disease . on re - examination of glybera in october 2011 , market authorization was granted by the commission in october 2009 for chondrocelect ( tigenix company ; european medicines agency , 2011b ) , indicated for the repair of knee cartilage defects . articular cartilage , if damaged , hardly heals , and traumatic loss of cartilaginous tissue therefore may lead to the subsequent development of oa - lesions ( e.g. chondrocelect is still the only ec authorized atmp : non - ec authorized chondrocytes for knee cartilage repair also exist . in this work we will further identify these products as either cci ( for ec authorized chondrocytes , atmp ) and aci ( for non - ec authorized chondrocytes ) . a simplified search in pubmed ( august 18 , 2011 ) autologous chondrocyte , limiting the search to controlled trials on human beings and further limited to papers published in english between 2008 ( implementation year of the regulation on atmps ) and mid august 2011 . from the 50 references , only four papers on three studies included clinical results from aci and mf : 45 papers were excluded because the study included only observational non - controlled data , because the study related to subgroups from the three main studies , because lack of ( knee ) clinical outcome results or because another comparator than mf was used . , 2010 ) which included the results of , the review authors took care of standardizing the clinical outcome results of the included trials which is particularly relevant because the variety of clinical and structural end - points ( the committee for advanced therapies , 2010 ) used in the included studies would otherwise be a hurdle to any meaningful quantitative analysis . for making itcs between cci and each of the various aci - products , the review authors included seven trial reports on > 900 patients in which cci and aci - products were compared to mf . in our opinion , for two trials , the results of the same patient population were presented at two different lengths of follow - up : to obtain independent trial data , only one manuscript ( the most recent one ) per patient population was kept , resulting in five datasets for further comparison . , 2011 ) with respect to modifiers of the relative treatment effect ( e.g. , important prognostic factors ) : because mf is the common comparator the appropriateness of the trial patient population with respect to the lesion size needs to be addressed . , 2010 in the review ) was excluded because it enrolled patients with cartilage lesions larger than 4 cm , which are commonly accepted to be unsuitable for mf treatment ( the committee for advanced therapies , 2010 ) . two of the four chondrocyte products could demonstrate significant clinical benefit as compared to mf ( cci , aci1 ) : the standardized mean treatment difference could be classified as large effect . the lower limit of the confidence interval of the cci standardized clinical effect still exceeded the large effect threshold ; both aci2 and aci3 products had a non - significant mean difference with mf but their confidence intervals still include large effect sizes . based on the individual study results versus mf , the itc between the chondrocyte products could be computed and are summarized as follows ( see statistical output from cadht - tool in appendix ) . from the six possible itcs four itcs yield significant differences : cci is significantly superior to aci1 ; cci , aci1 , and aci2 are significantly superior to aci3 . advanced therapy medicinal products are human cells and tissues or products with a genetic mode of action ; they generate huge expectations but are also associated to new significant threats [ the committee for advanced therapies ( cat ) and the cat scientific secretariat , 2010 ] including tumorigenicity , cell ( de)differentiation , and patient integration . the atmp regulation 1394/2007 aims to facilitate the patient access to these products and to foster the competitiveness of european pharmaceutical companies in the field , while guaranteeing the highest level of health protection for patients . the atmp - portfolio for which the cat has been involved in certification , classification , and ma is available via the monthly cat - reports ( european medicines agency , 2012 ) : by the end of 2011 only one certification procedure for quality and non - clinical data has been requested ( 2009 ) and adopted ( 2010 ) . six applications for ma of four products have been evaluated leading to one authorized atmp . fortunately , the number of submitted atmp - classification requests is sufficiently large to identify the actual main areas for atmp - development in europe . among the 46 atmp - classified products ( available from website ) : seventeen ( 37.0% ) are somatic cell therapy products . the split per therapeutic area is as follows : the oncology area captures 12 atmps mainly classified as somatic cell ( 8) therapy . , pancreatitis ) and tissue - engineered products for use in severe cardiovascular disease ( e.g. , myocardial infarction ) or skin disease ( mainly treatment of burn wounds ) are in development ; applications are studied in metabolic disease , ophthalmology , bone , and cartilage disease , but also in muscular , and neurological disease ( e.g. after 4 years the net outcome of the atmp regulation 1394/2007 is still limited as only one atmp has been authorized and only one request for certification has been finalized , despite the fact that already 46 compounds have been classified as atmp . in the us , the center for biologics evaluation and research ( cber ; food and drug administration , 2012a ) within fda regulates biological products for human use , both investigational and licensed . cber regulates biological products including gene therapy , human tissue , and cells under applicable federal laws , including the public health service act and the federal food , drug and cosmetic act . by now , no gene therapy has been authorized to market but various human tissue ( food and drug administration , 2012b ) and cell products are authorized including the autologous cellular immunotherapy product provenge ( dendreon company ) for treatment of prostate cancer and the cultured chondrocytes product carticel ( genzyme company ) . exemptions , specific to the european regulation include a temporary exemption ( till end of 2012 ) for products existing prior to the implementation of the regulation and a permanent hospital exemption for non - routinely made products for individual patients . in this work we aimed to verify whether different development tracks for these complex medicinal products are not putting the patient health protection ( defined as ensuring efficacy , safety , and pharmaceutical quality ) at risk . the only atmp authorized in the community market is a medicinal product containing chondrocytes for knee cartilage regeneration . based on the literature search on the ec authorized and non - ec authorized chondrocyte products , itc could be made between four different products . the regulation 1394 is based on the assumption that ec authorized and exempted atmps provide similar health patient benefit . but based on our indirect results , the probability of a healthy patient outcome is strongly dependent on the product administered ; this puts the exemption rule of the regulation under pressure . from a pharmacological point of view , similar ( the highest ) health patient benefit among exempted and non - exempted atmps is assuming there would be a class effect ( acknowledging this may be a difficult concept for autologous products ) . but even for a class of conventional medicinal products like ace - inhibitors , quantitative differences altering the therapeutic benefits for specific patient populations may exist as shown by furberg ( 2000 ) ; the author s conclusion was that untested drugs of a class should be considered unproven drugs . if this is the case for conventional medicinal products , how confident can we then be in assuming that the highest health benefit will be ensured between more complex medicinal products ? finally , more specifically in the orthopedic surgery field , the review by vavken and samartzis ( 2010 ) did mention : also aci can hardly be seen as one , standardized treatment , due to technical differences , and inter - patient variation in cell quality . based on the above , in the therapeutic area of orthopedic surgery , there is evidence to say clinically relevant differences in health outcome exist between the authorized atmp and the exemption products . only one medicinal product could be investigated despite the regulation being applicable from the end of 2008 . the evidence on different health outcome benefits between the atmp and its exemption products is based on itc from one recent systematic review in the orthopedic surgery field . there is a lack of controlled trials with acceptable quality in this area ( hanzlik et al . we analyzed efficacy and did not focus on other elements of patient health benefit like safety or pharmaceutical quality . investigating differences in safety issues might be very hard because of the small number of patients enrolled in the considered studies ( maximum of 118 patients randomized ) : the estimate of the incidence of adverse events for any compound will lack precision . differences in pharmaceutical quality could not be investigated because of lack of public available documentation . it is a clear advantage of ec centrally authorized medicinal products that important regulatory documentation is easily available : the summary of product characteristics ( spc ) and the european public assessment report ( epar ) provide relevant insights to patient and health care worker on efficacy , safety , and pharmaceutical quality of the medicinal product . the extent of documentation on non - ec authorized products is differing between member states , delivering companies or hospitals , and may be hard to obtain for any external party . we did not analyze how exemptions to atmps may affect the internal market functioning because of the limited experience with only one atmp available . obviously differences in development track will yield differences in the necessary r&d resources , which may result in substantial product price differences : this is a hurdle for the applicant submitting a centrally authorized atmp when lower priced exempted alternatives are on the market . in many member states a price premium can only be granted if added therapeutic value compared to the alternative has been demonstrated . but this direct comparison will probably lack , as it is the case for the 1st atmp containing cultured chondrocytes : the design section of the cat - reflection paper ( the committee for advanced therapies , 2010 ) in this field clearly specifies licensed products as valid comparators that can be used in controlled trials . it further states however , the use of a non - authorized medicinal product is problematic as it has not been validated for clinical use and the quality of the product has not been assessed . as the european pharmaceutical forum and the european transparency directive ( the council of the european communities , 1989 ; european commission , 2012 ) explicitly ask competent authorities on pricing and reimbursement to take decisions based on objective and verifiable criteria and to be well aligned with the estimated value of medicines , the question may arise how these member states will determine the relative value of an atmp in comparison to exempted alternatives with assumed but unproven effect . in conclusion , based on the analysis of the relative efficacy of the only ec authorized atmp and its exempted alternatives , there is evidence against the regulation 1394/2007 assumption of ensuring the highest level of patient health protection is maintained among each of these products . the ethical need for exemption must be balanced with the upcoming evidence of differences in health outcome between ec authorized and non - ec authorized atmp products . the scheduled review by the commission by the end of 2012 of the application of the regulation should include an assessment of the likelihood of similar health outcome among atmps with different development tracks .

attention is known to alter neural processing at multiple levels of both the peripheral and central nervous systems , and both auditory and visual attention have been conceptualized as operating in both top - down mechanisms reflect goal - based control in order to direct attention to particular targets or to sustain attention over time . in contrast , bottom - up mechanisms have traditionally been defined by the phenomenon of reflexive attentional orienting , as when attention is drawn without intent by highly salient sensory stimuli such as a sudden loud noise or flash of light . recently , however , some investigators have more broadly considered bottom - up effects as relevant for any incoming stimuli , with the relative saliency of the stimulus influencing whether it is ultimately encoded into memory . two recent theoretical models address the question of what roles stimulus saliency might play in first successfully encoding information into memory and then later retrieving it . as discussed below , the embedded processes model and the attention - to - memory model , while similar , also highlight the potentially divergent roles that attention at encoding may play in later recall . brain mapping studies based on these models have only started to identify the neural substrates that underlie these cognitive processes in different domains . in real life situations , numerous bits of information co - given that memory is a limited capacity system [ 911 ] , the information encoded into memory must be restricted to what is relevant . according to the this process first searches through a set of potential memory items , then selects the most salient items and brings them into the focus of attention [ 1214 ] . however , even though the attentional scanner is responsible for selecting the appropriate stimuli , its capacity is limited , and it therefore can maintain focus on only small amounts of information at a time . cowan and colleagues have speculated that regions in and around the temporal - parietal junction are key to focusing attention . more recently , others have found that the majority of functional neuroimaging data on working memory is consistent with the tenets of this model , with ventral posterior ( inferior parietal cortex and intraparietal sulcus ) regions associated with attentional focus and working memory maintenance , and lateral prefrontal regions involved in the executive control of attention and further manipulation of information , once it is in working memory [ 16 , 17 ] . if this interpretation is correct , then manipulations of attention at encoding should preferentially affect activation in parietal regions . another recent paradigm , the attention - to - memory model [ 8 , 1820 ] , shares functional features with the previous model , but it also emphasizes a distinction between the roles of ventral and dorsal parietal cortex in memory retrieval . successful recognition memory for intentionally studied items is predicted by increased activation of dorsal posterior parietal cortex at stimulus encoding , whereas activation in ventral posterior parietal cortex at encoding predicts success in incidental memory and perceptual priming tests [ 21 , 22 ] . this latter observation suggests a role for this ventral region not only in the bottom - up encoding of highly salient or unexpected sensory stimuli , but also in the successful retrieval of unintentionally encoded memories . according to these prevailing theories , retrieval of incidental or perceptual memory representations requires a bottom - up shift in selective attention , whereas retrieval of intentional memories requires a top - down mechanism that reflects the individual 's conscious intent to focus attention on incoming signals [ 21 , 22 ] importantly , however , the vast majority of studies exploring the intersection of attention and episodic memory and thus the theories on which these studies are based are heavily grounded in the use of visual paradigms [ 2330 ] . even studies that focus specifically on verbal encoding have mostly employed reading rather than listening tasks [ 3137 ] . it is therefore necessary to examine the effects of attention on item retrieval in listening tasks in order to establish whether the dorsal / ventral distinction observed in parietal cortex is specific to visual stimuli , or instead reflects a more generalized organization in this area of the brain . to examine these ideas in greater depth , it is necessary to learn more about the role of attention in establishing a selective focus on task - relevant sensory stimuli . moreover , in order to understand how limited attentional resources are allocated , it is also important to study the encoding of items both within the focus of attention as well as those peripheral to it . our goal in this study was to devise a method to modulate attention toward spoken words during a simple listening task . based on earlier studies , we predicted that words not intentionally within the focus of attention would be encoded into memory to some degree . during the encoding phase of our study , participants initially heard a list of words belonging to two semantic classes ( animals or foods ) , and these words were presented by two different talkers ( female or male ) . participants were given explicit instructions to remember only the animal words presented in the female voice ( the targets ) . importantly , this instruction resulted in a focus on the female voice , which had the consequence of placing the nontarget food words presented in that voice also within the focus of attention . we predicted that these words would also carry higher salience at encoding than words heard in the male voice , creating what we refer to as the high - attention condition in this study . following this rationale , the food words presented in the male voice would not be fully in the primary focus of attention ( the low - attention condition ) . we contrasted these two encoding conditions with responses to novel food words that were not previously heard at any time during the experiment . we reasoned that processing of these new words should reflect only the role of attention in initial word encoding , but not a later role in retrieval . using this paradigm coupled with functional magnetic resonance imaging ( fmri ) , we were able to disambiguate the neural activation patterns associated with attention at first encoding from those reflecting the effects of attention at encoding on subsequent retrieval . we recruited 14 native english - speaking adults ( 9 women ; ages 1849 ; mean 24 years ) living in the tucson area . exclusion criteria were a history of speech , language , or other neurological disorders , and all volunteers reported good general health with no contraindications for mri scanning . the study was approved by the university of arizona institutional review board , and written informed consent was obtained from all participants . for one participant , behavioral response data are not available due to computer error . word stimuli were single , concrete nouns ( one to three syllables ) that were either names of animals or foods . some words were recorded in an unfamiliar female voice , and some in an unfamiliar male voice . stimulus durations ranged from 204903 ms ( mean = 532 ms ) and were presented in an event - related format with an average interstimulus interval ( isi ) of 871 157 ms . isi was jittered and null events were included to facilitate estimation of the hemodynamic responses for deconvolution analysis ( see below ) . ordering of words belonging to the different stimulus categories described below was pseudorandom , and participants listened to them through mri - compatible stereo headphones ( resonance technology inc . , northridge , calif , usa ) . a prescan practice phase was followed by an encoding phase and a test phase ( both with scanning ) . these were then followed by a surprise postscan memory test that specifically measured the listener 's capacity to remember any words that were encoded during the earlier phases of the experiment , whether or not the listener was asked to remember them . the results of the test phase and scan , along with results from the postscan behavioral test , are the subject of this report . participants listened to the animal and food words that were presented in either the male or female voice during a prescan practice period and during the encoding phase . using a block design , the encoding phase consisted of two scans that were counterbalanced for order across participants . in one scan , approximately one - third of the total words used for this study were presented binaurally , with the male and female voices presented sequentially . in a second scan , two - thirds of the words were presented dichotically , with the female voice heard in one ear and the male voice heard in the other ( order counterbalanced across participants ) . the ratio of targets ( animal word + female voice ) to nontargets was 1 : 3 in both tasks . although all participants were explicitly instructed to focus on and remember the animal words presented in the female voice , these were not actually of interest for the purposes of this study . rather , this instruction was used deliberately to assure that the primary focus of attention was on the female voice . as a result , food words presented in the female voice were associated with a higher level of attention ( defined as the high - attention condition ) and food words presented in the male voice were associated with a lower level of attention ( the low - attention condition ) . , participants heard words presented by a second female speaker whose voice had not been heard earlier . this precluded the possibility that the voice in the memory task could provide a cue to the encoding context . participants were asked to respond yes via button press if the word presented was one of their target words ( animal words originally spoken in the female voice ) and no to all other words . therefore , both the decision ( not a target ) and response demands ( selecting the no button ) associated with all words analyzed in this study were identical . , participants mostly heard nontarget words . a combination of high - attention and low - attention food words ( 58 trials each ) as well as 58 new food words that had not been presented previously were pseudorandomized and presented in a fixed order to all participants . to lessen fatigue , participants were tested over the course of two separate scans ( each lasting ~9 min with a brief rest in between ) , during which a combined total of 232 words ( randomized with 58 nulls ) were presented . data from the two scans were then concatenated for further analysis . in order to assure that there was an effect of the attentional manipulation at encoding , we administered a surprise memory test within 15 minutes of the final scan . participants were presented verbally with a list of 40 food items and asked to recall whether they had heard each word at any time during the experiment . the list consisted of 10 high - attention , 10 low - attention , and 10 new food words that were all heard during the study phase and scan . a fourth category , 10 food items not presented in any of the scans , was added to measure false - alarm rate . these 40 words were presented verbally in a fixed pseudorandom order by the experimenter and the participants verbally responded yes or no to indicate whether they remembered having heard these words at any time during the experiment . scans were acquired with a 3.0 t ge signa vh / i scanner ( general electric medical systems , madison , wis , usa ) equipped with a quad - head rf coil . first , t1-weighted , fast - spin echo ( fse ) axial images covering the entire brain were acquired in 26 slices with an in - plane resolution of 3.44 3.44 5 mm . next , two functional t2 * -weighted scans were acquired using a spiral in / out pulse sequence ( tr = 2.3 s , te = 30 ms , flip angle = 90 , 26 slices at 5 mm with no gap , matrix = 64 64 , fov = 22 cm ) . finally , high - resolution spoiled gradient - echo ( spgr ) images were obtained in the sagittal plane ( tr = 30 , te = min , flip angle = 30 , 124 slices at 1.5 mm , matrix = 256 256 , fov = 25 cm ) and aligned with the fse images for improved regional localization and coregistration of functional data across participants after transforming the images into talairach space . due to scanner - related problems , we were unable to use the spgr images from three participants , and instead relied on the first set of axial fse anatomical images for alignment with the functional scans . structural and functional brain images were analyzed for each participant individually with afni followed by a group analysis . blood - oxygenation - level - dependent ( bold ) contrast images were coregistered with anatomical data after preprocessing using standard procedures for slice - time correction , removing linear signal drift and correcting for head motion . all volumes were realigned to the base volume and spatially smoothed using a 6 mm gaussian kernel . data were then normalized to a scale of 0100% , and functional images were coregistered to the structural data followed by transformation into standard talairach space . the first four volumes in each run were also discarded to allow for t1 equilibration , and the two runs were then concatenated for further analysis . a general linear model using a gamma - spline hemodynamic response function was used to estimate magnitude parameters for events of interest for each stimulus condition in each individual . in order to capture the amplitude of the bold activity over time the first was time locked to the stimulus onset , and each of the seven subsequent models was offset by one - tr ( 2.3 s ) increments from stimulus onset . stimulus functions were then convolved with the fmri time - series data from each individual . parameter estimates for the resulting regressors for each condition were calculated using the least - squares fit of the models to the time - series data . finally , a group analysis was performed with repeated - measures anova ( treating individuals as a random effect ) to help confirm key cortical regions that showed differential neural activity for each of the four conditions . monte carlo simulation ( 8 mm fwhm blur ; 1000 iterations ) was used to correct the group data for multiple comparisons . voxel - wise ( uncorrected ) threshold was p = 0.005 , and minimum corrected cluster volumes in original space were 32 contiguous voxels at p < 0.05 . to identify regions of interest ( roi ) associated with positive bold signal across the high - attention , low - attention , and new categories , we first combined the three group - averaged datasets . this allowed us to develop roi that were associated with activation in any of the three conditions of interest . we were particularly interested in areas in and around the dorsolateral prefrontal cortex and the temporoparietal junction due to their proposed involvement in executive control as well as phonological short - term storage and language processing [ 12 , 43 , 44 ] . however , since the proposed involvement of these regions is based almost entirely on visual rather than auditory paradigms , and there is growing evidence for bilateral involvement in language processing [ 45 , 46 ] , we examined the following roi in both hemispheres . masks corresponding to distinct anatomical regions of interest were developed , and roi were defined based on the threshold - corrected regions of significant activation . brain regions showing significant positive bold activation are listed in table 1 and described as follows ( approximate brodmann areas in parentheses ) : frontal lobe ( dorsal to ventral ) : medial frontal gyrus ( 6/32 ) , middle frontal gyrus ( 9/46 ) , inferior frontal gyrus ( 47/pars orbitalis ) , and anterior insular cortex ( 13 ) . parietal lobe ( inferior parietal lobule ) : angular gyrus ( 39 ) and supramarginal gyrus ( 40 ) . we looked for additional roi along the anterior - posterior axis of the superior temporal lobe due to its routine involvement in speech and language tasks [ 4751 ] , but found only one with significant activation : superior temporal gyrus including and extending around the transverse temporal gyrus ( ba41/42 ) . sub - cortical roi included basal ganglia ( caudate body ) and thalamic nuclei ( anterior ) . finally , several roi were localized to the anterior and posterior lobes of the cerebellum . the surprise memory test was intended to provide proof of concept that the attentional manipulation at encoding actually produced an effect . specifically , high - attention words should be better remembered than low - attention words , and each of these should be better attended than new words . conversely , participants should not indicate recognition for words introduced as foils that were not presented earlier at any time in the experiment . figure 1 shows the result of the group memory analysis ( full variance model : f3,48 = 17.44 , n = 13 listeners ; p < 0.001 , two - tailed ) . as predicted , the strongest memory scores were for responses to the high - attention words . these words showed the best hit rate at 93.8 2.4% ( mean sem ) , compared to 70.8 4.6% for low - attention words , and 51.0 6.5% for new words ( all means significantly different based on post - hoc , two - tailed anova ; see figure 1 ) . additionally , words introduced as foils during the postscan memory test were correctly rejected 86.2 3.3% of the time , indicating a low false alarm rate . a temporal analysis of the bold responses for each stimulus condition was performed by modeling the fmri time series over a range of time lags , as shown in figure 2 . although this analysis lacks the temporal precision of electrophysiological methods , it can provide valuable information about the relative timing of neural events associated with each test condition . as stated above , eight separate hemodynamic response models were constructed for each stimulus type , and the peak of each model was time - shifted by one tr in order to capture peak responses across an 8-tr ( 18.4 s ) time window . we then calculated the bold responses from the peak activation at each time lag , as shown in figure 2 . as expected , strong bold activity was observed in the superior temporal lobe in the vicinity of primary auditory cortex , and this activity did not vary with the three stimulus conditions ( table 1 ) . in addition to the expected early activity in left superior temporal gyrus , bold responses associated with all three stimulus categories were , on average , 20% greater in right superior temporal gyrus , which is consistent with the other right - lateralized activation patterns discussed below . as shown in figure 2(a ) , identification of words in all three categories was associated with activity in left middle frontal gyrus ( a portion of dorsolateral prefrontal cortex dlpfc ) and this activity was closely mirrored in anterior insular cortex ( figure 2(b ) ) . furthermore , the time course of the bold response in these two left - hemisphere regions was similar for all three word categories regardless of the initial encoding condition . in contrast , right dlpfc showed a distinctly different response pattern , with high - attention words yielding significantly greater responses relative to low - attention words , and the latter yielding significantly greater responses relative to new words . this pattern indicates a strong differential effect of attention at encoding in right dlpfc ( ba9/46 ) compared to left dlpfc ( figure 2(a ) ) and bilateral insula ( figure 2(b ) ) . importantly , this result is consistent with earlier studies using written words that linked activity in this region of right dlpfc with post - retrieval processing and/or monitoring functions [ 37 , 5254 ] . if right dlpfc is indeed involved selectively in post - retrieval processing , we would also expect , in accord with the attention - to - memory model , that words strongly attended at encoding might evoke a greater memory response than words only weakly attended at encoding . this graded pattern of activity was , in fact , observed for the high- and low - attention words , as shown in figure 2(a ) . conversely , this region would not be expected to activate in response to new words because these words were not yet encoded into memory . in accord with this hypothesis , only negligible positive bold activity was observed in right dlpfc in response to new words ( figure 2(a ) , right column ) . as shown in figure 2(c ) , another bilateral set of clusters was located in the inferior frontal gyrus ( ifg ) . this area is believed to be an important rostral component of the so - called ventral frontoparietal stream that is frequently observed in studies involving the detection of novel or low - frequency events , particularly when they are unexpected . although this pathway has been observed in numerous visuospatial attention studies , our findings using auditory language stimuli are also consistent with this anatomical framework . figure 2 shows how the three stimulus conditions in our study differentially activated the ventral frontoparietal stream . the focal point for these clusters in the frontal lobes was found in pars orbitalis ( ba47 ) , and in accord with the ventral frontoparietal model , activity was also lateralized to the right hemisphere ( figure 2(c ) , right column ) . both the high- and low - attention conditions were associated with comparable activation in left and right ifg . however , in left ifg , a bold response on a similar time course was absent for the new words , indicating that the early response in ifg to previously encoded words may specifically reflect memory for these previously presented items . despite the absence of an early bold response in left ifg for new words , these items were instead associated with a late - onset response in the right hemisphere ( figure 2(c ) , right column ) . in other words , responses to the new words differed from the previously attended words both in terms of hemispheric lateralization and in the late time course of activation in ifg ( ba47 ) , suggesting that this late activation more likely reflects the initial encoding of new words into memory . with respect to language processing , converging evidence suggests a key role for posterior parietal and temporal regions in verbal memory and attention [ 12 , 31 , 37 , 44 ] . one memory model predicts that items correctly identified as previously encountered ( old ) will trigger increased activity in left intraparietal cortex , relative to missed old and correctly rejected novel items . however , as noted above , selective attention is another cognitive function that is commonly associated with posterior parietal cortex , and it remains unclear how the influence of attention may affect the neural networks underlying recollection and familiarity , especially in the auditory domain . it has been proposed that inferior parietal lobule , a region within ventral posterior parietal cortex , may serve a specialized function in the expression of attention , as proposed in both the embedded - processes model [ 14 , 15 ] and the attention - to - memory ( atom ) model [ 8 , 1820 ] . these models allowed us to generate informed predictions about the neural substrates underlying a possible attention - dependent memory effect in our study . if ventral posterior parietal cortex serves specifically as the focus of attention as in the embedded - processes model , we would expect activation in this region to be differentiated according to the level of attention directed toward each stimulus at encoding , as shown in figure 1 . another prediction is that any region responsible for the reactivation of focused attention prior to memory retrieval should display activity earlier in the hemodynamic response than regions responsible for post - stimulus processing steps , such as semantic analysis . as shown in figure 2(d ) and table 1 , the principal locus of activation in posterior parietal cortex was found bilaterally in the supramarginal gyrus ( smg ) . this activity , furthermore , was associated specifically with listening to and identifying the high - attention words , but not the low - attention words . this is consistent with the notion that activation in and around the smg is related to attentional scanning that placed our high - attention words into the focus of attention according to the embedded - processes model . to examine the spatial location of the group activation in greater detail this participant - by - participant anatomical localization revealed that the peak activation was located in the left smg for 86% ( 12 of 14 individuals ) , and in the right smg for 64% of the participants . as shown in table 2 , activation in the left hemisphere was completely isolated to the smg in 36% of listeners , whereas in 50% , the activity also spread from the smg into the adjoining intraparietal sulcus ( ips ) . in contrast , the primary focus of activation in the right hemisphere was more variable , localizing to the smg for 50% of the participants , to the angular gyrus ( ag ) for 29% of participants , and to the ips for 21% of participants . this greater variation likely reflects the greater variability of the anatomical landmarks within the posterior right hemisphere for our participants . in contrast to these spatial activation patterns for previously encoded high - attention words , the principal locus of activation in posterior parietal cortex for new words was associated with delayed activity in ba39 ; specifically ag and the adjacent portion of posterior middle temporal gyrus ( pmtg ) in both hemispheres ( figure 2(e ) ; table 3 ) . in the left parietal lobe , the activity was centered within the ag for 64% of the participants ( 9 of 14 individuals ) with additional activity that spreads around the peak activation into pmtg . in the right parietal lobe , the primary focus was centered more often in pmtg ( 64% ) than in ag ( 36% ) ( table 3 ) . thus the bold activation associated with new words was distinctly more ventral and posterior than the parietal activation pattern for previously encoded high - attention words . moreover , the time course of new word - related activity in right posterior parietal cortex was similar to that observed in right ifg for new words ( compare figures 2(c ) and 2(e ) ) . this finding suggests that the timing of activation in right ba39 may be an important determinant in how different stimulus contexts are represented in this region . the timing difference may reflect two distinct functional roles for this component of the frontoparietal attention network in processing previously encoded ( high - attention ) words and newly encoded ( new ) words . one possibility is that the delayed response in ba39 may reflect the increased time required to access semantic content associated with these new words , or alternatively , activation may be associated more directly with processing novel events . based on these results , we propose a neuroanatomical framework that involves two subnetworks in the ventral frontoparietal attention stream , as shown in figure 3 . the relatively early onset activation for previously encoded words in ba40 and ba47 suggests an early attentional role for this subnetwork ( figure 3 , red pathway ) , while the delayed time course of new word responses in ba39 and ba47 could reflect the activity of a separate ventral pathway associated principally with bottom - up processing of novel word stimuli ( green pathway ) . our results are therefore consistent with previous findings that ventral parietal cortex plays a pivotal role in language - based tasks [ 37 , 5557 ] . inferior parietal activity has been found more reliably in studies of working memory than in those of explicit recall [ 15 , 5860 ] , and our results are also consistent with these findings . moreover , during the encoding phase of our study ( figure 1 ) , right ba40 was activated at the time our spoken stimulus words were first encoded . the results of the memory phase in the present study show that right ba40 was reactivated at retrieval , a finding strongly in line with the transfer - appropriate processing model [ 10 , 25 , 2931 , 6163 ] . we propose that early selective activation of right ba40 serves to initiate incidental memory for the studied words by bringing them back into the focus of attention that was established earlier during encoding [ 13 , 6466 ] . in summary , our data support a functional framework in which the brain regions that are engaged in identifying words are also sensitive to the level of attention at the time the words were initially encoded . a structural model generated from our findings our data are consistent with a focus of attention centered in right smg ( ba40 ) , but we also identified a frontal region in right dlpfc ( ba9/46 ) that is consistent with an attention - to - memory function in postretrieval processing . other frontal regions ( left dlpfc ) were insensitive to the attentional manipulation at encoding , responding similarly to both the two studied word categories as well as to the newly encoded ( new ) words . the pattern in left dlpfc is therefore consistent with other cognitive processes such as executive control of attention that would not be expected to vary across our word categories . a key result was that once a spoken word was unintentionally encoded , subsequent retrieval of that word varied as a function of the initial level of attention directed toward the word at the time of encoding . this is consistent with the notion that attention is critical not only for efficient encoding of words into memory , but it also helps to preserve salient information that is required for the successful retrieval of that information from memory .

attention is crucial for encoding information into memory , and current dual - process models seek to explain the roles of attention in both recollection memory and incidental - perceptual memory processes . the present study combined an incidental memory paradigm with event - related functional mri to examine the effect of attention at encoding on the subsequent neural activation associated with unintended perceptual memory for spoken words . at encoding , we systematically varied attention levels as listeners heard a list of single english nouns . we then presented these words again in the context of a recognition task and assessed the effect of modulating attention at encoding on the bold responses to words that were either attended strongly , weakly , or not heard previously . mri revealed activity in right - lateralized inferior parietal and prefrontal regions , and positive bold signals varied with the relative level of attention present at encoding . temporal analysis of hemodynamic responses further showed that the time course of bold activity was modulated differentially by unintentionally encoded words compared to novel items . our findings largely support current models of memory consolidation and retrieval , but they also provide fresh evidence for hemispheric differences and functional subdivisions in right frontoparietal attention networks that help shape auditory episodic recall .

1. Introduction 2. Materials and Methods 3. Results and Discussion 4. Conclusion

attention is known to alter neural processing at multiple levels of both the peripheral and central nervous systems , and both auditory and visual attention have been conceptualized as operating in both top - down mechanisms reflect goal - based control in order to direct attention to particular targets or to sustain attention over time . in contrast , bottom - up mechanisms have traditionally been defined by the phenomenon of reflexive attentional orienting , as when attention is drawn without intent by highly salient sensory stimuli such as a sudden loud noise or flash of light . recently , however , some investigators have more broadly considered bottom - up effects as relevant for any incoming stimuli , with the relative saliency of the stimulus influencing whether it is ultimately encoded into memory . two recent theoretical models address the question of what roles stimulus saliency might play in first successfully encoding information into memory and then later retrieving it . as discussed below , the embedded processes model and the attention - to - memory model , while similar , also highlight the potentially divergent roles that attention at encoding may play in later recall . more recently , others have found that the majority of functional neuroimaging data on working memory is consistent with the tenets of this model , with ventral posterior ( inferior parietal cortex and intraparietal sulcus ) regions associated with attentional focus and working memory maintenance , and lateral prefrontal regions involved in the executive control of attention and further manipulation of information , once it is in working memory [ 16 , 17 ] . if this interpretation is correct , then manipulations of attention at encoding should preferentially affect activation in parietal regions . another recent paradigm , the attention - to - memory model [ 8 , 1820 ] , shares functional features with the previous model , but it also emphasizes a distinction between the roles of ventral and dorsal parietal cortex in memory retrieval . successful recognition memory for intentionally studied items is predicted by increased activation of dorsal posterior parietal cortex at stimulus encoding , whereas activation in ventral posterior parietal cortex at encoding predicts success in incidental memory and perceptual priming tests [ 21 , 22 ] . this latter observation suggests a role for this ventral region not only in the bottom - up encoding of highly salient or unexpected sensory stimuli , but also in the successful retrieval of unintentionally encoded memories . according to these prevailing theories , retrieval of incidental or perceptual memory representations requires a bottom - up shift in selective attention , whereas retrieval of intentional memories requires a top - down mechanism that reflects the individual 's conscious intent to focus attention on incoming signals [ 21 , 22 ] importantly , however , the vast majority of studies exploring the intersection of attention and episodic memory and thus the theories on which these studies are based are heavily grounded in the use of visual paradigms [ 2330 ] . it is therefore necessary to examine the effects of attention on item retrieval in listening tasks in order to establish whether the dorsal / ventral distinction observed in parietal cortex is specific to visual stimuli , or instead reflects a more generalized organization in this area of the brain . to examine these ideas in greater depth , it is necessary to learn more about the role of attention in establishing a selective focus on task - relevant sensory stimuli . our goal in this study was to devise a method to modulate attention toward spoken words during a simple listening task . based on earlier studies , we predicted that words not intentionally within the focus of attention would be encoded into memory to some degree . during the encoding phase of our study , participants initially heard a list of words belonging to two semantic classes ( animals or foods ) , and these words were presented by two different talkers ( female or male ) . participants were given explicit instructions to remember only the animal words presented in the female voice ( the targets ) . importantly , this instruction resulted in a focus on the female voice , which had the consequence of placing the nontarget food words presented in that voice also within the focus of attention . we predicted that these words would also carry higher salience at encoding than words heard in the male voice , creating what we refer to as the high - attention condition in this study . following this rationale , the food words presented in the male voice would not be fully in the primary focus of attention ( the low - attention condition ) . we contrasted these two encoding conditions with responses to novel food words that were not previously heard at any time during the experiment . we reasoned that processing of these new words should reflect only the role of attention in initial word encoding , but not a later role in retrieval . using this paradigm coupled with functional magnetic resonance imaging ( fmri ) , we were able to disambiguate the neural activation patterns associated with attention at first encoding from those reflecting the effects of attention at encoding on subsequent retrieval . exclusion criteria were a history of speech , language , or other neurological disorders , and all volunteers reported good general health with no contraindications for mri scanning . word stimuli were single , concrete nouns ( one to three syllables ) that were either names of animals or foods . stimulus durations ranged from 204903 ms ( mean = 532 ms ) and were presented in an event - related format with an average interstimulus interval ( isi ) of 871 157 ms . ordering of words belonging to the different stimulus categories described below was pseudorandom , and participants listened to them through mri - compatible stereo headphones ( resonance technology inc . these were then followed by a surprise postscan memory test that specifically measured the listener 's capacity to remember any words that were encoded during the earlier phases of the experiment , whether or not the listener was asked to remember them . participants listened to the animal and food words that were presented in either the male or female voice during a prescan practice period and during the encoding phase . in a second scan , two - thirds of the words were presented dichotically , with the female voice heard in one ear and the male voice heard in the other ( order counterbalanced across participants ) . rather , this instruction was used deliberately to assure that the primary focus of attention was on the female voice . as a result , food words presented in the female voice were associated with a higher level of attention ( defined as the high - attention condition ) and food words presented in the male voice were associated with a lower level of attention ( the low - attention condition ) . this precluded the possibility that the voice in the memory task could provide a cue to the encoding context . participants were asked to respond yes via button press if the word presented was one of their target words ( animal words originally spoken in the female voice ) and no to all other words . in order to assure that there was an effect of the attentional manipulation at encoding , we administered a surprise memory test within 15 minutes of the final scan . participants were presented verbally with a list of 40 food items and asked to recall whether they had heard each word at any time during the experiment . the list consisted of 10 high - attention , 10 low - attention , and 10 new food words that were all heard during the study phase and scan . these 40 words were presented verbally in a fixed pseudorandom order by the experimenter and the participants verbally responded yes or no to indicate whether they remembered having heard these words at any time during the experiment . finally , high - resolution spoiled gradient - echo ( spgr ) images were obtained in the sagittal plane ( tr = 30 , te = min , flip angle = 30 , 124 slices at 1.5 mm , matrix = 256 256 , fov = 25 cm ) and aligned with the fse images for improved regional localization and coregistration of functional data across participants after transforming the images into talairach space . due to scanner - related problems , we were unable to use the spgr images from three participants , and instead relied on the first set of axial fse anatomical images for alignment with the functional scans . data were then normalized to a scale of 0100% , and functional images were coregistered to the structural data followed by transformation into standard talairach space . in order to capture the amplitude of the bold activity over time the first was time locked to the stimulus onset , and each of the seven subsequent models was offset by one - tr ( 2.3 s ) increments from stimulus onset . stimulus functions were then convolved with the fmri time - series data from each individual . voxel - wise ( uncorrected ) threshold was p = 0.005 , and minimum corrected cluster volumes in original space were 32 contiguous voxels at p < 0.05 . to identify regions of interest ( roi ) associated with positive bold signal across the high - attention , low - attention , and new categories , we first combined the three group - averaged datasets . this allowed us to develop roi that were associated with activation in any of the three conditions of interest . however , since the proposed involvement of these regions is based almost entirely on visual rather than auditory paradigms , and there is growing evidence for bilateral involvement in language processing [ 45 , 46 ] , we examined the following roi in both hemispheres . masks corresponding to distinct anatomical regions of interest were developed , and roi were defined based on the threshold - corrected regions of significant activation . brain regions showing significant positive bold activation are listed in table 1 and described as follows ( approximate brodmann areas in parentheses ) : frontal lobe ( dorsal to ventral ) : medial frontal gyrus ( 6/32 ) , middle frontal gyrus ( 9/46 ) , inferior frontal gyrus ( 47/pars orbitalis ) , and anterior insular cortex ( 13 ) . parietal lobe ( inferior parietal lobule ) : angular gyrus ( 39 ) and supramarginal gyrus ( 40 ) . we looked for additional roi along the anterior - posterior axis of the superior temporal lobe due to its routine involvement in speech and language tasks [ 4751 ] , but found only one with significant activation : superior temporal gyrus including and extending around the transverse temporal gyrus ( ba41/42 ) . the surprise memory test was intended to provide proof of concept that the attentional manipulation at encoding actually produced an effect . conversely , participants should not indicate recognition for words introduced as foils that were not presented earlier at any time in the experiment . these words showed the best hit rate at 93.8 2.4% ( mean sem ) , compared to 70.8 4.6% for low - attention words , and 51.0 6.5% for new words ( all means significantly different based on post - hoc , two - tailed anova ; see figure 1 ) . a temporal analysis of the bold responses for each stimulus condition was performed by modeling the fmri time series over a range of time lags , as shown in figure 2 . although this analysis lacks the temporal precision of electrophysiological methods , it can provide valuable information about the relative timing of neural events associated with each test condition . as stated above , eight separate hemodynamic response models were constructed for each stimulus type , and the peak of each model was time - shifted by one tr in order to capture peak responses across an 8-tr ( 18.4 s ) time window . we then calculated the bold responses from the peak activation at each time lag , as shown in figure 2 . as expected , strong bold activity was observed in the superior temporal lobe in the vicinity of primary auditory cortex , and this activity did not vary with the three stimulus conditions ( table 1 ) . in addition to the expected early activity in left superior temporal gyrus , bold responses associated with all three stimulus categories were , on average , 20% greater in right superior temporal gyrus , which is consistent with the other right - lateralized activation patterns discussed below . as shown in figure 2(a ) , identification of words in all three categories was associated with activity in left middle frontal gyrus ( a portion of dorsolateral prefrontal cortex dlpfc ) and this activity was closely mirrored in anterior insular cortex ( figure 2(b ) ) . furthermore , the time course of the bold response in these two left - hemisphere regions was similar for all three word categories regardless of the initial encoding condition . this pattern indicates a strong differential effect of attention at encoding in right dlpfc ( ba9/46 ) compared to left dlpfc ( figure 2(a ) ) and bilateral insula ( figure 2(b ) ) . importantly , this result is consistent with earlier studies using written words that linked activity in this region of right dlpfc with post - retrieval processing and/or monitoring functions [ 37 , 5254 ] . if right dlpfc is indeed involved selectively in post - retrieval processing , we would also expect , in accord with the attention - to - memory model , that words strongly attended at encoding might evoke a greater memory response than words only weakly attended at encoding . conversely , this region would not be expected to activate in response to new words because these words were not yet encoded into memory . in accord with this hypothesis , only negligible positive bold activity was observed in right dlpfc in response to new words ( figure 2(a ) , right column ) . although this pathway has been observed in numerous visuospatial attention studies , our findings using auditory language stimuli are also consistent with this anatomical framework . the focal point for these clusters in the frontal lobes was found in pars orbitalis ( ba47 ) , and in accord with the ventral frontoparietal model , activity was also lateralized to the right hemisphere ( figure 2(c ) , right column ) . however , in left ifg , a bold response on a similar time course was absent for the new words , indicating that the early response in ifg to previously encoded words may specifically reflect memory for these previously presented items . despite the absence of an early bold response in left ifg for new words , these items were instead associated with a late - onset response in the right hemisphere ( figure 2(c ) , right column ) . in other words , responses to the new words differed from the previously attended words both in terms of hemispheric lateralization and in the late time course of activation in ifg ( ba47 ) , suggesting that this late activation more likely reflects the initial encoding of new words into memory . with respect to language processing , converging evidence suggests a key role for posterior parietal and temporal regions in verbal memory and attention [ 12 , 31 , 37 , 44 ] . one memory model predicts that items correctly identified as previously encountered ( old ) will trigger increased activity in left intraparietal cortex , relative to missed old and correctly rejected novel items . however , as noted above , selective attention is another cognitive function that is commonly associated with posterior parietal cortex , and it remains unclear how the influence of attention may affect the neural networks underlying recollection and familiarity , especially in the auditory domain . it has been proposed that inferior parietal lobule , a region within ventral posterior parietal cortex , may serve a specialized function in the expression of attention , as proposed in both the embedded - processes model [ 14 , 15 ] and the attention - to - memory ( atom ) model [ 8 , 1820 ] . if ventral posterior parietal cortex serves specifically as the focus of attention as in the embedded - processes model , we would expect activation in this region to be differentiated according to the level of attention directed toward each stimulus at encoding , as shown in figure 1 . another prediction is that any region responsible for the reactivation of focused attention prior to memory retrieval should display activity earlier in the hemodynamic response than regions responsible for post - stimulus processing steps , such as semantic analysis . this is consistent with the notion that activation in and around the smg is related to attentional scanning that placed our high - attention words into the focus of attention according to the embedded - processes model . to examine the spatial location of the group activation in greater detail this participant - by - participant anatomical localization revealed that the peak activation was located in the left smg for 86% ( 12 of 14 individuals ) , and in the right smg for 64% of the participants . in contrast , the primary focus of activation in the right hemisphere was more variable , localizing to the smg for 50% of the participants , to the angular gyrus ( ag ) for 29% of participants , and to the ips for 21% of participants . in contrast to these spatial activation patterns for previously encoded high - attention words , the principal locus of activation in posterior parietal cortex for new words was associated with delayed activity in ba39 ; specifically ag and the adjacent portion of posterior middle temporal gyrus ( pmtg ) in both hemispheres ( figure 2(e ) ; table 3 ) . in the left parietal lobe , the activity was centered within the ag for 64% of the participants ( 9 of 14 individuals ) with additional activity that spreads around the peak activation into pmtg . thus the bold activation associated with new words was distinctly more ventral and posterior than the parietal activation pattern for previously encoded high - attention words . moreover , the time course of new word - related activity in right posterior parietal cortex was similar to that observed in right ifg for new words ( compare figures 2(c ) and 2(e ) ) . this finding suggests that the timing of activation in right ba39 may be an important determinant in how different stimulus contexts are represented in this region . one possibility is that the delayed response in ba39 may reflect the increased time required to access semantic content associated with these new words , or alternatively , activation may be associated more directly with processing novel events . based on these results , we propose a neuroanatomical framework that involves two subnetworks in the ventral frontoparietal attention stream , as shown in figure 3 . the relatively early onset activation for previously encoded words in ba40 and ba47 suggests an early attentional role for this subnetwork ( figure 3 , red pathway ) , while the delayed time course of new word responses in ba39 and ba47 could reflect the activity of a separate ventral pathway associated principally with bottom - up processing of novel word stimuli ( green pathway ) . inferior parietal activity has been found more reliably in studies of working memory than in those of explicit recall [ 15 , 5860 ] , and our results are also consistent with these findings . the results of the memory phase in the present study show that right ba40 was reactivated at retrieval , a finding strongly in line with the transfer - appropriate processing model [ 10 , 25 , 2931 , 6163 ] . we propose that early selective activation of right ba40 serves to initiate incidental memory for the studied words by bringing them back into the focus of attention that was established earlier during encoding [ 13 , 6466 ] . in summary , our data support a functional framework in which the brain regions that are engaged in identifying words are also sensitive to the level of attention at the time the words were initially encoded . a structural model generated from our findings our data are consistent with a focus of attention centered in right smg ( ba40 ) , but we also identified a frontal region in right dlpfc ( ba9/46 ) that is consistent with an attention - to - memory function in postretrieval processing . other frontal regions ( left dlpfc ) were insensitive to the attentional manipulation at encoding , responding similarly to both the two studied word categories as well as to the newly encoded ( new ) words . the pattern in left dlpfc is therefore consistent with other cognitive processes such as executive control of attention that would not be expected to vary across our word categories . a key result was that once a spoken word was unintentionally encoded , subsequent retrieval of that word varied as a function of the initial level of attention directed toward the word at the time of encoding . this is consistent with the notion that attention is critical not only for efficient encoding of words into memory , but it also helps to preserve salient information that is required for the successful retrieval of that information from memory .

while atrial fibrillation ( af ) is the most common associated cardiac arrhythmia making up to 12% of strokes among the general population , asymptomatic paroxysmal af ( paf ) is often suspected as the underlying cause of cs . the progressive nature of af is well established and known to lead to electro - anatomical remodeling and atrial fibrosis . atrial fibrosis develops from complex interactions among several cellular and neurohumoral mediators , with the resultant fibrotic tissue causing a low a voltage response and other electrophysiological changes in the left atrium ( la ) that act as a substrate for the occurrence of af . while electrical remodeling may shorten atrial refractoriness and contributes to an increase in the stability of af , atrial structural remodeling occurs because of heart failure and other underlying cardiovascular diseases . moreover , la fibrosis has been associated with an increased risk of developing a stroke in the absence of af or paf , as shown by magnetic resonance imaging ( mri ) . speckle tracking echocardiography , a new imaging technique that offers offline calculation of myocardial velocities and deformation parameters such as strain and strain rate ( sr ) , provides important insights into systolic and diastolic function , ischemia , myocardial mechanics , and many other pathophysiological heart processes [ 6 - 9 ] . a sensitive , noninvasive , reproducible , and quantitative assessment of la longitudinal deformation , which is closely related to la physiology and anatomy , is achieved by means of this technique [ 6 - 9 ] . la longitudinal deformation indices ( global peak la strain [ pla - s ] , global peak la early diastolic strain rate [ pla - sre ] , and global peak la late diastolic strain rate [ pla - sra ] ) appear to provide the most accurate physiologic and anatomic analysis of la . as decreased la longitudinal deformation has been closely associated with paf , its detection allows the identification of patients with a high risk of af recurrence after catheter ablation procedures , irrespective of la enlargement . furthermore , decreased la longitudinal deformation predicts the extent of la fibrosis independent of other echocardiographic parameters , including rhythm . therefore , characterization of la fibrosis using speckle - tracking echocardiography may aid in the diagnosis of cs . recently , an association between a decreased la ejection fraction ( la ef ) and an increased risk of paf in cs has been suggested . furthermore , la active relaxation and contraction is lower in paf patients than in those with sinus rhythm , regardless of la enlargement and aging . p wave dispersion ( pwd ) , defined as the difference between the maximum ( pmax ) and the minimum ( pmin ) p wave duration on electrocardiography ( ecg ) , has been regarded as an independent risk factor for the development of af . as pwd may be easily measured using a single ecg , it is considered an electrocardiographic marker of atrial electromechanical dyssynchrony caused by the heterogeneous propagation of sinus impulses . on the other hand , pwd determined within the first day after of an acute ischemic stroke serves as a surrogate marker to predict paf and the risk of recurrent strokes . the aim of this study was to investigate associations between la electro - structural remodeling and the occurrence of cs . in this cross - sectional study , we have used speckle tracking echocardiography and ecg to assess la structural and electrical remodeling , and examined its association with the occurrence of cs . all consecutive patients presenting with a stroke of unknown origin despite extensive routine diagnostic testing and diagnosed with cs between 2009 and 2010 , were included in the study . we excluded patients who had a previous cerebrovascular event , a toast classification of high- and medium - risk sources of cardioembolism , or malignant arterial hypertension ; uncontrolled diabetes mellitus ; a history of effort angina , acute coronary syndrome , or revascularization procedures ; evidence of a positive exercise stress test before the acute stroke ; segmental wall abnormalities at echocardiography ; presence or 24-hourholter detected af , atrial flutter , or other major arrhythmias ; moderate - to - severe valvulopathies , existence of inter - atrial block on 12-lead ecg , or inter - atrial septum abnormalities ; patients who had a malign clinical course in the neurology intensive care unit , including severe kidney , respiratory , cardiac or hepatic failure , and inadequate acoustic windows were excluded . patients who required intubation because of respiratory insufficiency , and those who required more than 3 days in a neurology intensive care unit were also excluded . all patients fulfilled both the inclusion and exclusion criteria . from those , 40 ( aged between 18 and 55 years ) who were admitted to our emergency department and hospitalized in the neurology intensive care unit with a diagnosis of acute ischemic stroke , and a control group comprised of 40 age- and sex - matched healthy individuals , participants in the control group underwent a complete routine clinical and cardiac laboratory evaluation for the detection of occult cardiac disease , including 12-lead ecg , transthoracic / transesophageal echocardiography ( tte / tee ) , extra cranial arteries duplex sonography , and single ecg-24-hour holter ecg monitoring before inclusion in the study . individuals with any cardiac structural pathology and arrhythmia , including patent foramen ovale , atrial septal aneurysm , or paroxysmal atrial fibrillation , were excluded from the study . the following routine diagnostic tests were performed in all patients : cranial computed tomography and magnetic resonance imaging of the brain , or both . duplex sonography of the extra cranial and intracranial arteries , a single ecg-24-hour holter ecg monitoring , and tte / tee were performed in patients within a median of 3 days after the acute stroke . electrocardiogram - guided echocardiographic measurements were carried out using a commercially available ultrasound device ( ie33 , philips medical system , bothell , washington , usa ) . images were obtained from parasternal and apical windows using 2d , m - mode , and doppler echocardiography . the lvef , as a standard index of global lv systolic function , was measured using the simpson s method . the ratio between the peaks of early ( e ) and late ( a ) diastolic lv filling velocities was used as the standard index of lv diastolic function . tissue doppler measurements were obtained at end - expiration with the sample volume placed on the atrial side of the mitral annulus at the basal inter - atrial septum in the apical four - chamber view . both early diastolic ( e ) , and late diastolic ( a ) the e / e ratio was also calculated to provide a reliable index of the lv filling pressure . at end - systole , just before the opening of the mitral valve ( at the end of the t wave on the ecg ) , the la maximum volume was indexed to body surface area ( lavimax ) . at end - diastole , just before mitral valve closure ( at the beginning of the qrs complex on the ecg ) , minimum la volume to body surface area ( lavimin ) were measured and indexed to body surface area , as previously described . for speckle tracking echocardiography analysis , apical 4- and 2-chamber view images were obtained using conventional 2-dimensional gray - scale echocardiography during breath - hold . three consecutive heart cycles were recorded in digital format for offline analysis using the commercially available software qlab 6.0 ( philips medical system , bothell , washington , usa ) . segments for which adequate tracking quality could not be obtained despite manual adjustment , were excluded from analysis , and patients in whom some segments were excluded due to the impossibility of adequate tracking , la deformation parameters were calculated by averaging the values measured in the remaining segments . la peak longitudinal strain and strain rate at basal segment from the apical views of the inter - atrial septum , la lateral wall , la anterior wall , and la inferior wall in the 5 3 mm region of interest were calculated as described in current guidelines . la reservoir strain during systole was obtained at the time of aortic valve closure , and la contractile strain rate during late diastole was obtained at the onset of the p wave on ecg . the reproducibility and feasibility of speckle tracking echocardiography measurement of la longitudinal strain and strain rates were considered acceptable . all standard 12-lead ecgs were obtained simultaneously at a 50-mm / s - paper speed and amplitude of 10 mm / mv . two cardiologists blind to the clinical status of the patients performed the p wave analysis with calipers and magnifying glasses to decrease the risk of measurement errors . the onset of the p wave was defined as the point in which the wave showed the first visible upward and downward departure from baseline for positive and negative waveforms , respectively . a return to the baseline maximum p wave duration ( pmax ) , measured from any of the 12 leads of the surface ecg , was used for the longest atrial conduction time . the difference between the maximum and minimum p wave duration was defined as pwd ( pwd = pmax - pmin ) . data have been presented as mean sd for continuous variables and as proportions for categorical variables . for continuous data , statistical differences were evaluated using a student s t test , or alternatively a mann - whitney u test for cases in which the assumptions of the student s t test were not satisfied . for categorical data , all statistical analyses were performed using spss statistical software , version 15.0 ( spss inc . , in this cross - sectional study , we have used speckle tracking echocardiography and ecg to assess la structural and electrical remodeling , and examined its association with the occurrence of cs . all consecutive patients presenting with a stroke of unknown origin despite extensive routine diagnostic testing and diagnosed with cs between 2009 and 2010 , were included in the study . we excluded patients who had a previous cerebrovascular event , a toast classification of high- and medium - risk sources of cardioembolism , or malignant arterial hypertension ; uncontrolled diabetes mellitus ; a history of effort angina , acute coronary syndrome , or revascularization procedures ; evidence of a positive exercise stress test before the acute stroke ; segmental wall abnormalities at echocardiography ; presence or 24-hourholter detected af , atrial flutter , or other major arrhythmias ; moderate - to - severe valvulopathies , existence of inter - atrial block on 12-lead ecg , or inter - atrial septum abnormalities ; patients who had a malign clinical course in the neurology intensive care unit , including severe kidney , respiratory , cardiac or hepatic failure , and inadequate acoustic windows were excluded . patients who required intubation because of respiratory insufficiency , and those who required more than 3 days in a neurology intensive care unit were also excluded . all patients fulfilled both the inclusion and exclusion criteria . from those , 40 ( aged between 18 and 55 years ) who were admitted to our emergency department and hospitalized in the neurology intensive care unit with a diagnosis of acute ischemic stroke , and a control group comprised of 40 age- and sex - matched healthy individuals , participants in the control group underwent a complete routine clinical and cardiac laboratory evaluation for the detection of occult cardiac disease , including 12-lead ecg , transthoracic / transesophageal echocardiography ( tte / tee ) , extra cranial arteries duplex sonography , and single ecg-24-hour holter ecg monitoring before inclusion in the study . individuals with any cardiac structural pathology and arrhythmia , including patent foramen ovale , atrial septal aneurysm , or paroxysmal atrial fibrillation , were excluded from the study . the following routine diagnostic tests were performed in all patients : cranial computed tomography and magnetic resonance imaging of the brain , or both . duplex sonography of the extra cranial and intracranial arteries , a single ecg-24-hour holter ecg monitoring , and tte / tee were performed in patients within a median of 3 days after the acute stroke . electrocardiogram - guided echocardiographic measurements were carried out using a commercially available ultrasound device ( ie33 , philips medical system , bothell , washington , usa ) . images were obtained from parasternal and apical windows using 2d , m - mode , and doppler echocardiography . the lvef , as a standard index of global lv systolic function , was measured using the simpson s method . the ratio between the peaks of early ( e ) and late ( a ) diastolic lv filling velocities was used as the standard index of lv diastolic function . tissue doppler measurements were obtained at end - expiration with the sample volume placed on the atrial side of the mitral annulus at the basal inter - atrial septum in the apical four - chamber view . both early diastolic ( e ) , and late diastolic ( a ) the e / e ratio was also calculated to provide a reliable index of the lv filling pressure . at end - systole , just before the opening of the mitral valve ( at the end of the t wave on the ecg ) , the la maximum volume was indexed to body surface area ( lavimax ) . at end - diastole , just before mitral valve closure ( at the beginning of the qrs complex on the ecg ) , minimum la volume to body surface area ( lavimin ) were measured and indexed to body surface area , as previously described . for speckle tracking echocardiography analysis , apical 4- and 2-chamber view images were obtained using conventional 2-dimensional gray - scale echocardiography during breath - hold . three consecutive heart cycles were recorded in digital format for offline analysis using the commercially available software qlab 6.0 ( philips medical system , bothell , washington , usa ) . segments for which adequate tracking quality could not be obtained despite manual adjustment , were excluded from analysis , and patients in whom some segments were excluded due to the impossibility of adequate tracking , la deformation parameters were calculated by averaging the values measured in the remaining segments . la peak longitudinal strain and strain rate at basal segment from the apical views of the inter - atrial septum , la lateral wall , la anterior wall , and la inferior wall in the 5 3 mm region of interest were calculated as described in current guidelines . la reservoir strain during systole was obtained at the time of aortic valve closure , and la contractile strain rate during late diastole was obtained at the onset of the p wave on ecg . the reproducibility and feasibility of speckle tracking echocardiography measurement of la longitudinal strain and strain rates were considered acceptable . all standard 12-lead ecgs were obtained simultaneously at a 50-mm / s - paper speed and amplitude of 10 mm / mv . two cardiologists blind to the clinical status of the patients performed the p wave analysis with calipers and magnifying glasses to decrease the risk of measurement errors . the onset of the p wave was defined as the point in which the wave showed the first visible upward and downward departure from baseline for positive and negative waveforms , respectively . a return to the baseline maximum p wave duration ( pmax ) , measured from any of the 12 leads of the surface ecg , was used for the longest atrial conduction time . the difference between the maximum and minimum p wave duration was defined as pwd ( pwd = pmax - pmin ) . data have been presented as mean sd for continuous variables and as proportions for categorical variables . for continuous data , statistical differences were evaluated using a student s t test , or alternatively a mann - whitney u test for cases in which the assumptions of the student s t test were not satisfied . for categorical data , all statistical analyses were performed using spss statistical software , version 15.0 ( spss inc . , chicago , il , usa ) . no significant differences with regard to age , gender , body mass index , body surface area , heart rate , blood pressure , left atrial minimal volume index , and left ventricular ejection fraction , were observed between the two groups . the pwd was 30.1 7.0 ms and 27.4 3.5 ms in the cs and control group , respectively ( p= 0.02 ) , whereas the la maximum volume index [ lavimax ] was 20.4 4.5 ml / m and 19.9 2.4 ml / m in the cs and control group , respectively ( p= 0.04 ) . the global peak la strain was [ pla - s ] ( la reservoir function ) 41.4 6.3% and 44.5 7.1% in the cs and control group , respectively ( p= 0.04 ) , whereas the global peak late diastolic strain rate values [ plasra ] ( la pump function ) were 2.5 0.4% and 2.9 0.5% in the cs and control group , respectively ( p= 0.001 ) . a strong negative correlation between global pla - s and lavimax ( r= -0.49 ; p < 0.01 ) , as well as a moderate correlation between pwd and global pla - s ( r= -0.52 ; p < 0.01 ) , was found in cs patients ( figures 1 , 2 ) . although there was no significant difference in left atrial tissue doppler velocities between patients and healthy controls , global pla - s was found to be significantly lower in cs patients ( table 2 ) . a significant difference was found in the pattern of la segment changes in cs patients ( table 2 and figure 3 ) . the la reservoir function ( la strain ) was smaller in cs patients than in healthy controls . the inter - atrial septal and inferior wall early diastolic strain rates ( la conduit function ) and the anterior late diastolic strain rate ( la pump function ) were significantly lower in cs patients than in healthy controls , whereas the interatrial septal late diastolic strain rate tended to be lower but the difference was not statistically significant . in this study we have demonstrated that electrical remodeling , shown by an increase in pwd on surface ecg , was associated with structural remodeling and decreased la deformation shown on echocardiography . these findings suggest that la structural , functional , and electrical impairments in conjunction with other risk factors i.e. increased thrombogenicity and a heightened proinflammatory response , may ultimately result in cardioembolism in patients without inter - atrial septal abnormalities . pwd was increased in cs patients mainly due to an increase in maximum p wave values rather than a decrease in minimum p wave durations on a 12-lead ecg , and it was associated with an increased risk of af development . although , the increase in pwd and structural changes observed in la may explain the occurrence of cs , whether these changes are an underlying cause or an associated finding in the early post - stroke period ( within 1 week ) in cs patients , remains unclear [ 18 - 20 ] . furthermore , af results in la remodeling through a process that involves the deposition of fibrotic tissue , leading to changes in the electrophysiological properties of the la substrate . detection and quantification of la structural remodeling as a possible determinant of fibrosis nevertheless , whether substrate changes seen in af patients are related to stroke and current stroke risk stratification schemes is not yet clear . the prolongation of intra- and inter - atrial conduction times and the inhomogeneous propagation of sinus impulses are well known electrophysiological characteristics seen in patients with paf . pwd has proven to be a sensitive and specific ecg predictor of af in various clinical settings , including patients with hypertension , coronary artery disease , undergoing coronary artery bypass surgery , or obstructive sleep apnea [ 20 - 23 ] . increases in pwd and pmax are important indicators of subsequent paf attacks and a tendency towards persistent atrial fibrillation . furthermore , an association between incremental la enlargement and an increased risk of af during follow - up has been suggested , with the risk of developing af being four times greater in patients with enlarged la . results from studies by abhayaratna et al . showed that increased lavimax was an independent predictor of death , heart failure , atrial fibrillation , and ischemic stroke . the role of la function in stroke pathogenesis has been supported by recent data showing that inter - atrial septal abnormalities lead to la dysfunction , which may in turn contribute to la thrombosis and subsequent ischemic stroke . moreover , goch et al . showed that patients with only atrial septum aneurysm ( asa ) had depressed la systolic function and increased left atrial appendage function , which might be a compensatory mechanism for la deterioration . jin et al . revealed that la active pump function was significantly depressed , and closely correlated with left atrial appendage emptying in cs patients with asa alone . therefore , impaired la and la appendage ( laa ) functions may be crucial pathophysiologic mechanisms for ischemic stroke in patients with asa alone . to our knowledge , la structure and function in cs patients without inter - atrial septal abnormalities have not been thoroughly investigated . the present study provides new insights into la remodeling in patients with cs , demonstrating that global pla - s , a newer parameter of atrial function , is more sensitive than the traditional echocardiographic markers of la size and function used to detect la remodeling . routine measurement of la strain might guarantee the detection of la remodeling , and provide useful additional information for cs diagnosis . thus , this novel imaging method may be considered a promising index for a more accurate quantification of la function , allowing the potential identification of la impairment , which may be a useful parameter for cs risk stratification . however , as this novel technique has insufficient resolution to measure the radial strain of the thin - walled la , la deformation assessment is only based on longitudinal strain . longitudinal strain is positive during the reservoir period , in which la myocardial fibers relax and stretch to adapt to the incoming blood flow , negative during the pump and most of the conduit phase , during which the la is emptying , and flat during diastasis , corresponding to the late phase of the conduit phase . therefore , measurements taken at end - systole , as well as early and late end - diastole , may provide information about the reservoir , conduit , and pump functions . furthermore , global pla - s has considerably high diagnostic accuracy while the e / e ratio correlates poorly with invasively measured la pressure . in patients with af who underwent catheter ablation , the systolic , as well as the early and late diastolic strains , were considerably lower in patients with persistent af than in patients with paf or normal subjects . similarly , lower strain and strain rate were associated with a higher rate of af reoccurrence after catheter ablation , and an improvement in la strain correlated with a reversion of la remodeling in patients who underwent catheter ablation for af . this latter finding may have essential prognostic and therapeutic implications concerning the post procedure cardioembolic risk and oral anticoagulation therapy . despite the obvious advantages of two - dimensional echocardiography , the difficulty of endocardial border tracing and its reliance on geometrical assumptions , which ignore lv and la geometrical differences between individuals , limits its application . however , la strain measurements may facilitate atrial remodeling assessment in various pathological conditions , as well as reverse remodeling after medical or more invasive therapy , such as cardiac resynchronization therapy , ablation for atrial fibrillation or flutter , hypertrophic cardiomyopathy , or coronary artery diseases [ 28 - 31 ] . in support of this view , karabay et al . recently demonstrated that la deformation parameters measured by speckle tracking were found to predict impaired la appendage ( laa ) functions and the presence of laa thrombus in ischemic stroke patients with suspected cardioembolism in sinus rhythm . in that study , la reservoir and pump functions were found to be significantly lower in patients with laa thrombus , and the la reservoir function showed the strongest correlation with laa morphologic parameters . pagola et al . recently claimed that measurement of pla - s in patients with cs might be a useful tool to detect patients with occult paf . furthermore , they concluded that pla - s analysis may play a role in the selection of patients with normal size la for prolonged cardiac monitorization , because most patients with paf showed low las in their study . although , their results were similar , the patients mean age of the study population was 62 years , whereas in our study it was only 42 years . since aging is associated with atrial fibrosis and atrial arrhythmias , we have excluded an aging effect on left atrial anatomy and function in cs patients in this study . la structural remodeling , as demonstrated by the histopathology findings , significantly influences global pla - s . therefore , assessment of la function and la ultrastructural changes may provide additional information for the prediction of cardiovascular events . in particular , atrial fibrosis has been strongly associated with the presence of heart disease and arrhythmias , including congestive heart failure and atrial fibrillation . moreover , an adverse prognosis linked to progressive la substrate remodeling , showed a correlation between stroke and high levels of la fibrosis , detected by delayed enhancement mri in patients with af . however , global pla - s measured by speckle tracking echocardiography was negatively correlated with la myocardial fibrosis grade , while poorer correlations with the la indexed volume , la ejection fraction , and e / e ratio have been shown . as global pla - s is also correlated with endocardial thickening , a histologic alteration appearing in the earlier stages of structural remodeling , it can be used to assess an increase in interstitial fibrosis in conditions which compromise the elastic properties of the atrial myocardium , inevitably leading to impairment of atrial compliance and reduction of la reservoir function . although our findings are in general agreement with the results of previous studies , there are several notable differences . compared with our study , most studies selected subjects from the general population who presented inter - atrial septal abnormalities . therefore , their results are not representative of all the underlying pathophysiologic mechanisms operating in the development of ischemic events in cs patients with no inter - atrial septal abnormalities . furthermore , in order to eliminate possible etiologies associated with paradoxical embolism and to prevent misleading results , we only included cs patients without pfo or asa on tee . the main limitation of our study the inability to demonstrate a clear relationship between la structure and la strain using histopathological or delayed enhancement mri . secondly , to exclude undetected paroxysmal af ( paf ) , in addition to a single 24-hour holter monitoring in all subjects , we performed standard 12-lead ecgs at every visit as well as at any time if the subjects reported palpitations . however , we were not able to exclude paf episodes reliably because they are often asymptomatic , and those undetected episodes may have contributed to confounding during the statistical analysis of la function . although the effect of various drugs , including anti - thrombotic drugs , angiotensin converting enzyme inhibitors or angiotensin receptor blockers , and statins , on la function or plasma biomarkers could not be fully controlled , as not all subjects were receiving the same drug therapies , there were no significant differences in medication regimens between the two study groups . some cardiac abnormalities that can not be detected by routine diagnostic methods may be a cause of af or paf and subsequent cs . acute cerebral events such as subarachnoid hemorrhage may cause stunned myocardium , a neurogenic cardiac dysfunction , as well as arrhythmia and ischemia changes during the acute phase . although the mechanisms responsible for those changes are unknown , the release of stress hormones , adrenaline , and nor - adrenaline , caused by major stressful events , is suspected . lastly , we can not completely exclude the existence of right - to - left shunt , as exclusion of pfo was only confirmed by tee using contrast bubbles . recently , transcranial doppler examination has been considered more sensitive than tee for the diagnosis of a right - to - left shunt . nevertheless , our hypothesis that la dysfunction itself may contribute to thrombosis in situ through an embolism with an extra - cardiac origin remains plausible . therefore , further interventional studies are warranted to investigate whether improvement of la function by reversion of la remodeling would protect from ischemic stroke in the general population . the main limitation of our study the inability to demonstrate a clear relationship between la structure and la strain using histopathological or delayed enhancement mri . secondly , to exclude undetected paroxysmal af ( paf ) , in addition to a single 24-hour holter monitoring in all subjects , we performed standard 12-lead ecgs at every visit as well as at any time if the subjects reported palpitations . however , we were not able to exclude paf episodes reliably because they are often asymptomatic , and those undetected episodes may have contributed to confounding during the statistical analysis of la function . although the effect of various drugs , including anti - thrombotic drugs , angiotensin converting enzyme inhibitors or angiotensin receptor blockers , and statins , on la function or plasma biomarkers could not be fully controlled , as not all subjects were receiving the same drug therapies , there were no significant differences in medication regimens between the two study groups . some cardiac abnormalities that can not be detected by routine diagnostic methods may be a cause of af or paf and subsequent cs . acute cerebral events such as subarachnoid hemorrhage may cause stunned myocardium , a neurogenic cardiac dysfunction , as well as arrhythmia and ischemia changes during the acute phase . although the mechanisms responsible for those changes are unknown , the release of stress hormones , adrenaline , and nor - adrenaline , caused by major stressful events , is suspected . lastly , we can not completely exclude the existence of right - to - left shunt , as exclusion of pfo was only confirmed by tee using contrast bubbles . recently , transcranial doppler examination has been considered more sensitive than tee for the diagnosis of a right - to - left shunt . nevertheless , our hypothesis that la dysfunction itself may contribute to thrombosis in situ through an embolism with an extra - cardiac origin remains plausible . therefore , further interventional studies are warranted to investigate whether improvement of la function by reversion of la remodeling would protect from ischemic stroke in the general population . increased pwd on surface ecg is associated with left atrial mechanical remodeling and dysfunction , both of which may be substrates for thromboembolism in cs patients .

background and purposeto investigate an association between left atrial ( la ) structural and p wave dispersion ( pwd ) during sinus rhythm , and electrical remodeling in cryptogenic stroke ( cs ) patients.methodsforty cs patients and 40 age- and sex - matched healthy controls were enrolled . p wave calculations were based on 12-lead electrocardiography ( ecg ) at a 50-mm / s - paper speed with an amplitude of 10 mm / mv . difference between the maximum and minimum p wave duration was the p wave dispersion ( pwd = pmax - pmin ) . la deformation was evaluated by speckle tracking echocardiography within 3 days of the acute event.resultspwd was 30.17.0 ms and 27.43.5 ms in cs and control group ( p=0.02 ) , whereas la maximum volume index [ lavimax ] was 20.44.5 ml / m2 and 19.92.4 ml / m2 in cs and control group , respectively ( p = 0.04 ) . while global peak la strain was [ pla - s ] ( la reservoir function ) 41.4 6.3% and 44.5 7.1% in cs and control group , ( p = 0.04 ) , global peak late diastolic strain rate values [ pla - sra ] ( la pump function ) were 2.5 0.4% and 2.9 0.5% in cs and control group , respectively ( p = 0.001 ) . a mild and a strong negative correlation between global pla - s and lavimax ( r=-0.49 ; p<0.01 ) , and between pwd and global pla - s ( r = -0.52 ; p < 0.01 ) , respectively , was observed in cs.conclusionsincreased pwd is associated with impaired la mechanical functions and enlargement , and involved in the pathophysiology of af or an af - like physiology in cs .

Introduction Methods Patients and data collection LA structural remodeling analysis using conventional and speckle tracking echocardiography LA electrical remodeling analysis using P wave dispersion Statistical analysis Results Discussion Limitations Conclusions

atrial fibrosis develops from complex interactions among several cellular and neurohumoral mediators , with the resultant fibrotic tissue causing a low a voltage response and other electrophysiological changes in the left atrium ( la ) that act as a substrate for the occurrence of af . while electrical remodeling may shorten atrial refractoriness and contributes to an increase in the stability of af , atrial structural remodeling occurs because of heart failure and other underlying cardiovascular diseases . moreover , la fibrosis has been associated with an increased risk of developing a stroke in the absence of af or paf , as shown by magnetic resonance imaging ( mri ) . speckle tracking echocardiography , a new imaging technique that offers offline calculation of myocardial velocities and deformation parameters such as strain and strain rate ( sr ) , provides important insights into systolic and diastolic function , ischemia , myocardial mechanics , and many other pathophysiological heart processes [ 6 - 9 ] . la longitudinal deformation indices ( global peak la strain [ pla - s ] , global peak la early diastolic strain rate [ pla - sre ] , and global peak la late diastolic strain rate [ pla - sra ] ) appear to provide the most accurate physiologic and anatomic analysis of la . as decreased la longitudinal deformation has been closely associated with paf , its detection allows the identification of patients with a high risk of af recurrence after catheter ablation procedures , irrespective of la enlargement . therefore , characterization of la fibrosis using speckle - tracking echocardiography may aid in the diagnosis of cs . recently , an association between a decreased la ejection fraction ( la ef ) and an increased risk of paf in cs has been suggested . furthermore , la active relaxation and contraction is lower in paf patients than in those with sinus rhythm , regardless of la enlargement and aging . p wave dispersion ( pwd ) , defined as the difference between the maximum ( pmax ) and the minimum ( pmin ) p wave duration on electrocardiography ( ecg ) , has been regarded as an independent risk factor for the development of af . in this cross - sectional study , we have used speckle tracking echocardiography and ecg to assess la structural and electrical remodeling , and examined its association with the occurrence of cs . we excluded patients who had a previous cerebrovascular event , a toast classification of high- and medium - risk sources of cardioembolism , or malignant arterial hypertension ; uncontrolled diabetes mellitus ; a history of effort angina , acute coronary syndrome , or revascularization procedures ; evidence of a positive exercise stress test before the acute stroke ; segmental wall abnormalities at echocardiography ; presence or 24-hourholter detected af , atrial flutter , or other major arrhythmias ; moderate - to - severe valvulopathies , existence of inter - atrial block on 12-lead ecg , or inter - atrial septum abnormalities ; patients who had a malign clinical course in the neurology intensive care unit , including severe kidney , respiratory , cardiac or hepatic failure , and inadequate acoustic windows were excluded . patients who required intubation because of respiratory insufficiency , and those who required more than 3 days in a neurology intensive care unit were also excluded . from those , 40 ( aged between 18 and 55 years ) who were admitted to our emergency department and hospitalized in the neurology intensive care unit with a diagnosis of acute ischemic stroke , and a control group comprised of 40 age- and sex - matched healthy individuals , participants in the control group underwent a complete routine clinical and cardiac laboratory evaluation for the detection of occult cardiac disease , including 12-lead ecg , transthoracic / transesophageal echocardiography ( tte / tee ) , extra cranial arteries duplex sonography , and single ecg-24-hour holter ecg monitoring before inclusion in the study . duplex sonography of the extra cranial and intracranial arteries , a single ecg-24-hour holter ecg monitoring , and tte / tee were performed in patients within a median of 3 days after the acute stroke . tissue doppler measurements were obtained at end - expiration with the sample volume placed on the atrial side of the mitral annulus at the basal inter - atrial septum in the apical four - chamber view . both early diastolic ( e ) , and late diastolic ( a ) the e / e ratio was also calculated to provide a reliable index of the lv filling pressure . at end - systole , just before the opening of the mitral valve ( at the end of the t wave on the ecg ) , the la maximum volume was indexed to body surface area ( lavimax ) . at end - diastole , just before mitral valve closure ( at the beginning of the qrs complex on the ecg ) , minimum la volume to body surface area ( lavimin ) were measured and indexed to body surface area , as previously described . segments for which adequate tracking quality could not be obtained despite manual adjustment , were excluded from analysis , and patients in whom some segments were excluded due to the impossibility of adequate tracking , la deformation parameters were calculated by averaging the values measured in the remaining segments . la peak longitudinal strain and strain rate at basal segment from the apical views of the inter - atrial septum , la lateral wall , la anterior wall , and la inferior wall in the 5 3 mm region of interest were calculated as described in current guidelines . la reservoir strain during systole was obtained at the time of aortic valve closure , and la contractile strain rate during late diastole was obtained at the onset of the p wave on ecg . the reproducibility and feasibility of speckle tracking echocardiography measurement of la longitudinal strain and strain rates were considered acceptable . all standard 12-lead ecgs were obtained simultaneously at a 50-mm / s - paper speed and amplitude of 10 mm / mv . two cardiologists blind to the clinical status of the patients performed the p wave analysis with calipers and magnifying glasses to decrease the risk of measurement errors . the onset of the p wave was defined as the point in which the wave showed the first visible upward and downward departure from baseline for positive and negative waveforms , respectively . a return to the baseline maximum p wave duration ( pmax ) , measured from any of the 12 leads of the surface ecg , was used for the longest atrial conduction time . the difference between the maximum and minimum p wave duration was defined as pwd ( pwd = pmax - pmin ) . , in this cross - sectional study , we have used speckle tracking echocardiography and ecg to assess la structural and electrical remodeling , and examined its association with the occurrence of cs . we excluded patients who had a previous cerebrovascular event , a toast classification of high- and medium - risk sources of cardioembolism , or malignant arterial hypertension ; uncontrolled diabetes mellitus ; a history of effort angina , acute coronary syndrome , or revascularization procedures ; evidence of a positive exercise stress test before the acute stroke ; segmental wall abnormalities at echocardiography ; presence or 24-hourholter detected af , atrial flutter , or other major arrhythmias ; moderate - to - severe valvulopathies , existence of inter - atrial block on 12-lead ecg , or inter - atrial septum abnormalities ; patients who had a malign clinical course in the neurology intensive care unit , including severe kidney , respiratory , cardiac or hepatic failure , and inadequate acoustic windows were excluded . patients who required intubation because of respiratory insufficiency , and those who required more than 3 days in a neurology intensive care unit were also excluded . from those , 40 ( aged between 18 and 55 years ) who were admitted to our emergency department and hospitalized in the neurology intensive care unit with a diagnosis of acute ischemic stroke , and a control group comprised of 40 age- and sex - matched healthy individuals , participants in the control group underwent a complete routine clinical and cardiac laboratory evaluation for the detection of occult cardiac disease , including 12-lead ecg , transthoracic / transesophageal echocardiography ( tte / tee ) , extra cranial arteries duplex sonography , and single ecg-24-hour holter ecg monitoring before inclusion in the study . duplex sonography of the extra cranial and intracranial arteries , a single ecg-24-hour holter ecg monitoring , and tte / tee were performed in patients within a median of 3 days after the acute stroke . tissue doppler measurements were obtained at end - expiration with the sample volume placed on the atrial side of the mitral annulus at the basal inter - atrial septum in the apical four - chamber view . both early diastolic ( e ) , and late diastolic ( a ) the e / e ratio was also calculated to provide a reliable index of the lv filling pressure . at end - systole , just before the opening of the mitral valve ( at the end of the t wave on the ecg ) , the la maximum volume was indexed to body surface area ( lavimax ) . at end - diastole , just before mitral valve closure ( at the beginning of the qrs complex on the ecg ) , minimum la volume to body surface area ( lavimin ) were measured and indexed to body surface area , as previously described . segments for which adequate tracking quality could not be obtained despite manual adjustment , were excluded from analysis , and patients in whom some segments were excluded due to the impossibility of adequate tracking , la deformation parameters were calculated by averaging the values measured in the remaining segments . la peak longitudinal strain and strain rate at basal segment from the apical views of the inter - atrial septum , la lateral wall , la anterior wall , and la inferior wall in the 5 3 mm region of interest were calculated as described in current guidelines . la reservoir strain during systole was obtained at the time of aortic valve closure , and la contractile strain rate during late diastole was obtained at the onset of the p wave on ecg . the reproducibility and feasibility of speckle tracking echocardiography measurement of la longitudinal strain and strain rates were considered acceptable . all standard 12-lead ecgs were obtained simultaneously at a 50-mm / s - paper speed and amplitude of 10 mm / mv . two cardiologists blind to the clinical status of the patients performed the p wave analysis with calipers and magnifying glasses to decrease the risk of measurement errors . the onset of the p wave was defined as the point in which the wave showed the first visible upward and downward departure from baseline for positive and negative waveforms , respectively . a return to the baseline maximum p wave duration ( pmax ) , measured from any of the 12 leads of the surface ecg , was used for the longest atrial conduction time . the difference between the maximum and minimum p wave duration was defined as pwd ( pwd = pmax - pmin ) . no significant differences with regard to age , gender , body mass index , body surface area , heart rate , blood pressure , left atrial minimal volume index , and left ventricular ejection fraction , were observed between the two groups . the pwd was 30.1 7.0 ms and 27.4 3.5 ms in the cs and control group , respectively ( p= 0.02 ) , whereas the la maximum volume index [ lavimax ] was 20.4 4.5 ml / m and 19.9 2.4 ml / m in the cs and control group , respectively ( p= 0.04 ) . the global peak la strain was [ pla - s ] ( la reservoir function ) 41.4 6.3% and 44.5 7.1% in the cs and control group , respectively ( p= 0.04 ) , whereas the global peak late diastolic strain rate values [ plasra ] ( la pump function ) were 2.5 0.4% and 2.9 0.5% in the cs and control group , respectively ( p= 0.001 ) . a strong negative correlation between global pla - s and lavimax ( r= -0.49 ; p < 0.01 ) , as well as a moderate correlation between pwd and global pla - s ( r= -0.52 ; p < 0.01 ) , was found in cs patients ( figures 1 , 2 ) . although there was no significant difference in left atrial tissue doppler velocities between patients and healthy controls , global pla - s was found to be significantly lower in cs patients ( table 2 ) . a significant difference was found in the pattern of la segment changes in cs patients ( table 2 and figure 3 ) . the la reservoir function ( la strain ) was smaller in cs patients than in healthy controls . the inter - atrial septal and inferior wall early diastolic strain rates ( la conduit function ) and the anterior late diastolic strain rate ( la pump function ) were significantly lower in cs patients than in healthy controls , whereas the interatrial septal late diastolic strain rate tended to be lower but the difference was not statistically significant . in this study we have demonstrated that electrical remodeling , shown by an increase in pwd on surface ecg , was associated with structural remodeling and decreased la deformation shown on echocardiography . these findings suggest that la structural , functional , and electrical impairments in conjunction with other risk factors i.e. pwd was increased in cs patients mainly due to an increase in maximum p wave values rather than a decrease in minimum p wave durations on a 12-lead ecg , and it was associated with an increased risk of af development . although , the increase in pwd and structural changes observed in la may explain the occurrence of cs , whether these changes are an underlying cause or an associated finding in the early post - stroke period ( within 1 week ) in cs patients , remains unclear [ 18 - 20 ] . furthermore , af results in la remodeling through a process that involves the deposition of fibrotic tissue , leading to changes in the electrophysiological properties of the la substrate . increases in pwd and pmax are important indicators of subsequent paf attacks and a tendency towards persistent atrial fibrillation . furthermore , an association between incremental la enlargement and an increased risk of af during follow - up has been suggested , with the risk of developing af being four times greater in patients with enlarged la . revealed that la active pump function was significantly depressed , and closely correlated with left atrial appendage emptying in cs patients with asa alone . to our knowledge , la structure and function in cs patients without inter - atrial septal abnormalities have not been thoroughly investigated . the present study provides new insights into la remodeling in patients with cs , demonstrating that global pla - s , a newer parameter of atrial function , is more sensitive than the traditional echocardiographic markers of la size and function used to detect la remodeling . routine measurement of la strain might guarantee the detection of la remodeling , and provide useful additional information for cs diagnosis . however , as this novel technique has insufficient resolution to measure the radial strain of the thin - walled la , la deformation assessment is only based on longitudinal strain . longitudinal strain is positive during the reservoir period , in which la myocardial fibers relax and stretch to adapt to the incoming blood flow , negative during the pump and most of the conduit phase , during which the la is emptying , and flat during diastasis , corresponding to the late phase of the conduit phase . furthermore , global pla - s has considerably high diagnostic accuracy while the e / e ratio correlates poorly with invasively measured la pressure . similarly , lower strain and strain rate were associated with a higher rate of af reoccurrence after catheter ablation , and an improvement in la strain correlated with a reversion of la remodeling in patients who underwent catheter ablation for af . recently demonstrated that la deformation parameters measured by speckle tracking were found to predict impaired la appendage ( laa ) functions and the presence of laa thrombus in ischemic stroke patients with suspected cardioembolism in sinus rhythm . in that study , la reservoir and pump functions were found to be significantly lower in patients with laa thrombus , and the la reservoir function showed the strongest correlation with laa morphologic parameters . recently claimed that measurement of pla - s in patients with cs might be a useful tool to detect patients with occult paf . furthermore , they concluded that pla - s analysis may play a role in the selection of patients with normal size la for prolonged cardiac monitorization , because most patients with paf showed low las in their study . since aging is associated with atrial fibrosis and atrial arrhythmias , we have excluded an aging effect on left atrial anatomy and function in cs patients in this study . la structural remodeling , as demonstrated by the histopathology findings , significantly influences global pla - s . however , global pla - s measured by speckle tracking echocardiography was negatively correlated with la myocardial fibrosis grade , while poorer correlations with the la indexed volume , la ejection fraction , and e / e ratio have been shown . as global pla - s is also correlated with endocardial thickening , a histologic alteration appearing in the earlier stages of structural remodeling , it can be used to assess an increase in interstitial fibrosis in conditions which compromise the elastic properties of the atrial myocardium , inevitably leading to impairment of atrial compliance and reduction of la reservoir function . therefore , their results are not representative of all the underlying pathophysiologic mechanisms operating in the development of ischemic events in cs patients with no inter - atrial septal abnormalities . furthermore , in order to eliminate possible etiologies associated with paradoxical embolism and to prevent misleading results , we only included cs patients without pfo or asa on tee . although the effect of various drugs , including anti - thrombotic drugs , angiotensin converting enzyme inhibitors or angiotensin receptor blockers , and statins , on la function or plasma biomarkers could not be fully controlled , as not all subjects were receiving the same drug therapies , there were no significant differences in medication regimens between the two study groups . some cardiac abnormalities that can not be detected by routine diagnostic methods may be a cause of af or paf and subsequent cs . although the effect of various drugs , including anti - thrombotic drugs , angiotensin converting enzyme inhibitors or angiotensin receptor blockers , and statins , on la function or plasma biomarkers could not be fully controlled , as not all subjects were receiving the same drug therapies , there were no significant differences in medication regimens between the two study groups . some cardiac abnormalities that can not be detected by routine diagnostic methods may be a cause of af or paf and subsequent cs . increased pwd on surface ecg is associated with left atrial mechanical remodeling and dysfunction , both of which may be substrates for thromboembolism in cs patients .

arnt - floxed ( arnt ) ( 9 ) and hif1-floxed ( hif1 ) ( 10 ) mice containing loxp sites flanking exons 6 and 1315 , of the arnt and hif1 genes , respectively , were crossed with mice harboring the cre recombinase under control of the ap2 promoter ( ap2-cre mice ) . all mice were on the c57bl/6 background , and only male mice were used for experiments . primers used to assess recombination and routine genotyping for the arnt and hif1 allele are listed in supplementary table 2 . male mice were housed in temperature- and light - controlled rooms and supplied with water and pelleted nih-31 chow diet ( 10% kcal consisting of fat ) ad libitum . in the hfd study , 6-week - old male mice were given an hfd ( 60% kcal consisting of fat ; bioserv , frenchtown , nj ) for 12 weeks . all animal studies were performed in accordance with institute of laboratory animal resources guidelines and approved by the national cancer institute animal care and use committee . for glucose tolerance test ( gtt ) , mice were fasted for 16 h , blood was drawn , and mice were injected intraperitoneally with 1 g / kg glucose . for insulin tolerance test ( itt ) , mice were fasted for 4 h , blood was drawn , and then mice were injected intraperitoneally with 1 unit / kg body wt insulin ( humulin r ; eli lilly , indianapolis , in ) . hyperinsulinemic - euglycemic clamps were performed in awake mice fasted for 12 h as previously described ( 11 ) with modifications . primed - continuous infusion of [ 3 - 3h]glucose was used : 2.5 ci bolus , 0.05 ci / min during the basal state and 0.1 ci / min during the clamp period . insulin ( humulin r ) was infused as a bolus of 18 mu / kg over a period of 3 min , followed by continuous insulin infusion at the rate of 3.5 mu / kg lean mass / min ( in hif1 mice ) and 9.4 m / kg lean mass / min ( in hif1 mice ) to raise plasma insulin concentration to 4 ng / ml . mice were fasted overnight , and then injected via the vena cava with 5 units of humulin r under anesthesia . liver , quadriceps , and white adipose tissue were collected after 5 min and stored at 80c until use . total akt and phospho - akt ( ser473 ) antibodies were from cell signaling technologies ( danvers , ma ) . body composition was measured in nonanesthetized mice using an echo3-in-1 nuclear magnetic resonance ( nmr ) analyzer ( echo medical systems , houston , tx ) . cumulative food intake was measured in 6- to 8-week - old male mice maintained on regular chow for 2 weeks and 10- to 12-week - old mice fed an hfd for 46 weeks . mice were housed individually in their home cages a week prior to recording food intake . metabolic efficiency was calculated as the ratio of weight gain to energy consumed during a 2-week period . total and resting metabolic rates were measured by indirect calorimetry using the oxymax system ( columbus instruments , columbus , oh ) ( 12 ) . mice had free access to food and water during the measurements and were allowed to adapt to metabolic cages for 24 h prior to data collection . following an adaptation period , data were recorded for 24 h at 24 and 30c for an additional 24 h. four mutant and four control mice were tested at the same time , and each mouse was tested every 20 min . motor activities were measured by an infrared beam interruption ( opto - varimex mini , columbus instruments ) , and resting was defined as time points with ambulation equal to zero . diet - induced thermogenesis and 3 adrenergic thermogenesis were measured as previously described ( 12,13 ) . fasted serum insulin was measured by use of an elisa kit ( crystal chem ) . fasted serum cholesterol , free fatty acids ( ffas ) , and triglycerides were measured using reagents from wako . quantitative pcr ( qpcr ) reactions were carried out on an abi prism 7900ht sequence detection system ( applied biosystems ) . the membranes were incubated with antibodies against total akt , phospho - akt ( ser473 ) , socs3 , total stat3 , phospho - stat3 ( cell signaling technologies , danvers , ma ) , and arnt and histone h ( santa cruz biotechnology , santa cruz , ca ) . the signals obtained were normalized to -actin ( millipore corp , temecula , ca ) for whole cell extracts . adipocytes and macrophage of stromal vascular fraction ( svf - m ) were isolated from epididymal white adipose tissue ( wat ) as previously described ( 14 ) . dye - coupled cdnas were purified with a minielute pcr purification kit ( qiagen ) and hybridized to an agilent 44 k mouse 60-mer oligo microarray ( agilent technologies ) . the procedures were repeated for replicate experiments with independent hybridization and processing and the data processed and analyzed by genespring gx software ( agilent technologies ) . paraffin - embedded tissue sections were stained with hematoxylin - eosin ( h - e ) using a standard protocol . quantification of adipocyte area was done on h - e stained sections using imagetool software . differences between groups were examined for statistical significance with student t test . a p value of < 0.05 was considered statistically significant . for glucose tolerance test ( gtt ) , mice were fasted for 16 h , blood was drawn , and mice were injected intraperitoneally with 1 g / kg glucose . for insulin tolerance test ( itt ) , mice were fasted for 4 h , blood was drawn , and then mice were injected intraperitoneally with 1 unit / kg body wt insulin ( humulin r ; eli lilly , indianapolis , in ) . hyperinsulinemic - euglycemic clamps were performed in awake mice fasted for 12 h as previously described ( 11 ) with modifications . primed - continuous infusion of [ 3 - 3h]glucose was used : 2.5 ci bolus , 0.05 ci / min during the basal state and 0.1 ci / min during the clamp period . insulin ( humulin r ) was infused as a bolus of 18 mu / kg over a period of 3 min , followed by continuous insulin infusion at the rate of 3.5 mu / kg lean mass / min ( in hif1 mice ) and 9.4 m / kg lean mass / min ( in hif1 mice ) to raise plasma insulin concentration to 4 ng / ml . mice were fasted overnight , and then injected via the vena cava with 5 units of humulin r under anesthesia . liver , quadriceps , and white adipose tissue were collected after 5 min and stored at 80c until use . total akt and phospho - akt ( ser473 ) antibodies were from cell signaling technologies ( danvers , ma ) . body composition was measured in nonanesthetized mice using an echo3-in-1 nuclear magnetic resonance ( nmr ) analyzer ( echo medical systems , houston , tx ) . cumulative food intake was measured in 6- to 8-week - old male mice maintained on regular chow for 2 weeks and 10- to 12-week - old mice fed an hfd for 46 weeks . mice were housed individually in their home cages a week prior to recording food intake . metabolic efficiency was calculated as the ratio of weight gain to energy consumed during a 2-week period . total and resting metabolic rates were measured by indirect calorimetry using the oxymax system ( columbus instruments , columbus , oh ) ( 12 ) . mice had free access to food and water during the measurements and were allowed to adapt to metabolic cages for 24 h prior to data collection . following an adaptation period , data were recorded for 24 h at 24 and 30c for an additional 24 h. four mutant and four control mice were tested at the same time , and each mouse was tested every 20 min . motor activities were measured by an infrared beam interruption ( opto - varimex mini , columbus instruments ) , and resting was defined as time points with ambulation equal to zero . diet - induced thermogenesis and 3 adrenergic thermogenesis were measured as previously described ( 12,13 ) . . fasted serum cholesterol , free fatty acids ( ffas ) , and triglycerides were measured using reagents from wako . quantitative pcr ( qpcr ) reactions were carried out on an abi prism 7900ht sequence detection system ( applied biosystems ) . the membranes were incubated with antibodies against total akt , phospho - akt ( ser473 ) , socs3 , total stat3 , phospho - stat3 ( cell signaling technologies , danvers , ma ) , and arnt and histone h ( santa cruz biotechnology , santa cruz , ca ) . the signals obtained were normalized to -actin ( millipore corp , temecula , ca ) for whole cell extracts . adipocytes and macrophage of stromal vascular fraction ( svf - m ) were isolated from epididymal white adipose tissue ( wat ) as previously described ( 14 ) . dye - coupled cdnas were purified with a minielute pcr purification kit ( qiagen ) and hybridized to an agilent 44 k mouse 60-mer oligo microarray ( agilent technologies ) . the procedures were repeated for replicate experiments with independent hybridization and processing and the data processed and analyzed by genespring gx software ( agilent technologies ) . paraffin - embedded tissue sections were stained with hematoxylin - eosin ( h - e ) using a standard protocol . quantification of adipocyte area was done on h - e stained sections using imagetool software . differences between groups were examined for statistical significance with student t test . a p value of < 0.05 was considered statistically significant . for examination of the role of hif transcription factors in obesity and insulin resistance , arnt and hif1 genes were disrupted in adipocytes . to estimate the extent of cell - specific disruption of the arnt and hif1 loci , pcr analysis was used . a pcr amplicon for the arnt - null allele amplified as a 340 base pair product and was detected in genomic dna isolated from adipocytes or adipose tissue of arnt mice and not in adipose dna isolated from arnt mice . in contrast , the floxed allele was the only band detected in adipose tissue from arnt mice and from all nonadipose tissues in arnt mice ( fig . the hif1-null amplicon amplified as a 355 base pair product was detected in genomic dna isolated from adipose tissue of hif1 mice , and was not detected in adipose tissue dna isolated from hif1 mice . the null allele was only detected in adipose tissue and not in liver , skeletal muscle , spleen , or kidney from hif1 mice ( fig . the expression of arnt mrna was specifically decreased in wat and brown adipose tissue ( bat ) by 50% in the arnt mice compared with arnt mice ; no decrease was evident from liver or skeletal muscle . in addition , qpcr showed nearly absent expression of arnt mrna in the adipocytes of arnt mice ( fig . similar results were obtained from tissues of hif1 mice , where an ~88% decrease in hif1 mrna from adipocytes was observed ( fig . to confirm that loss of arnt was of functional significance , the extent of activation of the arnt - dependent aryl hydrocarbon receptor ( ahr ) pathway upon 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) challenge was determined . induction of the ahr target gene cyp1a1 was markedly attenuated in wat and bat of arnt mice ( supplementary fig . however , no significant difference in the extent of induction of cyp1a1 mrna was noted in liver or skeletal muscle compared with arnt mice ( supplementary fig . these results demonstrate adipocyte - specific knockout of the arnt and hif1 genes in mice . adipocyte - specific disruption of the arnt and hif1a genes via cre - loxp mediated recombination . a and b : pcr diagnostic for ap2-cre mediated recombination of the arnt or hif1 allele in genomic dna isolated from adipocytes or tissue of arnt and arnt or hif1 and hif1 mice . c and d : qpcr analysis of arnt mrna expression in the tissues ( left panel ) or adipocytes ( right panel ) from arnt and arnt or hif1 and hif1 mice . * p < 0.05 , * * p < 0.01 compared with floxed littermates . to explore the role of hif1 in fat metabolism and glucose homeostasis , male mice were fed either a chow diet or hfd . when fed a chow diet , arnt and hif1 mice grew at a rate similar to that of arnt and hif1 mice , respectively . however , 12 weeks of hfd led to weight gain in arnt and hif1 mice , while arnt and hif1 mice were resistant to the hfd - induced weight gain ( supplementary fig . nmr measurements confirmed that the body fat mass and the ratio of fat and body mass of arnt and hif1 mice fed an hfd were decreased compared with arnt and hif1 mice , respectively ( supplementary fig . the adipocyte size in arnt and hif1 mice was significantly decreased compared with arnt and hif1 mice , respectively , after 12 weeks of hfd ( supplementary fig . 2c ; fig . 2c ) . to explore the mechanism of reduced adiposity in arnt and hif1 mice , cumulative food intake , metabolic efficiency , and metabolic rates were measured in young mice maintained on chow diet and in mice fed an hfd for 47 weeks , which is before the difference between control and mutant mice became apparent . there were no significant differences in weight , cumulative food intake , or metabolic efficiency between hif1 and hif1 mice maintained on chow diet ( supplementary fig . indirect calorimetry performed at 24c and thermoneutrality ( 30c ) on the same set of mice did not reveal significant differences in metabolic rate , respiratory exchange ratio ( rer ) , or activity between hif1 and hif1 mice fed chow diet ( supplementary fig . a short 4-day exposure to hfd caused comparable increases in body weight and resting metabolic rate in hif1 and hif1 mice , indicating that adipose - specific inactivation of hif1 did not alter the acute thermogenic response to hfd ( supplementary fig . these results indicate that it takes a long time for the adipose tissue to get big enough to be hypoxic and that hif1 expression was significantly induced , resulting in activation of hif1 target genes such as vegf and glut1 ( supplementary fig . after 46 weeks on an hfd , the cumulative food intake remained similar between hif1 and hif1 mice ; however , the metabolic efficiency was lower in the hif1 mice , suggesting an increase in the metabolic rate ( fig . indirect calorimetry performed at 24c on week 7 of hfd did not reveal a significant difference in resting or total oxygen consumption between the genotypes , while at 30c these parameters tended to be higher in hif1 mice compared with controls ( supplementary fig . hif1 mice had reduced resting and total rer at 30c during resting phase ( daytime ) and active phase ( nighttime ) , suggesting increased fatty acid oxidation ( fig . all of these changes in hif1 mice could contribute to the decreased metabolic efficiency and weight gain on an hfd compared with hif1 mice . however , it is unlikely that resistance to hfd was caused by activated bat thermogenesis because the response to mild cold measured as the difference in metabolic rate at thermoneutral and room temperature and the response to a maximal dose of the 3-adrenergic agonist cl316243 , which specifically stimulates bat thermogenesis , were similar in both strains ( supplementary fig . disruption of hif1 protected mice from hfd - induced obesity . a : typical growth curves of hif1 and hif1 mice maintained on chow diet ( left panel ) or hfd ( right panel ) ( n = 68/group ) . b : body composition by nmr to show the fat mass and fat mass ratio in hif1 and hif1 mice after 12 weeks of hfd ( n = 5/group ) . c : representative h - e stained wat sections and quantification of adipocyte size from hif1 and hif1 mice after 12 weeks of hfd ( n = 5/group ) . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . ( a high - quality digital representation of this figure is available in the online issue . ) energy balance in hif1 and hif1 mice . a and b : cumulative food intake and metabolic efficiency for 2 weeks in hif1 and hif1 mice after 46 weeks of hfd . indirect calorimetry ( c f ) was performed after 7 weeks of hfd at 24c and 30c ( thermoneutrality ) during resting phase ( daytime [ d ] ) and active phase ( nighttime [ n ] ) ( n = 68/group ) . . to explore the role of adipocyte hif1 deficiency in obesity - induced insulin resistance , gtts and itts were performed . when mice were fed a chow diet , there were no significant differences in gtt and itt between arnt and arnt mice ( supplementary fig . however , gtt revealed that after 11 weeks of hfd challenge , arnt and hif1 mice displayed significantly reduced blood glucose compared with arnt and hif1 mice after glucose loading ( supplementary fig . itt showed a significant improvement in insulin sensitivity by adipose hif1 disruption ( supplementary fig . 4c ) . moreover , fed glucose , fasted glucose , and fasted serum insulin levels were significantly lower in arnt and hif1 mice compared with arnt and hif1 mice , respectively , after 12 weeks of hfd . the calculated homeostasis model assessment ( homa ) measure of insulin resistance was significantly decreased in arnt and hif1 mice ( table 1 ) . arnt and hif1 mice also had reduced fasted serum triglycerides and ffa levels consistent with improved glucose tolerance and insulin sensitivity in these mice ( table 1 ) . although body mass was similar between hif1 and hif1 mice after 4 weeks , 6 weeks , and 8 weeks of hfd , gtt and itt revealed that glucose tolerance and insulin sensitivity were improved in hif1 mice from 4 weeks on an hfd ( supplementary fig . after 6 weeks of hfd challenge , fasted glucose was similar while fasted insulin levels and homa index in hif1 mice were significantly decreased ( supplementary fig . these results indicated that hif1 disruption in adipocytes improved hfd - induced glucose tolerance and insulin resistance before the onset of the decrease in body mass . a and b : blood glucose levels and serum insulin levels in 2-h gtt 12 weeks after hfd ( n = 68/group ) . inset graphs in a and b depict the respective analysis of the area under the curve ( auc ) . e and f : plasma glucose and insulin levels in the basal state and during the clamp . h : basal and clamp endogenous glucose production ( egp ) , gir , whole - body glucose disposal ( rd ) , and glucose uptake in skeletal muscle , wat , and bat . the hyperinsulinemic - euglycemic clamp ( d h ) was performed after 15 weeks of hfd ( n = 6/group ) . metabolic parameters after 12 weeks of hfd p < 0.01 compared with controls . a hyperinsulinemic - euglycemic clamp was performed in hif1 and hif1 mice after 15 weeks of hfd to further characterize in vivo insulin action . in the basal state , hif1 mice had significantly reduced plasma glucose and insulin levels ; basal endogenous glucose production ( egp ) was similar between genotypes ( fig . insulin was infused to maintain plasma insulin levels at 4 ng / ml ( fig . 4f ) , and the glucose infusion rate ( gir ) was adjusted in order to maintain blood glucose levels in hif1 and hif1 mice at similar levels ( fig . 4 g and h ) , which confirmed improved whole - body insulin sensitivity in hif1 mice . during the clamp , insulin induced a more marked suppression of egp in hif1 mice , suggesting increased insulin sensitivity in the liver . whole - body glucose disposal ( rd ) and glucose uptake into skeletal muscle and adipose tissue were significantly increased in hif1 mice ( fig . , these data suggest that adipose selective inactivation of hif1 caused increased insulin sensitivity in major insulin target tissues , liver , skeletal muscle , and fat . insulin action was further investigated in wat , liver , and skeletal muscle ( 15 ) . both arnt and hif1 deficiency in adipocytes improved insulin signaling pathways in wat , liver , and skeletal muscle , as revealed by increased phosphorylation of akt ( ser473 ) ( fig . these findings indicated that hif1 deficiency in adipocytes improved hfd - induced glucose tolerance and insulin resistance , which is in support of the hyperinsulinemic - euglycemic clamp studies . insulin - stimulated akt phosphorylation ( ser473 ) in wat ( a ) , liver ( b ) , and skeletal muscle ( c ) of arnt and arnt and hif1 and hif1 mice . after 12 weeks of hfd , hif1 was significantly elevated , resulting in activation of target genes such as glut1 and vegf ( supplementary fig . hif1 target genes and adiponectin expression were similar between hif1a and hif1 mice ( supplementary fig . in addition , there was no significant nuclear hif1 protein expression in wat from chow - fed wild - type mice , while it was significantly induced in hfd - fed mice and diminished in hif1 mice ( supplementary fig . thus , the phenotype of hif1 mice is similar to hif1a mice on a chow diet but different on an hfd . in contrast to hif1 protein , hfd treatment did not elevate hif2 protein expression and there was no increase in hif1 mice ( supplementary fig . gene expression profiling of wat after 12 weeks of hfd in arnt and arnt mice or hif1 and hif1 mice showed that the hif1 target genes glut1 and vegf were decreased in arnt and hif1 mice ( supplementary table 1 ; supplementary fig . expression of genes involved in adipogenesis and glucose metabolism , pparg , c / ebp , cfd , and glut4 , were upregulated in wat of arnt and hif1 mice compared with arnt and hif1 mice , respectively , on an hfd . importantly , total serum adiponectin , the high molecular weight ( hmw ) form of adiponectin , and the ratio of hmw to total adiponectin were increased in arnt and hif1 mice on a 12-week hfd ( supplementary fig . hmw adiponectin and ratio of hmw to total adiponectin began to be increased following 6 weeks of hfd before the decrease in body weight ( supplementary fig . previous studies showed that overexpression of a constitutively active form of hif1 initiates adipose tissue fibrosis and insulin resistance ( 16 ) . in agreement with this finding , expression of the fibrosis related genes lox , col1a1 , col3a1 , and loxl1 were decreased in arnt or hif1 mice ( supplementary table 1 ; supplementary fig . expression of the inflammation - related genes tnf and pai-1 was downregulated in arnt and hif1 mice ( supplementary table 1 ; supplementary fig . 7a ; fig . macrophage marker f4/80 staining of adipose tissue sections revealed that macrophage infiltration to wat decreased significantly in arnt and hif1 mice . expression of mrnas encoding the macrophage markers f4/80 and cd68 were also decreased in arnt mice and hif1 mice ( supplementary table 1 ; supplementary fig . 8a and b ) . because it was reported that the ap2 promoter is expressed in macrophage ( 17 ) , the possibility that hif1 was disrupted in macrophage was investigated by determining arnt gene disruption in peritoneal macrophage ( p - m ) from arnt mice . loss of arnt in peritoneal macrophage of arnt mice was minimal in contrast to much lower arnt mrna levels in arnt mouse adipocytes ( fig . b : total adiponectin levels , hmw adiponectin , and the ratio of hmw to total adiponectin . e : western blot analysis of socs3 expression and stat3 phosphorylation ( left panel ) and quantitation of socs3 expression and stat3 phosphorylation ( right panel ) . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . a : qpcr analysis of arnt mrna expression in the peritoneal macrophages ( p - m ) ( b ) and qpcr analysis of hif1 , socs3 , and adiponectin mrna expression in adipocytes and svf - m ( c ) of hif1 and hif1 mice after 12 weeks of hfd . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . previous studies demonstrated that socs3 mrna is elevated in wat of diet - induced obesity ( dio ) mice ( 18,19 ) . further , haploinsufficiency of socs3 significantly protected mice against the development of dio and associated metabolic complications ( 20 ) . consistent with these findings , socs3 mrna was also found to be downregulated in arnt and hif1 mice , as revealed by microarray analysis ( supplementary table 1 ) . expression of socs3 mrna in arnt and hif1 mice was significantly lower than that in arnt and hif1 mice , respectively ( supplementary fig . adipocytes and macrophage of stromal vascular fraction ( svf - m ) were prepared from wat of hif1 and hif1 mice after 12 weeks of hfd . in adipocytes , no significant expression of hif1 mrna in hif1 mice was found . after 12 weeks of hfd , expression of socs3 in adipocytes of hif1 mice was ~80% decreased compared with that in hif1 mice . consistent with data obtained in p - m , the expression of hif1 , socs3 , and adiponectin ( adiponectin expression being nearly undetectable in svf - m ) in svf - m were not significantly different between hif1 and hif1 mice after 12 weeks of hfd ( fig . previous studies revealed that socs3 can inhibit adiponectin production via jak2-stat3dependent mechanisms in adipocytes ( 21 ) . since inactivation of stat3 may be involved in the inhibition of adiponectin production by socs3 , tyrosine phosphorylation of stat3 was assessed in wat from hif1 and hif1 mice . indeed , tyrosine phosphorylation of stat3 was significantly induced in parallel with increased adiponectin level in hif1 mice ( fig . these results suggest that the socs3 and adiponectin pathway is involved , in part , in the improvement of hfd - induced insulin resistance in hif1 mice . for examination of the role of hif transcription factors in obesity and insulin resistance , arnt and hif1 genes were disrupted in adipocytes . to estimate the extent of cell - specific disruption of the arnt and hif1 loci , pcr analysis was used . a pcr amplicon for the arnt - null allele amplified as a 340 base pair product and was detected in genomic dna isolated from adipocytes or adipose tissue of arnt mice and not in adipose dna isolated from arnt mice . in contrast , the floxed allele was the only band detected in adipose tissue from arnt mice and from all nonadipose tissues in arnt mice ( fig . the hif1-null amplicon amplified as a 355 base pair product was detected in genomic dna isolated from adipose tissue of hif1 mice , and was not detected in adipose tissue dna isolated from hif1 mice . the null allele was only detected in adipose tissue and not in liver , skeletal muscle , spleen , or kidney from hif1 mice ( fig . the expression of arnt mrna was specifically decreased in wat and brown adipose tissue ( bat ) by 50% in the arnt mice compared with arnt mice ; no decrease was evident from liver or skeletal muscle . in addition , qpcr showed nearly absent expression of arnt mrna in the adipocytes of arnt mice ( fig . similar results were obtained from tissues of hif1 mice , where an ~88% decrease in hif1 mrna from adipocytes was observed ( fig . to confirm that loss of arnt was of functional significance , the extent of activation of the arnt - dependent aryl hydrocarbon receptor ( ahr ) pathway upon 2,3,7,8-tetrachlorodibenzo - p - dioxin ( tcdd ) challenge was determined . induction of the ahr target gene cyp1a1 was markedly attenuated in wat and bat of arnt mice ( supplementary fig . however , no significant difference in the extent of induction of cyp1a1 mrna was noted in liver or skeletal muscle compared with arnt mice ( supplementary fig . these results demonstrate adipocyte - specific knockout of the arnt and hif1 genes in mice . adipocyte - specific disruption of the arnt and hif1a genes via cre - loxp mediated recombination . a and b : pcr diagnostic for ap2-cre mediated recombination of the arnt or hif1 allele in genomic dna isolated from adipocytes or tissue of arnt and arnt or hif1 and hif1 mice . c and d : qpcr analysis of arnt mrna expression in the tissues ( left panel ) or adipocytes ( right panel ) from arnt and arnt or hif1 and hif1 mice . to explore the role of hif1 in fat metabolism and glucose homeostasis , male mice were fed either a chow diet or hfd . when fed a chow diet , arnt and hif1 mice grew at a rate similar to that of arnt and hif1 mice , respectively . however , 12 weeks of hfd led to weight gain in arnt and hif1 mice , while arnt and hif1 mice were resistant to the hfd - induced weight gain ( supplementary fig . nmr measurements confirmed that the body fat mass and the ratio of fat and body mass of arnt and hif1 mice fed an hfd were decreased compared with arnt and hif1 mice , respectively ( supplementary fig . the adipocyte size in arnt and hif1 mice was significantly decreased compared with arnt and hif1 mice , respectively , after 12 weeks of hfd ( supplementary fig . 2c ; fig . 2c ) . to explore the mechanism of reduced adiposity in arnt and hif1 mice , cumulative food intake , metabolic efficiency , and metabolic rates were measured in young mice maintained on chow diet and in mice fed an hfd for 47 weeks , which is before the difference between control and mutant mice became apparent . there were no significant differences in weight , cumulative food intake , or metabolic efficiency between hif1 and hif1 mice maintained on chow diet ( supplementary fig . indirect calorimetry performed at 24c and thermoneutrality ( 30c ) on the same set of mice did not reveal significant differences in metabolic rate , respiratory exchange ratio ( rer ) , or activity between hif1 and hif1 mice fed chow diet ( supplementary fig . a short 4-day exposure to hfd caused comparable increases in body weight and resting metabolic rate in hif1 and hif1 mice , indicating that adipose - specific inactivation of hif1 did not alter the acute thermogenic response to hfd ( supplementary fig . these results indicate that it takes a long time for the adipose tissue to get big enough to be hypoxic and that hif1 expression was significantly induced , resulting in activation of hif1 target genes such as vegf and glut1 ( supplementary fig . after 46 weeks on an hfd , the cumulative food intake remained similar between hif1 and hif1 mice ; however , the metabolic efficiency was lower in the hif1 mice , suggesting an increase in the metabolic rate ( fig . indirect calorimetry performed at 24c on week 7 of hfd did not reveal a significant difference in resting or total oxygen consumption between the genotypes , while at 30c these parameters tended to be higher in hif1 mice compared with controls ( supplementary fig . hif1 mice had reduced resting and total rer at 30c during resting phase ( daytime ) and active phase ( nighttime ) , suggesting increased fatty acid oxidation ( fig . all of these changes in hif1 mice could contribute to the decreased metabolic efficiency and weight gain on an hfd compared with hif1 mice . however , it is unlikely that resistance to hfd was caused by activated bat thermogenesis because the response to mild cold measured as the difference in metabolic rate at thermoneutral and room temperature and the response to a maximal dose of the 3-adrenergic agonist cl316243 , which specifically stimulates bat thermogenesis , were similar in both strains ( supplementary fig . 3h ) . disruption of hif1 protected mice from hfd - induced obesity . a : typical growth curves of hif1 and hif1 mice maintained on chow diet ( left panel ) or hfd ( right panel ) ( n = 68/group ) . b : body composition by nmr to show the fat mass and fat mass ratio in hif1 and hif1 mice after 12 weeks of hfd ( n = 5/group ) . c : representative h - e stained wat sections and quantification of adipocyte size from hif1 and hif1 mice after 12 weeks of hfd ( n = 5/group ) . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . ( a high - quality digital representation of this figure is available in the online issue . ) a and b : cumulative food intake and metabolic efficiency for 2 weeks in hif1 and hif1 mice after 46 weeks of hfd . indirect calorimetry ( c f ) was performed after 7 weeks of hfd at 24c and 30c ( thermoneutrality ) during resting phase ( daytime [ d ] ) and active phase ( nighttime [ n ] ) ( n = 68/group ) . to explore the role of adipocyte hif1 deficiency in obesity - induced insulin resistance , gtts and itts were performed . when mice were fed a chow diet , there were no significant differences in gtt and itt between arnt and arnt mice ( supplementary fig . however , gtt revealed that after 11 weeks of hfd challenge , arnt and hif1 mice displayed significantly reduced blood glucose compared with arnt and hif1 mice after glucose loading ( supplementary fig . itt showed a significant improvement in insulin sensitivity by adipose hif1 disruption ( supplementary fig . 4c ) . moreover , fed glucose , fasted glucose , and fasted serum insulin levels were significantly lower in arnt and hif1 mice compared with arnt and hif1 mice , respectively , after 12 weeks of hfd . the calculated homeostasis model assessment ( homa ) measure of insulin resistance was significantly decreased in arnt and hif1 mice ( table 1 ) . arnt and hif1 mice also had reduced fasted serum triglycerides and ffa levels consistent with improved glucose tolerance and insulin sensitivity in these mice ( table 1 ) . although body mass was similar between hif1 and hif1 mice after 4 weeks , 6 weeks , and 8 weeks of hfd , gtt and itt revealed that glucose tolerance and insulin sensitivity were improved in hif1 mice from 4 weeks on an hfd ( supplementary fig . 5a and b ) . after 6 weeks of hfd challenge , fasted glucose was similar while fasted insulin levels and homa index in hif1 mice were significantly decreased ( supplementary fig . these results indicated that hif1 disruption in adipocytes improved hfd - induced glucose tolerance and insulin resistance before the onset of the decrease in body mass . hif1 disruption in adipocytes improved hfd - induced glucose intolerance and insulin resistance . a and b : blood glucose levels and serum insulin levels in 2-h gtt 12 weeks after hfd ( inset graphs in a and b depict the respective analysis of the area under the curve ( auc ) . e and f : plasma glucose and insulin levels in the basal state and during the clamp . h : basal and clamp endogenous glucose production ( egp ) , gir , whole - body glucose disposal ( rd ) , and glucose uptake in skeletal muscle , wat , and bat . the hyperinsulinemic - euglycemic clamp ( d h ) was performed after 15 weeks of hfd ( n = 6/group ) . metabolic parameters after 12 weeks of hfd p < 0.01 compared with controls . a hyperinsulinemic - euglycemic clamp was performed in hif1 and hif1 mice after 15 weeks of hfd to further characterize in vivo insulin action . in the basal state , hif1 mice had significantly reduced plasma glucose and insulin levels ; basal endogenous glucose production ( egp ) was similar between genotypes ( fig . insulin was infused to maintain plasma insulin levels at 4 ng / ml ( fig . 4f ) , and the glucose infusion rate ( gir ) was adjusted in order to maintain blood glucose levels in hif1 and hif1 mice at similar levels ( fig . 4 g and h ) , which confirmed improved whole - body insulin sensitivity in hif1 mice . during the clamp , insulin induced a more marked suppression of egp in hif1 mice , suggesting increased insulin sensitivity in the liver . whole - body glucose disposal ( rd ) and glucose uptake into skeletal muscle and adipose tissue were significantly increased in hif1 mice ( fig . these data suggest that adipose selective inactivation of hif1 caused increased insulin sensitivity in major insulin target tissues , liver , skeletal muscle , and fat . insulin action was further investigated in wat , liver , and skeletal muscle ( 15 ) . both arnt and hif1 deficiency in adipocytes improved insulin signaling pathways in wat , liver , and skeletal muscle , as revealed by increased phosphorylation of akt ( ser473 ) ( fig . these findings indicated that hif1 deficiency in adipocytes improved hfd - induced glucose tolerance and insulin resistance , which is in support of the hyperinsulinemic - euglycemic clamp studies . insulin - stimulated akt phosphorylation ( ser473 ) in wat ( a ) , liver ( b ) , and skeletal muscle ( c ) of arnt and arnt and hif1 and hif1 mice . after 12 weeks of hfd , hif1 was significantly elevated , resulting in activation of target genes such as glut1 and vegf ( supplementary fig . hif1 target genes and adiponectin expression were similar between hif1a and hif1 mice ( supplementary fig . in addition , there was no significant nuclear hif1 protein expression in wat from chow - fed wild - type mice , while it was significantly induced in hfd - fed mice and diminished in hif1 mice ( supplementary fig . thus , the phenotype of hif1 mice is similar to hif1a mice on a chow diet but different on an hfd . in contrast to hif1 protein , hfd treatment did not elevate hif2 protein expression and there was no increase in hif1 mice ( supplementary fig . gene expression profiling of wat after 12 weeks of hfd in arnt and arnt mice or hif1 and hif1 mice showed that the hif1 target genes glut1 and vegf were decreased in arnt and hif1 mice ( supplementary table 1 ; supplementary fig . expression of genes involved in adipogenesis and glucose metabolism , pparg , c / ebp , cfd , and glut4 , were upregulated in wat of arnt and hif1 mice compared with arnt and hif1 mice , respectively , on an hfd . importantly , total serum adiponectin , the high molecular weight ( hmw ) form of adiponectin , and the ratio of hmw to total adiponectin were increased in arnt and hif1 mice on a 12-week hfd ( supplementary fig . hmw adiponectin and ratio of hmw to total adiponectin began to be increased following 6 weeks of hfd before the decrease in body weight ( supplementary fig . previous studies showed that overexpression of a constitutively active form of hif1 initiates adipose tissue fibrosis and insulin resistance ( 16 ) . in agreement with this finding , expression of the fibrosis related genes lox , col1a1 , col3a1 , and loxl1 were decreased in arnt or hif1 mice ( supplementary table 1 ; supplementary fig . expression of the inflammation - related genes tnf and pai-1 was downregulated in arnt and hif1 mice ( supplementary table 1 ; supplementary fig . 7a ; fig . macrophage marker f4/80 staining of adipose tissue sections revealed that macrophage infiltration to wat decreased significantly in arnt and hif1 mice . expression of mrnas encoding the macrophage markers f4/80 and cd68 were also decreased in arnt mice and hif1 mice ( supplementary table 1 ; supplementary fig . 8a and b ) . because it was reported that the ap2 promoter is expressed in macrophage ( 17 ) , the possibility that hif1 was disrupted in macrophage was investigated by determining arnt gene disruption in peritoneal macrophage ( p - m ) from arnt mice . loss of arnt in peritoneal macrophage of arnt mice was minimal in contrast to much lower arnt mrna levels in arnt mouse adipocytes ( fig . b : total adiponectin levels , hmw adiponectin , and the ratio of hmw to total adiponectin . e : western blot analysis of socs3 expression and stat3 phosphorylation ( left panel ) and quantitation of socs3 expression and stat3 phosphorylation ( right panel ) . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . a : qpcr analysis of arnt mrna expression in the peritoneal macrophages ( p - m ) ( b ) and qpcr analysis of hif1 , socs3 , and adiponectin mrna expression in adipocytes and svf - m ( c ) of hif1 and hif1 mice after 12 weeks of hfd . data are means sd . * p < 0.05 , * * p < 0.01 compared with floxed littermates . previous studies demonstrated that socs3 mrna is elevated in wat of diet - induced obesity ( dio ) mice ( 18,19 ) . further , haploinsufficiency of socs3 significantly protected mice against the development of dio and associated metabolic complications ( 20 ) . consistent with these findings , socs3 mrna was also found to be downregulated in arnt and hif1 mice , as revealed by microarray analysis ( supplementary table 1 ) . expression of socs3 mrna in arnt and hif1 mice was significantly lower than that in arnt and hif1 mice , respectively ( supplementary fig . adipocytes and macrophage of stromal vascular fraction ( svf - m ) were prepared from wat of hif1 and hif1 mice after 12 weeks of hfd . in adipocytes , no significant expression of hif1 mrna in hif1 mice was found . after 12 weeks of hfd , expression of socs3 in adipocytes of hif1 mice was ~80% decreased compared with that in hif1 mice . consistent with data obtained in p - m , the expression of hif1 , socs3 , and adiponectin ( adiponectin expression being nearly undetectable in svf - m ) in svf - m were not significantly different between hif1 and hif1 mice after 12 weeks of hfd ( fig . previous studies revealed that socs3 can inhibit adiponectin production via jak2-stat3dependent mechanisms in adipocytes ( 21 ) . since inactivation of stat3 may be involved in the inhibition of adiponectin production by socs3 , tyrosine phosphorylation of stat3 was assessed in wat from hif1 and hif1 mice . indeed , tyrosine phosphorylation of stat3 was significantly induced in parallel with increased adiponectin level in hif1 mice ( fig . these results suggest that the socs3 and adiponectin pathway is involved , in part , in the improvement of hfd - induced insulin resistance in hif1 mice . however , the role of hypoxia in adipose tissue during obesity and insulin resistance remains unclear . in the current study , adipocyte - specific disruption of hif1 or its heterodimerization partner , arnt , hif1 signaling may regulate socs3 and adiponectin , thus providing a mechanistic clue by which hif1 exacerbates whole - body insulin resistance . these findings indicate a central role for adipocyte hif1 signaling in the pathogenesis of obesity and insulin resistance . in the obese mouse model , hypoxia occurs specifically in wat , and the response to adipose hypoxia may lead to insulin resistance ( 23 ) . hif1 , the main mediator of the hypoxia response in adipose tissue , is almost undetectable in lean mice but significantly increased in obese mice resulting in induction of the hif1 target genes glut1 and pdk1 ( 2 ) . moreover , overexpression of a constitutively - active hif1 in adipose tissue leads to glucose intolerance and insulin resistance ( 16 ) . these studies are consistent with results of the present work showing that ablation of arnt or hif1 in adipose tissue improved hfd - induced obesity and insulin resistance . in arnt and hif1 mice , adipogenesis - related genes such as pparg , c / ebp , cfd , and glut4 were increased and adipocyte size and mass reduced after hfd challenge , consistent with the notion that enhanced adipogenesis does not correlate with obesity ( 24,25 ) . these results provide in vivo evidence that hypoxia inhibits adipogenesis in an hif1-dependent manner ( 26 ) . the reduced adiposity in arnt and hif1 mice was independent of food intake and may be due in part to increased energy expenditure . others found that in obese mice , adiponectin treatment increased energy expenditure and decreased mouse fat mass by upregulating expression of uncoupling protein 2 ( ucp2 ) and increasing fatty acid oxidation ( 2730 ) . in addition , decreased vascular endothelial growth factor ( vegf ) mrna in arnt and hif1 mice wat suggests that lower vegf as a result of loss of hif1 may also contribute to the decreased adiposity in arnt and hif1 mice . indeed , vegf inhibitors were shown to decrease adipose mass following hfd ( 31 ) . it has previously been claimed that ap2 expression is induced in activated macrophages ( 17 ) . however , in hif1 mice the knockout efficiency of hif1 in svf - m was very low consistent with recent reports showing that the efficiency of cre recombination in macrophage was much less than that in adipocytes ( 14,3234 ) . loss of hif1 in adipose tissue can improve whole - body glucose intolerance after hfd , mainly due to enhanced insulin signaling in wat , liver , and skeletal muscle . inflammatory factors such as tumor necrosis factor- , interleukin-6 , and lipopolysaccharide also inhibit insulin signaling via upregulation of socs3 ( 18,35 ) . socs3 can bind to phosphorylated tyrosine 960 of the insulin receptor ( 36 ) and inhibit insulin receptor autophosphorylation ( 37 ) , irs1 phosphorylation , and downstream insulin signaling ( 35 ) . therefore , decreased socs3 in arnt and hif1 mice may account for the improved insulin signaling in wat . the increased adiponectin expression and secretion was accompanied by a decrease of socs3 and activation of stat3 in arnt and hif1 mice . this can explain the present findings that insulin sensitivity in liver and muscle was improved in arnt and hif1 mice compared with arnt and hif1 mice . in addition , hmw adiponectin levels and the ratio of hmw to total adiponectin were increased in arnt and hif1 mice . indeed , hmw adiponectin levels , or the ratio of hmw adiponectin to total adiponectin , are more meaningful markers than total adiponectin levels for predicting insulin resistance and the development of metabolic syndrome ( 38 ) . overexpression of socs3 in adipose tissue inhibited local insulin action but improved systemic glucose metabolism , and adiponectin production was increased after hfd ( 39 ) . whereas these data seem paradoxical given the current results , others reported that socs3 is elevated in wat under the pathological conditions of insulin resistance ( 18,19 ) and when socs3 is overexpressed socs3 levels rise far higher than under normal physiological conditions . in the current study , socs3 expression was decreased in arnt and hif1 mice after hfd . in addition , consistent with the present results , a recent study showed that pioglitazone exerts its effect to improve whole - body insulin sensitivity through the suppression of socs3 , which is associated with an increase in stat3 phosphorylation and adiponectin production in wat ( 21 ) . moreover , arnt and hif1 mice also have reduced serum triglycerides and ffas , which is consistent with studies showing that ffas and triglycerides impair insulin signaling and induce insulin resistance mainly in liver and muscle ( 40,41 ) . it is also of interest that the proinflammatory factors tumor necrosis factor- and plasminogen activator inhibitor-1 were decreased in arnt and hif1 mice . these factors are also associated with improved insulin sensitivity of the whole body in arnt and hif1 mice after hfd . in conclusion , the current study clearly shows that hif1 in adipose tissue plays an important role in the metabolism of lipid and glucose . the hif1 effects on insulin sensitivity may be due in part to its direct or indirect regulation of socs3 in wat , whereas the mechanism of its effect on adiposity and obesity is still not clear and requires further investigation . because loss of hif1 activity improves metabolic function , compounds that inhibit hif1 function in adipose tissue might have significant therapeutic potential in reducing obesity and insulin resistance .

objectiveobesity , insulin resistance , and type 2 diabetes form a tightly correlated cluster of metabolic disorders in which adipose is one of the first affected tissues . the role of hypoxia and hypoxia - inducible factor 1 ( hif1 ) in the development of high - fat diet ( hfd)induced obesity and insulin resistance was investigated using animal models.research design and methodsmice with adipocyte - specific targeted disruption of the genes encoding the hif1 obligatory subunits hif1 or arnt ( hif1 ) were generated using an ap2-cre transgene with the cre / loxp system . the mice were fed an hfd for 12 weeks and their metabolic phenotypes were determined . gene expression patterns in adipose tissues were also determined by microarray and quantitative pcr.resultson an hfd , adipocyte - specific arnt knockout mice and adipocyte - specific hif1 knockout mice exhibit similar metabolic phenotypes , including reduced fat formation , protection from hfd - induced obesity , and insulin resistance compared with similarly fed wild - type controls . the cumulative food intake remained similar ; however , the metabolic efficiency was lower in adipocyte - specific hif1 knockout mice . moreover , indirect calorimetry revealed respiratory exchange ratios were reduced in adipocyte - specific hif1 knockout mice . hyperinsulinemic - euglycemic clamp studies demonstrated that targeted disruption of hif1 in adipocytes enhanced whole - body insulin sensitivity . the improvement of insulin resistance is associated with decreased expression of socs3 and induction of adiponectin.conclusionsinhibition of hif1 in adipose tissue ameliorates obesity and insulin resistance . this study reveals that hif1 could provide a novel potential therapeutic target for obesity and type 2 diabetes .

RESEARCH DESIGN AND METHODS Metabolic assays. In vivo insulin stimulation and analysis of insulin signaling. Body composition, food intake, and metabolic rate. Biochemical assays. RNA and protein analysis. Isolation of adipocytes and macrophage of stromal vascular fraction and microarray analysis. Histology. Data analysis. RESULTS Generation and characterization of None HIF1 deficiency in adipocytes improves HFD-induced glucose intolerance and insulin resistance. Expression of genes altered in ARNT- and HIF1-deficient adipose tissue. DISCUSSION

arnt - floxed ( arnt ) ( 9 ) and hif1-floxed ( hif1 ) ( 10 ) mice containing loxp sites flanking exons 6 and 1315 , of the arnt and hif1 genes , respectively , were crossed with mice harboring the cre recombinase under control of the ap2 promoter ( ap2-cre mice ) . in the hfd study , 6-week - old male mice were given an hfd ( 60% kcal consisting of fat ; bioserv , frenchtown , nj ) for 12 weeks . hyperinsulinemic - euglycemic clamps were performed in awake mice fasted for 12 h as previously described ( 11 ) with modifications . cumulative food intake was measured in 6- to 8-week - old male mice maintained on regular chow for 2 weeks and 10- to 12-week - old mice fed an hfd for 46 weeks . cumulative food intake was measured in 6- to 8-week - old male mice maintained on regular chow for 2 weeks and 10- to 12-week - old mice fed an hfd for 46 weeks . for examination of the role of hif transcription factors in obesity and insulin resistance , arnt and hif1 genes were disrupted in adipocytes . a pcr amplicon for the arnt - null allele amplified as a 340 base pair product and was detected in genomic dna isolated from adipocytes or adipose tissue of arnt mice and not in adipose dna isolated from arnt mice . in contrast , the floxed allele was the only band detected in adipose tissue from arnt mice and from all nonadipose tissues in arnt mice ( fig . the hif1-null amplicon amplified as a 355 base pair product was detected in genomic dna isolated from adipose tissue of hif1 mice , and was not detected in adipose tissue dna isolated from hif1 mice . the expression of arnt mrna was specifically decreased in wat and brown adipose tissue ( bat ) by 50% in the arnt mice compared with arnt mice ; no decrease was evident from liver or skeletal muscle . however , no significant difference in the extent of induction of cyp1a1 mrna was noted in liver or skeletal muscle compared with arnt mice ( supplementary fig . these results demonstrate adipocyte - specific knockout of the arnt and hif1 genes in mice . adipocyte - specific disruption of the arnt and hif1a genes via cre - loxp mediated recombination . to explore the role of hif1 in fat metabolism and glucose homeostasis , male mice were fed either a chow diet or hfd . however , 12 weeks of hfd led to weight gain in arnt and hif1 mice , while arnt and hif1 mice were resistant to the hfd - induced weight gain ( supplementary fig . nmr measurements confirmed that the body fat mass and the ratio of fat and body mass of arnt and hif1 mice fed an hfd were decreased compared with arnt and hif1 mice , respectively ( supplementary fig . to explore the mechanism of reduced adiposity in arnt and hif1 mice , cumulative food intake , metabolic efficiency , and metabolic rates were measured in young mice maintained on chow diet and in mice fed an hfd for 47 weeks , which is before the difference between control and mutant mice became apparent . there were no significant differences in weight , cumulative food intake , or metabolic efficiency between hif1 and hif1 mice maintained on chow diet ( supplementary fig . these results indicate that it takes a long time for the adipose tissue to get big enough to be hypoxic and that hif1 expression was significantly induced , resulting in activation of hif1 target genes such as vegf and glut1 ( supplementary fig . after 46 weeks on an hfd , the cumulative food intake remained similar between hif1 and hif1 mice ; however , the metabolic efficiency was lower in the hif1 mice , suggesting an increase in the metabolic rate ( fig . all of these changes in hif1 mice could contribute to the decreased metabolic efficiency and weight gain on an hfd compared with hif1 mice . disruption of hif1 protected mice from hfd - induced obesity . to explore the role of adipocyte hif1 deficiency in obesity - induced insulin resistance , gtts and itts were performed . moreover , fed glucose , fasted glucose , and fasted serum insulin levels were significantly lower in arnt and hif1 mice compared with arnt and hif1 mice , respectively , after 12 weeks of hfd . although body mass was similar between hif1 and hif1 mice after 4 weeks , 6 weeks , and 8 weeks of hfd , gtt and itt revealed that glucose tolerance and insulin sensitivity were improved in hif1 mice from 4 weeks on an hfd ( supplementary fig . these results indicated that hif1 disruption in adipocytes improved hfd - induced glucose tolerance and insulin resistance before the onset of the decrease in body mass . h : basal and clamp endogenous glucose production ( egp ) , gir , whole - body glucose disposal ( rd ) , and glucose uptake in skeletal muscle , wat , and bat . the hyperinsulinemic - euglycemic clamp ( d h ) was performed after 15 weeks of hfd ( n = 6/group ) . 4 g and h ) , which confirmed improved whole - body insulin sensitivity in hif1 mice . during the clamp , insulin induced a more marked suppression of egp in hif1 mice , suggesting increased insulin sensitivity in the liver . whole - body glucose disposal ( rd ) and glucose uptake into skeletal muscle and adipose tissue were significantly increased in hif1 mice ( fig . , these data suggest that adipose selective inactivation of hif1 caused increased insulin sensitivity in major insulin target tissues , liver , skeletal muscle , and fat . these findings indicated that hif1 deficiency in adipocytes improved hfd - induced glucose tolerance and insulin resistance , which is in support of the hyperinsulinemic - euglycemic clamp studies . in addition , there was no significant nuclear hif1 protein expression in wat from chow - fed wild - type mice , while it was significantly induced in hfd - fed mice and diminished in hif1 mice ( supplementary fig . expression of genes involved in adipogenesis and glucose metabolism , pparg , c / ebp , cfd , and glut4 , were upregulated in wat of arnt and hif1 mice compared with arnt and hif1 mice , respectively , on an hfd . previous studies showed that overexpression of a constitutively active form of hif1 initiates adipose tissue fibrosis and insulin resistance ( 16 ) . expression of mrnas encoding the macrophage markers f4/80 and cd68 were also decreased in arnt mice and hif1 mice ( supplementary table 1 ; supplementary fig . a : qpcr analysis of arnt mrna expression in the peritoneal macrophages ( p - m ) ( b ) and qpcr analysis of hif1 , socs3 , and adiponectin mrna expression in adipocytes and svf - m ( c ) of hif1 and hif1 mice after 12 weeks of hfd . previous studies demonstrated that socs3 mrna is elevated in wat of diet - induced obesity ( dio ) mice ( 18,19 ) . further , haploinsufficiency of socs3 significantly protected mice against the development of dio and associated metabolic complications ( 20 ) . in adipocytes , no significant expression of hif1 mrna in hif1 mice was found . after 12 weeks of hfd , expression of socs3 in adipocytes of hif1 mice was ~80% decreased compared with that in hif1 mice . consistent with data obtained in p - m , the expression of hif1 , socs3 , and adiponectin ( adiponectin expression being nearly undetectable in svf - m ) in svf - m were not significantly different between hif1 and hif1 mice after 12 weeks of hfd ( fig . these results suggest that the socs3 and adiponectin pathway is involved , in part , in the improvement of hfd - induced insulin resistance in hif1 mice . for examination of the role of hif transcription factors in obesity and insulin resistance , arnt and hif1 genes were disrupted in adipocytes . in contrast , the floxed allele was the only band detected in adipose tissue from arnt mice and from all nonadipose tissues in arnt mice ( fig . the hif1-null amplicon amplified as a 355 base pair product was detected in genomic dna isolated from adipose tissue of hif1 mice , and was not detected in adipose tissue dna isolated from hif1 mice . the expression of arnt mrna was specifically decreased in wat and brown adipose tissue ( bat ) by 50% in the arnt mice compared with arnt mice ; no decrease was evident from liver or skeletal muscle . however , no significant difference in the extent of induction of cyp1a1 mrna was noted in liver or skeletal muscle compared with arnt mice ( supplementary fig . these results demonstrate adipocyte - specific knockout of the arnt and hif1 genes in mice . adipocyte - specific disruption of the arnt and hif1a genes via cre - loxp mediated recombination . to explore the role of hif1 in fat metabolism and glucose homeostasis , male mice were fed either a chow diet or hfd . however , 12 weeks of hfd led to weight gain in arnt and hif1 mice , while arnt and hif1 mice were resistant to the hfd - induced weight gain ( supplementary fig . nmr measurements confirmed that the body fat mass and the ratio of fat and body mass of arnt and hif1 mice fed an hfd were decreased compared with arnt and hif1 mice , respectively ( supplementary fig . to explore the mechanism of reduced adiposity in arnt and hif1 mice , cumulative food intake , metabolic efficiency , and metabolic rates were measured in young mice maintained on chow diet and in mice fed an hfd for 47 weeks , which is before the difference between control and mutant mice became apparent . there were no significant differences in weight , cumulative food intake , or metabolic efficiency between hif1 and hif1 mice maintained on chow diet ( supplementary fig . indirect calorimetry performed at 24c and thermoneutrality ( 30c ) on the same set of mice did not reveal significant differences in metabolic rate , respiratory exchange ratio ( rer ) , or activity between hif1 and hif1 mice fed chow diet ( supplementary fig . these results indicate that it takes a long time for the adipose tissue to get big enough to be hypoxic and that hif1 expression was significantly induced , resulting in activation of hif1 target genes such as vegf and glut1 ( supplementary fig . after 46 weeks on an hfd , the cumulative food intake remained similar between hif1 and hif1 mice ; however , the metabolic efficiency was lower in the hif1 mice , suggesting an increase in the metabolic rate ( fig . all of these changes in hif1 mice could contribute to the decreased metabolic efficiency and weight gain on an hfd compared with hif1 mice . disruption of hif1 protected mice from hfd - induced obesity . a and b : cumulative food intake and metabolic efficiency for 2 weeks in hif1 and hif1 mice after 46 weeks of hfd . to explore the role of adipocyte hif1 deficiency in obesity - induced insulin resistance , gtts and itts were performed . moreover , fed glucose , fasted glucose , and fasted serum insulin levels were significantly lower in arnt and hif1 mice compared with arnt and hif1 mice , respectively , after 12 weeks of hfd . the calculated homeostasis model assessment ( homa ) measure of insulin resistance was significantly decreased in arnt and hif1 mice ( table 1 ) . although body mass was similar between hif1 and hif1 mice after 4 weeks , 6 weeks , and 8 weeks of hfd , gtt and itt revealed that glucose tolerance and insulin sensitivity were improved in hif1 mice from 4 weeks on an hfd ( supplementary fig . these results indicated that hif1 disruption in adipocytes improved hfd - induced glucose tolerance and insulin resistance before the onset of the decrease in body mass . hif1 disruption in adipocytes improved hfd - induced glucose intolerance and insulin resistance . h : basal and clamp endogenous glucose production ( egp ) , gir , whole - body glucose disposal ( rd ) , and glucose uptake in skeletal muscle , wat , and bat . the hyperinsulinemic - euglycemic clamp ( d h ) was performed after 15 weeks of hfd ( n = 6/group ) . a hyperinsulinemic - euglycemic clamp was performed in hif1 and hif1 mice after 15 weeks of hfd to further characterize in vivo insulin action . 4 g and h ) , which confirmed improved whole - body insulin sensitivity in hif1 mice . whole - body glucose disposal ( rd ) and glucose uptake into skeletal muscle and adipose tissue were significantly increased in hif1 mice ( fig . these findings indicated that hif1 deficiency in adipocytes improved hfd - induced glucose tolerance and insulin resistance , which is in support of the hyperinsulinemic - euglycemic clamp studies . in addition , there was no significant nuclear hif1 protein expression in wat from chow - fed wild - type mice , while it was significantly induced in hfd - fed mice and diminished in hif1 mice ( supplementary fig . thus , the phenotype of hif1 mice is similar to hif1a mice on a chow diet but different on an hfd . expression of genes involved in adipogenesis and glucose metabolism , pparg , c / ebp , cfd , and glut4 , were upregulated in wat of arnt and hif1 mice compared with arnt and hif1 mice , respectively , on an hfd . importantly , total serum adiponectin , the high molecular weight ( hmw ) form of adiponectin , and the ratio of hmw to total adiponectin were increased in arnt and hif1 mice on a 12-week hfd ( supplementary fig . previous studies showed that overexpression of a constitutively active form of hif1 initiates adipose tissue fibrosis and insulin resistance ( 16 ) . in agreement with this finding , expression of the fibrosis related genes lox , col1a1 , col3a1 , and loxl1 were decreased in arnt or hif1 mice ( supplementary table 1 ; supplementary fig . expression of mrnas encoding the macrophage markers f4/80 and cd68 were also decreased in arnt mice and hif1 mice ( supplementary table 1 ; supplementary fig . because it was reported that the ap2 promoter is expressed in macrophage ( 17 ) , the possibility that hif1 was disrupted in macrophage was investigated by determining arnt gene disruption in peritoneal macrophage ( p - m ) from arnt mice . a : qpcr analysis of arnt mrna expression in the peritoneal macrophages ( p - m ) ( b ) and qpcr analysis of hif1 , socs3 , and adiponectin mrna expression in adipocytes and svf - m ( c ) of hif1 and hif1 mice after 12 weeks of hfd . previous studies demonstrated that socs3 mrna is elevated in wat of diet - induced obesity ( dio ) mice ( 18,19 ) . after 12 weeks of hfd , expression of socs3 in adipocytes of hif1 mice was ~80% decreased compared with that in hif1 mice . consistent with data obtained in p - m , the expression of hif1 , socs3 , and adiponectin ( adiponectin expression being nearly undetectable in svf - m ) in svf - m were not significantly different between hif1 and hif1 mice after 12 weeks of hfd ( fig . these results suggest that the socs3 and adiponectin pathway is involved , in part , in the improvement of hfd - induced insulin resistance in hif1 mice . however , the role of hypoxia in adipose tissue during obesity and insulin resistance remains unclear . in the current study , adipocyte - specific disruption of hif1 or its heterodimerization partner , arnt , hif1 signaling may regulate socs3 and adiponectin , thus providing a mechanistic clue by which hif1 exacerbates whole - body insulin resistance . these findings indicate a central role for adipocyte hif1 signaling in the pathogenesis of obesity and insulin resistance . hif1 , the main mediator of the hypoxia response in adipose tissue , is almost undetectable in lean mice but significantly increased in obese mice resulting in induction of the hif1 target genes glut1 and pdk1 ( 2 ) . moreover , overexpression of a constitutively - active hif1 in adipose tissue leads to glucose intolerance and insulin resistance ( 16 ) . these studies are consistent with results of the present work showing that ablation of arnt or hif1 in adipose tissue improved hfd - induced obesity and insulin resistance . in arnt and hif1 mice , adipogenesis - related genes such as pparg , c / ebp , cfd , and glut4 were increased and adipocyte size and mass reduced after hfd challenge , consistent with the notion that enhanced adipogenesis does not correlate with obesity ( 24,25 ) . however , in hif1 mice the knockout efficiency of hif1 in svf - m was very low consistent with recent reports showing that the efficiency of cre recombination in macrophage was much less than that in adipocytes ( 14,3234 ) . loss of hif1 in adipose tissue can improve whole - body glucose intolerance after hfd , mainly due to enhanced insulin signaling in wat , liver , and skeletal muscle . indeed , hmw adiponectin levels , or the ratio of hmw adiponectin to total adiponectin , are more meaningful markers than total adiponectin levels for predicting insulin resistance and the development of metabolic syndrome ( 38 ) . overexpression of socs3 in adipose tissue inhibited local insulin action but improved systemic glucose metabolism , and adiponectin production was increased after hfd ( 39 ) . in addition , consistent with the present results , a recent study showed that pioglitazone exerts its effect to improve whole - body insulin sensitivity through the suppression of socs3 , which is associated with an increase in stat3 phosphorylation and adiponectin production in wat ( 21 ) . these factors are also associated with improved insulin sensitivity of the whole body in arnt and hif1 mice after hfd . in conclusion , the current study clearly shows that hif1 in adipose tissue plays an important role in the metabolism of lipid and glucose . the hif1 effects on insulin sensitivity may be due in part to its direct or indirect regulation of socs3 in wat , whereas the mechanism of its effect on adiposity and obesity is still not clear and requires further investigation . because loss of hif1 activity improves metabolic function , compounds that inhibit hif1 function in adipose tissue might have significant therapeutic potential in reducing obesity and insulin resistance .

genome sequencing has revealed that most higher eukaryotes have a relatively limited set of genes , whose numbers do not correlate with organismal complexity . the ability of a limited number of genes to achieve diverse protein functions depends on a series of post - translational modifications ( ptms ) , including phosphorylation , glycosylation , acylation , methylation , lipidation , protein ligation , sulfation , and myriad oxidations , that result in controllable and conditional functions of proteins . intracellularly , serine and threonine residues are modified by phosphorylation , regulated by protein kinases and phosphatases that collectively account for 2.5% of human genes , and by oglcnacylation , controlled by oglcnac transferases and oglcnacases ( figure 1 ) . frequently , the same residues that are phosphorylated are also observed under different conditions to be oglcnacylated . interestingly , in many cases , phosphorylation and oglcnacylation are observed to have opposing functional effects . thus , improved understanding of the differential structural effects of phosphorylation and oglcnacylation is of broad potential application in cellular biology . intracellular post - translational modifications of ser and thr by phosphorylation and oglcnacylation . the diethylphosphate triester of ser / thr is neutral and sterically similar to oglcnac . the protein tau is a 441 amino acid ( largest isoform ) natively disordered microtubule - binding protein that is most prominent in neurons . hyperphosphorylated forms of tau aggregate as fibrils and precipitate as the major protein components of the neurofibrillary tangles ( nfts ) observed in alzheimer s disease and other neurodegenerative disorders , including frontotemporal dementia , pick s disease , and chronic traumatic encephalopathy ( cte ) ( collectively termed tauopathies ) . the protein tau consists of a number of functional domains ( figure 2 ) , including 4 hydrophobic tubulin - binding domains ( tbds ) ( residues 242367 ) that are directly responsible for both binding microtubules and for tau aggregation , an n - terminal hydrophobic region which dynamically interacts with the tbds , and a proline - rich domain ( residues 174241 ) , which also contains a second hydrophobic region ( residues 220231 ) . the proline - rich domain of tau serves as a linker between the n - terminal sequence and the tubulin - binding domains . the majority of phosphorylation sites identified to be important to hyperphosphorylation - mediated tau aggregation are in the proline - rich domain , with additional phosphorylation sites located c - terminal to the tubulin - binding domains in a region that also interacts with the tbd to stabilize it . red , hydrophobic regions a and b ; green , proline - rich domain ( prd ) ; blue , tubulin - binding domain ( tbd ) repeats 14 ( r1 , r2 , r3 , r4 ) . the most commonly used boundaries of the tbds are indicated ; notably , residues 242251 of r1 have poor homology to analogous residues in r2 , r3 , and r4 thus defined . the tbd boundaries have alternatively been defined as r1 residues 252282 , r2 283313 , r3 314344 , r4 345376 . by this definition most phosphorylation sites associated with tau aggregation in alzheimer s disease are in the proline - rich domain and in the c - terminal domain . bottom : sequences of tau - derived proline - rich peptides examined in this study . all peptides were acetylated at the n - terminus and contained c - terminal amides . residues in blue indicate sites modified by phosphorylation , diethylphosphorylation , or oglcnacylation . residue numbers are based on the largest ( 441 residue ) isoform of tau . in contrast to these peptides , addition of either an n - terminal or c - terminal tyr to tau174183 resulted in substantial changes to its cd spectrum . knowledge of the structural effects of post - translational modifications of tau is important to understand the mechanisms of pathological protein misfolding induced by hyperphosphorylation observed in the alzheimer s diseased brain , as well as to understand how to maintain tau in a soluble , nonaggregated form . data on natively disordered proteins , including tau , indicate that conformations observed in peptides are similar to those observed in the larger protein contexts , because of the absence of stable tertiary and quaternary structures . because of the challenges of both structure determination in natively disordered proteins and of reliable preparation of homogeneous samples of expressed proteins with defined patterns of multiple protein post - translational modifications , particularly for oglcnacylation , we have used peptide models to understand the local structural effects of natively disordered regions of proteins . we previously investigated the structural effects of phosphorylation on peptides derived from the proline - rich domain of tau . in that work , we found that phosphorylation of tau peptides induced a structural change promoting polyproline helix ( ppii ) . oglcnacylation of tau , which has been identified on a series of sites in the proline - rich domain that are also sites of phosphorylation , has been found to be protective against hyperphosphorylation and neurofibrillary tangle formation . indeed , inhibitors of oglcnacase , the enzyme that removes the oglcnac group and thus can functionally inhibit phosphorylation by preventing access of the kinase substrate , are under investigation as potential therapeutics in alzheimer s disease . the mechanism of oglcnacylation - mediated protection against nft formation could be via prevention of phosphorylation , and additionally or alternatively oglcnacylation could promote a structure change that is different than that of phosphorylation . in view of the general observation of phosphorylation and oglcnacylation occurring on similar sites , particularly within natively disordered regions of proteins , we sought to examine within a biologically relevant sequence context the relative structural effects of phosphorylation and oglcnacylation compared to the unmodified ser / thr residues . a series of peptides derived from the proline - rich domain of tau was synthesized , and their structures were analyzed as free hydroxyls and as phosphorylated and oglcnacylated amino acids , by circular dichroism and nmr ( figure 2 ) . all residues that contained post - translational modifications have been previously identified as sites of these post - translational modifications in tau . the protected fmoc -oglcnac serine and threonine amino acids were synthesized via a modification of the methodology of arsequell et al . the protected oglcnacylated amino acids were incorporated in peptides , and the peptides were subjected to tfa cleavage / deprotection and purified by hplc . the purified peptides containing protected oglcnac hydroxyls were then subjected to deesterification via naome / meoh and purified to generate peptides with defined patterns of oglcnacylation on multiple residues ( scheme 2 ) . in contrast to the common use of glu as a mimic of phosphoserine / phosphothreonine , there is no readily accessible mimic of oglcnacylated serine / threonine . the diethylphosphate modification of serine and threonine , which is readily incorporated into peptides using phosphoramidites by standard chemistry employed for peptide phosphorylation , generates a derivatized side chain that is neutral and sterically similar to oglcnac . however , in contrast to oglcnacylated ser / thr , which typically require expensive amino acids ( fmoc - ac3seroglcnac and fmoc - ac3throglcnac cost $ 600/100 mg , a quantity sufficient for the incorporation of just one oglcnacylated residue in a single peptide synthesized at small scale ) and/or substantial synthetic manipulation , peptides with the diethylphosphate modification are readily synthesized on solid phase from inexpensive , commercially available reagents ( figure 1 ) . diethylphosphate , which exhibits substantial steric effects in model peptides , is thus potentially a practical mimic of the steric effects of oglcnacylation . therefore , in addition to synthesizing the oglcnacylated peptides , we also synthesized peptides with the diethylphosphates at sites of oglcnacylation to investigate them as potential readily accessible mimics of oglcnacylation . polyproline helix ( ppii ) is a predicted major conformation of protein proline - rich domains . the ppii content in peptides can be quantified using circular dichroism ( cd ) , via the weak positive band at 228 nm . in larger peptides , the intensity of this band can be obscured by the substantially more intense signal from -helices ( [ ]max 33 000 deg cm dmol for -helix , compared to [ ]max = + 5000 deg cm dmol for polyproline helix ) ; thus , even nascent -helices and -structure can substantially obscure the cd signal of polyproline helix in larger peptides . ppii is also characterized by a negative band at 200 nm , although other structures can contribute to the intensity of this band , and thus analysis of this band is nonquantitative . polyproline helix can also be identified by changes in the max in cd and in nmr via coupling constants . in general , it is difficult to identify polyproline helix in larger peptides and proteins by either cd or nmr because of similarities to data in random coil , though polyproline helix can be definitively identified and is observed to thermally melt to a random coil conformation , which has a different cd signature . because of these complications , smaller peptides often have substantial advantages for the definitive identification of polyproline helix . in polyproline helix propensity scales , serine and threonine have low ppii propensities , due to the possibility of multiple side chain / main chain hydrogen bonds and 1 conformational heterogeneity . the lowest ppii propensities are observed for aromatic amino acids and for sterically hindered -branched amino acids ( thr , ile , val , and particularly the highly sterically congested tert - leucine ( tle ) ) , indicating that steric hindrance near the protein backbone strongly opposes ppii . all peptides were analyzed by circular dichroism . in tau174183 ( figure 3 ) , which contains the alzheimer s disease phosphoepitope pthr175/pthr181 , the phosphorylated peptide was observed to have greater ppii ( larger signal at 228 nm , a defined maximum indicative of ppii ) than the nonphosphorylated peptide . interestingly , the cd of the phosphorylated peptide was similar to the peptide with both threonines changed to the phosphomimic ( and ppii - favoring residue ) glu , although the structural effect of replacement of thr by glu was less than that of thr phosphorylation ( figure 3b ) . in contrast , the oglcnacylated peptide exhibited reduced ppii compared to the nonphosphorylated peptide , indicating that oglcnacylation disfavors ppii . the effect of diethylphosphorylation was qualitatively similar to that of oglcnacylation , though greater in magnitude . the structural change of oglcnacylation was also observable in a red shift of the max of the cd minimum , from 201 nm in the phosphorylated peptide to 203 nm in the oglcnacylated peptide and to 204 nm in the diethylphosphorylated peptide , as well as in a greater mean residue ellipticity at 190 nm for the oglcnacylated peptide . data on the oglcnac and diethylphosphate peptides were similar to those of a polyproline helix negative control , with the threonines replaced by the sterically demanding tert - leucine . cd spectra of tau174183 peptides ( ac ktppapktpp - nh2 ) in water with 5 mm phosphate buffer and 25 mm kf at ph 7.5 . ( a ) thr , green squares ; phosphothreonine , red circles ; throglcnac , blue diamonds ; thropo3et2 , black triangles . ( b ) peptides with thr ( green squares ) or with both thr residues replaced by either glu ( ac keppapkepp - nh2 ) ( magenta open circles ) or tert - leucine ( tle ) ( ac ktleppapktlepp - nh2 ) ( purple open diamonds ) . similar opposing effects of phosphorylation versus oglcnacylation were also observed in other proline - rich peptides exhibiting residual ppii structure ( tau211219 , tau229238 ) , with phosphorylation inducing ppii and oglcnacylation opposing ppii ( figure 4a , b ) . these peptides include the sites of several major tau phosphoepitopes ( pthr212 , pser214 , and pthr231 ) that are observed pathologically in alzheimer s disease . the reduction in ppii for the oglcnacylated and diethylphosphorylated peptides , particularly compared to the phosphorylated peptides , was observable in reduced mean residue ellipticity at 228 nm , in a red shift in the minimum around 200 nm , and in increased mean residue ellipticity at 190 nm . cd spectra of ( a ) tau211219 , ( b ) tau229238 , and ( c ) tau196209 peptides in water with 5 mm phosphate buffer and 25 mm kf at ph 7.5 . unmodified ser / thr , green squares ; phosphoserine / phosphothreonine , red circles ; ser / thr oglcnac , blue diamonds ; ser / thr opo3et2 , black triangles . in contrast , the structural effects of oglcnacylation and phosphorylation were not distinct in tau196209 , although , interestingly , peptides with both post - translational modifications were different from the unmodified peptide ( figure 4c ) . in tau196209 , both oglcnacylation and phosphorylation disrupted a nascent -helix cd signature ( minimum in cd 220 nm ) in the unmodified peptides . this sequence is less proline - rich than the other peptides examined ( 3 pro in 14 residues ) , and includes three consecutive pg(s / t ) repeats . pgspg(s / t ) sequences in the pdb are observed as -helix nucleation sites , with the ser side chain and spg(s / t ) main chain oxygens acting as hydrogen bond acceptors to nucleate ( n - cap ) the n - terminus of an -helix ( e.g. , glutaminyl cyclase ( pdb 3si0 ) , interleukin-5 receptor ( pdb 3qt2 ) ) . phosphorylation has been observed to disrupt -helix formation when the phosphorylation site is at an internal site in -helices . in contrast to the results above , in this case , the diethylphosphate , which induced increased -helix , was structurally divergent from oglcnacylation . the observation here of similar effects of phosphorylation and oglcnacylation on -helicity emphasizes the importance of structural context in understanding the effects of phosphorylation and oglcnacylation . indeed , while in many cases phosphorylation and oglcnacylation are functionally opposing , in some cases oglcnacylation and phosphorylation result in similar functional effects in proteins . in order to identify whether the structural effects of post - translational modifications seen in smaller peptides were also observed in a broader structural context , we examined the peptide tau211238 . this peptide contains six phosphorylation sites , incorporating two proline - rich regions ( residues 211219 and 229238 ) separated by a hydrophobic segment ( the b domain of figure 2 , including the highly hydrophobic vavv motif ) . by circular dichroism , phosphorylation of tau211238 induced an increase in mean residue ellipticity at 228 nm , consistent with induced polyproline helix upon tau phosphorylation in data seen in smaller peptides above ( figure 5 ) . the magnitude of the increase in mean residue ellipticity at 228 nm was substantially less than seen in smaller peptides , and can not be definitively structurally assigned in the larger peptide , but is consistent with an equivalent change in structure to polyproline helix within the proline - rich segments . the magnitude of the change in mean residue ellipticity is smaller because of the larger number of residues in the peptide , including residues not affected by the local structural organization induced by phosphorylation ( signal dilution by other residues in the peptide , including b domain residues that adopt an extended conformation in tau ) . the data from these peptides confirm induced polyproline helix upon phosphorylation in larger peptides but emphasize the difficulty in identifying polyproline helix in larger peptides and the special utility of smaller peptides for definitively identifying polyproline helix . cd spectra of unmodified ( green squares ) and phosphorylated ( red circles ) tau211238 at 0.5 c in water with 5 mm phosphate buffer ph 8 and 25 mm kf . one additional peptide , tau234251 ( figure 2 ) , was examined by circular dichroism . landrieu and lippens identified via c chemical shift index analysis that enzymatic phosphorylation of a tau protein fragment ( residues 208324 ) by cdk2/cyclina3 , including phosphorylation at ser235 , resulted in an increase in c chemical shift consistent with a small induction of -helix in residues 236239 of tau . analysis of the tau sequence suggests a short segment between residues 235 and 246 with the potential to form an -helix , with the sequence bounded by prolines at residues 236 and 247 serving as -helix start and stop signals . the c - terminal prolines at residues 247 , 249 , and 251 are expected to strongly prevent -helix propagation beyond residue 246 , although the p247vpmp251 sequence could potentially function as a hydrophobic -helix c - cap . cd experiments revealed a very weak -helical signature in the nonphosphorylated peptide , with modestly increased -helicity in trifluoroethanol ( tfe ) ( figure 6 , figure s6 , supporting information ) . these data are consistent with analysis of griesinger , mandelkow , zweckstetter , and co - workers , who found that residues 240251 of nonphosphorylated tau do not adopt a well - defined conformation , and that residues 232239 predominantly adopt a polyproline helix conformation , as we observed above and previously for tau229238 and tau229242 . phosphorylation of ser235 resulted in a small increase in -helicity of this peptide , consistent with the results of landrieu and lippens , with a greater -helical induction observed in the -helix - promoting solvent tfe . in contrast , phosphorylation at both ser235 and ser237 , in addition to exhibiting a weak -helical signature , induced a small positive band at 225 nm consistent with the local induction of polyproline helix around these residues that was seen in tau229238 . overall , these results are consistent with the expected low -helicity of this peptide sequence , whose -helicity is hampered by a short sequence of potential -helical character ( 12 residues ; pro has good -helical propensity only at the first and second residues of an -helix ) , multiple residues with low -helix propensity ( 3 ser and a thr within the central 10 residues between the prolines ) , and a c - terminal proline residue , which prevents continuation of the hydrogen - bonding pattern of the -helix and substantially reduces -helical content of short -helical peptides . notably , the -helical content and induced -helicity could be substantially greater in the dynamic presence of transient tertiary structure present in tau . cd spectra of unmodified ( green squares ) , monophosphorylated at ser235 ( magenta open circles ) , and doubly phosphorylated ( at ser235/ser237 ) ( red circles ) tau234251 at 25 c in water with 5 mm phosphate buffer ph 8 and 25 mm kf . to understand the structural basis for the observed opposing conformational effects of oglcnacylation versus phosphorylation in proline - rich motifs , in addition to analysis of tau peptides , the effects of post - translational modifications were examined within the simple tau174183-derived model peptide ac - kxpp - nh2 ( x = ser , thr , or phosphorylated or oglcnacylated ser or thr ) , whose sequence ( with thr ) is repeated twice in tau174183 ( k174tpp177 and k180tpp183 ) and which is homologous to the r230tpp233 sequence in tau229238 . this peptide was also applied to examine the effects of threonine versus serine modification . in addition , the structural effects of phosphorylation versus oglcnacylation were also examined within the model peptide ac - gppxppgy - nh2 context , which was previously used to identify polyproline helix propensity , and in the related proline - rich peptide ac - gpkxppgy - nh2 , which contains the ktpp sequence present in tau174183 ( for these peptides , x = throh , thropo3 , thropo3et2 , and throglcnac ) . in these proline - rich model peptides , similar conformational effects of post - translational modifications were observed by cd as were found in proline - rich tau peptides , with phosphorylation increasing ppii and oglcnacylation and diethylphosphorylation opposing ppii ( figure 7 , figures s7s17 , tables s5s7 , supporting information ) . notably , comparison of the cd spectra of ktpp and kspp peptides ( figure 7 , figures s7s11 , tables s5 and s6 , supporting information ) revealed a substantially larger structural change for thr phosphorylation than ser phosphorylation ( []224 = + 5310 and + 2230 deg cm dmol for pthr thr and pser ser , respectively ) . the larger change in structure upon thr phosphorylation than ser phosphorylation was both due to lower population of ppii for the peptide with thr than with ser and due to greater ppii with pthr than with pser . these data suggest that substantially larger structural changes are induced because of threonine phosphorylation than because of serine phosphorylation . cd spectra of ( a ) ac - ktpp - nh2 and ( b ) ac - kspp - nh2 peptides at 25 c in water with 5 mm phosphate ph 8 and 25 mm kf . unmodified ser / thr , green squares ; phosphoserine / phosphothreonine , red circles ; ser / thr oglcnac , blue diamonds ; ser / thr opo3et2 , black triangles . data on these peptides at 2 c , where greater ppii is observed , are in the supporting information ( figure s11 ) . data on polyproline helix model peptides ac - gpptppgy - nh2 and ac - gpktppgy - nh2 peptides were similarly consistent with data in tau peptides . data from ac - gppptppgy - nh2 also indicated no effect of 2 mm mgcl2 on the cd spectra ( and thus , no substantial effect of mg on structure ) of phosphorylated peptides . in addition , a greater mean residue ellipticity at 228 nm was observed at 2 c than at 25 c , consistent with the interpretation that these cd data are specifically indicative of ppii content in the peptides , as was previously seen in other proline - rich and polyproline helix - containing peptides . a series of homonuclear ( 1-d h and tocsy ) and heteronuclear ( h n hsqc , h c hsqc , and h c hmbc ) nmr experiments was conducted on tau - derived peptides and proline - rich model peptides to identify residue - specific and post - translational - modification - specific changes in structure ( figures 811 , tables 14 ) . nmr data in this series of peptides were consistent with cd data , indicating that phosphorylation in proline - rich sequences induces structural changes leading to more compact and more ordered conformations , whereas oglcnacylation and diethylphosphorylation exhibited evidence of more extended conformations , as expected for sterically demanding amino acids in proline - rich domains . 1-d h nmr spectroscopy indicated substantial divergence of the peptides that was a function of post - translational modification : across all peptides , relative to unmodified ser / thr , phosphorylation induced downfield amide chemical shifts and smaller jn for phosphorylated residues ; in contrast , oglcnacylation of thr residues induced upfield amide chemical shifts and amide chemical shifts similar to those of ser for seroglcnac . in addition , experiments on nonphosphorylated and phosphorylated tau211238 indicated that the large amide chemical shift changes and conformational restriction observed for phosphoresidues in tau211219 and tau229238 peptides were also observed in the larger peptide context ( figures s54 and s55 , supporting information ) . of particular note , in all peptides , phosphorylated residues exhibited substantially downfield ( = 9.49.8 ppm as thropo3 , 8.79.2 ppm as seropo3 ) amide proton chemical shifts , with amide proton chemical shifts substantially more downfield for the dianionic than the monoanionic phosphates ( monoanionic pser / pthr = 8.358.75 ppm ) ( figures 8 and 9 , table 2 , table s36 , supporting information ) , as has been observed previously in some peptides and proteins . 1-d nmr spectra ( amide region ) of peptides with ser / thr , ser / thr(oglcnac ) , ser / thr(opo3h ) ( ph 4 ) , ser / thr(opo3(h ) ) ( ph 6.5 ) , and ser / thr(opo3 ) ( ph 8) . experiments were conducted at 298 k in 90% h2o/10% d2o with 5 mm phosphate ( ph 4 or as indicated ) and 25 mm nacl . gs and gt indicate the resonances of the seroglcnac and throglcnac , respectively , backbone amide protons . ( a ) tau174183 peptides ; ( b ) tau211219 peptides ; ( c ) tau229238 peptides ; ( d ) ac - ktpp - nh2 peptides . n hsqc spectra of ( a ) tau174183 , ( b ) tau211219 , ( c ) tau229238 , and ( d ) ac - ktpp - nh2 peptides . green , peptides with unmodified ser / thr ; blue , peptides with thr(oglcnac ) ; magenta , peptides with ser / thr(opo3h ) ( ph 4 ) ; red , peptides with ser / thr(opo3 ) ( ph 8) . jn values < 6 hz correlate with compact and ordered conformations , values > 8 hz indicate extended conformations , and values between 6 and 8 hz are observed in disordered peptides . in general , smaller jn values indicate more compact conformations , while larger jn values indicate more extended conformations . tert - leucine jn values in tau174183(thr tle ) are 8.8 and 7.9 hz . in ac - gppxppgy - nh2 peptides , the jn value of glu is 6.3 hz , the third most - restricted value for canonical amino acids after ala ( 5.7 hz ) and asp ( 6.2 hz ) , while that of tert - leucine is 8.3 hz , larger than all canonical amino acids ( val 8.0 hz , ile 7.9 hz ) and indicative of a strong preference for the extended conformation . data on phosphorylated peptides were obtained at ph 4 ( ropo3h ) or ph 8 ( ropo3 ) ( typical phosphoserine / phosphothreonine pka 5.56.0 ) . additional nmr data for all peptides are in the supporting information . full tabulated nmr data and statistical analysis are in the supporting information . phosphorylated ser / thr residues in these peptides were particularly conformationally restricted . jn values correlate with the backbone torsion angle via a parametrized karplus equation , with values between 6 and 8 hz consistent with disorder or averaging of multiple conformations , values > 8 hz indicative of ordered , extended conformations , and values < 6 hz indicative of ordered , compact conformations , and more broadly with smaller values indicating more compact conformations , larger values indicating more extended conformations , and values further from random coil values indicating greater extent of order . the jn values observed are indicative of special conformational order for the phosphorylated residues : across all peptides , dianionic phosphothreonine exhibits a mean jn = 3.5 hz , corresponding to = 55 , compared to a random coil value for thr ( jn = 7.2 hz , average = 83 ) ; dianionic phosphoserine exhibits a mean jn = 5.4 hz , corresponding to average = 70 , compared to a random coil value for ser ( jn = 6.8 hz , average = 80 ) ) ( table 1 ) . notably , the substantial conformational order induced by phosphorylation was dependent on the dianionic phosphates : only small increases in order were induced by monoanionic phosphoserine ( jn = 6.4 hz ) , phosphothreonine ( jn = 6.6 hz ) , and glutamic acid ( jn = 6.2 hz ) . interestingly , the small coupling constants for dianionic phosphorylated amino acids observed herein are also consistent with a growing number of examples of proteins in which phosphorylation induces -helix formation when at its n - terminus . h n hsqc experiments indicated that , in addition to large downfield changes in the chemical shifts of amide protons , the serine / threonine amide nitrogens exhibited large downfield changes in chemical shift upon phosphorylation across all peptides ( figure 9 , table 3 ; tabulated data table s39 , supporting information ) . in tau174183 , the dianionic phosphothreonine amide nitrogens were 7.2 ppm downfield of the amides of threonine and 6.6 ppm downfield of the amides of monoanionic phosphothreonine . in contrast , oglcnacylated threonine exhibited amide nitrogen chemical shifts only 0.6 ppm downfield of those of threonine . the large divergence in amide nitrogen chemical shift between monoanionic and dianionic phosphothreonine is not consistent with differences in the electron - withdrawing nature of the different protonation states of these phosphates , suggesting that these differences are due to particular structure induced by dianionic phosphothreonine , consistent with differences in jn values between monoanionic and dianionic phosphopeptides . h n hsqc data from other proline - rich peptides ( tau211219 , tau229238 , and ac - ktpp - nh2 ) exhibited similar trends , with large downfield changes in amide hydrogen and amide nitrogen resonances for dianionic phosphoresidues compared to the monoanionic phosphoresidues or unmodified ser / thr [ amide nitrogens : tau211219 mean = + 0.1 and + 1.0 ppm for monoanionic phosphoserine and phosphothreonine , respectively , compared to unmodified ser / thr , versus mean = + 2.9 and + 5.5 ppm for dianionic phosphoserine and phosphothreonine , respectively , compared to unmodified ser / thr ; tau229238 mean = + 2.6 ppm ( pser(opo3 ) ) and + 5.6 ppm ( thr(opo3 ) ) ppm for the dianionic phosphoresidues compared to the unmodified ser / thr ; ac - ktpp - nh2 = + 1.1 ppm ( pthr(opo3h ) ) and = + 6.5 ppm ( pthr(opo3 ) ) relative to unmodified thr ; ac - gpptppgy - nh2 , = + 0.1 ppm ( pthr(opo3h ) ) and = + 6.2 ppm ( pthr(opo3 ) ) relative to unmodified thr ] . collectively , these data demonstrate very large induced changes in the electronic environment around the amide nitrogens of dianionic phosphoserine and dianionic phosphothreonine residues compared to ser / thr , ser / thr(oglcnac ) , or to monoanionic phosphoserine or phosphothreonine . = ( ser / thr(opo3 ) ) ( ser / thr(oh ) ) . notably , small downfield changes in amide nitrogen chemical shift are associated with the polyproline helix conformation ( = + 1.1 ppm for change from random coil to polyproline helix ) . while the changes in phosphoserine / phosphothreonine amide chemical shifts are too large to be explained by secondary structure , most other resonances in these peptides also exhibited small downfield changes in n amide chemical shift upon phosphorylation , consistent with the increased ppii seen by cd ( figure 10 ; tabulated data in the supporting information ) . in contrast , the amides of oglcnacylated tau174183 exhibited small upfield changes in n chemical shift , consistent with the reduced ppii seen in this peptide . ( a d ) h c hsqc spectra ( hc region ) of ( a ) tau174183 , ( b ) tau211219 , ( c ) tau229238 , and ( d ) ac - ktpp - nh2 peptides . ( e , f ) h c hmbc spectra ( hc = o region ) of ( e ) tau174183 and ( f ) ac - ktpp - nh2 peptides . green , peptides with unmodified ser / thr ; red , peptides with ser / thr(opo3 ) ( ph 8) c hsqc experiments were conducted on tau peptides to further characterize the residue - specific effects of protein phosphorylation ( figure 10 , table 4 ) . in addition , to determine the effects of phosphorylation on the backbone carbonyls , h c hmbc experiments were conducted on nonphosphorylated and phosphorylated tau174183 and the model peptide ac - ktpp - nh2 ( figure 10e , f ) . ppii , in contrast to -helix or -sheet , does not exhibit large changes in h ( = 0.03 ppm ) , c ( = + 0.3 ppm ) , or c = o ( = + 0.1 ppm ) chemical shift compared to random coil . these experiments revealed large changes in the h and c chemical shifts of phosphorylated residues , as expected because of the electronic change of the side chain , though with substantially larger chemical shift changes at phosphothreonine than at phosphoserine ( table 3 ) . in particular , as the dianion , phosphorylation of serine induced downfield shifts in h ( = + 0.13 ppm ) and upfield shifts in c ( = 0.3 ppm ) , in contrast to upfield shifts in h ( = 0.21 ppm ) and downfield shifts in c ( = + 1.3 ppm ) for phosphorylation of threonine . data from other residues , within the context of the small inherent changes in chemical shift for ppii , also indicated that the structural changes upon phosphorylation propagated to residues beyond the phosphorylated residues ( figure 10 ) . one defining feature stabilizing the polyproline helix is an n * interaction between adjacent carbonyls . the observation of changes in the carbonyl chemical shifts across all residues ( including the n - terminal acetyls ) is consistent with phosphorylation - induced changes in the environment around the backbone carbonyls of all residues in the peptides . = ( ser / thr(opo3 ) ) ( ser / thr(oh ) ) . peptides with phosphorylated amino acids also exhibited relatively slow amide hydrogen exchange considering the absence of tertiary structure or hydrogen - bonded secondary structure , with most amide protons observable at ph 8 for phosphorylated proline - rich peptides ( figure 8 , supporting information ) . for the peptide ac - kt(opo3)pp - nh2 , slow amide exchange , small jn values , and downfield pthr amide chemical shifts at ph 8 were persistent even at elevated temperature ( up to 323 k ) and at high salt concentrations ( up to 1 m nacl ) ( figure 11 ) . these data are consistent with a strong interaction that is in slow exchange and that can not be readily screened electrostatically , suggesting that the induced structure is not primarily due to a simple lysine - phosphate or arginine - phosphate electrostatic interaction of the phosphorylated residue with the prior basic residue . these data on dianionic phosphorylated amino acids are particularly striking , since in disordered peptides , amide protons are generally not observed or are substantially exchange - broadened at ph 8 . slow amide exchange on the nmr time scale at ph 8 is generally associated with more stable structures ( e.g. , those involving hydrogen bonding , an interaction that could slow amide exchange at the pser / pthr amides , though not other amides ) . while ppii does not exhibit hydrogen bonding , ppii is stabilized by n * interactions between adjacent carbonyls , which could also potentially slow exchange of the amide protons . ( a ) temperature - dependent h nmr spectra of ac - kt(opo3)pp - nh2 . experiments were conducted in 90% h2o/10% d2o with 5 mm phosphate buffer and 25 mm nacl at ph 8.0 . experiments were conducted at 277 ( top ) , 298 , 308 , 323 , and 338 k ( bottom ) . experiments at 277 , 298 , and 308 k were conducted on a 600 mhz cryoprobe instrument , while experiments at 323 and 338 k were conducted on a 400 mhz instrument . ( b ) salt - dependent h nmr spectra of ac - kt(opo3)pp - nh2 . experiments were conducted in 90% h2o/10% d2o in 5 mm phosphate buffer at ph 8.0 with 25 ( top ) , 125 , 225 , 325 , and 1000 ( bottom ) mm nacl . interestingly , by nmr , the effects of modifications on serine versus threonine were divergent in magnitude ( tables 14 ) , as had been seen by cd above in ac - kxpp - nh2 peptides ( figure 5 ) . greater overall conformational restriction ( jn ) was observed at phosphothreonine than at phosphoserine , as well as a larger change in jn ( and thus in the main chain torsion angle ) between the nonphosphorylated and phosphorylated peptides ( mean jn = 3.7 hz for thr , versus 1.4 hz for ser ) . phosphothreonine amide protons also exhibited greater downfield shifts than phosphoserine amides ( mean amide 9.63 ppm for dianionic pthr , versus 8.99 ppm for dianionic pser , compared to 8.23 and 8.32 ppm for thr and ser ; mean = + 1.40 ppm for threonine phosphorylation , versus mean = + 0.67 ppm for serine phosphorylation ( table 2 ) ) , as well as greater downfield shifts in amide nitrogens ( mean amide 124.3 ppm for dianionic pthr , versus 120.8 ppm for dianionic pser , compared to 118.1 and 118.2 ppm for thr ( mean = + 6.2 ppm ) and ser ( mean = + 2.6 ppm ) , respectively ) ( table 3 ) . larger changes in chemical shifts were also observed for threonine phosphorylation than serine phosphorylation on h , c , and c ( table 2 , table 4 , tables s37 and s40 , supporting information ) . collectively , these data are consistent with a stronger phosphate - amide interaction in phosphothreonine than phosphoserine and greater induced structural changes for phosphothreonine than phosphoserine . notably , threonine and serine residues are differentially phosphorylated and dephosphorylated in vivo , and evolution of thr and ser residues occurs at different rates , suggesting native functional differences between thr and ser . we have described the direct comparison of the structural effects of phosphorylation versus oglcnacylation , competing intracellular protein post - translational modifications that often occur on the same ser / thr residues , on peptide conformation within a typical natively disordered protein context . this work was specifically applied within the context of the tau protein , where hyperphosphorylation is associated with protein misfolding and aggregation , but oglcnacylation is protective against protein misfolding . we found that within proline - rich sequences phosphorylation promotes conformational order and ppii formation , whereas oglcnacylation or modification with the practical oglcnac mimic of diethylphosphorylation leads to conformational preference against ppii and a more disordered or extended conformation . the effects of phosphorylation and oglcnacylation are divergent : whereas phosphorylation induces significant conformational order , particularly on threonine residues , the effects of oglcnacylation are more subtle , confirming the strong conformational biases of serine and threonine against ppii . interestingly , in the rna polymerase ii c - terminal domain repeat ( sysptsps ) , phosphorylation promotes binding to the pin1 ww domain as a polyproline helix , whereas thr oglcnacylation induces a more extended conformation , consistent with results on tau peptides herein . as phosphorylation and oglcnacylation are competing protein intracellular post - translational modifications , which generally occur on disordered protein sequences , these data suggest potentially general opposing modes of structural changes due to these post - translational modifications . hyperphosphorylation of tau is associated with conformational changes leading to tau misfolding and aggregation as the neurofibrillary tangles of alzheimer s disease . aggregation of tau is mediated by the hydrophobic tubulin - binding domains ( tbds ) . the tbds may be stabilized against aggregation by binding to microtubules , or via association with the n - terminus and c - terminus in a global hairpin conformation that masks the hydrophobic residues of the tbds . phosphorylation or pseudophosphorylation induces structural changes that open this hairpin conformation , exposing the hydrophobic tbds to promote aggregation , while oglcnacylation is protective against aggregation , potentially due to maintaining or stabilizing the global hairpin , though to date no structural data exist to suggest this latter possibility . the importance of the n - terminus in protecting the tbds from aggregation is emphasized by the ability of n - terminal tau peptides to inhibit tau aggregation , consistent with a critical role of the structure of the proline - rich linker between these domains in maintaining soluble tau . thus , changes in secondary structure in tau s proline - rich domain may mediate global changes in tau structure and function . notably , phosphorylation at ser - pro and thr - pro sites within the proline - rich domain inhibits the ability of tau to promote tubulin polymerization . the most striking data herein are the high degree of order observed at phosphothreonine residues , with jn values that indicate very stable non - random coil conformation at these residues . overall , these data indicate that phosphorylation of the tau proline - rich domain induces significant conformational changes that result in ordering of the proline - rich domain , particularly as observed by nmr in the dianionic state , whereas the effects of oglcnacylation are somewhat similar to the free ser / thr hydroxyls and specifically oppose the effects of phosphorylation , consistent with the observed effects of oglcnacylation in opposing tau aggregation . phosphorylation of threonine residues was found herein to induce greater structural changes than serine phosphorylation , although both phosphorylation events induced more ordered conformations , including induced ppii , restriction of , and slower amide exchange . the greater structural change at thr was due to a combination of both more disorder for nonphosphorylated thr than ser and more induced order for pthr than pser , with particular conformational restriction of and evidence consistent with a phosphate - amide hydrogen bond . to identify a possible structural basis for these observations , phosphothreonine residues in several high resolution crystal structures ( protein protein interactions ( see below ) and globular proteins ) were analyzed . the crystallographic data revealed a significant degree of conformational restriction in some phosphothreonine residues , consistent with the data herein . the conformation of phosphothreonine 197 in protein kinase a ( pdb 1rdq , the highest resolution ( 1.26 ) protein with pthr in the pdb ) exhibits pthr in a ppii conformation ( = 67 , = + 134 ) , 1 = 53 ( g ) , 2 = + 119 ( surprisingly , eclipsing c h / o p bonds ) , a hydrogen bond between the phosphate and the phosphothreonine amide , and close interaction between the n 1 carbonyl ( conjugated to the hydrogen - bonded pthr amide ) and the pthr carbonyl ( o c distance 2.88 , substantially less than the 3.22 sum of van der waals radii , as well as an o c o angle of 107 , similar to the brgi dunitz trajectory ) , as would be expected by a ppii - favoring n * interaction ( figure 12 ) . notably , thr197 is a critical and conserved residue in the activation loop of pka and related protein kinases . thr197 phosphorylation induces a substantial disorder - to - order transition in pka and increase in pka activity and stability , with thr197 exhibiting no electron density in nonphosphorylated pka . the side chain conformational restriction observed crystallographically in phosphothreonine residues was also observed by nmr in ac - kt(opo3)pp - nh2 , which exhibited jhh = 9.5 hz for phosphothreonine , near the maximum of the karplus curve and indicating 1 60 ( g rotamer ) ( compared to nonphosphorylated thr jhh = 6.4 hz , as expected for disorder ; figure s66 , supporting information ) , as well as jhp = 9 hz , which is close to ideal for an eclipsing 2 c h / o p bond ( jhp = 9 hz was also seen in tau174183 ; see figures s20 and s77 , supporting information ) . collectively , the data observed herein across multiple phosphothreonine - containing peptides , including induction of polyproline helix by cd , small jn (= restricted ) for phosphothreonine , large downfield changes in amide h and n chemical shifts , and slow amide hydrogen exchange , are consistent with this crystallographically observed conformation of phosphothreonine , suggesting a potentially general mode for conformational restriction by phosphothreonine residues in both disordered and globular proteins . structure of phosphothreonine residue 197 in protein kinase a ( pdb 1rdq , 1.26 resolution ) . a similar conformation of phosphothreonine ( , = 64 , + 131 ; 1 = g ; eclipsing c h / o p bonds ( 2 = + 118 ) ; phosphate - amide hydrogen bond ; n * interaction ) was observed in a tau peptide containing pthr231 bound to a monoclonal antibody ( pdb 4glr ) , or of a pser phosphopeptide bound to pin1 ( pdb 1f8a ) . we previously observed that nature often employs the polyproline helix for phosphoprotein recognition by phosphoserine / phosphothreonine - binding domains , including binding of phosphopeptides in a polyproline helix by ww , fha , polo - box , and brct domains , though not by 1433 domains . in addition , recent analysis of phosphorylation sites in intrinsically disordered proteins suggests a greater propensity for ppii around phosphorylation sites . serine and threonine have natively low ppii propensities , suggesting the possibility of phosphorylation - mediated switches to ppii . in proline - rich sequences , eukaryotic proteins also exhibit low frequencies of asp and glu residues , but high frequencies of ser and thr residues . these data suggest that phosphorylation can provide both conformational preference for ppii and anion specificity via electrostatics to protein - binding domains that recognize phosphorylated proteins via a polyproline helix conformation . notably , tau phosphorylated at residue 231 is observed in a polyproline helix both when bound to the pin1 ww domain ( pdb 1i8h ) and to an antiphosphotau antibody ( pdb 4glr ) . in this case , antibody recognition , which involves no inherent conformational bias in recognition epitopes , utilized ppii for high affinity ( kd = 1.1 nm ) and high specificity ( > 500-fold specificity ) recognition of the phosphorylated peptide over the nonphosphorylated peptide . in sum , these data suggest that polyproline helix provides the possibility of increased specificity and affinity in folding and recognition of phosphorylated ser / thr , over both nonphosphorylated or oglcnacylated ser / thr . phosphorylation and oglcnacylation are the most significant intracellular post - translational modifications of serine and threonine . we found opposing structural effects of phosphorylation and oglcnacylation in proline - rich sequences , which are among the most common sequences in eukaryotes , but similar effects of both modifications in opposing the unmodified hydroxyl in sequences with a nascent -helix . these data provide a plausible structural basis for the observation that oglcnacylation of tau opposes neurofibrillary tangle formation , because of its confirmation of the disordered structure of sequences with unmodified serine and threonine residues , while phosphorylation is associated with neurofibrillary tangle formation , potentially because of a disorder to order transition that promotes opening of the global hairpin conformation of tau . more generally , these data provide a context for interpreting sequence - specific structural effects of these post - translational modifications , with broad potential application to understanding the intracellular effects of phosphorylation and oglcnacylation . across all peptides , dianionic phosphoserine and dianionic phosphothreonine adopted ordered structures , including induction of polyproline helix . we found particular conformational restriction in phosphothreonine residues , with a highly ordered structure adopted . notably , phosphoproteomics experiments have revealed that over 25% of phosphorylation sites are at ser - pro or thr - pro sequences , suggesting that the results observed herein in tau peptides and in proline - rich model peptides may have broad applicability in understanding the effects of phosphorylation on protein structure , particularly in regions of protein disorder . all peptides were acetylated at the n - terminus and contained c - terminal amides . cd spectra were collected on a jasco j-810 spectropolarimeter in a 1 mm cell at 25 c . peptide concentrations were 15400 m in water containing 5 mm phosphate buffer ( ph 8.0 or as indicated ) and 25 mm kf . nmr spectra of peptides were collected at 298 k on a brker avc 600 mhz nmr spectrometer equipped with a triple resonance cryoprobe . p nmr spectra were recorded on a brker drx 400 mhz nmr spectrometer equipped with a bbo probe . peptides were dissolved in buffer containing 5 mm phosphate ( ph 4.0 , 6.5 , 7.2 , or 8.0 ) with 25 mm nacl , 100 m tsp , and 90% h2o/10% d2o . jn and jhp were determined directly from the 1-d h nmr spectra and proton - coupled p nmr spectra , respectively .

phosphorylation and oglcnacylation are dynamic intracellular protein post - translational modifications that frequently are alternatively observed on the same serine and threonine residues . phosphorylation and oglcnacylation commonly occur in natively disordered regions of proteins , and often have opposing functional effects . in the microtubule - associated protein tau , hyperphosphorylation is associated with protein misfolding and aggregation as the neurofibrillary tangles of alzheimer s disease , whereas oglcnacylation stabilizes the soluble form of tau . a series of peptides derived from the proline - rich domain ( residues 174251 ) of tau was synthesized , with free ser / thr hydroxyls , phosphorylated ser / thr ( pser / pthr ) , oglcnacylated ser / thr , and diethylphosphorylated ser / thr . phosphorylation and oglcnacylation were found by cd and nmr to have opposing structural effects on polyproline helix ( ppii ) formation , with phosphorylation favoring ppii , oglcnacylation opposing ppii , and the free hydroxyls intermediate in structure , and with phosphorylation structural effects greater than oglcnacylation . for tau196209 , phosphorylation and oglcnacylation had similar structural effects , opposing a nascent -helix . phosphomimic glu exhibited ppii - favoring structural effects . structural changes due to thr phosphorylation were greater than those of ser phosphorylation or glu , with particular conformational restriction as the dianion , with mean 3jn = 3.5 hz ( pthr ) versus 5.4 hz ( pser ) , compared to 7.2 , 6.8 , and 6.2 hz for thr , ser , and glu , respectively , values that correlate with the backbone torsion angle . dianionic phosphothreonine induced strong phosphothreonine amide protection and downfield amide chemical shifts ( mean = 9.63 ppm ) , consistent with formation of a stable phosphate - amide hydrogen bond . these data suggest potentially greater structural importance of threonine phosphorylation than serine phosphorylation due to larger induced structural effects .

Introduction Results Discussion Conclusions Experimental Section

the ability of a limited number of genes to achieve diverse protein functions depends on a series of post - translational modifications ( ptms ) , including phosphorylation , glycosylation , acylation , methylation , lipidation , protein ligation , sulfation , and myriad oxidations , that result in controllable and conditional functions of proteins . interestingly , in many cases , phosphorylation and oglcnacylation are observed to have opposing functional effects . hyperphosphorylated forms of tau aggregate as fibrils and precipitate as the major protein components of the neurofibrillary tangles ( nfts ) observed in alzheimer s disease and other neurodegenerative disorders , including frontotemporal dementia , pick s disease , and chronic traumatic encephalopathy ( cte ) ( collectively termed tauopathies ) . the protein tau consists of a number of functional domains ( figure 2 ) , including 4 hydrophobic tubulin - binding domains ( tbds ) ( residues 242367 ) that are directly responsible for both binding microtubules and for tau aggregation , an n - terminal hydrophobic region which dynamically interacts with the tbds , and a proline - rich domain ( residues 174241 ) , which also contains a second hydrophobic region ( residues 220231 ) . by this definition most phosphorylation sites associated with tau aggregation in alzheimer s disease are in the proline - rich domain and in the c - terminal domain . knowledge of the structural effects of post - translational modifications of tau is important to understand the mechanisms of pathological protein misfolding induced by hyperphosphorylation observed in the alzheimer s diseased brain , as well as to understand how to maintain tau in a soluble , nonaggregated form . because of the challenges of both structure determination in natively disordered proteins and of reliable preparation of homogeneous samples of expressed proteins with defined patterns of multiple protein post - translational modifications , particularly for oglcnacylation , we have used peptide models to understand the local structural effects of natively disordered regions of proteins . we previously investigated the structural effects of phosphorylation on peptides derived from the proline - rich domain of tau . oglcnacylation of tau , which has been identified on a series of sites in the proline - rich domain that are also sites of phosphorylation , has been found to be protective against hyperphosphorylation and neurofibrillary tangle formation . in view of the general observation of phosphorylation and oglcnacylation occurring on similar sites , particularly within natively disordered regions of proteins , we sought to examine within a biologically relevant sequence context the relative structural effects of phosphorylation and oglcnacylation compared to the unmodified ser / thr residues . a series of peptides derived from the proline - rich domain of tau was synthesized , and their structures were analyzed as free hydroxyls and as phosphorylated and oglcnacylated amino acids , by circular dichroism and nmr ( figure 2 ) . similar opposing effects of phosphorylation versus oglcnacylation were also observed in other proline - rich peptides exhibiting residual ppii structure ( tau211219 , tau229238 ) , with phosphorylation inducing ppii and oglcnacylation opposing ppii ( figure 4a , b ) . in contrast , the structural effects of oglcnacylation and phosphorylation were not distinct in tau196209 , although , interestingly , peptides with both post - translational modifications were different from the unmodified peptide ( figure 4c ) . the magnitude of the increase in mean residue ellipticity at 228 nm was substantially less than seen in smaller peptides , and can not be definitively structurally assigned in the larger peptide , but is consistent with an equivalent change in structure to polyproline helix within the proline - rich segments . to understand the structural basis for the observed opposing conformational effects of oglcnacylation versus phosphorylation in proline - rich motifs , in addition to analysis of tau peptides , the effects of post - translational modifications were examined within the simple tau174183-derived model peptide ac - kxpp - nh2 ( x = ser , thr , or phosphorylated or oglcnacylated ser or thr ) , whose sequence ( with thr ) is repeated twice in tau174183 ( k174tpp177 and k180tpp183 ) and which is homologous to the r230tpp233 sequence in tau229238 . in these proline - rich model peptides , similar conformational effects of post - translational modifications were observed by cd as were found in proline - rich tau peptides , with phosphorylation increasing ppii and oglcnacylation and diethylphosphorylation opposing ppii ( figure 7 , figures s7s17 , tables s5s7 , supporting information ) . these data suggest that substantially larger structural changes are induced because of threonine phosphorylation than because of serine phosphorylation . in addition , a greater mean residue ellipticity at 228 nm was observed at 2 c than at 25 c , consistent with the interpretation that these cd data are specifically indicative of ppii content in the peptides , as was previously seen in other proline - rich and polyproline helix - containing peptides . a series of homonuclear ( 1-d h and tocsy ) and heteronuclear ( h n hsqc , h c hsqc , and h c hmbc ) nmr experiments was conducted on tau - derived peptides and proline - rich model peptides to identify residue - specific and post - translational - modification - specific changes in structure ( figures 811 , tables 14 ) . nmr data in this series of peptides were consistent with cd data , indicating that phosphorylation in proline - rich sequences induces structural changes leading to more compact and more ordered conformations , whereas oglcnacylation and diethylphosphorylation exhibited evidence of more extended conformations , as expected for sterically demanding amino acids in proline - rich domains . 1-d h nmr spectroscopy indicated substantial divergence of the peptides that was a function of post - translational modification : across all peptides , relative to unmodified ser / thr , phosphorylation induced downfield amide chemical shifts and smaller jn for phosphorylated residues ; in contrast , oglcnacylation of thr residues induced upfield amide chemical shifts and amide chemical shifts similar to those of ser for seroglcnac . of particular note , in all peptides , phosphorylated residues exhibited substantially downfield ( = 9.49.8 ppm as thropo3 , 8.79.2 ppm as seropo3 ) amide proton chemical shifts , with amide proton chemical shifts substantially more downfield for the dianionic than the monoanionic phosphates ( monoanionic pser / pthr = 8.358.75 ppm ) ( figures 8 and 9 , table 2 , table s36 , supporting information ) , as has been observed previously in some peptides and proteins . 1-d nmr spectra ( amide region ) of peptides with ser / thr , ser / thr(oglcnac ) , ser / thr(opo3h ) ( ph 4 ) , ser / thr(opo3(h ) ) ( ph 6.5 ) , and ser / thr(opo3 ) ( ph 8) . jn values correlate with the backbone torsion angle via a parametrized karplus equation , with values between 6 and 8 hz consistent with disorder or averaging of multiple conformations , values > 8 hz indicative of ordered , extended conformations , and values < 6 hz indicative of ordered , compact conformations , and more broadly with smaller values indicating more compact conformations , larger values indicating more extended conformations , and values further from random coil values indicating greater extent of order . the jn values observed are indicative of special conformational order for the phosphorylated residues : across all peptides , dianionic phosphothreonine exhibits a mean jn = 3.5 hz , corresponding to = 55 , compared to a random coil value for thr ( jn = 7.2 hz , average = 83 ) ; dianionic phosphoserine exhibits a mean jn = 5.4 hz , corresponding to average = 70 , compared to a random coil value for ser ( jn = 6.8 hz , average = 80 ) ) ( table 1 ) . h n hsqc data from other proline - rich peptides ( tau211219 , tau229238 , and ac - ktpp - nh2 ) exhibited similar trends , with large downfield changes in amide hydrogen and amide nitrogen resonances for dianionic phosphoresidues compared to the monoanionic phosphoresidues or unmodified ser / thr [ amide nitrogens : tau211219 mean = + 0.1 and + 1.0 ppm for monoanionic phosphoserine and phosphothreonine , respectively , compared to unmodified ser / thr , versus mean = + 2.9 and + 5.5 ppm for dianionic phosphoserine and phosphothreonine , respectively , compared to unmodified ser / thr ; tau229238 mean = + 2.6 ppm ( pser(opo3 ) ) and + 5.6 ppm ( thr(opo3 ) ) ppm for the dianionic phosphoresidues compared to the unmodified ser / thr ; ac - ktpp - nh2 = + 1.1 ppm ( pthr(opo3h ) ) and = + 6.5 ppm ( pthr(opo3 ) ) relative to unmodified thr ; ac - gpptppgy - nh2 , = + 0.1 ppm ( pthr(opo3h ) ) and = + 6.2 ppm ( pthr(opo3 ) ) relative to unmodified thr ] . collectively , these data demonstrate very large induced changes in the electronic environment around the amide nitrogens of dianionic phosphoserine and dianionic phosphothreonine residues compared to ser / thr , ser / thr(oglcnac ) , or to monoanionic phosphoserine or phosphothreonine . while the changes in phosphoserine / phosphothreonine amide chemical shifts are too large to be explained by secondary structure , most other resonances in these peptides also exhibited small downfield changes in n amide chemical shift upon phosphorylation , consistent with the increased ppii seen by cd ( figure 10 ; tabulated data in the supporting information ) . in particular , as the dianion , phosphorylation of serine induced downfield shifts in h ( = + 0.13 ppm ) and upfield shifts in c ( = 0.3 ppm ) , in contrast to upfield shifts in h ( = 0.21 ppm ) and downfield shifts in c ( = + 1.3 ppm ) for phosphorylation of threonine . greater overall conformational restriction ( jn ) was observed at phosphothreonine than at phosphoserine , as well as a larger change in jn ( and thus in the main chain torsion angle ) between the nonphosphorylated and phosphorylated peptides ( mean jn = 3.7 hz for thr , versus 1.4 hz for ser ) . phosphothreonine amide protons also exhibited greater downfield shifts than phosphoserine amides ( mean amide 9.63 ppm for dianionic pthr , versus 8.99 ppm for dianionic pser , compared to 8.23 and 8.32 ppm for thr and ser ; mean = + 1.40 ppm for threonine phosphorylation , versus mean = + 0.67 ppm for serine phosphorylation ( table 2 ) ) , as well as greater downfield shifts in amide nitrogens ( mean amide 124.3 ppm for dianionic pthr , versus 120.8 ppm for dianionic pser , compared to 118.1 and 118.2 ppm for thr ( mean = + 6.2 ppm ) and ser ( mean = + 2.6 ppm ) , respectively ) ( table 3 ) . larger changes in chemical shifts were also observed for threonine phosphorylation than serine phosphorylation on h , c , and c ( table 2 , table 4 , tables s37 and s40 , supporting information ) . we have described the direct comparison of the structural effects of phosphorylation versus oglcnacylation , competing intracellular protein post - translational modifications that often occur on the same ser / thr residues , on peptide conformation within a typical natively disordered protein context . this work was specifically applied within the context of the tau protein , where hyperphosphorylation is associated with protein misfolding and aggregation , but oglcnacylation is protective against protein misfolding . as phosphorylation and oglcnacylation are competing protein intracellular post - translational modifications , which generally occur on disordered protein sequences , these data suggest potentially general opposing modes of structural changes due to these post - translational modifications . hyperphosphorylation of tau is associated with conformational changes leading to tau misfolding and aggregation as the neurofibrillary tangles of alzheimer s disease . overall , these data indicate that phosphorylation of the tau proline - rich domain induces significant conformational changes that result in ordering of the proline - rich domain , particularly as observed by nmr in the dianionic state , whereas the effects of oglcnacylation are somewhat similar to the free ser / thr hydroxyls and specifically oppose the effects of phosphorylation , consistent with the observed effects of oglcnacylation in opposing tau aggregation . phosphorylation of threonine residues was found herein to induce greater structural changes than serine phosphorylation , although both phosphorylation events induced more ordered conformations , including induced ppii , restriction of , and slower amide exchange . the greater structural change at thr was due to a combination of both more disorder for nonphosphorylated thr than ser and more induced order for pthr than pser , with particular conformational restriction of and evidence consistent with a phosphate - amide hydrogen bond . the conformation of phosphothreonine 197 in protein kinase a ( pdb 1rdq , the highest resolution ( 1.26 ) protein with pthr in the pdb ) exhibits pthr in a ppii conformation ( = 67 , = + 134 ) , 1 = 53 ( g ) , 2 = + 119 ( surprisingly , eclipsing c h / o p bonds ) , a hydrogen bond between the phosphate and the phosphothreonine amide , and close interaction between the n 1 carbonyl ( conjugated to the hydrogen - bonded pthr amide ) and the pthr carbonyl ( o c distance 2.88 , substantially less than the 3.22 sum of van der waals radii , as well as an o c o angle of 107 , similar to the brgi dunitz trajectory ) , as would be expected by a ppii - favoring n * interaction ( figure 12 ) . the side chain conformational restriction observed crystallographically in phosphothreonine residues was also observed by nmr in ac - kt(opo3)pp - nh2 , which exhibited jhh = 9.5 hz for phosphothreonine , near the maximum of the karplus curve and indicating 1 60 ( g rotamer ) ( compared to nonphosphorylated thr jhh = 6.4 hz , as expected for disorder ; figure s66 , supporting information ) , as well as jhp = 9 hz , which is close to ideal for an eclipsing 2 c h / o p bond ( jhp = 9 hz was also seen in tau174183 ; see figures s20 and s77 , supporting information ) . in sum , these data suggest that polyproline helix provides the possibility of increased specificity and affinity in folding and recognition of phosphorylated ser / thr , over both nonphosphorylated or oglcnacylated ser / thr . phosphorylation and oglcnacylation are the most significant intracellular post - translational modifications of serine and threonine . we found opposing structural effects of phosphorylation and oglcnacylation in proline - rich sequences , which are among the most common sequences in eukaryotes , but similar effects of both modifications in opposing the unmodified hydroxyl in sequences with a nascent -helix . these data provide a plausible structural basis for the observation that oglcnacylation of tau opposes neurofibrillary tangle formation , because of its confirmation of the disordered structure of sequences with unmodified serine and threonine residues , while phosphorylation is associated with neurofibrillary tangle formation , potentially because of a disorder to order transition that promotes opening of the global hairpin conformation of tau . more generally , these data provide a context for interpreting sequence - specific structural effects of these post - translational modifications , with broad potential application to understanding the intracellular effects of phosphorylation and oglcnacylation .

vulvar cancer has an incidence of 12 per 100,000 women per year and represents 35 % of all gynecological malignancies [ 13 ] . squamous cell carcinoma ( scc ) is the predominant malignancy at this site , accounting for approximately 8590 % of vulvar cancers [ 4 , 5 ] . treatment of advanced stage ( figo iii and iv ) vulvar cancer patients may be ineffective , even with chemotherapy and radiotherapy [ 6 , 7 ] . therefore , new approaches are required including immunotherapy and greater knowledge of the factors influencing prognosis . tumor infiltrating lymphocytes ( tils ) cd8 + lymphocytes represent an important subpopulation of cytotoxic t lymphocytes and are the most likely effector tils of adaptive anti - tumor immunity . innate anti - tumor immunity is mediated by cells or soluble factors that naturally exist in the tumor microenvironment . among hematopoietic cells , natural killer ( nk ) ( cd3cd56 + ) and nk / t ( cd3+cd56 + ) cells have the natural ability to eliminate tumor - cell targets [ 10 , 11 ] . the cytotoxic function of all immune cells critically depends on serine proteases known as granzymes . as granzyme b is one of the most abundant granzymes , granzyme b - mediated cytotoxicity has been intensively studied [ 12 , 13 ] . grb - induced cell death has traditionally been viewed as a primary mechanism that is used by adaptive ( cd8 + ) as well as innate ( nk / nkt ) effectors to eliminate harmful target cells including tumor cells . recently , the status of adaptive immunity represented by cd8 + , cd4 + and foxp3 + t cells was found to lack prognostic significance in vscc [ 1416 ] . therefore , the purpose of this study was to clarify the prognostic roles of innate immune effectors represented by cd56 + cells and granzyme b - dependent cytolysis . this retrospective study was approved by the polish ministry of science and higher education review board . the board determined that further informed consent was not required , as all patients provided informed consent for tissue sampling prior to surgical treatment , including written consent for the storage of their information in the hospital database and the use of their information for research . the clinico - pathological characteristics of this group were carefully described in our two previous manuscripts ( section : materials and methods ) analyzing the expression of indoleamine 2,3-dioxygenase ( ido ) and the infiltration of cd8 + , cd4 + and foxp3 + t lymphocytes in vscc [ 14 , 15 ] . the median age of the patients was 69.5 years ( range 3685 ) , the median duration of follow - up was 51.23 months ( range 6.33135.5 ) , and the median overall survival was 41.16 months ( range 1.798.43 ) [ 14 , 15 ] . the immunohistochemical staining and histological analyses were performed on paraffin - embedded material consisting of 76 primary tumors and additional lymph node metastases from 35 patients . a mouse anti - human monoclonal antibody against cd56 ( cat . no ncl - cd56 - 1b ; concentration 1:400 ) and a mouse anti - human polyclonal antibody against granzyme b ( cat . no 760 - 4283 ; ready to use ) were obtained from ventana medical systems , inc . serial sections with a thickness of 4 m were cut , deparaffinized and subjected to a heat - induced epitope retrieval step before incubation with the primary antibodies . sections were immersed in target retrieval solution ( ph 6.0 for cd56 , dako cytomation , denmark ) and heated in a pressure cooker . appropriate positive ( normal cd56 and grb status ) and negative controls ( the primary antibody was replaced with normal mouse igg at an appropriate dilution ) were included for each case . the immunohistochemical results were evaluated by two independent pathologists who did not have access to the clinical data . the degree of immune cell infiltration was determined for more than 10 high - power ( 400 ) microscopic fields for each tissue sample . then , five areas with the densest lymphocyte distribution ( hot spots ) were selected and microphotographs were taken . the numbers of cd56 + and grb+ cells were counted exclusively within the primary tumor cancer cell nest . for each case , the mean index of cd56 + and grb+ cells per single high - power field was counted and then statistically analyzed . patients were divided into cd56 low- and high - intensity groups ( low cd56 + and high cd56 + , respectively ) and grb low- and high - intensity groups ( low grb+ and high grb+ , respectively ) based on median cell number ( cut - off point ) to determine differences in overall survival among these groups [ 12 , 14 , 15 , 1719 ] . the statistical analysis was performed using the chi - square test or fisher s exact probability test , followed by the kruskal overall survival curves were estimated by the kaplan meier method and compared by the two - sided log - rank test . all analyses were performed using the statistical software statistica 10 ( stat soft inc . ) . the clinico - pathological characteristics of this group were carefully described in our two previous manuscripts ( section : materials and methods ) analyzing the expression of indoleamine 2,3-dioxygenase ( ido ) and the infiltration of cd8 + , cd4 + and foxp3 + t lymphocytes in vscc [ 14 , 15 ] . the median age of the patients was 69.5 years ( range 3685 ) , the median duration of follow - up was 51.23 months ( range 6.33135.5 ) , and the median overall survival was 41.16 months ( range 1.798.43 ) [ 14 , 15 ] . the immunohistochemical staining and histological analyses were performed on paraffin - embedded material consisting of 76 primary tumors and additional lymph node metastases from 35 patients . a mouse anti - human monoclonal antibody against cd56 ( cat . no ncl - cd56 - 1b ; concentration 1:400 ) and a mouse anti - human polyclonal antibody against granzyme b ( cat . no 760 - 4283 ; ready to use ) were obtained from ventana medical systems , inc . serial sections with a thickness of 4 m were cut , deparaffinized and subjected to a heat - induced epitope retrieval step before incubation with the primary antibodies . sections were immersed in target retrieval solution ( ph 6.0 for cd56 , dako cytomation , denmark ) and heated in a pressure cooker . appropriate positive ( normal cd56 and grb status ) and negative controls ( the primary antibody was replaced with normal mouse igg at an appropriate dilution ) were included for each case . the immunohistochemical results were evaluated by two independent pathologists who did not have access to the clinical data . the degree of immune cell infiltration was determined for more than 10 high - power ( 400 ) microscopic fields for each tissue sample . then , five areas with the densest lymphocyte distribution ( hot spots ) were selected and microphotographs were taken . the quantitative analysis was performed with multiscan 14.2 software . the numbers of cd56 + and grb+ cells were counted exclusively within the primary tumor cancer cell nest . for each case , the mean index of cd56 + and grb+ cells per single high - power field was counted and then statistically analyzed . patients were divided into cd56 low- and high - intensity groups ( low cd56 + and high cd56 + , respectively ) and grb low- and high - intensity groups ( low grb+ and high grb+ , respectively ) based on median cell number ( cut - off point ) to determine differences in overall survival among these groups [ 12 , 14 , 15 , 1719 ] . the statistical analysis was performed using the chi - square test or fisher s exact probability test , followed by the kruskal overall survival curves were estimated by the kaplan meier method and compared by the two - sided log - rank test . all analyses were performed using the statistical software statistica 10 ( stat soft inc . ) . 1microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest the median number of ie grb+ cells was 3 ( range 0.0013.33 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) and in the locally limited stage ( 41 non - metastatic cases ) had primary cancer nests that were similarly infiltrated by ( ie ) grb+ cells ( median 3.17 vs. 2.63 , p = 0.07 ) . in 11 cases , no ie grb+ cells were observed ( 14.47 % , grb - negative tumors ) . grb - negative tumors exhibited a higher degree of morphological differentiation and were less frequently associated with lymph node metastases ( table 1).table 1comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cellsclinicopathological featureie grb p ie cd56 p negative ( n = 11)positive ( n = 61)negative ( n = 10)positive ( n = 65)age /median/62700.13474690.554depth of invasion /median/67.030.2046.6370.291g1/g2 + g38/316/45 0.049 4/622/430.731g1/g2/g38/1/216/28/17 0.009 4/3/322/26/170.832pt10/1/055/5/10.9098/2/060/4/10.306meta+/meta2/932/29 0.05 5/529/361.0figo stage i / ii / iii / iv8/1/2/028/1/28/40.1304/1/4/135/1/26/30.374recurrence + /1/1014/470.4392/813/521.0overall survivalnsnsbold values indicate statistical significance comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cells bold values indicate statistical significance the median number of grb+ cells infiltrating lymph node metastases was 0.83 ( range 0.008.33 ) . a lack of intracarcinomatous grb+ cells in nodal metastases was observed in 6 of 28 cases ( 21.43 % ) , whereas the primary tumors from these patients contained ie grb+ cells . the lack of grb+ cells within metastases does not influence overall survival ( p = 0.512 ) . cd56 + t cells were detected within cancer cell nests and sporadically in the mesenchymal stroma ( fig . the median number of ( ie ) cd56 + cells was 2 ( range 0.0037 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) had primary cancer nests that were more infiltrated by ( ie ) cd56 + cells than patients in the locally limited stage ( 41 non - metastatic cases ) ( median 2 vs. 1.67 , p = 0.05 ) . a lack of ie cd56 + infiltrates was observed in 10 of 76 cases ( 13.16 % ) . no differences were observed in all compared clinicopathological features between cases with and without ( ie ) cd56 + infiltrates ( table 1 ) . the median number of cd56 + t cells infiltrating lymph node metastases was 1.16 ( range 0.0010 ) . a lack of cd56 + infiltrates within nodal metastases was observed in 8 of 35 cases ( 22.86 % ) . only 2 patients had primary nests that were not infiltrated by ie cd56 + cells . the lack of cd56 + cells in lymph node metastases had no impact on overall survival ( p = 0.3012 ) . the number of ie cd56 + ( per hpf ) was correlated with the depth of invasion and recurrence , while the intensity of the ie grb+ lymphocytes was correlated with age and tumor grade based on a 2-tier scale : g1 versus g2 + g3 ( table 2).table 2correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patientsclinicopathological featureie cd56 + p ie gzb+ p age r = 0.1260.280 r = 0.333 0.004 pt r = 0.0010.992 r = 0.0210.862depth of invasion r = 0.339 0.003 r = 0.1960.102g1/g2/g3 r = 0.0230.849 r = 0.2070.080g1/g2 + g3 r = 0.0930.432 r = 0.304 0.009 pn r = 0.2220.0570.2160.069figo stage r = 0.2080.075 r = 0.2260.056recurrence + / r = 0.295 0.011 r = 0.1090.362bold values indicate statistical significance correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patients bold values indicate statistical significance patients were divided into grb low- and high - intensity groups ( low grb+ and high grb + , respectively ) based on the median value of ie grb+ cells . with the exception of age ( high grb+ patients were older than low grb+ patients ) , no differences were observed between groups in the pathological features analyzed here ( table 3).table 3differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point)clinicopathological featurelow ( ie)grb+high ( ie)grb+plow ( ie)cd56+high ( ie)cd56+page /median/6571 0.011 69690.996depth of invasion /median/77.40.3556.257.750.01g1/g2/g315/9/89/20/110.07017/15/139/14/70.509g1/g2 i / ii / iii / iv19/1/11/117/1/19/30.49627/1/15/212/1/15/20.410recurrence + /5/2710/300.3925/4010/200.036bold values indicate statistical significance differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point ) bold values indicate statistical significance while no differences in overall survival ( os ) were observed between high grb+ and low grb+ cases in the general cohort ( fig . 2a ) ( f cox p = 0.479 ) , more ie grb+ cells than the median were correlated with longer os among cases with local disease ( f cox p = 0.028 ) ( fig . 2kaplan meier survival curves for overall survival of patients . a low cd56+/high cd56 + infiltrates within cancer nests in the general population d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases kaplan meier survival curves for overall survival of patients . a low cd56+/high cd56 + infiltrates within cancer nests in the general population . d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases patients were divided into cd56 low- and high - intensity groups ( low cd56 + and high cd56 + , respectively ) based on the median value of ie cd56 + cells . no differences were observed in all analyzed clinico - pathological features among different cd56 infiltration categories in our cohort ( table 3 ) . while no differences in overall survival were observed between these groups in the general cohort ( f cox p = 0.141 ) ( fig . 2c ) , intensities of ie cd56 + cells exceeding the median ( high - cd56 + ) were correlated with longer os among patients with metastatic dissemination ( f cox p = 0.009 ) ( fig . 1microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest the median number of ie grb+ cells was 3 ( range 0.0013.33 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) and in the locally limited stage ( 41 non - metastatic cases ) had primary cancer nests that were similarly infiltrated by ( ie ) grb+ cells ( median 3.17 vs. 2.63 , p = 0.07 ) . in 11 cases , no ie grb+ cells were observed ( 14.47 % , grb - negative tumors ) . grb - negative tumors exhibited a higher degree of morphological differentiation and were less frequently associated with lymph node metastases ( table 1).table 1comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cellsclinicopathological featureie grb p ie cd56 p negative ( n = 11)positive ( n = 61)negative ( n = 10)positive ( n = 65)age /median/62700.13474690.554depth of invasion /median/67.030.2046.6370.291g1/g2 + g38/316/45 0.049 4/622/430.731g1/g2/g38/1/216/28/17 0.009 4/3/322/26/170.832pt10/1/055/5/10.9098/2/060/4/10.306meta+/meta2/932/29 0.05 5/529/361.0figo stage i / ii / iii / iv8/1/2/028/1/28/40.1304/1/4/135/1/26/30.374recurrence + /1/1014/470.4392/813/521.0overall survivalnsnsbold values indicate statistical significance comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cells bold values indicate statistical significance the median number of grb+ cells infiltrating lymph node metastases was 0.83 ( range 0.008.33 ) . a lack of intracarcinomatous grb+ cells in nodal metastases was observed in 6 of 28 cases ( 21.43 % ) , whereas the primary tumors from these patients contained ie grb+ cells . the lack of grb+ cells within metastases does not influence overall survival ( p = 0.512 ) . cd56 + t cells were detected within cancer cell nests and sporadically in the mesenchymal stroma ( fig . the median number of ( ie ) cd56 + cells was 2 ( range 0.0037 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) had primary cancer nests that were more infiltrated by ( ie ) cd56 + cells than patients in the locally limited stage ( 41 non - metastatic cases ) ( median 2 vs. 1.67 , p = 0.05 ) . a lack of ie cd56 + infiltrates was observed in 10 of 76 cases ( 13.16 % ) . no differences were observed in all compared clinicopathological features between cases with and without ( ie ) cd56 + infiltrates ( table 1 ) . the median number of cd56 + t cells infiltrating lymph node metastases was 1.16 ( range 0.0010 ) . a lack of cd56 + infiltrates within nodal metastases was observed in 8 of 35 cases ( 22.86 % ) . only 2 patients had primary nests that were not infiltrated by ie cd56 + cells . the lack of cd56 + cells in lymph node metastases had no impact on overall survival ( p = 0.3012 ) . the number of ie cd56 + ( per hpf ) was correlated with the depth of invasion and recurrence , while the intensity of the ie grb+ lymphocytes was correlated with age and tumor grade based on a 2-tier scale : g1 versus g2 + g3 ( table 2).table 2correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patientsclinicopathological featureie cd56 + p ie gzb+ p age r = 0.1260.280 r = 0.333 0.004 pt r = 0.0010.992 r = 0.0210.862depth of invasion r = 0.339 0.003 r = 0.1960.102g1/g2/g3 r = 0.0230.849 r = 0.2070.080g1/g2 + g3 r = 0.0930.432 r = 0.304 0.009 pn r = 0.2220.0570.2160.069figo stage i / ii / iii / iv r = 0.2080.075 r = 0.2260.056recurrence + / r = 0.295 0.011 r = 0.1090.362bold values indicate statistical significance correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patients bold values indicate statistical significance patients were divided into grb low- and high - intensity groups ( low grb+ and high grb + , respectively ) based on the median value of ie grb+ cells . with the exception of age ( high grb+ patients were older than low grb+ patients ) , no differences were observed between groups in the pathological features analyzed here ( table 3).table 3differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point)clinicopathological featurelow ( ie)grb+high ( ie)grb+plow ( ie)cd56+high ( ie)cd56+page /median/6571 0.011 69690.996depth of invasion /median/77.40.3556.257.750.01g1/g2/g315/9/89/20/110.07017/15/139/14/70.509g1/g2 + g315/179/310.04417/289/210.622pt30/1/135/5/00.20241/3/127/3/00.632meta+/meta12/2022/180.16117/2817/130.155figo stage i / ii / iii / iv19/1/11/117/1/19/30.49627/1/15/212/1/15/20.410recurrence + /5/2710/300.3925/4010/200.036bold values indicate statistical significance differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point ) bold values indicate statistical significance while no differences in overall survival ( os ) were observed between high grb+ and low grb+ cases in the general cohort ( fig . cox p = 0.479 ) , more ie grb+ cells than the median were correlated with longer os among cases with local disease ( f cox p = 0.028 ) ( fig . . 2kaplan meier survival curves for overall survival of patients . a low cd56+/high cd56 + infiltrates within cancer nests in the general population . d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases kaplan meier survival curves for overall survival of patients . a low cd56+/high cd56 + infiltrates within cancer nests in the general population . patients were divided into cd56 low- and high - intensity groups ( low cd56 + and high cd56 + , respectively ) based on the median value of ie cd56 + cells . no differences were observed in all analyzed clinico - pathological features among different cd56 infiltration categories in our cohort ( table 3 ) . while no differences in overall survival were observed between these groups in the general cohort ( f cox p = 0.141 ) ( fig . 2c ) , intensities of ie cd56 + cells exceeding the median ( high - cd56 + ) were correlated with longer os among patients with metastatic dissemination ( f cox p = 0.009 ) ( fig . the distribution of cd56 + cells and grb expressing cells at the primary site differed in the vscc cases analyzed here . cd56 + cells were mostly detected within the cancer nests , while the grb+ cells were predominant in the tumor stroma . only activated cytotoxic lymphocytes and nk / nkt cells are able to express granzyme b [ 12 , 13 ] . the higher stromal distribution of grb+ cells suggests that the vast majority of immune effectors are activated outside the cancer nests , chiefly through mechanisms unrelated to the recognition of vscc antigens . moreover , the large discrepancy between ie grb + infiltrates ( median 3 ) and the cumulative number of intraepithelial immune effectors ( median 2 for ie cd56 + and median 19 for ie cd8 + infiltrates ) recently described by our group for this cohort could suggest that the cytolytic power of the immune system has been lost outside rather than inside cancer nests . this could explain the lack of prognostic significance of cd8 + lymphocytes in vscc [ 15 , 16 ] and suggests that the number of tils present in the stroma probably provides a better indication of coexisting inflammatory processes than the immunogenicity of cancer cells . in contrast , tils within cancer nests are certainly connected with direct spontaneous immune reactions against cancer [ 20 , 21 ] , and therefore , only these intraepithelial infiltrates ( ie ) were analyzed further . we found that the median numbers of ie grb+ and ie cd56 + cells were comparable ( 3 and 2 per hpf within the primary cancer nest and 0.83 and 1.016 per hpf within lymph node metastases , respectively ) . this could suggest that these cells are the same cells , but considering the current results together with our previous data on ie cd8 + t lymphocytes in this cohort , we believe that only a small subset of ie cd56 + cells could be granzyme b positive . this is supported by further results indicating that ie cd56 + and ie grb+ infiltrates have separate correlations with clinico - pathological features in patients with vscc . the intensity of ie cd56 + cells was significantly correlated with depth of invasion and recurrence , while the intensity of ie grb+ lymphocytes was correlated with age and tumor grade . patients in the advanced stage of the disease had primary cancer nests that were more infiltrated by ie cd56 + cells than cases in the locally limited stage . however , neither high ie cd56 + nor high ie grb+ infiltrates had an impact on overall survival in vscc patients . further , to analyze the prognostic significance of these cells , we decided to subdivide the cohort into 2 groups : non - metastatic and metastatic . from the immunological point of view , patients with local and disseminated disease represent unsuccessful and successful immune escape , respectively . therefore , subdividing the general cohort into these two subtypes and conducting separate survival analyses seems to be justified . it is unclear whether the immune surveillance performed by the cytotoxic function of the activated effectors of adaptive ( cd8 + t cells ) and innate ( nk / nkt cells ) immunity is efficient until tumors develop mechanisms of immune escape . various proportions of the adaptive and innate effectors could be involved in granzyme b - dependent cytotoxicity because of the diverse expression of mhc antigens , costimulatory molecules and antigen - presenting cell ( apc ) statuses in different cancers , as well as the different number of regulatory cells within tumor tissue . this could explain why the effectors of adaptive ( cd8 + ) immunity did not reveal prognostic significance when analyzed separately . natural killer cells are considered to play a major role in the early stages of tumor development [ 11 , 24 , 25 ] . previous reports showed that while innate immunity represents the first line of pathogen defense , most human tumor cells are resistant to perforin - mediated nk cell lysis , and nk cells are rarely found among tils in advanced cancer cases . interestingly , the correlation between the intensity of ie cd56 + ( nk / nkt ) cells and survival was observed among metastatic vscc patients , while the prognostic significance of granzyme b - dependent killing was not observed in this subgroup . either these immune cells defending against metastases have already released grb and are now grb( ) , or they represent a distinct subset of nk / nkt cells mediating tumor destruction by mechanisms other than grb - mediated cytotoxicity . the second concept is consistent with more recent data suggesting that the primary biological role of nks might not be the elimination of tumor targets , but rather the facilitation of dendritic - cell ( dc)t - cell interactions . as a consequence , this process may drive the immune responses against tumor - associated antigens ( taas ) . this hypothesis could be supported by the fact that a diminished infiltration of cd4 + t cells is observed among advanced vscc cases ( authors unpublished data ) . cd4 + t - cell - deficient intratumoral infiltrates may impair the adaptive immune response , which is primarily regulated by dcs . recent data suggest that in addition to granzyme - mediated lysis , nk / nkt cells constitutively express several ligands of the tnf - family and can induce apoptosis in a broad variety of tumor - cell targets . this mechanism might be of greater biological importance than secretory , granule - mediated killing , largely because of the expression of tnf - family ligands by tumor cells resulting in their sensitivity to apoptotic death . interestingly , patients with disseminated cancer were significantly older than patients without metastases in the cohort analyzed here . it is known that immune anti - tumor responses can be influenced by the gradual deterioration of the immune system with age . a significant expansion in the number of nk cells was observed upon aging , but some controversy remains concerning the efficacy of their cytotoxic function , which has been described as comparable to or decreased [ 32 , 33 ] compared with younger individuals . ie grb+ infiltrates , which indicate the cytolytic power of combined adaptive and innate immune effectors , and ie cd56 + infiltrates , which indicate nk / nkt cells of unknown functional status , correlate with longer os among non - metastatic and metastatic vscc cases , respectively . both of these significant results suggest that immunological effects contribute to improved clinical outcomes in vscc . the authors have contributed to read and approved this manuscript and declare that there are no conflicts of interest to be disclosed .

objectiveadaptive immune effectors do not influence prognosis in vulvar squamous cell carcinoma ( vscc ) . therefore , we tried to clarify the prognostic role of innate immunity and granzyme b - dependent cytotoxicity as defined by intratumoral infiltrates of natural killer cells ( cd56 + ) and lymphocytes expressing granzyme b ( grb+).methodswe analyzed 76 primary vsccs and 35 lymph node metastases that were obtained from 76 patients with a full clinical history . the distribution and density of grb+ and cd56 + cells within cancer tissues were evaluated by immunohistochemistry and correlated with clinicopathological features , commonly recognized prognostic factors and overall survival ( os).resultscd56 + cells were mostly detected within the cancer nests , while grb+ cells were predominant in the tumor stroma . intraepithelial ( ie ) cd56 + infiltrates at the primary site were correlated with depth of invasion ( r = 0.339 , p = 0.003 ) and recurrence ( r = 0.295 , p = 0.011 ) , while ie grb+ infiltrates were correlated with tumor grade ( r = 0.304 , p = 0.009 ) and age ( r = 0.333 , p = 0.004 ) . the primary cancer nests of metastatic patients were infiltrated more by intraepithelial ( ie ) cd56 + cells than were those of the non - metastatic patients ( p = 0.05 ) . the median os was 41.16 months ( range 1.798.43 ) . high ie grb+ infiltrates predicted longer os among patients without metastases ( p = 0.028 ) . high ie cd56 + infiltrates were correlated with longer os in metastatic cases ( p = 0.009).conclusionthe combined cytotoxicity of innate and adaptive immune effectors infiltrating cancer nests ( ie grb+ ) predicts an improved clinical outcome among non - metastatic vscc patients . the functional status of prognostic ie cd56 + infiltrates in immune escaped ( metastatic ) tumors requires further investigation .

Introduction Materials and methods Patients and specimens Antibodies Immunohistochemistry Evaluation and classification of CD56+ and GrB+ cells Statistical analysis Results Immunohistochemistry for GrB+ cells Immunohistochemistry for CD56+ cells Correlation of IE CD56+ and IE GrB+ cells with clinicopathological features Prognostic significance of IE GrB+ cells at the primary site Prognostic significance of IE CD56+ cells at the primary site Discussion Conclusion Conflict of interest

therefore , the purpose of this study was to clarify the prognostic roles of innate immune effectors represented by cd56 + cells and granzyme b - dependent cytolysis . the median age of the patients was 69.5 years ( range 3685 ) , the median duration of follow - up was 51.23 months ( range 6.33135.5 ) , and the median overall survival was 41.16 months ( range 1.798.43 ) [ 14 , 15 ] . the median age of the patients was 69.5 years ( range 3685 ) , the median duration of follow - up was 51.23 months ( range 6.33135.5 ) , and the median overall survival was 41.16 months ( range 1.798.43 ) [ 14 , 15 ] . the numbers of cd56 + and grb+ cells were counted exclusively within the primary tumor cancer cell nest . 1microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest the median number of ie grb+ cells was 3 ( range 0.0013.33 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) and in the locally limited stage ( 41 non - metastatic cases ) had primary cancer nests that were similarly infiltrated by ( ie ) grb+ cells ( median 3.17 vs. 2.63 , p = 0.07 ) . grb - negative tumors exhibited a higher degree of morphological differentiation and were less frequently associated with lymph node metastases ( table 1).table 1comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cellsclinicopathological featureie grb p ie cd56 p negative ( n = 11)positive ( n = 61)negative ( n = 10)positive ( n = 65)age /median/62700.13474690.554depth of invasion /median/67.030.2046.6370.291g1/g2 + g38/316/45 0.049 4/622/430.731g1/g2/g38/1/216/28/17 0.009 4/3/322/26/170.832pt10/1/055/5/10.9098/2/060/4/10.306meta+/meta2/932/29 0.05 5/529/361.0figo stage i / ii / iii / iv8/1/2/028/1/28/40.1304/1/4/135/1/26/30.374recurrence + /1/1014/470.4392/813/521.0overall survivalnsnsbold values indicate statistical significance comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cells bold values indicate statistical significance the median number of grb+ cells infiltrating lymph node metastases was 0.83 ( range 0.008.33 ) . the lack of grb+ cells within metastases does not influence overall survival ( p = 0.512 ) . the median number of ( ie ) cd56 + cells was 2 ( range 0.0037 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) had primary cancer nests that were more infiltrated by ( ie ) cd56 + cells than patients in the locally limited stage ( 41 non - metastatic cases ) ( median 2 vs. 1.67 , p = 0.05 ) . no differences were observed in all compared clinicopathological features between cases with and without ( ie ) cd56 + infiltrates ( table 1 ) . the lack of cd56 + cells in lymph node metastases had no impact on overall survival ( p = 0.3012 ) . the number of ie cd56 + ( per hpf ) was correlated with the depth of invasion and recurrence , while the intensity of the ie grb+ lymphocytes was correlated with age and tumor grade based on a 2-tier scale : g1 versus g2 + g3 ( table 2).table 2correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patientsclinicopathological featureie cd56 + p ie gzb+ p age r = 0.1260.280 r = 0.333 0.004 pt r = 0.0010.992 r = 0.0210.862depth of invasion r = 0.339 0.003 r = 0.1960.102g1/g2/g3 r = 0.0230.849 r = 0.2070.080g1/g2 + g3 r = 0.0930.432 r = 0.304 0.009 pn r = 0.2220.0570.2160.069figo stage r = 0.2080.075 r = 0.2260.056recurrence + / r = 0.295 0.011 r = 0.1090.362bold values indicate statistical significance correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patients bold values indicate statistical significance patients were divided into grb low- and high - intensity groups ( low grb+ and high grb + , respectively ) based on the median value of ie grb+ cells . with the exception of age ( high grb+ patients were older than low grb+ patients ) , no differences were observed between groups in the pathological features analyzed here ( table 3).table 3differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point)clinicopathological featurelow ( ie)grb+high ( ie)grb+plow ( ie)cd56+high ( ie)cd56+page /median/6571 0.011 69690.996depth of invasion /median/77.40.3556.257.750.01g1/g2/g315/9/89/20/110.07017/15/139/14/70.509g1/g2 i / ii / iii / iv19/1/11/117/1/19/30.49627/1/15/212/1/15/20.410recurrence + /5/2710/300.3925/4010/200.036bold values indicate statistical significance differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point ) bold values indicate statistical significance while no differences in overall survival ( os ) were observed between high grb+ and low grb+ cases in the general cohort ( fig . 2a ) ( f cox p = 0.479 ) , more ie grb+ cells than the median were correlated with longer os among cases with local disease ( f cox p = 0.028 ) ( fig . a low cd56+/high cd56 + infiltrates within cancer nests in the general population d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases kaplan meier survival curves for overall survival of patients . d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases patients were divided into cd56 low- and high - intensity groups ( low cd56 + and high cd56 + , respectively ) based on the median value of ie cd56 + cells . 2c ) , intensities of ie cd56 + cells exceeding the median ( high - cd56 + ) were correlated with longer os among patients with metastatic dissemination ( f cox p = 0.009 ) ( fig . 1microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest microphotograph of immunohistochemical staining for cd56 + cells ( a ) and grb+ cells ( b ) within the primary cancer nest the median number of ie grb+ cells was 3 ( range 0.0013.33 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) and in the locally limited stage ( 41 non - metastatic cases ) had primary cancer nests that were similarly infiltrated by ( ie ) grb+ cells ( median 3.17 vs. 2.63 , p = 0.07 ) . grb - negative tumors exhibited a higher degree of morphological differentiation and were less frequently associated with lymph node metastases ( table 1).table 1comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cellsclinicopathological featureie grb p ie cd56 p negative ( n = 11)positive ( n = 61)negative ( n = 10)positive ( n = 65)age /median/62700.13474690.554depth of invasion /median/67.030.2046.6370.291g1/g2 + g38/316/45 0.049 4/622/430.731g1/g2/g38/1/216/28/17 0.009 4/3/322/26/170.832pt10/1/055/5/10.9098/2/060/4/10.306meta+/meta2/932/29 0.05 5/529/361.0figo stage i / ii / iii / iv8/1/2/028/1/28/40.1304/1/4/135/1/26/30.374recurrence + /1/1014/470.4392/813/521.0overall survivalnsnsbold values indicate statistical significance comparison of clinicopathological features between patients having primary tumors negative and positive for grb+ as well as cd56 + cells bold values indicate statistical significance the median number of grb+ cells infiltrating lymph node metastases was 0.83 ( range 0.008.33 ) . a lack of intracarcinomatous grb+ cells in nodal metastases was observed in 6 of 28 cases ( 21.43 % ) , whereas the primary tumors from these patients contained ie grb+ cells . the lack of grb+ cells within metastases does not influence overall survival ( p = 0.512 ) . the median number of ( ie ) cd56 + cells was 2 ( range 0.0037 ) per single high - power field ( hpf ) . patients in the advanced stage of the disease ( 35 metastatic cases ) had primary cancer nests that were more infiltrated by ( ie ) cd56 + cells than patients in the locally limited stage ( 41 non - metastatic cases ) ( median 2 vs. 1.67 , p = 0.05 ) . no differences were observed in all compared clinicopathological features between cases with and without ( ie ) cd56 + infiltrates ( table 1 ) . the lack of cd56 + cells in lymph node metastases had no impact on overall survival ( p = 0.3012 ) . the number of ie cd56 + ( per hpf ) was correlated with the depth of invasion and recurrence , while the intensity of the ie grb+ lymphocytes was correlated with age and tumor grade based on a 2-tier scale : g1 versus g2 + g3 ( table 2).table 2correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patientsclinicopathological featureie cd56 + p ie gzb+ p age r = 0.1260.280 r = 0.333 0.004 pt r = 0.0010.992 r = 0.0210.862depth of invasion r = 0.339 0.003 r = 0.1960.102g1/g2/g3 r = 0.0230.849 r = 0.2070.080g1/g2 + g3 r = 0.0930.432 r = 0.304 0.009 pn r = 0.2220.0570.2160.069figo stage i / ii / iii / iv r = 0.2080.075 r = 0.2260.056recurrence + / r = 0.295 0.011 r = 0.1090.362bold values indicate statistical significance correlation of ie cd56 + and ie grb+ infiltrates with clinicopathological features of vscc patients bold values indicate statistical significance patients were divided into grb low- and high - intensity groups ( low grb+ and high grb + , respectively ) based on the median value of ie grb+ cells . with the exception of age ( high grb+ patients were older than low grb+ patients ) , no differences were observed between groups in the pathological features analyzed here ( table 3).table 3differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point)clinicopathological featurelow ( ie)grb+high ( ie)grb+plow ( ie)cd56+high ( ie)cd56+page /median/6571 0.011 69690.996depth of invasion /median/77.40.3556.257.750.01g1/g2/g315/9/89/20/110.07017/15/139/14/70.509g1/g2 + g315/179/310.04417/289/210.622pt30/1/135/5/00.20241/3/127/3/00.632meta+/meta12/2022/180.16117/2817/130.155figo stage i / ii / iii / iv19/1/11/117/1/19/30.49627/1/15/212/1/15/20.410recurrence + /5/2710/300.3925/4010/200.036bold values indicate statistical significance differences in clinicopathological features between patients having primary tumors infiltrated with low and high numbers of intraepithelial grb+ and cd56 + cells ( median as a cutoff point ) bold values indicate statistical significance while no differences in overall survival ( os ) were observed between high grb+ and low grb+ cases in the general cohort ( fig . cox p = 0.479 ) , more ie grb+ cells than the median were correlated with longer os among cases with local disease ( f cox p = 0.028 ) ( fig . d low grb+/high grb+ infiltrates within cancer nests in non - metastatic cases kaplan meier survival curves for overall survival of patients . a low cd56+/high cd56 + infiltrates within cancer nests in the general population . 2c ) , intensities of ie cd56 + cells exceeding the median ( high - cd56 + ) were correlated with longer os among patients with metastatic dissemination ( f cox p = 0.009 ) ( fig . the distribution of cd56 + cells and grb expressing cells at the primary site differed in the vscc cases analyzed here . cd56 + cells were mostly detected within the cancer nests , while the grb+ cells were predominant in the tumor stroma . the higher stromal distribution of grb+ cells suggests that the vast majority of immune effectors are activated outside the cancer nests , chiefly through mechanisms unrelated to the recognition of vscc antigens . moreover , the large discrepancy between ie grb + infiltrates ( median 3 ) and the cumulative number of intraepithelial immune effectors ( median 2 for ie cd56 + and median 19 for ie cd8 + infiltrates ) recently described by our group for this cohort could suggest that the cytolytic power of the immune system has been lost outside rather than inside cancer nests . we found that the median numbers of ie grb+ and ie cd56 + cells were comparable ( 3 and 2 per hpf within the primary cancer nest and 0.83 and 1.016 per hpf within lymph node metastases , respectively ) . this is supported by further results indicating that ie cd56 + and ie grb+ infiltrates have separate correlations with clinico - pathological features in patients with vscc . the intensity of ie cd56 + cells was significantly correlated with depth of invasion and recurrence , while the intensity of ie grb+ lymphocytes was correlated with age and tumor grade . patients in the advanced stage of the disease had primary cancer nests that were more infiltrated by ie cd56 + cells than cases in the locally limited stage . however , neither high ie cd56 + nor high ie grb+ infiltrates had an impact on overall survival in vscc patients . various proportions of the adaptive and innate effectors could be involved in granzyme b - dependent cytotoxicity because of the diverse expression of mhc antigens , costimulatory molecules and antigen - presenting cell ( apc ) statuses in different cancers , as well as the different number of regulatory cells within tumor tissue . interestingly , the correlation between the intensity of ie cd56 + ( nk / nkt ) cells and survival was observed among metastatic vscc patients , while the prognostic significance of granzyme b - dependent killing was not observed in this subgroup . ie grb+ infiltrates , which indicate the cytolytic power of combined adaptive and innate immune effectors , and ie cd56 + infiltrates , which indicate nk / nkt cells of unknown functional status , correlate with longer os among non - metastatic and metastatic vscc cases , respectively .

the study population consisted of 539 consecutive patients with diabetes who presented to our outpatient clinic or were admitted to our hospital for cardiac evaluation ( exercise electrocardiogram , stress echocardiography , or invasive coronary angiography ) because of suspected cad ( new - onset chest pain , abnormal stress test , multiple cardiovascular risk factors including diabetes ) between january 2006 and september 2007 . in all , a total of 90 patients were excluded because of known cad ( 42 patients , of whom 27 had previous myocardial infarction and 15 had previous coronary revascularization ) or other known cardiovascular diseases ( 48 patients , of whom 10 had heart failure , 4 had congenital heart disease , 20 had valvular disease , 7 had cardiomyopathy , 7 had aortic aneurysm ) . other exclusion criteria were contraindications to contrast agents ( five patients ) , impaired renal function ( creatinine clearance < 60 ml / min ) ( eight patients ) , inability to sustain a 15-s breath hold ( two patients ) , and arrhythmias ( five patients ) . a previous diagnosis of diabetes had been confirmed by a diabetologist using serial fasting plasma glucose evaluations . sixty - nine patients were classified as having type 1 diabetes and 321 had type 2 diabetes . the study was approved by our institution s scientific and ethical committees , and all patients gave written informed consent . a structured interview was performed and a clinical history was acquired ; the following cardiac risk factors were assessed before mdct - ca : diabetes ( glucose level of 7 mmol / l or the need for medications ) ( 11 ) , hypercholesterolemia ( cholesterol level 5 mmol / l or treatment with lipid - lowering drugs ) ( 12 ) , hypertension ( blood pressure 140/90 mmhg or use of antihypertensive medications ) ( 13 ) , positive family history of cad ( presence of cad in first - degree relatives younger than 55 years [ men ] or 65 years [ women ] ) , ( 14 ) and current smoking . pretest probability of cad was determined using the diamond - forrester method ( 15 ) . metoprolol was administered intravenously before mdct - ca with a titration dose up to 20 mg in patients with a heart rate > 65 beats per minute . in all patients , mdct - ca was performed using a 64-slice scanner ( vct , ge medical system , milwaukee , wi ; 64 0.625 mm collimation , 330-ms gantry rotation time , 100 kvp tube voltage , and 600 ma tube current ) . electrocardiographic gating for a maximum gantry delivery between 40 and 80% during the r - r interval . a bolus of 80 ml of high - concentration contrast ( iomeron 400 mg / ml , bracco , milan , italy ) was administered intravenously at 5 ml / s , followed by 50 ml of saline injected at same infusion rate . image data sets were analyzed using multiplanar reconstruction on postprocessing workstations ( cardioq3 package , advantage workstation version 4.2 , ge healthcare , milwaukee , wi ) . the effective dose for mdct - ca was calculated as the product of the dose - length product multiplied by a conversion coefficient for the chest ( k = 0.014 msv / mgy cm ) . all mdct - ca examinations were evaluated by two expert readers who were unaware of the patients clinical data . in the case of disagreement , coronary arteries were divided into 16 segments according to american heart association classification ( 16 ) . patients were excluded when proximal or mid - segment or more than 3 segments were not interpretable ( 4 ) . coronary plaques were defined as structures > 1 mm within artery lumen , adjacent to artery lumen , or both and clearly distinguishable from vessel lumen and surrounding pericardial tissue ( 4 ) . one coronary plaque was assigned per coronary segment , even in the presence of multiple plaques . plaque type was determined using the following classification : 1 ) noncalcified ( plaques having a lower density compared with the contrast - enhanced vessel lumen ) ; 2 ) calcified ( high - density plaques ) ; and 3 ) mixed ( noncalcified and calcified components within a single plaque ) . if a segment contained calcified and noncalcified plaques , we classified the plaque as calcified . vessel segments were graded on the basis of visually estimated obstruction of coronary lumen as normal , nonobstructive lesions ( < 50% ) and obstructive lesions ( 50% ) . patients were divided into three groups : normal ( no coronary plaques ) , nonobstructive cad ( lesions < 50% ) , and obstructive cad ( lesions 50% ) . moreover , coronary arteries were analyzed using two evaluation methods : the presence of obstructive lesions in major epicardial vessels and coronary plaque scores ( 4 ) . to this purpose , we first analyzed mdct - ca scans , and the number of major epicardial vessels exhibiting 50% stenosis was recorded . patients with obstructive cad in diagonal or obtuse marginal branches were included in the left anterior descending and left circumflex artery obstructive cad groups , respectively . in case of obstructive stenosis of the left main coronary artery ( lmca ) , patients were assigned to this category even if they had other diseased vessels . second , we analyzed the extent of atherosclerotic burden using two coronary artery plaque scores ( 4 ) : 1 ) the segment - involvement score ( sis ) , that is , the number of segments ( minimum = 0 ; maximum = 16 ) with at least one plaque , irrespective of the degree of stenosis ; and 2 ) the segment - stenosis score ( sss ) , that is , the overall extent of coronary artery plaque . with the latter score , each coronary segment was graded as having no to severe plaque ( i.e. , score from 0 to 3 , with no plaque = 0 , stenosis < 50% = 1 , 5069% stenosis = 2 , and 70% stenosis = 3 ) , based on the extent of obstruction of coronary lumen diameter . the sss of the 16 coronary segments were summed to yield a total score , ranging from 0 to 48 . for both scores , a cut off of 5 was used to differentiate patients with a low or high probability of cardiac events ( 4 ) . follow - up , either a clinical visit or telephone interview , was performed by researchers who were blinded to mdct - ca data . our hospital records and outsourced clinical documents were screened for clinical events to confirm the information obtained . to avoid missing cardiac events , correspondence with treating physicians outcome measures were a composite of hard cardiac events ( cardiac death , nonfatal myocardial infarction , and unstable angina requiring hospitalization ) and all cardiac events ( cardiac death , nonfatal myocardial infarction , unstable angina requiring hospitalization , and revascularization ) . all deaths were reviewed and classified as cardiac ( death caused by acute myocardial infarction , ventricular arrhythmias , or refractory heart failure ) or noncardiac . all revascularizations were classified as early ( elective revascularization within 60 days after mdct - ca ) or late . only late revascularizations were considered as cardiac events , whereas patients with elective early revascularization were excluded from the analysis . the diagnosis of nonfatal myocardial infarction was based on the presence of typical chest pain , elevated cardiac enzymes , and typical electrocardiographic changes ( 17 ) . unstable angina was defined as acute chest pain with or without the presence of electrocardiographic abnormalities and no cardiac enzyme elevation ( 18 ) . statistical analysis was performed using sas ( version 9.1.3 , sas institute inc . , cary , nc ) and spss software ( version 13.0 , spss inc . , continuous variables are presented as mean sd , and discrete variables as absolute numbers and percentages . to compare patient characteristics and mdct - ca data , or fisher exact tests were used for categorical variables and the student t test , continuous variables were expressed as median ( 25th to 75th percentile range ) and compared using the nonparametric mann - whitney u test . to identify the association between mdct - ca variables and outcomes , first , univariate analysis of clinical characteristics and mdct - ca variables was performed to identify potential predictors . hazard ratios ( hrs ) were calculated with 95% cis as an estimate of the risk associated with a particular variable . to determine independent predictors of the composite end points , multivariate analysis of mdct - ca variables with p 0.05 in univariate analysis was performed , which was corrected for baseline characteristics ( male sex , age , hypercholesterolemia , hypertension , family history of cad , smoking ) . moreover , another multivariate analysis of mdct - ca variables stratified by age ( < 65 vs. 65 years ) and by sex was performed . cumulative event - free survival rates as a function over time were obtained using the kaplan - meier method . hard and all cardiac event - free survival curves were compared using the log - rank test . the study population consisted of 539 consecutive patients with diabetes who presented to our outpatient clinic or were admitted to our hospital for cardiac evaluation ( exercise electrocardiogram , stress echocardiography , or invasive coronary angiography ) because of suspected cad ( new - onset chest pain , abnormal stress test , multiple cardiovascular risk factors including diabetes ) between january 2006 and september 2007 . in all , a total of 90 patients were excluded because of known cad ( 42 patients , of whom 27 had previous myocardial infarction and 15 had previous coronary revascularization ) or other known cardiovascular diseases ( 48 patients , of whom 10 had heart failure , 4 had congenital heart disease , 20 had valvular disease , 7 had cardiomyopathy , 7 had aortic aneurysm ) . other exclusion criteria were contraindications to contrast agents ( five patients ) , impaired renal function ( creatinine clearance < 60 ml / min ) ( eight patients ) , inability to sustain a 15-s breath hold ( two patients ) , and arrhythmias ( five patients ) . a previous diagnosis of diabetes had been confirmed by a diabetologist using serial fasting plasma glucose evaluations . sixty - nine patients were classified as having type 1 diabetes and 321 had type 2 diabetes . the study was approved by our institution s scientific and ethical committees , and all patients gave written informed consent . a structured interview was performed and a clinical history was acquired ; the following cardiac risk factors were assessed before mdct - ca : diabetes ( glucose level of 7 mmol / l or the need for medications ) ( 11 ) , hypercholesterolemia ( cholesterol level 5 mmol / l or treatment with lipid - lowering drugs ) ( 12 ) , hypertension ( blood pressure 140/90 mmhg or use of antihypertensive medications ) ( 13 ) , positive family history of cad ( presence of cad in first - degree relatives younger than 55 years [ men ] or 65 years [ women ] ) , ( 14 ) and current smoking . pretest probability of cad was determined using the diamond - forrester method ( 15 ) . metoprolol was administered intravenously before mdct - ca with a titration dose up to 20 mg in patients with a heart rate > 65 beats per minute . in all patients , mdct - ca was performed using a 64-slice scanner ( vct , ge medical system , milwaukee , wi ; 64 0.625 mm collimation , 330-ms gantry rotation time , 100 kvp tube voltage , and 600 ma tube current ) . electrocardiographic gating for a maximum gantry delivery between 40 and 80% during the r - r interval . a bolus of 80 ml of high - concentration contrast ( iomeron 400 mg / ml , bracco , milan , italy ) was administered intravenously at 5 ml / s , followed by 50 ml of saline injected at same infusion rate . image data sets were analyzed using multiplanar reconstruction on postprocessing workstations ( cardioq3 package , advantage workstation version 4.2 , ge healthcare , milwaukee , wi ) . the effective dose for mdct - ca was calculated as the product of the dose - length product multiplied by a conversion coefficient for the chest ( k = 0.014 msv / mgy cm ) . all mdct - ca examinations were evaluated by two expert readers who were unaware of the patients clinical data . in the case of disagreement , coronary arteries were divided into 16 segments according to american heart association classification ( 16 ) . patients were excluded when proximal or mid - segment or more than 3 segments were not interpretable ( 4 ) . coronary plaques were defined as structures > 1 mm within artery lumen , adjacent to artery lumen , or both and clearly distinguishable from vessel lumen and surrounding pericardial tissue ( 4 ) . one coronary plaque was assigned per coronary segment , even in the presence of multiple plaques . plaque type was determined using the following classification : 1 ) noncalcified ( plaques having a lower density compared with the contrast - enhanced vessel lumen ) ; 2 ) calcified ( high - density plaques ) ; and 3 ) mixed ( noncalcified and calcified components within a single plaque ) . if a segment contained calcified and noncalcified plaques , we classified the plaque as calcified . vessel segments were graded on the basis of visually estimated obstruction of coronary lumen as normal , nonobstructive lesions ( < 50% ) and obstructive lesions ( 50% ) . patients were divided into three groups : normal ( no coronary plaques ) , nonobstructive cad ( lesions < 50% ) , and obstructive cad ( lesions 50% ) . moreover , coronary arteries were analyzed using two evaluation methods : the presence of obstructive lesions in major epicardial vessels and coronary plaque scores ( 4 ) . to this purpose , we first analyzed mdct - ca scans , and the number of major epicardial vessels exhibiting 50% stenosis was recorded . patients with obstructive cad in diagonal or obtuse marginal branches were included in the left anterior descending and left circumflex artery obstructive cad groups , respectively . in case of obstructive stenosis of the left main coronary artery ( lmca ) , patients were assigned to this category even if they had other diseased vessels . second , we analyzed the extent of atherosclerotic burden using two coronary artery plaque scores ( 4 ) : 1 ) the segment - involvement score ( sis ) , that is , the number of segments ( minimum = 0 ; maximum = 16 ) with at least one plaque , irrespective of the degree of stenosis ; and 2 ) the segment - stenosis score ( sss ) , that is , the overall extent of coronary artery plaque . with the latter score , each coronary segment was graded as having no to severe plaque ( i.e. , score from 0 to 3 , with no plaque = 0 , stenosis < 50% = 1 , 5069% stenosis = 2 , and 70% stenosis = 3 ) , based on the extent of obstruction of coronary lumen diameter . the sss of the 16 coronary segments were summed to yield a total score , ranging from 0 to 48 . for both scores , a cut off of 5 was used to differentiate patients with a low or high probability of cardiac events ( 4 ) . follow - up , either a clinical visit or telephone interview , was performed by researchers who were blinded to mdct - ca data . our hospital records and outsourced clinical documents were screened for clinical events to confirm the information obtained . to avoid missing cardiac events , correspondence with treating physicians outcome measures were a composite of hard cardiac events ( cardiac death , nonfatal myocardial infarction , and unstable angina requiring hospitalization ) and all cardiac events ( cardiac death , nonfatal myocardial infarction , unstable angina requiring hospitalization , and revascularization ) . all deaths were reviewed and classified as cardiac ( death caused by acute myocardial infarction , ventricular arrhythmias , or refractory heart failure ) or noncardiac . all revascularizations were classified as early ( elective revascularization within 60 days after mdct - ca ) or late . only late revascularizations were considered as cardiac events , whereas patients with elective early revascularization were excluded from the analysis . the diagnosis of nonfatal myocardial infarction was based on the presence of typical chest pain , elevated cardiac enzymes , and typical electrocardiographic changes ( 17 ) . unstable angina was defined as acute chest pain with or without the presence of electrocardiographic abnormalities and no cardiac enzyme elevation ( 18 ) . statistical analysis was performed using sas ( version 9.1.3 , sas institute inc . , cary , nc ) and spss software ( version 13.0 , spss inc . , continuous variables are presented as mean sd , and discrete variables as absolute numbers and percentages . to compare patient characteristics and mdct - ca data , or fisher exact tests were used for categorical variables and the student t test , continuous variables were expressed as median ( 25th to 75th percentile range ) and compared using the nonparametric mann - whitney u test . to identify the association between mdct - ca variables and outcomes , first , univariate analysis of clinical characteristics and mdct - ca variables was performed to identify potential predictors . hazard ratios ( hrs ) were calculated with 95% cis as an estimate of the risk associated with a particular variable . to determine independent predictors of the composite end points , multivariate analysis of mdct - ca variables with p 0.05 in univariate analysis was performed , which was corrected for baseline characteristics ( male sex , age , hypercholesterolemia , hypertension , family history of cad , smoking ) . moreover , another multivariate analysis of mdct - ca variables stratified by age ( < 65 vs. 65 years ) and by sex was performed . cumulative event - free survival rates as a function over time were obtained using the kaplan - meier method . hard and all cardiac event - free survival curves were compared using the log - rank test . of the 429 patients prospectively enrolled , 24 were excluded because mdct - ca images were not interpretable . of the remaining 405 patients , 15 were lost to follow - up ( 3 with normal coronary arteries , 3 with nonobstructive cad , and 9 with obstructive cad ) , whereas 390 ( 98% ) had a complete follow - up ( mean 62 9 months , up to 72 months ) . among these 390 patients , indications for mdct - ca were chest pain ( 35% ) , multiple cardiac risk factors including diabetes ( 38% ) , and equivocal or abnormal stress test ( 27% ) . blood glucose levels were controlled by diet in 40 patients , whereas oral antidiabetic medication and insulin were used in 281 and 69 patients , respectively . patients lost to follow - up had no significant difference in clinical characteristics or mdct - ca results . we recorded 279 events , of which 117 were hard events ( 9 cardiac deaths , 66 nonfatal myocardial infarctions , and 42 cases of unstable angina ) and 142 late revascularizations . prevalence of male sex and hypertension were significantly higher in patients with events than in those without events ( table 1 ) . clinical characteristics of the study population , mdct - ca results , and patient clinical outcomes the mean effective dose of mdct - ca examinations was 8.7 3.5 msv . sis , sss , number of segments with obstructive plaques , and prevalence of obstructive cad , three - vessel disease ( vd ) , and lmca disease were significantly higher in patients with events than in patients without events . univariate clinical predictors of events were male sex , hypertension , high pretest likelihood of cad , and medical therapy with -blockers , aspirin , and statins ( table 2 ) . univariate mdct - ca predictors of events also are reported in table 2 . regarding obstructive cad , hr was 3.41 for hard events and 7.93 for all events . hrs were increased particularly in patients with three - vd ( 5.79 for hard events and 11.62 for all events ) and lmca disease ( 10.57 for hard events and 22.83 for all events ) . clinical characteristics and mdct - ca results and univariate predictors of events significant independent predictors of hard events were 50% three - vd ( hr 5.21 [ 95% ci 1.3320.33 ] ; p = 0.01 ) , 50% lmca disease ( 5.35 [ 1.3920.52 ] ; p = 0.01 ) , and the number of segments with noncalcified ( 1.84 [ 1.492.27 ] ; p < 0.0001 ) , mixed ( 1.39 [ 1.121.72 ] ; p = 0.003 ) , and calcified plaques ( 1.62 [ 1.331.96 ] , p < 0.0001 ) . significant independent predictors of all events were 50% one - vd ( 3.94 [ 1.4910.45 ] ; p = 0.006 ) , 50% two - vd ( 4.82 [ 2.1710.73 ] ; p = 0.0001 ) , 50% three - vd ( 7.93 [ 4.5613.79 ] ; p < 0.0001 ) , 50% lmca disease ( 7.92 [ 2.6223.88 ] ; p = 0.005 ) , and number of segments with mixed ( 1.40 [ 1.221.61 ] ; p < 0.0001 ) and calcified ( 1.18 [ 1.041.35 ] ; p = 0.01 ) plaques . when stratified by age ( < 65 vs. 65 years ) , younger patients experienced a higher hr for hard events for obstructive cad ( 10.13 [ 4.2624.07 ] ; p < 0.0001 vs. 2.96 [ 1.455.99 ] ; p = 0.003 ) . when stratified by sex , women experienced a higher hr for obstructive cad for hard events ( 9.12 [ 4.1121.12 ] ; p < 0.0001 vs. 2.78 [ 1.654.71 ] ; p = 0.0001 ) and for all events ( 17.19 [ 5.3655.1 ] ; p < 0.0001 vs. 4.04 [ 2.267.26 ] ; p < 0.0001 ) . kaplan - meier survival curves about patients with normal coronary arteries , nonobstructive cad , and obstructive cad and about coronary plaque scores are shown in figs . 1 and 2 , respectively . the 62-month cumulative hard and all event - free survival rates were 78% and 56% , respectively , in patients with nonobstructive cad and 60% and 16% , respectively , in those with obstructive cad ( log - rank p = 0.0001 ) ( fig . the relationship between atherosclerotic burden , expressed as sis and sss , and event - free survival rate is reported in fig . cumulative event - free survival was 50% with sis 5 , 16% with sis > 5 , 67% with sss 5 , and 11% with sss > 5 ( log - rank p = 0.0001 ) . regarding hard events , cumulative event - free survival was 77% with sis 5 , 54% with sis > 5 , 88% with sss 5 , and 54% with sss > 5 ( log - rank p = 0.0001 ) . moreover , we evaluated the relationship between cad extension , expressed as the number of major epicardial vessels exhibiting 50% stenosis , and event - free survival rate . regarding all events , cumulative event - free survival was 20% with one - vd , 12% with two - vd , 18% with three - vd , and 0% with lmca disease ( log - rank p = 0.0001 ) . excluding revascularization procedures , cumulative event - free survival was 73% with one - vd , 62% with two - vd , 50% with three - vd , and 25% with lmca disease ( log - rank p = 0.0001 ) . ( b ) in patients with normal coronary arteries , nonobstructive cad , and obstructive cad . ( b ) in patients with sis 5 and > 5 and for all events ( c ) and hard events ( d ) in patients with sss 5 and sss > 5 . sis , sss , number of segments with obstructive plaques , and prevalence of obstructive cad , three - vessel disease ( vd ) , and lmca disease were significantly higher in patients with events than in patients without events . univariate clinical predictors of events were male sex , hypertension , high pretest likelihood of cad , and medical therapy with -blockers , aspirin , and statins ( table 2 ) . univariate mdct - ca predictors of events also are reported in table 2 . regarding obstructive cad , hrs were increased particularly in patients with three - vd ( 5.79 for hard events and 11.62 for all events ) and lmca disease ( 10.57 for hard events and 22.83 for all events ) . significant independent predictors of hard events were 50% three - vd ( hr 5.21 [ 95% ci 1.3320.33 ] ; p = 0.01 ) , 50% lmca disease ( 5.35 [ 1.3920.52 ] ; p = 0.01 ) , and the number of segments with noncalcified ( 1.84 [ 1.492.27 ] ; p < 0.0001 ) , mixed ( 1.39 [ 1.121.72 ] ; p = 0.003 ) , and calcified plaques ( 1.62 [ 1.331.96 ] , p < 0.0001 ) . significant independent predictors of all events were 50% one - vd ( 3.94 [ 1.4910.45 ] ; p = 0.006 ) , 50% two - vd ( 4.82 [ 2.1710.73 ] ; p = 0.0001 ) , 50% three - vd ( 7.93 [ 4.5613.79 ] ; p < 0.0001 ) , 50% lmca disease ( 7.92 [ 2.6223.88 ] ; p = 0.005 ) , and number of segments with mixed ( 1.40 [ 1.221.61 ] ; p < 0.0001 ) and calcified ( 1.18 [ 1.041.35 ] ; p = 0.01 ) plaques . when stratified by age ( < 65 vs. 65 years ) , younger patients experienced a higher hr for hard events for obstructive cad ( 10.13 [ 4.2624.07 ] ; p < 0.0001 vs. 2.96 [ 1.455.99 ] ; p = 0.003 ) . when stratified by sex , women experienced a higher hr for obstructive cad for hard events ( 9.12 [ 4.1121.12 ] ; p < 0.0001 vs. 2.78 [ 1.654.71 ] ; p = 0.0001 ) and for all events ( 17.19 [ 5.3655.1 ] ; p < 0.0001 vs. 4.04 [ 2.267.26 ] ; p < 0.0001 ) . kaplan - meier survival curves about patients with normal coronary arteries , nonobstructive cad , and obstructive cad and about coronary plaque scores are shown in figs . 1 and 2 , respectively . the 62-month cumulative hard and all event - free survival rates were 78% and 56% , respectively , in patients with nonobstructive cad and 60% and 16% , respectively , in those with obstructive cad ( log - rank p = 0.0001 ) ( fig . the relationship between atherosclerotic burden , expressed as sis and sss , and event - free survival rate is reported in fig . cumulative event - free survival was 50% with sis 5 , 16% with sis > 5 , 67% with sss 5 , and 11% with sss > 5 ( log - rank p = 0.0001 ) . regarding hard events , cumulative event - free survival was 77% with sis 5 , 54% with sis > 5 , 88% with sss 5 , and 54% with sss > 5 ( log - rank p = 0.0001 ) . moreover , we evaluated the relationship between cad extension , expressed as the number of major epicardial vessels exhibiting 50% stenosis , and event - free survival rate . regarding all events , cumulative event - free survival was 20% with one - vd , 12% with two - vd , 18% with three - vd , and 0% with lmca disease ( log - rank p = 0.0001 ) . excluding revascularization procedures , cumulative event - free survival was 73% with one - vd , 62% with two - vd , 50% with three - vd , and 25% with lmca disease ( log - rank p = 0.0001 ) . kaplan - meier curves for all events ( a ) and hard events ( b ) in patients with normal coronary arteries , nonobstructive cad , and obstructive cad . kaplan - meier curves for all events ( a ) and hard events ( b ) in patients with sis 5 and > 5 and for all events ( c ) and hard events ( d ) in patients with sss 5 and sss > 5 . in patients with diabetes , cad is the major cause of morbidity , mortality , and medical costs ( 19 ) . indeed , management guidelines in europe and the u.s . therefore , early cad diagnosis in patients with diabetes is of the utmost importance in the attempt to prevent disease progression and clinical events . the american diabetes association recently convened an expert panel that reviewed the issue of cad screening in patients with diabetes aimed at identifying high - risk patients in whom the outcome may be improved through more aggressive risk factor modification , medical surveillance , or revascularization ( 19 ) . unfortunately , the sensitivity of clinical risk assessment is limited in patients with diabetes , mainly because typical symptoms of ischemia often are absent and cardiac stress tests have limited negative predictive value ( 2 ) . indeed , the diagnostic accuracy of exercise electrocardiogram , as well as the specificity of stress echocardiography and nuclear perfusion imaging ( 20,21 ) , are lower in patients with diabetes compared with nondiabetic patients ( 2 ) . mdct - ca currently is considered a reliable diagnostic method for the evaluation of patients with suspected cad because of the high diagnostic performance in ruling out the disease and in detecting obstructive lesions ( 3 ) . several mdct - ca studies demonstrated an increased prevalence of obstructive and nonobstructive cad and fewer normal coronary arteries in patients with diabetes in comparison with nondiabetics ( 22,23 ) . however , only two studies with midterm follow - up evaluated the prognostic value of this imaging modality in patients with diabetes ( 9,10 ) . compared with them , our study has a longer follow - up with a larger group of patients with diabetes who underwent mdct - ca for suspected cad . moreover , patients with any type of known cardiac disease were excluded , making our study population significantly more homogeneous compared with the patients enrolled in previous studies . the main findings of our study are the following : 1 ) mdct - ca is able to provide long - term prognostic information in patients with diabetes with suspected cad and may predict hard cardiac events ; 2 ) patients with diabetes without evidence of cad seen on mdct - ca have an excellent prognosis , with no cardiac events at 62-month follow - up . it is noteworthy that detection of obstructive cad using mdct - ca was a strong predictor of cardiac events in univariate analysis ( hr 3.41 and 7.93 for hard and all events , respectively ) . kaplan - meier survival curves confirmed this finding , showing an event - free survival of 60% for hard events and moreover , mdct - ca allowed prognostic grading according to one - vd , two - vd , three - vd , and lmca disease classification . in both univariate and multivariate analysis , hrs for hard and accordingly , survival free of hard events was reduced progressively from 73% in patients with one - vd to 25% in those with lmca disease . another major finding of this study pertains to patients with diabetes with nonobstructive cad as seen on mdct - ca . among these patients , noninvasive testing is usually negative because this type of lesion rarely triggers myocardial ischemia , suggesting a prognosis similar to that found in patients with normal coronary arteries . however , our data indicate that these patients have a worse long - term outcome compared with patients with diabetes without cad . indeed , kaplan - meier survival curves showed an event - free survival of 78% for hard events and thus , their risk of cardiac event is intermediate , between that found in patients with normal coronary arteries and patients with obstructive cad . first of all , a recent study by van velzen et al . ( 24 ) demonstrated that mdct - ca has an accuracy of 100% in comparison with intravascular ultrasound in the detection of nonobstructive cad . on the other hand , previous studies indicated that plaque composition also may be a predictor of adverse events . they demonstrated that vulnerable plaques may be present across the full spectrum of stenosis severity , suggesting that nonobstructive lesions may also contribute to cardiac events ( 25 ) . in our study , the number of noncalcified plaques had higher hrs for hard events at univariate and multivariate analysis than the number of calcified and mixed plaques . because nonobstructive plaques are more frequent than obstructive plaques , it is conceivable that moderate stenoses are more frequently associated with acute coronary occlusion ( 25 ) . another possible reason for the high prognostic value of nonobstructive cad found in our study may be the long - term follow - up . in fact , we can not rule out that some moderate stenoses at the time of mdct - ca examination could have become obstructive over time . nevertheless , early identification of nonobstructive cad with mdct - ca in patients with diabetes is clinically important because it may lead to a more aggressive strategy of cardiovascular risk factor control and modification of clinical follow - up . this is in agreement with the recommendations of the american diabetes association expert panel suggesting more aggressive risk factor modification and medical surveillance in high - risk patients with diabetes ( 19 ) . moreover , mdct - ca also demonstrated the ability to stratify the prognosis of patients with diabetes according to their atherosclerotic burden . ( 9 ) were able to stratify cardiac events using the mdct - ca atherosclerotic burden in patients with diabetes . our study also demonstrates that mdct - ca is able to predict events on the basis of atherosclerotic burden evaluated using coronary artery plaque scores . indeed , event - free survival significantly decreased at 62-month follow - up , from 67% for sss 5 to 11% for sss > 5 , considering revascularizations , and from 88% for sss 5 to 54% for sss > 5 , excluding revascularizations . another remarkable finding is that the atherosclerotic burden maintains a similar prognostic value using sis . another important result of our study relates to patients with diabetes with normal coronary arteries at mdct - ca . in agreement with a previous study that enrolled a smaller number of less homogeneous patients with diabetes and had shorter follow - up ( 10 ) , our study found that the absence of cad , found in 90 patients with diabetes , is associated with an event - free survival of 100% for both hard and all events at 62-month follow - up . the excellent outcome observed in this subset of patients is clinically relevant because it suggests that mdct - ca , in contrast to other imaging modalities , can help to identify the truly low - risk patients with diabetes . although nuclear stress imaging and stress echocardiography , the two stress imaging tests used most widely in clinical practice , demonstrated good prognostic value in the general population with suspected cad ( 19 ) , their performance was found to be lower in studies that evaluated patients with diabetes . the prognostic value of single - photon emission computed tomography imaging in patients with diabetes was assessed in seven large studies . similar to what has been shown in nondiabetic patients , their results confirmed the higher event rate in the presence of an abnormal scan compared with a normal scan . however , the event rate was higher compared with the general population , even in the presence of a normal scan ( 19 ) . similar results were reported by another five large studies that evaluated the prognostic value of stress echocardiography in patients with diabetes ( 19 ) . in one of these studies , kamalesh et al . ( 2 ) demonstrated that patients with diabetes with a negative stress echocardiogram had a significantly higher incidence of events compared with nondiabetics ( 19 ) . ( 26 ) , who found that patients with diabetes with normal exercise echocardiography had an increase in cardiac events ( up to 7.6% ) at 5-year follow - up , despite an event - free survival at 1 year . interestingly , in our study , patients with diabetes with normal coronary arteries seen on mdct - ca were event free at 5-year ( mean 62 9 months , up to 72 months ) follow - up . therefore , this imaging modality can be a useful tool to reassure patients with diabetes with suspected cad regarding their outcome , with a warranty period of at least 5 years in the presence of a normal result . in conclusion , these results , if confirmed in further studies with larger patient populations , suggest that mdct - ca could be used in patients with diabetes as a screening diagnostic modality to stratify the cardiovascular prognosis . we believe that this approach could be particularly useful in specific subsets of patients with diabetes with unknown cad and equivocal or uninterpretable stress tests or in case of a discrepancy between clinical presentation and stress test results . indeed , mdct - ca already has demonstrated its utility in these clinical settings ( 27 ) . moreover , the new generation of scanners has demonstrated the ability to evaluate coronary arteries with high diagnostic performance and a lower radiation exposure ( < 2 msv ) compared with that of the previous generation of scanners and traditional invasive coronary angiography ( 28 ) . first , this is a relatively small , single - center study evaluating mainly caucasian patients . thus , the results may not necessarily reflect the patient population of other centers or countries , especially in terms of potential age - related differences in the study population . second , we recognize that incomplete follow - up may result in underreporting of cardiac events , and we are not able to exclude the possibility that a few patients did not report some events . however , the percentage of patients with complete follow - up was remarkably high ( 98% ) . third , mdct - ca allowed the identification of patients with obstructive cad , likely resulting in an increased revascularization rate , which constituted a large proportion of the composite all cardiac event end point . however , in our study , mdct - ca was performed in addition to a standard diagnostic work - up . moreover , the decision regarding revascularization was based on symptoms , the presence of ischemia on noninvasive testing , and invasive coronary angiography results ; patients with early elective revascularizations were excluded from survival analysis and the presence of obstructive cad at mdct - ca was strongly associated with hard cardiac events . fourth , although our study demonstrated an important prognostic role of mdct - ca anatomical evaluation alone , assessment of the amount and distribution of regional ischemia documented by nuclear imaging would have been important to know the potential complementary prognostic value of mdct - ca and nuclear stress tests in our patients . fifth , although this study demonstrated that mdct - ca is able to predict the prognosis of diabetic patients on the basis of the presence / extent of cad and plaque type , coronary imaging by mdct - ca is not , as in case of invasive angiography , able to predict which plaque may progress to destabilization and rupture , potentially causing a clinical event . finally , in the subset of newly diagnosed type 2 patients with diabetes , a comparison between the prognostic value of mdct - ca and that of the uk prospective diabetes study risk engine would have been useful to demonstrate a potential additional value of mdct - ca .

objectiveto assess the prognostic role of multidetector computed tomography coronary angiography ( mdct - ca ) in patients with diabetes with suspected coronary artery disease ( cad ) . use of mdct - ca is increasing in patients with suspected cad . however , data supporting its prognostic value in patients with diabetes are limited.research design and methodsbetween january 2006 and september 2007 , 429 consecutive diabetic patients were prospectively studied with mdct - ca for detecting the presence and assessing the extent of cad ( disease extension and coronary plaque scores ) . patients were classified according to the presence of normal coronary arteries and nonobstructive ( < 50% ) and obstructive ( 50% ) coronary lesions . the composite rates of hard cardiac events ( cardiac death , nonfatal myocardial infarction , unstable angina ) and all cardiac events ( including revascularization ) were the end points of the study.resultstwenty-four patients were excluded because mdct - ca data were not able to be interpreted . of the remaining 405 patients , clinical follow - up ( mean 62 9 months ) was obtained in 390 ( 98% ) . multivariate analysis showed that predictors of hard and all events were obstructive cad , three - vessel cad , and left main coronary artery ( lmca ) disease . cumulative event - free survival was 100% for hard and all events in patients with normal coronary arteries , 78% for hard events and 56% for all events in patients with nonobstructive cad , and 60% for hard events and 16% for all events in patients with obstructive cad . three - vessel cad and lmca disease were associated with a higher rate of hard cardiac events.conclusionsmdct-ca provides long - term prognostic information for patients with diabetes with suspected cad , showing excellent prognosis when there is no evidence of atherosclerosis and allowing risk stratification when cad is present .

RESEARCH DESIGN AND METHODS Patients and study protocol MDCT-CA scan protocol, image reconstruction, and patient preparation MDCT-CA data analysis Follow-up Statistical analysis RESULTS MDCT-CA results Univariate predictors of events Multivariate predictors of events Survival analysis CONCLUSIONS

the study population consisted of 539 consecutive patients with diabetes who presented to our outpatient clinic or were admitted to our hospital for cardiac evaluation ( exercise electrocardiogram , stress echocardiography , or invasive coronary angiography ) because of suspected cad ( new - onset chest pain , abnormal stress test , multiple cardiovascular risk factors including diabetes ) between january 2006 and september 2007 . in all , a total of 90 patients were excluded because of known cad ( 42 patients , of whom 27 had previous myocardial infarction and 15 had previous coronary revascularization ) or other known cardiovascular diseases ( 48 patients , of whom 10 had heart failure , 4 had congenital heart disease , 20 had valvular disease , 7 had cardiomyopathy , 7 had aortic aneurysm ) . a structured interview was performed and a clinical history was acquired ; the following cardiac risk factors were assessed before mdct - ca : diabetes ( glucose level of 7 mmol / l or the need for medications ) ( 11 ) , hypercholesterolemia ( cholesterol level 5 mmol / l or treatment with lipid - lowering drugs ) ( 12 ) , hypertension ( blood pressure 140/90 mmhg or use of antihypertensive medications ) ( 13 ) , positive family history of cad ( presence of cad in first - degree relatives younger than 55 years [ men ] or 65 years [ women ] ) , ( 14 ) and current smoking . patients were divided into three groups : normal ( no coronary plaques ) , nonobstructive cad ( lesions < 50% ) , and obstructive cad ( lesions 50% ) . moreover , coronary arteries were analyzed using two evaluation methods : the presence of obstructive lesions in major epicardial vessels and coronary plaque scores ( 4 ) . in case of obstructive stenosis of the left main coronary artery ( lmca ) , patients were assigned to this category even if they had other diseased vessels . to avoid missing cardiac events , correspondence with treating physicians outcome measures were a composite of hard cardiac events ( cardiac death , nonfatal myocardial infarction , and unstable angina requiring hospitalization ) and all cardiac events ( cardiac death , nonfatal myocardial infarction , unstable angina requiring hospitalization , and revascularization ) . to determine independent predictors of the composite end points , multivariate analysis of mdct - ca variables with p 0.05 in univariate analysis was performed , which was corrected for baseline characteristics ( male sex , age , hypercholesterolemia , hypertension , family history of cad , smoking ) . the study population consisted of 539 consecutive patients with diabetes who presented to our outpatient clinic or were admitted to our hospital for cardiac evaluation ( exercise electrocardiogram , stress echocardiography , or invasive coronary angiography ) because of suspected cad ( new - onset chest pain , abnormal stress test , multiple cardiovascular risk factors including diabetes ) between january 2006 and september 2007 . in all , a total of 90 patients were excluded because of known cad ( 42 patients , of whom 27 had previous myocardial infarction and 15 had previous coronary revascularization ) or other known cardiovascular diseases ( 48 patients , of whom 10 had heart failure , 4 had congenital heart disease , 20 had valvular disease , 7 had cardiomyopathy , 7 had aortic aneurysm ) . a structured interview was performed and a clinical history was acquired ; the following cardiac risk factors were assessed before mdct - ca : diabetes ( glucose level of 7 mmol / l or the need for medications ) ( 11 ) , hypercholesterolemia ( cholesterol level 5 mmol / l or treatment with lipid - lowering drugs ) ( 12 ) , hypertension ( blood pressure 140/90 mmhg or use of antihypertensive medications ) ( 13 ) , positive family history of cad ( presence of cad in first - degree relatives younger than 55 years [ men ] or 65 years [ women ] ) , ( 14 ) and current smoking . patients were divided into three groups : normal ( no coronary plaques ) , nonobstructive cad ( lesions < 50% ) , and obstructive cad ( lesions 50% ) . in case of obstructive stenosis of the left main coronary artery ( lmca ) , patients were assigned to this category even if they had other diseased vessels . to avoid missing cardiac events , correspondence with treating physicians outcome measures were a composite of hard cardiac events ( cardiac death , nonfatal myocardial infarction , and unstable angina requiring hospitalization ) and all cardiac events ( cardiac death , nonfatal myocardial infarction , unstable angina requiring hospitalization , and revascularization ) . to determine independent predictors of the composite end points , multivariate analysis of mdct - ca variables with p 0.05 in univariate analysis was performed , which was corrected for baseline characteristics ( male sex , age , hypercholesterolemia , hypertension , family history of cad , smoking ) . moreover , another multivariate analysis of mdct - ca variables stratified by age ( < 65 vs. 65 years ) and by sex was performed . of the 429 patients prospectively enrolled , 24 were excluded because mdct - ca images were not interpretable . of the remaining 405 patients , 15 were lost to follow - up ( 3 with normal coronary arteries , 3 with nonobstructive cad , and 9 with obstructive cad ) , whereas 390 ( 98% ) had a complete follow - up ( mean 62 9 months , up to 72 months ) . we recorded 279 events , of which 117 were hard events ( 9 cardiac deaths , 66 nonfatal myocardial infarctions , and 42 cases of unstable angina ) and 142 late revascularizations . clinical characteristics of the study population , mdct - ca results , and patient clinical outcomes the mean effective dose of mdct - ca examinations was 8.7 3.5 msv . sis , sss , number of segments with obstructive plaques , and prevalence of obstructive cad , three - vessel disease ( vd ) , and lmca disease were significantly higher in patients with events than in patients without events . regarding obstructive cad , hr was 3.41 for hard events and 7.93 for all events . hrs were increased particularly in patients with three - vd ( 5.79 for hard events and 11.62 for all events ) and lmca disease ( 10.57 for hard events and 22.83 for all events ) . clinical characteristics and mdct - ca results and univariate predictors of events significant independent predictors of hard events were 50% three - vd ( hr 5.21 [ 95% ci 1.3320.33 ] ; p = 0.01 ) , 50% lmca disease ( 5.35 [ 1.3920.52 ] ; p = 0.01 ) , and the number of segments with noncalcified ( 1.84 [ 1.492.27 ] ; p < 0.0001 ) , mixed ( 1.39 [ 1.121.72 ] ; p = 0.003 ) , and calcified plaques ( 1.62 [ 1.331.96 ] , p < 0.0001 ) . significant independent predictors of all events were 50% one - vd ( 3.94 [ 1.4910.45 ] ; p = 0.006 ) , 50% two - vd ( 4.82 [ 2.1710.73 ] ; p = 0.0001 ) , 50% three - vd ( 7.93 [ 4.5613.79 ] ; p < 0.0001 ) , 50% lmca disease ( 7.92 [ 2.6223.88 ] ; p = 0.005 ) , and number of segments with mixed ( 1.40 [ 1.221.61 ] ; p < 0.0001 ) and calcified ( 1.18 [ 1.041.35 ] ; p = 0.01 ) plaques . when stratified by sex , women experienced a higher hr for obstructive cad for hard events ( 9.12 [ 4.1121.12 ] ; p < 0.0001 vs. 2.78 [ 1.654.71 ] ; p = 0.0001 ) and for all events ( 17.19 [ 5.3655.1 ] ; p < 0.0001 vs. 4.04 [ 2.267.26 ] ; p < 0.0001 ) . kaplan - meier survival curves about patients with normal coronary arteries , nonobstructive cad , and obstructive cad and about coronary plaque scores are shown in figs . the 62-month cumulative hard and all event - free survival rates were 78% and 56% , respectively , in patients with nonobstructive cad and 60% and 16% , respectively , in those with obstructive cad ( log - rank p = 0.0001 ) ( fig . regarding hard events , cumulative event - free survival was 77% with sis 5 , 54% with sis > 5 , 88% with sss 5 , and 54% with sss > 5 ( log - rank p = 0.0001 ) . regarding all events , cumulative event - free survival was 20% with one - vd , 12% with two - vd , 18% with three - vd , and 0% with lmca disease ( log - rank p = 0.0001 ) . excluding revascularization procedures , cumulative event - free survival was 73% with one - vd , 62% with two - vd , 50% with three - vd , and 25% with lmca disease ( log - rank p = 0.0001 ) . ( b ) in patients with normal coronary arteries , nonobstructive cad , and obstructive cad . ( b ) in patients with sis 5 and > 5 and for all events ( c ) and hard events ( d ) in patients with sss 5 and sss > 5 . sis , sss , number of segments with obstructive plaques , and prevalence of obstructive cad , three - vessel disease ( vd ) , and lmca disease were significantly higher in patients with events than in patients without events . regarding obstructive cad , hrs were increased particularly in patients with three - vd ( 5.79 for hard events and 11.62 for all events ) and lmca disease ( 10.57 for hard events and 22.83 for all events ) . significant independent predictors of hard events were 50% three - vd ( hr 5.21 [ 95% ci 1.3320.33 ] ; p = 0.01 ) , 50% lmca disease ( 5.35 [ 1.3920.52 ] ; p = 0.01 ) , and the number of segments with noncalcified ( 1.84 [ 1.492.27 ] ; p < 0.0001 ) , mixed ( 1.39 [ 1.121.72 ] ; p = 0.003 ) , and calcified plaques ( 1.62 [ 1.331.96 ] , p < 0.0001 ) . significant independent predictors of all events were 50% one - vd ( 3.94 [ 1.4910.45 ] ; p = 0.006 ) , 50% two - vd ( 4.82 [ 2.1710.73 ] ; p = 0.0001 ) , 50% three - vd ( 7.93 [ 4.5613.79 ] ; p < 0.0001 ) , 50% lmca disease ( 7.92 [ 2.6223.88 ] ; p = 0.005 ) , and number of segments with mixed ( 1.40 [ 1.221.61 ] ; p < 0.0001 ) and calcified ( 1.18 [ 1.041.35 ] ; p = 0.01 ) plaques . when stratified by sex , women experienced a higher hr for obstructive cad for hard events ( 9.12 [ 4.1121.12 ] ; p < 0.0001 vs. 2.78 [ 1.654.71 ] ; p = 0.0001 ) and for all events ( 17.19 [ 5.3655.1 ] ; p < 0.0001 vs. 4.04 [ 2.267.26 ] ; p < 0.0001 ) . kaplan - meier survival curves about patients with normal coronary arteries , nonobstructive cad , and obstructive cad and about coronary plaque scores are shown in figs . the 62-month cumulative hard and all event - free survival rates were 78% and 56% , respectively , in patients with nonobstructive cad and 60% and 16% , respectively , in those with obstructive cad ( log - rank p = 0.0001 ) ( fig . regarding hard events , cumulative event - free survival was 77% with sis 5 , 54% with sis > 5 , 88% with sss 5 , and 54% with sss > 5 ( log - rank p = 0.0001 ) . regarding all events , cumulative event - free survival was 20% with one - vd , 12% with two - vd , 18% with three - vd , and 0% with lmca disease ( log - rank p = 0.0001 ) . excluding revascularization procedures , cumulative event - free survival was 73% with one - vd , 62% with two - vd , 50% with three - vd , and 25% with lmca disease ( log - rank p = 0.0001 ) . kaplan - meier curves for all events ( a ) and hard events ( b ) in patients with normal coronary arteries , nonobstructive cad , and obstructive cad . kaplan - meier curves for all events ( a ) and hard events ( b ) in patients with sis 5 and > 5 and for all events ( c ) and hard events ( d ) in patients with sss 5 and sss > 5 . mdct - ca currently is considered a reliable diagnostic method for the evaluation of patients with suspected cad because of the high diagnostic performance in ruling out the disease and in detecting obstructive lesions ( 3 ) . several mdct - ca studies demonstrated an increased prevalence of obstructive and nonobstructive cad and fewer normal coronary arteries in patients with diabetes in comparison with nondiabetics ( 22,23 ) . however , only two studies with midterm follow - up evaluated the prognostic value of this imaging modality in patients with diabetes ( 9,10 ) . compared with them , our study has a longer follow - up with a larger group of patients with diabetes who underwent mdct - ca for suspected cad . the main findings of our study are the following : 1 ) mdct - ca is able to provide long - term prognostic information in patients with diabetes with suspected cad and may predict hard cardiac events ; 2 ) patients with diabetes without evidence of cad seen on mdct - ca have an excellent prognosis , with no cardiac events at 62-month follow - up . it is noteworthy that detection of obstructive cad using mdct - ca was a strong predictor of cardiac events in univariate analysis ( hr 3.41 and 7.93 for hard and all events , respectively ) . kaplan - meier survival curves confirmed this finding , showing an event - free survival of 60% for hard events and moreover , mdct - ca allowed prognostic grading according to one - vd , two - vd , three - vd , and lmca disease classification . in both univariate and multivariate analysis , hrs for hard and accordingly , survival free of hard events was reduced progressively from 73% in patients with one - vd to 25% in those with lmca disease . another major finding of this study pertains to patients with diabetes with nonobstructive cad as seen on mdct - ca . among these patients , noninvasive testing is usually negative because this type of lesion rarely triggers myocardial ischemia , suggesting a prognosis similar to that found in patients with normal coronary arteries . indeed , kaplan - meier survival curves showed an event - free survival of 78% for hard events and thus , their risk of cardiac event is intermediate , between that found in patients with normal coronary arteries and patients with obstructive cad . nevertheless , early identification of nonobstructive cad with mdct - ca in patients with diabetes is clinically important because it may lead to a more aggressive strategy of cardiovascular risk factor control and modification of clinical follow - up . ( 9 ) were able to stratify cardiac events using the mdct - ca atherosclerotic burden in patients with diabetes . indeed , event - free survival significantly decreased at 62-month follow - up , from 67% for sss 5 to 11% for sss > 5 , considering revascularizations , and from 88% for sss 5 to 54% for sss > 5 , excluding revascularizations . another important result of our study relates to patients with diabetes with normal coronary arteries at mdct - ca . in agreement with a previous study that enrolled a smaller number of less homogeneous patients with diabetes and had shorter follow - up ( 10 ) , our study found that the absence of cad , found in 90 patients with diabetes , is associated with an event - free survival of 100% for both hard and all events at 62-month follow - up . although nuclear stress imaging and stress echocardiography , the two stress imaging tests used most widely in clinical practice , demonstrated good prognostic value in the general population with suspected cad ( 19 ) , their performance was found to be lower in studies that evaluated patients with diabetes . the prognostic value of single - photon emission computed tomography imaging in patients with diabetes was assessed in seven large studies . ( 26 ) , who found that patients with diabetes with normal exercise echocardiography had an increase in cardiac events ( up to 7.6% ) at 5-year follow - up , despite an event - free survival at 1 year . interestingly , in our study , patients with diabetes with normal coronary arteries seen on mdct - ca were event free at 5-year ( mean 62 9 months , up to 72 months ) follow - up . therefore , this imaging modality can be a useful tool to reassure patients with diabetes with suspected cad regarding their outcome , with a warranty period of at least 5 years in the presence of a normal result . third , mdct - ca allowed the identification of patients with obstructive cad , likely resulting in an increased revascularization rate , which constituted a large proportion of the composite all cardiac event end point . moreover , the decision regarding revascularization was based on symptoms , the presence of ischemia on noninvasive testing , and invasive coronary angiography results ; patients with early elective revascularizations were excluded from survival analysis and the presence of obstructive cad at mdct - ca was strongly associated with hard cardiac events . fourth , although our study demonstrated an important prognostic role of mdct - ca anatomical evaluation alone , assessment of the amount and distribution of regional ischemia documented by nuclear imaging would have been important to know the potential complementary prognostic value of mdct - ca and nuclear stress tests in our patients . fifth , although this study demonstrated that mdct - ca is able to predict the prognosis of diabetic patients on the basis of the presence / extent of cad and plaque type , coronary imaging by mdct - ca is not , as in case of invasive angiography , able to predict which plaque may progress to destabilization and rupture , potentially causing a clinical event . finally , in the subset of newly diagnosed type 2 patients with diabetes , a comparison between the prognostic value of mdct - ca and that of the uk prospective diabetes study risk engine would have been useful to demonstrate a potential additional value of mdct - ca .

several pyridazine compounds exhibit antimicrobial , potent analgesic , cox inhibitor , antidiabetic , antihypertensive , herbicidal , anticancer , and antifungal activities . further , various pyridazinones have been used as intermediates for drugs and agrochemicals and blood platelet aggregation inhibitors . the synthesis of pyridazine frameworks has been achieved primarily by the addition of hydrazine or its derivative to an appropriate 1,4-diketones and 1,4-ketoacids [ 1113 ] . other various pyridazines particularly amino - pyridazines have been prepared from polyfunctionalized nitriles , especially via the jaap - klingemaan reaction [ 1418 ] . the literature also showed the preparation of pyridazines and pyridazinones involving active methylene species , benzil , and hydrazine . however , the methods employed harsh bases [ 1921 ] or acids in presence of hazardous solvents , and also the reactions require long period of time to complete . therefore , there is a need for developing a milder and safer solvent - free procedure for the synthesis of substituted pyridazines especially because of the rise in demand for environmentally benign organic synthesis . to address the challenge of green synthesis , multicomponent reactions ( mcrs ) provide a solution since they are more efficient , cost effective , and less wasteful than traditional methods . such synthetic approach , however , when teamed with microwave ( mw ) irradiation , facilitates the reaction better as mw gives very efficient thermal management and atom efficiency thus resulting in faster reaction with an increased product yield . in another development , in recent years , the use of inorganic solid supports as catalysts for the synthesis of various biologically active molecules has increased tremendously . among these inorganic solid supports , potassium hydroxide coated with alumina ( koh - alumina ) has been a versatile reagent for various reactions and transformations such as in transesterification and biodiesel production [ 2326 ] , ester hydrolysis , selective alkylation [ 2830 ] , michael addition , cyanoethylation , and gas phase dehydrogenations . it has also been found that koh - alumina exhibited the highest basicity and superior catalytic activity among the alumina - supported alkaline catalysts during transesterification processes . moreover , koh - alumina can be prepared easily and is inexpensive . in view of these advantages in the applications of heterogeneous catalysts in the synthesis of heterocyclic compounds , we have chosen koh - alumina ( 10% in alumina ) for the synthesis of some substituted pyridazines . therefore , based on our previous work on pyridazine synthesis and in conjunction with our current research aimed at development of synthetic methodologies using solid support catalysts through mcr 's [ 3436 ] , we report herein the three - component neat synthesis of 3,4,5-triarylpyridazines and other substituted pyridazines using koh - alumina ( 10 mol% ) by the microwave irradiation technique ( scheme 1 ) . initially , the three - component synthesis was optimized by irradiating a mixture of acetophenone ( 2a ) ( 0.2 ml , 1.50 mmol ) , benzil ( 1 ) ( 0.32 g , 1.50 mmol ) and hydrazine hydrate ( 3 ) ( 0.10 ml , 2.00 mmol ) in presence of 5 mol% koh - alumina in a microwave reactor at 100c for three minutes which afforded the product 4a in 57% yield . the same reaction when irradiated for ten minutes gave 4a in 64% yield . by varying the amount of the catalyst and irradiation time , optimization was finally arrived at 10 mol% of koh - alumina which significantly resulted in 89% of the product 4a ( table 1 ) . in another attempt , the catalyst recovered from the reaction after filtration , and washing with ethyl acetate was used further for the condensation of acetophenone ( 2a ) ( 0.20 ml , 1.50 mmol ) , benzil ( 1 ) ( 0.32 g , 1.50 mmol ) , and hydrazine hydrate ( 3 ) ( 0.10 ml , 2.00 mmol ) . interestingly , the reaction was observed to complete within 15 min of irradiation giving 4a in 61% yield . the results of the reactions using recycled koh - alumina are shown in table 1 . thus , the present method was employed for the synthesis of a series of 3,4,5-triarylpyridazines involving different aromatic ketones ( 4a g , table 2 ) . irrespective of the presence of different substituents in the ortho and para positions on the ring of various aromatic aldehydes , the reactions proceeded well to furnish the desired products in good yields ( 4a g , table 2 ) . unfortunately , the reaction performed with meta substituted aromatic ketones gave only unisolable intermediates and failed to furnish the desired products . on the other hand , polyaromatic acetophenones such as 2-acetylnaphthalene ( 2h ) underwent reaction smoothly with benzil ( 1 ) and hydrazine hydrate ( 3 ) to afford the desired product 4h in 77% yield ( entry 9 , table 2 ) . similarly , the scope of this methodology was extended to synthesize other substituted pyridazines involving different active methylene carbonyl compounds such as ethyl cyano acetate ( 5a ) , diethyl malonate ( 5b ) , ethyl acetoacetate ( 5c ) , and acetyl acetone ( 5d ) ( scheme 2 ) . in all the cases the reactions proceeded fairly well and afforded the desired products in good yields ( 6a f ) , ( table 3 ) . the reactions were clean and all the products were purified by simple work - up and crystallization except for products 4d , 4 g , 4h , and 6b which were purified by column chromatography using ethyl acetate and hexane . all the synthesized compounds were characterized by h nmr , c nmr , ir , mass , and elemental analyses which were found to be in good agreement with the expected data . from the mechanistic point of view , the formation of the triarlpyridazines 4 probably takes place through the addition of hydrazine hydrate to the 1 , 4-dicarbonyl species ( 8) formed in situ by reaction between the acetophenone ( 2 ) and 1,2-dicarbonyl compound ( 1 ) in a similar fashion as reported earlier . the overall plausible mechanism for the formation of the triarylpyridazines is depicted in scheme 3 . in summary , we have established a mild and efficient method for the synthesis of highly functionalized substituted pyridazines and other substituted pyridazinones using koh - alumina ( 10 mol% ) . more importantly , the methodology presented here offers milder , more efficient , and particularly an environmentally friendly approach towards the synthesis of pyridazines by the use of potassium hydroxide impregnated on alumina as a recyclable catalyst . koh - alumina was prepared according to the procedure reported by sukata , however , as 10% of koh adsorbed on neutral alumina . the thin layer chromatography was performed on acme 's silica or merck precoated silica gel and the components were visualized in iodine chamber or by potassium permanganate spray technique . flash column chromatography was performed on merck silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent . the h and c nmr were recorded with bruker avance ii 400 ft - nmr machine with tms as the internal standard . mass spectra were recorded with waters zq-4000 equipped with esi and apci mass detector , and chn was analyzed on perkin - elmer pe 2400 series ii . a thoroughly mixed aromatic ketone ( 2 ) ( 1.50 mmol ) , 1,2-dicarbonyl compound ( 1 ) ( 1.50 mmol ) , hydrazine hydrate ( 3 ) ( 0.1 ml , 2.00 mmol ) in presence of 10 mol% koh - alumina was irradiated in a chem discover microwave reactor at 100c ( power 200 w ) at regular intervals of 60 sec for 510 min . on completion of the reaction ( monitored by thin layer chromatography ) , the reaction mixture was diluted with ethyl acetate and filtered on a sintered funnel . it was further washed down with ethyl acetate ( 5 ml 4 ) . the filtrate was then worked up with cold water , and the organic layer was separated and dried with anhydrous na2so4 . the organic filtrate was evaporated in vacuo to afford the crude product which was crystallized from ethanol ( 4a , 4b , 4c , 4e , and 4f ) or purified by flash column chromatography ( 4d , 4 g , and 4h ) over silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent to afford the 3,4,6-triarylpyridazines . a thoroughly mixed 1,2-dicarbonyl compound ( 1 ) ( 1.50 mmol ) and hydrazine hydrate ( 3 ) ( 2.00 mmol ) was irradiated in a chem discover microwave reactor at 100c ( power 200 w ) for 5 minutes . the mixture was cooled and then introduced therein the active methylene species ( 5 ) ( 1.50 mmol ) and koh - alumina ( 10 mol% ) . the components were mixed thoroughly and subjected to microwave irradiation at 100c ( power 200 w ) for 36 minutes . on completion of the reaction ( monitored by thin layer chromatography ) , the reaction mixture was diluted with ethyl acetate and filtered on a sintered funnel . it was further washed down with ethyl acetate ( 5 ml 4 ) . the filtrate was then worked up with cold water and the organic layer was separated and dried with anhydrous na2so4 . the organic filtrate was evaporated in vacuo to afford the crude product which was crystallized from ethanol ( 6a , 6c , 6d , 6e , and 6f ) or purified by flash column chromatography ( 6b ) over silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent to afford the pure product . 3,4,6-triphenylpyridazine ( 4a , table 2)white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 ) : h 7.277.89 ( m , 13h , ar - h ) , 8.20 ( s , 1h , ar - h ) , 8.20 ( d , 2h , j = 6.8 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 124.5 , 126.6 , 127.6 , 128.3 , 128.6 , 129.5 , 129.6 , 135.4 , 136.0 , 136.6 , 139.2 , 157.2 , 157.7 ppm ; ir ( kbr ) : max 1075 , 1177 , 1394 , 1444 , 1488 , 1582 , 2854 , 2924 , 3063 cm ; ms ( es ) for c22h16n2 308.1 found 308.9 ( m + h ) , 331.0 ( m + na ) ; chn calcd . for c22h16n2 c , 85.69 ; h , 5.23 ; n , 9.08 found c , 85.71 ; h , 5.38 ; n , 9.32 . white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 ) : h 7.277.89 ( m , 13h , ar - h ) , 8.20 ( s , 1h , ar - h ) , 8.20 ( d , 2h , j = 6.8 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 124.5 , 126.6 , 127.6 , 128.3 , 128.6 , 129.5 , 129.6 , 135.4 , 136.0 , 136.6 , 139.2 , 157.2 , 157.7 ppm ; ir ( kbr ) : max 1075 , 1177 , 1394 , 1444 , 1488 , 1582 , 2854 , 2924 , 3063 cm ; ms ( es ) for c22h16n2 308.1 found 308.9 ( m + h ) , 331.0 ( m + na ) ; chn calcd . for c22h16n2 c , 85.69 ; h , 5.23 ; n , 9.08 found c , 85.71 ; h , 5.38 ; n , 9.32 . 3,4-diphenyl-6-p - tolylpyridazine ( 4b , table 2)light yellow solid ; mp 160162c ; h nmr ( 400 mhz , cdcl3 ) : h 2.43 ( s , 3h , ch3 ) , 7.257.35 ( m , 10h , ar - h ) , 7.48 ( d , 2h , j = 6.8 hz , ar - h ) , 7.83 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 8.0 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 21.4 , 124.7 , 126.9 , 128.1 , 128.7 , 129.0 , 129.8 , 130.0 , 133.0 , 136.5 , 137.1 , 139.6 , 140.4 , 157.6 , 157.9 ppm ; ir ( kbr ) : max 1079 , 1222 , 1367 , 1419 , 1592 , 2867 , 2923 , 3019 cm ; ms ( es ) for c23h18n2 322.1 found 323.0 ( m + h ) , 345.0 ( m + na ) ; chn calcd . for c23h18n2 c , 85.68 ; h , 5.63 ; n , 8.69 found c,85.55 ; h , 5.65 ; n , 8.44 . light yellow solid ; mp 160162c ; h nmr ( 400 mhz , cdcl3 ) : h 2.43 ( s , 3h , ch3 ) , 7.257.35 ( m , 10h , ar - h ) , 7.48 ( d , 2h , j = 6.8 hz , ar - h ) , 7.83 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 8.0 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 21.4 , 124.7 , 126.9 , 128.1 , 128.7 , 129.0 , 129.8 , 130.0 , 133.0 , 136.5 , 137.1 , 139.6 , 140.4 , 157.6 , 157.9 ppm ; ir ( kbr ) : max 1079 , 1222 , 1367 , 1419 , 1592 , 2867 , 2923 , 3019 cm ; ms ( es ) for c23h18n2 322.1 found 323.0 ( m + h ) , 345.0 ( m + na ) ; chn calcd . for c23h18n2 c , 85.68 ; h , 5.63 ; n , 8.69 found c,85.55 ; h , 5.65 ; n , 8.44 . 6-(2-methoxyphenyl)-3,4-diphenylpyridazine ( 4c , table 2)white solid ; mp 137139c ; h nmr ( 400 mhz , cdcl3 ) : h 3.85 ( s , 3h , och3 ) , 7.007.30 ( m , 12h , ar - h ) , 7.43 ( s , 1h , ar - h ) , 8.03 ( d , 2h , j = 7.2 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 55.8 , 111.4 , 121.4 , 124.7 , 128.2 , 128.7 , 128.9 , 129.2 , 130.1 , 131.5 , 131.6 , 136.3 , 137.0 , 139.1 , 156.9 , 157.3 , 157.7 ppm ; ir ( kbr ) : max 1076 , 1199 , 1206 , 1371 , 1496 , 1509 , 2877 , 2943 , 3016 cm ; ms ( es ) for c23h18n2o 338.1 found 339.0 ( m + h ) , 361.0 ( m + na ) ; chn calcd . for c23h18n2o c , 81.63 ; h , 5.36 ; n , 8.28 found c , 81.59 ; h , 5.16 ; n , 8.35 . white solid ; mp 137139c ; h nmr ( 400 mhz , cdcl3 ) : h 3.85 ( s , 3h , och3 ) , 7.007.30 ( m , 12h , ar - h ) , 7.43 ( s , 1h , ar - h ) , 8.03 ( d , 2h , j = 7.2 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 55.8 , 111.4 , 121.4 , 124.7 , 128.2 , 128.7 , 128.9 , 129.2 , 130.1 , 131.5 , 131.6 , 136.3 , 137.0 , 139.1 , 156.9 , 157.3 , 157.7 ppm ; ir ( kbr ) : max 1076 , 1199 , 1206 , 1371 , 1496 , 1509 , 2877 , 2943 , 3016 cm ; ms ( es ) for c23h18n2o 338.1 found 339.0 ( m + h ) , 361.0 ( m + na ) ; chn calcd . for c23h18n2o c , 81.63 ; h , 5.36 ; n , 8.28 found c , 81.59 ; h , 5.16 ; n , 8.35 . 6-(4-methoxyphenyl)-3,4-diphenylpyridazine ( 4d , table 2)yellow solid ; mp 164166c ; h nmr ( 400 mhz , cdcl3 ) : h 3.87 ( s , 3h , och3 ) , 7.04 ( d , 2h , j = 8.8 hz , ar - h ) , 7.217.35 ( m , 8h , ar - h ) , 7.47 ( d , 2h , j = 6.8 hz , ar - h ) , 7.79 ( s , 1h , ar - h ) , 8.15 ( d , 2h , j = 8.4 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 55.39 , 114.4 , 124.2 , 128.2 , 128.3 , 128.4 , 128.6 , 128.7 , 129.0 , 130.0 , 136.7 , 137.2 , 139.5 , 157.2 , 157.6 , 161.3 ppm ; ir ( kbr ) : max 1067 , 1208 , 1310 , 1487 , 1562 , 2877 , 2931 , 3012 cm ; ms ( es ) for c23h18n2o 338.1 found 339.0 ( m + h ) , 361.0 ( m + na ) ; chn calcd . for c23h18n2o c , 81.63 ; h , 5.36 ; n , 8.28 found c , 81.58 ; h , 5.29 ; n , 8.11 . yellow solid ; mp 164166c ; h nmr ( 400 mhz , cdcl3 ) : h 3.87 ( s , 3h , och3 ) , 7.04 ( d , 2h , j = 8.8 hz , ar - h ) , 7.217.35 ( m , 8h , ar - h ) , 7.47 ( d , 2h , j = 6.8 hz , ar - h ) , 7.79 ( s , 1h , ar - h ) , 8.15 ( d , 2h , j = 8.4 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 55.39 , 114.4 , 124.2 , 128.2 , 128.3 , 128.4 , 128.6 , 128.7 , 129.0 , 130.0 , 136.7 , 137.2 , 139.5 , 157.2 , 157.6 , 161.3 ppm ; ir ( kbr ) : max 1067 , 1208 , 1310 , 1487 , 1562 , 2877 , 2931 , 3012 cm ; ms ( es ) for c23h18n2o 338.1 found 339.0 ( m + h ) , 361.0 ( m + na ) ; chn calcd . for c23h18n2o c , 81.63 ; h , 5.36 ; n , 8.28 found c , 81.58 ; h , 5.29 ; n , 8.11 . 6-(4-bromophenyl)-3,4-diphenylpyridazine ( 4e , table 2)white solid ; mp 147149c ; h nmr ( 400 mhz , cdcl3 ) : h 7.057.45 ( m , 12h , ar - h ) , 7.74 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 6.8 hz ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 122.6 , 125.2 , 126.9 , 127.2 , 128.2 , 128.8 , 128.9 , 129.1 , 130.1 , 130.2 , 131.6 , 131.8 , 135.8 , 136.4 , 137.0 , 139.9 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1075 , 1296 , 1400 , 1488 , 1571 , 2934 , 3011 cm ; ms ( es ) for c22h15brn2 386.0 found 387.0 ( m + h ) , 409.0 ( m + na ) ; chn calcd . for c22h15brn2 c , 68.23 ; h , 3.90 ; n , 7.23 found c , 68.21 ; h , 3.73 ; n , 7.11 . white solid ; mp 147149c ; h nmr ( 400 mhz , cdcl3 ) : h 7.057.45 ( m , 12h , ar - h ) , 7.74 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 6.8 hz ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 122.6 , 125.2 , 126.9 , 127.2 , 128.2 , 128.8 , 128.9 , 129.1 , 130.1 , 130.2 , 131.6 , 131.8 , 135.8 , 136.4 , 137.0 , 139.9 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1075 , 1296 , 1400 , 1488 , 1571 , 2934 , 3011 cm ; ms ( es ) for c22h15brn2 386.0 found 387.0 ( m + h ) , 409.0 ( m + na ) ; chn calcd . for c22h15brn2 c , 68.23 ; h , 3.90 ; n , 7.23 found c , 68.21 ; h , 3.73 ; n , 7.11 . 6-(4-chlorophenyl)-3,4-diphenylpyridazine ( 4f , table 2)light yellow solid ; mp 179181c ; h nmr ( 400 mhz , cdcl3 ) : h 7.067.44 ( m , 12h , ar - h ) , 7.73 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 7.6 hz ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 125.1 , 126.4 , 126.5 , 127.1 , 127.2 , 128.2 , 128.6 , 128.9 , 129.1 , 129.5 , 129.7 , 130.1 , 130.2 , 135.9 , 136.5 , 139.8 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1076 , 1200 , 1301 , 1481 , 1546 , 2879 , 2932 , 3088 cm ; ms ( es ) for c22h15cln2 342.1 found 343.0 ( m + h ) , 365.0 ( m + na ) ; chn calcd . for c22h15cln2 c , 77.08 ; h , 4.41 ; n , 8.17 found c , 77.26 ; h , 4.59 ; n , 8.06 . light yellow solid ; mp 179181c ; h nmr ( 400 mhz , cdcl3 ) : h 7.067.44 ( m , 12h , ar - h ) , 7.73 ( s , 1h , ar - h ) , 8.08 ( d , 2h , j = 7.6 hz ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 125.1 , 126.4 , 126.5 , 127.1 , 127.2 , 128.2 , 128.6 , 128.9 , 129.1 , 129.5 , 129.7 , 130.1 , 130.2 , 135.9 , 136.5 , 139.8 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1076 , 1200 , 1301 , 1481 , 1546 , 2879 , 2932 , 3088 cm ; ms ( es ) for c22h15cln2 342.1 found 343.0 ( m + h ) , 365.0 ( m + na ) ; chn calcd . for c22h15cln2 c , 77.08 ; h , 4.41 ; n , 8.17 found c , 77.26 ; h , 4.59 ; n , 8.06 . 6-(4-nitrophenyl)-3,4-diphenylpyridazine ( 4 g , table 2)white solid ; mp 164166c ; h nmr ( 400 mhz , cdcl3 ) : h 7.167.44 ( m , 11h , ar - h ) , 7.56 ( t , 1h , j = 7.2 hz , ar - h ) , 7.79 ( s , 1h , ar - h ) , 8.07 ( d , 2h , j = 8.0 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 123.0 , 123.6 , 126.1 , 127.3 , 128.2 , 128.9 , 129.0 , 129.1 , 129.3 , 129.6 , 134.4 , 135.6 , 140.9 , 148.1 , 157.6 , 158.1 ppm ; ir ( kbr ) : max 1076 , 1230 , 1309 , 1441 , 1541 , 2877 , 2932 , 3081 cm ; ms ( es ) for c22h15n3o2 353.1 found 354.0 ( m + h ) , 376.0 ( m + na ) ; chn calcd . for c22h15n3o2 c , 74.78 ; h , 4.28 ; n , 11.89 found c , 74.53 ; h , 4.40 ; n , 11.76 . white solid ; mp 164166c ; h nmr ( 400 mhz , cdcl3 ) : h 7.167.44 ( m , 11h , ar - h ) , 7.56 ( t , 1h , j = 7.2 hz , ar - h ) , 7.79 ( s , 1h , ar - h ) , 8.07 ( d , 2h , j = 8.0 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 123.0 , 123.6 , 126.1 , 127.3 , 128.2 , 128.9 , 129.0 , 129.1 , 129.3 , 129.6 , 134.4 , 135.6 , 140.9 , 148.1 , 157.6 , 158.1 ppm ; ir ( kbr ) : max 1076 , 1230 , 1309 , 1441 , 1541 , 2877 , 2932 , 3081 cm ; ms ( es ) for c22h15n3o2 353.1 found 354.0 ( m + h ) , 376.0 ( m + na ) ; chn calcd . for c22h15n3o2 c , 74.78 ; h , 4.28 ; n , 11.89 found c , 74.53 ; h , 4.40 ; n , 11.76 . 6-(naphthalen-2-yl)-3,4-diphenylpyridazine ( 4h , table 2)white solid ; mp 190192c ; h nmr ( 400 mhz , cdcl3 ) : h 7.107.73 ( m , 18h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 124.2 , 125.4 , 125.8 , 126.1 , 127.3 , 127.9 , 128.3 , 128.8 , 129.0 , 129.1 , 130.2 , 130.3 , 133.1 , 135.6 , 136.9 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1080 , 1234 , 1290 , 1438 , 1521 , 2861 , 2932 , 2995 , 3043 cm ; ms ( es ) for c26h18n2 358.1 found 359.0 ( m + h ) , 381.0 ( m + na ) ; chn calcd . for c26h18n2 c , 87.12 ; h , 5.06 ; n , 7.82 found c , 87.31 ; h , 5.21 ; n , 7.71 . white solid ; mp 190192c ; h nmr ( 400 mhz , cdcl3 ) : h 7.107.73 ( m , 18h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 124.2 , 125.4 , 125.8 , 126.1 , 127.3 , 127.9 , 128.3 , 128.8 , 129.0 , 129.1 , 130.2 , 130.3 , 133.1 , 135.6 , 136.9 , 157.7 , 158.2 ppm ; ir ( kbr ) : max 1080 , 1234 , 1290 , 1438 , 1521 , 2861 , 2932 , 2995 , 3043 cm ; ms ( es ) for c26h18n2 358.1 found 359.0 ( m + h ) , 381.0 ( m + na ) ; chn calcd . for c26h18n2 c , 87.12 ; h , 5.06 ; n , 7.82 found c , 87.31 ; h , 5.21 ; n , 7.71 . 2,3-dihydro-3-oxo-5,6-diphenylpyridazine-4-carbonitrile ( 6a , table 3)white solid ; mp 270272c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 6.917.94 ( m , 10h , ar - h ) , 11.58 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.3 , 116.5 , 127.8 , 128.9 , 129.1 , 129.6 , 130.2 , 131.4 , 132.7 , 133.6 , 135.3 , 146.9 , 158.2 ppm ; ir ( kbr ) : max 1010 , 1089 , 1210 , 1464 , 1511 , 1693 , 2256 , 2868 , 2932 , 3412 cm ; ms ( es ) for c17h11n3o 273.1 found 274.0 ( m + h ) , 296.0 ( m + na ) ; chn calcd . for c17h11n3o c , 74.71 ; h , 4.06 ; n , 15.38 found c , 74.86 ; h , 4.21 ; n , 15.12 . white solid ; mp 270272c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 6.917.94 ( m , 10h , ar - h ) , 11.58 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.3 , 116.5 , 127.8 , 128.9 , 129.1 , 129.6 , 130.2 , 131.4 , 132.7 , 133.6 , 135.3 , 146.9 , 158.2 ppm ; ir ( kbr ) : max 1010 , 1089 , 1210 , 1464 , 1511 , 1693 , 2256 , 2868 , 2932 , 3412 cm ; ms ( es ) for c17h11n3o 273.1 found 274.0 ( m + h ) , 296.0 ( m + na ) ; chn calcd . for c17h11n3o c , 74.71 ; h , 4.06 ; n , 15.38 found c , 74.86 ; h , 4.21 ; n , 15.12 . 2,3-dihydro-3-oxopyridazine-4-carbonitrile ( 6b , table 3)white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 7.24 ( s , 1h , ar - h ) , 7.63 ( s , 1h , ar - h ) , 11.57 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.4 , 118.3 , 138.7 , 152.3 , 168.8 ppm ; ir ( kbr ) : max 1089 , 1212 , 1400 , 1526 , 1676 , 2247 , 2881 , 2932 , 3310 cm ; ms ( es ) for c5h3n3o 121.0 found 122.0 ( m + h ) , 144.0 ( m + na ) ; chn calcd . for c5h3n3o c , 49.59 ; h , 2.50 ; n , 34.70 found c , 49.53 ; h , 2.41 ; n , 34.62 . white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 7.24 ( s , 1h , ar - h ) , 7.63 ( s , 1h , ar - h ) , 11.57 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.4 , 118.3 , 138.7 , 152.3 , 168.8 ppm ; ir ( kbr ) : max 1089 , 1212 , 1400 , 1526 , 1676 , 2247 , 2881 , 2932 , 3310 cm ; ms ( es ) for c5h3n3o 121.0 found 122.0 ( m + h ) , 144.0 ( m + na ) ; chn calcd . for c5h3n3o c , 49.59 ; h , 2.50 ; n , 34.70 found c , 49.53 ; h , 2.41 ; n , 34.62 . 2,3-dihydro-5,6-dimethyl-3-oxopyridazine-4-carbonitrile ( 6c , table 3)white solid ; mp 209211c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 2.33 ( s , 3h , ch3 ) , 2.50 ( s , 3h , ch3 ) , 11.25 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 9.9 , 27.6 , 116.7 , 126.2 , 152.8 , 157.4 , 167.9 ppm ; ir ( kbr ) : max 1046 , 1287 , 1412 , 1547 , 1671 , 2219 , 2931 , 3349 cm ; ms ( es ) for c7h7n3o 149.1 found 150.0 ( m + h ) , 172.0 ( m + na ) ; chn calcd . for c7h7n3o c , 56.37 ; h , 4.73 ; n , 28.17 found c , 56.21 ; h , 4.68 ; n , 28.32 . white solid ; mp 209211c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 2.33 ( s , 3h , ch3 ) , 2.50 ( s , 3h , ch3 ) , 11.25 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 9.9 , 27.6 , 116.7 , 126.2 , 152.8 , 157.4 , 167.9 ppm ; ir ( kbr ) : max 1046 , 1287 , 1412 , 1547 , 1671 , 2219 , 2931 , 3349 cm ; ms ( es ) for c7h7n3o 149.1 found 150.0 ( m + h ) , 172.0 ( m + na ) ; chn calcd . for c7h7n3o c , 56.37 ; h , 4.73 ; n , 28.17 found c , 56.21 ; h , 4.68 ; n , 28.32 . ethyl 2,3-dihydro-3-oxo-5,6-diphenylpyridazine-4-carboxylate ( 6d , table 3)white solid ; mp 217219c ; h nmr ( 400 mhz , cdcl3 ) : h 0.90 ( t , 3h , j = 7.2 hz , ch3 ) , 4.05 ( q , 2h , j = 7.2 hz , ch2 ) , 7.037.27 ( m , 10h , ar - h ) , 12.56 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 13.7 , 62.0 , 128.0 , 128.3 , 128.7 , 129.1 , 129.2 , 133.7 , 133.8 , 134.7 , 143.3 , 147.7 , 158.7 , 163.6 ppm ; ir ( kbr ) : max 1101 , 1200 , 1432 , 1500 , 1672 , 1768 , 2867 , 2931 , 3401 cm ; ms ( es ) for c19h16n2o3 320.1 found 321.0 ( m + h ) , 343.0 ( m + na ) ; chn calcd . for c19h16n2o3 c , 71.24 ; h , 5.03 ; n , 8.74 found c , 71.43 ; h , 5.17 ; n , 8.68 . white solid ; mp 217219c ; h nmr ( 400 mhz , cdcl3 ) : h 0.90 ( t , 3h , j = 7.2 hz , ch3 ) , 4.05 ( q , 2h , j = 7.2 hz , ch2 ) , 7.037.27 ( m , 10h , ar - h ) , 12.56 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 13.7 , 62.0 , 128.0 , 128.3 , 128.7 , 129.1 , 129.2 , 133.7 , 133.8 , 134.7 , 143.3 , 147.7 , 158.7 , 163.6 ppm ; ir ( kbr ) : max 1101 , 1200 , 1432 , 1500 , 1672 , 1768 , 2867 , 2931 , 3401 cm ; ms ( es ) for c19h16n2o3 320.1 found 321.0 ( m + h ) , 343.0 ( m + na ) ; chn calcd . for c19h16n2o3 c , 71.24 ; h , 5.03 ; n , 8.74 found c , 71.43 ; h , 5.17 ; n , 8.68 . ethyl 3-methyl-5,6-diphenylpyridazine-4-carboxylate ( 6e , table 3)white solid ; mp 7779c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 0.95 ( t , 3h , j = 7.4 hz , ch3 ) , 2.65 ( s , 3h , ch3 ) , 4.05 ( q , 2h , j = 7.6 hz , ch2 ) , 7.147.46 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 14.6 , 23.7 , 61.4 , 126.7 , 126.9 , 127.5 , 128.3 , 128.8 , 129.3 , 130.1 , 132.1 , 132.7 , 134.6 , 137.8 , 139.5 , 141.0 , 152.6 , 196.3 ppm ; ir ( kbr ) : max 1087 , 1100 , 1280 , 1434 , 1510 , 1769 , 2862 , 2932 , 3084 cm ; ms ( es ) for c20h18n2o2 318.1 found 319.0 ( m for c20h18n2o2 c , 75.45 ; h , 5.70 ; n , 8.80 found c , 75.57 ; h , 5.69 ; n , 8.82 . white solid ; mp 7779c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 0.95 ( t , 3h , j = 7.4 hz , ch3 ) , 2.65 ( s , 3h , ch3 ) , 4.05 ( q , 2h , j = 7.6 hz , ch2 ) , 7.147.46 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 14.6 , 23.7 , 61.4 , 126.7 , 126.9 , 127.5 , 128.3 , 128.8 , 129.3 , 130.1 , 132.1 , 132.7 , 134.6 , 137.8 , 139.5 , 141.0 , 152.6 , 196.3 ppm ; ir ( kbr ) : max 1087 , 1100 , 1280 , 1434 , 1510 , 1769 , 2862 , 2932 , 3084 cm ; ms ( es ) for c20h18n2o2 318.1 found 319.0 ( m + h ) , 341.0 ( m + na ) ; chn calcd . for c20h18n2o2 c , 75.45 ; h , 5.70 ; n , 8.80 found c , 75.57 ; h , 5.69 ; n , 8.82 . 1-(3-methyl-5,6-diphenylpyridazin-4-yl)ethanone ( 6f , table 3)white solid ; mp 132134c ; h nmr ( 400 mhz , cdcl3 ) : h 1.82 ( s , 3h , ch3 ) , 2.59 ( s , 3h , ch3 ) , 7.187.47 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 21.9 , 27.6 , 126.7 , 126.8 , 128.5 , 129.3 , 129.7 , 130.0 , 134.6 , 135.8 , 136.6 , 138.4 , 138.8 , 151.2 , 152.3 , 186.9 ppm ; ir ( kbr ) : max 1201 , 1240 , 1433 , 1520 , 1692 , 2888 , 2932 , 3100 cm ; ms ( es ) for c19h16n2o 288.1 found 289.0 ( m + h ) , 311.0 ( m + na ) ; chn calcd . for c19h16n2o c , 79.14 ; h , 5.59 ; n , 9.72 found c , 79.33 ; h , 5.51 ; n , 9.77 . white solid ; mp 132134c ; h nmr ( 400 mhz , cdcl3 ) : h 1.82 ( s , 3h , ch3 ) , 2.59 ( s , 3h , ch3 ) , 7.187.47 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 21.9 , 27.6 , 126.7 , 126.8 , 128.5 , 129.3 , 129.7 , 130.0 , 134.6 , 135.8 , 136.6 , 138.4 , 138.8 , 151.2 , 152.3 , 186.9 ppm ; ir ( kbr ) : max 1201 , 1240 , 1433 , 1520 , 1692 , 2888 , 2932 , 3100 cm ; ms ( es ) for c19h16n2o 288.1 found 289.0 ( m + h ) , 311.0 ( m + na ) ; chn calcd . for c19h16n2o c , 79.14 ; h , 5.59 ; n , 9.72 found c , 79.33 ; h , 5.51 ; n , 9.77 .

the work described herein employs potassium hydroxide impregnated alumina ( koh - alumina ) as a mild , efficient , and recyclable catalyst for a one - pot solvent - free and environmentally safer synthesis of 3,4,6-triarylpyridazines and some substituted pyridazines from active methylene carbonyl species , 1,2-dicarbonyls , and hydrazine hydrate by microwave ( mw ) irradiation . the method offers highly convergent , inexpensive , and functionality - tolerable procedure for rapid access to important pyridazine compounds in good yields .

1. Introduction 2. Conclusion 3. Experimental Section

several pyridazine compounds exhibit antimicrobial , potent analgesic , cox inhibitor , antidiabetic , antihypertensive , herbicidal , anticancer , and antifungal activities . the synthesis of pyridazine frameworks has been achieved primarily by the addition of hydrazine or its derivative to an appropriate 1,4-diketones and 1,4-ketoacids [ 1113 ] . other various pyridazines particularly amino - pyridazines have been prepared from polyfunctionalized nitriles , especially via the jaap - klingemaan reaction [ 1418 ] . the literature also showed the preparation of pyridazines and pyridazinones involving active methylene species , benzil , and hydrazine . however , the methods employed harsh bases [ 1921 ] or acids in presence of hazardous solvents , and also the reactions require long period of time to complete . therefore , there is a need for developing a milder and safer solvent - free procedure for the synthesis of substituted pyridazines especially because of the rise in demand for environmentally benign organic synthesis . to address the challenge of green synthesis , multicomponent reactions ( mcrs ) provide a solution since they are more efficient , cost effective , and less wasteful than traditional methods . such synthetic approach , however , when teamed with microwave ( mw ) irradiation , facilitates the reaction better as mw gives very efficient thermal management and atom efficiency thus resulting in faster reaction with an increased product yield . in another development , in recent years , the use of inorganic solid supports as catalysts for the synthesis of various biologically active molecules has increased tremendously . among these inorganic solid supports , potassium hydroxide coated with alumina ( koh - alumina ) has been a versatile reagent for various reactions and transformations such as in transesterification and biodiesel production [ 2326 ] , ester hydrolysis , selective alkylation [ 2830 ] , michael addition , cyanoethylation , and gas phase dehydrogenations . it has also been found that koh - alumina exhibited the highest basicity and superior catalytic activity among the alumina - supported alkaline catalysts during transesterification processes . moreover , koh - alumina can be prepared easily and is inexpensive . in view of these advantages in the applications of heterogeneous catalysts in the synthesis of heterocyclic compounds , we have chosen koh - alumina ( 10% in alumina ) for the synthesis of some substituted pyridazines . therefore , based on our previous work on pyridazine synthesis and in conjunction with our current research aimed at development of synthetic methodologies using solid support catalysts through mcr 's [ 3436 ] , we report herein the three - component neat synthesis of 3,4,5-triarylpyridazines and other substituted pyridazines using koh - alumina ( 10 mol% ) by the microwave irradiation technique ( scheme 1 ) . initially , the three - component synthesis was optimized by irradiating a mixture of acetophenone ( 2a ) ( 0.2 ml , 1.50 mmol ) , benzil ( 1 ) ( 0.32 g , 1.50 mmol ) and hydrazine hydrate ( 3 ) ( 0.10 ml , 2.00 mmol ) in presence of 5 mol% koh - alumina in a microwave reactor at 100c for three minutes which afforded the product 4a in 57% yield . by varying the amount of the catalyst and irradiation time , optimization was finally arrived at 10 mol% of koh - alumina which significantly resulted in 89% of the product 4a ( table 1 ) . in another attempt , the catalyst recovered from the reaction after filtration , and washing with ethyl acetate was used further for the condensation of acetophenone ( 2a ) ( 0.20 ml , 1.50 mmol ) , benzil ( 1 ) ( 0.32 g , 1.50 mmol ) , and hydrazine hydrate ( 3 ) ( 0.10 ml , 2.00 mmol ) . interestingly , the reaction was observed to complete within 15 min of irradiation giving 4a in 61% yield . the results of the reactions using recycled koh - alumina are shown in table 1 . thus , the present method was employed for the synthesis of a series of 3,4,5-triarylpyridazines involving different aromatic ketones ( 4a g , table 2 ) . irrespective of the presence of different substituents in the ortho and para positions on the ring of various aromatic aldehydes , the reactions proceeded well to furnish the desired products in good yields ( 4a g , table 2 ) . on the other hand , polyaromatic acetophenones such as 2-acetylnaphthalene ( 2h ) underwent reaction smoothly with benzil ( 1 ) and hydrazine hydrate ( 3 ) to afford the desired product 4h in 77% yield ( entry 9 , table 2 ) . similarly , the scope of this methodology was extended to synthesize other substituted pyridazines involving different active methylene carbonyl compounds such as ethyl cyano acetate ( 5a ) , diethyl malonate ( 5b ) , ethyl acetoacetate ( 5c ) , and acetyl acetone ( 5d ) ( scheme 2 ) . in all the cases the reactions proceeded fairly well and afforded the desired products in good yields ( 6a f ) , ( table 3 ) . the reactions were clean and all the products were purified by simple work - up and crystallization except for products 4d , 4 g , 4h , and 6b which were purified by column chromatography using ethyl acetate and hexane . all the synthesized compounds were characterized by h nmr , c nmr , ir , mass , and elemental analyses which were found to be in good agreement with the expected data . from the mechanistic point of view , the formation of the triarlpyridazines 4 probably takes place through the addition of hydrazine hydrate to the 1 , 4-dicarbonyl species ( 8) formed in situ by reaction between the acetophenone ( 2 ) and 1,2-dicarbonyl compound ( 1 ) in a similar fashion as reported earlier . in summary , we have established a mild and efficient method for the synthesis of highly functionalized substituted pyridazines and other substituted pyridazinones using koh - alumina ( 10 mol% ) . more importantly , the methodology presented here offers milder , more efficient , and particularly an environmentally friendly approach towards the synthesis of pyridazines by the use of potassium hydroxide impregnated on alumina as a recyclable catalyst . koh - alumina was prepared according to the procedure reported by sukata , however , as 10% of koh adsorbed on neutral alumina . flash column chromatography was performed on merck silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent . mass spectra were recorded with waters zq-4000 equipped with esi and apci mass detector , and chn was analyzed on perkin - elmer pe 2400 series ii . a thoroughly mixed aromatic ketone ( 2 ) ( 1.50 mmol ) , 1,2-dicarbonyl compound ( 1 ) ( 1.50 mmol ) , hydrazine hydrate ( 3 ) ( 0.1 ml , 2.00 mmol ) in presence of 10 mol% koh - alumina was irradiated in a chem discover microwave reactor at 100c ( power 200 w ) at regular intervals of 60 sec for 510 min . it was further washed down with ethyl acetate ( 5 ml 4 ) . the filtrate was then worked up with cold water , and the organic layer was separated and dried with anhydrous na2so4 . the organic filtrate was evaporated in vacuo to afford the crude product which was crystallized from ethanol ( 4a , 4b , 4c , 4e , and 4f ) or purified by flash column chromatography ( 4d , 4 g , and 4h ) over silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent to afford the 3,4,6-triarylpyridazines . a thoroughly mixed 1,2-dicarbonyl compound ( 1 ) ( 1.50 mmol ) and hydrazine hydrate ( 3 ) ( 2.00 mmol ) was irradiated in a chem discover microwave reactor at 100c ( power 200 w ) for 5 minutes . the mixture was cooled and then introduced therein the active methylene species ( 5 ) ( 1.50 mmol ) and koh - alumina ( 10 mol% ) . the components were mixed thoroughly and subjected to microwave irradiation at 100c ( power 200 w ) for 36 minutes . on completion of the reaction ( monitored by thin layer chromatography ) , the reaction mixture was diluted with ethyl acetate and filtered on a sintered funnel . the organic filtrate was evaporated in vacuo to afford the crude product which was crystallized from ethanol ( 6a , 6c , 6d , 6e , and 6f ) or purified by flash column chromatography ( 6b ) over silica gel ( 60120 mesh ) using ethyl acetate - hexane ( 3 : 7 ) as the eluent to afford the pure product . for c23h18n2o c , 81.63 ; h , 5.36 ; n , 8.28 found c , 81.59 ; h , 5.16 ; n , 8.35 . for c22h15brn2 c , 68.23 ; h , 3.90 ; n , 7.23 found c , 68.21 ; h , 3.73 ; n , 7.11 . for c22h15cln2 c , 77.08 ; h , 4.41 ; n , 8.17 found c , 77.26 ; h , 4.59 ; n , 8.06 . 6-(4-nitrophenyl)-3,4-diphenylpyridazine ( 4 g , table 2)white solid ; mp 164166c ; h nmr ( 400 mhz , cdcl3 ) : h 7.167.44 ( m , 11h , ar - h ) , 7.56 ( t , 1h , j = 7.2 hz , ar - h ) , 7.79 ( s , 1h , ar - h ) , 8.07 ( d , 2h , j = 8.0 hz , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 123.0 , 123.6 , 126.1 , 127.3 , 128.2 , 128.9 , 129.0 , 129.1 , 129.3 , 129.6 , 134.4 , 135.6 , 140.9 , 148.1 , 157.6 , 158.1 ppm ; ir ( kbr ) : max 1076 , 1230 , 1309 , 1441 , 1541 , 2877 , 2932 , 3081 cm ; ms ( es ) for c22h15n3o2 353.1 found 354.0 ( m + h ) , 376.0 ( m + na ) ; chn calcd . for c17h11n3o c , 74.71 ; h , 4.06 ; n , 15.38 found c , 74.86 ; h , 4.21 ; n , 15.12 . 2,3-dihydro-3-oxopyridazine-4-carbonitrile ( 6b , table 3)white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 7.24 ( s , 1h , ar - h ) , 7.63 ( s , 1h , ar - h ) , 11.57 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.4 , 118.3 , 138.7 , 152.3 , 168.8 ppm ; ir ( kbr ) : max 1089 , 1212 , 1400 , 1526 , 1676 , 2247 , 2881 , 2932 , 3310 cm ; ms ( es ) for c5h3n3o 121.0 found 122.0 ( m + h ) , 144.0 ( m + na ) ; chn calcd . white solid ; mp 182184c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 7.24 ( s , 1h , ar - h ) , 7.63 ( s , 1h , ar - h ) , 11.57 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 110.4 , 118.3 , 138.7 , 152.3 , 168.8 ppm ; ir ( kbr ) : max 1089 , 1212 , 1400 , 1526 , 1676 , 2247 , 2881 , 2932 , 3310 cm ; ms ( es ) for c5h3n3o 121.0 found 122.0 ( m + h ) , 144.0 ( m + na ) ; chn calcd . for c5h3n3o c , 49.59 ; h , 2.50 ; n , 34.70 found c , 49.53 ; h , 2.41 ; n , 34.62 . ethyl 2,3-dihydro-3-oxo-5,6-diphenylpyridazine-4-carboxylate ( 6d , table 3)white solid ; mp 217219c ; h nmr ( 400 mhz , cdcl3 ) : h 0.90 ( t , 3h , j = 7.2 hz , ch3 ) , 4.05 ( q , 2h , j = 7.2 hz , ch2 ) , 7.037.27 ( m , 10h , ar - h ) , 12.56 ( s , 1h , nh ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 13.7 , 62.0 , 128.0 , 128.3 , 128.7 , 129.1 , 129.2 , 133.7 , 133.8 , 134.7 , 143.3 , 147.7 , 158.7 , 163.6 ppm ; ir ( kbr ) : max 1101 , 1200 , 1432 , 1500 , 1672 , 1768 , 2867 , 2931 , 3401 cm ; ms ( es ) for c19h16n2o3 320.1 found 321.0 ( m + h ) , 343.0 ( m + na ) ; chn calcd . ethyl 3-methyl-5,6-diphenylpyridazine-4-carboxylate ( 6e , table 3)white solid ; mp 7779c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 0.95 ( t , 3h , j = 7.4 hz , ch3 ) , 2.65 ( s , 3h , ch3 ) , 4.05 ( q , 2h , j = 7.6 hz , ch2 ) , 7.147.46 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 14.6 , 23.7 , 61.4 , 126.7 , 126.9 , 127.5 , 128.3 , 128.8 , 129.3 , 130.1 , 132.1 , 132.7 , 134.6 , 137.8 , 139.5 , 141.0 , 152.6 , 196.3 ppm ; ir ( kbr ) : max 1087 , 1100 , 1280 , 1434 , 1510 , 1769 , 2862 , 2932 , 3084 cm ; ms ( es ) for c20h18n2o2 318.1 found 319.0 ( m for c20h18n2o2 c , 75.45 ; h , 5.70 ; n , 8.80 found c , 75.57 ; h , 5.69 ; n , 8.82 . white solid ; mp 7779c ; h nmr ( 400 mhz , cdcl3 + dmso - d6 ) : h 0.95 ( t , 3h , j = 7.4 hz , ch3 ) , 2.65 ( s , 3h , ch3 ) , 4.05 ( q , 2h , j = 7.6 hz , ch2 ) , 7.147.46 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 + dmso - d6 ) : c 14.6 , 23.7 , 61.4 , 126.7 , 126.9 , 127.5 , 128.3 , 128.8 , 129.3 , 130.1 , 132.1 , 132.7 , 134.6 , 137.8 , 139.5 , 141.0 , 152.6 , 196.3 ppm ; ir ( kbr ) : max 1087 , 1100 , 1280 , 1434 , 1510 , 1769 , 2862 , 2932 , 3084 cm ; ms ( es ) for c20h18n2o2 318.1 found 319.0 ( m + h ) , 341.0 ( m + na ) ; chn calcd . for c20h18n2o2 c , 75.45 ; h , 5.70 ; n , 8.80 found c , 75.57 ; h , 5.69 ; n , 8.82 . 1-(3-methyl-5,6-diphenylpyridazin-4-yl)ethanone ( 6f , table 3)white solid ; mp 132134c ; h nmr ( 400 mhz , cdcl3 ) : h 1.82 ( s , 3h , ch3 ) , 2.59 ( s , 3h , ch3 ) , 7.187.47 ( m , 10h , ar - h ) ppm ; c nmr ( 100 mhz , cdcl3 ) : c 21.9 , 27.6 , 126.7 , 126.8 , 128.5 , 129.3 , 129.7 , 130.0 , 134.6 , 135.8 , 136.6 , 138.4 , 138.8 , 151.2 , 152.3 , 186.9 ppm ; ir ( kbr ) : max 1201 , 1240 , 1433 , 1520 , 1692 , 2888 , 2932 , 3100 cm ; ms ( es ) for c19h16n2o 288.1 found 289.0 ( m + h ) , 311.0 ( m + na ) ; chn calcd . for c19h16n2o c , 79.14 ; h , 5.59 ; n , 9.72 found c , 79.33 ; h , 5.51 ; n , 9.77 .

n- , and ki - ras ( with ki - ras existing as the predominant ki - ras4b and the alternatively spliced ki - ras4a isoforms ) that play a central role in cell signaling ( barbacid , 1987 ; malumbres and barbacid , 2003 ) . ras proteins are anchored on the cytoplasmic side of the cell membrane , where they mediate signal transduction downstream from tyrosine kinase membrane receptors to a variety of effector molecules , stimulating a cascade of parallel phosphorylation reaction pathways that ultimately culminate with the activation of nuclear transcription factors . raf / mek / mapk , pi3k / akt , and ral gds play major roles in mediating signals relating to cell proliferation , cell survival , cell adhesion , and cell motility ( fan and bertino , 1997 ; campbell et al . , 1998 ; gille and downward , 1999 ; zuber et al . , 2000 ; each of the ras isoforms appears to differentially regulate its downstream effectors in vivo , resulting in marked differences in the strength and type of signal produced ( hancock , 2003 ; ehrhardt et al . , 2004 ; moon , 2006 ; omerovic et al . , 2008 ) . this differential signaling appears to be mediated partly by the trafficking pathways used by each ras isoform to reach the plasma membrane , as well as the location of each isoform in the plasma membrane itself ( chiu et al . , 2002 ; hancock , 2003 ; moon , 2006 ) ; n- and ha - ras associate with lipid rafts in the plasma membrane , whereas ki - ras appears to be located in non - raft domains . the ras pathway is one of the most prevalent oncogenic alterations in both human and experimentally induced animal tumors ( bos , 1989 ; conti , 1992 ; malumbres and barbacid , 2003 ) . in vivo , oncogenic mutations have been shown to occur at exons 12 , 13 , and 61 , resulting in any 1 of 19 possible point mutations for each ras isoform . when stimulated by upstream signaling molecules , wild type ras proteins interact with guanine nucleotide exchange factors to replace gdp with gtp , resulting in an activated protein conformation . ras activity is terminated by interaction with gtpase activating protein , which stimulates the gtpase activity of the protein and converts gtp back to gdp , thereby restoring the inactive form of ras . mutations in ras inhibit the gtpase activity and lock the protein in the active gtp bound conformation ( barbacid , 1987 ; bos , 1989 ; malumbres and barbacid , 2003 ) in particular , mutations in the ki - ras gene have been shown to play a key role in the pathogenesis of a variety of human tumors , with mutations occurring in 95% of pancreatic tumors , 50% of colon tumors , and 30% of lung adenocarcinomas ( barbacid , 1987 ; bos et al . , 1987 ; conti , 1992 ; malumbres and barbacid , 2003 ) . of these cancers , lung and colon cancer are the first and second leading cause of cancer - related deaths in the u.s . , respectively ( jemal et al . , 2010 ) . among the candidate genes implicated in the initiation of these cancers , ki - ras has received considerable attention as mutations in ki - ras appear in early neoplastic lesions in both human and experimentally induced murine lung and colon tumors , and influence both tumor progression and drug resistance ( cerny et al . , 1992 ; reynolds et al . , 1992 ; hruban et al . , 1993 ; westra et al . , 1993 , 1996 ; li et al . , 1994a ; miller , 1994 ; gryfe et al . , 1997 ; grady and markowitz , 2002 ; agbunag and bar - sagi , 2004 ; fleming et al . , 2005 ) . the spectrum of ras mutations differs by organ site and allele frequency , probably as a result of different environmental exposures and tissue specific differences in ras expression . the sanger institute s cosmic database ( catalog of somatic mutations in cancer ; http://www.sanger.ac.uk/genetics/cgp/cosmic/add_info/ ) integrates data from the published literature on type and frequency of somatic mutations in human cancers . using the database search tools , we examined the spectrum and frequency of ki - ras mutations ( table 1 ) . consistent with the literature , ki - ras mutations were observed most frequently in cancers of the lung , large intestine ( including colon , rectal , and anal ) , pancreas , and biliary tract ( including bile duct and gall bladder ) . based on a collective mutational analysis involving > 15,000 tumors , the most frequent alterations observed were point mutations at codons 12 , 13 , and 61 . a spectrum of predominant mutant alleles were observed , and their relative frequencies are shown in table 1 as the percentage of all mutant alleles observed for a given tumor type . consistent with historical observations , asp , val , and cys emerged as the predominant mutant ki - ras alleles . however , that the alleles distributed with large variation within each cancer type may reflect the non - redundant functions of the different alleles in tumorigenesis . large variation across cancer types was also observed for some alleles , such as cys , asp , and asp , suggesting that mutant allele functionality may also depend , to some extent , on the tumor tissue of origin . relative frequencies of the major ki - ras mutations by cancer type : analysis of the sanger cosmic database . the relative percentage of each mutant allele within a cancer type ( i.e. , of the four predominant ki - ras mutation - bearing cancer types ) is shown ; * includes colon , rectal , and anal cancer ; includes cancers of the bile duct and gallbladder . although some studies have provided evidence for mutation specific effects of different mutant ras alleles , most studies and therapeutic approaches have treated ras mutations as a single entity we believe that the different ras mutations exhibit subtle differences in their ability to signal to their downstream effectors , which may impact their relative contribution to the carcinogenic process , their role as driver mutations , and tumor responsiveness to novel therapeutic agents that target ras or its downstream effectors . thus , this manuscript reviews the evidence obtained from biochemical , cell culture , and animal model data , as well as the limited number of human studies available , documenting the differential response of cells to different mutant ras alleles . studies on the potential differences in the mutagenicity / oncogenicity of different ras mutant alleles began shortly after the identification of ras as a transforming oncogene . initial studies demonstrated that different ha - ras mutant alleles exhibited differences in their ability to transform mouse fibroblasts ( fasano et al . ( 1984 ) found that the val mutation was the most potent in terms of the induction of focus formation in the nih3t3 assay , with arg , asp , ser , asp , and ser exhibiting transforming efficiencies that were 60 , 50 , 40 , 20 , and 0.1% relative to the val mutant . ( 1984 ) , found somewhat similar results in rat-1 cells with mutants to both ki - ras and ha - ras . ( 1984 ) , these authors found that alleles of both ki- and ha - ras containing the val and arg mutations exhibited greater transforming activity than alleles with the cys , asp , asn , and ser mutations . der et al . ( 1986 ) examined 17 different codon 61 mutations in the ha - ras gene and found that the transforming activity in nih 3t3 cells varied by more than 300-fold between the different mutant alleles . several groups ( gibbs et al . , 1984a , b ; mcgrath et al . , 1984 ; sweet et al . , 1984 ; manne et al . , 1985 ) , using purified ha - ras produced in e. coli , demonstrated that the valine 12 mutant exhibited 5 to 10-fold lower gtpase activity than the normal wild type allele . it was initially thought that the relative level of gtpase activity might account for the differences in transforming potential between the different mutant alleles . ( 1986 ) did not find any correlation between the transforming potency and gtpase activity of the different alleles . subsequent to these early studies , the biochemical activity of ras and its interactions with its downstream receptors has continued to be the focus of intensive investigations . several laboratories have demonstrated that mutations in specific amino acids have very significant effects on both guanine nucleotide exchange factors and ras gtpase activity ( nielsen et al . , 2001 ; however , many of these mutational analyses have been limited to examining amino acids that cause conformational changes in regions of the ras genes associated with gtpase activity , such as the a59 g variant ( lukman et al . , 2010 ) , rather than the common mutagenic variant alleles associated with tumorigenesis . more recently , redox agents such as reactive oxygen species and reactive nitrogen species have been shown to enhance the rate of guanine nucleotide exchange as a result of the formation of a thiyl radical on cys ha - ras ( del villar et al . , 1996 ; kjeldgaard et al . , 1996 ; reuther and der , 2000 ; ford et al . , 2002 ; jourdheuil et al . , 2003 ; , 2003 ; heo et al . , 2005 , 2006 ; davis et al . , this raises the possibility that codon 12 , 13 , and 61 mutations could affect ras gtpase activity by either ( 1 ) increasing oxidative stress and thereby increasing thiylation of cys or ( 2 ) altering the accessibility of the cys residue to modification by reactive oxygen species . as will be discussed below , the cys allele has been shown in a variety of experimental systems to exhibit less potent tumorigenic activity than other mutations in codon 12 . it is thus possible that the cys variant could provide a new redox active cysteine motif in the ras protein that differentially responds to the increased oxidative stress of the cancer cell environment in a different manner than other mutant ras alleles . clearly , none of these potential mechanisms are mutually exclusive and suggest new areas of inquiry into the mechanisms of the observed differential effects of ras mutants on tumorigenicity . studies from this and other laboratories have demonstrated an association between the histological stage of both murine and human lung tumors and the presence of specific mutant ras alleles in tumor tissue . ( 1990 ) demonstrated that , following treatment of adult a / j mice with urethane , mouse lung adenocarcinomas ( acs ) had a high incidence of glu arg ( caa cga ) mutations relative to the smaller adenomas ( ads ) , which preferentially exhibited glu leu ( caa cta ) mutations . li et al . ( 1994b ) also demonstrated that lung acs showed a higher incidence of mutations at the second base of codon 61 following treatment of newborn mice with nitrochrysene and its metabolites . utilizing a transplacental treatment protocol , whereby mice are exposed in utero to a single dose of the chemical carcinogen 3-methylcholanthrene , we have demonstrated in three independent studies using different strains of mice that different ras mutations are associated with tumor stage ( leone - kabler et al . , 1997 ; gressani et al . , 1999 ; jennings - gee et al . , 2006 ) . treatment of pregnant mice with 3-methylcholanthrene resulted in a high incidence of lung tumors in the offspring 612 months after birth . mice harboring a val , arg , asp , or arg mutant ki - ras gene were more likely to contain later stage tumors than mice with the cys or wild type allele , which exhibited mostly benign ads and hyperplasias . further work with this model also suggested that the type of mutation induced in ki - ras following in utero exposure to the chemical carcinogen was associated with specific types of damage ( hypermethylation vs. base pair mutations ) at the ink4a gene locus ( mizesko et al . , 2001 ) . interestingly , the mutant ki - ras alleles associated with progression to later stage tumors in our transplacental mouse studies were the same ones associated with a trend for poorer patient outcomes in a clinical study of human lung cancer ( keohavong et al . , 1996 ) . a clinical study examining the prognostic significance of ki - ras mutations in lung cancer patients found that patients containing cys , arg , and asp mutations at codon 12 appeared to have a poorer prognosis than those containing hydrophobic amino acid substitutions such as val or ala ( siegfried et al . , 1997 ) . however , the sample sizes for this analysis were small and the authors did not find an overall association of ki - ras mutations and poorer patient survival , as has been noted in several other studies ( reviewed in rodenhuis and slebos , 1992 ) . ( 1993a , b ) reported that the ki - ras asp mutant allele was associated with the metastatic properties of colon tumors . in addition to the ability of different ras mutant alleles to initiate tumor formation , the above mentioned results obtained in vivo also suggest differences in tumor progression imparted by the different ras variant alleles . in the in vivo studies cited above , a consistent finding was that more aggressive tumors ( i.e. , acs ) were more prevalent with one type of ras mutation , often the val allele , whereas benign lesions such as ads or hyperplasias were more prevalent with other types of ras variants , most notably the cys mutation . these results suggest that specific ras mutant alleles can impart a greater growth advantage than other alleles , and the often disparate results may be due to context and organ dependent effects of the different alleles ( guerra et al . , 2003 ) . ( 2006 ) have extended these findings and also confirmed the relatively weak transforming activity of the ki - ras cys allele observed in our studies . these authors transfected nih3t3 cells with the asp , val , and cys alleles of ki - ras . when the transforming properties of the alleles were assessed , the ki - ras val allele exhibited a more aggressive tumorigenic phenotype than the ki - ras asp allele , which was attributed to the inability of the asp allele to signal through the raf / mek / erk pathway . when the transfected cells were injected into nude mice , the ki - ras cys containing cells failed to establish tumors . along the same lines , studies by this group utilizing nih3t3 cells found that the ki - ras val allele , in contrast to the wild type and ki - ras val allele , exhibited increased glycolysis ( vizan et al . , 2005 ) . in addition , cells transfected with the val mutant allele were resistant to the induction of apoptosis induced by confluence and exhibited a much greater ability to grow in soft agar . the val mutant clones exhibited elevated levels of p - akt , increased expression of bcl-2 , e - cadherin , -catenin , and focal adhesion kinase , and decreased expression of rhoa ( guerrero et al . , 2000 ) 2008 ) found that the val and asp variants of ki - ras enhanced cell survival and resistance to oxidative stress . although they did not specifically address this in their paper , the val allele exhibited greater protection against formaldehyde- and h2o2-mediated toxicity and reduced caspase 3/7 activity relative to the asp allele . thus , a generally consistent finding across the animal studies , cell culture experiments , and human patient samples suggests that the cys allele is associated with a relatively weak or no transforming activity , while val and asp alleles were associated with more aggressive oncogenic properties . these transforming properties were not only associated with increased tumor formation , but seemed to also play a role in tumor progression . the somewhat conflicting studies with human samples most likely result from the multitude of alterations that occur in human tumors by the time they are diagnosed . it is very likely that tumors with weakly transforming ras alleles may require additional alterations at other oncogenic loci in order to drive tumorigenesis . thus , in some cases , a ras mutation such as the val allele could act as a driver mutation whereas in other cases , such as the cys allele , mutations in other genes may be the key drivers of tumorigenesis . a major limitation of studies utilizing human tissue is the fact that human tumors contain several mutations in a variety of oncogenic loci . if a weakly oncogenic ki - ras mutation occurs in a particular patient s tumor , it is likely that by the time the tumor is isolated , mutations in other critical driver genes would have occurred . to our knowledge , no studies have been published which attempt to correlate specific mutations in ki - ras with the presence or absence of other known driver mutations . thus , attempts to associate specific ras mutations with patient outcome or survival may not be able to distinguish the relative contribution of a specific ki - ras mutation to the tumorigenic process . in order to characterize the role of ki - ras mutations in the initiation of lung tumorigenesis these included : ( 1 ) regulation of a murine ki - ras asp cdna transgene via a doxycyline ( dox ) regulated promoter ( fisher et al . , 2001 ) ; ( 2 ) sporadic activation of a human ki - ras val cdna transgene via a cre - lox mediated recombination construct ( meuwissen et al . , 2001 ; ( 3 ) spontaneous activation of the murine ki - ras asp gene via a somatic recombination system ( johnson et al . , 2001 ) ; and ( 4 ) sporadic activation of the same murine ki - ras asp gene mutation via a cre based lox - stop - lox construct ( jackson et al . , 2001 ) . expression of these mutant ki - ras alleles were shown to be a strong oncogenic stimulus for lung epithelial cells , resulting in significant increases in lung tumorigenicity . in each of these models , 100% of the mice developed aggressive acs and died within 210 months from their lung tumor burden . in contrast to these results our laboratory , utilizing a transgenic mouse in which the mutant human ki - ras cys allele is regulated in a dox - inducible and lung - specific manner ( floyd et al . , 2005 ) , found that these mice developed hyperplasias and small , benign adenomas ( ads ) after 12 months of dox treatment that rarely progressed to the carcinoma stage . the cys transgene appeared to signal to a subset of its downstream effectors , exhibiting increases in proliferative and both anti- and pro - apoptotic signals , as well as up - regulation of cell cycle inhibitory molecules . mice harboring this mutant ki - ras allele exhibited increased signaling through the ras / raf / erk / cyclin d1 , p38 , and ral / gds pathways , but no alterations in signaling through the jnk and pi3k / akt pathways ( floyd et al . , 2006 ; dance - barnes et al . , 2008 ) . interestingly , the relatively benign tumor phenotype observed in ki - ras cys mice was also observed in mice with activated and wild type raf genes ( kerkhoff et al . , 2000 ) . these authors developed a strain of mice expressing an activated human c - raf-1 transgene specifically in the lung ( spc - c - raf-1-bxb mice ) that also exhibited a relatively benign tumor phenotype , despite increased phosphorylation of downstream effector molecules in the erk pathway . because mutant raf is downstream of ras and thus signals to a specific subset of ras effector molecules , these experiments provide further in vivo confirmation of the need for the activation of multiple ras downstream effector molecules to mediate the full transformation of lung epithelial cells and provide the most definitive evidence to date of the potential for different ki - ras mutations to exhibit differential effects on the carcinogenic process . with the limitations that these studies often involved different inducible gene systems , different strains of mice , and the unknowns of the potential effects of integration of the transgene in the mouse genome , future studies will need to take advantage of targeted knock - in technologies to create a series of transgenic mouse strains that replace the endogenous mouse ki - ras gene with various mutated alleles and allow the mutants to be expressed from the natural murine ki - ras promoter . these types of studies will be critical in dissecting out the effect of different ras mutations on downstream signaling pathways and tumorigenicity . several studies using human tissue samples and cell lines ( bhattacharjee et al . , 2001 ; beer et al . , 2002 ; miura et al . , 2002 ; virtanen et al . , 2002 ; wikman et al . , 2002 ; 2005 ) have utilized rna and cdna microarray analyses to document the complex array of alterations in signaling pathways that accompany lung tumorigenesis . yao et al . ( 2002 ) isolated lung tumors 6 and 14 months following treatment of a / j mice with a single injection of n - methylnitrosourea at 6 weeks of age . using the affymetrix atlas mouse cdna expression array , consisting of 588 known mouse genes , they were able to identify 19 genes that showed differential expression between ads and acs , as well as 10 genes that exhibited similar alterations in expression levels between the two tumor types . subsequent studies from the same group , using the more extensive affymetrix mu74av2 chip , which can interrogate 36,000 full - length mouse genes and est clusters from the unigene database , identified 50 genes that were either up - or down - regulated in ads vs. acs , including genes involved in cell cycle control , differentiation , and apoptosis ( bonner et al . , 2004 ) . in addition , when the murine tumors were compared with lung acs isolated from human patients , the murine ads and acs clustered with two groups of human acs that differed in their differentiation status , with murine ads clustering with the well differentiated human acs and murine acs clustering with the less differentiated tumors . these investigators also identified 39 genes that were similarly regulated in murine and human lung acs , further emphasizing the appropriateness of the mouse as a model for human lung cancer . similarly , jacks group ( sweet - cordero et al . , 2005 ) used the data obtained from this study in combination with their own analysis of mouse tumors from transgenic ki - ras mice , which express the ki - ras asp transgene sporadically as a result of spontaneous recombination ( johnson et al . , 2001 ) , and compared the results obtained with the murine lung tumors with human arrays from a variety of sources , including their own analyses as well as those from beer et al . these investigators were able to demonstrate patterns of gene expression that were common to both human and murine lung acs ( sweet - cordero et al . , 2005 ) . most interesting , these researchers demonstrated that a ki - ras expression signature in human acs that was not identifiable by statistical analysis unless the mouse data was included in the integrated analyses , due to the high degree of variability inherent in human lung tumor tissues , results which have been confirmed by creighton et al . interestingly , in all of these studies mutated ras genes were treated as a single entity , as the comparison that was made was samples containing mutated ras genes vs. those that did not . thus , to date , it is unclear to what extent these signatures reflect the differential effects of different ras mutant alleles on downstream signaling pathways . accounting for this added complexity may allow a further refinement of the signatures down to the subset of ras stimulated genes that are the most critical for tumor initiation and maintenance . clearly , different ras mutant alleles may engage different subsets of ras downstream effector pathways , which may influence the gene expression profiles . this could be the reason that statistical significance for ras mutated vs. wild type tumors could not be reached until the mouse data , which had a more homogenous ki - ras mutational spectrum , were added . recent studies have utilized synthetic lethal and genome wide inhibitory rna approaches to identify ras effector molecules that are critical for tumorigenesis ( kassie et al . , 2008 ; barbie et al . , 2009 ; luo et al . , 2009 ; scholl et al . , 2009 ; singh et al . , 2009 ; vicent et al . , these studies have clearly shown the dependency of tumor cells on a subset of ras downstream effector pathways when mutated ras genes are present . all of these studies have been conducted with the alleles that animal and in vitro studies have identified as the most oncogenic ras mutations . future studies will thus need to compare the use of synthetic lethal approaches with different ras mutants . this will allow an understanding of which mutant ras alleles signal to these critical pathways and will be an important approach in developing novel therapeutic agents . a number of studies have shown that mutations in ki - ras are prognostic factors for poor patient outcome in lung cancer ( mitsudomi et al . , 1991 ; rodenhuis and slebos , 1992 ; rosell et al . , 1993 , 1996 ; patients whose tumors contained ki - ras mutations often exhibited poorer overall survival and reduced time to disease progression . as noted above , one study found that lung cancer patients whose tumors contained the cys , arg , or asp mutations at codon 12 appeared to have a poorer prognosis than those containing hydrophobic amino acid substitutions such as val or ala ( siegfried et al . , 1997 ) . however , a recent study in stage iii colon cancer patients failed to find an association with disease - free or overall survival ( ogino et al . , 2009 ) . a limitation of both of these studies was the fact that mutations in ki - ras were treated as a single entity , so that all patients with mutations were compared to patients with wild type ki - ras . ( 2011 ) found that lung tumors of patients harboring the cys mutations were less sensitive to cisplatin therapy but exhibited increased sensitivity to taxol and pemetrexed relative to the val and asp mutant alleles . the asp mutation demonstrated increased resistance to taxol and enhanced sensitivity to sorafenib . while none of the ras mutants exhibited differential sensitivity to the tyrosine kinase inhibitors erlotinib , a study examining colorectal cancer patients with chemotherapy - refractory metastatic disease who harbored the ki - ras asp mutation found a small but statistically significant increase in overall survival and progression - free survival relative to patients harboring other ki - ras mutations ( de et al . , 2010 ) . these results clearly suggest that different ras mutations may have a significant impact on patient response to therapeutic interventions , and that the ras mutational profile may need to be considered in future clinical trials assessing the effects of novel agents in tumors harboring ras mutations . recent studies have identified novel mutations in ras genes , whose influence on tumorigenicity are first being assessed . mutations in exon 4 of ki - ras coding for lys and ala were associated with an increased probability of disease - free survival despite increases in ras - gtp ( janakiraman et al . , 2010 ) . ( 2008 ) demonstrated that the minor ki - ras4a isoform may be the critical form of ki - ras that is responsible for lung carcinogenesis . the asn and thr mutations , when transfected into nih3t3 cells , produced a similar number of transformed foci as the asp mutation but fewer colonies than the val and asp mutant alleles . the phe and gln mutant alleles , similar to the wild type allele , had little transforming activity . when expression arrays were performed for cell lines harboring the different alleles , differential expression of a number of genes associated with tumor proliferation and cell survival were observed , consistent with the different alleles stimulating different gene expression programs . this work is among the first to document mutant - specific alterations in gene transcriptional programming , and may yield further insights into the effector pathways of allele - specific signaling . finally , a ki - ras single nucleotide polymorphism in the 3 untranslated region of the gene that disrupts binding of the let7 microrna has been shown to be a potential prognostic marker of ovarian cancer risk , as 25% of unselected ovarian cancer cases and 61% of hereditary ovarian cancer patients lacking mutations in the brca genes harbored this ras variant allele ( ratner et al . , 2010 ) . interestingly , hwang and cohen ( 1997 ) found that deletion or inversion of a splicing enhancer region located in the 3 untranslated region of the ha - ras gene reduced the transcription of ras mrna and thus decreased the transforming activity of this variant allele . thus , the list of mutant ras alleles with the differential ability to influence tumorigenicity continues to grow . how each of these mutant alleles influences carcinogenicity and the mechanisms by which they do so are an area of research that should be a major emphasis for future studies . since the identification of mutant ras genes as transforming oncogenes , investigators have attempted to identify a mechanistic basis for the allele - specific differences that could account for the observed differences in transforming ability observed in vitro and in vivo . these studies have so far failed to identify a specific gene pathway or pathways that account for the differential effects of different ras mutants . the majority of evidence obtained to date and described above suggests that , in particular , the cys mutation may exhibit less potent transforming properties than the val and asp mutants , but a rigorous comparison of all possible ras mutations of biological significance in the same experimental system has not been conducted . in addition , the list of clinically relevant and biologically active ras mutations continues to grow . some of the reason for the lack of a clear mechanistic basis for the observed phenotypic differences imparted by different mutant ras alleles may be due to very subtle effects of the mutant ras alleles on multiple gene pathways ( either at the transcriptome or proteome level ) and context as well as cell and tissue specific effects of mutant ras genes . recent advances in technology should enable us to determine the basis of these differences going forward . biochemical and kinetic studies demonstrating the redox sensitivity of the cys residue ( heo et al . , 2005 ; heo and campbell , 2006 ; raines et al . , 2007 ) suggest that specific mutant forms of ras could exhibit differential sensitivity to the redox state of the cell , which could influence tumor progression by either direct effects on ras gtpase activity or indirect effects via alterations of protein interactions between ras and its downstream effectors . as illustrated by the expression profiling studies described above ( smith et al . , 2010 ) , applications of transcriptomic and proteomic expression profiling hold much promise for delineating the allele - specific effects of different ras mutant alleles . these studies need to be done in both human tumor samples as well as novel transgenic models . the development of knock - in mouse models for each of the ras mutant alleles would be a critically important advance in the field . because of the tissue specific and dose related effects of ras gene expression , it will be important to test these alleles in the same genetic background , driven from the endogenous ras promoters , to eliminate some of the uncertainties associated with traditional transgenic constructs . while these studies should initially be done with ki - ras , which is mutated more frequently than other ras family members in human cancer , extending this research to other ras family members will allow the development of a comprehensive understanding of ras signaling networks . this will provide important information in terms of developing targeted therapies to the alleles most responsible for driving tumorigenesis as opposed to those that have much less significant contributions to maintenance of the tumorigenic phenotype . it is likely that the answer will not be simple and that more than one mechanism will influence the behavior of different mutant ras alleles . since ras is the most commonly mutated gene in human cancers and signals to a wide variety of molecules involved in proliferation , cell death , cell survival , oxidative stress , angiogenesis , inflammation , and drug resistance ( campbell et al . , 1998 ; hancock , 2003 ; malumbres and barbacid , 2003 ; young et al . , 2009 ) , it is likely that patients whose tumors harbor specific ras mutations will exhibit differences in survival , tumor aggressiveness , and response to chemotherapy agents . an understanding of the properties of each of the ras mutants in specific tissues will aid in future attempts to personalize cancer treatment regimens and assure the best possible outcomes for patients . 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 . ac , adenocarcinomas ; ad , adenomas ; ccsp , clara cell secretory protein ; dox , doxycycline ; ihc , immunohistochemistry ; rt - pcr , reverse transcription - polymerase chain reaction ; rtta , reverse tetracycline trans - activator ; tet , tetracycline .

mutation in ras proteins is one of the most common genetic alterations observed in human and experimentally induced rodent cancers . in vivo , oncogenic mutations have been shown to occur at exons 12 , 13 , and 61 , resulting in any 1 of 19 possible point mutations in a given tumor for a specific ras isoform . while some studies have suggested a possible role of different mutant alleles in determining tumor severity and phenotype , no general consensus has emerged on the oncogenicity of different mutant alleles in tumor formation and progression . part of this may be due to a lack of a single , signature pathway that shows significant alterations between different mutations . rather , it is likely that subtle differences in the activation , or lack thereof , of downstream effectors by different ras mutant alleles may determine the eventual outcome in terms of tumor phenotype . this paper reviews our current understanding of the potential role of different ras mutations on tumorigenesis , highlights studies in model cell culture and in vivo systems , and discusses the potential of expression array and computational network modeling to dissect out differences in activated ras genes in conferring a transforming phenotype .

Introduction None None Differential Expression of Ki- Emerging Research Conclusion and Future Perspectives Conflict of Interest Statement Abbreviations

n- , and ki - ras ( with ki - ras existing as the predominant ki - ras4b and the alternatively spliced ki - ras4a isoforms ) that play a central role in cell signaling ( barbacid , 1987 ; malumbres and barbacid , 2003 ) . ras proteins are anchored on the cytoplasmic side of the cell membrane , where they mediate signal transduction downstream from tyrosine kinase membrane receptors to a variety of effector molecules , stimulating a cascade of parallel phosphorylation reaction pathways that ultimately culminate with the activation of nuclear transcription factors . , 2000 ; each of the ras isoforms appears to differentially regulate its downstream effectors in vivo , resulting in marked differences in the strength and type of signal produced ( hancock , 2003 ; ehrhardt et al . this differential signaling appears to be mediated partly by the trafficking pathways used by each ras isoform to reach the plasma membrane , as well as the location of each isoform in the plasma membrane itself ( chiu et al . , 2002 ; hancock , 2003 ; moon , 2006 ) ; n- and ha - ras associate with lipid rafts in the plasma membrane , whereas ki - ras appears to be located in non - raft domains . the ras pathway is one of the most prevalent oncogenic alterations in both human and experimentally induced animal tumors ( bos , 1989 ; conti , 1992 ; malumbres and barbacid , 2003 ) . in vivo , oncogenic mutations have been shown to occur at exons 12 , 13 , and 61 , resulting in any 1 of 19 possible point mutations for each ras isoform . when stimulated by upstream signaling molecules , wild type ras proteins interact with guanine nucleotide exchange factors to replace gdp with gtp , resulting in an activated protein conformation . mutations in ras inhibit the gtpase activity and lock the protein in the active gtp bound conformation ( barbacid , 1987 ; bos , 1989 ; malumbres and barbacid , 2003 ) in particular , mutations in the ki - ras gene have been shown to play a key role in the pathogenesis of a variety of human tumors , with mutations occurring in 95% of pancreatic tumors , 50% of colon tumors , and 30% of lung adenocarcinomas ( barbacid , 1987 ; bos et al . among the candidate genes implicated in the initiation of these cancers , ki - ras has received considerable attention as mutations in ki - ras appear in early neoplastic lesions in both human and experimentally induced murine lung and colon tumors , and influence both tumor progression and drug resistance ( cerny et al . the spectrum of ras mutations differs by organ site and allele frequency , probably as a result of different environmental exposures and tissue specific differences in ras expression . the sanger institute s cosmic database ( catalog of somatic mutations in cancer ; http://www.sanger.ac.uk/genetics/cgp/cosmic/add_info/ ) integrates data from the published literature on type and frequency of somatic mutations in human cancers . consistent with the literature , ki - ras mutations were observed most frequently in cancers of the lung , large intestine ( including colon , rectal , and anal ) , pancreas , and biliary tract ( including bile duct and gall bladder ) . based on a collective mutational analysis involving > 15,000 tumors , the most frequent alterations observed were point mutations at codons 12 , 13 , and 61 . a spectrum of predominant mutant alleles were observed , and their relative frequencies are shown in table 1 as the percentage of all mutant alleles observed for a given tumor type . however , that the alleles distributed with large variation within each cancer type may reflect the non - redundant functions of the different alleles in tumorigenesis . large variation across cancer types was also observed for some alleles , such as cys , asp , and asp , suggesting that mutant allele functionality may also depend , to some extent , on the tumor tissue of origin . relative frequencies of the major ki - ras mutations by cancer type : analysis of the sanger cosmic database . , of the four predominant ki - ras mutation - bearing cancer types ) is shown ; * includes colon , rectal , and anal cancer ; includes cancers of the bile duct and gallbladder . although some studies have provided evidence for mutation specific effects of different mutant ras alleles , most studies and therapeutic approaches have treated ras mutations as a single entity we believe that the different ras mutations exhibit subtle differences in their ability to signal to their downstream effectors , which may impact their relative contribution to the carcinogenic process , their role as driver mutations , and tumor responsiveness to novel therapeutic agents that target ras or its downstream effectors . thus , this manuscript reviews the evidence obtained from biochemical , cell culture , and animal model data , as well as the limited number of human studies available , documenting the differential response of cells to different mutant ras alleles . studies on the potential differences in the mutagenicity / oncogenicity of different ras mutant alleles began shortly after the identification of ras as a transforming oncogene . initial studies demonstrated that different ha - ras mutant alleles exhibited differences in their ability to transform mouse fibroblasts ( fasano et al . ( 1984 ) found that the val mutation was the most potent in terms of the induction of focus formation in the nih3t3 assay , with arg , asp , ser , asp , and ser exhibiting transforming efficiencies that were 60 , 50 , 40 , 20 , and 0.1% relative to the val mutant . ( 1986 ) examined 17 different codon 61 mutations in the ha - ras gene and found that the transforming activity in nih 3t3 cells varied by more than 300-fold between the different mutant alleles . it was initially thought that the relative level of gtpase activity might account for the differences in transforming potential between the different mutant alleles . , 2001 ; however , many of these mutational analyses have been limited to examining amino acids that cause conformational changes in regions of the ras genes associated with gtpase activity , such as the a59 g variant ( lukman et al . more recently , redox agents such as reactive oxygen species and reactive nitrogen species have been shown to enhance the rate of guanine nucleotide exchange as a result of the formation of a thiyl radical on cys ha - ras ( del villar et al . , this raises the possibility that codon 12 , 13 , and 61 mutations could affect ras gtpase activity by either ( 1 ) increasing oxidative stress and thereby increasing thiylation of cys or ( 2 ) altering the accessibility of the cys residue to modification by reactive oxygen species . as will be discussed below , the cys allele has been shown in a variety of experimental systems to exhibit less potent tumorigenic activity than other mutations in codon 12 . it is thus possible that the cys variant could provide a new redox active cysteine motif in the ras protein that differentially responds to the increased oxidative stress of the cancer cell environment in a different manner than other mutant ras alleles . studies from this and other laboratories have demonstrated an association between the histological stage of both murine and human lung tumors and the presence of specific mutant ras alleles in tumor tissue . utilizing a transplacental treatment protocol , whereby mice are exposed in utero to a single dose of the chemical carcinogen 3-methylcholanthrene , we have demonstrated in three independent studies using different strains of mice that different ras mutations are associated with tumor stage ( leone - kabler et al . treatment of pregnant mice with 3-methylcholanthrene resulted in a high incidence of lung tumors in the offspring 612 months after birth . a clinical study examining the prognostic significance of ki - ras mutations in lung cancer patients found that patients containing cys , arg , and asp mutations at codon 12 appeared to have a poorer prognosis than those containing hydrophobic amino acid substitutions such as val or ala ( siegfried et al . in addition to the ability of different ras mutant alleles to initiate tumor formation , the above mentioned results obtained in vivo also suggest differences in tumor progression imparted by the different ras variant alleles . in the in vivo studies cited above , a consistent finding was that more aggressive tumors ( i.e. these results suggest that specific ras mutant alleles can impart a greater growth advantage than other alleles , and the often disparate results may be due to context and organ dependent effects of the different alleles ( guerra et al . ( 2006 ) have extended these findings and also confirmed the relatively weak transforming activity of the ki - ras cys allele observed in our studies . thus , a generally consistent finding across the animal studies , cell culture experiments , and human patient samples suggests that the cys allele is associated with a relatively weak or no transforming activity , while val and asp alleles were associated with more aggressive oncogenic properties . these transforming properties were not only associated with increased tumor formation , but seemed to also play a role in tumor progression . it is very likely that tumors with weakly transforming ras alleles may require additional alterations at other oncogenic loci in order to drive tumorigenesis . thus , in some cases , a ras mutation such as the val allele could act as a driver mutation whereas in other cases , such as the cys allele , mutations in other genes may be the key drivers of tumorigenesis . a major limitation of studies utilizing human tissue is the fact that human tumors contain several mutations in a variety of oncogenic loci . if a weakly oncogenic ki - ras mutation occurs in a particular patient s tumor , it is likely that by the time the tumor is isolated , mutations in other critical driver genes would have occurred . to our knowledge , no studies have been published which attempt to correlate specific mutations in ki - ras with the presence or absence of other known driver mutations . thus , attempts to associate specific ras mutations with patient outcome or survival may not be able to distinguish the relative contribution of a specific ki - ras mutation to the tumorigenic process . in order to characterize the role of ki - ras mutations in the initiation of lung tumorigenesis these included : ( 1 ) regulation of a murine ki - ras asp cdna transgene via a doxycyline ( dox ) regulated promoter ( fisher et al . , 2001 ) ; ( 2 ) sporadic activation of a human ki - ras val cdna transgene via a cre - lox mediated recombination construct ( meuwissen et al . expression of these mutant ki - ras alleles were shown to be a strong oncogenic stimulus for lung epithelial cells , resulting in significant increases in lung tumorigenicity . the cys transgene appeared to signal to a subset of its downstream effectors , exhibiting increases in proliferative and both anti- and pro - apoptotic signals , as well as up - regulation of cell cycle inhibitory molecules . interestingly , the relatively benign tumor phenotype observed in ki - ras cys mice was also observed in mice with activated and wild type raf genes ( kerkhoff et al . these authors developed a strain of mice expressing an activated human c - raf-1 transgene specifically in the lung ( spc - c - raf-1-bxb mice ) that also exhibited a relatively benign tumor phenotype , despite increased phosphorylation of downstream effector molecules in the erk pathway . because mutant raf is downstream of ras and thus signals to a specific subset of ras effector molecules , these experiments provide further in vivo confirmation of the need for the activation of multiple ras downstream effector molecules to mediate the full transformation of lung epithelial cells and provide the most definitive evidence to date of the potential for different ki - ras mutations to exhibit differential effects on the carcinogenic process . with the limitations that these studies often involved different inducible gene systems , different strains of mice , and the unknowns of the potential effects of integration of the transgene in the mouse genome , future studies will need to take advantage of targeted knock - in technologies to create a series of transgenic mouse strains that replace the endogenous mouse ki - ras gene with various mutated alleles and allow the mutants to be expressed from the natural murine ki - ras promoter . these types of studies will be critical in dissecting out the effect of different ras mutations on downstream signaling pathways and tumorigenicity . ( 2002 ) isolated lung tumors 6 and 14 months following treatment of a / j mice with a single injection of n - methylnitrosourea at 6 weeks of age . subsequent studies from the same group , using the more extensive affymetrix mu74av2 chip , which can interrogate 36,000 full - length mouse genes and est clusters from the unigene database , identified 50 genes that were either up - or down - regulated in ads vs. acs , including genes involved in cell cycle control , differentiation , and apoptosis ( bonner et al . most interesting , these researchers demonstrated that a ki - ras expression signature in human acs that was not identifiable by statistical analysis unless the mouse data was included in the integrated analyses , due to the high degree of variability inherent in human lung tumor tissues , results which have been confirmed by creighton et al . interestingly , in all of these studies mutated ras genes were treated as a single entity , as the comparison that was made was samples containing mutated ras genes vs. those that did not . thus , to date , it is unclear to what extent these signatures reflect the differential effects of different ras mutant alleles on downstream signaling pathways . accounting for this added complexity may allow a further refinement of the signatures down to the subset of ras stimulated genes that are the most critical for tumor initiation and maintenance . clearly , different ras mutant alleles may engage different subsets of ras downstream effector pathways , which may influence the gene expression profiles . recent studies have utilized synthetic lethal and genome wide inhibitory rna approaches to identify ras effector molecules that are critical for tumorigenesis ( kassie et al . , these studies have clearly shown the dependency of tumor cells on a subset of ras downstream effector pathways when mutated ras genes are present . all of these studies have been conducted with the alleles that animal and in vitro studies have identified as the most oncogenic ras mutations . a number of studies have shown that mutations in ki - ras are prognostic factors for poor patient outcome in lung cancer ( mitsudomi et al . a limitation of both of these studies was the fact that mutations in ki - ras were treated as a single entity , so that all patients with mutations were compared to patients with wild type ki - ras . while none of the ras mutants exhibited differential sensitivity to the tyrosine kinase inhibitors erlotinib , a study examining colorectal cancer patients with chemotherapy - refractory metastatic disease who harbored the ki - ras asp mutation found a small but statistically significant increase in overall survival and progression - free survival relative to patients harboring other ki - ras mutations ( de et al . these results clearly suggest that different ras mutations may have a significant impact on patient response to therapeutic interventions , and that the ras mutational profile may need to be considered in future clinical trials assessing the effects of novel agents in tumors harboring ras mutations . recent studies have identified novel mutations in ras genes , whose influence on tumorigenicity are first being assessed . mutations in exon 4 of ki - ras coding for lys and ala were associated with an increased probability of disease - free survival despite increases in ras - gtp ( janakiraman et al . finally , a ki - ras single nucleotide polymorphism in the 3 untranslated region of the gene that disrupts binding of the let7 microrna has been shown to be a potential prognostic marker of ovarian cancer risk , as 25% of unselected ovarian cancer cases and 61% of hereditary ovarian cancer patients lacking mutations in the brca genes harbored this ras variant allele ( ratner et al . interestingly , hwang and cohen ( 1997 ) found that deletion or inversion of a splicing enhancer region located in the 3 untranslated region of the ha - ras gene reduced the transcription of ras mrna and thus decreased the transforming activity of this variant allele . since the identification of mutant ras genes as transforming oncogenes , investigators have attempted to identify a mechanistic basis for the allele - specific differences that could account for the observed differences in transforming ability observed in vitro and in vivo . these studies have so far failed to identify a specific gene pathway or pathways that account for the differential effects of different ras mutants . the majority of evidence obtained to date and described above suggests that , in particular , the cys mutation may exhibit less potent transforming properties than the val and asp mutants , but a rigorous comparison of all possible ras mutations of biological significance in the same experimental system has not been conducted . some of the reason for the lack of a clear mechanistic basis for the observed phenotypic differences imparted by different mutant ras alleles may be due to very subtle effects of the mutant ras alleles on multiple gene pathways ( either at the transcriptome or proteome level ) and context as well as cell and tissue specific effects of mutant ras genes . recent advances in technology should enable us to determine the basis of these differences going forward . , 2007 ) suggest that specific mutant forms of ras could exhibit differential sensitivity to the redox state of the cell , which could influence tumor progression by either direct effects on ras gtpase activity or indirect effects via alterations of protein interactions between ras and its downstream effectors . , 2010 ) , applications of transcriptomic and proteomic expression profiling hold much promise for delineating the allele - specific effects of different ras mutant alleles . the development of knock - in mouse models for each of the ras mutant alleles would be a critically important advance in the field . because of the tissue specific and dose related effects of ras gene expression , it will be important to test these alleles in the same genetic background , driven from the endogenous ras promoters , to eliminate some of the uncertainties associated with traditional transgenic constructs . while these studies should initially be done with ki - ras , which is mutated more frequently than other ras family members in human cancer , extending this research to other ras family members will allow the development of a comprehensive understanding of ras signaling networks . this will provide important information in terms of developing targeted therapies to the alleles most responsible for driving tumorigenesis as opposed to those that have much less significant contributions to maintenance of the tumorigenic phenotype . it is likely that the answer will not be simple and that more than one mechanism will influence the behavior of different mutant ras alleles . since ras is the most commonly mutated gene in human cancers and signals to a wide variety of molecules involved in proliferation , cell death , cell survival , oxidative stress , angiogenesis , inflammation , and drug resistance ( campbell et al . , 2009 ) , it is likely that patients whose tumors harbor specific ras mutations will exhibit differences in survival , tumor aggressiveness , and response to chemotherapy agents . an understanding of the properties of each of the ras mutants in specific tissues will aid in future attempts to personalize cancer treatment regimens and assure the best possible outcomes for patients . 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 .

to convert a closed double - stranded dna or rna helix into two open single strands , so that other protein machinery can manipulate the polynucleotides , the cells require helicases . several dna and rna helicases have been isolated from all kingdoms of life , from virus to man [ 58 ] . detailed structural information , biological mechanisms , and clear outlook on inhibitors of therapeutic relevance as antiviral agents are recently provided by xi et al . several ssrna ( positive sense single - stranded rna ) helicases have been studied in detail including those from dengue fever virus ( dfv ) , west nile virus ( wnv ) , and japanese encephalitis virus ( jev ) . more in general , a recent article on anti - flaviviridae chemotherapy has been published by ghosh and basu , who expand the original information regarding the role of helicases in flaviviridae previously reported by borowski . this enzyme is a promising target to develop new therapies and preventative agents , since ssrna viruses belonging to families like flaviviridae , coronaviridae , and picornaviridae cause clinically significant diseases both in humans and animals , determining life lost , economical loss , and higher productivity costs . examples are the bovine viral diarrhea virus ( bvdv ) , a serious welfare problem that significantly damages the farm business , and the hepatitis c virus [ hcv ] , that is now a global public health issue , being a major cause of human hepatitis . actually , with the exception of yfv , no vaccine exists against the various flaviviridae members therefore , new therapies and preventative agents are strongly needed . viruses belonging to picornaviridae family cause a variety of illnesses , including meningitis , cold , heart infection , conjunctivitis , and hepatitis . this family includes nine genera , some of which comprise major human pathogens , namely , enterovirus ( including poliovirus , coxsackievirus , echovirus ) , rhinovirus ( approximately 105 serotypes ) , and hepatovirus ( hepatitis a virus ) . at present , no specific antiviral therapy is available for the treatment of picornaviridae infections . finally , severe acute respiratory syndrome coronavirus ( sars - cov ) , an enveloped virus , has recently infected thousand of humans , with about 800 deaths , and no vaccine or specific antiviral therapy is known against this virus . no retroviruses or ssrna viruses have been reported to encode the synthesis of a helicase ; they might simply utilize helicases encoded by the host cell instead of their own proteins , as recently shown for hiv replication , which requires the human ddx3 dead - box rna helicase [ 17 , 18 ] . in ssrna viruses , the rna helicases are implicated in several functions including rna genome replication , ribosome biogenesis , messengers rna transcription , pre - mrna splicing , rna maturation , rna export and degradation , as well as rna translation [ 19 , 20 ] . basing on certain signature motifs in the amino acid sequence , gorbalenya and koonin have shown that all helicases can be classified in several genetic families . all but two of the helicase families can be grouped into one of three larger superfamilies , designed as superfamily 1 ( sf1 ) , superfamily 2 ( sf2 ) , and superfamily 3 ( sf3 ) . of the remaining 2 families , one is similar to the dnab helicase of e. coli and the other resembles the e. coli rho helicase that is used in transcriptional termination . only the dnab - like family , sometimes called family 4 ( f4 ) or superfamily 4 ( sf4 ) , contains viral proteins . all helicases bind ntp using two structurally common amino acidic sequences named motif i and motif ii , described by walker et al . and subramanya et al . motif i ( also known as walker a motif / boxes a ) is a phosphate - binding p - loop that also interact with the ribose , while motif ii ( also known as walker b / boxes b ) is a mg co - factor binding loop . the atp - binding site of helicase is completed by an arginine finger and a catalytic base , which accepts a proton from the attacking water molecule . in related proteins , this catalytic base has been demonstrated to be a conserved glutamate near the walker b motif [ 27 , 28 ] . arginine amino acids often interact with the beta and gamma phosphates of the bound atp , stabilizing the transition state [ 29 , 30 ] , figure 1 . all helicases can also be classified according to their movement relative to the nucleic acid strand to which they are primarily associated or to their quaternary structures . thus , a helicase can be classified basing on each of the three above schemes . for example , the helicase encoded by hcv ( hepatitis c virus ) is an sf2 , nonring , 35 rna helicase . the functional importance of helicases means that inhibitors or modulators for these enzymes are potentially important as therapeutic agents . over the past decade , significant progress has been made in the development of highly selective inhibitors as antiviral and anticancer drugs for clinical uses . developing nontoxic helicase inhibitors as antiviral drugs is considerably more difficult than developing drugs designed to inhibit other viral enzymes . in fact , in contrast with proteases and polymerases , the helicases - dependent reactions are still not fully elucidated . furthermore , the helicase atp - binding site is conserved not only in all the classes of helicases , but also in other proteins necessary for the cellular lifecycle , such as small gtpases , kinases , the aaa family ( atpases associated with various cellular activities ) , and even the mitochondrial atp synthase ( f1 atpase ) . thus , compounds that inhibit helicases via their atp - binding sites could have toxic effects on the host cells . many viral pathogens encode rna helicases which have been demonstrated essential for viral replication and pathogenesis [ 3133 ] . between them areemerging or re - emerging viruses with pandemic potential , such as sars - cov ( severe acute respiratory syndrome - coronavirus ) , dengue , west nile , and japanese encephalitis viruses , viruses that have a stable spread worldwide , such as hcv ( hepatitis c virus),viruses that do not have a large spread , but can generate serious health problems because of lack or limited availability of effective drugs , such as cvb ( human coxsackie b virus ) . emerging or re - emerging viruses with pandemic potential , such as sars - cov ( severe acute respiratory syndrome - coronavirus ) , dengue , west nile , and japanese encephalitis viruses , viruses that have a stable spread worldwide , such as hcv ( hepatitis c virus ) , viruses that do not have a large spread , but can generate serious health problems because of lack or limited availability of effective drugs , such as cvb ( human coxsackie b virus ) . general strategies to design specific and selective drugs for the treatment of viral infections targeting helicase could act via one or more of the following mechanisms : inhibition of the ntpase activity by interferences with atp binding and therefore by limiting the energy required for the unwinding and translocation , inhibition of the ntpase activity by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state , inhibition of nucleic acids binding to the helicase , inhibition of coupling of atp hydrolysis to unwinding , inhibition of unwinding by sterically blocking helicase translocation , development of small molecule antagonists against essential protein - protein interactions involving helicases . inhibition of the ntpase activity by interferences with atp binding and therefore by limiting the energy required for the unwinding and translocation , inhibition of the ntpase activity by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state , inhibition of nucleic acids binding to the helicase , inhibition of coupling of atp hydrolysis to unwinding , inhibition of unwinding by sterically blocking helicase translocation , development of small molecule antagonists against essential protein - protein interactions involving helicases . some characteristics of helicase families of pathogen viruses belonging to flaviviridae , coronaviridae , and picornaviridae families are reported in table 1 [ 9 , 10 , 34 ] . the flaviviridae is a large family of related positive - strand rna viruses that currently consists of three genera : flavivirus , pestivirus ( from the latin pestis , plague ) , and hepacivirus ( from the greek hepatos , liver ) . in addition , the family includes two groups of viruses , gbv - a and gbv - c , that are currently unassigned to a specific genus and await formal classification . within this family are comprised viruses that cause significant diseases in human and animal populations . from flavivirus genus is dengue virus ( denv ) with its associated dengue hemorrhagic fever ( dhf ) and dengue shock syndrome ( dss ) , japanese encephalitis virus ( jev ) , west nile virus ( wnv ) , yellow fever virus ( yfv ) , and tick - borne encephalitis virus ( tbev ) . the pestiviruses are animal pathogens of major economic importance for the livestock industry , like bovine viral diarrhea virus ( bvdv ) , border disease virus ( bdv ) of sheep , and classical swine fever virus ( csfv ) . the hepacivirus genus includes only the hepatitis c virus ( hcv ) , an important human pathogen . hcv , identified in 1989 , is a major cause of human hepatitis , globally , and infects about 3% of the world 's population . hepacivirus is spread primarily by direct contact with human blood ; hence , the major causes of infection are use of unscreened blood transfusions and reuse of needles and syringes that have not been adequately sterilised . the world health organization ( who ) estimates that over 170 million people worldwide are presently infected with this virus . most infections become persistent and about 60% of cases progress towards chronic liver disease , that can lead to development of cirrhosis , hepatocellular carcinoma , and liver failure [ 39 , 40 ] . pegylated interferon in combination with ribavirin is used in the clinic for hepatitis due to hcv . unfortunately , this therapy requires lengthy periods of administration and is often associated with severe and adverse events . furthermore , this drug has limited efficacy and the sustained virological response rate is of 4050% in genotype hcv-1 infected patients , and of 80% in those infected with genotypes 2 and 3 [ 41 , 42 ] . this emphasizes that new therapies are clearly needed , since for the treatment of this infection , and generally for diseases caused by viruses belonging to the flaviviridae family , therapeutic strategies really effective and selective are not available . all of the 12 hcv genotypes , which have nucleotide sequences that differ by as much as 30% , produce a single polyprotein of about 3,000 amino acids , which is subsequently processed by viral and host proteases into four structural proteins and six nonstructural proteins ( altogether 10 mature proteins ) . as summarized in figure 2 , the structural proteins ( s proteins : core , e1 , e2 , and p7 ) generate the viral capsid and envelope proteins and are cleaved by host - signal peptidases , while the six nonstructural proteins ( ns proteins : ns2 , ns3 , ns4a , ns4b , ns5a , and ns5b ) are responsible for genome replication and are largely generated by hcv - encoded protease . hcv helicase is part of the bi - functional ns3 protein , carrying three different enzymatic activities : helicase , ntpase , and serine protease activities . ns3 helicase is essential for viral replication , and this makes it one of the most promising target for the antiviral therapy . the known hcv helicase inhibitors can be classified on the base of their mechanism of action , into the first four groups of those above cited : inhibitors of ntpase activity by interference with ntp binding , inhibitors of ntpase activity by allosteric mechanism , competitive inhibitors of rna binding , inhibitors of the coupling of ntp hydrolysis at the unwinding reaction . inhibitors of ntpase activity by interference with ntp binding , inhibitors of ntpase activity by allosteric mechanism , competitive inhibitors of rna binding , inhibitors of the coupling of ntp hydrolysis at the unwinding reaction . the hydrolysis of atp supplies the energy that allows the helicase to adopt various nucleotide ligation states that allosterically cause conformational changes in the nucleic acid binding site to drive the movement of the helicase along the length of the nucleic acid chain . so , competitive ntpase inhibitors may lead to decreased atpase activity and therefore to reduction of the unwinding rate . consequently , non-(or slowly ) hydrolysable atp - analogs seemed to be effective tools for inhibiting the helicase activity , like adenosine-5-thiotriphosphate ( atp--s ) , which is used to determine a low level of unwinding of hcv dsrna [ 44 , 45 ] . however , ribavirin 5-triphosphate ( rtp ) , that inhibits the hcv ntpase / helicase by a competitive mechanism in regard to atp , and ribavirin 5-diphosphate ( rdp ) , both reported in figure 3 , even showing ic50 values in the micromolar range , demonstrates to determine only a weakly enzymatic inhibition . the same behavior has been put in evidence for paclitaxel , compound structurally nonrelated to ntp . this derivative is able to block the ntp - binding site ( ic50 = 22 m ) and to inhibit the atpase activity ( ic50 = 17 m ) in a competitive way , but is not able to inhibit the helicase activity at concentration lower than 1 mm the partial unwinding activity mediated by these competitive ntpase inhibitors is common to all members of the class , and the concentrations needed for the helicase inhibition usually exceed the ic50 value by 35 times . at these concentrations , most potent benzotriazole helicase inhibitors were identified during the course of a random screening study [ 49 , 50 ] . in particular , 4 , 5 , 6 , 7-tetrabromobenzotriazole ( tbbt ) ( 4 ) , known as a potent and highly selective inhibitor of protein kinase 2 , and 5,6-dichloro-1-(-d - ribofuranosyl ) benzotriazole ( drbt ) ( 5 ) displayed ic50 values of 20 and 1.5 m , respectively ( figure 4 ) . on the contrary , the corresponding imidazole derivative of drbt , the 5 , 6-dichloro-1-(-d - ribofuranosyl ) benzimidazole ( drbi ) , against ntpase / helicase of a large number of members of the flaviviridae family ( hcv , wnv , denv , and jev ) resulted to be completely inactive . . synthesized and studied a new series of substituted ( alkyl , hydroxy alkyl , chloro alkyl , ribofuranose ) 1h - benzimidazole and 1h - benzotriazole derivatives shown in figures 5 and 6 [ 50 , 51 ] . tbbt ( more less drbt ) resulted effective in hcv subgenomic replicon system in a comparable way to the inhibition reported in the enzymatic essays , showing a property that has been detected only for a handful group of hcv inhibitors . it has been reported that the starting compounds 1h - benzotriazole ( 6 ) and 1h - benzimidazole ( 17 ) , screened for their effect against the hcv - helicase , showedvery low activity ( ic50 200 m and 500 m , respectively ) when measured with a dna substrate , no activity when measured either with an rna substrate or against the flavivirus enzymes of wnv , denv , and jev ( ic50 > 500 m ) . very low activity ( ic50 200 m and 500 m , respectively ) when measured with a dna substrate , no activity when measured either with an rna substrate or against the flavivirus enzymes of wnv , denv , and jev ( ic50 > 500 m ) . on the contrary , the whole halogenation of 1h - benzotriazole ( 6 ) with bromine atoms , to afford the above cited 4 , caused either a 10-fold or 9-fold more effective inhibition of the hcv helicase when determined with a dna substrate or an rna substrate , respectively , and of 25-fold in the case of the jev enzyme ( ic50 20 m ) . the corresponding bromination of 1h - benzimidazole ( 17 ) afforded the derivative ( 18 ) , which resulted to be less effective than 4 and 22.5 times more potent than parent 17 against hcv helicase . when 1- or 2-alkyl benzotriazoles were screened for their effect on the hcv - helicase activity using the dna substrate , the 2-alkylated derivatives ( 1012 ) resulted to be significantly more potent inhibitors of the enzyme ( 2- to 7- times ) than the respective 1-alkylated analogues ( 79 ) . on the other hand , enhancement of the activity was observed when the aliphatic chain was elongated in both 1-alkylated benzotriazoles ( 79 ) and 1-alkylated benzimidazoles ( 1921 ) than the respective 2-alkylated analogues . in the case of the benzimidazole derivatives ( 1921 ) , however , the inhibitory activity was very low and ranged between 250 and 500 m . furthermore , the hcv helicase activity of the alkylated benzimidazoles tested using the rna substrate , as well as using other viral ntpase / helicases , displayed no inhibitory activity . this behaviour suggests that these inhibitors do not act by blocking the ntp binding sites of the enzymes and that occupation of an allosteric nucleoside binding site should be considered , as previously suggested by porter . furthermore , in this study the authors observed that replacement of the alkyl side - chain by a substituent endowed with higher hydrophilicity ( hydroxyethyl derivatives 13 and 14 in figure 5 ) or with higher hydrophobicity ( chloroethyl derivatives 15 and 16 in figure 5 ) dramatically decreases the activity of the tetrabromobenzotriazoles . consequently , it seems that a small hydrophobic alkyl moiety ( methyl or ethyl ) at position 2- of the tetrabromobenzotriazole could play a crucial role in the inhibition of the hcv ntpase / helicase . introduction of a ribofuranosyl ring in both benzotriazole and tetrabromobenzotriazole improves the water solubility but leads to a decrease of the inhibitory activity against hcv and all the enzymes tested . the same substituent in the position 1 of the 5,6-dichlorobenzotriazole drbt ( 5 ) was , as above reported , very effective in inhibiting the hcv and wnv helicases ( ic50 1.5 m and 3.0 m , respectively ) but ineffective against jev helicase . on the contrary , replacement of chlorine atoms of the benzotriazole ring with either bromine atoms or methyl groups ( compounds 2830 , figure 7 ) showed lower activity compared to drbt . in an extension of this study , an additional class of nucleoside analogues known as ring - expanded nucleosides ( ren or fat ) involving 6-aminoimidazo [ 4,5-e ] [ 1 , 3 ] diazepine-4,8-dione ring were reported to be active against the helicase unwinding reaction . a number of rens , such as compounds 31 and 32 of figure 8 , displayed ic50 values in the micromolar range . in view of the observed tight complex between some nucleosides and rna and/or dna substrates of a helicase , the mechanism of ren action might involve binding to the minor or major groove of the helical nucleic acid substrate . the fat nucleosides 31 , 32 , and tbbt ( 4 ) and nogalamycin ( see compound 76 ) have been recently used to construct a pharmacophore model for designing new japanese encephalitis virus ns3 helicase / ntpase inhibitors , using a refined structure of this enzyme . on the other hand , the ren 5-triphosphates , such as compounds 33 and 34 of figure 9 , did not influence the unwinding reaction while exerting their inhibitory effect ( ic50 0.55 m and 1.5 m , respectively ) on the atpase activity of the enzyme . as reported in figure 9 , compounds 33 and 34 , containing the 5 : 7-fused heterocyclic systems , imidazo [ 4,5-e ] [ 1 , 3 ] diazepine and imidazo [ 4,5-e ] [ 1 , 2 , 4 ] triazepine , respectively , were synthesized from the corresponding nucleosides 36 and 37 , employing the ludwig 's procedure . the nucleosides 36 and 37 , in turn , were synthesized by vorbrggen ribosylation [ 5760 ] of the respective heterocycles 35 and 38 [ 61 , 62 ] . therefore , in exploring the potential anti - flaviviridae activity of the ring system contained in 31 , the same authors focused on different substituents ( alkyl , arylalkyl , and aromatic groups ) at position 6 , along with variations of sugar moieties at position 1 ( ribose , 2-deoxyribose , or acyclic derivatives ) as well as their attachment to the base ( or configuration ) . the general method for the synthesis of the designed nucleosides ( 4159 ) was involved , as reported in figure 10 , the vorbrggen ribosylation [ 53 , 54 ] of dimethyl imidazole-4,5-dicarboxylate ( 39 ) [ 64 , 65 ] , followed by condensation of the resulting imidazole nucleoside ( 40 ) with the appropriately substituted guanidine derivatives . the modulation effect exerted by rens can result in an inhibition or activation . in the first case , the mechanism may involve the interaction of rens with a dna or an rna substrate through binding to the major or minor groove of the double - helix . in the case of activation , the mechanism may involve an allosteric binding site that can be occupied by nucleoside / nucleotide - type molecules including , but not limited to rens . the occupation of this allosteric site on the enzyme is dependent upon the high level of atp ( ntp ) concentration in the reaction mixture . rens obtained with the above procedures were screened for inhibition of ntpase / helicase of the wnv . one of the most promising among these early inhibitors is 1-(2-o - methyl--d - ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5h,6h)-dione ( hmc - ho4 ) ( 60 ) , figure 11 , which produces a promising antiviral effect ( ec50 = 2530 m ) . at all the concentrations of hmc - ho4 , atp hydrolysis is stimulated , suggesting that the inhibitor somehow uncouples the atpase and helicase functions . in that regard , rens may represent a starting point for the development of highly selective inhibitors of wnv ntpase / helicase . an other recent starting point is represented by triphenylmethane derivatives , as reported from chen et al . . compound ( 61 ) of figure 12 , where the triphenylmethane moiety is linked to a 2-(3-bromo-4-hydroxyphenyl)propane , was identified as a good inhibitor that suppresses hcv rna replication in the hcv replicon cells through both the inhibition of atp hydrolysis and the rna substrate binding . the partial inhibition mediated by the competitive ntpase inhibitors may be avoided by utilizing compounds chemically unrelated to ntp , which reduce the accessibility to the ntp - binding site in a noncompetitive manner . an example is the calmodulin antagonist trifluoperazine ( 62 , figure 13 ) . although the molecule is known to interact with domain 1 of hcv helicase , it is uncertain if inhibition results from conformational changes or from blockage of the atp - binding site . even some tropolones have been screened as inhibitors of hcv helicase - catalyzed dna unwinding . have described several derivatives bearing a side chain that connect the seven - member ring system to some n - heterocycles . the most active compound , 3,5,7-tri[(40-methylpiperazin-10-yl)methyl]tropolone ( 63 ) , inhibited rna replication by 50% at 46.9 m ( ec50 ) , showing an ic50 = 3.4 m and a cc50 > 1000 m ( si > 21 ) , whereas the most efficient was 3,5,7-tri[(30-methylpiperidin-10-yl)methyl]tropolone ( 64 ) , with an ec50 of 35.6 m , which unfortunately exhibited a lower si ( 9.8 ) derived by a cc50 = 348 m . these tropolone derivatives , reported in figure 14 , are the first antihelicase compounds that inhibit hcv replication with the ability to cause the appearance of resistant mutants , suggesting that inhibition of replication is the result of inhibition of the enzyme activity . the inhibition is believed to result from the competition of the polynucleotides with dna or rna substrates , an effect that could be mimicked by synthetic macromolecules . with the aim of discovering new anti - hcv agents , viropharma synthesized several benzimidazole derivatives , two of them ( compounds 65 and 66 , figure 15 ) showing high activity against hcv helicase . although the exact mechanism of 65 and 66 is still not clear , they might compete with nucleic acids in the manner above mentioned . in particular , the benzene ring and the nh group could interact by hydrophobic interaction and hydrogen bound , respectively . in the attempt to extend the sar analysis , some new dimers containing benzimidazole , benzoxazole , pyridinoxazole , and benzothiazole rings , attached to symmetrical linkers , were synthesized by phoon et al . preliminary studies of these compounds showed a significant decrease in potency when the benzimidazole moiety was replaced by the benzoxazole or benzothiazole rings ( compounds 67 ) . on the other hand , the aminobenzimidazole - diamides ( 68 ) and aminophenyl benzimidazole - diureas ( 69 ) derivatives displayed , at 25 g / ml , 613 , and 2028 percent inhibitory activity , respectively . likewise , the linker was also implicated in the inhibitory activity since replacement of the diamide linkage possessed by 65 with the diurea linkage ( compounds 69 ) led to reduced potency . thus , the sar data indicate that the benzimidazole ring , the benzene group at the c2 position of the benzimidazole moiety , and the nature of the linker are essential for the activity . the synthesis of these analogues is outlined in figure 17 . aminophenols and thiophenols , or the corresponding pyridine derivatives , reacted easily with p - aminobenzoic acid in the presence of polyphosphoric acid to afford the corresponding oxazole and thiazole derivatives ( 72 ) . subsequent coupling of 72 and 2-aminobenzoimidazole with the opportune acid dichlorides furnished the products 6769 . belon et al . recently described how a prototype in the symmetrical benzimidazolephenyl series , the n , n - bis[4-(1h - benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide ( bip)2b , ( 69a , derivative of 69 with y = phenyl , figure 18 ) , binds directly the hcv ns3 helicase in the same binding site for rna in a competitive manner . furthermore , they reported that 69a interacts with ns3 encoded non only by various hcv genotypes , but even by dengue virus ( dv ) , japanese encephalitis virus ( jev ) and , even if less tightly , the related human helicase . also small peptides specifically inhibit hcv helicases , even in cells bearing hcv replicon . between them , a peptide expressed in bacteria , composed of 14 amino acids ( p14 , rrgrtgrgrrgiyr ) , demonstrated to be the best enzyme inhibitor . p14 has the same amino - acidic sequence as that surrounding the putative hcv helicase arginine finger and inhibits the replication of hcv replicon in cells with an ec50 = 83 m , while reduces the dna unwinding with an ic50 of 0.2 m . a new selective inhibitor of the hcv helicase , qu663 ( compound 73 of figure 19 ) , discovered by maga and coworkers , showed a potent and selective inhibition with ki of 0.75 m . the study of the inhibition mechanism has revealed that qu663 is a specific inhibitor of the strand - displacement activity , without affecting the ability of ns3 helicase to hydrolyse atp . qu663 could function as a competitive inhibitor with respect to nucleic acid substrate by decreasing the affinity of the enzyme for the substrate . therefore , qu663 inhibits the unwinding activity of ns3 in a competitive manner with respect to the dna substrate , making it a promising candidate for a novel class of anti - hcv drugs . recently , a new rational approach for the design of selective inhibitors of the hcv ns3 helicase brought the discovery of a novel hcv helicase inhibitor that potentially could compete for the nucleic acid binding site , occupying the ns3/rna binding cler . in consequence of this de novo drug design , the predicted ( e)-methyl 4-((5-(3-oxobut-1-enyl)-1h - pyrrole-2-carboxamido)methyl)benzoate ( 74 , figure 20 ) was synthesized and tested in the hcv replicon system . it inhibits hcv replicons with an ec50 of 9 m , but showing a cc50 = 30 m . dna and rna intercalating compounds are potential helicase inhibitors by increasing the energy required for duplex / intercalator complex unwinding [ 7779 ] . in particular , two anthracycline derivatives , doxorubicin and nogalamycin ( compounds 75 and 76 , figure 21 ) , have been shown to be effective inhibitors of the unwinding reaction . the limits in their application for the treatment of chronic viral infections is their high cytotoxicity and weak penetration into the cell . thus , if intercalative modulation of the dna or rna substrates is to be considered as a possible antiviral therapy , less toxic and more selective derivatives must be identified . as previously seen , the antibiotic nogalamycin ( 76 ) , that interacts with allosteric binding site , has been recently used to obtain a structure - based pharmacophore model for jev ns3 helicase / ntpase . in the aim to find less toxic compounds , a large group of amidinoanthracyclines , with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics , were screened against hcv helicase . from this studies emerged one of the most potent and selective inhibitors of helicase activity described in the literature . the derivative 77 , showed in figure 22 , acts not only via intercalation into the double - stranded dna substrate , but also impeding formation of the active helicase complex via competition with the enzyme for access to the substrate . tested in the subgenomic hcv replicon system , 77 it showed an ec50 of 0.13 m and a cc50 = 4.3 m . an other class of compounds that probably acts via intercalation into double - stranded nucleic acids with strong specificity for rna are the acridone derivatives , but a direct interaction with the viral ns3 helicase can not be excluded . a large group of acridones were tested from stankiewicz - drogon et al . using the direct fluorometric helicase activity assay to determine their inhibitory properties towards the ns3 helicase of hcv . from a preliminary study , n-(pyridin-4-yl)-amide ( 78 ) and n-(pyridin-2-yl)-amide ( 79 ) of acridone-4-carboxylic acid emerged to be efficient rna replication inhibitors with a good specificity in subgenomic replicon system and low cytotoxicity ( figure 23 ) . even the thiazolpiperazinyl acridone derivative 80 demonstrated to act as a potent agent against hcv replicons ( ec50 = 3 m ) and as a selective inhibitor of the hcv ns3 helicase , albeit with low potency ( ic50 = 110 m ) . comparing acridone derivatives 78 and 79 with 80 , we can see that the amide bonding formed after the derivatization of acridone-4-carboxylic acid with amines seemed to increase affinity and selectivity for the ns3 enzyme . finally , with the intent to improve the antiviral activity of acridones , stankiewicz - drogon et al . prepared a new class of compounds , namely , 5-methoxyacridone-4-carboxylic acids ( maca ) . from this group , compound 81 ( figure 24 ) came out not only as an efficient inhibitor of the ns3 helicase in the in vitro assay but also as a potent inhibitor of hcv replication endowed with low cytotoxicity for human hepatoma cells . an enveloped single - stranded positive - sense rna ( ssrna ) virus , sars coronavirus ( sars - cov ) , has been recently identified as the etiological agent of severe acute respiratory syndrome ( sars ) in humans [ 8488 ] . about ten thousand cases of sars worldwide , including 800 deaths , were reported in 2003 ( who data ) . although this initial global outbreak , sars appears to has been successfully contained , but it remains a serious concern because no vaccine or effective drug treatment is actually available . recently , tanner and coworkers have found that bananin and three of their derivatives , figure 25 , function as non - competitive sars - cov helicase inhibitors ( with ic50 values in the micromolar range ) at a site different from the atp and nucleic acid binding site , causing inhibition probably through an allosteric mechanism . in foetal rhesus kidney-4cells infected with sars - cov , bananin inhibited the viral replication ( ic50= 10 m ) with low host cellular toxicity ( cc50= 390 m ) . finally , in the last years various molecules have been detected showing an interesting and promising anti - coronaviridae activity . unfortunately , for many of them , was not identified a clear molecular target or mechanism of action . the fact remains that the eventual target could be the ns3 helicase . with this in mind , we report briefly the new classes of compounds that have emerged in recent published works . among them glycopeptide antibiotics , which seem to interfere with the coronavirus entry process but do not exclude an unknown cellular target ; pyridine n - oxide derivatives ; plant lectins , which most probably interfere with the glycans on the spike protein during virus entry and virus release ; phenanthroindolizines and phenanthroquinolizidines ; tetrahydroquinoline oxocarbazate derivatives as inhibitor of human cathepsin l and as entry blockers . picornaviridae family includes 9 genera , 3 of which are human pathogens : enterovirus ( containing poliovirus , enterovirus , coxsackievirus , echovirus ) , rhinovirus ( approximately 105 serotypes ) , and hepatovirus ( hepatitis a virus ) . at present the viruses belonging to this family , all having a single - stranded positive - sense rna ( ssrna ) genome , cause a dramatic variety of illnesses , including meningitis , colds , heart infection , conjunctivitis , and hepatitis . recently carta and coworkers reported the synthesis and antiviral screening of a series of n-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides ( 86(1-yl ) , 87(2-yl ) and n , n-bis-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides ( 88(1-yl ) , 89(2-yl ) ) ( see figure 26 ) . compounds were evaluated in vitro for cytotoxicity and antiviral activity against a wide spectrum of ssrna viruses , like bovine viral diarrhea virus ( bvdv ) , yellow fever virus ( yfv ) , coxsakie virus b ( cvb-2 ) , polio virus ( sb-1 ) , and human immunodeficiency virus ( hiv-1 ) . only cvb-2 and sb-1 were inhibited by n-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamide derivatives . in particular , two of them emerged for their selectivity : 87e , which was the most active against cvb-2 ( ec50 = 10 m and cc50 > 100 m ) and 86h , which was the most active against sb-1 ( ec50 = 30 m and cc50 = 90 m ) , figure 27 . n-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides ( 86a e , g , h and 87a g ) were prepared by condensation of the amino derivatives 90 , 91 with the appropriate anhydrides 92 under stirring at 100c for 2 h , as shown in figure 28 . the n , n-bis[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides ( 88a d , g , h and 88a d89a g , i k ) were in turn prepared , as reported in figure 29 , by condensation of the amines 1(2)-(4-aminophenyl)benzotriazoles ( 90 , 91 ) with the suitable diacyl dichlorides ( 93 ) . among n , n-bis-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides , the bis-5,6-dimethyl - derivatives ( 89d f ) exhibited good activity against enteroviruses ( ec50 were 711 m against cvb-2 and 1952 m against sb-1 ) and the bis-5,6-dichloro - benzotriazol-2-yl derivatives ( 80i k ) showed very selective activity against cvb-2 ( ec50 = 411 m ) resulting to be completely inactive against all the other viruses screened . n-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamides ( 86 and 87 ) were evaluated in silico against the 3d model of the sb-1 helicase , as exemplified by compounds 86h ( a ) and 89f ( b ) in figure 30 . the portion of the enzyme containing the binding site interacting with the inhibitors consists of two loops , part of two -sheets , and part of three helices . it is important to notice that , with respect to the n-[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkylcarboxamide series , all the n , n-bis[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamide derivatives bind helicase sb-1 in a different manner , as expected , due to their different shapes and dimensions . this is well quantified by the value of the solvent accessible volume of this new class of inhibitors , which on average has almost doubled compared to that of the previous molecular series ( e.g. , 1672 versus 891 , resp . ) . accordingly , it is impossible for the protein pocket to host n , n-bis[4-(1h(2h)-benzotriazol-1(2)-yl ) phenyl]alkyldicarboxamides with the same binding mode , and the results form the docking study reveal that only one of the two identical inhibitors moieties can be positioned well within the binding pocket . however , in correspondence of the most favored binding mode for the most active compounds , the formation of a new , small network of h - bonds between 89f and enzyme was observed . in particular , the analysis of the trajectories of the md simulations for the 89f / helicase complex as an example indicates that there is a constant presence of an h - bond which involves the carbonyl oxygen atom of the asn179 side chain and the triazole n(1 ) atom of the drug , characterized by an average dynamic length ( adl ) of 3.0 . at the same time , it is possible to verify the formation of other two h - bond interactions , the former between the c = o backbone group of ser221 and the nh group of the amidic moiety of 89f ( adl = 1.6 ) , and the latter between the carbonyl oxygen atom of the c = o group of the same amidic moiety of 89f and the side chain hydroxyl group of ser221 ( adl = 2.6 ) . unfortunately , homology standard techniques were not able to produce a reliable 3d model for the cvb-2 virus helicase , due to very low sequence identities found during alignment processes . in the absence of a 3d model for the cvb-2 helicase , the activity of 89i k can be explained adopting a 2d alignment analysis . the putative binding site proposed by carta and coworkers for sb-1 is composed by 30 residues and , according to their 2d alignment , the binding site for cvb-2 differs for 7 residues only . following their analysis , and a preliminary visual inspection based on the swapping of ser to arg in the polio helicase , they concluded that this is the most important residue in the case of compounds 89i k , featuring chlorine atoms as substituents . in fact , the positively charged side chain of arg237 is placed at an average distance of 3.5 from the cl atoms , thus sensibly resulting is strong electrostatic interactions between the inhibitor and the protein . these speculations , which may account in part for the selectivity of these compounds with respect to cvb-2 , clearly await further confirmation from the simulations performed on the corresponding protein 3d models .

many viral pathogens encode the motor proteins named rna helicases which display various functions in genome replication . general strategies to design specific and selective drugs targeting helicase for the treatment of viral infections could act via one or more of the following mechanisms : inhibition of the ntpase activity , by interferences with atp binding and therefore by limiting the energy required for the unwinding and translocation , or by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state ; inhibition of nucleic acids binding to the helicase ; inhibition of coupling of atp hydrolysis to unwinding ; inhibition of unwinding by sterically blocking helicase translocation . recently , by in vitro screening studies , it has been reported that several benzotriazole , imidazole , imidazodiazepine , phenothiazine , quinoline , anthracycline , triphenylmethane , tropolone , pyrrole , acridone , small peptide , and bananin derivatives are endowed with helicase inhibition of pathogen viruses belonging to flaviviridae , coronaviridae , and picornaviridae families .

1. Introduction 2. Viral RNA Helicases As Antiviral Drug Targets 3. Flaviviridae 4. Coronaviridae 5. Picornaviridae

several dna and rna helicases have been isolated from all kingdoms of life , from virus to man [ 58 ] . this enzyme is a promising target to develop new therapies and preventative agents , since ssrna viruses belonging to families like flaviviridae , coronaviridae , and picornaviridae cause clinically significant diseases both in humans and animals , determining life lost , economical loss , and higher productivity costs . examples are the bovine viral diarrhea virus ( bvdv ) , a serious welfare problem that significantly damages the farm business , and the hepatitis c virus [ hcv ] , that is now a global public health issue , being a major cause of human hepatitis . viruses belonging to picornaviridae family cause a variety of illnesses , including meningitis , cold , heart infection , conjunctivitis , and hepatitis . at present , no specific antiviral therapy is available for the treatment of picornaviridae infections . no retroviruses or ssrna viruses have been reported to encode the synthesis of a helicase ; they might simply utilize helicases encoded by the host cell instead of their own proteins , as recently shown for hiv replication , which requires the human ddx3 dead - box rna helicase [ 17 , 18 ] . in ssrna viruses , the rna helicases are implicated in several functions including rna genome replication , ribosome biogenesis , messengers rna transcription , pre - mrna splicing , rna maturation , rna export and degradation , as well as rna translation [ 19 , 20 ] . all but two of the helicase families can be grouped into one of three larger superfamilies , designed as superfamily 1 ( sf1 ) , superfamily 2 ( sf2 ) , and superfamily 3 ( sf3 ) . of the remaining 2 families , one is similar to the dnab helicase of e. coli and the other resembles the e. coli rho helicase that is used in transcriptional termination . in related proteins , this catalytic base has been demonstrated to be a conserved glutamate near the walker b motif [ 27 , 28 ] . arginine amino acids often interact with the beta and gamma phosphates of the bound atp , stabilizing the transition state [ 29 , 30 ] , figure 1 . furthermore , the helicase atp - binding site is conserved not only in all the classes of helicases , but also in other proteins necessary for the cellular lifecycle , such as small gtpases , kinases , the aaa family ( atpases associated with various cellular activities ) , and even the mitochondrial atp synthase ( f1 atpase ) . many viral pathogens encode rna helicases which have been demonstrated essential for viral replication and pathogenesis [ 3133 ] . general strategies to design specific and selective drugs for the treatment of viral infections targeting helicase could act via one or more of the following mechanisms : inhibition of the ntpase activity by interferences with atp binding and therefore by limiting the energy required for the unwinding and translocation , inhibition of the ntpase activity by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state , inhibition of nucleic acids binding to the helicase , inhibition of coupling of atp hydrolysis to unwinding , inhibition of unwinding by sterically blocking helicase translocation , development of small molecule antagonists against essential protein - protein interactions involving helicases . inhibition of the ntpase activity by interferences with atp binding and therefore by limiting the energy required for the unwinding and translocation , inhibition of the ntpase activity by allosteric mechanism and therefore by stabilizing the conformation of the enzyme in low helicase activity state , inhibition of nucleic acids binding to the helicase , inhibition of coupling of atp hydrolysis to unwinding , inhibition of unwinding by sterically blocking helicase translocation , development of small molecule antagonists against essential protein - protein interactions involving helicases . some characteristics of helicase families of pathogen viruses belonging to flaviviridae , coronaviridae , and picornaviridae families are reported in table 1 [ 9 , 10 , 34 ] . the flaviviridae is a large family of related positive - strand rna viruses that currently consists of three genera : flavivirus , pestivirus ( from the latin pestis , plague ) , and hepacivirus ( from the greek hepatos , liver ) . the pestiviruses are animal pathogens of major economic importance for the livestock industry , like bovine viral diarrhea virus ( bvdv ) , border disease virus ( bdv ) of sheep , and classical swine fever virus ( csfv ) . hcv , identified in 1989 , is a major cause of human hepatitis , globally , and infects about 3% of the world 's population . this emphasizes that new therapies are clearly needed , since for the treatment of this infection , and generally for diseases caused by viruses belonging to the flaviviridae family , therapeutic strategies really effective and selective are not available . as summarized in figure 2 , the structural proteins ( s proteins : core , e1 , e2 , and p7 ) generate the viral capsid and envelope proteins and are cleaved by host - signal peptidases , while the six nonstructural proteins ( ns proteins : ns2 , ns3 , ns4a , ns4b , ns5a , and ns5b ) are responsible for genome replication and are largely generated by hcv - encoded protease . hcv helicase is part of the bi - functional ns3 protein , carrying three different enzymatic activities : helicase , ntpase , and serine protease activities . ns3 helicase is essential for viral replication , and this makes it one of the most promising target for the antiviral therapy . the known hcv helicase inhibitors can be classified on the base of their mechanism of action , into the first four groups of those above cited : inhibitors of ntpase activity by interference with ntp binding , inhibitors of ntpase activity by allosteric mechanism , competitive inhibitors of rna binding , inhibitors of the coupling of ntp hydrolysis at the unwinding reaction . inhibitors of ntpase activity by interference with ntp binding , inhibitors of ntpase activity by allosteric mechanism , competitive inhibitors of rna binding , inhibitors of the coupling of ntp hydrolysis at the unwinding reaction . the hydrolysis of atp supplies the energy that allows the helicase to adopt various nucleotide ligation states that allosterically cause conformational changes in the nucleic acid binding site to drive the movement of the helicase along the length of the nucleic acid chain . so , competitive ntpase inhibitors may lead to decreased atpase activity and therefore to reduction of the unwinding rate . consequently , non-(or slowly ) hydrolysable atp - analogs seemed to be effective tools for inhibiting the helicase activity , like adenosine-5-thiotriphosphate ( atp--s ) , which is used to determine a low level of unwinding of hcv dsrna [ 44 , 45 ] . however , ribavirin 5-triphosphate ( rtp ) , that inhibits the hcv ntpase / helicase by a competitive mechanism in regard to atp , and ribavirin 5-diphosphate ( rdp ) , both reported in figure 3 , even showing ic50 values in the micromolar range , demonstrates to determine only a weakly enzymatic inhibition . this derivative is able to block the ntp - binding site ( ic50 = 22 m ) and to inhibit the atpase activity ( ic50 = 17 m ) in a competitive way , but is not able to inhibit the helicase activity at concentration lower than 1 mm the partial unwinding activity mediated by these competitive ntpase inhibitors is common to all members of the class , and the concentrations needed for the helicase inhibition usually exceed the ic50 value by 35 times . on the contrary , the corresponding imidazole derivative of drbt , the 5 , 6-dichloro-1-(-d - ribofuranosyl ) benzimidazole ( drbi ) , against ntpase / helicase of a large number of members of the flaviviridae family ( hcv , wnv , denv , and jev ) resulted to be completely inactive . tbbt ( more less drbt ) resulted effective in hcv subgenomic replicon system in a comparable way to the inhibition reported in the enzymatic essays , showing a property that has been detected only for a handful group of hcv inhibitors . it has been reported that the starting compounds 1h - benzotriazole ( 6 ) and 1h - benzimidazole ( 17 ) , screened for their effect against the hcv - helicase , showedvery low activity ( ic50 200 m and 500 m , respectively ) when measured with a dna substrate , no activity when measured either with an rna substrate or against the flavivirus enzymes of wnv , denv , and jev ( ic50 > 500 m ) . on the contrary , the whole halogenation of 1h - benzotriazole ( 6 ) with bromine atoms , to afford the above cited 4 , caused either a 10-fold or 9-fold more effective inhibition of the hcv helicase when determined with a dna substrate or an rna substrate , respectively , and of 25-fold in the case of the jev enzyme ( ic50 20 m ) . when 1- or 2-alkyl benzotriazoles were screened for their effect on the hcv - helicase activity using the dna substrate , the 2-alkylated derivatives ( 1012 ) resulted to be significantly more potent inhibitors of the enzyme ( 2- to 7- times ) than the respective 1-alkylated analogues ( 79 ) . furthermore , the hcv helicase activity of the alkylated benzimidazoles tested using the rna substrate , as well as using other viral ntpase / helicases , displayed no inhibitory activity . furthermore , in this study the authors observed that replacement of the alkyl side - chain by a substituent endowed with higher hydrophilicity ( hydroxyethyl derivatives 13 and 14 in figure 5 ) or with higher hydrophobicity ( chloroethyl derivatives 15 and 16 in figure 5 ) dramatically decreases the activity of the tetrabromobenzotriazoles . consequently , it seems that a small hydrophobic alkyl moiety ( methyl or ethyl ) at position 2- of the tetrabromobenzotriazole could play a crucial role in the inhibition of the hcv ntpase / helicase . in view of the observed tight complex between some nucleosides and rna and/or dna substrates of a helicase , the mechanism of ren action might involve binding to the minor or major groove of the helical nucleic acid substrate . on the other hand , the ren 5-triphosphates , such as compounds 33 and 34 of figure 9 , did not influence the unwinding reaction while exerting their inhibitory effect ( ic50 0.55 m and 1.5 m , respectively ) on the atpase activity of the enzyme . the nucleosides 36 and 37 , in turn , were synthesized by vorbrggen ribosylation [ 5760 ] of the respective heterocycles 35 and 38 [ 61 , 62 ] . therefore , in exploring the potential anti - flaviviridae activity of the ring system contained in 31 , the same authors focused on different substituents ( alkyl , arylalkyl , and aromatic groups ) at position 6 , along with variations of sugar moieties at position 1 ( ribose , 2-deoxyribose , or acyclic derivatives ) as well as their attachment to the base ( or configuration ) . the general method for the synthesis of the designed nucleosides ( 4159 ) was involved , as reported in figure 10 , the vorbrggen ribosylation [ 53 , 54 ] of dimethyl imidazole-4,5-dicarboxylate ( 39 ) [ 64 , 65 ] , followed by condensation of the resulting imidazole nucleoside ( 40 ) with the appropriately substituted guanidine derivatives . in the first case , the mechanism may involve the interaction of rens with a dna or an rna substrate through binding to the major or minor groove of the double - helix . the occupation of this allosteric site on the enzyme is dependent upon the high level of atp ( ntp ) concentration in the reaction mixture . rens obtained with the above procedures were screened for inhibition of ntpase / helicase of the wnv . compound ( 61 ) of figure 12 , where the triphenylmethane moiety is linked to a 2-(3-bromo-4-hydroxyphenyl)propane , was identified as a good inhibitor that suppresses hcv rna replication in the hcv replicon cells through both the inhibition of atp hydrolysis and the rna substrate binding . although the molecule is known to interact with domain 1 of hcv helicase , it is uncertain if inhibition results from conformational changes or from blockage of the atp - binding site . these tropolone derivatives , reported in figure 14 , are the first antihelicase compounds that inhibit hcv replication with the ability to cause the appearance of resistant mutants , suggesting that inhibition of replication is the result of inhibition of the enzyme activity . on the other hand , the aminobenzimidazole - diamides ( 68 ) and aminophenyl benzimidazole - diureas ( 69 ) derivatives displayed , at 25 g / ml , 613 , and 2028 percent inhibitory activity , respectively . thus , the sar data indicate that the benzimidazole ring , the benzene group at the c2 position of the benzimidazole moiety , and the nature of the linker are essential for the activity . a new selective inhibitor of the hcv helicase , qu663 ( compound 73 of figure 19 ) , discovered by maga and coworkers , showed a potent and selective inhibition with ki of 0.75 m . the study of the inhibition mechanism has revealed that qu663 is a specific inhibitor of the strand - displacement activity , without affecting the ability of ns3 helicase to hydrolyse atp . qu663 could function as a competitive inhibitor with respect to nucleic acid substrate by decreasing the affinity of the enzyme for the substrate . therefore , qu663 inhibits the unwinding activity of ns3 in a competitive manner with respect to the dna substrate , making it a promising candidate for a novel class of anti - hcv drugs . recently , a new rational approach for the design of selective inhibitors of the hcv ns3 helicase brought the discovery of a novel hcv helicase inhibitor that potentially could compete for the nucleic acid binding site , occupying the ns3/rna binding cler . dna and rna intercalating compounds are potential helicase inhibitors by increasing the energy required for duplex / intercalator complex unwinding [ 7779 ] . in particular , two anthracycline derivatives , doxorubicin and nogalamycin ( compounds 75 and 76 , figure 21 ) , have been shown to be effective inhibitors of the unwinding reaction . the limits in their application for the treatment of chronic viral infections is their high cytotoxicity and weak penetration into the cell . from this studies emerged one of the most potent and selective inhibitors of helicase activity described in the literature . the derivative 77 , showed in figure 22 , acts not only via intercalation into the double - stranded dna substrate , but also impeding formation of the active helicase complex via competition with the enzyme for access to the substrate . from this group , compound 81 ( figure 24 ) came out not only as an efficient inhibitor of the ns3 helicase in the in vitro assay but also as a potent inhibitor of hcv replication endowed with low cytotoxicity for human hepatoma cells . recently , tanner and coworkers have found that bananin and three of their derivatives , figure 25 , function as non - competitive sars - cov helicase inhibitors ( with ic50 values in the micromolar range ) at a site different from the atp and nucleic acid binding site , causing inhibition probably through an allosteric mechanism . at present the viruses belonging to this family , all having a single - stranded positive - sense rna ( ssrna ) genome , cause a dramatic variety of illnesses , including meningitis , colds , heart infection , conjunctivitis , and hepatitis . compounds were evaluated in vitro for cytotoxicity and antiviral activity against a wide spectrum of ssrna viruses , like bovine viral diarrhea virus ( bvdv ) , yellow fever virus ( yfv ) , coxsakie virus b ( cvb-2 ) , polio virus ( sb-1 ) , and human immunodeficiency virus ( hiv-1 ) . the n , n-bis[4-(1h(2h)-benzotriazol-1(2)-yl)phenyl]alkyldicarboxamides ( 88a d , g , h and 88a d89a g , i k ) were in turn prepared , as reported in figure 29 , by condensation of the amines 1(2)-(4-aminophenyl)benzotriazoles ( 90 , 91 ) with the suitable diacyl dichlorides ( 93 ) . the portion of the enzyme containing the binding site interacting with the inhibitors consists of two loops , part of two -sheets , and part of three helices . accordingly , it is impossible for the protein pocket to host n , n-bis[4-(1h(2h)-benzotriazol-1(2)-yl ) phenyl]alkyldicarboxamides with the same binding mode , and the results form the docking study reveal that only one of the two identical inhibitors moieties can be positioned well within the binding pocket . however , in correspondence of the most favored binding mode for the most active compounds , the formation of a new , small network of h - bonds between 89f and enzyme was observed . in particular , the analysis of the trajectories of the md simulations for the 89f / helicase complex as an example indicates that there is a constant presence of an h - bond which involves the carbonyl oxygen atom of the asn179 side chain and the triazole n(1 ) atom of the drug , characterized by an average dynamic length ( adl ) of 3.0 . at the same time , it is possible to verify the formation of other two h - bond interactions , the former between the c = o backbone group of ser221 and the nh group of the amidic moiety of 89f ( adl = 1.6 ) , and the latter between the carbonyl oxygen atom of the c = o group of the same amidic moiety of 89f and the side chain hydroxyl group of ser221 ( adl = 2.6 ) .

caloric restriction ( cr ; or dietary restriction [ dr ] ) is the only known intervention that extends the life span of organisms as divergent as yeast , worms , flies , fish , and primates [ 1 , 2 ] , an observation which might indicate the existence of a universal , conserved mechanism of aging . despite almost 80 years of research since mccay 's initial discovery that caloric restriction without malnutrition extended the life span of rats , the mechanisms underlying its retardation of the rate of aging are still incompletely understood [ 47 ] . since decreased nutrient intake also lowers the incidence of many age - related maladies such as diabetes , cancer , and cardiovascular diseases in several organisms , intense efforts at identifying the molecular processes underlying these beneficial effects are underway in the aging researcher community . decreased signaling through nutrient - sensing pathways , for example , protein kinase a ( pka ) , target - of - rapamycin ( tor ) , or insulin - like growth factor ( igf ) pathways [ 2 , 9 ] , is in several model organisms required for life span extension upon cr . however , the many targets and their highly interconnected nature have prevented the identification of targets important for aging . the views of some researchers in the field were , at least until rather recently , that life span extension by cr depends on the combined activity of many gene products acting through multiple pathways . in contrast to this hypothesis , two recent reports have pointed to two unique target mechanisms for how cr postpones replicative aging in yeast by counteracting detrimental processes acting at the genesis of aging [ 11 , 12 ] . we demonstrated that cr , through reduced pka signaling , activates the peroxiredoxin ( prx ) tsa1 , an antioxidant protein reducing h2o2 , to prolong yeast life span . strikingly , whereas wild - type cells responded to cr ( and reduced pka activity ) by an increased life span , tsa1 mutants did not , identifying tsa1 as a key enzyme extending life span during cr . in line with this , cr stimulated tsa1 activity through increasing the levels of the prx reducing enzyme srx1 , which reduces hyperoxidized ( sulfinylated ) tsa1 , and ectopically increasing srx1 levels was sufficient to retard aging in calorie - replete medium ( figure 1 ) . interestingly , the cr - induced increase in srx1 levels did not involve increased transcript levels , but rather appeared to result from gcn2-dependent increased translation of the srx1 mrna ( figure 1(b ) ) . increased srx1 levels are expected to increase both the recycling of hyperoxidized tsa1 and as a consequence also the ability of tsa1 to reduce peroxide ( peroxidase activity ) . however , prx are not only h2o2-reducing enzymes but also function in h2o2-signaling and in proteostasis ( see below ) and it is currently not clear which facet(s ) of peroxiredoxin function that is / are required for cr - induced longevity . interestingly , a recent report from the gottschling lab identified increased vacuolar ph as an early - age promoter of age - induced mitochondrial depolarization and fragmentation leading to replicative aging in yeast ( figure 2(a ) , ) . caloric restriction , as well as reducing the activities of conserved nutrient signaling pathways ( e.g. , ras - camp - pka and torc1 ) , delayed an age - induced loss of vacuolar acidity suggesting that the control of vacuolar ph also constitutes a target process regulated by cr ( figure 2 , arrows i and ii , ) . mutating subunits of the vacuolar proton - translocating atpase ( v - atpase ) leads to increased vacuolar ph and accelerated aging ( figure 2(a ) ) . conversely , restoring vacuolar acidity in aging cells retarded aging ( figure 2(b ) ) , suggesting that the control of vacuolar ph critically regulates aging . the authors furthermore observed that increasing vacuolar ph decreased the import of cytosolic amino acids into the vacuole through the h - neutral amino acid antiporter avt1 , and both mitochondrial dysfunction and replicative aging were in part coupled to this reduced transport ( figure 2(a ) ) . how increased levels of cytosolic amino acids induce mitochondrial dysfunction and replicative aging was not addressed . as we will discuss later , however , both v - atpase and a cytosolic neutral amino acid ( leucine ) have been implicated in the regulation of nutrient signaling [ 1315 ] , raising the question whether v - atpase and cytosolic amino acids affect aging via feedback regulation of nutrient signaling pathways . the purpose of this paper is to review the physiological impact of both prx - mediated stress defense and vacuolar ph control and to pinpoint possible common denominators between these two apparently distinct target mechanisms proposed for cr - mediated life span extension . we will then point to outstanding questions that need to be resolved to further understand the beneficial impact of caloric restriction on aging and age - related diseases . to achieve this , we will first review the roles of ros and peroxiredoxins in aging and in cr - mediated longevity . next , we will shortly consider nutrient and glucose signaling in general and through the lenses of the free radical theory of aging in particular , since these signaling pathways are conserved mediators of cr life span extension in most organisms . following this , we will discuss the cellular roles of v - atpases including recent observations that these enzymes , via regulating intracellular ph homeostasis , are implicated in the regulation of nutrient signaling . on a final note , we will discuss points of intersection between v - atpases and prxs identified here , which include recent papers implicating both pathways in iron metabolism . in aerobic organisms , incomplete reduction of oxygen in the mitochondrial respiratory chain leads to the production of reactive molecules called reactive oxygen species ( ros ) . these reactive molecules mainly include hydrogen peroxide ( h2o2 ) , superoxide anion ( o2 ) , and hydroxyl radical ( oh ) and they may damage macromolecules , including proteins , lipids , and nucleic acids . the most widely accepted aging theory , the free radical theory , proposed by denham harman in 1956 , states that damage caused on biomolecules by ros / free radicals might be an underlying cause of the aging process . consistent with this hypothesis , h2o2 levels have been shown to increase with the replicative age of yeast mother cells [ 20 , 21 ] and with the chronological age of both rats and mice [ 22 , 23 ] . whereas h2o2 in itself mainly oxidizes cysteine or methionine residues , its conversion into the highly reactive oh via metal - catalyzed oxidation reactions , may inflict widespread damage . macromolecular damage has been observed to increase with age in many organisms [ 18 , 2426 ] . furthermore , a correlation between the amount of oxidized proteins and the rate of aging was reported in flies and in cultured human fibroblasts [ 18 , 27 ] . the levels of oxidized proteins have also been shown to increase with age in rat liver and brain extracts . in many organisms studied , cr post - pones the increased production of ros seen with age [ 28 , 29 ] , increases the resistance to oxidative insults [ 12 , 30 , 31 ] , and reduces oxidative damage [ 32 , 33 ] . on the other hand , observations that too low levels of ros lead to deficiencies in proliferation and immune responses , suggest that a balanced level of oxidants is necessary for the normal functions of cells and organisms . illustrating this , proliferation and growth factor signaling in multicellular organisms involve controlled production of o2 or h2o2 by , for example , nadph oxidase enzymes . superoxide anion is instable due to its reactivity and it can not diffuse through membranes , which makes it a poor signaling molecule [ 17 , 36 ] . in contrast , the half - life of h2o2 is longer and since it is an uncharged molecule it can diffuse through membranes . therefore , it is not surprising that most ros signaling involves signal transmission via h2o2 . ros signaling has been implicated in a variety of cellular events , for example , development and growth , oxidative stress resistance and even in the retardation of aging . in particular , adaptive responses to moderately increased ros levels have been proposed to induce antioxidant defences and hence resistance to ros and longevity upon cr [ 38 , 39 ] . in a recent study , thiol - peroxidases were shown to be the main receptors for h2o2 signaling in yeast cells , since the absence of all the eight thioredoxin - dependent peroxidases ( five prxs and 3 glutathione - peroxidase homologs ) resulted in an inability to sense and respond to externally added h2o2 . however , for most of the thiol peroxidases / prxs , the signal transduction pathways regulated are not known . a dual role in peroxide scavenging and signaling has been proposed for thiol peroxidases in several organisms [ 4143 ] . stimulation of longevity by ros signaling is suggested by the observation that treatment of c. elegans worms with low levels of the superoxide anion generator paraquat increased lifespan by more than 50% . such a prolongevity role of ros is , however , only seen at moderately increased ros levels , since higher doses of paraquat reduced life span and low levels of the drug were toxic in worms lacking detectable sod activity . growing yeast cells under cr ( 0.5% glucose or lower ) appears to lead to both higher respiration and higher mitochondrial ros production , as suggests the life span of cells lacking the mitochondrial sod , sod2 , that is normal at high glucose levels but drops precipitously at glucose levels of 0.5% or lower . this deficiency could be suppressed by the addition of the antioxidant ascorbic acid supporting the idea that sod2 mutants suffer excessive ros levels at reduced glucose levels . similarly , cr has been observed to increase respiration and mitochondrial ros production in mice and worms and these increases were reported to be required for the induction of both antioxidant defences and longevity [ 47 , 48 ] . in summary , ros appear to extend life - span , presumably by inducing adaptive ros signaling , at low levels , but are toxic at higher levels . cr may mitigate oxidative damage by stimulating the cellular ros defence capability through an increase in mitochondrial ros production . prxs are an evolutionarily highly conserved antioxidant enzyme family which members reduce intracellular peroxide levels through a cysteine - based mechanism [ 40 , 49 ] . prxs were first discovered in yeast , e. coli and s. typhimurium [ 50 , 51 ] but are now found to be widespread across phylogeny with one or more members typically being abundantly expressed in many aerobic organisms . prxs are divided into groups based on the number and the position(s ) of catalytic cysteine residues . the major subclass is the 2-cys prxs that form homodimers and catalyze the reduction of peroxide via two conserved catalytic cysteine residues [ 52 , 53 ] . catalysis is initiated by the reduction of h2o2 by a conserved n - terminal cysteine residue ( the peroxidatic cysteine ) which , in turn , oxidizes to the sulfenic acid form ( cys - soh ) . in 2-cys prxs , the peroxidatic cys - soh then condenses with the second catalytic cysteine residue present in the other monomer to form a disulfide that is reduced by thioredoxin , thus completing the catalytic cycle . however , the peroxidatic cys - soh might react again with h2o2 , when the levels of it are high , which leads to the formation of a sulfinic acid ( cys - sooh ) , and enzyme inactivation ( figure 1 ) . such inactive form of prx is slowly reactivated by atp - dependent reduction of the sulfinic acid by sulfiredoxin ( srx1 , ) . prx deficiency accelerates aging in yeast , worms , flies , and rodents [ 42 , 5557 ] . in addition , increasing the levels of a neuronal prx in flies or increasing sulfiredoxin levels in yeast , extends life span ( figure 1(b ) ) , suggesting that the anti - aging function of prxs is conserved . in addition , mice deficient in prxi age prematurely and develop several malignant cancers indicating that prxs prevent a prevalent group of age - related diseases . yeast cells lacking tsa1 suffer from increased mutation rates and genome instability , which at least to some extent has been linked to increased oxidative dna damage [ 58 , 60 ] . five of these are prxs proper ( tsa1 , tsa2 , ahp1 , dot5 , and prx1 ) and the other three prx - like enzymes with sequence homology to glutathione peroxidases ( gpx1 , gpx2 , and gpx3 ) [ 40 , 61 , 62 ] . although these prxs are thought to constitute an important part of the antioxidant arsenal , s. cerevisiae cells lacking the eight yeast thioredoxin - dependent peroxidases are viable , do not appear to contain increased levels of ros and can withstand a certain level of oxidative stress . interestingly , this mutant still suffers from a reduced replicative lifespan which might therefore be caused by the lack of functions other than direct ros detoxification . however , these data somewhat contradict earlier reports that yeast cells lacking all five prxs contain increased ros levels and are genomically unstable . to settle this issue , specifically in the context of aging , it will be important to monitor intracellular ros levels using modern , more sensitive and specific ros sensors [ 64 , 65 ] in aging prx - sufficient and -deficient cells [ 20 , 21 ] . similar to tsa1 , the 2-cys prxs prx1 , and prx2 are also very abundant in mammalian cells , which despite poor catalytic efficiency might indicate that they are important in h2o2 scavenging . tsa2 is a 2-cys prx 86% sequence identical to tsa1 but that is expressed only at ~80-fold lower levels than tsa1 . in agreement with its low expression , cells lacking tsa2 are not deficient in ros defence , but in fact , paradoxically , appear to grow better than wild - type cells in the presence of h2o2 . however , in cells lacking tsa1 , the levels of tsa2 are increased and cells lacking both tsa1 and tsa2 are more sensitive to h2o2 than the tsa1 single mutant , indicating that the two cytosolic prxs cooperate in peroxide defence . 2-cys prxs are multifunctional enzymes which , in addition to their peroxidase activity required to cope with oxidative stress , can act as chaperone holdases to counteract protein damage and as h2o2 signaling devices [ 71 , 72 ] . during peroxidase catalysis , further oxidation of the peroxidatic cysteine of a 2-cys - prx from sulfenic acid into sulfinic interestingly , hyperoxidation stabilizes the formation of high - molecular - weight prx complexes ( dodecameric and higher order decameric derivative forms ) [ 73 , 74 ] , which carry increased chaperone activity . similar alterations in quaternary structure were observed upon increased temperature or reduced ph and were also found to stimulate the ability of prxs to prevent the aggregation of model proteins in vitro [ 74 , 76 ] . reversion of the sulfinylated form of tsa1 by atp- and sulfiredoxin - dependent reduction indicates that sulfinylation is a redox - switch that alters the function of prxs from a peroxidase to a chaperone . prxs have been shown to interact with a multitude of signaling proteins and the redox - dependent oligomerization of 2-cys - prxs may therefore be important also in the regulation of signaling . interestingly , accumulation of the hyperoxidized forms of the yeast and rat prxs , tsa1 , and prxiii , have been reported upon aging [ 12 , 77 ] , indicating that prx inactivation may be a common phenotype in aging organisms ( figure 1(a ) ) . it is , however , not clear whether increased hyperoxidation with age is due to increased levels of h2o2 [ 2023 ] or deficient srx1-mediated prx de - sulfinylation and whether this in turn controls a switch to the chaperone - function or modulates prx - dependent h2o2 signaling [ 40 , 78 ] . in summary , prxs are multifunctional enzymes that retard aging and it is currently unknown which of their functions is / are important to retard aging . it appears that the signaling role of tsa1 may be discarded , however , since cells lacking tsa1 do not appear to display altered gene expression [ 12 , 40 ] yet age at a faster rate and fail to extend life - span upon cr . the function of yeast tsa1 in cr - mediated longevity appears to require srx1 since the recycling of hyperoxidized tsa1 is necessary and sufficient for cr life span extension . however , it is currently not clear what is the beneficial effect of tsa1 recycling during aging ; the consequential increased tsa1 peroxidase activity or a function linked to enzyme chaperone activity ? given the unique ability of prx peroxidase activity to scavenge low levels of endogenous peroxide to protect the genome , the determination of mutation rates and/or dna damage in aging cr cells would give an indication of the importance of the scavenging function during aging . the three conserved nutrient sensing kinases that mediate life span extension by cr in yeast are the protein kinase a ( pka ) , the target - of - rapamycin - complex 1 ( torc1 ) kinase , and the akt / protein kinase b / ribosomal s6 kinase homologue sch9 [ 4 , 79 , 80 ] . partly reduced activites in any of these three pathways mimic the effect of calorie restriction on aging . accordingly , partial inactivation of pka catalytic subunits , camp synthesis , or regulatory proteins of the pathway ( e.g. , cdc35/cyr1 , cdc25 , gpr1 , and gpa2 in yeast ) are frequently used as calorie restriction mimetics and mitigate aging and/or age - related diseases in mice [ 9 , 81 , 82 ] , drosophila , and yeast ( [ 4 , 84 ] , figure 1(b ) ) . since both the torc1/sch9 and ras - camp - pka pathways control mitochondrial activity and stress - defenses , nutrient signaling clearly impinges on pathways relevant in the context of the free radical theory of aging . for example , in both yeast cells and mouse adipose tissue torc1 negatively controls respiration [ 10 , 85 ] . similarly , the reprogramming of yeast metabolism upon glucose depletion and growth on respiratory carbon sources requires reduced activity of the ras - camp - pka pathway , which inhibits of mitochondrial biogenesis and mitochondrial activity . many yeast antioxidant enzymes are also under negative control of the ras - camp - pka pathway , for example , cytosolic catalase , all five yeast prxs [ 12 , 8789 ] and the mitochondrial sod . most of the studies on cr - mediated longevity in yeast have focused on the impact of reduced glucose levels on aging and a short review of yeast glucose signaling can therefore be found below ( box 1 ) . in conclusion , the major regulatory impact of the nutrient signaling pathways on mitochondrial activity and on antioxidant defence pathways indicate that the mechanisms underlying their effects on the rate of aging and cr - induced longevity is compatible with the free radical theory of aging . in agreement with this , decreased nutrient signaling increases the resistance to oxidative stress , presumably at least in part because increased ros defences more than offset an increased mitochondrial activity . box 1 ( yeast glucose signaling)glucose is the most abundant monosaccharide in nature and a rich carbon source that is preferred as a primary energy source by a variety of organisms , from yeast to humans . in s. cerevisiae , it has been shown that upon glucose addition the expression of about 40% of the genes in the genome is altered , indicating a substantial reprogramming of gene expression . the response to glucose is mainly regulated by the ras - camp - pka pathway . yeast has a g protein coupled receptor ( gpcr ) ( gpr1 ) that can accommodate glucose or sucrose as ligands to activate the g - protein gpa2 . gpa2 in turn stimulates the pka signaling pathway via adenylate cyclase ( cyr1 ) and camp levels ( figure 1 , ) . however , the most important part of the response to glucose is thought to involve the stimulation of ras activity ( figure 1 , [ 94 , 95 ] ) , the mechanism of which still remains elusive despite extensive research . ras2 ( and to a minor extent gpa2 ) activation more or less fully recapitulates the transcriptional response to glucose addition through stimulating pka activity , arguing that pka activation is sufficient for the response to glucose . whereas glucose still stimulates camp synthesis in the absence of gpr1 or gpa2 , the camp increase seen upon glucose addition is lost in cells deficient in ras . of note , glucose phosphorylation is required for ras activation and camp synthesis indicating that the ras proteins may respond to the levels of a glucose metabolite . glucose is the most abundant monosaccharide in nature and a rich carbon source that is preferred as a primary energy source by a variety of organisms , from yeast to humans . in s. cerevisiae , it has been shown that upon glucose addition the expression of about 40% of the genes in the genome is altered , indicating a substantial reprogramming of gene expression . the response to glucose yeast has a g protein coupled receptor ( gpcr ) ( gpr1 ) that can accommodate glucose or sucrose as ligands to activate the g - protein gpa2 . gpa2 in turn stimulates the pka signaling pathway via adenylate cyclase ( cyr1 ) and camp levels ( figure 1 , ) . however , the most important part of the response to glucose is thought to involve the stimulation of ras activity ( figure 1 , [ 94 , 95 ] ) , the mechanism of which still remains elusive despite extensive research . ras2 ( and to a minor extent gpa2 ) activation more or less fully recapitulates the transcriptional response to glucose addition through stimulating pka activity , arguing that pka activation is sufficient for the response to glucose . whereas glucose still stimulates camp synthesis in the absence of gpr1 or gpa2 , the camp increase seen upon glucose addition of note , glucose phosphorylation is required for ras activation and camp synthesis indicating that the ras proteins may respond to the levels of a glucose metabolite . recent data on yeast nutrient sensing suggest that nutrient signaling is intimately connected to v - atpase function and intracellular ph homeostasis . v - atpase deficiencies in aging cells thus likely directly impinge on the activation of nutrient signaling pathways . to better understand this connection , we will next shortly review the cellular roles of v - atpases . in addition to being localized at the vacuolar membrane , where they ( v - atpases ) acidify the vacuole , they are also found at the plasma membrane of several mammalian cell types [ 97100 ] , where they pump protons towards extracellular space . in yeast cells , ph homeostasis is maintained by two proton pumps ; pma1 , which resides at the plasma membrane , and v - atpases , which reside within the membranes of multiple organelles ( in particular the vacuole and the golgi ) . these two h - atpase pump systems seem to function coordinately since any defect in the v - atpase causes altered localization of the plasma membrane h - atpase , pma1 , presumably to maintain intracellular ph homeostasis . all eukaryotic v - atpases consist of two subcomplexes , v0 , and v1 , that are formed from 6 and 8 subunits , respectively . deletion of any v - atpase subunit is lethal in all organisms except fungi . in yeast cells , vma phenotype , which is characterized by an inability to grow on respiratory carbon sources , a sensitivity to increased extracellular ph , to heavy metals and to oxidants . accordingly , several genes encoding vacuolar proteins , for example , those important for vacuolar acidification , were identified in a genome - wide screen for genes important for oxidant tolerance suggesting that v - atpase function is required for proper antioxidant defences . in yeast , the addition of glucose to starved cells leads to a rapid and very transient cytosolic acidification ( 30 seconds , [ 106 , 107 ] ) , which is thought to be caused by the initiation of glycolysis , followed by its alkalinization through the h - atpase activities of pma1 and the v - atpase upon which growth recommences and camp levels increase . cytosolic alkalinity correlates closely with glycolytic rates and would eventually be expected to inactivate v - atpase function because of reduced substrate availability ( figure 2(a ) , ) . mutants lacking v - atpase activity , grown at steady state in high glucose , display acidic cytosolic ph [ 14 , 102 ] , in agreement with a role for v - atpase in cytosolic alkalinization . upon glucose withdrawal , the v - atpase is inactivated by disassembly of its two subunits , which lowers cytosolic ph and reduces the activity of the pka pathway . presumably , upon reduced glucose ( cr ) , the v - atpase is subsequently reactivated to promote vacuolar acidification and/or to maintain cytosolic ph homeostasis ( figure 2(b ) , ) . in agreement with this , cytosolic ph drops continuously as yeast cells grow and consume glucose , but cytosolic and vacuolar ph have not been measured during replicative aging of cr cells . in addition , although the measurement of vacuolar ph was used to argue for a role for v - atpase mediated vacuolar acidification in cr - mediated longevity , cytosolic ph was never measured and thus the full scope of increased v - atpase activity in aging cr cells was not assessed . these data thus raise the question whether v - atpase - mediated regulation of cytosolic ph contributes to cr life span extension by influencing nutrient signaling ( figure 2 ) . importantly , cytosolic ph appears intimately connected to the cellular nutrient status since both glucose starved cells and cells grown on respiratory carbon sources show reduced cytosolic ph . in fact , cytosolic ph was proposed to control the cell division rate both in yeast cells and in xenopus oocytes . thus , to better understand life span extension upon cr it would be important to monitor also cytosolic ph as cells age . the assembly / disassembly of v - atpases is fast and constitutes an important regulatory mechanism of v - atpase function . the role of the ras - camp - pka pathway in these events is , however , controversial . as mentioned above , v - atpase assembly is stimulated by glucose in vivo and this process was reported independent on camp - pka and the conventional glucose signaling pathways , but dependent on glucose metabolism beyond glucose-6-phosphate . v - atpase complex integrity independent on the ras - camp - pka pathway is supported by the unaltered disassembly of the vma5 subunit of the v1 domain upon glucose withdrawal in ira2 mutant cells , as observed by microscopical observation of a vma5-rfp fusion protein . in contrast , a genetic screen in yeast for mutants lacking v - atpase disassembly upon glucose withdrawal identified loss of ira1 , ira2 and bcy1 gene functions , which negatively regulate ras - camp - pka ( figure 1 , table 1 ) . furthermore , vacuolar acidity seems reduced in ira2 mutant cells in vivo , also suggesting that ira2 is essential for proper v - atpase function ( figure 2(a ) , table 1 ) . thus it is not clear at this point if v - atpase is acting upstream of or downstream of the ras - camp - pka pathway or if it participates in a complex feedback loop involving ras - camp - pka ( figure 2 ) . reciprocal regulation of pka and v - atpases has been proposed to reinforce pka activation and v - atpase assembly upon glucose sensing but the roles of glucose and the ras - camp - pka pathway in v - atpase assembly and disassembly clearly need further clarification . as discussed earlier , part of the increased longevity and the maintenance of mitochondrial function achieved by vacuolar acidification could be linked to the uptake of neutral amino acids into the vacuole ( figure 2(b ) , ) . supporting this , overproduction of the vacuolar neutral amino acid antiporter avt1 reduced age - induced mitochondrial dysfunction and extended life span . in addition , overexpression of the v - atpase subunit vma1 only partly suppressed age - induced mitochondrial dysfunction in cells lacking avt1 ( figure 2(b ) , ) . in this respect it is interesting that v - atpase and the proton - driven transport of lysosomal amino acids have been implicated in the activation of mammalian torc1 in response to amino acids . in yeast , torc1 is activated by cytosolic leucine , the most frequently utilized ( and neutral ) amino acid ( figure 2(b ) , arrow iv ) , by a mechanism involving the cytoplasmic leucyl - trna synthetase leurs charged by leucine and the gtpase gtr1 . it is thus conceivable that the reduced v - atpase - function and vacuolar proton - gradient in aging cells promotes both pka and torc1 activation via inefficient transport of neutral amino acids into the vacuole . in summary , cr , via reduced glucose intake , may repress pka activity through increased v - atpase function and reduced cytosolic ph ( figure 2(b ) , arrow iii ) . accordingly , maintained v - atpase activity would extend life span via feedback regulation of both ras - pka- and torc1 pathways , the latter via reducing cytosolic leucine levels . similarly , amino acid - restriction could be expected to efficiently reduce torc1 activity through increased v - atpase function and avt1 controlled amino acid transport into the vacuole ( figure 2(b ) , arrows ii & iv ) , thus also repressing ras - camp - pka activity ( figure 2(b ) , arrow iii ) . future studies should address the roles of v - atpase in feedback regulation of nutrient signaling pathways and in the cross - talk between the pathways . the compilation of data from different organisms identified vacuolar functions among several conserved genes essential for cr to extend life span . in addition , a recent study examining the life spans of 166 yeast deletion mutants upon cr suggested that both loss of vacuolar ph control or antioxidant defences negatively affect life span during cr , suggesting that both processes are essential for cr life span extension . it is clear from the studies reviewed here that both ras - pka and torc1 interact reciprocally with v - atpase functions . furthermore , the expression and activites of prxs is regulated by nutrient signaling pathways during cr or changes in carbon source in the media [ 12 , 88 ] . regarding potential connections between v - atpase and prxs , the ph - regulated oligomerization and chaperone activity of a schistosoma mansoni prx in vitro is certainly intriguing but the occurrence of prx oligomerisation at acidic ph is currently unknown in vivo . as mentioned above , in starved yeast cells pulsed with glucose , cytosolic ph has been reported initially to reach as low as 5.3 , which is still significantly higher than the ph where oligomerisation of the s. mansoni prx was observed in vitro ( ph 4.2 ) . thus , it will be interesting to know whether prxs from other organisms also oligomerize at acidic ph and at in vivo relevant values . another still intriguing connection between v - atpase and oxidative stress , and hence the prxs , is the observation that a yeast mutant lacking v - atpase function ( vma2 ) is exquisitely sensitive to h2o2 and less affected by other oxidants ( table 1 ) when grown under conditions that minimize the vma phenotype ( i.e. , acidic extracellular ph ) . under these conditions vma2 cells also display elevated dcdf - da staining , that might indicate elevated intracellular ros , and increased protein carbonylation . in addition , vma2tsa1 mutants display a severe synthetic growth phenotype indicating that they share a function important in cell physiology . iron metabolism may be yet another connection between v - atpases and prxs . in cells lacking vma2 , microarray analyses revealed prominent induction of genes that are involved in iron metabolism under the control of the transcription factor aft1 . the link between v - atpase and iron metabolism was further supported by the synthetic lethality of a strain lacking both vma2 and aft1 . in yeast , iron is indirectly sensed through the amount of fe / s - cluster proteins that are matured in the mitochondrial matrix [ 119 , 120 ] , which itself is a function of cellular iron availability and of mitochondrial fe / s - biogenesis . in a follow - up study , the vma2 mutant was , in accordance with a defect in mitochondrial fe / s - cluster biogenesis , shown to contain total iron levels similar to the wild - type but , importantly , to display reduced activity of aconitase , a mitochondrial fe - s cluster enzyme . importantly , adding a weak acid to wild - type cells which , like deficient v - atpase function , is expected to acidify the cytosol , and mitochondria was sufficent to induce aft1 transcription . these data indicate that v - atpase - regulated ph homeostasis is crucial for mitochondrial fe - s cluster biosynthesis / biogenesis . however , fe - s clusters have been reported to become unstable at low ph [ 121 , 122 ] indicating that v - atpase - regulated ph homeostasis also may impinge on fe / s - cluster stability . interestingly , deficient mitochondrial fe - s cluster biogenesis and growth arrest in daughters of replicatively aged yeast mother cells was earlier linked to the loss of mitochondrial dna and mitochondrial membrane potential , suggesting that mitochondrial fe - s clusters become instable with age as a result of mitochondrial dna damage . reciprocal crosstalk between mitochondria and v - atpase regulated ph homeostasis is suggested by the observation that defects associated with the loss of mitochondrial dna ( e.g. , slow growth and defective mitochondrial protein import ) can be suppressed by loss of v - atpase function ( table 1 ) . taken together , these data raise the possibility that reduced vacuolar acidity in aging cells may be an adaptation to suppress certain phenotypes associated with mitochondrial deficiency , but which on the contrary might exacerbate yet others , such as reduced mitochondrial fe / s cluster biogenesis . future studies are clearly necessary to identify mechanisms causing the loss of v - atpase function with age as well as the intricate interplay between v - atpase mediated ph homeostasis and mitochondrial function . interestingly , tsa2 transcription , but not the transcription of other antioxidant genes , is increased upon decreased v - atpase function ( in cells treated with concanamycin a or cells lacking vma2 , table 1 , [ 112 , 115 ] ) . in line with a role in iron metabolism , increased tsa2 transcription in a vma2 mutant could be suppressed by supplementing cells with iron . in addition , in cells lacking tsa2 ( tsa2 and vma2tsa2 ) , but not in peroxidase - negative mutants , expression of the aft1-target gene fit2 is increased ( 4-fold and 2-fold , resp . ) , indicating that tsa2 represses aft1-activity in a manner independent of tsa2 catalytic activity . furthermore , tsa1 and its peroxidatic cysteine appear necessary to support aerobic growth of cells lacking high affinity iron transport across the mitochondrial membrane by mrs3mrs4 deficiency ( table 1 ) . interestingly , mrs3- and mrs4-deficient cells require a repressor of the iron regulon , fra1 , for aerobic growth and suppression of the aerobic growth defect of mrs3mrs4 deficient cells by fra1 required tsa1 ( table 1 ) . taken together , repression of aft1 transcription by tsa2 and the interactions of both tsa1 and tsa2 with the iron regulatory protein fra1 indicate a link between prx and iron metabolism that will need to be elucidated at the molecular level . hence , it seems that both vacuolar ph control and prx activity are linked to iron homeostasis and fe / s cluster biogenesis and a further characterization of their impact on these vital processes appears important . more specifically , a better understanding of the role of prxs in longevity requires more careful assessment of their described roles in peroxide scavenging and in signaling in aging cells . since v - atpases appear to impinge on mitochondrial fe / s - cluster stability and both cytosolic prxs in yeast appear to interact closely with iron metabolism , a better understanding of their roles in these processes might also help to unravel how prxs and v - atpases contribute to slow down the rate of aging during caloric restriction .

calorie restriction ( cr ) is an intervention extending the life spans of many organisms . the mechanisms underlying cr - dependent retardation of aging are still poorly understood . despite mechanisms involving conserved nutrient signaling pathways proposed , few target processes that can account for cr - mediated longevity have so far been identified . recently , both peroxiredoxins and vacuolar - atpases were reported to control cr - mediated retardation of aging downstream of conserved nutrient signaling pathways . in this review , we focus on peroxiredoxin - mediated stress - defence and vacuolar - atpase regulated acidification and pinpoint common denominators between the two mechanisms proposed for how cr extends life span . both the activities of peroxiredoxins and vacuolar - atpases are stimulated upon cr through reduced activities in conserved nutrient signaling pathways and both seem to stimulate cellular resistance to peroxide - stress . however , whereas vacuolar - atpases have recently been suggested to control both ras - camp - pka- and torc1-mediated nutrient signaling , neither the physiological benefits of a proposed role for peroxiredoxins in h2o2-signaling nor downstream targets regulated are known . both peroxiredoxins and vacuolar - atpases do , however , impinge on mitochondrial iron - metabolism and further characterization of their impact on iron homeostasis and peroxide - resistance might therefore increase our understanding of the beneficial effects of cr on aging and age - related diseases .

1. Introduction 2. Reactive Oxygen Species (ROS) in Aging and in Caloric Restriction-Mediated Life Span Extension: Damaging Agents or Signal Transducers? 3. Peroxiredoxins in ROS Defence and in the Retardation of Aging 4. Calorie Restriction, Glucose Signaling, and the Link to the Free Radical Theory of Aging 5. Roles for Intracellular pH Homeostasis and Vacuolar ATPases in Nutrient Signaling 6. A Link between Calorie Restriction, Vacuolar Acidification, and Peroxiredoxins in the Regulation of Aging

caloric restriction ( cr ; or dietary restriction [ dr ] ) is the only known intervention that extends the life span of organisms as divergent as yeast , worms , flies , fish , and primates [ 1 , 2 ] , an observation which might indicate the existence of a universal , conserved mechanism of aging . despite almost 80 years of research since mccay 's initial discovery that caloric restriction without malnutrition extended the life span of rats , the mechanisms underlying its retardation of the rate of aging are still incompletely understood [ 47 ] . since decreased nutrient intake also lowers the incidence of many age - related maladies such as diabetes , cancer , and cardiovascular diseases in several organisms , intense efforts at identifying the molecular processes underlying these beneficial effects are underway in the aging researcher community . decreased signaling through nutrient - sensing pathways , for example , protein kinase a ( pka ) , target - of - rapamycin ( tor ) , or insulin - like growth factor ( igf ) pathways [ 2 , 9 ] , is in several model organisms required for life span extension upon cr . the views of some researchers in the field were , at least until rather recently , that life span extension by cr depends on the combined activity of many gene products acting through multiple pathways . in contrast to this hypothesis , two recent reports have pointed to two unique target mechanisms for how cr postpones replicative aging in yeast by counteracting detrimental processes acting at the genesis of aging [ 11 , 12 ] . we demonstrated that cr , through reduced pka signaling , activates the peroxiredoxin ( prx ) tsa1 , an antioxidant protein reducing h2o2 , to prolong yeast life span . strikingly , whereas wild - type cells responded to cr ( and reduced pka activity ) by an increased life span , tsa1 mutants did not , identifying tsa1 as a key enzyme extending life span during cr . interestingly , the cr - induced increase in srx1 levels did not involve increased transcript levels , but rather appeared to result from gcn2-dependent increased translation of the srx1 mrna ( figure 1(b ) ) . however , prx are not only h2o2-reducing enzymes but also function in h2o2-signaling and in proteostasis ( see below ) and it is currently not clear which facet(s ) of peroxiredoxin function that is / are required for cr - induced longevity . caloric restriction , as well as reducing the activities of conserved nutrient signaling pathways ( e.g. , ras - camp - pka and torc1 ) , delayed an age - induced loss of vacuolar acidity suggesting that the control of vacuolar ph also constitutes a target process regulated by cr ( figure 2 , arrows i and ii , ) . mutating subunits of the vacuolar proton - translocating atpase ( v - atpase ) leads to increased vacuolar ph and accelerated aging ( figure 2(a ) ) . as we will discuss later , however , both v - atpase and a cytosolic neutral amino acid ( leucine ) have been implicated in the regulation of nutrient signaling [ 1315 ] , raising the question whether v - atpase and cytosolic amino acids affect aging via feedback regulation of nutrient signaling pathways . the purpose of this paper is to review the physiological impact of both prx - mediated stress defense and vacuolar ph control and to pinpoint possible common denominators between these two apparently distinct target mechanisms proposed for cr - mediated life span extension . we will then point to outstanding questions that need to be resolved to further understand the beneficial impact of caloric restriction on aging and age - related diseases . to achieve this , we will first review the roles of ros and peroxiredoxins in aging and in cr - mediated longevity . next , we will shortly consider nutrient and glucose signaling in general and through the lenses of the free radical theory of aging in particular , since these signaling pathways are conserved mediators of cr life span extension in most organisms . following this , we will discuss the cellular roles of v - atpases including recent observations that these enzymes , via regulating intracellular ph homeostasis , are implicated in the regulation of nutrient signaling . on a final note , we will discuss points of intersection between v - atpases and prxs identified here , which include recent papers implicating both pathways in iron metabolism . furthermore , a correlation between the amount of oxidized proteins and the rate of aging was reported in flies and in cultured human fibroblasts [ 18 , 27 ] . in many organisms studied , cr post - pones the increased production of ros seen with age [ 28 , 29 ] , increases the resistance to oxidative insults [ 12 , 30 , 31 ] , and reduces oxidative damage [ 32 , 33 ] . ros signaling has been implicated in a variety of cellular events , for example , development and growth , oxidative stress resistance and even in the retardation of aging . in particular , adaptive responses to moderately increased ros levels have been proposed to induce antioxidant defences and hence resistance to ros and longevity upon cr [ 38 , 39 ] . however , for most of the thiol peroxidases / prxs , the signal transduction pathways regulated are not known . such a prolongevity role of ros is , however , only seen at moderately increased ros levels , since higher doses of paraquat reduced life span and low levels of the drug were toxic in worms lacking detectable sod activity . growing yeast cells under cr ( 0.5% glucose or lower ) appears to lead to both higher respiration and higher mitochondrial ros production , as suggests the life span of cells lacking the mitochondrial sod , sod2 , that is normal at high glucose levels but drops precipitously at glucose levels of 0.5% or lower . similarly , cr has been observed to increase respiration and mitochondrial ros production in mice and worms and these increases were reported to be required for the induction of both antioxidant defences and longevity [ 47 , 48 ] . however , the peroxidatic cys - soh might react again with h2o2 , when the levels of it are high , which leads to the formation of a sulfinic acid ( cys - sooh ) , and enzyme inactivation ( figure 1 ) . such inactive form of prx is slowly reactivated by atp - dependent reduction of the sulfinic acid by sulfiredoxin ( srx1 , ) . in addition , increasing the levels of a neuronal prx in flies or increasing sulfiredoxin levels in yeast , extends life span ( figure 1(b ) ) , suggesting that the anti - aging function of prxs is conserved . in addition , mice deficient in prxi age prematurely and develop several malignant cancers indicating that prxs prevent a prevalent group of age - related diseases . although these prxs are thought to constitute an important part of the antioxidant arsenal , s. cerevisiae cells lacking the eight yeast thioredoxin - dependent peroxidases are viable , do not appear to contain increased levels of ros and can withstand a certain level of oxidative stress . however , in cells lacking tsa1 , the levels of tsa2 are increased and cells lacking both tsa1 and tsa2 are more sensitive to h2o2 than the tsa1 single mutant , indicating that the two cytosolic prxs cooperate in peroxide defence . during peroxidase catalysis , further oxidation of the peroxidatic cysteine of a 2-cys - prx from sulfenic acid into sulfinic interestingly , hyperoxidation stabilizes the formation of high - molecular - weight prx complexes ( dodecameric and higher order decameric derivative forms ) [ 73 , 74 ] , which carry increased chaperone activity . reversion of the sulfinylated form of tsa1 by atp- and sulfiredoxin - dependent reduction indicates that sulfinylation is a redox - switch that alters the function of prxs from a peroxidase to a chaperone . it is , however , not clear whether increased hyperoxidation with age is due to increased levels of h2o2 [ 2023 ] or deficient srx1-mediated prx de - sulfinylation and whether this in turn controls a switch to the chaperone - function or modulates prx - dependent h2o2 signaling [ 40 , 78 ] . in summary , prxs are multifunctional enzymes that retard aging and it is currently unknown which of their functions is / are important to retard aging . it appears that the signaling role of tsa1 may be discarded , however , since cells lacking tsa1 do not appear to display altered gene expression [ 12 , 40 ] yet age at a faster rate and fail to extend life - span upon cr . the function of yeast tsa1 in cr - mediated longevity appears to require srx1 since the recycling of hyperoxidized tsa1 is necessary and sufficient for cr life span extension . however , it is currently not clear what is the beneficial effect of tsa1 recycling during aging ; the consequential increased tsa1 peroxidase activity or a function linked to enzyme chaperone activity ? the three conserved nutrient sensing kinases that mediate life span extension by cr in yeast are the protein kinase a ( pka ) , the target - of - rapamycin - complex 1 ( torc1 ) kinase , and the akt / protein kinase b / ribosomal s6 kinase homologue sch9 [ 4 , 79 , 80 ] . partly reduced activites in any of these three pathways mimic the effect of calorie restriction on aging . , cdc35/cyr1 , cdc25 , gpr1 , and gpa2 in yeast ) are frequently used as calorie restriction mimetics and mitigate aging and/or age - related diseases in mice [ 9 , 81 , 82 ] , drosophila , and yeast ( [ 4 , 84 ] , figure 1(b ) ) . since both the torc1/sch9 and ras - camp - pka pathways control mitochondrial activity and stress - defenses , nutrient signaling clearly impinges on pathways relevant in the context of the free radical theory of aging . similarly , the reprogramming of yeast metabolism upon glucose depletion and growth on respiratory carbon sources requires reduced activity of the ras - camp - pka pathway , which inhibits of mitochondrial biogenesis and mitochondrial activity . many yeast antioxidant enzymes are also under negative control of the ras - camp - pka pathway , for example , cytosolic catalase , all five yeast prxs [ 12 , 8789 ] and the mitochondrial sod . most of the studies on cr - mediated longevity in yeast have focused on the impact of reduced glucose levels on aging and a short review of yeast glucose signaling can therefore be found below ( box 1 ) . in conclusion , the major regulatory impact of the nutrient signaling pathways on mitochondrial activity and on antioxidant defence pathways indicate that the mechanisms underlying their effects on the rate of aging and cr - induced longevity is compatible with the free radical theory of aging . in agreement with this , decreased nutrient signaling increases the resistance to oxidative stress , presumably at least in part because increased ros defences more than offset an increased mitochondrial activity . the response to glucose is mainly regulated by the ras - camp - pka pathway . however , the most important part of the response to glucose is thought to involve the stimulation of ras activity ( figure 1 , [ 94 , 95 ] ) , the mechanism of which still remains elusive despite extensive research . however , the most important part of the response to glucose is thought to involve the stimulation of ras activity ( figure 1 , [ 94 , 95 ] ) , the mechanism of which still remains elusive despite extensive research . recent data on yeast nutrient sensing suggest that nutrient signaling is intimately connected to v - atpase function and intracellular ph homeostasis . v - atpase deficiencies in aging cells thus likely directly impinge on the activation of nutrient signaling pathways . to better understand this connection , we will next shortly review the cellular roles of v - atpases . these two h - atpase pump systems seem to function coordinately since any defect in the v - atpase causes altered localization of the plasma membrane h - atpase , pma1 , presumably to maintain intracellular ph homeostasis . in yeast , the addition of glucose to starved cells leads to a rapid and very transient cytosolic acidification ( 30 seconds , [ 106 , 107 ] ) , which is thought to be caused by the initiation of glycolysis , followed by its alkalinization through the h - atpase activities of pma1 and the v - atpase upon which growth recommences and camp levels increase . mutants lacking v - atpase activity , grown at steady state in high glucose , display acidic cytosolic ph [ 14 , 102 ] , in agreement with a role for v - atpase in cytosolic alkalinization . upon glucose withdrawal , the v - atpase is inactivated by disassembly of its two subunits , which lowers cytosolic ph and reduces the activity of the pka pathway . presumably , upon reduced glucose ( cr ) , the v - atpase is subsequently reactivated to promote vacuolar acidification and/or to maintain cytosolic ph homeostasis ( figure 2(b ) , ) . in agreement with this , cytosolic ph drops continuously as yeast cells grow and consume glucose , but cytosolic and vacuolar ph have not been measured during replicative aging of cr cells . in addition , although the measurement of vacuolar ph was used to argue for a role for v - atpase mediated vacuolar acidification in cr - mediated longevity , cytosolic ph was never measured and thus the full scope of increased v - atpase activity in aging cr cells was not assessed . these data thus raise the question whether v - atpase - mediated regulation of cytosolic ph contributes to cr life span extension by influencing nutrient signaling ( figure 2 ) . thus , to better understand life span extension upon cr it would be important to monitor also cytosolic ph as cells age . the role of the ras - camp - pka pathway in these events is , however , controversial . as mentioned above , v - atpase assembly is stimulated by glucose in vivo and this process was reported independent on camp - pka and the conventional glucose signaling pathways , but dependent on glucose metabolism beyond glucose-6-phosphate . v - atpase complex integrity independent on the ras - camp - pka pathway is supported by the unaltered disassembly of the vma5 subunit of the v1 domain upon glucose withdrawal in ira2 mutant cells , as observed by microscopical observation of a vma5-rfp fusion protein . in contrast , a genetic screen in yeast for mutants lacking v - atpase disassembly upon glucose withdrawal identified loss of ira1 , ira2 and bcy1 gene functions , which negatively regulate ras - camp - pka ( figure 1 , table 1 ) . thus it is not clear at this point if v - atpase is acting upstream of or downstream of the ras - camp - pka pathway or if it participates in a complex feedback loop involving ras - camp - pka ( figure 2 ) . reciprocal regulation of pka and v - atpases has been proposed to reinforce pka activation and v - atpase assembly upon glucose sensing but the roles of glucose and the ras - camp - pka pathway in v - atpase assembly and disassembly clearly need further clarification . supporting this , overproduction of the vacuolar neutral amino acid antiporter avt1 reduced age - induced mitochondrial dysfunction and extended life span . in addition , overexpression of the v - atpase subunit vma1 only partly suppressed age - induced mitochondrial dysfunction in cells lacking avt1 ( figure 2(b ) , ) . it is thus conceivable that the reduced v - atpase - function and vacuolar proton - gradient in aging cells promotes both pka and torc1 activation via inefficient transport of neutral amino acids into the vacuole . accordingly , maintained v - atpase activity would extend life span via feedback regulation of both ras - pka- and torc1 pathways , the latter via reducing cytosolic leucine levels . similarly , amino acid - restriction could be expected to efficiently reduce torc1 activity through increased v - atpase function and avt1 controlled amino acid transport into the vacuole ( figure 2(b ) , arrows ii & iv ) , thus also repressing ras - camp - pka activity ( figure 2(b ) , arrow iii ) . future studies should address the roles of v - atpase in feedback regulation of nutrient signaling pathways and in the cross - talk between the pathways . the compilation of data from different organisms identified vacuolar functions among several conserved genes essential for cr to extend life span . in addition , a recent study examining the life spans of 166 yeast deletion mutants upon cr suggested that both loss of vacuolar ph control or antioxidant defences negatively affect life span during cr , suggesting that both processes are essential for cr life span extension . it is clear from the studies reviewed here that both ras - pka and torc1 interact reciprocally with v - atpase functions . furthermore , the expression and activites of prxs is regulated by nutrient signaling pathways during cr or changes in carbon source in the media [ 12 , 88 ] . regarding potential connections between v - atpase and prxs , the ph - regulated oligomerization and chaperone activity of a schistosoma mansoni prx in vitro is certainly intriguing but the occurrence of prx oligomerisation at acidic ph is currently unknown in vivo . another still intriguing connection between v - atpase and oxidative stress , and hence the prxs , is the observation that a yeast mutant lacking v - atpase function ( vma2 ) is exquisitely sensitive to h2o2 and less affected by other oxidants ( table 1 ) when grown under conditions that minimize the vma phenotype ( i.e. the link between v - atpase and iron metabolism was further supported by the synthetic lethality of a strain lacking both vma2 and aft1 . however , fe - s clusters have been reported to become unstable at low ph [ 121 , 122 ] indicating that v - atpase - regulated ph homeostasis also may impinge on fe / s - cluster stability . reciprocal crosstalk between mitochondria and v - atpase regulated ph homeostasis is suggested by the observation that defects associated with the loss of mitochondrial dna ( e.g. future studies are clearly necessary to identify mechanisms causing the loss of v - atpase function with age as well as the intricate interplay between v - atpase mediated ph homeostasis and mitochondrial function . hence , it seems that both vacuolar ph control and prx activity are linked to iron homeostasis and fe / s cluster biogenesis and a further characterization of their impact on these vital processes appears important . more specifically , a better understanding of the role of prxs in longevity requires more careful assessment of their described roles in peroxide scavenging and in signaling in aging cells . since v - atpases appear to impinge on mitochondrial fe / s - cluster stability and both cytosolic prxs in yeast appear to interact closely with iron metabolism , a better understanding of their roles in these processes might also help to unravel how prxs and v - atpases contribute to slow down the rate of aging during caloric restriction .

constipation is very common in the general population ( belsey et al . , 2010 ) and affects the quality of human life . colonic and rectal sensory - motor function and biomechanical properties are now strongly implicated in the pathogenesis of constipation [ 3 , 4 ] . however , the pathophysiological mechanisms underlying chronic constipation remain to be explored [ 5 , 6 ] . furthermore constipation is difficult to treat and has a high recurrence rate [ 7 , 8 ] . the traditional chinese medicine has good clinical effects on constipation . during the past several years , the authors have through clinical observation found that the chang run tong ( crt ) produced by china - japan friendship hospital could effectively treat senile constipation . from chinese medicine point of view , crt regulates qi , relieves stagnation , and lubricates the bowel . however , from the western medicine point of view , the mechanism of crt in the treatment of constipation is unclear . hence , biomechanical properties such as the stress - strain relationship are of particular importance . the remodeling of the mechanical properties reflects the changes in the tissue structure that determine a specific motor dysfunction . a previous study has demonstrated that experimental diabetes could induce colon morphological and biomechanical remodeling . following the development of diabetes , the colonic wall became thicker and the stiffness of the wall increased in a time - dependent manner . therefore , the streptozotocin - induced diabetic rat model is a good model to study the effect of drugs in the treatment of constipation on the morphological and biomechanical remodeling of the gi tract caused by diseases . using this model , we recently demonstrated that crt could partly restore the morphometric and biomechanical remodeling of colon in streptozotocin- ( stz- ) induced diabetic rats . advanced glycation end products ( ages ) are formed physiologically , increased with aging , and accelerated in diabetes . ages can lead to structural and functional changes by direct target protein or through their receptor ( rage ) . ages and rage have been demonstrated to play an important role in diabetic complications including the complications of the gastrointestinal tract . tgf-1 has been considered to be core factor in the occurrence and development of diabetic nephropathy ( dn ) [ 17 , 18 ] . in recent years , the relationship between tgf-1 and other diabetic complications has been gradually noticed [ 19 , 20 ] . but there is lack of study about the relationship between tgf-1 and tgf-1 receptor with the diabetic colonic histomorphological and biomechanical remodeling . some studies have demonstrated that the association existed in the expressions of age / rage and tgf- during the development of diabetes [ 21 , 22 ] . in order to investigate the mechanism of crt in the improvement on the morphometric and biomechanical remodeling of the colon in streptozotocin- ( stz- ) induced diabetic rats , the expressions of advanced glycation end product ( age ) , age receptor ( rage ) , transforming growth factor-1 ( tgf-1 ) , and tgf- receptor were detected in the colon wall . the association between those proteins expression and the histomorphometric and biomechanical parameters was analyzed as well . forty male sprague - dawley rats weighing 220250 g were included in this study . thirty rats were made diabetic by a single tail - vein injection of 40 mg / kg stz ( sigma - aldrich , china ) . twenty - seven stz - induced diabetic rats were subdivided into three groups ( n = 9 in each group ) , namely , the diabetic control group ( dm ) , the high - dose crt group ( t1 ) , and the low - dose crt group ( t2 ) . another ten rats of similar age and body weight from the same vendor were used as the nondiabetic control group ( con ) . crt is composed of radix angelicae sinensis , radix cyathulae , herba cistanches , rhizoma alismatis , rhizoma cimicifugae , fructus aurantii immaturus , rhizoma atractylodis macrocephalae , semen arecae prepareta , and hemp seed provided by china - japan hospital , the ministry of health of the people 's republic of china . the medicine was directly injected into the stomach lumen by gastric gavage once daily from the beginning of the experiment . the body weight and blood glucose levels were measured at 2-week intervals after the start of the experiment . the experimental period was 60 d. at the ending of the experiment , the rats fasted overnight and then were anesthetized with 4% chloral hydrate ( 10 ml / kg , ip ) . following laparotomy , the whole colon was harvested . after the lumen of the segments was gently cleaned with saline , the length and the wet weight were measured . the colonic segment was divided into three parts : a 2 cm long tissue was cut from proximal end of the segments and fixed in 10% formalin for immunohistochemistry examination . then a 1 cm long part was cut and used for the zero - stress state experiment and the remaining part was used for the distension test . the results of zero - stress state and the distension test have been reported in our previous paper . the parameters of morphometric properties , residual strains , and stress - strain of the wall in colonic segments were adopted from our previous paper and used for correlation analysis with the expressions of different proteins used in the present paper . the tissue samples for immunohistochemistry were fixed in 10% phosphate - buffered formalin for 24 h and embedded in paraffin . five - micron sections were cut perpendicular to the mucosa surface and placed in a water bath at 40c . thereafter , sections were transferred onto pretreated microscopic slides which electrostatically attracted formalin fixed tissue and bound them to the slides . after drying the slides completely at room temperature , they were treated in an oven at 37c overnight to enhance the attachment of tissue to the slides . the sections were deparaffinized two times in xylene , 15 min per time , and rehydrated in 100% , 95% , 90% , 80% , 70% , 60% , and 50% ethanol two times , respectively , 3 sec per time , followed by rinsing for 10 min and washing in 0.01 m pbs ( ph 7.4 ) . after treatment with h2o2 ( 3% in ethanol , room temperature , 15 min ) and proteinase k ( 100 g / ml , 100 l , 37c , 20 min ) , the sections were incubated with 5% bsa - pbs buffer ( room temperature , 30 min ) for blocking nonspecific staining . afterwards , the sections were incubated with the primary anti - age antibody ( abcam , 1 : 100 , diluted in 1% bsa - pbs ) or normal mouse igg ( 250 g / ml ) pretreated with excessive cml ( 1 : 250 , diluted in 1% bsa - pbs , negative control ) over night at 4c . the sections were then washed and incubated with link ( biotinylated anti - rabbit and anti - mouse immunoglobulin ) and streptavidin peroxidase ( streptavidin conjugated with horseradish peroxidase ) , respectively , at room temperature for 10 min ( both are part of reagents of lsab2 system - hrp , products of dako company , denmark ) . then the peroxidase activity was visualized by incubating the sections in substrate working solution containing hydrogen peroxide and 3,3-diaminobenzidine tetrahydrochloride at room temperature for 5 min . the sections were rinsed for 10 min , counterstained with mayer hematoxylin for 1 min , treated in hcl - ethanol for 3 sec , and dehydrated in 80% , 90% , 95% , and 100% ethanol for 3 sec , respectively . then the slides were immersed in xylene for 15 min two times and mounted . the primary anti - rage antibody was produced in rabbits immunized with a synthetic peptide corresponding to a sequence at the n - terminal of human rage ( sigma ) . only two amino acids are different from the related rat sequence . instead of treating sections with proteinase k for rage immunostaining , the sections were boiled in 10 mm citrate buffer ( ph 6.0 ) for 18 min for retrieving antigen . normal rat lung was used as positive control since rage is highly expressed in the lung . the primary antibody was diluted ( 1 : 80 ) with 1% bsa - pbs and normal rabbit serum ( diluted 1 : 60 ) pretreated with excessive soluble rage was used as negative control . the primary tgf-1 antibody ( ba0290 ) and tgf-1 receptor antibody ( ba0526 - 2 ) were obtained from wuhan boster biological engineering co. , ltd . they were all diluted ( 1 : 100 ) with 1% bsa - pbs . the second antibody is hrp - goat anti - rabbit igg and was diluted ( 1 : 150 ) with 1% bsa - pbs . the sections were placed in 3% h2o2 ( ar1108 ) at room temperature for 510 minutes to inactivate endogenous enzymes . hot fix antigen : the sections were immersed in 0.01 m citrate buffer ( ar0024 , ph 6.0 ) or 0.02 m pbs ( ar0030 , ph 7.27.6 ) and heated to boiling using electricity or microwave for retrieving antigen . the sections were incubated with 5% bsa blocking solution ( ar0004 ) ( 37c , 30 minutes ) for blocking nonspecific staining . then the excess liquid was shanked off from the slides ( do not wash ) and incubated with diluted primary antibody over night at 4c or 2 hours at 37c . the slides were rinsed with pbs ( ph 7.27.6 ) for 3 times ( each time lasts 5 minutes ) . then the slides were incubated with corresponding second antibodies ( 37c , 30 minutes ) . the slides were rinsed again for 3 times ( each time lasts 5 minutes ) . the slides were incubated with sabc ( 37c , 30 minutes ) and washed for 4 times ( each time lasts 5 minutes ) with pbs ( ph 7.27.6 ) . then the peroxidase activity was visualized by incubating the sections with dab visualized kit ( ar1022 , taking 1 drop from each of a , b , and c reagent and mixing into 1 ml of distilled water ) for about 15 minutes at room temperature . age , rage , tgf-1 , and tgf-1 receptor are shown brown staining , but such color does not appear in the negative control slides , indicating that the staining is specific . to minimize errors , 6 to 10 photographs from different locations of the same layer in each slide were randomly taken . the region of interest ( roi ) was defined using sigmascan pro 4.0 image analysis software . finally the images were exported as binary images . the total area and area fraction of age , rage , tgf-1 , and tgf-1 receptor positive staining were calculated by a matlab program ( matlab 6.5 , the mathworks inc . , usa ) . then the fraction of age , rage , tgf-1 , and tgf-1 receptor in mucosa , muscle , and submucosa layers were computed as fraction of protein expressions = immunopositive area / total measured area . the data were representative of a normal distribution and accordingly the results were expressed as means sem . student 's t - test and analysis of variance ( anova ) were used to detect differences between parameters and groups ( sigmastat 2.0 ) . linear regression analysis was used to demonstrate possible association between the age , rage , tgf-1 , and tgf-1 receptor expressions and histomorphometric and biomechanical parameters . the blood glucose , body weight , the wet weight per unit length , no - load wall thickness , and cross section wall area measured at the end of the experiment are shown in table 1 . the blood glucose level was about 4-fold higher in the dm group compared with that of the con group ( p < 0.01 ) . the body weight in the dm group was nearly 50% lower than that in the con group . compared with the dm group , the blood glucose level in the t1 group ( p < 0.05 ) was lower but that in the t2 group ( p > 0.05 ) was higher . the body weight did not differ among the dm , t1 , and t2 groups ( p > 0.05 ) . the wet weight per unit length , no - load wall thickness , and cross section wall area of the colonic segments were significantly higher in the diabetic group compared with those of the con group ( p < 0.01 ) . after treatment with t1 , these parameters significantly decreased in the two segments ( p < 0.05 and p < 0.01 ) ; however , they did not significantly change in the t2 group ( p > 0.05 ) with the exception of the wall thickness ( p < 0.05 ) . the biomechanical parameters of the colon segment obtained from previous study were shown in table 2 . the opening angles were significantly higher in the dm group compared with those in the con group ( p < 0.01 ) . treatment with a high dosage of crt decreased the opening angle significantly ( p < 0.05 ) ; the opening angle did not change in the t2 group ( p > 0.05 ) . a similar trend was found for the inner and outer residual strains ; that is , the absolute values of the residual strain were significantly higher in the dm group compared with those in the con group ( p < 0.05 , p < 0.01 ) . treatment with a high dosage of crt ( t1 group ) partially reversed the changes of residual strain ( p < 0.05 ) . computation of constant showed a significant difference between the dm group and the con group ( p < 0.05 ) . high - dosage crt ( t1 ) treatment significantly decreased the stiffness of the colonic wall in both circumferential ( p < 0.05 ) and longitudinal ( p < 0.05 ) directions . low - dosage crt treatment ( t2 ) did not show improvement in the stiffening of the colonic wall caused by diabetes ( p > 0.05 ) . the fraction of age , rage , tgf-1 , and tgf-1 receptor expressions in the different layers of the colon in different groups were shown in table 3 . generally the expression of all proteins was stronger in the muscle layer than other layers . the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers were upregulated in the dm group than in con group . treatment with a high dosage of crt ( t1 group ) partially reversed the abnormal expressions of different proteins in the different layers of the colon wall ; however they did not significantly change in the t2 group . the detail results including p values were shown in table 3 . in order to analyze the expressions of age , rage , tgf-1 , and tgf-1 receptor with other parameters , the linear regression analysis was separately done on the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers of the colon wall with blood glucose level , body weight , wet weight per unit length of the colon , wall thickness , wall cross - sectional area , opening angle , inner residual strain , outer residual strain , circumferential material constant , and longitudinal material constant . it was shown that expressions of age , rage , and tgf-1 receptor correlated with most of the other parameters age and rage in different layers were highly correlated with glucose level , wall thickness , wall area , opening angle , and outer residual strain . age and rage in muscle layer correlated with circumferential material constant , whereas rage in muscle and submucosa layers correlated with longitudinal material constant . tgf-1 receptor in different layers correlated with most parameters , whereas it was only found that the tgf-1 in muscle layer correlated with glucose level , body weight , and circumferential and longitudinal material constant . the details of correlation equations and values of r and p were shown in table 4 . in order to determine the main contributor of age , rage , tgf-1 , and tgf-1 receptor expressions in different layers in association with other parameters , and to see interrelation among age , rage , tgf-1 , and tgf-1 receptor expressions , the multiple linear correlation analysis was done . table 5 showed the relation between some parameters with the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers . outer residual strain correlated with all proteins expressions in the mucosa layer and with rage expressions in the muscle and submucosa layers . opening angle was mainly determined by the rage in mucosa layer , age in muscle layer , and tgf-1 receptor in muscle and submucosa layers . circumferential material constant seems more related to tgf-1 in mucosa and age in muscle and submucosa layers , whereas longitudinal constant seems more related to age in mucosa , tgf-1 in muscle , and rage in submucosa layers . the details of correlation equations and values of r , f , and p were shown in table 5 . interrelation among age , rage , tgf-1 , and tgf-1 receptor expressions in different layers was shown in table 6 . age and tgf-1 in different layers mostly correlated with their receptors : rage and tgf-1 receptor . however it is interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . the details of correlation equations and values of r , f , and p were shown in table 6 . previously we demonstrated that crt could restore the morphometric and biomechanical remodeling of colon in streptozotocin- ( stz- ) induced diabetic rats . the main finding found at the present study was that the expression of age , rage , tgf-1 , and tgf-1 receptor was significantly higher in different colon layers in the dm group than in con group . the expressions of those proteins were highly correlated to the histomorphometric and biomechanical remodeling parameters . furthermore , the expressions of age , rage , tgf- , and tgf- receptor were significantly decreased in the t1 group but not in the t2 group . diabetic gi complications are common in long - standing diabetes . although many studies have demonstrated that diabetic gi complications involved multifactors , the mechanisms are not well understood . at present study we demonstrated that the expressions of age , rage , tgf-1 , and tgf-1 receptor were upregulated in the different layers of diabetic colon wall . it indicated that the abnormal expressions of these proteins are related to diabetic gi complications . ages and rage accumulated during the development of dm are associated with cardiovascular complication , retinopathy , and nephropathy as well as gi complications . recently chen and coworkers [ 31 , 32 ] have found that age and rage are upregulated in the diabetic small intestine and colon for both type 1 and type 2 diabetic animal models . the present study confirms these findings . the accumulated ages may affect the tissue structural changes and neuromuscular function of diabetic gi tract through receptor - dependent and receptor - independent pathways [ 33 , 34 ] . the former modulates cellular functions through ligation of specific cell surface receptors such as rage . the latter alters the extracellular matrix architecture by nonenzymatic glycation and the formation of protein cross - links . transforming growth factor- ( tgf- ) 1 is a ubiquitously expressed cytokine belonging to a large superfamily of activins / bone morphogenetic proteins . this mediator plays an active role in the processes of wound healing and synthesis of ecm molecules . it was reported that plasma levels of tgf-1 were elevated in niddm patients and might contribute to the occurrence of diabetic complications . indeed , many studies have demonstrated that tgf- strongly contributes to diabetic nephropathy [ 17 , 18 ] , diabetic retinopathy [ 39 , 40 ] , and diabetic neuropathy . in the present study , we first demonstrated that the tgf-1 and tgf-1 receptor were upregulated in the diabetic colon wall . although no detail molecular pathway was demonstrated in the present study , the increasing tgf-1 may through ligand binding activate tgf-1 receptors and then activate smad proteins through phosphorylation according to a review article about the role of tgf- in gastrointestinal pathophysiology and modulation of ulcer healing . analysis for interrelation among age , rage , tgf-1 , and tgf-1 receptor showed that age and tgf-1 in different layers mostly correlated with their receptors : rage and tgf-1 receptor . it seems easy to understand that the effects of age and tgf-1 are through their corresponding receptors . however it is interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . there are some studies to investigate the complicated interaction between age and tgf-1 with their receptors in the pathological progression of diabetic nephropathy [ 43 , 44 ] and interstitial fibrosis induced by imbalances in extracellular matrix homeostasis . however interpretation for the relation between rage and tgf-1 receptor in the diabetic gi complication is needed to explore . we have previously demonstrated that the histomorphological and biomechanical remodeling occurred in the diabetic rat model ; that is , diabetes generated pronounced increases in the colon wall thickness , wall cross - sectional area , the thickness of all layers , the opening angle , the absolute values of residual strain , and the circumferential and longitudinal stiffness of the colon wall . however , the mechanism of such remodeling is not so clear . it was shown from the study of relation between ages and vascular wall stiffness that glycation - induced intermolecular cross - links contributed to diabetic vascular stiffening . also study found that high serum age concentrations are associated with increased arterial stiffness and thickness in patients with diabetes . more recently one study demonstrated that chronic cml ingestion induced arterial stiffness and aging in a rage - dependent manner in the mice . the abovementioned studies demonstrated that the morphological and biomechanical remodeling of arteries in diabetes is closely associated with age and rage . we , in a previous study , demonstrated that the most histomorphometric and biomechanical parameters of intestinal wall in the gk diabetic rats were associated with the expression of age and rage in villi , especially that the highest association was found between the mechanical constant and villous age . at the same time , it was also found that the mechanical constant was associated with age and rage expressions in the crypt . in the present study , we further confirmed that diabetes - induced morphological and biomechanical remodeling of rat colon were also closely associated with the abnormal expressions of age and rage in the different layers of colon wall . data is lacking in relation to the association between tgf-1 and tgf-1 receptor and gastrointestinal morphological and biomechanical remodeling in diabetes . fleenor and coworkers reported that arterial stiffening with ageing is associated with tgf-1-related changes in adventitial collagen . few studies demonstrated that tgf-1 increased f - actin levels in single chondrocytes leading to stiffening of cells [ 51 , 52 ] . in the present study , we first demonstrated that tgf-1 in muscle layer correlated with circumferential and longitudinal material constant , whereas tgf-1 receptor in different layers correlated with most morphological and biomechanical parameters . it is also interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . this may indicate that either tgf-1 is an independent contributing factor or tgf-1 and age are cocontributors for the morphological and biomechanical remodeling of colon in diabetes . the detail molecular pathways for the effect of age and tgf-1 on colonic remodeling in diabetes are needed to further explore . studies have shown that herba cistanches had laxative activity in the intestine and promoted bowel movement . semen arecae prepareta promoted gastrointestinal movement , excited cholinergic m receptor , and promoted spontaneous contraction of isolated ileum . fructus cannabis could shorten the defecation time and significantly increase frequency of bowel movements [ 56 , 57 ] . therefore , the crt could regulate gi motility and improve the symptoms in patients with constipation . previous study has demonstrated that the experimental diabetes could induce colon morphological and biomechanical remodeling . such remodeling plays an important role in diabetic gi complications including the constipation . in order to investigate the mechanism of crt to treat the constipation whether or not through the improvement of the morphometric and biomechanical remodeling , we selected stz - induced diabetic rat model to test the crt on the morphometric and colonic remodeling induced by diabetes . we have demonstrated that high - dose crt could partly reverse the diabetes - induced morphological and biomechanical remodeling of colon ( tables 1 and 2 ) . however the mechanism of the crt effect is not clear . in the present study we demonstrated that the expressions of age , rage , tgf-1 , and the crt ( high dose ) could significantly decrease the expressions of age , rage , tgf-1 , and tgf-1 receptor in the diabetic colonic wall . furthermore , we found that the expressions of age , rage , tgf-1 , and tgf-1 receptor were associated with most morphological and biomechanical parameters of rat colon . in one previous study , we demonstrated that tangweian jianji ( high dose ) treatment partly restored the morphometric and biomechanical remodeling of the upper gastrointestinal tract in diabetic rats , and one mechanism was through decreasing the mrna level of rage in the gi wall . therefore we believed that the corrective effect of crt ( high dose ) on the expressions of age , rage , tgf- , and tgf- receptor is one of the molecular pathways for the improvement of crt on the colon remodeling in diabetic rats . furthermore , we demonstrated that high dose of crt could significantly decrease the blood glucose level in the diabetic rats . the evidence of correlation analysis showed that the blood glucose level highly associated with the expressions of age and rage . therefore , the effect of crt on the colonic wall remodeling of diabetic rats is likely to be partly from lowing blood glucose level . the colonic wall remodeling in diabetes will alter the relative position of the mechanosensitive afferents ( zero setting of the mechanosensitive afferents ) and their environment . improving the colonic wall remodeling induced diabetes will improve colon sensory - motor function and further improve the symptoms of the patients in the clinic . stz - induced diabetes upregulated the expression of age , rage , tgf-1 , and tgf-1 receptor in different colon layers of rats . crt could reverse the abnormal expressions of age , rage , tgf-1 , and tgf-1 receptor in the diabetic colon . the expressions of those proteins were highly associated with histomorphometric and biomechanical parameters of colon . the evidence showed that the corrective effect of crt ( high dose ) on the expressions of age , rage , tgf-1 , and tgf-1 receptor is one of the molecular pathways for the improvement of crt on the colon remodeling in diabetic rats . in the future , the detail molecular pathways for the effect of age and tgf-1 on colonic remodeling in diabetes and the relation between rage and tgf-1 receptor in the diabetic gi complication are needed to explore .

previous study demonstrated that chang run tong ( crt ) could partly restore the colon remodeling in streptozotocin- ( stz- ) induced diabetic rats . here we investigated the mechanisms of such effects of crt . diabetes was induced by a single injection of 40 mg / kg of stz . crt was poured into the stomach by gastric lavage once daily for 60 days . the remodeling parameters were obtained from diabetic ( dm ) , crt treated diabetic ( t1 , 50 g / kg ; t2 , 25 g / kg ) , and normal ( con ) rats . expressions of advanced glycation end product ( age ) , age receptor , transforming growth factor-1 ( tgf-1 ) , and tgf-1 receptor in the colon wall were immunochemically detected and quantitatively analyzed . the association between the expressions of those proteins and the remodeling parameters was analyzed . the expressions of those proteins were significantly higher in different colon layers in the dm group ( p < 0.05 , p < 0.01 ) and highly correlated to the remodeling parameters . furthermore , the expressions of those proteins were significantly decreased in the t1 group ( p < 0.05 , p < 0.01 ) but not in the t2 group ( p > 0.05 ) . the corrective effect on the expressions of those proteins is likely to be one molecular pathway for the improvement of crt on the diabetes - induced colon remodeling .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion 5. Conclusion and Perspectives of the Study

however , the pathophysiological mechanisms underlying chronic constipation remain to be explored [ 5 , 6 ] . during the past several years , the authors have through clinical observation found that the chang run tong ( crt ) produced by china - japan friendship hospital could effectively treat senile constipation . from chinese medicine point of view , crt regulates qi , relieves stagnation , and lubricates the bowel . however , from the western medicine point of view , the mechanism of crt in the treatment of constipation is unclear . the remodeling of the mechanical properties reflects the changes in the tissue structure that determine a specific motor dysfunction . a previous study has demonstrated that experimental diabetes could induce colon morphological and biomechanical remodeling . following the development of diabetes , the colonic wall became thicker and the stiffness of the wall increased in a time - dependent manner . therefore , the streptozotocin - induced diabetic rat model is a good model to study the effect of drugs in the treatment of constipation on the morphological and biomechanical remodeling of the gi tract caused by diseases . using this model , we recently demonstrated that crt could partly restore the morphometric and biomechanical remodeling of colon in streptozotocin- ( stz- ) induced diabetic rats . advanced glycation end products ( ages ) are formed physiologically , increased with aging , and accelerated in diabetes . tgf-1 has been considered to be core factor in the occurrence and development of diabetic nephropathy ( dn ) [ 17 , 18 ] . but there is lack of study about the relationship between tgf-1 and tgf-1 receptor with the diabetic colonic histomorphological and biomechanical remodeling . some studies have demonstrated that the association existed in the expressions of age / rage and tgf- during the development of diabetes [ 21 , 22 ] . in order to investigate the mechanism of crt in the improvement on the morphometric and biomechanical remodeling of the colon in streptozotocin- ( stz- ) induced diabetic rats , the expressions of advanced glycation end product ( age ) , age receptor ( rage ) , transforming growth factor-1 ( tgf-1 ) , and tgf- receptor were detected in the colon wall . the association between those proteins expression and the histomorphometric and biomechanical parameters was analyzed as well . thirty rats were made diabetic by a single tail - vein injection of 40 mg / kg stz ( sigma - aldrich , china ) . twenty - seven stz - induced diabetic rats were subdivided into three groups ( n = 9 in each group ) , namely , the diabetic control group ( dm ) , the high - dose crt group ( t1 ) , and the low - dose crt group ( t2 ) . another ten rats of similar age and body weight from the same vendor were used as the nondiabetic control group ( con ) . crt is composed of radix angelicae sinensis , radix cyathulae , herba cistanches , rhizoma alismatis , rhizoma cimicifugae , fructus aurantii immaturus , rhizoma atractylodis macrocephalae , semen arecae prepareta , and hemp seed provided by china - japan hospital , the ministry of health of the people 's republic of china . the medicine was directly injected into the stomach lumen by gastric gavage once daily from the beginning of the experiment . the experimental period was 60 d. at the ending of the experiment , the rats fasted overnight and then were anesthetized with 4% chloral hydrate ( 10 ml / kg , ip ) . after the lumen of the segments was gently cleaned with saline , the length and the wet weight were measured . then a 1 cm long part was cut and used for the zero - stress state experiment and the remaining part was used for the distension test . the parameters of morphometric properties , residual strains , and stress - strain of the wall in colonic segments were adopted from our previous paper and used for correlation analysis with the expressions of different proteins used in the present paper . the sections were deparaffinized two times in xylene , 15 min per time , and rehydrated in 100% , 95% , 90% , 80% , 70% , 60% , and 50% ethanol two times , respectively , 3 sec per time , followed by rinsing for 10 min and washing in 0.01 m pbs ( ph 7.4 ) . after treatment with h2o2 ( 3% in ethanol , room temperature , 15 min ) and proteinase k ( 100 g / ml , 100 l , 37c , 20 min ) , the sections were incubated with 5% bsa - pbs buffer ( room temperature , 30 min ) for blocking nonspecific staining . afterwards , the sections were incubated with the primary anti - age antibody ( abcam , 1 : 100 , diluted in 1% bsa - pbs ) or normal mouse igg ( 250 g / ml ) pretreated with excessive cml ( 1 : 250 , diluted in 1% bsa - pbs , negative control ) over night at 4c . the sections were then washed and incubated with link ( biotinylated anti - rabbit and anti - mouse immunoglobulin ) and streptavidin peroxidase ( streptavidin conjugated with horseradish peroxidase ) , respectively , at room temperature for 10 min ( both are part of reagents of lsab2 system - hrp , products of dako company , denmark ) . the primary tgf-1 antibody ( ba0290 ) and tgf-1 receptor antibody ( ba0526 - 2 ) were obtained from wuhan boster biological engineering co. , ltd . hot fix antigen : the sections were immersed in 0.01 m citrate buffer ( ar0024 , ph 6.0 ) or 0.02 m pbs ( ar0030 , ph 7.27.6 ) and heated to boiling using electricity or microwave for retrieving antigen . age , rage , tgf-1 , and tgf-1 receptor are shown brown staining , but such color does not appear in the negative control slides , indicating that the staining is specific . the total area and area fraction of age , rage , tgf-1 , and tgf-1 receptor positive staining were calculated by a matlab program ( matlab 6.5 , the mathworks inc . then the fraction of age , rage , tgf-1 , and tgf-1 receptor in mucosa , muscle , and submucosa layers were computed as fraction of protein expressions = immunopositive area / total measured area . linear regression analysis was used to demonstrate possible association between the age , rage , tgf-1 , and tgf-1 receptor expressions and histomorphometric and biomechanical parameters . the blood glucose , body weight , the wet weight per unit length , no - load wall thickness , and cross section wall area measured at the end of the experiment are shown in table 1 . the blood glucose level was about 4-fold higher in the dm group compared with that of the con group ( p < 0.01 ) . the body weight in the dm group was nearly 50% lower than that in the con group . compared with the dm group , the blood glucose level in the t1 group ( p < 0.05 ) was lower but that in the t2 group ( p > 0.05 ) was higher . the body weight did not differ among the dm , t1 , and t2 groups ( p > 0.05 ) . the wet weight per unit length , no - load wall thickness , and cross section wall area of the colonic segments were significantly higher in the diabetic group compared with those of the con group ( p < 0.01 ) . after treatment with t1 , these parameters significantly decreased in the two segments ( p < 0.05 and p < 0.01 ) ; however , they did not significantly change in the t2 group ( p > 0.05 ) with the exception of the wall thickness ( p < 0.05 ) . the biomechanical parameters of the colon segment obtained from previous study were shown in table 2 . the opening angles were significantly higher in the dm group compared with those in the con group ( p < 0.01 ) . treatment with a high dosage of crt decreased the opening angle significantly ( p < 0.05 ) ; the opening angle did not change in the t2 group ( p > 0.05 ) . a similar trend was found for the inner and outer residual strains ; that is , the absolute values of the residual strain were significantly higher in the dm group compared with those in the con group ( p < 0.05 , p < 0.01 ) . treatment with a high dosage of crt ( t1 group ) partially reversed the changes of residual strain ( p < 0.05 ) . computation of constant showed a significant difference between the dm group and the con group ( p < 0.05 ) . high - dosage crt ( t1 ) treatment significantly decreased the stiffness of the colonic wall in both circumferential ( p < 0.05 ) and longitudinal ( p < 0.05 ) directions . low - dosage crt treatment ( t2 ) did not show improvement in the stiffening of the colonic wall caused by diabetes ( p > 0.05 ) . the fraction of age , rage , tgf-1 , and tgf-1 receptor expressions in the different layers of the colon in different groups were shown in table 3 . the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers were upregulated in the dm group than in con group . treatment with a high dosage of crt ( t1 group ) partially reversed the abnormal expressions of different proteins in the different layers of the colon wall ; however they did not significantly change in the t2 group . in order to analyze the expressions of age , rage , tgf-1 , and tgf-1 receptor with other parameters , the linear regression analysis was separately done on the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers of the colon wall with blood glucose level , body weight , wet weight per unit length of the colon , wall thickness , wall cross - sectional area , opening angle , inner residual strain , outer residual strain , circumferential material constant , and longitudinal material constant . it was shown that expressions of age , rage , and tgf-1 receptor correlated with most of the other parameters age and rage in different layers were highly correlated with glucose level , wall thickness , wall area , opening angle , and outer residual strain . tgf-1 receptor in different layers correlated with most parameters , whereas it was only found that the tgf-1 in muscle layer correlated with glucose level , body weight , and circumferential and longitudinal material constant . in order to determine the main contributor of age , rage , tgf-1 , and tgf-1 receptor expressions in different layers in association with other parameters , and to see interrelation among age , rage , tgf-1 , and tgf-1 receptor expressions , the multiple linear correlation analysis was done . table 5 showed the relation between some parameters with the expressions of age , rage , tgf-1 , and tgf-1 receptor in different layers . opening angle was mainly determined by the rage in mucosa layer , age in muscle layer , and tgf-1 receptor in muscle and submucosa layers . interrelation among age , rage , tgf-1 , and tgf-1 receptor expressions in different layers was shown in table 6 . age and tgf-1 in different layers mostly correlated with their receptors : rage and tgf-1 receptor . however it is interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . previously we demonstrated that crt could restore the morphometric and biomechanical remodeling of colon in streptozotocin- ( stz- ) induced diabetic rats . the main finding found at the present study was that the expression of age , rage , tgf-1 , and tgf-1 receptor was significantly higher in different colon layers in the dm group than in con group . the expressions of those proteins were highly correlated to the histomorphometric and biomechanical remodeling parameters . furthermore , the expressions of age , rage , tgf- , and tgf- receptor were significantly decreased in the t1 group but not in the t2 group . although many studies have demonstrated that diabetic gi complications involved multifactors , the mechanisms are not well understood . at present study we demonstrated that the expressions of age , rage , tgf-1 , and tgf-1 receptor were upregulated in the different layers of diabetic colon wall . indeed , many studies have demonstrated that tgf- strongly contributes to diabetic nephropathy [ 17 , 18 ] , diabetic retinopathy [ 39 , 40 ] , and diabetic neuropathy . in the present study , we first demonstrated that the tgf-1 and tgf-1 receptor were upregulated in the diabetic colon wall . although no detail molecular pathway was demonstrated in the present study , the increasing tgf-1 may through ligand binding activate tgf-1 receptors and then activate smad proteins through phosphorylation according to a review article about the role of tgf- in gastrointestinal pathophysiology and modulation of ulcer healing . analysis for interrelation among age , rage , tgf-1 , and tgf-1 receptor showed that age and tgf-1 in different layers mostly correlated with their receptors : rage and tgf-1 receptor . it seems easy to understand that the effects of age and tgf-1 are through their corresponding receptors . however it is interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . there are some studies to investigate the complicated interaction between age and tgf-1 with their receptors in the pathological progression of diabetic nephropathy [ 43 , 44 ] and interstitial fibrosis induced by imbalances in extracellular matrix homeostasis . however interpretation for the relation between rage and tgf-1 receptor in the diabetic gi complication is needed to explore . we have previously demonstrated that the histomorphological and biomechanical remodeling occurred in the diabetic rat model ; that is , diabetes generated pronounced increases in the colon wall thickness , wall cross - sectional area , the thickness of all layers , the opening angle , the absolute values of residual strain , and the circumferential and longitudinal stiffness of the colon wall . however , the mechanism of such remodeling is not so clear . more recently one study demonstrated that chronic cml ingestion induced arterial stiffness and aging in a rage - dependent manner in the mice . we , in a previous study , demonstrated that the most histomorphometric and biomechanical parameters of intestinal wall in the gk diabetic rats were associated with the expression of age and rage in villi , especially that the highest association was found between the mechanical constant and villous age . in the present study , we further confirmed that diabetes - induced morphological and biomechanical remodeling of rat colon were also closely associated with the abnormal expressions of age and rage in the different layers of colon wall . data is lacking in relation to the association between tgf-1 and tgf-1 receptor and gastrointestinal morphological and biomechanical remodeling in diabetes . in the present study , we first demonstrated that tgf-1 in muscle layer correlated with circumferential and longitudinal material constant , whereas tgf-1 receptor in different layers correlated with most morphological and biomechanical parameters . it is also interesting to notice that rage and tgf-1 receptor in muscle layers were strongly correlated with each other . the detail molecular pathways for the effect of age and tgf-1 on colonic remodeling in diabetes are needed to further explore . semen arecae prepareta promoted gastrointestinal movement , excited cholinergic m receptor , and promoted spontaneous contraction of isolated ileum . previous study has demonstrated that the experimental diabetes could induce colon morphological and biomechanical remodeling . in order to investigate the mechanism of crt to treat the constipation whether or not through the improvement of the morphometric and biomechanical remodeling , we selected stz - induced diabetic rat model to test the crt on the morphometric and colonic remodeling induced by diabetes . we have demonstrated that high - dose crt could partly reverse the diabetes - induced morphological and biomechanical remodeling of colon ( tables 1 and 2 ) . in the present study we demonstrated that the expressions of age , rage , tgf-1 , and the crt ( high dose ) could significantly decrease the expressions of age , rage , tgf-1 , and tgf-1 receptor in the diabetic colonic wall . furthermore , we found that the expressions of age , rage , tgf-1 , and tgf-1 receptor were associated with most morphological and biomechanical parameters of rat colon . in one previous study , we demonstrated that tangweian jianji ( high dose ) treatment partly restored the morphometric and biomechanical remodeling of the upper gastrointestinal tract in diabetic rats , and one mechanism was through decreasing the mrna level of rage in the gi wall . therefore we believed that the corrective effect of crt ( high dose ) on the expressions of age , rage , tgf- , and tgf- receptor is one of the molecular pathways for the improvement of crt on the colon remodeling in diabetic rats . furthermore , we demonstrated that high dose of crt could significantly decrease the blood glucose level in the diabetic rats . the evidence of correlation analysis showed that the blood glucose level highly associated with the expressions of age and rage . therefore , the effect of crt on the colonic wall remodeling of diabetic rats is likely to be partly from lowing blood glucose level . the colonic wall remodeling in diabetes will alter the relative position of the mechanosensitive afferents ( zero setting of the mechanosensitive afferents ) and their environment . stz - induced diabetes upregulated the expression of age , rage , tgf-1 , and tgf-1 receptor in different colon layers of rats . crt could reverse the abnormal expressions of age , rage , tgf-1 , and tgf-1 receptor in the diabetic colon . the expressions of those proteins were highly associated with histomorphometric and biomechanical parameters of colon . the evidence showed that the corrective effect of crt ( high dose ) on the expressions of age , rage , tgf-1 , and tgf-1 receptor is one of the molecular pathways for the improvement of crt on the colon remodeling in diabetic rats . in the future , the detail molecular pathways for the effect of age and tgf-1 on colonic remodeling in diabetes and the relation between rage and tgf-1 receptor in the diabetic gi complication are needed to explore .

muscular dystrophies ( mds ) are a group of fatal inherited diseases ( ~30 ) , associated with progressive muscle wasting caused by muscle membrane fragility , abnormal metabolic control , increased oxidative and energetic stress and abnormal proliferation of satellite cells ( campbell , 1995 ; cohn and campbell , 2000 ; constantin et al . , 2006 ; vercherat et al . , 2009 ; wallace and mcnally , 2009 ) . the most prevalent and severe duchenne md ( dmd ) and the milder becker md ( bmd ) both result from mutations in the dystrophin ( dys ) gene . dys , the largest gene in the human genome ( 1.2 mb , 79 exons ) , encodes a structural cytoplasmic protein that is the key component of the larger membrane - associated multiprotein dystrophin glycoprotein complex ( dgc ; kunkel et al . , 1986 ; hoffman et al . , 1987 ) . the dgc classically consists of dystrophin , dystroglycans and , sarcoglycans , , , and , sarcospan , and other proteins , such as -dystrobrevin , syntrophins 1 , 1 , 2 , 1 , 2 , calveolin-3 , growth factor receptor bound protein-2 ( grb2 ) , and neuronal nitric oxide synthase ( nnos ) have been shown to be associated with the dgc ( durbeej and campbell , 2002 ) . the dgc has multiple roles in muscle : mechanical stabilization of the muscle sarcolemma via anchoring the extracellular matrix ( ecm ) to the cytoskeleton , signal transduction between the internal and external environments of the muscle cell and provides a scaffold responsible for the membrane localization of signaling proteins ( ervasti and sonnemann , 2008 ) . the dgc , via syntrophin ( syn ) anchors a variety of signaling molecules to their functional sites at the membrane . for example , it localizes nnos at the sarcolemma consequently regulating intramuscular generation of nitric oxide ( no ; wehling et al . , 2001 ) . nnos signaling nitrosylates the histone deacetylases ( hdacs ) that regulate gene transcription in muscle progenitor cells by controlling the activity of myogenic ( myod , myf5 , myogenin , and mrf4 ) and mef2 family proteins ( mckinsey et al . hdacs are displaced from the chromatin that then becomes hyperacetylated leading to transcriptional activation . since dys deficiency leads to downregulation of nnos signaling ( brenman et al . , 1995 ) , it indirectly plays a role in control of the balance between acetylation and deacetylation of muscle differentiation genes and has an impact on the regenerative potential of muscle stem cells . it is not clear if the dgc is just a scaffold for syn nnos or , since it has been shown that the dgc is also a signal - transducing module involved in cross talk between the internal and external environments of the muscle cell ( moore and winder , 2010 ) , it can also actively control epigenetic characteristics of muscle cells via the nnos signaling pathway . muscles can withstand rigorous mechanical stress and , if damaged , be repaired by the progeny of satellite cells ( figure 1 ) . satellite cells are quiescent muscle progenitor cells that reside along the basal lamina of the muscle fiber and proliferate only in response to specific signals , for example muscle injury ( charge and rudnicki , 2004 ) . for continuous and sufficient muscle regeneration , satellite cells must be easily activated to proliferate but also should return to quiescence to maintain their stem cell characteristics ( dhawan and rando , 2005 ) . when muscle fibers need to be replaced , signals are sent to otherwise quiescent satellite cells , which activates their proliferation and generates proliferating myoblasts . these myoblasts proliferate to increase in number and upon activation differentiate and fuse to form myotubes . these myotubes then cluster into myofibers which again cluster to form muscle cells or myocytes . this program must be tightly regulated to ensure that muscle is replenished as necessary and that satellite cells are not lost in an untimely manner ( buckingham , 2006 ) . in order for a cell to be a myocyte it must express specific transcription factors , including mef2 , myod , and myogenin among others ( sabourin and rudnicki , 2000 ) . before myoblasts express these factors they are in a proliferative state that requires repression of terminal differentiation genes and cell cycle inhibitors . in order for differentiation of myoblasts to occur , this requires the coordinated agreement of cell cycle inhibition and initiation of muscle specific gene expression . in dystrophic patients , dys insufficiency causes the muscle sarcolemma to become fragile leading to damage that can not be easily repaired due to inadequate response of satellite cells . this results in chronic inflammation of muscle tissue by penetration of immune cells and eventually leads to the replacement of myofibers with adipose and fibrotic tissue ( porter et al . , 2002 ) . age - dependent muscle degeneration in md patients is associated with defects in multiple processes that occur in different cell types such as activation of satellite cell proliferation , progenitor cell maintenance , myoblast differentiation , muscle cell homeostasis , immune cell recruitment , etc . these processes are also required during normal muscle development and growth and the transcriptional circuitry and signaling pathways controlling these events have been extensively studied using many model organisms . in this review we will discuss how recent studies have also revealed a new level of regulation of muscle gene expression that is mediated by mirnas . since mirnas have great potential for therapeutics , understanding their basic biology and specific functions in healthy and diseased muscle tissue is of great importance . nnos pathway regulates the expression of mirnas required for muscle tissue maintenance and regeneration ( cacchiarelli et al . , 2010 indeed , it has been found that mrna levels are altered in the human dmd pathology , which correlates with mis - expressed mirnas ( eisenberg et al . , 2007 ) . specifically nnos signaling controls modification of hdac2 , which in normal situations prevents binding to promoter regions that would lead to transcriptional activation . when dys is absent this signaling is disturbed , which also results in mirna dysregulation ( figure 3 ) . interestingly it was found that most of the mis - expressed mirnas found in dmd patients are returned to normal expression levels when hdacs were inhibited by restoring no signaling ( cacchiarelli et al . , 2010 ) . this is in accord with a previous study that showed that class i hdac inhibitors and no - inhibited hdacs effectively ameliorate md in mouse models ( colussi et al . , 2008 ) . nnos signaling regulates the epigenetic profile of muscle cells via nitrosylation of hdac2 that influences gene expression by altering the acetylation status of histones . mir-221 and mir-222 share the same seed sequence and both target cell cycle inhibitors , p27 and p57 . mir-1 and mir-133 are transcriptionally controlled by the nitrosylation state of hdac2 , which is regulated by nnos . mir-133 targets srf during proliferation , which in a self regulatory manner promotes expression of mir-133 . mir-1 targets g6pd and hdac4 during differentiation , and since hdac4 regulates the transcription of muscle differentiation genes , dys syn nnos signaling indirectly promotes differentiation . in a self - promoting regulatory loop , the expression of muscle differentiation genes results in mir-1 expression . the skeletal muscle specific mirna , mir-206 is also upregulated by muscle gene expression and aids in cell cycle inhibition by targeting dna pola1 and pax7 . utrophin is another target of mir-206 providing another link between it and the dgc . in dystrophic muscle when dys is absent , dys syn nnos signaling is disrupted inhibiting nitrosylation of hdac2 . consequently , levels of mir-133/mir-1 and mir-29 , all of which are repressed by non - nitrosylated hdac2 , are down . the regulatory roles of mir-133/mir-1 are disturbed leading to inhibition of muscle differentiation gene expression and an increase in oxidative cellular stress . the decrease in mir-29 levels correlates with an increase in the amount of collagen and elastin that are indicative of fat and fibrotic tissue . due to reasons that are not quite clear at this time , levels of mir-206 and mir-31 are increased altering the equilibrium between the proliferation and differentiation states . mirnas circled in green are upregulated and mirnas circled in red are down regulated , bold letters indicate upregulated protein levels , gray circles with diagonal lines and gray lines indicate deficiencies . not only does the dgc play a role in mirna regulation , but it is also regulated via mirnas . nnos pathway possibly by targeting the 3-utr of 1-syntrophin ( de arcangelis et al . , 2010 ) . since syntrophins bind to dys and act as a scaffold for nnos , mir-222 could control the timing of no signaling during differentiation . mir-222 has the same seed sequence as mir-221 , both are controlled by the ras mapk pathway and are downregulated upon differentiation of myoblasts ( cardinali et al . , 2009 ) . additionally , mir-221 and mir-222 promote cell cycle progression by down - regulating the cyclin - dependent kinase inhibitors p27 ( cdkn1b / kip1 ) and p57 ( cdkn1c / kip2 ) that are essential for the maintenance of the proliferative state ( le sage et al . , 2007 ; medina et al . , 2008 since both mir-221 and mir-222 are significantly upregulated in mds ( eisenberg et al . , 2007 2010 ) , the cell cycle kinetics of muscle progenitor cells could also be affected . another mirna that is known to regulate muscle terminal differentiation genes is mir-31 , which directly targets the 3-utr or dys consequently inactivating the dys in normal muscle , mir-31 activity is detected in early myoblasts to suppress precocious expression of late differentiation markers , while in dystrophic muscles mir-31 levels are elevated probably due to activation of the regenerative program in differentiating satellite cells and myoblasts . increased mir-31 levels in these cells subsequently reduce the amount of dys , the lack of which correlates with a differentiation delay ( greco et al . importantly , in dystrophic conditions , when dys synthesis is rescued through exon skipping , inhibition of mir-31 significantly improved dys production ( cacchiarelli et al . , 2011 ) . as a result , once nitrosylated , hdac2 no longer inhibits expression of select mirnas ( cacchiarelli et al . , 2010 ) , for example muscle specific mir-1 that marks differentiating myoblasts ( chen et al . , 2006 ) . mir-1 is evolutionarily conserved from invertebrates to vertebrates and via targeting another histone deacetylase , hdac4 ( chen et al . , 2006 ) multiple hdac4 transcriptionally regulated genes promote muscle differentiation , including mef2 ( lu et al . , 2000 ; insights from research in drosophila showed that mir-1 is necessary for post - mitotic growth but not establishment of muscles ( sokol and ambros , 2005 ) . mir-1 expression is regulated initially by twist , a mesoderm fate determining transcription factor , then in later stages by mef2 ( sokol and ambros , 2005 ) , a central regulator of myogenesis . this puts mir-1 in a positive feedback loop where its expression is initiated and promoted by the muscle differentiation program ( myogenic factors and dys syn nnos signaling ) and then , mir-1 acts to reassure terminal differentiation through downregulation of hdac4 , a repressor of myogenic genes . in mdx mouse and human dmd patients , when dys is absent and no signaling is perturbed , mir-1 is downregulated in differentiating myoblasts ( mccarthy and esser , 2007 ; greco et al . , 2009 ; cacchiarelli et al . , myofibers generated under such conditions are not robust and can be lost as part of the disease pathology . transcriptomic analysis carried out on the dystrophic mouse revealed an altered gene expression profile that might affect proper myofiber differentiation ( ghahramani seno et al . , 2010 ) . additionally , mir-1 targets glucose-6-phosphate dehydrogenase ( g6pd ) that controls oxidative stress by maintaining levels of oxidized and reduced glutathione , a major antioxidant ( cacchiarelli et al . , 2010 ) . since dystrophic muscle is sensitive to different stresses and has altered levels of reactive oxygen / nitrogen species ( martins et al . , 2008 ; shkryl et al . , 2009 ; kucherenko et al . , 2011 ; marrone et al . , 2011 ) , finding new ways to protect against oxidative stress is of a great significance . importantly , mirnas have been shown in many organisms to be involved in stress response ( biggar and storey , 2011 ; dorn , 2011 ) . once muscle differentiation genes start to be expressed they in turn can regulate expression of mirnas . mir-1 and mir-133 are transcribed together from a single primary transcript ( chen et al . , 2006 ) ; however , they perform antagonistic functions in muscle regeneration . for instance , during proliferation mir-133 is upregulated to target serum response factor ( srf ) , which is important during muscle differentiation ( gauthier - rouviere et al . , 1996 ; wang et al . , 2002 ; li et al . , 2005 ; chen et al . , 2006 ) . in a negative feedforward loop srf also promotes the expression of mir-133 ( chen et al . , 2006 ) . in addition , induction of mir-1/133 and mir-206 ( discussed later ) transcription is regulated by other factors that drive myogenesis including myod and mef2 ( kim et al . , 2006 ; rao et al . , 2006 ; rosenberg et al . , 2006 ) ; however , the amount of mir-133 decreases during differentiation . it is interesting that mir-133 , an enhancer of proliferation , and mir-1 , an enhancer of differentiation , are involved in temporally separated developmental processes but are transcribed together . this supports the idea that there is posttranscriptional regulation of these mirnas by an as yet unknown mechanism . for example , primary mirna ( pri - mirna ) could be processed by auxiliary factors . it has been shown that an rna processing protein ( hnrnp a1 ) promotes production of one mirna over the other members of the cluster via binding to the terminal loop of the pri - mirna ( michlewski et al . , 2008 ) . despite initial beliefs that the terminal loops of pri - mirnas are unimportant , a large number of pri - mirnas including transcripts of mir-1 and mir-133 have conserved terminal loops , which suggests that mir-1/133 in muscle could be regulated posttranscriptionally . nonetheless , similar to mir-1 , in dystrophic muscle mir-133 is downregulated ( mccarthy and esser , 2007 ; cacchiarelli et al . , 2010 ) supporting that proliferation and differentiation of satellite cells are disrupted in dmd patients . when early muscle transcription factors start to be expressed they promote expression of several mirnas , which in turn fine - tune signals required to reprogram cells to differentiate and fuse into myotubes . skeletal muscle specific mir-206 is upregulated in activated myoblasts to aid differentiation and halt proliferation via cell cycle arrest ( kim et al . , 2006 ) . this is accomplished by inhibiting dna synthesis by targeting pola1 , a dna polymerase ( kim et al . , 2006 ) . additionally , mir-206 targets the satellite cell self renewal factor , pax7 ( cacchiarelli et al . , 2010 ) , and the muscle differentiation inhibitor hdac4 ( williams et al . , 2009 ) . though mir-206 can be positively regulated by muscle differentiation genes similar to mir-1/133 , in dystrophic muscle the levels of this mirna are higher than normal ( mccarthy et al . interestingly , utrophin , a dys homolog , which due to a compensatory mechanism is upregulated in the mdx mouse , is another target of mir-206 ( rosenberg et al . , 2006 ) . however , increased levels of mir-206 as a part of the dmd pathology do not agree with increased levels of utrophin ( mccarthy et al . , 2007 ) , suggesting that compensatory mechanisms act independent from mir-206 . unlike mir-1/133 , mir-206 is not repressed by hdac2 , but by hdac1 ( cacchiarelli et al . , 2010 ) . syn nnos pathway ; however , mir-206 contributes to the imbalance of the regeneration program in dystrophic muscle . even though different types of mds have specific mechanisms of muscle degeneration , inflammation is a hallmark symptom of dystrophic muscle . accordingly , an increase in mir-222 and mir-223 levels in dystrophic muscle is believed to be due to inflammation ( eisenberg et al . inflammation is associated with fat and fibrotic replacement of muscle fibers and degeneration ( porter et al . , 2002 ) . to elucidate the differences seen in different types of muscular disorders a comprehensive study was carried out in which muscle specimens were analyzed representing 10 different human conditions ( eisenberg et al . , 2007 ) . what was learned from this study is that ( 1 ) there is a large amount of variation in mirna levels in disease states , ( 2 ) there are shared misregulated mirnas across muscular disorders ( mir-146b and mir - mir-221 ) and ( 3 ) only a small number of mirnas are unique to an individual disorder such as dmd ( mir-95 , mir-30d , mir-486 , mir-331 , mir-193b , mir-30a_5p , mir-30e_5p , and mir-26a ) . previously it was thought that improper fate determination of satellite cell progeny generated extra fibrotic tissue in dystrophic muscle ( shefer et al . , 2004 ; however , recently it has been shown that in addition to satellite muscle progenitor cells , there are also mesenchymal progenitor cells living among myofibers that give rise to adipocytes and fibroblasts which generate fat / fibrotic tissue ( joe et al . , 2010 ; uezumi et al . , 2010 ) . these cells have been termed fibro / adipogenic progenitors ( natarajan et al . , 2010 ) . what is amazing about these cells is that they are activated during muscle regeneration similar to satellite cells , but in a non - autonomous manner receive signals from the surrounding muscle environment . if muscle regeneration is producing normal healthy muscle fibers then adipogenesis is inhibited . however , if muscle regeneration is compromised then adipogenesis proceeds and fibrotic tissue will arise in the interstitial space between myofibers ( figure 4 ) . certain proteins are indicative of fibrotic tissue such as collagen and elastin . recently it has been shown that their mrnas are targeted by the mir-29 family , which helps to modify ecm organization ( van rooij et al . , 2008 ) . mir-29 is an enhancer of differentiation and is repressed in myoblasts by yin yang 1 ( yy1 ) , a protein that is positively regulated by nf-b and is a member of the polycomb group that acts as a transcriptional silencer ( wang et al . , 2008 ) . during myogenesis expression of mir-29 is upregulated by srf and mef2 , and in a self regulatory manner , mir-29 suppresses yy1 and hdac4 by targeting their 3-utrs ( wang et al . , 2008 ; li et al . , 2009 ) . nnos pathway where , similar to mir-1 , nitrosylation of hdac2 allows for its transcription ( cacchiarelli et al . , 2010 ) . in mdx mouse and human dmd patients there is therefore an expected decrease in mir-29 levels which promotes an increase in fibrotic tissue ( eisenberg et al . if muscle is regenerating normally then fibro / adipogenic progenitor cells will receive a non - cell autonomous signal from the muscle environment and die . the signal from the degenerative muscle environment stimulates fibro / adipogenic progenitor cells to give rise to adipocites and fibroblasts that will differentiate into fat and fibrotic tissue . all of these findings lead to the conclusion that in dystrophic muscle the alteration in mirna levels is the result of an imbalanced regeneration program , where not only activation of satellite cell proliferation but also myoblast differentiation are impaired ( figure 3 ) . the overall result is that damaged muscle fibers do not properly regenerate resulting in fat and fibrotic tissue replacement with accompanying inflammation . each of these processes is accompanied by misregulation of certain mirnas that can be considered in a multistep treatment of these fatal neuromuscular disorders . as discussed above , mirna dysregulation contributes to a variety of md pathologies and the mirna regulatory network is disturbed in dystrophic muscle . in order to return the diseased tissue to normal mode , multiple processes have to be adjusted , and mirnas are important functional units controlling different aspects of muscle regeneration and maintenance . in addition , mirnas have been shown to be perfect players in fine - tuning different developmental processes : stem cell division and maintenance , differentiation , cell cycle control , etc ( shcherbata et al . , 2006 ; inui et al . , 2010 ) . therefore re - establishing the normal mirna levels the proteomic fingerprint can be altered by variation of mirna levels , which can be achieved in a positive or a negative manner . mirnas can be inhibited by in vivo silencing via use of chemically modified antisense nucleotides locked nucleid acids ( lna ; kumar et al . , 1998 ) , 2-o - methyl - rna oligonucleotides ( 2-o - me - rna ; matera and ward , 1993 ) , microrna sponges ( ebert et al . , 2007 ) , or cholesterol conjugated rna analogs ( antagomirs ; krutzfeldt et al . , 2005 ) . inversely mirna levels can be increased by in vivo injection or viral delivery resulting in reduced expression of upregulated disease genes ( fasanaro et al . , 2010 ; pfeifer and lehmann , 2010 ) . ( 2011 ) . in mdx mice symptoms of muscle degeneration can be reversed by the introduction of viral vectors that allow for transcriptional exon skipping that removes a non - sense mutation in the dys gene . however , these therapies have not been able to restore full muscle function due to low dys levels . dys is a target for mir-31 and any replacement of dys via genetic manipulation could potentially be inhibited by the presence of this mirna . the authors were able to significantly increase dys levels in exon skipping treated dmd myoblasts by downregulation of mir-31 using a mir-31 sponge and an anti - mir-31 lna oligonucleotide . these results set a precedent for the use of mirnas as therapeutic targets to alleviate muscular dystrophy . it is unknown at this time if there are other mrna targets of mir-31 , alteration of which would give rise to deleterious side effects of this treatment . fibrotic degeneration , which is characteristic in mdx mice , was also successfully decreased by increasing mir-29 levels ( cacchiarelli et al . , 2010 ) . electroporation of mdx muscles with mir-29 had a therapeutic effect resulting in significant reduction of collagen deposition and an increase in both collagen and elastin mrnas . these data prove that mirnas are crucial regulators of the ecm that can possibly regulate the signals sent from the muscle environment to fibro / adipogenic progenitors , and targeting / promoting such mirnas could inhibit fibrosis allowing for more successful muscle regeneration . though it seems obvious from the discussion presented thus far that dys nnos signaling plays a key role in balancing the epigenetic network of muscle regeneration , it must be considered that therapies restoring no signaling alone may not be fully curative . patients with the milder bmd in some cases are missing the domain of dys essential for dys syn nnos signaling ( wells et al . , 2003 ; lai et al . , 2009 ) . since these patients do not succumb to their illness , the malfunction of nnos signaling is still compatible with life . key muscle regenerative mirnas regulated by nnos signaling are probable therapeutic targets ; however other isolated mirnas should be given attention for disease treatment too . screening for misregulated mirnas has been done by several groups in dystrophic animal models and dmd patients which allowed to compile a comprehensive list of differentially regulated mirnas that can be clustered intro three functional categories : regeneration , degeneration , and inflammation ( eisenberg et al . , 2007 ; regenerative mirnas are upregulated during embryonic development and post ischemic regeneration ( greco et al . , 2009 ; suh and blelloch , 2011 ) , supporting their role in proliferation and differentiation of muscle cell progenitors . this indicates that functional tissue replacement is based on satellite cell populations , and for mirna - based therapies to be effective progenitor cell maintenance and division should be taken into consideration . muscle satellite cells are quiescent until they become proliferative upon response to muscle damage or heavy use . it will be of interest to determine what role mirnas have in the initial activation of satellite cells , since it has been shown that mirna signatures differ from self - renewing to quiescent stem cells and from proliferative to differentiating progenitor cells ( arnold et al . initially it was believed that the role of the dgc was to simply provide structural support at costameres and without this support the muscle cell would tear and waste away . however , that is obviously not the only avenue with which disease progresses . the dgc is right in the middle of the muscle differentiation program , since without the dys this means that formed myotubes are compromised and contribute to tissue replacement by fat and fibrosis . the plethora of mirnas implicated in the dmd pathology present a substantial and complex level of regulation that opens diverse avenues for future research and therapies . 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 .

mammalian musculature is a very robust and dynamic tissue that goes through many rounds of degeneration and regeneration in an individual s lifetime . there is a biological program that maintains muscle progenitor cells that , when activated , give rise to intermediate myoblast progeny that consequently differentiate into mature muscle cells . recent works have provided a picture of the role that micrornas ( mirnas ) play in maintaining aspects of this program . intriguingly , a subset of these mirnas is de - regulated in muscular dystrophies ( mds ) , a group of fatal inherited neuromuscular disorders that are often associated with deficiencies in the dystrophin ( dys ) complex . apparently , transcriptional expression of many of the muscle specific genes and mirnas is dependent on chromatin state regulated by the dys syn nnos pathway . this puts dystrophin at the epicenter of a highly regulated program of muscle gene expression in which mirnas help to coordinate networking between multiple phases of muscle maintenance , degeneration , and regeneration . therefore , understanding the role of mirnas in physiology of normal and diseased muscle tissue could be useful for future applications in improving the md therapies and could open new clinical perspectives .

Function of the Dystrophin Glycoprotein Complex Muscle Maintenance and Regeneration The DysSynnNOS Pathway and miRNA Levels are Reciprocally Regulated miRNAs Balance the Epigenetic Network of Dystrophic Muscle miRNAs Regulate Tissue Replacement in Compromised Dystrophic Muscle New Options for miRNAs in Muscular Dystrophy Therapies Conflict of Interest Statement

muscular dystrophies ( mds ) are a group of fatal inherited diseases ( ~30 ) , associated with progressive muscle wasting caused by muscle membrane fragility , abnormal metabolic control , increased oxidative and energetic stress and abnormal proliferation of satellite cells ( campbell , 1995 ; cohn and campbell , 2000 ; constantin et al . the most prevalent and severe duchenne md ( dmd ) and the milder becker md ( bmd ) both result from mutations in the dystrophin ( dys ) gene . dys , the largest gene in the human genome ( 1.2 mb , 79 exons ) , encodes a structural cytoplasmic protein that is the key component of the larger membrane - associated multiprotein dystrophin glycoprotein complex ( dgc ; kunkel et al . the dgc classically consists of dystrophin , dystroglycans and , sarcoglycans , , , and , sarcospan , and other proteins , such as -dystrobrevin , syntrophins 1 , 1 , 2 , 1 , 2 , calveolin-3 , growth factor receptor bound protein-2 ( grb2 ) , and neuronal nitric oxide synthase ( nnos ) have been shown to be associated with the dgc ( durbeej and campbell , 2002 ) . the dgc has multiple roles in muscle : mechanical stabilization of the muscle sarcolemma via anchoring the extracellular matrix ( ecm ) to the cytoskeleton , signal transduction between the internal and external environments of the muscle cell and provides a scaffold responsible for the membrane localization of signaling proteins ( ervasti and sonnemann , 2008 ) . the dgc , via syntrophin ( syn ) anchors a variety of signaling molecules to their functional sites at the membrane . for example , it localizes nnos at the sarcolemma consequently regulating intramuscular generation of nitric oxide ( no ; wehling et al . nnos signaling nitrosylates the histone deacetylases ( hdacs ) that regulate gene transcription in muscle progenitor cells by controlling the activity of myogenic ( myod , myf5 , myogenin , and mrf4 ) and mef2 family proteins ( mckinsey et al . , 1995 ) , it indirectly plays a role in control of the balance between acetylation and deacetylation of muscle differentiation genes and has an impact on the regenerative potential of muscle stem cells . it is not clear if the dgc is just a scaffold for syn nnos or , since it has been shown that the dgc is also a signal - transducing module involved in cross talk between the internal and external environments of the muscle cell ( moore and winder , 2010 ) , it can also actively control epigenetic characteristics of muscle cells via the nnos signaling pathway . muscles can withstand rigorous mechanical stress and , if damaged , be repaired by the progeny of satellite cells ( figure 1 ) . satellite cells are quiescent muscle progenitor cells that reside along the basal lamina of the muscle fiber and proliferate only in response to specific signals , for example muscle injury ( charge and rudnicki , 2004 ) . these myotubes then cluster into myofibers which again cluster to form muscle cells or myocytes . this program must be tightly regulated to ensure that muscle is replenished as necessary and that satellite cells are not lost in an untimely manner ( buckingham , 2006 ) . in order for a cell to be a myocyte it must express specific transcription factors , including mef2 , myod , and myogenin among others ( sabourin and rudnicki , 2000 ) . before myoblasts express these factors they are in a proliferative state that requires repression of terminal differentiation genes and cell cycle inhibitors . in order for differentiation of myoblasts to occur , this requires the coordinated agreement of cell cycle inhibition and initiation of muscle specific gene expression . in dystrophic patients , dys insufficiency causes the muscle sarcolemma to become fragile leading to damage that can not be easily repaired due to inadequate response of satellite cells . this results in chronic inflammation of muscle tissue by penetration of immune cells and eventually leads to the replacement of myofibers with adipose and fibrotic tissue ( porter et al . age - dependent muscle degeneration in md patients is associated with defects in multiple processes that occur in different cell types such as activation of satellite cell proliferation , progenitor cell maintenance , myoblast differentiation , muscle cell homeostasis , immune cell recruitment , etc . in this review we will discuss how recent studies have also revealed a new level of regulation of muscle gene expression that is mediated by mirnas . since mirnas have great potential for therapeutics , understanding their basic biology and specific functions in healthy and diseased muscle tissue is of great importance . nnos pathway regulates the expression of mirnas required for muscle tissue maintenance and regeneration ( cacchiarelli et al . , 2010 indeed , it has been found that mrna levels are altered in the human dmd pathology , which correlates with mis - expressed mirnas ( eisenberg et al . interestingly it was found that most of the mis - expressed mirnas found in dmd patients are returned to normal expression levels when hdacs were inhibited by restoring no signaling ( cacchiarelli et al . nnos signaling regulates the epigenetic profile of muscle cells via nitrosylation of hdac2 that influences gene expression by altering the acetylation status of histones . mir-1 and mir-133 are transcriptionally controlled by the nitrosylation state of hdac2 , which is regulated by nnos . mir-133 targets srf during proliferation , which in a self regulatory manner promotes expression of mir-133 . mir-1 targets g6pd and hdac4 during differentiation , and since hdac4 regulates the transcription of muscle differentiation genes , dys syn nnos signaling indirectly promotes differentiation . in a self - promoting regulatory loop , the expression of muscle differentiation genes results in mir-1 expression . the skeletal muscle specific mirna , mir-206 is also upregulated by muscle gene expression and aids in cell cycle inhibition by targeting dna pola1 and pax7 . in dystrophic muscle when dys is absent , dys syn nnos signaling is disrupted inhibiting nitrosylation of hdac2 . the regulatory roles of mir-133/mir-1 are disturbed leading to inhibition of muscle differentiation gene expression and an increase in oxidative cellular stress . the decrease in mir-29 levels correlates with an increase in the amount of collagen and elastin that are indicative of fat and fibrotic tissue . due to reasons that are not quite clear at this time , levels of mir-206 and mir-31 are increased altering the equilibrium between the proliferation and differentiation states . mirnas circled in green are upregulated and mirnas circled in red are down regulated , bold letters indicate upregulated protein levels , gray circles with diagonal lines and gray lines indicate deficiencies . nnos pathway possibly by targeting the 3-utr of 1-syntrophin ( de arcangelis et al . mir-222 has the same seed sequence as mir-221 , both are controlled by the ras mapk pathway and are downregulated upon differentiation of myoblasts ( cardinali et al . additionally , mir-221 and mir-222 promote cell cycle progression by down - regulating the cyclin - dependent kinase inhibitors p27 ( cdkn1b / kip1 ) and p57 ( cdkn1c / kip2 ) that are essential for the maintenance of the proliferative state ( le sage et al . , 2007 2010 ) , the cell cycle kinetics of muscle progenitor cells could also be affected . another mirna that is known to regulate muscle terminal differentiation genes is mir-31 , which directly targets the 3-utr or dys consequently inactivating the dys in normal muscle , mir-31 activity is detected in early myoblasts to suppress precocious expression of late differentiation markers , while in dystrophic muscles mir-31 levels are elevated probably due to activation of the regenerative program in differentiating satellite cells and myoblasts . importantly , in dystrophic conditions , when dys synthesis is rescued through exon skipping , inhibition of mir-31 significantly improved dys production ( cacchiarelli et al . , 2010 ) , for example muscle specific mir-1 that marks differentiating myoblasts ( chen et al . mir-1 expression is regulated initially by twist , a mesoderm fate determining transcription factor , then in later stages by mef2 ( sokol and ambros , 2005 ) , a central regulator of myogenesis . this puts mir-1 in a positive feedback loop where its expression is initiated and promoted by the muscle differentiation program ( myogenic factors and dys syn nnos signaling ) and then , mir-1 acts to reassure terminal differentiation through downregulation of hdac4 , a repressor of myogenic genes . in mdx mouse and human dmd patients , when dys is absent and no signaling is perturbed , mir-1 is downregulated in differentiating myoblasts ( mccarthy and esser , 2007 ; greco et al . , myofibers generated under such conditions are not robust and can be lost as part of the disease pathology . transcriptomic analysis carried out on the dystrophic mouse revealed an altered gene expression profile that might affect proper myofiber differentiation ( ghahramani seno et al . additionally , mir-1 targets glucose-6-phosphate dehydrogenase ( g6pd ) that controls oxidative stress by maintaining levels of oxidized and reduced glutathione , a major antioxidant ( cacchiarelli et al . , 2011 ) , finding new ways to protect against oxidative stress is of a great significance . once muscle differentiation genes start to be expressed they in turn can regulate expression of mirnas . in a negative feedforward loop srf also promotes the expression of mir-133 ( chen et al . in addition , induction of mir-1/133 and mir-206 ( discussed later ) transcription is regulated by other factors that drive myogenesis including myod and mef2 ( kim et al . it is interesting that mir-133 , an enhancer of proliferation , and mir-1 , an enhancer of differentiation , are involved in temporally separated developmental processes but are transcribed together . this supports the idea that there is posttranscriptional regulation of these mirnas by an as yet unknown mechanism . for example , primary mirna ( pri - mirna ) could be processed by auxiliary factors . it has been shown that an rna processing protein ( hnrnp a1 ) promotes production of one mirna over the other members of the cluster via binding to the terminal loop of the pri - mirna ( michlewski et al . despite initial beliefs that the terminal loops of pri - mirnas are unimportant , a large number of pri - mirnas including transcripts of mir-1 and mir-133 have conserved terminal loops , which suggests that mir-1/133 in muscle could be regulated posttranscriptionally . when early muscle transcription factors start to be expressed they promote expression of several mirnas , which in turn fine - tune signals required to reprogram cells to differentiate and fuse into myotubes . skeletal muscle specific mir-206 is upregulated in activated myoblasts to aid differentiation and halt proliferation via cell cycle arrest ( kim et al . this is accomplished by inhibiting dna synthesis by targeting pola1 , a dna polymerase ( kim et al . , 2010 ) , and the muscle differentiation inhibitor hdac4 ( williams et al . though mir-206 can be positively regulated by muscle differentiation genes similar to mir-1/133 , in dystrophic muscle the levels of this mirna are higher than normal ( mccarthy et al . interestingly , utrophin , a dys homolog , which due to a compensatory mechanism is upregulated in the mdx mouse , is another target of mir-206 ( rosenberg et al . however , increased levels of mir-206 as a part of the dmd pathology do not agree with increased levels of utrophin ( mccarthy et al . , 2007 ) , suggesting that compensatory mechanisms act independent from mir-206 . syn nnos pathway ; however , mir-206 contributes to the imbalance of the regeneration program in dystrophic muscle . even though different types of mds have specific mechanisms of muscle degeneration , inflammation is a hallmark symptom of dystrophic muscle . inflammation is associated with fat and fibrotic replacement of muscle fibers and degeneration ( porter et al . to elucidate the differences seen in different types of muscular disorders a comprehensive study was carried out in which muscle specimens were analyzed representing 10 different human conditions ( eisenberg et al . what was learned from this study is that ( 1 ) there is a large amount of variation in mirna levels in disease states , ( 2 ) there are shared misregulated mirnas across muscular disorders ( mir-146b and mir - mir-221 ) and ( 3 ) only a small number of mirnas are unique to an individual disorder such as dmd ( mir-95 , mir-30d , mir-486 , mir-331 , mir-193b , mir-30a_5p , mir-30e_5p , and mir-26a ) . , 2004 ; however , recently it has been shown that in addition to satellite muscle progenitor cells , there are also mesenchymal progenitor cells living among myofibers that give rise to adipocytes and fibroblasts which generate fat / fibrotic tissue ( joe et al . however , if muscle regeneration is compromised then adipogenesis proceeds and fibrotic tissue will arise in the interstitial space between myofibers ( figure 4 ) . recently it has been shown that their mrnas are targeted by the mir-29 family , which helps to modify ecm organization ( van rooij et al . mir-29 is an enhancer of differentiation and is repressed in myoblasts by yin yang 1 ( yy1 ) , a protein that is positively regulated by nf-b and is a member of the polycomb group that acts as a transcriptional silencer ( wang et al . during myogenesis expression of mir-29 is upregulated by srf and mef2 , and in a self regulatory manner , mir-29 suppresses yy1 and hdac4 by targeting their 3-utrs ( wang et al . nnos pathway where , similar to mir-1 , nitrosylation of hdac2 allows for its transcription ( cacchiarelli et al . in mdx mouse and human dmd patients there is therefore an expected decrease in mir-29 levels which promotes an increase in fibrotic tissue ( eisenberg et al . if muscle is regenerating normally then fibro / adipogenic progenitor cells will receive a non - cell autonomous signal from the muscle environment and die . the signal from the degenerative muscle environment stimulates fibro / adipogenic progenitor cells to give rise to adipocites and fibroblasts that will differentiate into fat and fibrotic tissue . all of these findings lead to the conclusion that in dystrophic muscle the alteration in mirna levels is the result of an imbalanced regeneration program , where not only activation of satellite cell proliferation but also myoblast differentiation are impaired ( figure 3 ) . each of these processes is accompanied by misregulation of certain mirnas that can be considered in a multistep treatment of these fatal neuromuscular disorders . in order to return the diseased tissue to normal mode , multiple processes have to be adjusted , and mirnas are important functional units controlling different aspects of muscle regeneration and maintenance . in addition , mirnas have been shown to be perfect players in fine - tuning different developmental processes : stem cell division and maintenance , differentiation , cell cycle control , etc ( shcherbata et al . , 1998 ) , 2-o - methyl - rna oligonucleotides ( 2-o - me - rna ; matera and ward , 1993 ) , microrna sponges ( ebert et al . , 2007 ) , or cholesterol conjugated rna analogs ( antagomirs ; krutzfeldt et al . inversely mirna levels can be increased by in vivo injection or viral delivery resulting in reduced expression of upregulated disease genes ( fasanaro et al . in mdx mice symptoms of muscle degeneration can be reversed by the introduction of viral vectors that allow for transcriptional exon skipping that removes a non - sense mutation in the dys gene . dys is a target for mir-31 and any replacement of dys via genetic manipulation could potentially be inhibited by the presence of this mirna . these results set a precedent for the use of mirnas as therapeutic targets to alleviate muscular dystrophy . it is unknown at this time if there are other mrna targets of mir-31 , alteration of which would give rise to deleterious side effects of this treatment . fibrotic degeneration , which is characteristic in mdx mice , was also successfully decreased by increasing mir-29 levels ( cacchiarelli et al . these data prove that mirnas are crucial regulators of the ecm that can possibly regulate the signals sent from the muscle environment to fibro / adipogenic progenitors , and targeting / promoting such mirnas could inhibit fibrosis allowing for more successful muscle regeneration . though it seems obvious from the discussion presented thus far that dys nnos signaling plays a key role in balancing the epigenetic network of muscle regeneration , it must be considered that therapies restoring no signaling alone may not be fully curative . patients with the milder bmd in some cases are missing the domain of dys essential for dys syn nnos signaling ( wells et al . key muscle regenerative mirnas regulated by nnos signaling are probable therapeutic targets ; however other isolated mirnas should be given attention for disease treatment too . screening for misregulated mirnas has been done by several groups in dystrophic animal models and dmd patients which allowed to compile a comprehensive list of differentially regulated mirnas that can be clustered intro three functional categories : regeneration , degeneration , and inflammation ( eisenberg et al . , 2009 ; suh and blelloch , 2011 ) , supporting their role in proliferation and differentiation of muscle cell progenitors . this indicates that functional tissue replacement is based on satellite cell populations , and for mirna - based therapies to be effective progenitor cell maintenance and division should be taken into consideration . it will be of interest to determine what role mirnas have in the initial activation of satellite cells , since it has been shown that mirna signatures differ from self - renewing to quiescent stem cells and from proliferative to differentiating progenitor cells ( arnold et al . initially it was believed that the role of the dgc was to simply provide structural support at costameres and without this support the muscle cell would tear and waste away . the dgc is right in the middle of the muscle differentiation program , since without the dys this means that formed myotubes are compromised and contribute to tissue replacement by fat and fibrosis . the plethora of mirnas implicated in the dmd pathology present a substantial and complex level of regulation that opens diverse avenues for future research and therapies . 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 .

human retinal pigment epithelial cell line ( arpe-19 ) was obtained from atcc ( manassas , va ) . cells were grown in 1:1 mixture ( vol./vol . ) of dulbecco 's modified eagle 's and ham 's nutrient mixture f-12 medium ( dmem f-12 , gibco , carlsbad , ca ) , 10 mm non - essential amino acids , 0.37% sodium bicarbonate , 0.058% l - glutamine , 10% fetal bovine serum and antibiotics ( 100 u / ml penicillin g , 0.1 mg / ml streptomycin sulfate , 10 g / ml gentamicin , 2.5 g / ml fungizone - amphotericin b ) . rat embryonal neurosensory precursor retinal ( r28 ) cells were derived from day 6 post - natal rat retina from the laboratory of dr . gail m. seigel , buffalo , ny . r28 cells express genes characteristic of neurons , as well as functional neuronal properties . the cell line was cultured in dulbecco 's modified eagle 's medium , high glucose ( dmem high glucose , gibco , carlsbad , ca ) with 10% fetal bovine serum , 1x minimum essential medium ( mem ) , 10 mm non - essential amino acids , 0.37% sodium bicarbonate and 10 g / ml gentamicin . human microvascular endothelial cells ( hmvec ) and their tissue culture reagents were obtained from cascade biologics , inc . the cells were acquired as proliferating quaternary cultures established from cryopreserved normal human microvascular endothelial cells isolated from adult human dermis . hmvec were grown in medium 131 supplemented with microvascular growth supplement ( mvgs ) to support the plating and proliferation of cells . medium 131 is a sterile , liquid tissue culture basal medium containing the essential and non - essential amino acids , vitamins , other organic compounds , trace minerals , and inorganic salts . this medium does not contain antibiotics , antimycotics , hormones , growth factors , or proteins and is bicarbonate buffered . mvgs contains fetal bovine serum ( 5% v / v final concentration ) , hydrocortisone , recombinant human fibroblast growth factor , heparin , recombinant human epidermal growth factor , and dibutyryl cyclic amp . medium 131 not supplemented with mvgs was used as serum - free medium for hmvec . before use , the tissue culture surfaces were coated with attachment factor ( af ) , a sterile 1x solution containing 0.1% gelatin . two groups of hmvec were evaluated : ( 1 ) proliferating hmvec , maintained in culture with 50 ng / ml of recombinant human vascular endothelial growth factor ( vegf , r and d systems , inc . minneapolis , mn ) and ( 2 ) non - proliferating hmvec , maintained in culture with 5 ng / ml of vegf . all cell types were plated onto 96-well elisa plates ( becton dickinson labware , franklin lakes , nj ) for wst-1 assay at 2 10 cells / well and incubated at 37c in 5% co2 to reach the confluence . after incubating all the cell types for 24 h to reach the confluence , the media in the wells were replaced with the respective serum - free media containing 0.1% bovine serum albumin ( bsa ; sigma - aldrich , st . cells were incubated in this serum - free media to render them quiescent ( non - proliferating ) condition . arpe-19 and r28 cells were treated with four concentrations of bevacizumab : 0.125 , 0.25 , 0.50 and 1 mg / ml for 5 days . avastin is commercially available as a 100 mg or 400 mg vial containing 25 mg / ml bevacizumab . the recommended intravitreal dose of bevacizumab is 1 to 1.25 mg ( 0.04 to 0.05 ml ) of the commercially available avastin . the average normal vitreous volume is about 4 ml , and therefore , the dose to which the retinal cells are expected to be exposed after intravitreal injection of bevacizumab is 0.25 - 0.3125 mg / ml . in our study in vitro bevacizumab safety was tested at the clinical dose ( 0.25 mg / ml ) , half the clinical dose ( 0.125 mg / ml ) and 2 and 4x the clinical dose ( 0.50 and 1 mg / ml , respectively ) . these concentrations were prepared using the serial dilutions of the drug in the respective serum - free culture medium . untreated arpe-19 and r28 cells incubated in serum - free medium for 5 days and arpe-19 and r28 cells treated 5 days with 1 mg / ml of human purified immunoglobulin ( igg ) ( sigma - aldrich , st . the use of igg allows us to compare the effect of bevacizumab ( a full length humanized monoclonal antibody specific for vegf ) versus that of human igg ( a non - specific human antibody ) on the cells . proliferating and non - proliferating hmvec were treated 5 days with the same four concentrations of bevacizumab . for both hmvec groups the drug was serially diluted using medium 131 supplemented with psa solution ( cascade biologics , portland , or ) . psa solution ( penicillin , streptomycin , and amphotericin b solution ) is a sterile , antibiotic / antimycotic solution . when added to the medium , the final mixture contained 100 u / ml penicillin g , 100 g / ml streptomycin sulfate , and 0.25 g / ml amphotericin b. proliferating hmvec control groups were treated with 50 ng / ml vegf alone or with 1 mg / ml of igg for 5 days . non - proliferating hmvec used as controls were treated with 5 ng / ml vegf alone or with 1 mg / ml of igg . for all the three cell lines , the media in both treated and control wells were replaced with the respective media every day for 5 consecutive days . in order to assess mitochondrial function , mitochondrial dehydrogenase ( succinate - tetrazolium - reductase ) activity was determined using the wst-1 ( 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ) colorimetric assay ( roche diagnostics , indianapolis , in ) after 5 days of treatment with bevacizumab . ten microliters of the formazan dye was added to each well and incubated for 2 h at 37c . the absorbance was then measured at 490 nm on a multi - well spectrophotometer ( perkin elmer , downers grove , il ) . digital images at 20 magnification were taken of treated and untreated arpe-19 cultures using a leica dm irb inverted fluorescence microscopy and an optronics digital system ( meyer instruments , houston , tx ) . data was subjected to anova statistical analysis using graphpad prism 3.0 version statistics program ( graphpad software inc . , san diego , ca ) . newman - keuls multiple comparison test was used to compare the data within each experiment . error bars in the graphs represent standard error of mean ( sem ) with experiments performed in triplicate . human retinal pigment epithelial cell line ( arpe-19 ) was obtained from atcc ( manassas , va ) . cells were grown in 1:1 mixture ( vol./vol . ) of dulbecco 's modified eagle 's and ham 's nutrient mixture f-12 medium ( dmem f-12 , gibco , carlsbad , ca ) , 10 mm non - essential amino acids , 0.37% sodium bicarbonate , 0.058% l - glutamine , 10% fetal bovine serum and antibiotics ( 100 u / ml penicillin g , 0.1 mg / ml streptomycin sulfate , 10 g / ml gentamicin , 2.5 g / ml fungizone - amphotericin b ) . rat embryonal neurosensory precursor retinal ( r28 ) cells were derived from day 6 post - natal rat retina from the laboratory of dr . gail m. seigel , buffalo , ny . r28 cells express genes characteristic of neurons , as well as functional neuronal properties . the cell line was cultured in dulbecco 's modified eagle 's medium , high glucose ( dmem high glucose , gibco , carlsbad , ca ) with 10% fetal bovine serum , 1x minimum essential medium ( mem ) , 10 mm non - essential amino acids , 0.37% sodium bicarbonate and 10 g / ml gentamicin . human microvascular endothelial cells ( hmvec ) and their tissue culture reagents were obtained from cascade biologics , inc . the cells were acquired as proliferating quaternary cultures established from cryopreserved normal human microvascular endothelial cells isolated from adult human dermis . hmvec were grown in medium 131 supplemented with microvascular growth supplement ( mvgs ) to support the plating and proliferation of cells . medium 131 is a sterile , liquid tissue culture basal medium containing the essential and non - essential amino acids , vitamins , other organic compounds , trace minerals , and inorganic salts . this medium does not contain antibiotics , antimycotics , hormones , growth factors , or proteins and is bicarbonate buffered . mvgs contains fetal bovine serum ( 5% v / v final concentration ) , hydrocortisone , recombinant human fibroblast growth factor , heparin , recombinant human epidermal growth factor , and dibutyryl cyclic amp . medium 131 not supplemented with mvgs was used as serum - free medium for hmvec . before use , the tissue culture surfaces were coated with attachment factor ( af ) , a sterile 1x solution containing 0.1% gelatin . two groups of hmvec were evaluated : ( 1 ) proliferating hmvec , maintained in culture with 50 ng / ml of recombinant human vascular endothelial growth factor ( vegf , r and d systems , inc . minneapolis , mn ) and ( 2 ) non - proliferating hmvec , maintained in culture with 5 ng / ml of vegf . all cell types were plated onto 96-well elisa plates ( becton dickinson labware , franklin lakes , nj ) for wst-1 assay at 2 10 cells / well and incubated at 37c in 5% co2 to reach the confluence . after incubating all the cell types for 24 h to reach the confluence , the media in the wells were replaced with the respective serum - free media containing 0.1% bovine serum albumin ( bsa ; sigma - aldrich , st . cells were incubated in this serum - free media to render them quiescent ( non - proliferating ) condition . arpe-19 and r28 cells were treated with four concentrations of bevacizumab : 0.125 , 0.25 , 0.50 and 1 mg / ml for 5 days . avastin is commercially available as a 100 mg or 400 mg vial containing 25 mg / ml bevacizumab . the recommended intravitreal dose of bevacizumab is 1 to 1.25 mg ( 0.04 to 0.05 ml ) of the commercially available avastin . the average normal vitreous volume is about 4 ml , and therefore , the dose to which the retinal cells are expected to be exposed after intravitreal injection of bevacizumab is 0.25 - 0.3125 mg / ml . in our study in vitro bevacizumab safety was tested at the clinical dose ( 0.25 mg / ml ) , half the clinical dose ( 0.125 mg / ml ) and 2 and 4x the clinical dose ( 0.50 and 1 mg / ml , respectively ) . these concentrations were prepared using the serial dilutions of the drug in the respective serum - free culture medium . untreated arpe-19 and r28 cells incubated in serum - free medium for 5 days and arpe-19 and r28 cells treated 5 days with 1 mg / ml of human purified immunoglobulin ( igg ) ( sigma - aldrich , st . the use of igg allows us to compare the effect of bevacizumab ( a full length humanized monoclonal antibody specific for vegf ) versus that of human igg ( a non - specific human antibody ) on the cells . proliferating and non - proliferating hmvec were treated 5 days with the same four concentrations of bevacizumab . for both hmvec groups the drug was serially diluted using medium 131 supplemented with psa solution ( cascade biologics , portland , or ) . psa solution ( penicillin , streptomycin , and amphotericin b solution ) is a sterile , antibiotic / antimycotic solution . when added to the medium , the final mixture contained 100 u / ml penicillin g , 100 g / ml streptomycin sulfate , and 0.25 g / ml amphotericin b. proliferating hmvec control groups were treated with 50 ng / ml vegf alone or with 1 mg / ml of igg for 5 days . non - proliferating hmvec used as controls were treated with 5 ng / ml vegf alone or with 1 mg / ml of igg . for all the three cell lines , the media in both treated and control wells were replaced with the respective media every day for 5 consecutive days . in order to assess mitochondrial function , mitochondrial dehydrogenase ( succinate - tetrazolium - reductase ) activity was determined using the wst-1 ( 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ) colorimetric assay ( roche diagnostics , indianapolis , in ) after 5 days of treatment with bevacizumab . ten microliters of the formazan dye was added to each well and incubated for 2 h at 37c . the absorbance was then measured at 490 nm on a multi - well spectrophotometer ( perkin elmer , downers grove , il ) . digital images at 20 magnification were taken of treated and untreated arpe-19 cultures using a leica dm irb inverted fluorescence microscopy and an optronics digital system ( meyer instruments , houston , tx ) . data was subjected to anova statistical analysis using graphpad prism 3.0 version statistics program ( graphpad software inc . , san diego , ca ) . newman - keuls multiple comparison test was used to compare the data within each experiment . error bars in the graphs represent standard error of mean ( sem ) with experiments performed in triplicate . bevacizumab at doses of 0.125 , 0.25 , 0.50 and 1 mg / ml ( 1/2 , 1 , 2 and 4 the equivalent clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of arpe-19 cells after 5 days [ fig . . the mean absorbance of the untreated arpe-19 cell control group was 1.154 0.070 , while the mean of the same cells with 1 mg / ml igg was 1.248 0.026 [ fig the mean absorbance of bevacizumab- treated arpe-19 cells after 5 day drug exposure of 0.125 , 0.25 , 0.50 and 1 mg / ml were 1.147 0.023 , 1.187 0.022 , 1.131 0.047 and 1.062 0.079 , respectively . the mean absorbance of arpe-19 cells treated with bevacizumab were not significantly different from those of controls ( p > 0.05 ) . phase contrast microscopy also showed that arpe-19 cells were healthy after bevacizumab treatment [ fig . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of human retinal pigment epithelial ( arpe-19 ) cells in culture . the mean mitochondrial dehydrogenase activity of arpe-19 cells treated with bevacizumab was not significantly different ( p > 0.05 ) compared to the controls phase contrast microscopy images of arpe-19 cells after 5 days exposure to bevacizumab shows healthy cells . ( b ) igg - treated ( 1 mg / ml ) control arpe-19 cells . ( c ) arpe-19 cells treated with 1 mg / ml bevacizumab . 40 bevacizumab at doses of 0.125 and 0.25 mg / ml ( 1/2 and 1 clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of r28 cells after 5 days [ fig . 0.50 and 1 mg / ml ( 2 and 4 clinical dose ) bevacizumab significantly reduced the mitochondrial dehydrogenase activity of r28 cells after 5 days exposure . the mean absorbance of the control untreated or 1 mg / ml igg - treated r28 cells were 0.637 0.055 and 0.603 0.035 , respectively . the mean absorbance of r28 cells , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.554 0.014 ; 0.540 0.021 ; 0.472 0.015 ( p < 0.05 ) and 0.420 0.015 ( p < 0.01 ) , respectively . the mean absorbance of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab were not significantly different ( p > 0.05 ) from those of the controls . in contrast , the mean absorbance of r28 cells treated with the higher doses of 0.50 and 1 mg / ml bevacizumab were significantly different ( p < 0.05 and p < 0.01 , respectively ) when compared to the controls . d ] . increased numbers of rounded cells are observed at 0.5 and 1.0 mg / ml compared to untreated and igg controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of rat neurosensory retina ( r28 ) cells . the mean mitochondrial dehydrogenase activity of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab was not significantly different ( p > 0.05 ) than the controls but at higher doses ( 0.50 and 1 mg / ml ) was significantly different than the controls . * p < 0.05 , * * p < 0.01 phase contrast microscopy images of r28 cells after 5 days exposure to bevacizumab . increased numbers of rounded cells are observed at 0.5 and 1.0 mg / ml compared to untreated and igg controls . r28 cells treated with ( c ) 0.50 mg / ml and , ( d ) 1 mg/ ml bevacizumab . 40 all four doses of bevacizumab significantly reduced the mitochondrial dehydrogenase activity of proliferating and non - proliferating hmvec after 5 days [ figs . 5 and 6 respectively ] . the mean absorbance of untreated or 1 mg / ml igg - treated proliferating hmvec cultures wells were 0.540 0.0206 and 0.405 0.016 , respectively . the mean absorbance of proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.350 0.0096 ( p < 0.01 ) ; 0.335 0.0124 ( p < 0.01 ) ; 0.345 0.010 ( p the mean absorbance of proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < 0.001 ) from those of the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of proliferating human microvascular endothelial ( hmvec ) . the mean mitochondrial dehydrogenase activity of proliferating hmvec treated with bevacizumab was significantly different than the controls for all the doses . * * p < 0.01 , * * * p < 0.001 effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of non - proliferating hmvec . the mean mitochondrial dehydrogenase activity of non - proliferating hmvec treated with bevacizumab was significantly different than the controls for all the doses . * * p < 0.01 , * * * p < 0.001 in untreated or 1 mg / ml igg non - proliferating hmvec cultures , the mean absorbance were 0.405 0.008 and 0.347 0.010 , respectively . the mean absorbance of non - proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.311 0.0076 ( p < 0.01 ) ; 0.295 0.006 ( p the mean absorbance of non - proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < bevacizumab at doses of 0.125 , 0.25 , 0.50 and 1 mg / ml ( 1/2 , 1 , 2 and 4 the equivalent clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of arpe-19 cells after 5 days [ fig . . the mean absorbance of the untreated arpe-19 cell control group was 1.154 0.070 , while the mean of the same cells with 1 mg / ml igg was 1.248 0.026 [ fig the mean absorbance of bevacizumab- treated arpe-19 cells after 5 day drug exposure of 0.125 , 0.25 , 0.50 and 1 mg / ml were 1.147 0.023 , 1.187 0.022 , 1.131 0.047 and 1.062 0.079 , respectively . the mean absorbance of arpe-19 cells treated with bevacizumab were not significantly different from those of controls ( p > 0.05 ) . phase contrast microscopy also showed that arpe-19 cells were healthy after bevacizumab treatment [ fig . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of human retinal pigment epithelial ( arpe-19 ) cells in culture . the mean mitochondrial dehydrogenase activity of arpe-19 cells treated with bevacizumab was not significantly different ( p > 0.05 ) compared to the controls phase contrast microscopy images of arpe-19 cells after 5 days exposure to bevacizumab shows healthy cells . ( b ) igg - treated ( 1 mg / ml ) control arpe-19 cells . bevacizumab at doses of 0.125 and 0.25 mg / ml ( 1/2 and 1 clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of r28 cells after 5 days [ fig . however , 0.50 and 1 mg / ml ( 2 and 4 clinical dose ) bevacizumab significantly reduced the mitochondrial dehydrogenase activity of r28 cells after 5 days exposure . the mean absorbance of the control untreated or 1 mg / ml igg - treated r28 cells were 0.637 0.055 and 0.603 0.035 , respectively . the mean absorbance of r28 cells , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.554 0.014 ; 0.540 0.021 ; 0.472 0.015 ( p < 0.05 ) and 0.420 0.015 ( p < 0.01 ) , respectively . the mean absorbance of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab were not significantly different ( p > 0.05 ) from those of the controls . in contrast , the mean absorbance of r28 cells treated with the higher doses of 0.50 and 1 mg / ml bevacizumab were significantly different ( p < 0.05 and p < 0.01 , respectively ) when compared to the controls . d ] . increased numbers of rounded cells are observed at 0.5 and 1.0 mg / ml compared to untreated and igg controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of rat neurosensory retina ( r28 ) cells . the mean mitochondrial dehydrogenase activity of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab was not significantly different ( p > 0.05 ) than the controls but at higher doses ( 0.50 and 1 mg / ml ) was significantly different than the controls . * p < 0.05 , * * p < 0.01 phase contrast microscopy images of r28 cells after 5 days exposure to bevacizumab . increased numbers of rounded cells are observed at 0.5 and 1.0 mg / ml compared to untreated and igg controls . r28 cells treated with ( c ) 0.50 mg / ml and , ( d ) 1 mg/ ml bevacizumab . all four doses of bevacizumab significantly reduced the mitochondrial dehydrogenase activity of proliferating and non - proliferating hmvec after 5 days [ figs . 5 and 6 respectively ] . the mean absorbance of untreated or 1 mg / ml igg - treated proliferating hmvec cultures wells were 0.540 0.0206 and 0.405 0.016 , respectively . the mean absorbance of proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.350 0.0096 ( p < 0.01 ) ; 0.335 0.0124 ( p < 0.01 ) ; 0.345 0.010 ( p < 0.01 ) and 0.304 0.003 ( p the mean absorbance of proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < 0.001 ) from those of the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of proliferating human microvascular endothelial ( hmvec ) . the mean mitochondrial dehydrogenase activity of proliferating hmvec treated with bevacizumab was significantly different than the controls for all the doses . * * p < 0.01 , * * * p < 0.001 effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of non - proliferating hmvec . the mean mitochondrial dehydrogenase activity of non - proliferating hmvec treated with bevacizumab was significantly different than the controls for all the doses . * * p < 0.01 , * * * p < 0.001 in untreated or 1 mg / ml igg non - proliferating hmvec cultures , the mean absorbance were 0.405 0.008 and 0.347 0.010 , respectively . the mean absorbance of non - proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.311 0.0076 ( p < 0.01 ) ; 0.295 0.006 ( p < 0.001 ) ; 0.285 0.0089 ( p < 0.001 ) and 0.248 0.0025 ( p the mean absorbance of non - proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < since the first report in 2005 of the efficacy of intravitreal bevacizumab by philip rosenfeld , the clinical utilization of the drug has become widespread . however , given that the drug was not originally designed for intraocular use , the normal portfolio of pre - clinical studies evaluating this important drug remains underdeveloped . the first in vitro study evaluating the toxicity of bevacizumab on retinal cells , published by our group , suggested that bevacizumab at concentrations at or above the dose normally used in clinical practice , is safe in a short term ( 24 h ) experiment determining the cell viability of the human retinal pigment epithelial , rat neurosensory retinal , and human microvascular endothelial cells 27 . another in vitro study confirmed our findings and found no bevacizumab cytotoxicity on rat ganglion cell line ( rgc5 ) , pig choroidal endothelial cells ( cec ) and arpe-19 cells following a one day drug exposure . however , after a two day incubation with 2.5 mg / ml bevacizumab ( 8 - 10 clinical dose ) , a moderate decrease in arpe-19 cell number and viability was observed . also , bevacizumab caused a dose - dependent suppression of dna synthesis in cec , but the drug had no relevant antiproliferative effect on rgc5 and arpe-19 cells at 0.8 mg / ml or less . the present study shows that all tested doses of bevacizumab ( up to 4x clinical dose ) did not significantly affect the mitochondrial function of human arpe-19 cells in culture after 5 days . , in which there was no antiproliferative effect with concentrations of 0.8 mg / ml or less bevacizumab on arpe-19 cells . however , following 2 days of drug exposure , arpe-19 cytotoxicity was detected at 10 times the clinical dose ( 2.5 mg / ml ) of bevacizumab while in contrast in this study no arpe-19 cell toxicity was observed at a longer exposure time using a lower dose of 1 mg / ml ( our maximum dose and 4 the clinical dose ) . in r28 cells , 0.125 mg / ml and 0.25 mg / ml ( 1/2 and 1 clinical dose ) bevacizumab did not significantly affect the mitochondrial function after the same incubation period . however , 0.50 and 1 mg / ml ( 2 and 4x clinical dose ) bevacizumab significantly decreased the mitochondrial function of r28 cells after 5 days . this suggests that rat neurosensory retina ( r28 ) cells are more sensitive to bevacizumab than human arpe-19 cells . in contrast , the spitzer group did not find cytotoxicity at 0.8 mg / ml ( 3 clinical dose ) or less bevacizumab when using the rgc5 cells after 2 days . this discrepancy may be due to the shorter period of drug exposure in the spitzer experiment . we also found in this study that all doses of bevacizumab significantly affected the mitochondrial function of proliferating and non - proliferating hmvec after 5 days . a dose - dependent decrease in mitochondrial dehydrogenase activity of hmvec with bevacizumab was found in this study and is similar to the findings of spitzer et al . , who reported the dose dependent inhibition of dna synthesis in cec using bevacizumab . in vitro studies on human umbilical vein endothelial cells ( huvec ) have demonstrated that bevacizumab completely blocks both proliferating and non - proliferating vegf - induced endothelial cells after a period of several days . the mechanism of action of bevacizumab is believed to be through neutralization of secreted vegf as there was no cell or complement - mediated cytotoxicity in either vegf - producing or -targeting cells . low concentrations ( 5 ng / ml ) of vegf have been shown to protect cultured human dermal microvascular endothelial cells from undergoing senescence without inducing the malignant transformation . in our study , this low concentration of vegf was used for maintaining the non - proliferating hmvec . in experimental studies in the monkeys , the half - life in vitreous of 125i - labeled full - length humanized rhumab her2 antibody ( 148 kda ) was 5.6 days , compared to 3.2 days for the 125i - labeled humanized rhumab vegf fab antibody ( 48.3 kda ) . thus , in our study , bevacizumab ( 149 kda ) induced cell toxicity was evaluated after 5 days , the approximate half - life of bevacizumab , a physiologically and pharmacokinetically relevant time - point . however , to prevent the adverse effect of serum deprivation on the cells during the 5 day experiments , media was replaced daily in all wells , treated and controls , with the respective media and the drug when appropriate . consequently , there may be a slightly higher drug exposure in this in vitro study than would be expected after an in vivo intravitreal injection . in conclusion , we found that bevacizumab , at standard clinical doses , is safe to the retinal cells in vitro . we noted an increased sensitivity to bevacizumab in the retinal neurosensory cells at supranormal doses but not in the rpe cells . finally , we noted significant effects of bevacizumab at all doses on vascular endothelial cells consistent with the selective action of this drug .

purpose : to evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial ( arpe-19 ) , rat neurosensory retinal ( r28 ) and human microvascular endothelial ( hmvec ) cells in culture.materials and methods : arpe-19 and r28 cells were treated with 0.125 , 0.25 , 0.50 and 1 mg / ml of bevacizumab . the hmvec cultures were treated with 0.125 , 0.25 , 0.50 and 1 mg / ml of bevacizumab or 1 mg / ml of immunoglobulin g ( control ) . mitochondrial function assessed by mitochondrial dehydrogenase activity ( mda ) was determined using the wst-1 assay.results:bevacizumab doses of 0.125 to 1 mg / ml for 5 days did not significantly affect the mda of arpe-19 cells . bevacizumab treatment at 0.125 and 0.25 mg / ml ( clinical dose ) did not significantly affect the mda of r28 cells ; however , 0.50 and 1 mg / ml doses significantly reduced the r28 cell mitochondrial function . all doses of bevacizumab significantly reduced the mda of proliferating and non - proliferating hmvec.conclusion:bevacizumab exposure for 5 days was safe at clinical doses in both arpe-19 and r28 retinal neurosensory cells in culture . by contrast , bevacizumab exposure at all doses show a significant dose - dependent decrease in mitochondrial activity in both the proliferating and non - proliferating hmvec in vitro . this suggests a selective action of bevacizumab on endothelial cells at clinical doses .

Materials and Methods Cell culture Exposure to bevacizumab Mitochondrial dehydrogenase activity: WST-1 assay Gross morphology: Phase contrast microscopy Statistical analysis Results ARPE-19 cells R28 Cells HMVEC Discussion

of dulbecco 's modified eagle 's and ham 's nutrient mixture f-12 medium ( dmem f-12 , gibco , carlsbad , ca ) , 10 mm non - essential amino acids , 0.37% sodium bicarbonate , 0.058% l - glutamine , 10% fetal bovine serum and antibiotics ( 100 u / ml penicillin g , 0.1 mg / ml streptomycin sulfate , 10 g / ml gentamicin , 2.5 g / ml fungizone - amphotericin b ) . human microvascular endothelial cells ( hmvec ) and their tissue culture reagents were obtained from cascade biologics , inc . minneapolis , mn ) and ( 2 ) non - proliferating hmvec , maintained in culture with 5 ng / ml of vegf . arpe-19 and r28 cells were treated with four concentrations of bevacizumab : 0.125 , 0.25 , 0.50 and 1 mg / ml for 5 days . in our study in vitro bevacizumab safety was tested at the clinical dose ( 0.25 mg / ml ) , half the clinical dose ( 0.125 mg / ml ) and 2 and 4x the clinical dose ( 0.50 and 1 mg / ml , respectively ) . untreated arpe-19 and r28 cells incubated in serum - free medium for 5 days and arpe-19 and r28 cells treated 5 days with 1 mg / ml of human purified immunoglobulin ( igg ) ( sigma - aldrich , st . proliferating and non - proliferating hmvec were treated 5 days with the same four concentrations of bevacizumab . when added to the medium , the final mixture contained 100 u / ml penicillin g , 100 g / ml streptomycin sulfate , and 0.25 g / ml amphotericin b. proliferating hmvec control groups were treated with 50 ng / ml vegf alone or with 1 mg / ml of igg for 5 days . non - proliferating hmvec used as controls were treated with 5 ng / ml vegf alone or with 1 mg / ml of igg . in order to assess mitochondrial function , mitochondrial dehydrogenase ( succinate - tetrazolium - reductase ) activity was determined using the wst-1 ( 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ) colorimetric assay ( roche diagnostics , indianapolis , in ) after 5 days of treatment with bevacizumab . human retinal pigment epithelial cell line ( arpe-19 ) was obtained from atcc ( manassas , va ) . human microvascular endothelial cells ( hmvec ) and their tissue culture reagents were obtained from cascade biologics , inc . minneapolis , mn ) and ( 2 ) non - proliferating hmvec , maintained in culture with 5 ng / ml of vegf . arpe-19 and r28 cells were treated with four concentrations of bevacizumab : 0.125 , 0.25 , 0.50 and 1 mg / ml for 5 days . in our study in vitro bevacizumab safety was tested at the clinical dose ( 0.25 mg / ml ) , half the clinical dose ( 0.125 mg / ml ) and 2 and 4x the clinical dose ( 0.50 and 1 mg / ml , respectively ) . untreated arpe-19 and r28 cells incubated in serum - free medium for 5 days and arpe-19 and r28 cells treated 5 days with 1 mg / ml of human purified immunoglobulin ( igg ) ( sigma - aldrich , st . proliferating and non - proliferating hmvec were treated 5 days with the same four concentrations of bevacizumab . when added to the medium , the final mixture contained 100 u / ml penicillin g , 100 g / ml streptomycin sulfate , and 0.25 g / ml amphotericin b. proliferating hmvec control groups were treated with 50 ng / ml vegf alone or with 1 mg / ml of igg for 5 days . non - proliferating hmvec used as controls were treated with 5 ng / ml vegf alone or with 1 mg / ml of igg . in order to assess mitochondrial function , mitochondrial dehydrogenase ( succinate - tetrazolium - reductase ) activity was determined using the wst-1 ( 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2h-5-tetrazolio]-1,3-benzene disulfonate ) colorimetric assay ( roche diagnostics , indianapolis , in ) after 5 days of treatment with bevacizumab . bevacizumab at doses of 0.125 , 0.25 , 0.50 and 1 mg / ml ( 1/2 , 1 , 2 and 4 the equivalent clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of arpe-19 cells after 5 days [ fig . the mean absorbance of the untreated arpe-19 cell control group was 1.154 0.070 , while the mean of the same cells with 1 mg / ml igg was 1.248 0.026 [ fig the mean absorbance of bevacizumab- treated arpe-19 cells after 5 day drug exposure of 0.125 , 0.25 , 0.50 and 1 mg / ml were 1.147 0.023 , 1.187 0.022 , 1.131 0.047 and 1.062 0.079 , respectively . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of human retinal pigment epithelial ( arpe-19 ) cells in culture . the mean mitochondrial dehydrogenase activity of arpe-19 cells treated with bevacizumab was not significantly different ( p > 0.05 ) compared to the controls phase contrast microscopy images of arpe-19 cells after 5 days exposure to bevacizumab shows healthy cells . 40 bevacizumab at doses of 0.125 and 0.25 mg / ml ( 1/2 and 1 clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of r28 cells after 5 days [ fig . 0.50 and 1 mg / ml ( 2 and 4 clinical dose ) bevacizumab significantly reduced the mitochondrial dehydrogenase activity of r28 cells after 5 days exposure . the mean absorbance of the control untreated or 1 mg / ml igg - treated r28 cells were 0.637 0.055 and 0.603 0.035 , respectively . the mean absorbance of r28 cells , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.554 0.014 ; 0.540 0.021 ; 0.472 0.015 ( p < 0.05 ) and 0.420 0.015 ( p < 0.01 ) , respectively . the mean absorbance of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab were not significantly different ( p > 0.05 ) from those of the controls . in contrast , the mean absorbance of r28 cells treated with the higher doses of 0.50 and 1 mg / ml bevacizumab were significantly different ( p < 0.05 and p < 0.01 , respectively ) when compared to the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of rat neurosensory retina ( r28 ) cells . the mean mitochondrial dehydrogenase activity of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab was not significantly different ( p > 0.05 ) than the controls but at higher doses ( 0.50 and 1 mg / ml ) was significantly different than the controls . 40 all four doses of bevacizumab significantly reduced the mitochondrial dehydrogenase activity of proliferating and non - proliferating hmvec after 5 days [ figs . the mean absorbance of untreated or 1 mg / ml igg - treated proliferating hmvec cultures wells were 0.540 0.0206 and 0.405 0.016 , respectively . the mean absorbance of proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.350 0.0096 ( p < 0.01 ) ; 0.335 0.0124 ( p < 0.01 ) ; 0.345 0.010 ( p the mean absorbance of proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < 0.001 ) from those of the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of proliferating human microvascular endothelial ( hmvec ) . * * p < 0.01 , * * * p < 0.001 effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of non - proliferating hmvec . the mean mitochondrial dehydrogenase activity of non - proliferating hmvec treated with bevacizumab was significantly different than the controls for all the doses . * * p < 0.01 , * * * p < 0.001 in untreated or 1 mg / ml igg non - proliferating hmvec cultures , the mean absorbance were 0.405 0.008 and 0.347 0.010 , respectively . the mean absorbance of non - proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.311 0.0076 ( p < 0.01 ) ; 0.295 0.006 ( p the mean absorbance of non - proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < bevacizumab at doses of 0.125 , 0.25 , 0.50 and 1 mg / ml ( 1/2 , 1 , 2 and 4 the equivalent clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of arpe-19 cells after 5 days [ fig . the mean absorbance of the untreated arpe-19 cell control group was 1.154 0.070 , while the mean of the same cells with 1 mg / ml igg was 1.248 0.026 [ fig the mean absorbance of bevacizumab- treated arpe-19 cells after 5 day drug exposure of 0.125 , 0.25 , 0.50 and 1 mg / ml were 1.147 0.023 , 1.187 0.022 , 1.131 0.047 and 1.062 0.079 , respectively . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of human retinal pigment epithelial ( arpe-19 ) cells in culture . the mean mitochondrial dehydrogenase activity of arpe-19 cells treated with bevacizumab was not significantly different ( p > 0.05 ) compared to the controls phase contrast microscopy images of arpe-19 cells after 5 days exposure to bevacizumab shows healthy cells . bevacizumab at doses of 0.125 and 0.25 mg / ml ( 1/2 and 1 clinical dose ) did not significantly affect the mitochondrial dehydrogenase activity of r28 cells after 5 days [ fig . however , 0.50 and 1 mg / ml ( 2 and 4 clinical dose ) bevacizumab significantly reduced the mitochondrial dehydrogenase activity of r28 cells after 5 days exposure . the mean absorbance of the control untreated or 1 mg / ml igg - treated r28 cells were 0.637 0.055 and 0.603 0.035 , respectively . the mean absorbance of r28 cells , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.554 0.014 ; 0.540 0.021 ; 0.472 0.015 ( p < 0.05 ) and 0.420 0.015 ( p < 0.01 ) , respectively . the mean absorbance of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab were not significantly different ( p > 0.05 ) from those of the controls . in contrast , the mean absorbance of r28 cells treated with the higher doses of 0.50 and 1 mg / ml bevacizumab were significantly different ( p < 0.05 and p < 0.01 , respectively ) when compared to the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of rat neurosensory retina ( r28 ) cells . the mean mitochondrial dehydrogenase activity of r28 cells treated with 0.125 and 0.25 mg / ml bevacizumab was not significantly different ( p > 0.05 ) than the controls but at higher doses ( 0.50 and 1 mg / ml ) was significantly different than the controls . all four doses of bevacizumab significantly reduced the mitochondrial dehydrogenase activity of proliferating and non - proliferating hmvec after 5 days [ figs . the mean absorbance of untreated or 1 mg / ml igg - treated proliferating hmvec cultures wells were 0.540 0.0206 and 0.405 0.016 , respectively . the mean absorbance of proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.350 0.0096 ( p < 0.01 ) ; 0.335 0.0124 ( p < 0.01 ) ; 0.345 0.010 ( p < 0.01 ) and 0.304 0.003 ( p the mean absorbance of proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < 0.001 ) from those of the controls . effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of proliferating human microvascular endothelial ( hmvec ) . * * p < 0.01 , * * * p < 0.001 effect of 5 days exposure of bevacizumab at 0.125 , 0.25 , 0.50 and 1 mg / ml on the mitochondrial dehydrogenase activity of non - proliferating hmvec . * * p < 0.01 , * * * p < 0.001 in untreated or 1 mg / ml igg non - proliferating hmvec cultures , the mean absorbance were 0.405 0.008 and 0.347 0.010 , respectively . the mean absorbance of non - proliferating hmvec , after exposure to bevacizumab concentrations of 0.125 , 0.25 , 0.50 and 1 mg / ml were 0.311 0.0076 ( p < 0.01 ) ; 0.295 0.006 ( p < 0.001 ) ; 0.285 0.0089 ( p < 0.001 ) and 0.248 0.0025 ( p the mean absorbance of non - proliferating hmvec treated with bevacizumab were significantly different ( p < 0.01 , p < since the first report in 2005 of the efficacy of intravitreal bevacizumab by philip rosenfeld , the clinical utilization of the drug has become widespread . the first in vitro study evaluating the toxicity of bevacizumab on retinal cells , published by our group , suggested that bevacizumab at concentrations at or above the dose normally used in clinical practice , is safe in a short term ( 24 h ) experiment determining the cell viability of the human retinal pigment epithelial , rat neurosensory retinal , and human microvascular endothelial cells 27 . however , after a two day incubation with 2.5 mg / ml bevacizumab ( 8 - 10 clinical dose ) , a moderate decrease in arpe-19 cell number and viability was observed . also , bevacizumab caused a dose - dependent suppression of dna synthesis in cec , but the drug had no relevant antiproliferative effect on rgc5 and arpe-19 cells at 0.8 mg / ml or less . the present study shows that all tested doses of bevacizumab ( up to 4x clinical dose ) did not significantly affect the mitochondrial function of human arpe-19 cells in culture after 5 days . however , following 2 days of drug exposure , arpe-19 cytotoxicity was detected at 10 times the clinical dose ( 2.5 mg / ml ) of bevacizumab while in contrast in this study no arpe-19 cell toxicity was observed at a longer exposure time using a lower dose of 1 mg / ml ( our maximum dose and 4 the clinical dose ) . in r28 cells , 0.125 mg / ml and 0.25 mg / ml ( 1/2 and 1 clinical dose ) bevacizumab did not significantly affect the mitochondrial function after the same incubation period . however , 0.50 and 1 mg / ml ( 2 and 4x clinical dose ) bevacizumab significantly decreased the mitochondrial function of r28 cells after 5 days . this suggests that rat neurosensory retina ( r28 ) cells are more sensitive to bevacizumab than human arpe-19 cells . in contrast , the spitzer group did not find cytotoxicity at 0.8 mg / ml ( 3 clinical dose ) or less bevacizumab when using the rgc5 cells after 2 days . we also found in this study that all doses of bevacizumab significantly affected the mitochondrial function of proliferating and non - proliferating hmvec after 5 days . a dose - dependent decrease in mitochondrial dehydrogenase activity of hmvec with bevacizumab was found in this study and is similar to the findings of spitzer et al . in vitro studies on human umbilical vein endothelial cells ( huvec ) have demonstrated that bevacizumab completely blocks both proliferating and non - proliferating vegf - induced endothelial cells after a period of several days .

regenerative medicine has continued to gain clinical significance by research evidence from both animal and human studies , clinical trials , and clinical practice . the therapeutic rationale for regenerative medicine is that cell - based therapies have the potential to return the patient to health with respect to that condition , without further treatment . it is now well recognized that damaged mammalian tissues do repair or self - regenerate and that during gestation injured or damaged neonatal tissues can be wholly recreated.1 while this inherent capacity decreases with age and chronic injury conditions , evidence for regenerative or self - repair pathways in adult mammals remains incontrovertible with adult stem cell populations now isolated successfully from a variety of tissues in the body , including bone marrow , adipose tissue , muscle , dermis , liver , neural , olfactory , and tendon . mesenchymal stem cells ( mscs ) are the focus for the development of many cell - based therapies for a diverse array of diseases , and are a subset of nonhematopoietic adult stem cells that originate from the mesoderm and exist in almost all adult tissues.2 the relative abundance of mscs throughout the body is understandable , if most mscs are of perivascular origin.3 mscs have been demonstrated to secrete a broad range of bioactive or trophic factors that are essential in the cellular microenvironment for survival , protection , immune modulation , and differentiation effects.4 trophic factors include c granulocyte ; interleukins-6,-7,-8 , and -11 ; macrophage colony - stimulating factors ; tumor necrosis factor - alpha ; hepatocyte growth factor ; granulocyte - stimulating factors ; macrophage - stimulating factors 11 ; vascular endothelial growth factor ; nerve growth factor ; brain - derived neurotrophic factor ; adipokines ; and several others.4 experimental evidence have also demonstrated that mscs have the capabilities of differentiating into adipocytic , chondrocytic , and osteocytic lineages on suitable stimulation.5 these cellular properties may make mscs suitable for cartilage and bone repair . indeed , clinically , bone marrow- and adipose - derived tissue stem cell populations have been used with degrees of success in fracture repair,6 osteonecrotic therapy,7 cartilage repair,8 spinal fusion,9 and arthritis.10 adipose tissue is a rich source of mscs , and they are identified as adipose - derived stromal stem cells ( ascs ) . on separation of the fat cells from the adipose tissue , the remaining concentrated noncultured heterogeneous population of mononuclear cells is termed stromal vascular fraction ( svf ) cells . this term is more than 4 decades old and , as a source of stem cells , was first described by zuk et al,11 who identified msc - like cells in svf . the concentration of native ascs , defined as fibroblastoid colony - forming units , has been observed to be ~0.06% to 4% in svf , providing the therapeutic rationale for use of this cell population.1214 svf also comprises other cell types including preadipocytes , endothelial progenitor cells , adipocytes , fibroblasts , pericytes , t - regulatory cells , monocytes , lymphocytes , vascular smooth muscle cells , and m2 macrophages.15 the heterogeneous content of svf has been reported to assist the modulation and vascularization of the targeted tissue , contribute to the reduction of inflammation , and favor the re - equilibration of tolerogenic mechanisms.16,17 svf as a cell source for msc regenerative therapy has many advantages ; it contains abundant mscs , is easily extracted from the patient s own autologous adipose tissue , the tissue supply is plentiful , is harvested with a minimally invasive procedure ( by mini - liposuction aspiration ) , and is able to be replenished . several methods are available for the separation of svf from lipoaspirate including ultrasonic cavitation and collagenase . the viability and differentiation capabilities of the cells in stromed have been confirmed by the adherence , proliferation , and differentiation of cells into adipocytic , chondrocytic , and osteocytic lineages ( unpublished data ) . cell therapy treatment using autologous svf and undertaken by a medical practitioner is a procedure permitted by the therapeutic goods administration of australia ( excluded goods order 2011 ) . informed consent was obtained from each patient , and the study protocol conformed to the principles of the 1975 declaration of helsinki . osteoarthritis is a common chronic musculoskeletal disorder of the joints affecting the cartilage , ligaments , joint lining , and surrounding bones . it is caused by the progressive deterioration in the integrity of the joint structures and is associated with inflammation of the supporting cartilaginous structures within the joint capsule , and stiffness , pain , and reduction in mobility and normal function . osteoarthritis can result in a reduced quality of life ( qol ) and higher mortality rates . it can affect both the working age population and the elderly and is a leading cause of disability in the elderly . age , sex , obesity , excessive joint loading , injuries to the joint , metabolic dysfunction , and genetic and environmental factors are known risk factors.18 the incidence of osteoarthritis is increasing rapidly in parallel with the rising aging and obese populations , and is placing a burden on health care budgets . despite its debilitating effects there are , as yet , no reliable or effective treatments that can prevent osteoarthritis or halt the disease progression . clinical observation and magnetic resonance imaging ( mri ) scans of the arthritic knee show clear joint deformities , poor bone structure and alignment , cartilage wear , ligament damage , muscle atrophy , and meniscal tears . the physical and clinical histories of osteoarthritis disorders point to a multitissue , multicell involvement in the etiology of the syndrome . this suggests that a multitreatment approach that recognizes the range of the underlying causes and symptoms in the osteoarthritic syndrome is critical to treatment . current treatment options are based on combinations of patient education ; physical exercise ; nonsteroidal anti - inflammatory medication to relieve pain and inflammation ; weight control ; and , eventually , invasive total joint replacement surgery . moderate exercise has been shown to be effective in improving physical function , muscle strength , and flexibility , and in reducing joint pain and stiffness . in addition , platelet - rich plasma ( prp ) preparations are increasingly being used in different clinical scenarios as a growth factor pool to treat a range of degenerative diseases ( including cartilage lesions and knee osteoarthritis ) , although efficacy is debatable.19 therefore , given the multietiological nature of the osteoarthritis syndrome and its effect on multiple tissues and structures within the entire joint , the combination of a stromed cell - based and prp therapy , supplemented with moderate exercise , holds promise for cartilage regeneration and repair , and , importantly pain and inflammation relief , leading to a reduction in medications . a single dose of freshly prepared , noncultured svf cells ( average range 510 to 1.1510 , with > 85% viability ) was prepared from the patient s own adipose tissue and injected along with 3 ml of autologous prp into the osteoarthritic knee . case study patients also received monthly injections of prp for 4 months after the initial injection , followed by the occasional prp injection . three or six months posttreatment , patients were enrolled in a moderate exercise program for a total of 4 months . svf cells and prp were prepared as follows : the lipoaspirate procedure was performed under local anesthetic only . three millimeter puncture holes were created subcutaneously on either side of the periumbilical region ( to avoid previous surgical incisions ) . this region was injected with 2 ml of 2% lignocaine with adrenaline after cleaning with betadine antiseptic . then , 150 ml tumescent fluid ( 400 mg lignocaine , 1 mg adrenaline , and 840 mg sodium bicarbonate per 1,000 ml normal saline ) was injected subcutaneously into each of the puncture hole sites via a 21 g 10 cm needle . this process resulted in a relatively painless and bloodless liposuction using a 10 cm long and 3 mm wide cannula connected to a 25 ml syringe . the puncture hole sites were not sutured but dressed with a compression bandage to be worn for the first 24 hours . the lipoaspirate was then extracted from the four syringes and processed using the cell - innovations pty ltd standard ultrasonic cavitation proprietary protocol under sterile conditions to obtain stromed.20 the cell solution was filtered through a 100 m filter ( merck millipore , billerica , ma , usa ) and pellet resuspended in 0.9% saline . svf yield was determined using a muse facs cell counter ( merck millipore ) with a nuclear count and cell viability dye ( merck millipore ) . an autologous concentration of platelets in a small plasma volume ( prp ) , having an abundance of growth factors to promote soft and hard tissue healing , was prepared from the case study patients by centrifugation of 36 ml of blood ( per knee joint ) with anticoagulant citrate dextrose solution a at 450 g for 10 minutes . posttreatment rehabilitation comprised of a 4-month supervised progressive resistance training program incorporating specific water- and land - based exercises designed to improve physical function , strength , qol and to reduce pain levels . supervised rehabilitation sessions were performed twice per week in month 1 , three times per week in month 2 , and four times per week in months 3 and 4 . the specific exercise program was as follows : month 1 , a water - based exercise program using body weight exercises each session , twice weekly ; month 2 , the introduction of weekly land - based exercises using body weights combined with additional water - based resistance exercises , twice weekly ; month 3 , increasing land - based resistance exercises to twice weekly , while continuing with twice weekly water - based resistance exercises ; month 4 , resistance training was progressively increased for both the land- and water - based exercises . prior to treatment , the patients were administered the knee injury and osteoarthritis outcome score ( koos ) questionnaire , and then monthly for 12 months . it measures five separately scored sub - scales or clinical conditions that include pain ; symptoms ; activities of daily living ( adl ) ; function in sport and recreation ( sport / rec ) ; and knee - related qol . ( gug ) test and the stair climbing test ( sct ) for measures of balance and mobility.21 the rate of perceived exertion ( rpe ) scale ( 010 ) was used to indicate physical strain , with 10 being the most difficult and 0 being the easiest.22 a 23-year - old male patient , 100 kg weight and 1.93 m height ( body mass index [ bmi ] 26.8 ) was assessed . clinical inspection and mri initial koos subscale scores for the left knee were recorded as pain 81 ; symptoms 86 ; adl 59 ; function in sport / rec 50 , and knee - related qol 0 . the right knee koos subscale scores were pain 75 ; symptoms 79 ; adl 71 ; function in sport / rec 45 , and knee - related qol 31 . pretreatment functional assessment by sct reported a rpe of 8 out of 10 for both knees , and a gug score of 8 ( left knee ) and 0 ( right knee ) . following baseline measurements of koos , the patient was given a single dose of svf ( 7.510 cells ) and prp per knee . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . moderate exercise therapy began at 6 months posttreatment for 4 months , consisting of both water- and land - based exercises . monthly assessment of the patient s knee and associated problems were recorded by use of the koos ( table 1 ) , sct ( figure 1 ) , and gug test ( figure 2 ) . at final follow - up , the patient improved to the maximum of a 100 koos score in all five subscales ( left knee ) and a 96 score for the right knee in all five subscales of pain , symptoms , adl , sport / rec function , and knee - related qol . the patient completely regained normal functional activity for both knees as measured by the rpe scale for the sct ( figure 1 ) , and for the left knee by the gug test ( figure 2 ) by 12 months . a 59-year - old female , 85 kg weight and 1.65 m in height ( bmi 31.2 ) , was assessed . clinical examination and mri confirmed osteoarthritic disorder of both knees , with a pretreatment , functional assessment of 7 for both knees by the sct . the gug test documented a rpe of 6 for both knees . the pretreatment koos subscale scores for both knees were pain 56 ; symptoms 25 ; adl 74 ; function in sport / rec 15 , and knee - related qol 50 . the patient was given a single dose of svf ( 5.010 ) cells in addition to 3 ml of prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . six months posttreatment the patient began an exercise program consisting of water- and land - based exercises . koos and functional test scores were monitored monthly to determine the effectiveness of the combined svf cells , prp , and moderate exercise therapy . twelve months posttreatment the left and right knees improved in all five subscales ( table 2 ) . the left knee scored 100 in all subscales , and the right knee improved to pain 97 ; symptoms 86 ; adl 100 ; sport / rec function 85 , and knee - related qol 94 . functional assessment on the rpe by the sct and gug test indicated a normal score of 0 for both knees at 12 months ( figures 1 and 2 , respectively ) . the patient was a 74-year - old male with a body weight of 94 kg and 1.78 m in height ( bmi 29.7 ) . clinical assessment showed osteoarthritic disorder of both knees with koos subscale scores of pain 28 ; symptoms 43 ; adl 34 ; function in sport / rec 10 , and knee - related qol 13 for the left knee . the right knee koos profile was pain 36 ; symptoms 43 ; adl 40 ; sport / rec function 10 , and a knee - related qol 19 . initial functional assessment by the sct recorded a rpe of 9 for both knees and 9 by the gug test ( both knees ) . following assessment , the patient was given a single dose of svf ( 10.010 ) cells obtained from the patient s own lipoaspirate and 3 ml of autologous prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . three months posttreatment the patient began a moderate exercise program consisting of water- and land - based exercises . the koos subscale scores for pain , symptoms , and adl improved to 99 for both knees ( table 3 ) . the function in sport / rec and knee - related qol improved to 60 and 94 , respectively ( both knees ) . the rpe for the sct and gug test were determined to be normal ( 0 ) at 12 months post combined treatment ( figures 1 and 2 , respectively ) . a 50-year - old female weighing 60 kg and 1.60 m height ( bmi 23.4 ) . clinical evaluation confirmed osteoarthritis of the right knee . pretreatment koos subscale scores of pain 64 ; symptoms 21 ; adl 81 ; function in sport / rec 10 , and knee - related qol 25 were reported ( table 4 ) . initial functional assessment by the sct and gug test confirmed an rpe of 8 of 5 , respectively for the right knee . the patient was given a single dose of svf ( 6.010 ) cells and 3 ml of prp to the right knee following extraction from the patient s own lipoaspirate . the patient received further injections of prp into the knee at the 2nd , 3rd , and 4th month . three months posttreatment the patient began an exercise program consisting of water- and land - based exercises . koos scores improved to normal ( 100 ) in all five subscales ( table 4 ) . measures of functional assessments using the sct and gug test indicated a normal exertion score of 0 , ( figures 1 and 2 , respectively ) . a 23-year - old male patient , 100 kg weight and 1.93 m height ( body mass index [ bmi ] 26.8 ) was assessed . clinical inspection and mri initial koos subscale scores for the left knee were recorded as pain 81 ; symptoms 86 ; adl 59 ; function in sport / rec 50 , and knee - related qol 0 . the right knee koos subscale scores were pain 75 ; symptoms 79 ; adl 71 ; function in sport / rec 45 , and knee - related qol 31 . pretreatment functional assessment by sct reported a rpe of 8 out of 10 for both knees , and a gug score of 8 ( left knee ) and 0 ( right knee ) . following baseline measurements of koos , the patient was given a single dose of svf ( 7.510 cells ) and prp per knee . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . moderate exercise therapy began at 6 months posttreatment for 4 months , consisting of both water- and land - based exercises . monthly assessment of the patient s knee and associated problems were recorded by use of the koos ( table 1 ) , sct ( figure 1 ) , and gug test ( figure 2 ) . at final follow - up , the patient improved to the maximum of a 100 koos score in all five subscales ( left knee ) and a 96 score for the right knee in all five subscales of pain , symptoms , adl , sport / rec function , and knee - related qol . the patient completely regained normal functional activity for both knees as measured by the rpe scale for the sct ( figure 1 ) , and for the left knee by the gug test ( figure 2 ) by 12 months . a 59-year - old female , 85 kg weight and 1.65 m in height ( bmi 31.2 ) , was assessed . clinical examination and mri confirmed osteoarthritic disorder of both knees , with a pretreatment , functional assessment of 7 for both knees by the sct . the pretreatment koos subscale scores for both knees were pain 56 ; symptoms 25 ; adl 74 ; function in sport / rec 15 , and knee - related qol 50 . the patient was given a single dose of svf ( 5.010 ) cells in addition to 3 ml of prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . six months posttreatment the patient began an exercise program consisting of water- and land - based exercises . koos and functional test scores were monitored monthly to determine the effectiveness of the combined svf cells , prp , and moderate exercise therapy . twelve months posttreatment the left and right knees improved in all five subscales ( table 2 ) . the left knee scored 100 in all subscales , and the right knee improved to pain 97 ; symptoms 86 ; adl 100 ; sport / rec function 85 , and knee - related qol 94 . functional assessment on the rpe by the sct and gug test indicated a normal score of 0 for both knees at 12 months ( figures 1 and 2 , respectively ) . the patient was a 74-year - old male with a body weight of 94 kg and 1.78 m in height ( bmi 29.7 ) . clinical assessment showed osteoarthritic disorder of both knees with koos subscale scores of pain 28 ; symptoms 43 ; adl 34 ; function in sport / rec 10 , and knee - related qol 13 for the left knee . the right knee koos profile was pain 36 ; symptoms 43 ; adl 40 ; sport / rec function 10 , and a knee - related qol 19 . initial functional assessment by the sct recorded a rpe of 9 for both knees and 9 by the gug test ( both knees ) . following assessment , the patient was given a single dose of svf ( 10.010 ) cells obtained from the patient s own lipoaspirate and 3 ml of autologous prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . three months posttreatment the patient began a moderate exercise program consisting of water- and land - based exercises . the koos subscale scores for pain , symptoms , and adl improved to 99 for both knees ( table 3 ) . the function in sport / rec and knee - related qol improved to 60 and 94 , respectively ( both knees ) . the rpe for the sct and gug test were determined to be normal ( 0 ) at 12 months post combined treatment ( figures 1 and 2 , respectively ) . a 50-year - old female weighing 60 kg and 1.60 m height ( bmi 23.4 ) . clinical evaluation confirmed osteoarthritis of the right knee . pretreatment koos subscale scores of pain 64 ; symptoms 21 ; adl 81 ; function in sport / rec 10 , and knee - related qol 25 were reported ( table 4 ) . initial functional assessment by the sct and gug test confirmed an rpe of 8 of 5 , respectively for the right knee . the patient was given a single dose of svf ( 6.010 ) cells and 3 ml of prp to the right knee following extraction from the patient s own lipoaspirate . the patient received further injections of prp into the knee at the 2nd , 3rd , and 4th month . three months posttreatment the patient began an exercise program consisting of water- and land - based exercises . koos scores improved to normal ( 100 ) in all five subscales ( table 4 ) . measures of functional assessments using the sct and gug test indicated a normal exertion score of 0 , ( figures 1 and 2 , respectively ) . osteoarthritis disorders affect the entire joint and result from the degradation of cartilage and subchondral bone in articulated joints . the resulting outcome for patients is pain and inflammation ( which may be severe ) , stiffness , tenderness , and in some cases effusion.23 subsequently , there is diminished functional activity , decreased qol , increased susceptibility to falls , and increased morbidity and mortality . as there are currently no cures for osteoarthritis , treatment is based on relieving symptoms and improving function . of the seven joints treated , all demonstrated an improvement to 94 in knee - related qol . six joints improved to near normal ( 96 ) for koos pain and symptoms subscales with three of the joints improving to normal ( 100 ) in all five koos subscales . all patients demonstrated improvement in all five koos subscales of > 810 points ( minimal important change ) for all joints.24 there are a number of limitations with this study , including that the changes observed wholly or partly , may be due to placebo effect . other confounding factors of the study are the relative importance of each individual component ( stromed , prp , and exercise ) to the clinical improvements , and whether only one or all of the therapies were required to obtain the benefits . it is also not possible with this study to unravel the effect ( both short - and long - term ) that posttreatment rehabilitation had on the outcome scores , with a large portion of the benefits observed prior to exercise commencement . it could be suggested that svf / prp treatment may have improved the long - term inflammatory environment , and that controlled exercise led to additional improvements in pain and activity levels through musculoskeletal readjustment and strengthening , although additional studies are required to explore this hypothesis . this case series , while encouraging , requires control clinical trials for combinatorial therapy in the treatment of knee osteoarthritis and an understanding of the structural , clinical , molecular , and cellular outcomes . this is the first reported case of using ultrasonified lipoaspirate ( stromed ) to treat osteoarthritis in a combinatorial approach utilizing prp and posttreatment rehabilitation exercise . overall , the data suggest that this approach may be effective in reducing pain and inflammation , and in restoring movements and functional activity for patients with osteoarthritis of the knee . combinational stromed therapy also demonstrated improvement in the patients knee - related qol and functional exertion . the use of stromed may have great potential to delay or even prevent future complex knee replacement surgery with all of its associated complications .

introductionknee osteoarthritis is associated with persistent joint pain , stiffness , joint deformities , ligament damage , and surrounding muscle atrophy . the complexity of the disease makes treatment difficult . there are no therapeutic drugs available to halt the disease progression , leaving patients dependent on pain medication , anti - inflammatory drugs , or invasive joint replacement surgery.case presentationsfour patients with a history of unresolved symptomatic knee osteoarthritis were investigated for the therapeutic outcome of combining an exercise rehabilitation program with intra - articular injections of autologous stromed ( ie , stromal vascular fraction cells concentrated by ultrasonic cavitation from lipoaspirate ) and platelet - rich plasma ( prp ) . the knee injury and osteoarthritis outcome score questionnaire ( koos ) was administered along with physical function tests over a 12-month period . the first patient achieved a maximum therapeutic outcome of 100 in all five koos subscales ( left knee ) , and 100 for four subscales ( right knee ) . the second patient scored 100 in all five koos subscales ( left knee ) , and greater than 84 in all subscales ( right knee ) . treatment of the third patient resulted in improved outcomes in both knees of > 93 for four koos subscales , and 60 for the function in sport and recreation subscale . the fourth patient improved to 100 in all five koos subscales . in all patients , the physical function get - up and go test and stair climbing test returned to normal ( a value of zero).conclusionthis case series indicates that improved outcomes may be obtained when autologous stromal vascular fraction ( stromed ) cell therapy is combined with traditional exercise practices and prp for osteoarthritis . of the seven joints treated : all patients scores of pain improved to > 96 ; and quality of life scores to > 93 . functional performance measures of mobility returned to normal . this simple treatment appears to be extremely effective for osteoarthritis disorders that have no drug treatment to halt disease progression .

Introduction Case presentations Case 1 Case 2 Case 3 Case 4 Conclusion

it is now well recognized that damaged mammalian tissues do repair or self - regenerate and that during gestation injured or damaged neonatal tissues can be wholly recreated.1 while this inherent capacity decreases with age and chronic injury conditions , evidence for regenerative or self - repair pathways in adult mammals remains incontrovertible with adult stem cell populations now isolated successfully from a variety of tissues in the body , including bone marrow , adipose tissue , muscle , dermis , liver , neural , olfactory , and tendon . mesenchymal stem cells ( mscs ) are the focus for the development of many cell - based therapies for a diverse array of diseases , and are a subset of nonhematopoietic adult stem cells that originate from the mesoderm and exist in almost all adult tissues.2 the relative abundance of mscs throughout the body is understandable , if most mscs are of perivascular origin.3 mscs have been demonstrated to secrete a broad range of bioactive or trophic factors that are essential in the cellular microenvironment for survival , protection , immune modulation , and differentiation effects.4 trophic factors include c granulocyte ; interleukins-6,-7,-8 , and -11 ; macrophage colony - stimulating factors ; tumor necrosis factor - alpha ; hepatocyte growth factor ; granulocyte - stimulating factors ; macrophage - stimulating factors 11 ; vascular endothelial growth factor ; nerve growth factor ; brain - derived neurotrophic factor ; adipokines ; and several others.4 experimental evidence have also demonstrated that mscs have the capabilities of differentiating into adipocytic , chondrocytic , and osteocytic lineages on suitable stimulation.5 these cellular properties may make mscs suitable for cartilage and bone repair . on separation of the fat cells from the adipose tissue , the remaining concentrated noncultured heterogeneous population of mononuclear cells is termed stromal vascular fraction ( svf ) cells . the concentration of native ascs , defined as fibroblastoid colony - forming units , has been observed to be ~0.06% to 4% in svf , providing the therapeutic rationale for use of this cell population.1214 svf also comprises other cell types including preadipocytes , endothelial progenitor cells , adipocytes , fibroblasts , pericytes , t - regulatory cells , monocytes , lymphocytes , vascular smooth muscle cells , and m2 macrophages.15 the heterogeneous content of svf has been reported to assist the modulation and vascularization of the targeted tissue , contribute to the reduction of inflammation , and favor the re - equilibration of tolerogenic mechanisms.16,17 svf as a cell source for msc regenerative therapy has many advantages ; it contains abundant mscs , is easily extracted from the patient s own autologous adipose tissue , the tissue supply is plentiful , is harvested with a minimally invasive procedure ( by mini - liposuction aspiration ) , and is able to be replenished . several methods are available for the separation of svf from lipoaspirate including ultrasonic cavitation and collagenase . the viability and differentiation capabilities of the cells in stromed have been confirmed by the adherence , proliferation , and differentiation of cells into adipocytic , chondrocytic , and osteocytic lineages ( unpublished data ) . cell therapy treatment using autologous svf and undertaken by a medical practitioner is a procedure permitted by the therapeutic goods administration of australia ( excluded goods order 2011 ) . informed consent was obtained from each patient , and the study protocol conformed to the principles of the 1975 declaration of helsinki . osteoarthritis is a common chronic musculoskeletal disorder of the joints affecting the cartilage , ligaments , joint lining , and surrounding bones . it is caused by the progressive deterioration in the integrity of the joint structures and is associated with inflammation of the supporting cartilaginous structures within the joint capsule , and stiffness , pain , and reduction in mobility and normal function . osteoarthritis can result in a reduced quality of life ( qol ) and higher mortality rates . age , sex , obesity , excessive joint loading , injuries to the joint , metabolic dysfunction , and genetic and environmental factors are known risk factors.18 the incidence of osteoarthritis is increasing rapidly in parallel with the rising aging and obese populations , and is placing a burden on health care budgets . despite its debilitating effects there are , as yet , no reliable or effective treatments that can prevent osteoarthritis or halt the disease progression . clinical observation and magnetic resonance imaging ( mri ) scans of the arthritic knee show clear joint deformities , poor bone structure and alignment , cartilage wear , ligament damage , muscle atrophy , and meniscal tears . the physical and clinical histories of osteoarthritis disorders point to a multitissue , multicell involvement in the etiology of the syndrome . this suggests that a multitreatment approach that recognizes the range of the underlying causes and symptoms in the osteoarthritic syndrome is critical to treatment . current treatment options are based on combinations of patient education ; physical exercise ; nonsteroidal anti - inflammatory medication to relieve pain and inflammation ; weight control ; and , eventually , invasive total joint replacement surgery . moderate exercise has been shown to be effective in improving physical function , muscle strength , and flexibility , and in reducing joint pain and stiffness . in addition , platelet - rich plasma ( prp ) preparations are increasingly being used in different clinical scenarios as a growth factor pool to treat a range of degenerative diseases ( including cartilage lesions and knee osteoarthritis ) , although efficacy is debatable.19 therefore , given the multietiological nature of the osteoarthritis syndrome and its effect on multiple tissues and structures within the entire joint , the combination of a stromed cell - based and prp therapy , supplemented with moderate exercise , holds promise for cartilage regeneration and repair , and , importantly pain and inflammation relief , leading to a reduction in medications . a single dose of freshly prepared , noncultured svf cells ( average range 510 to 1.1510 , with > 85% viability ) was prepared from the patient s own adipose tissue and injected along with 3 ml of autologous prp into the osteoarthritic knee . case study patients also received monthly injections of prp for 4 months after the initial injection , followed by the occasional prp injection . then , 150 ml tumescent fluid ( 400 mg lignocaine , 1 mg adrenaline , and 840 mg sodium bicarbonate per 1,000 ml normal saline ) was injected subcutaneously into each of the puncture hole sites via a 21 g 10 cm needle . the puncture hole sites were not sutured but dressed with a compression bandage to be worn for the first 24 hours . the lipoaspirate was then extracted from the four syringes and processed using the cell - innovations pty ltd standard ultrasonic cavitation proprietary protocol under sterile conditions to obtain stromed.20 the cell solution was filtered through a 100 m filter ( merck millipore , billerica , ma , usa ) and pellet resuspended in 0.9% saline . an autologous concentration of platelets in a small plasma volume ( prp ) , having an abundance of growth factors to promote soft and hard tissue healing , was prepared from the case study patients by centrifugation of 36 ml of blood ( per knee joint ) with anticoagulant citrate dextrose solution a at 450 g for 10 minutes . the specific exercise program was as follows : month 1 , a water - based exercise program using body weight exercises each session , twice weekly ; month 2 , the introduction of weekly land - based exercises using body weights combined with additional water - based resistance exercises , twice weekly ; month 3 , increasing land - based resistance exercises to twice weekly , while continuing with twice weekly water - based resistance exercises ; month 4 , resistance training was progressively increased for both the land- and water - based exercises . prior to treatment , the patients were administered the knee injury and osteoarthritis outcome score ( koos ) questionnaire , and then monthly for 12 months . it measures five separately scored sub - scales or clinical conditions that include pain ; symptoms ; activities of daily living ( adl ) ; function in sport and recreation ( sport / rec ) ; and knee - related qol . ( gug ) test and the stair climbing test ( sct ) for measures of balance and mobility.21 the rate of perceived exertion ( rpe ) scale ( 010 ) was used to indicate physical strain , with 10 being the most difficult and 0 being the easiest.22 a 23-year - old male patient , 100 kg weight and 1.93 m height ( body mass index [ bmi ] 26.8 ) was assessed . clinical inspection and mri initial koos subscale scores for the left knee were recorded as pain 81 ; symptoms 86 ; adl 59 ; function in sport / rec 50 , and knee - related qol 0 . the right knee koos subscale scores were pain 75 ; symptoms 79 ; adl 71 ; function in sport / rec 45 , and knee - related qol 31 . pretreatment functional assessment by sct reported a rpe of 8 out of 10 for both knees , and a gug score of 8 ( left knee ) and 0 ( right knee ) . following baseline measurements of koos , the patient was given a single dose of svf ( 7.510 cells ) and prp per knee . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . monthly assessment of the patient s knee and associated problems were recorded by use of the koos ( table 1 ) , sct ( figure 1 ) , and gug test ( figure 2 ) . at final follow - up , the patient improved to the maximum of a 100 koos score in all five subscales ( left knee ) and a 96 score for the right knee in all five subscales of pain , symptoms , adl , sport / rec function , and knee - related qol . the patient completely regained normal functional activity for both knees as measured by the rpe scale for the sct ( figure 1 ) , and for the left knee by the gug test ( figure 2 ) by 12 months . clinical examination and mri confirmed osteoarthritic disorder of both knees , with a pretreatment , functional assessment of 7 for both knees by the sct . the gug test documented a rpe of 6 for both knees . the pretreatment koos subscale scores for both knees were pain 56 ; symptoms 25 ; adl 74 ; function in sport / rec 15 , and knee - related qol 50 . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . six months posttreatment the patient began an exercise program consisting of water- and land - based exercises . koos and functional test scores were monitored monthly to determine the effectiveness of the combined svf cells , prp , and moderate exercise therapy . twelve months posttreatment the left and right knees improved in all five subscales ( table 2 ) . the left knee scored 100 in all subscales , and the right knee improved to pain 97 ; symptoms 86 ; adl 100 ; sport / rec function 85 , and knee - related qol 94 . the patient was a 74-year - old male with a body weight of 94 kg and 1.78 m in height ( bmi 29.7 ) . clinical assessment showed osteoarthritic disorder of both knees with koos subscale scores of pain 28 ; symptoms 43 ; adl 34 ; function in sport / rec 10 , and knee - related qol 13 for the left knee . the right knee koos profile was pain 36 ; symptoms 43 ; adl 40 ; sport / rec function 10 , and a knee - related qol 19 . following assessment , the patient was given a single dose of svf ( 10.010 ) cells obtained from the patient s own lipoaspirate and 3 ml of autologous prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . the koos subscale scores for pain , symptoms , and adl improved to 99 for both knees ( table 3 ) . the function in sport / rec and knee - related qol improved to 60 and 94 , respectively ( both knees ) . the rpe for the sct and gug test were determined to be normal ( 0 ) at 12 months post combined treatment ( figures 1 and 2 , respectively ) . clinical evaluation confirmed osteoarthritis of the right knee . pretreatment koos subscale scores of pain 64 ; symptoms 21 ; adl 81 ; function in sport / rec 10 , and knee - related qol 25 were reported ( table 4 ) . initial functional assessment by the sct and gug test confirmed an rpe of 8 of 5 , respectively for the right knee . the patient received further injections of prp into the knee at the 2nd , 3rd , and 4th month . koos scores improved to normal ( 100 ) in all five subscales ( table 4 ) . clinical inspection and mri initial koos subscale scores for the left knee were recorded as pain 81 ; symptoms 86 ; adl 59 ; function in sport / rec 50 , and knee - related qol 0 . the right knee koos subscale scores were pain 75 ; symptoms 79 ; adl 71 ; function in sport / rec 45 , and knee - related qol 31 . pretreatment functional assessment by sct reported a rpe of 8 out of 10 for both knees , and a gug score of 8 ( left knee ) and 0 ( right knee ) . following baseline measurements of koos , the patient was given a single dose of svf ( 7.510 cells ) and prp per knee . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . monthly assessment of the patient s knee and associated problems were recorded by use of the koos ( table 1 ) , sct ( figure 1 ) , and gug test ( figure 2 ) . at final follow - up , the patient improved to the maximum of a 100 koos score in all five subscales ( left knee ) and a 96 score for the right knee in all five subscales of pain , symptoms , adl , sport / rec function , and knee - related qol . the patient completely regained normal functional activity for both knees as measured by the rpe scale for the sct ( figure 1 ) , and for the left knee by the gug test ( figure 2 ) by 12 months . clinical examination and mri confirmed osteoarthritic disorder of both knees , with a pretreatment , functional assessment of 7 for both knees by the sct . the pretreatment koos subscale scores for both knees were pain 56 ; symptoms 25 ; adl 74 ; function in sport / rec 15 , and knee - related qol 50 . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . koos and functional test scores were monitored monthly to determine the effectiveness of the combined svf cells , prp , and moderate exercise therapy . twelve months posttreatment the left and right knees improved in all five subscales ( table 2 ) . the left knee scored 100 in all subscales , and the right knee improved to pain 97 ; symptoms 86 ; adl 100 ; sport / rec function 85 , and knee - related qol 94 . the patient was a 74-year - old male with a body weight of 94 kg and 1.78 m in height ( bmi 29.7 ) . clinical assessment showed osteoarthritic disorder of both knees with koos subscale scores of pain 28 ; symptoms 43 ; adl 34 ; function in sport / rec 10 , and knee - related qol 13 for the left knee . the right knee koos profile was pain 36 ; symptoms 43 ; adl 40 ; sport / rec function 10 , and a knee - related qol 19 . initial functional assessment by the sct recorded a rpe of 9 for both knees and 9 by the gug test ( both knees ) . following assessment , the patient was given a single dose of svf ( 10.010 ) cells obtained from the patient s own lipoaspirate and 3 ml of autologous prp . the patient received further injections of prp into the knees at the 2nd , 3rd , and 4th month . the koos subscale scores for pain , symptoms , and adl improved to 99 for both knees ( table 3 ) . the function in sport / rec and knee - related qol improved to 60 and 94 , respectively ( both knees ) . the rpe for the sct and gug test were determined to be normal ( 0 ) at 12 months post combined treatment ( figures 1 and 2 , respectively ) . clinical evaluation confirmed osteoarthritis of the right knee . pretreatment koos subscale scores of pain 64 ; symptoms 21 ; adl 81 ; function in sport / rec 10 , and knee - related qol 25 were reported ( table 4 ) . initial functional assessment by the sct and gug test confirmed an rpe of 8 of 5 , respectively for the right knee . the patient was given a single dose of svf ( 6.010 ) cells and 3 ml of prp to the right knee following extraction from the patient s own lipoaspirate . the patient received further injections of prp into the knee at the 2nd , 3rd , and 4th month . koos scores improved to normal ( 100 ) in all five subscales ( table 4 ) . osteoarthritis disorders affect the entire joint and result from the degradation of cartilage and subchondral bone in articulated joints . the resulting outcome for patients is pain and inflammation ( which may be severe ) , stiffness , tenderness , and in some cases effusion.23 subsequently , there is diminished functional activity , decreased qol , increased susceptibility to falls , and increased morbidity and mortality . as there are currently no cures for osteoarthritis , treatment is based on relieving symptoms and improving function . of the seven joints treated , all demonstrated an improvement to 94 in knee - related qol . six joints improved to near normal ( 96 ) for koos pain and symptoms subscales with three of the joints improving to normal ( 100 ) in all five koos subscales . all patients demonstrated improvement in all five koos subscales of > 810 points ( minimal important change ) for all joints.24 there are a number of limitations with this study , including that the changes observed wholly or partly , may be due to placebo effect . other confounding factors of the study are the relative importance of each individual component ( stromed , prp , and exercise ) to the clinical improvements , and whether only one or all of the therapies were required to obtain the benefits . it is also not possible with this study to unravel the effect ( both short - and long - term ) that posttreatment rehabilitation had on the outcome scores , with a large portion of the benefits observed prior to exercise commencement . this case series , while encouraging , requires control clinical trials for combinatorial therapy in the treatment of knee osteoarthritis and an understanding of the structural , clinical , molecular , and cellular outcomes . this is the first reported case of using ultrasonified lipoaspirate ( stromed ) to treat osteoarthritis in a combinatorial approach utilizing prp and posttreatment rehabilitation exercise . overall , the data suggest that this approach may be effective in reducing pain and inflammation , and in restoring movements and functional activity for patients with osteoarthritis of the knee .

male wistar rats ( 150200 g ) were obtained from b&k universal , sollentuna , sweden . animals were housed under a 12-h light/12-h dark cycle and had free access to water and standard rodent chow and were fasted ( 5 h ) before each experiment . rats were anesthetized with an intraperitoneal injection of sodium pentobarbital ( 5 mg/100 g body wt i.p . ) . all incubation media were prepared from pregassed ( 95% o2/5% co2 ) krebs - henseleit buffer ( khb ) containing 5 mmol / l hepes and 0.1% bsa ( radioimmunoassay grade ) . six healthy men with no known family history of metabolic disorder ( aged 39 11 years and bmi 25.8 0.8 kg / m ) volunteered for the muscle biopsy procedure . all subjects were instructed to avoid strenuous exercise for 72 h before the muscle biopsy . the study protocol was approved by the ethics committee of the karolinska institutet , and informed consent was received from all subjects before participation . open biopsies were taken from vastus lateralis muscle under local anesthesia ( 5 mg / ml mepivakain chloride ) , as previously described ( 20,11 ) . muscle strips were dissected , mounted on plexiglass clamps , and incubated in vitro in pregassed ( 95% o2 and 5% co2 ) khb in a shaking water bath at 35c . isolated rat epitrochlearis muscles were incubated in pregassed khb containing 15 mmol / l mannitol and 5 mmol / l glucose for 20 min . muscles were transferred to fresh khb and incubated with either 120 nmol / l insulin or 1 mmol muscles were then transferred to fresh khb containing 20 mmol / l mannitol and incubated for 10 min to rinse excess glucose , followed by incubation for 12 min in khb containing 5 mmol / l 3-o - methyl - d-[h]-glucose ( 800 ci / mmol ) and muscles were blotted of excess fluid , frozen in liquid nitrogen , and stored at 80c . glucose transport rate was analyzed by the accumulation of intracellular 3-o - methyl [ h ] glucose ( 21 ) . muscles were homogenized and protein phosphorylation was determined as previously described ( 22 ) by western blot analysis using anti muscles were incubated at 18c for 8 min in the presence of 1 mmol / l glucose - photolabel-15 ( gp15 ) ( 23,24 ) and then irradiated for 2 min in a rayonet photochemical reactor ( southern new england ultraviolet , branford , ct ) using 300-nm lamps . muscles were rinsed four times in ice - cold pbs and either frozen directly in liquid nitrogen or transferred for continued incubation . gp15-tagged glut4 was identified as described ( 12 ) for the similar but shorter photoaffinity labeling compound bio - lc - atb - bmpa . for internalization - trafficking time course experiments , rat epitrochlearis muscles were first incubated in khb containing 15 mmol / l mannitol and 5 mmol / l glucose at 30c for 20 min . muscles were then transferred to fresh khb and incubated with either 120 nmol / l insulin for 30 min or 1 mmol / l aicar for 50 min . insulin and aicar concentrations were maintained at these constant levels throughout the remainder of the protocol . before gp15 photoaffinity tagging of glut4 , muscles were transferred to fresh khb containing 20 mmol / l mannitol for 10 min to rinse away glucose . after irradiation , muscles were washed once in khb at 18c to remove excess gp15 label and were then transferred to fresh khb and incubated at 30c to initiate internalization of the biotin - tagged glut4 . at the specified internalization time points , 80 g / ml avidin ( pierce chemical , rockford , il ) was added for 6 min at 30c to block transporters that were still at the cell surface . muscles were rinsed four times in ice - cold pbs , frozen directly in liquid nitrogen , and stored at 80c until analysis . the maximum signal of glut4 , which internalized and escaped the surface quenching , was determined in each experiment from insulin- or aicar - stimulated samples that were returned to the basal state and allowed to internalize the biotinylated glut4 for 60 min . initial glut4 signal was determined in samples incubated without the addition of avidin . for exocytosis experiments , human vastus lateralis muscles or rat epitrochlearis muscles were incubated at 35 or 30c , respectively . after 20 min insulin - stimulated muscles were photoaffinity labeled with gp15 and subsequently washed with mes buffer ( khb with mes replacing hepes ) , ph 6.0 , for 5 min to terminate insulin stimulation . muscles were maintained in khb for 40 min to obtain maximal internalization . to begin the determination of exocytosis thereafter , muscles were transferred to khb with or without 120 nmol / l insulin or 1 mmol / l aicar for specified times . the rate of loss of the internal transporters as they became quenched by the surface avidin was determined at the indicated time points . values for net internalization of glut4 under steady - state conditions were curve fitted using a two - parameter equation : f = ( 1.0 e).exp(k.t ) + e , where f is the fraction of glut4 remaining at the surface , e is the equilibrium position for internalization , k is the net internalization rate constant , and t is time . the contributions from exocytosis ( kex ) and endocytosis ( ken ) to the progress to equilibrium were determined using the following equation : f = { kex + ken.exp[t.(kex + ken)]}/(kex + ken ) ( 11 ) . values for unidirectional exocytosis of glut4 were curve fitted to a single exponential equation : f = exp( k.t ) , where f is the fraction of glut4 remaining internal , and k is the exocytosis rate constant , and t is time . individual experiments were analyzed , and the resulting parameters were used to obtain mean and se values , which were then compared in unpaired t tests . male wistar rats ( 150200 g ) were obtained from b&k universal , sollentuna , sweden . animals were housed under a 12-h light/12-h dark cycle and had free access to water and standard rodent chow and were fasted ( 5 h ) before each experiment . rats were anesthetized with an intraperitoneal injection of sodium pentobarbital ( 5 mg/100 g body wt i.p . ) . all incubation media were prepared from pregassed ( 95% o2/5% co2 ) krebs - henseleit buffer ( khb ) containing 5 mmol / l hepes and 0.1% bsa ( radioimmunoassay grade ) . six healthy men with no known family history of metabolic disorder ( aged 39 11 years and bmi 25.8 0.8 kg / m ) volunteered for the muscle biopsy procedure . all subjects were instructed to avoid strenuous exercise for 72 h before the muscle biopsy . the study protocol was approved by the ethics committee of the karolinska institutet , and informed consent was received from all subjects before participation . open biopsies were taken from vastus lateralis muscle under local anesthesia ( 5 mg / ml mepivakain chloride ) , as previously described ( 20,11 ) . muscle strips were dissected , mounted on plexiglass clamps , and incubated in vitro in pregassed ( 95% o2 and 5% co2 ) khb in a shaking water bath at 35c . isolated rat epitrochlearis muscles were incubated in pregassed khb containing 15 mmol / l mannitol and 5 mmol / l glucose for 20 min . muscles were transferred to fresh khb and incubated with either 120 nmol / l insulin or 1 mmol / l aicar for the times specified in the figure legends . muscles were then transferred to fresh khb containing 20 mmol / l mannitol and incubated for 10 min to rinse excess glucose , followed by incubation for 12 min in khb containing 5 mmol / l 3-o - methyl - d-[h]-glucose ( 800 ci / mmol ) and muscles were blotted of excess fluid , frozen in liquid nitrogen , and stored at 80c . glucose transport rate was analyzed by the accumulation of intracellular 3-o - methyl [ h ] glucose ( 21 ) . muscles were homogenized and protein phosphorylation was determined as previously described ( 22 ) by western blot analysis using anti phospho - ser473 akt , anti muscles were incubated at 18c for 8 min in the presence of 1 mmol / l glucose - photolabel-15 ( gp15 ) ( 23,24 ) and then irradiated for 2 min in a rayonet photochemical reactor ( southern new england ultraviolet , branford , ct ) using 300-nm lamps . muscles were rinsed four times in ice - cold pbs and either frozen directly in liquid nitrogen or transferred for continued incubation . gp15-tagged glut4 was identified as described ( 12 ) for the similar but shorter photoaffinity labeling compound bio - lc - atb - bmpa . for internalization - trafficking time course experiments , rat epitrochlearis muscles were first incubated in khb containing 15 mmol / l mannitol and 5 mmol / l glucose at 30c for 20 min . muscles were then transferred to fresh khb and incubated with either 120 nmol / l insulin for 30 min or 1 mmol / l aicar for 50 min . insulin and aicar concentrations were maintained at these constant levels throughout the remainder of the protocol . before gp15 photoaffinity tagging of glut4 , muscles were transferred to fresh khb containing 20 mmol / l mannitol for 10 min to rinse away glucose . after irradiation , muscles were washed once in khb at 18c to remove excess gp15 label and were then transferred to fresh khb and incubated at 30c to initiate internalization of the biotin - tagged glut4 . at the specified internalization time points , 80 g / ml avidin ( pierce chemical , rockford , il ) was added for 6 min at 30c to block transporters that were still at the cell surface . muscles were rinsed four times in ice - cold pbs , frozen directly in liquid nitrogen , and stored at 80c until analysis . the maximum signal of glut4 , which internalized and escaped the surface quenching , was determined in each experiment from insulin- or aicar - stimulated samples that were returned to the basal state and allowed to internalize the biotinylated glut4 for 60 min . for exocytosis experiments , human vastus lateralis muscles or rat epitrochlearis muscles were incubated at 35 or 30c , respectively . after 20 min insulin - stimulated muscles were photoaffinity labeled with gp15 and subsequently washed with mes buffer ( khb with mes replacing hepes ) , ph 6.0 , for 5 min to terminate insulin stimulation . muscles were maintained in khb for 40 min to obtain maximal internalization . to begin the determination of exocytosis , 160 g / ml avidin was added for 6 min . thereafter , muscles were transferred to khb with or without 120 nmol / l insulin or 1 mmol / l aicar for specified times . the rate of loss of the internal transporters as they became quenched by the surface avidin was determined at the indicated time points . values for net internalization of glut4 under steady - state conditions were curve fitted using a two - parameter equation : f = ( 1.0 e).exp(k.t ) + e , where f is the fraction of glut4 remaining at the surface , e is the equilibrium position for internalization , k is the net internalization rate constant , and t is time . the contributions from exocytosis ( kex ) and endocytosis ( ken ) to the progress to equilibrium were determined using the following equation : f = { kex + ken.exp[t.(kex + ken)]}/(kex + ken ) ( 11 ) . values for unidirectional exocytosis of glut4 were curve fitted to a single exponential equation : f = exp( k.t ) , where f is the fraction of glut4 remaining internal , and k is the exocytosis rate constant , and t is time . individual experiments were analyzed , and the resulting parameters were used to obtain mean and se values , which were then compared in unpaired t tests . insulin stimulation of epitrochlearis muscle leads to a rapid increase in glucose transport activity that reaches an equilibrium level of stimulation three- to fourfold above basal levels within 20 min ( fig . conversely , in response to aicar , the maximum level of stimulation is slower in onset and only reaches a level of stimulation 2.5-fold above basal after 60 min . the maximum levels of stimulation by insulin and aicar are maintained at a steady state for a further 30 min . at the steady state , the stimulation of glucose transport activity by insulin is slightly higher than that induced by aicar . we assessed whether changes in the kinetics of glut4 trafficking induced by these treatments could account for the differences observed in glucose transport activity . we used an impermeable probe ( gp15 ) to tag glut4 transporters at the cell surface . gp15 consists of a glucose moiety that is recognized by the glut4 binding site and a biotin tag that can interact with avidin or streptavidin . the biotin and glucose moieties are separated by a very long spacer that allows access of avidin to the tagged transporter ( 23,24 ) . we found that the higher increase in glucose transport activity following insulin stimulation versus aicar stimulation is matched by a slightly but insignificantly higher level of steady - state glut4 labeling by gp15 ( fig . comparison of the stimulation of glucose transport and cell - surface glut4 levels by insulin and aicar in rat epitrochlearis muscle . a : transport rates were determined from measurements of 3-o - methyl - d-[h]-glucose uptake into muscle specimens incubated for the indicated times in the presence of 120 nmol / l insulin or 1 mmol / l aicar . b : levels of cell surface glut4 were determined by labeling muscle strips with gp15 photolabel following 30 min of incubation with 120 nmol / l insulin ( ) and 50 min of incubation with 1 mmol / l aicar ( ) . labeled samples were solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and then detected and quantified by blotting with glut4 antibody . results are means se of six ( insulin ) and five ( aicar ) experiments . in addition to the observed differences in the rate of onset of the maximal stimulation of glucose transport activity , insulin and aicar produce a markedly different pattern of early signaling changes in the intermediate serine kinases , akt / protein kinase b , and ampk . insulin , but not aicar , action leads to a marked stimulation of akt phosphorylation ( fig . conversely , aicar , but not insulin , action leads to a robust stimulation of ampk phosphorylation ( fig . the slight increase in ampk phosphorylation following insulin treatment is not significant over a series of experiments or when corrected for total ampk levels . the maximally rapid time frame for insulin - stimulated increase in akt phosphorylation was within 210 min , and the aicar - stimulated increase in ampk phosphorylation also occurred rapidly and reached a maximum within 30 min ( 25 ) ( h.k.r.k . this increase is more rapid than the increase to maximal levels of glucose transport activity ( fig . ampk thr172 ( b ) , and p coa carboxylase ser79 ( c ) in lysates of rat epitrochlearis and p akt ser 473 ( d ) in human vastus lateralis muscle . muscle lysates were resolved on sds - page , transferred to nitrocellulose membranes , and incubated overnight with phospho - specific antibodies . conditions are as follows : basal , initial basal ; insulin 1 , 12 nmol / l insulin stimulation for 52 min ; recovery 1 , 102-min recovery after removal of initial insulin stimulation ; insulin 2 , restimulation with 120 nmol / l insulin for 50 min after recovery ; aicar , restimulation with 1 mmol / l aicar for 50 min after recovery ; and recovery 2 , 162-min recovery after removal of initial insulin stimulation . the maintenance of steady - state and maximal levels of glucose transport activity and cell - surface glut4 content ( fig . 1 ) could be due to a pulse of glut4 translocation , which is then followed by a cessation of subsequent movement as the cell signaling processes continue . alternatively , glut4 could continue to recycle under steady - state conditions , and the cell - surface glut4 level could be maintained by a net balance of a fast exocytosis and a fast endocytosis rate of translocation . in adipocytes the rate of net internalization of gp15-tagged glut4 under steady - state stimulation by either insulin or aicar has been measured by separating the glut4 that remains at the cell surface from that which has been internalized . to do this 3a ) is reduced by the avidin addition at the beginning of the time course ( second lane in fig . 3a ) . at subsequent times of incubation , glut4 internalizes and escapes the extracellular avidin . this gives a steady rise in the internal signal from the tagged glut4 at 560 min ( fig . the steady - state insulin stimulation is associated with rapid internalization that is almost complete within 10 min . the final level of net internalization occurs when approximately half the initial cell - surface glut4 is internalized ( fig . conversely , the steady - state aicar stimulation is associated with a slower initial net internalization , particularly over the first 5 min where the fraction of internalization is lower than that occurring in insulin - stimulated muscle . additionally , the rate of progress to the equilibrium level is not complete until 3060 min ( fig . is associated with a slower rate constant for net internalization between aicar- and insulin - stimulated muscles ( 0.059 vs. 0.134 min , respectively ) . furthermore , the final equilibrium level of internalized glut4 is reached at a lower cell - surface fraction ( 0.29 ) than that associated with insulin stimulation ( 0.52 ) ( fig . 3d ) indicates that aicar treatment is associated with a slower endocytosis rate constant ( 0.042 vs. 0.068 min ) and a much slower exocytosis rate constant ( 0.017 vs. 0.067 min ) . these kinetic parameters predict that the levels of glucose transport activity and cell - surface glut4 in aicar - treated muscle will be 55% of those in insulin - treated muscle . 1 . internalization of gp15-tagged glut4 under steady - state stimulation with insulin or aicar in rat epitrochlearis muscle . muscle strips were incubated with 120 nmol / l insulin or 1 mmol / l aicar and then labeled with gp15 . the gp15-tagged glut4 was then allowed to internalize for the indicated times . to separately identify internalized glut4 , avidin was added to the medium to quench the signal from cell surface located glut4 . internal glut4 was then solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . b : by comparison of the initial glut4 signal with the signal at the indicated times , the level of cell - surface glut4 was calculated . c and d : curve fitting to data from separate experiments was used to calculate rate constants as described in research design and methods . the curves in b were computed from the average rate constants in c. results are the means se of four experiments . the steady - state data reveal important differences in the kinetics of glut4 trafficking following insulin or aicar treatment . however , the net internalization of glut4 under these conditions is a complex kinetic process that involves both endocytosis of glut4 and its recycling ( exocytosis ) . to further address whether aicar- or insulin - stimulated glucose transport and signaling activities occur by a convergent mechanism , we simplified our study design in subsequent experiments to only measure the exocytosis rate constant . to more directly measure the kinetic parameters of exocytosis , a procedure was adopted in which gp15-tagged glut4 was internalized and then the unidirectional return to the cell surface was monitored . to internalize the gp15-tagged glut4 , it was necessary to first label insulin - stimulated muscle and then reverse the stimulated state by washing with a low ph buffer that disassociates insulin from its receptor ( 28 ) . to check that this procedure is adequately effective , we monitored the reversal of akt phosphorylation ( fig . the return to basal levels of phosphorylation is incomplete ; however , it is only necessary that the reversal process is sufficient to allow a detectable portion of gp15-glut4 to be internalized , and this was found to be adequate ( fig . insulin action was then terminated by washing with a ph 6.0 buffer , and the tagged glut4 was allowed to internalize for 40 min . muscle specimens were maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . avidin was added at the cell surface throughout the restimulation time courses ; this reduced the signal for glut4 that exocytosed to the cell surface , thereby reducing the signal from internal glut4 . the remaining internal glut4 was then solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . c : curve fitting for internal glut4 was carried out for the basal , insulin , and aicar treatments and data from separate experiments were used to calculate rate constants as described in research design and methods . b : the curves were computed from the average rate constants in c. results are means se . data are derived from seven , five , and three experiments for basal , insulin , and aicar treatments , respectively . basal experiments had at least three time points , but these were varied to allow comparison with early and late time points for the insulin or aicar stimulations . * p < 0.05 . the movement of glut4 from the internal compartment to the plasma membrane was rendered unidirectional by the addition of avidin at the cell exterior throughout the time course used to study the exocytosis . as the glut4 reaches the cell surface , its signal is quenched by avidin binding . the glut4 signal that remains in the cell , therefore , represents the fraction of glut4 remaining internally , and this decreases with time ( fig . this allows curve fitting to a single exponential function even when the scatter in the data points is quite large ( fig . the scatter is an unavoidable consequence of working with single muscle samples for each time course data point . insulin treatment increases the rate constant for exocytosis sixfold ( 0.010 in the basal state to 0.067 min in the insulin - stimulated state ; p < 0.05 ) . this exocytosis rate constant is in reasonable agreement with the steady - state exocytosis rate constant in aicar - treated cells ( 0.017 min ) . a limitation of the fitting of a single exponential function is that it assumes that the internal glut4 signal decreases continuously to zero . however , it may not ( 29,30 ) . in the basal and aicar - treated cells , the release of glut4 may occur in a pulse and the internal glut4 signal may reach a plateau point at which no further release occurs . the fitting method we have employed may lead to an underestimate of the rate constant for this partial release . the more limited exocytosis response to aicar can not be due to a slow onset in ampk activation , as we have compared basal and aicar at times of exocytosis up to 120 min . furthermore , the steady - state parameters were obtained following an extensive treatment with aicar . taking into consideration both the steady - state and the direct exocytosis measurements , we can not exclude the possibility of an aicar - mediated increase in exocytosis ; however , this effect is clearly smaller , or more limited as a pulse of release of glut4 , than that occurring in insulin - treated muscle . the exocytosis approach characterized in the isolated rat epitrochlearis muscle system was applied to human muscle strips . we confirmed that the reversal procedure adopted for the rat epitrochlearis was also applicable to human muscle ( fig . a comparison of basal and insulin - treated muscle revealed clear insulin stimulation in the gp15-tagged glut4 movement out of the internal compartment and toward the cell surface ( fig . 5c ) revealed that the exocytosis rate constant was increased six- to sevenfold ( from 0.011 to 0.075 min ) . these rate - constant values and the six- to sevenfold stimulation are very similar to the changes occurring in rat epitrochlearis muscle . this suggests that the same kinetic process is present , even under conditions in which the levels of glucose transport are quite different . the six- to sevenfold increase in glucose transport activity does not occur in human muscle , but this is not necessarily inconsistent with a large change in glut4 translocation , as additional factors may influence the glucose transport activity . the effect of aicar on glut4 exocytosis in human muscle is less clear , partly because of the large scatter in data points derived from the samples available in the study ( fig . 5c ) indicated that there may be some slight , but statistically insignificant , stimulation of exocytosis above basal levels following aicar treatment of the muscle strips . the observed exocytosis rate constant is , however , identical to that derived from the steady - state exocytosis experiment in aicar - treated rat muscle . insulin action was then terminated by washing with a ph 6.0 buffer , and the tagged glut4 was allowed to internalize for 40 min . the strips were then maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . avidin was added at the cell surface throughout the restimulation time courses ; this reduced the signal for glut4 that exocytosed to the cell surface , thereby reducing the signal from internal glut4 . the remaining internal glut4 was then solubilized and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . c : curve fitting was carried out for the basal , insulin , and aicar treatments , and data from separate experiments were used to calculate rate constants as described in research design and methods . b : the curves were computed from the average rate constants in c. results are means se . where no error bar is shown , n = 2 . data are derived from four , four , and two experiments for basal , insulin , or aicar treatment , respectively . in basal experiments , time points were varied to allow comparison with early and late time points for the insulin and aicar stimulations . * insulin stimulation of epitrochlearis muscle leads to a rapid increase in glucose transport activity that reaches an equilibrium level of stimulation three- to fourfold above basal levels within 20 min ( fig . conversely , in response to aicar , the maximum level of stimulation is slower in onset and only reaches a level of stimulation 2.5-fold above basal after 60 min . the maximum levels of stimulation by insulin and aicar are maintained at a steady state for a further 30 min . at the steady state , the stimulation of glucose transport activity by insulin is slightly higher than that induced by aicar . we assessed whether changes in the kinetics of glut4 trafficking induced by these treatments could account for the differences observed in glucose transport activity . we used an impermeable probe ( gp15 ) to tag glut4 transporters at the cell surface . gp15 consists of a glucose moiety that is recognized by the glut4 binding site and a biotin tag that can interact with avidin or streptavidin . the biotin and glucose moieties are separated by a very long spacer that allows access of avidin to the tagged transporter ( 23,24 ) . we found that the higher increase in glucose transport activity following insulin stimulation versus aicar stimulation is matched by a slightly but insignificantly higher level of steady - state glut4 labeling by gp15 ( fig . comparison of the stimulation of glucose transport and cell - surface glut4 levels by insulin and aicar in rat epitrochlearis muscle . a : transport rates were determined from measurements of 3-o - methyl - d-[h]-glucose uptake into muscle specimens incubated for the indicated times in the presence of 120 nmol / l insulin or 1 mmol / l aicar . b : levels of cell surface glut4 were determined by labeling muscle strips with gp15 photolabel following 30 min of incubation with 120 nmol / l insulin ( ) and 50 min of incubation with 1 mmol / l aicar ( ) . labeled samples were solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and then detected and quantified by blotting with glut4 antibody . results are means se of six ( insulin ) and five ( aicar ) experiments . in addition to the observed differences in the rate of onset of the maximal stimulation of glucose transport activity , insulin and aicar produce a markedly different pattern of early signaling changes in the intermediate serine kinases , akt / protein kinase b , and ampk . insulin , but not aicar , action leads to a marked stimulation of akt phosphorylation ( fig . conversely , aicar , but not insulin , action leads to a robust stimulation of ampk phosphorylation ( fig . the slight increase in ampk phosphorylation following insulin treatment is not significant over a series of experiments or when corrected for total ampk levels . the maximally rapid time frame for insulin - stimulated increase in akt phosphorylation was within 210 min , and the aicar - stimulated increase in ampk phosphorylation also occurred rapidly and reached a maximum within 30 min ( 25 ) ( h.k.r.k . this increase is more rapid than the increase to maximal levels of glucose transport activity ( fig . ampk thr172 ( b ) , and p coa carboxylase ser79 ( c ) in lysates of rat epitrochlearis and p akt ser 473 ( d ) in human vastus lateralis muscle . muscle lysates were resolved on sds - page , transferred to nitrocellulose membranes , and incubated overnight with phospho - specific antibodies . conditions are as follows : basal , initial basal ; insulin 1 , 12 nmol / l insulin stimulation for 52 min ; recovery 1 , 102-min recovery after removal of initial insulin stimulation ; insulin 2 , restimulation with 120 nmol / l insulin for 50 min after recovery ; aicar , restimulation with 1 mmol / l aicar for 50 min after recovery ; and recovery 2 , 162-min recovery after removal of initial insulin stimulation . the maintenance of steady - state and maximal levels of glucose transport activity and cell - surface glut4 content ( fig . 1 ) could be due to a pulse of glut4 translocation , which is then followed by a cessation of subsequent movement as the cell signaling processes continue . alternatively , glut4 could continue to recycle under steady - state conditions , and the cell - surface glut4 level could be maintained by a net balance of a fast exocytosis and a fast endocytosis rate of translocation . in adipocytes , glut4 appears to continuously recycle during insulin - stimulated signaling ( 13,26,27 ) . the rate of net internalization of gp15-tagged glut4 under steady - state stimulation by either insulin or aicar has been measured by separating the glut4 that remains at the cell surface from that which has been internalized . to do this 3a ) is reduced by the avidin addition at the beginning of the time course ( second lane in fig . 3a ) . at subsequent times of incubation , glut4 internalizes and escapes the extracellular avidin . this gives a steady rise in the internal signal from the tagged glut4 at 560 min ( fig . the steady - state insulin stimulation is associated with rapid internalization that is almost complete within 10 min . the final level of net internalization occurs when approximately half the initial cell - surface glut4 is internalized ( fig . conversely , the steady - state aicar stimulation is associated with a slower initial net internalization , particularly over the first 5 min where the fraction of internalization is lower than that occurring in insulin - stimulated muscle . additionally , the rate of progress to the equilibrium level is not complete until 3060 min ( fig . this is associated with a slower rate constant for net internalization between aicar- and insulin - stimulated muscles ( 0.059 vs. 0.134 min , respectively ) . furthermore , the final equilibrium level of internalized glut4 is reached at a lower cell - surface fraction ( 0.29 ) than that associated with insulin stimulation ( 0.52 ) ( fig . 3d ) indicates that aicar treatment is associated with a slower endocytosis rate constant ( 0.042 vs. 0.068 min ) and a much slower exocytosis rate constant ( 0.017 vs. 0.067 min ) . these kinetic parameters predict that the levels of glucose transport activity and cell - surface glut4 in aicar - treated muscle will be 55% of those in insulin - treated muscle . 1 . internalization of gp15-tagged glut4 under steady - state stimulation with insulin or aicar in rat epitrochlearis muscle . muscle strips were incubated with 120 nmol / l insulin or 1 mmol / l aicar and then labeled with gp15 . the gp15-tagged glut4 was then allowed to internalize for the indicated times . to separately identify internalized glut4 , avidin was added to the medium to quench the signal from cell surface located glut4 . internal glut4 was then solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . b : by comparison of the initial glut4 signal with the signal at the indicated times , the level of cell - surface glut4 was calculated . c and d : curve fitting to data from separate experiments was used to calculate rate constants as described in research design and methods . the curves in b were computed from the average rate constants in c. results are the means se of four experiments . * p < 0.05 . the steady - state data reveal important differences in the kinetics of glut4 trafficking following insulin or aicar treatment . however , the net internalization of glut4 under these conditions is a complex kinetic process that involves both endocytosis of glut4 and its recycling ( exocytosis ) . to further address whether aicar- or insulin - stimulated glucose transport and signaling activities occur by a convergent mechanism , we simplified our study design in subsequent experiments to only measure the exocytosis rate constant . to more directly measure the kinetic parameters of exocytosis , a procedure was adopted in which gp15-tagged glut4 was internalized and then the unidirectional return to the cell surface was monitored . to internalize the gp15-tagged glut4 , it was necessary to first label insulin - stimulated muscle and then reverse the stimulated state by washing with a low ph buffer that disassociates insulin from its receptor ( 28 ) . to check that this procedure is adequately effective , we monitored the reversal of akt phosphorylation ( fig . the return to basal levels of phosphorylation is incomplete ; however , it is only necessary that the reversal process is sufficient to allow a detectable portion of gp15-glut4 to be internalized , and this was found to be adequate ( fig . insulin action was then terminated by washing with a ph 6.0 buffer , and the tagged glut4 was allowed to internalize for 40 min . muscle specimens were maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . avidin was added at the cell surface throughout the restimulation time courses ; this reduced the signal for glut4 that exocytosed to the cell surface , thereby reducing the signal from internal glut4 . the remaining internal glut4 was then solubilized , and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . c : curve fitting for internal glut4 was carried out for the basal , insulin , and aicar treatments and data from separate experiments were used to calculate rate constants as described in research design and methods . b : the curves were computed from the average rate constants in c. results are means se . data are derived from seven , five , and three experiments for basal , insulin , and aicar treatments , respectively . basal experiments had at least three time points , but these were varied to allow comparison with early and late time points for the insulin or aicar stimulations . * p < 0.05 . the movement of glut4 from the internal compartment to the plasma membrane was rendered unidirectional by the addition of avidin at the cell exterior throughout the time course used to study the exocytosis . as the glut4 reaches the cell surface , its signal is quenched by avidin binding . the glut4 signal that remains in the cell , therefore , represents the fraction of glut4 remaining internally , and this decreases with time ( fig . this allows curve fitting to a single exponential function even when the scatter in the data points is quite large ( fig . the scatter is an unavoidable consequence of working with single muscle samples for each time course data point . insulin treatment increases the rate constant for exocytosis sixfold ( 0.010 in the basal state to 0.067 min in the insulin - stimulated state ; p < 0.05 ) . this exocytosis rate constant is in reasonable agreement with the steady - state exocytosis rate constant in aicar - treated cells ( 0.017 min ) . a limitation of the fitting of a single exponential function is that it assumes that the internal glut4 signal decreases continuously to zero . however , it may not ( 29,30 ) . in the basal and aicar - treated cells , the release of glut4 may occur in a pulse and the internal glut4 signal may reach a plateau point at which no further release occurs . the fitting method we have employed may lead to an underestimate of the rate constant for this partial release . the more limited exocytosis response to aicar can not be due to a slow onset in ampk activation , as we have compared basal and aicar at times of exocytosis up to 120 min . furthermore , the steady - state parameters were obtained following an extensive treatment with aicar . taking into consideration both the steady - state and the direct exocytosis measurements , we can not exclude the possibility of an aicar - mediated increase in exocytosis ; however , this effect is clearly smaller , or more limited as a pulse of release of glut4 , than that occurring in insulin - treated muscle . the exocytosis approach characterized in the isolated rat epitrochlearis muscle system was applied to human muscle strips . we confirmed that the reversal procedure adopted for the rat epitrochlearis was also applicable to human muscle ( fig . a comparison of basal and insulin - treated muscle revealed clear insulin stimulation in the gp15-tagged glut4 movement out of the internal compartment and toward the cell surface ( fig . 5c ) revealed that the exocytosis rate constant was increased six- to sevenfold ( from 0.011 to 0.075 min ) . these rate - constant values and the six- to sevenfold stimulation are very similar to the changes occurring in rat epitrochlearis muscle . this suggests that the same kinetic process is present , even under conditions in which the levels of glucose transport are quite different . the six- to sevenfold increase in glucose transport activity does not occur in human muscle , but this is not necessarily inconsistent with a large change in glut4 translocation , as additional factors may influence the glucose transport activity . the effect of aicar on glut4 exocytosis in human muscle is less clear , partly because of the large scatter in data points derived from the samples available in the study ( fig . 5c ) indicated that there may be some slight , but statistically insignificant , stimulation of exocytosis above basal levels following aicar treatment of the muscle strips . the observed exocytosis rate constant is , however , identical to that derived from the steady - state exocytosis experiment in aicar - treated rat muscle . muscle strips were incubated with 12 nmol / l insulin and then labeled with gp15 . insulin action was then terminated by washing with a ph 6.0 buffer , and the tagged glut4 was allowed to internalize for 40 min . the strips were then maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . avidin was added at the cell surface throughout the restimulation time courses ; this reduced the signal for glut4 that exocytosed to the cell surface , thereby reducing the signal from internal glut4 . the remaining internal glut4 was then solubilized and the biotin - tagged glut4 was precipitated on immobilized streptavidin , resolved on sds - page gels , and detected and quantified by immunoblot analysis with a glut4 antibody . c : curve fitting was carried out for the basal , insulin , and aicar treatments , and data from separate experiments were used to calculate rate constants as described in research design and methods . b : the curves were computed from the average rate constants in c. results are means se . where no error bar is shown , n = 2 . data are derived from four , four , and two experiments for basal , insulin , or aicar treatment , respectively . in basal experiments , time points were varied to allow comparison with early and late time points for the insulin and aicar stimulations . * analysis of the trafficking kinetic data parameters for glut4 translocation in skeletal muscle is essential to resolve the site of convergence with the insulin - signaling steps . if the site of insulin action is more fully resolved , this would help define where defects in glut4 traffic occur in insulin - resistant states and type 2 diabetes . by applying and extending use of our glut4-labeling technique , we have provided some of the first evidence that glut4 exocytosis in rat and human skeletal muscle is the major site of insulin action and that the magnitude of the stimulation of the exocytosis rate constant is sufficient to account for the observed insulin stimulation of glucose transport . studies on relatively simpler cell - culture systems have revealed that glut4 traffics through multiple compartments ( 31,32 ) . in skeletal muscle , additional complexities in the trafficking route occur because of the large fibrous cell structures and the presence of a more complex transverse - tubule network of membranes . recent studies on green fluorescent protein tagged glut4 in skeletal muscle have highlighted the importance of the transverse - tubule system and have led to the hypothesis that glut4 is not translocated over long distances in skeletal muscle but is recruited from glut4 satellite compartments localized close to the transverse tubules ( 17,18 ) . our photolabeling approach is complementary to studies on gfp - glut4 , because we tag endogenous glut4 , rather than expressed recombinant protein . however , the approach does not provide information on any potential differences in trafficking at the sarcolemma or the transverse tubules . previous autoradiography studies using a tritiated version of a glut4 photolabel have provided evidence that the label does penetrate and label glut4 in the transverse - tubule locations in skeletal muscle ( 33 ) . additionally , molecules as large as a fragment antigen binding of an antibody can permeate into transverse - tubule spaces in heart muscle ( 34 ) . the six- to sevenfold stimulation of the glut4 exocytosis rate in the present study is greater than the fold stimulation of glucose transport . this effect is consistent with data on cell - surface glut4 labeling in rat ( 35 ) and human ( 12 ) skeletal muscle . this may occur because glucose transport is dependent on additional transporters such as glut1 that respond less effectively to insulin . changes in glut4 endocytosis and storage will also influence the level of stimulation of transport activity . the absolute values of the rate constants obtained from rat ( 0.01 for basal and 0.067 min for insulin at 30c ) and human ( 0.011 and 0.075 min at 37c ) skeletal muscle studies are remarkably close to adipose cell studies . for example , basal and insulin - stimulated rate constants for glut4 exocytosis in 3t3-l1 adipocytes are 0.01 and 0.09 min , respectively , at 37c ( 27 ) . when the gp15 photolabel in rat adipocytes was used , slightly higher values were obtained ( 23 ) . the similarity of these rate constants suggests that the same fundamental process rate limits glut4 movement in these diverse systems and that this process is relatively unaltered by divergent cell architecture and membrane tubule complexities in these differing cell types . there are several technical problems associated with its use , including its slow onset of action . even when glut4 exocytosis is studied over 120 min , there is no clear evidence that this process is stimulated . in cardiac myocytes , ampk activation following hypoxia , mitochondrial inhibition ( 36 ) , or metformin mitochondrial inhibition and a combination of aicar treatment and pkc stimulation also lead to inhibition of glut4 internalization in l6 muscle cells ( 38 ) . we find that the initial rate of net internalization and endocytosis of glut4 in rat skeletal muscle is also reduced , but these changes do not reach statistical significance . the reduction in net internalization that is evident at early time points is masked in the steady - state experiments by the progress to a lower equilibrium level for the equilibration of the gp15-tagged glut4 during aicar treatment . the endocytosis of glut4 is always a more difficult kinetic parameter to obtain and interpret than the unidirectional exocytosis parameter . this is because internalization is a net process , involving both endocytosis and re - exocytosis , and some recycling of glut4 will inevitably occur . these limitations may account for some of the controversies that surround the issue of whether insulin markedly reduces glut4 endocytosis ( 16,39,40 ) . whether the observed 50% reduction in glut4 net internalization is sufficient to account for the full magnitude of the aicar stimulation of glucose transport ( a two- to threefold stimulation ) the slow onset of the increase in glucose transport is also consistent with a slowing of glut4 endocytosis . our data suggest that a single site of convergence of ampk- and akt - signaling pathways on a single intermediate such as as160 ( 7,4143 ) can not fully and simultaneously account for regulation of glut4 traffic at the level of both exocytosis and endocytosis . indeed , as160 action is reported to be localized to the exocytosis rather than the endocytosis arm of glut4 traffic ( 40 ) . the steady - state recycling experiments are of interest and mechanistic importance because they show that in muscle , glut4 continues to recycle in both the insulin- and aicar - stimulated states . in contrast to earlier ( 44,45 ) , but not later , versions of the translocation hypothesis ( 13 ) , glut4 is not released as a transitory pulse to the cell surface where it would then be resident while the stimulus remains . instead , glut4 continues to recycle even when the stimulus is maintained ( 13,27 ) . however , the observed recycling could be consistent with a variant of the pulse model in which glut4 is released in a pulse from a reservoir compartment to the cell surface and then on to the endosome system , within which it continues to recycle while the stimulus remains ( 29,30 ) . in conclusion , our demonstration that exocytosis is the major site of insulin action on glut4 traffic in rat and human skeletal muscle suggests that a focus on this limb of the glut4 traffic pathway in insulin - resistant conditions such as type 2 diabetes will be necessary and appropriate to resolve defects in glucose metabolism in future studies . because of the technical challenges and limitations described above , the photolabeling approach will only give a partial and incomplete analysis in future studies . nevertheless , the approach described is complementary to more dynamic studies in which glut4 is analyzed at a detailed subcellular level ( 18 ) .

objectivein skeletal muscle , insulin stimulates glucose transport activity three- to fourfold , and a large part of this stimulation is associated with a net translocation of glut4 from an intracellular compartment to the cell surface . we examined the extent to which insulin or the amp - activated protein kinase activator aicar can lead to a stimulation of the exocytosis limb of the glut4 translocation pathway and thereby account for the net increase in glucose transport activity.research design and methodsusing a biotinylated photoaffinity label , we tagged endogenous glut4 and studied the kinetics of exocytosis of the tagged protein in rat and human skeletal muscle in response to insulin or aicar . isolated epitrochlearis muscles were obtained from male wistar rats . vastus lateralis skeletal muscle strips were prepared from open muscle biopsies obtained from six healthy men ( age 39 11 years and bmi 25.8 0.8 kg / m2).resultsin rat epitrochlearis muscle , insulin exposure leads to a sixfold stimulation of the glut4 exocytosis rate ( with basal and insulin - stimulated rate constants of 0.010 and 0.067 min1 , respectively ) . in human vastus lateralis muscle , insulin stimulates glut4 translocation by a similar sixfold increase in the exocytosis rate constant ( with basal and insulin - stimulated rate constants of 0.011 and 0.075 min1 , respectively ) . in contrast , aicar treatment does not markedly increase exocytosis in either rat or human muscle.conclusionsinsulin stimulation of the glut4 exocytosis rate constant is sufficient to account for most of the observed increase in glucose transport activity in rat and human muscle .

RESEARCH DESIGN AND METHODS Epitrochlearis muscle isolation. Open muscle biopsy. Glucose transport. Akt and AMP-activated protein kinase phosphorylation. GP15 photoaffinity tagging of GLUT4. Steady-state internalization of GLUT4. Exocytosis of GLUT4. Curve fitting and analysis of rate constants. RESULTS Comparison of insulin and AICAR treatment on glucose transport activity in rat epitrochlearis muscle. Steady-state GLUT4 trafficking in insulin- and AICAR-stimulated rat epitrochlearis muscle. Exocytosis of GLUT4 in insulin- and AICAR-stimulated rat epitrochlearis muscle. Exocytosis of GLUT4 in insulin- and AICAR-stimulated human vastus lateralis muscle. DISCUSSION

male wistar rats ( 150200 g ) were obtained from b&k universal , sollentuna , sweden . six healthy men with no known family history of metabolic disorder ( aged 39 11 years and bmi 25.8 0.8 kg / m ) volunteered for the muscle biopsy procedure . the maximum signal of glut4 , which internalized and escaped the surface quenching , was determined in each experiment from insulin- or aicar - stimulated samples that were returned to the basal state and allowed to internalize the biotinylated glut4 for 60 min . for exocytosis experiments , human vastus lateralis muscles or rat epitrochlearis muscles were incubated at 35 or 30c , respectively . to begin the determination of exocytosis thereafter , muscles were transferred to khb with or without 120 nmol / l insulin or 1 mmol / l aicar for specified times . values for net internalization of glut4 under steady - state conditions were curve fitted using a two - parameter equation : f = ( 1.0 e).exp(k.t ) + e , where f is the fraction of glut4 remaining at the surface , e is the equilibrium position for internalization , k is the net internalization rate constant , and t is time . values for unidirectional exocytosis of glut4 were curve fitted to a single exponential equation : f = exp( k.t ) , where f is the fraction of glut4 remaining internal , and k is the exocytosis rate constant , and t is time . male wistar rats ( 150200 g ) were obtained from b&k universal , sollentuna , sweden . six healthy men with no known family history of metabolic disorder ( aged 39 11 years and bmi 25.8 0.8 kg / m ) volunteered for the muscle biopsy procedure . muscles were transferred to fresh khb and incubated with either 120 nmol / l insulin or 1 mmol / l aicar for the times specified in the figure legends . the maximum signal of glut4 , which internalized and escaped the surface quenching , was determined in each experiment from insulin- or aicar - stimulated samples that were returned to the basal state and allowed to internalize the biotinylated glut4 for 60 min . for exocytosis experiments , human vastus lateralis muscles or rat epitrochlearis muscles were incubated at 35 or 30c , respectively . values for net internalization of glut4 under steady - state conditions were curve fitted using a two - parameter equation : f = ( 1.0 e).exp(k.t ) + e , where f is the fraction of glut4 remaining at the surface , e is the equilibrium position for internalization , k is the net internalization rate constant , and t is time . values for unidirectional exocytosis of glut4 were curve fitted to a single exponential equation : f = exp( k.t ) , where f is the fraction of glut4 remaining internal , and k is the exocytosis rate constant , and t is time . insulin stimulation of epitrochlearis muscle leads to a rapid increase in glucose transport activity that reaches an equilibrium level of stimulation three- to fourfold above basal levels within 20 min ( fig . we assessed whether changes in the kinetics of glut4 trafficking induced by these treatments could account for the differences observed in glucose transport activity . we found that the higher increase in glucose transport activity following insulin stimulation versus aicar stimulation is matched by a slightly but insignificantly higher level of steady - state glut4 labeling by gp15 ( fig . comparison of the stimulation of glucose transport and cell - surface glut4 levels by insulin and aicar in rat epitrochlearis muscle . in addition to the observed differences in the rate of onset of the maximal stimulation of glucose transport activity , insulin and aicar produce a markedly different pattern of early signaling changes in the intermediate serine kinases , akt / protein kinase b , and ampk . conversely , aicar , but not insulin , action leads to a robust stimulation of ampk phosphorylation ( fig . the maximally rapid time frame for insulin - stimulated increase in akt phosphorylation was within 210 min , and the aicar - stimulated increase in ampk phosphorylation also occurred rapidly and reached a maximum within 30 min ( 25 ) ( h.k.r.k . ampk thr172 ( b ) , and p coa carboxylase ser79 ( c ) in lysates of rat epitrochlearis and p akt ser 473 ( d ) in human vastus lateralis muscle . 1 ) could be due to a pulse of glut4 translocation , which is then followed by a cessation of subsequent movement as the cell signaling processes continue . alternatively , glut4 could continue to recycle under steady - state conditions , and the cell - surface glut4 level could be maintained by a net balance of a fast exocytosis and a fast endocytosis rate of translocation . in adipocytes the rate of net internalization of gp15-tagged glut4 under steady - state stimulation by either insulin or aicar has been measured by separating the glut4 that remains at the cell surface from that which has been internalized . conversely , the steady - state aicar stimulation is associated with a slower initial net internalization , particularly over the first 5 min where the fraction of internalization is lower than that occurring in insulin - stimulated muscle . is associated with a slower rate constant for net internalization between aicar- and insulin - stimulated muscles ( 0.059 vs. 0.134 min , respectively ) . 3d ) indicates that aicar treatment is associated with a slower endocytosis rate constant ( 0.042 vs. 0.068 min ) and a much slower exocytosis rate constant ( 0.017 vs. 0.067 min ) . internalization of gp15-tagged glut4 under steady - state stimulation with insulin or aicar in rat epitrochlearis muscle . the steady - state data reveal important differences in the kinetics of glut4 trafficking following insulin or aicar treatment . to further address whether aicar- or insulin - stimulated glucose transport and signaling activities occur by a convergent mechanism , we simplified our study design in subsequent experiments to only measure the exocytosis rate constant . to more directly measure the kinetic parameters of exocytosis , a procedure was adopted in which gp15-tagged glut4 was internalized and then the unidirectional return to the cell surface was monitored . insulin action was then terminated by washing with a ph 6.0 buffer , and the tagged glut4 was allowed to internalize for 40 min . c : curve fitting for internal glut4 was carried out for the basal , insulin , and aicar treatments and data from separate experiments were used to calculate rate constants as described in research design and methods . the movement of glut4 from the internal compartment to the plasma membrane was rendered unidirectional by the addition of avidin at the cell exterior throughout the time course used to study the exocytosis . the glut4 signal that remains in the cell , therefore , represents the fraction of glut4 remaining internally , and this decreases with time ( fig . insulin treatment increases the rate constant for exocytosis sixfold ( 0.010 in the basal state to 0.067 min in the insulin - stimulated state ; p < 0.05 ) . the more limited exocytosis response to aicar can not be due to a slow onset in ampk activation , as we have compared basal and aicar at times of exocytosis up to 120 min . taking into consideration both the steady - state and the direct exocytosis measurements , we can not exclude the possibility of an aicar - mediated increase in exocytosis ; however , this effect is clearly smaller , or more limited as a pulse of release of glut4 , than that occurring in insulin - treated muscle . the exocytosis approach characterized in the isolated rat epitrochlearis muscle system was applied to human muscle strips . a comparison of basal and insulin - treated muscle revealed clear insulin stimulation in the gp15-tagged glut4 movement out of the internal compartment and toward the cell surface ( fig . 5c ) revealed that the exocytosis rate constant was increased six- to sevenfold ( from 0.011 to 0.075 min ) . these rate - constant values and the six- to sevenfold stimulation are very similar to the changes occurring in rat epitrochlearis muscle . the six- to sevenfold increase in glucose transport activity does not occur in human muscle , but this is not necessarily inconsistent with a large change in glut4 translocation , as additional factors may influence the glucose transport activity . the effect of aicar on glut4 exocytosis in human muscle is less clear , partly because of the large scatter in data points derived from the samples available in the study ( fig . 5c ) indicated that there may be some slight , but statistically insignificant , stimulation of exocytosis above basal levels following aicar treatment of the muscle strips . the observed exocytosis rate constant is , however , identical to that derived from the steady - state exocytosis experiment in aicar - treated rat muscle . the strips were then maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . c : curve fitting was carried out for the basal , insulin , and aicar treatments , and data from separate experiments were used to calculate rate constants as described in research design and methods . data are derived from four , four , and two experiments for basal , insulin , or aicar treatment , respectively . * insulin stimulation of epitrochlearis muscle leads to a rapid increase in glucose transport activity that reaches an equilibrium level of stimulation three- to fourfold above basal levels within 20 min ( fig . we assessed whether changes in the kinetics of glut4 trafficking induced by these treatments could account for the differences observed in glucose transport activity . we found that the higher increase in glucose transport activity following insulin stimulation versus aicar stimulation is matched by a slightly but insignificantly higher level of steady - state glut4 labeling by gp15 ( fig . comparison of the stimulation of glucose transport and cell - surface glut4 levels by insulin and aicar in rat epitrochlearis muscle . in addition to the observed differences in the rate of onset of the maximal stimulation of glucose transport activity , insulin and aicar produce a markedly different pattern of early signaling changes in the intermediate serine kinases , akt / protein kinase b , and ampk . conversely , aicar , but not insulin , action leads to a robust stimulation of ampk phosphorylation ( fig . the maximally rapid time frame for insulin - stimulated increase in akt phosphorylation was within 210 min , and the aicar - stimulated increase in ampk phosphorylation also occurred rapidly and reached a maximum within 30 min ( 25 ) ( h.k.r.k . ampk thr172 ( b ) , and p coa carboxylase ser79 ( c ) in lysates of rat epitrochlearis and p akt ser 473 ( d ) in human vastus lateralis muscle . 1 ) could be due to a pulse of glut4 translocation , which is then followed by a cessation of subsequent movement as the cell signaling processes continue . alternatively , glut4 could continue to recycle under steady - state conditions , and the cell - surface glut4 level could be maintained by a net balance of a fast exocytosis and a fast endocytosis rate of translocation . the rate of net internalization of gp15-tagged glut4 under steady - state stimulation by either insulin or aicar has been measured by separating the glut4 that remains at the cell surface from that which has been internalized . conversely , the steady - state aicar stimulation is associated with a slower initial net internalization , particularly over the first 5 min where the fraction of internalization is lower than that occurring in insulin - stimulated muscle . this is associated with a slower rate constant for net internalization between aicar- and insulin - stimulated muscles ( 0.059 vs. 0.134 min , respectively ) . 3d ) indicates that aicar treatment is associated with a slower endocytosis rate constant ( 0.042 vs. 0.068 min ) and a much slower exocytosis rate constant ( 0.017 vs. 0.067 min ) . internalization of gp15-tagged glut4 under steady - state stimulation with insulin or aicar in rat epitrochlearis muscle . the steady - state data reveal important differences in the kinetics of glut4 trafficking following insulin or aicar treatment . to further address whether aicar- or insulin - stimulated glucose transport and signaling activities occur by a convergent mechanism , we simplified our study design in subsequent experiments to only measure the exocytosis rate constant . to more directly measure the kinetic parameters of exocytosis , a procedure was adopted in which gp15-tagged glut4 was internalized and then the unidirectional return to the cell surface was monitored . c : curve fitting for internal glut4 was carried out for the basal , insulin , and aicar treatments and data from separate experiments were used to calculate rate constants as described in research design and methods . the movement of glut4 from the internal compartment to the plasma membrane was rendered unidirectional by the addition of avidin at the cell exterior throughout the time course used to study the exocytosis . the glut4 signal that remains in the cell , therefore , represents the fraction of glut4 remaining internally , and this decreases with time ( fig . insulin treatment increases the rate constant for exocytosis sixfold ( 0.010 in the basal state to 0.067 min in the insulin - stimulated state ; p < 0.05 ) . the more limited exocytosis response to aicar can not be due to a slow onset in ampk activation , as we have compared basal and aicar at times of exocytosis up to 120 min . taking into consideration both the steady - state and the direct exocytosis measurements , we can not exclude the possibility of an aicar - mediated increase in exocytosis ; however , this effect is clearly smaller , or more limited as a pulse of release of glut4 , than that occurring in insulin - treated muscle . the exocytosis approach characterized in the isolated rat epitrochlearis muscle system was applied to human muscle strips . a comparison of basal and insulin - treated muscle revealed clear insulin stimulation in the gp15-tagged glut4 movement out of the internal compartment and toward the cell surface ( fig . 5c ) revealed that the exocytosis rate constant was increased six- to sevenfold ( from 0.011 to 0.075 min ) . these rate - constant values and the six- to sevenfold stimulation are very similar to the changes occurring in rat epitrochlearis muscle . the six- to sevenfold increase in glucose transport activity does not occur in human muscle , but this is not necessarily inconsistent with a large change in glut4 translocation , as additional factors may influence the glucose transport activity . the effect of aicar on glut4 exocytosis in human muscle is less clear , partly because of the large scatter in data points derived from the samples available in the study ( fig . 5c ) indicated that there may be some slight , but statistically insignificant , stimulation of exocytosis above basal levels following aicar treatment of the muscle strips . the observed exocytosis rate constant is , however , identical to that derived from the steady - state exocytosis experiment in aicar - treated rat muscle . the strips were then maintained in the basal state or stimulated with either 120 nmol / l insulin or 1 mmol / l aicar for the indicated times . c : curve fitting was carried out for the basal , insulin , and aicar treatments , and data from separate experiments were used to calculate rate constants as described in research design and methods . data are derived from four , four , and two experiments for basal , insulin , or aicar treatment , respectively . * analysis of the trafficking kinetic data parameters for glut4 translocation in skeletal muscle is essential to resolve the site of convergence with the insulin - signaling steps . by applying and extending use of our glut4-labeling technique , we have provided some of the first evidence that glut4 exocytosis in rat and human skeletal muscle is the major site of insulin action and that the magnitude of the stimulation of the exocytosis rate constant is sufficient to account for the observed insulin stimulation of glucose transport . in skeletal muscle , additional complexities in the trafficking route occur because of the large fibrous cell structures and the presence of a more complex transverse - tubule network of membranes . recent studies on green fluorescent protein tagged glut4 in skeletal muscle have highlighted the importance of the transverse - tubule system and have led to the hypothesis that glut4 is not translocated over long distances in skeletal muscle but is recruited from glut4 satellite compartments localized close to the transverse tubules ( 17,18 ) . the six- to sevenfold stimulation of the glut4 exocytosis rate in the present study is greater than the fold stimulation of glucose transport . the absolute values of the rate constants obtained from rat ( 0.01 for basal and 0.067 min for insulin at 30c ) and human ( 0.011 and 0.075 min at 37c ) skeletal muscle studies are remarkably close to adipose cell studies . for example , basal and insulin - stimulated rate constants for glut4 exocytosis in 3t3-l1 adipocytes are 0.01 and 0.09 min , respectively , at 37c ( 27 ) . in cardiac myocytes , ampk activation following hypoxia , mitochondrial inhibition ( 36 ) , or metformin mitochondrial inhibition and a combination of aicar treatment and pkc stimulation also lead to inhibition of glut4 internalization in l6 muscle cells ( 38 ) . the reduction in net internalization that is evident at early time points is masked in the steady - state experiments by the progress to a lower equilibrium level for the equilibration of the gp15-tagged glut4 during aicar treatment . whether the observed 50% reduction in glut4 net internalization is sufficient to account for the full magnitude of the aicar stimulation of glucose transport ( a two- to threefold stimulation ) the slow onset of the increase in glucose transport is also consistent with a slowing of glut4 endocytosis . in contrast to earlier ( 44,45 ) , but not later , versions of the translocation hypothesis ( 13 ) , glut4 is not released as a transitory pulse to the cell surface where it would then be resident while the stimulus remains . however , the observed recycling could be consistent with a variant of the pulse model in which glut4 is released in a pulse from a reservoir compartment to the cell surface and then on to the endosome system , within which it continues to recycle while the stimulus remains ( 29,30 ) . in conclusion , our demonstration that exocytosis is the major site of insulin action on glut4 traffic in rat and human skeletal muscle suggests that a focus on this limb of the glut4 traffic pathway in insulin - resistant conditions such as type 2 diabetes will be necessary and appropriate to resolve defects in glucose metabolism in future studies .

neuroinflammation is the mechanism of cns inflammation that occurs in response to trauma , infections , and/or neurodegenerative diseases . in neuroinflammation , cellular and molecular immune components such as specialised macrophages ( microglia ) , cytokines , complement , and pattern - recognition receptors are the contributing players . these proinflammatory mediators are either produced locally within the cns or recruited from the peripheral system following disruption of the blood - brain barrier . this in turn leads to the activation of the glial cells , such as microglia and astrocytes . the effect of neuroinflammation is considered neuroprotective when the inflammatory activity is for a shorter period of time whereas chronic neuroinflammation is associated with harmful consequences for the cns . some of the components of first line of defence include epithelium ( skin , gut , and lungs ) that acts as a physical barrier and also produces several kinds of antimicrobial enzymes and peptides , namely , lysozyme , defensins , mucin , lectin . other components of innate immunity include the pattern - recognition receptors ( prrs ) such as toll - like receptors ( tlrs ) , nucleotide - binding , and oligomerisation domain , leucine - rich repeats containing ( nod)-like receptors ( nlrs ) ; and scavenger receptors ( srs ) . present on phagocytic and antigen - presenting cells , these receptors recognise not only exogenous pathogen - associated molecular pattern ( pamp ) but also endogenous modified molecules called damage - associated molecular pattern ( damp ) . the innate immune system launches inflammatory and regulatory responses via prrs , phagocytes ( macrophages ) , complement system , cytokines , and chemokines in order to counteract infection , injury , and maintenance of tissue homeostasis . here the origin of these innate immune cells is debatable but it is now widely believed that they are of myeloid lineage . in mice studies , it has been found that microglia originate from primitive ( yolk sac ) myeloid progenitors that migrate to cns independent of definitive progenitors and circulation ( i.e. , bone marrow ) . these cells are found in brain , spinal cord , retina , and optic nerve . this is the shape they have when in resting state . in this state , these cells constantly monitor and survey their area . the microglial cells in resting form have been shown to be involved in other functions such as neurogenesis , neuroprotection and synaptic pruning , which has been found to be complement dependent . upon environmental stimulation / challenges , the microglia become activated and the morphology changes to an amoeboid appearance where they retract the ramifications . activation of microglia by tlrs and nlrs is considered to be classical form of microglial activation where innate immune responses include production of proinflammatory cytokines like tumour necrosis factor ( tnf)- , interleukin ( il)-1 and il-6 , and chemokines . classical activation also leads to adaptive immune response by expressing major histocompatibility class ii molecules and interaction with t cells . tnf- stimulation increases phagocytic activity of microglia , and deficiency of tnf receptors has been found to reduce microglial activation . tnf- is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases like alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) . il-1 induces expression of tnf- and il-6 and is implicated in neuroinflammatory processes in traumatic brain injury ( tbi ) , ad , and pd . activated microglia have also been implicated in neurotransmission . in order to regulate the immune responses , anti - inflammatory cytokines il-10 and transforming growth factor beta are produced by microglia [ 1820 ] . microglia also produce inhibitor of nuclear factor (nf- ) , mitogen - activated protein kinase ( mapk ) phosphatases , and suppressor of cytokine signalling proteins , which help in immune activation regulation . glucocorticoids have also been considered to play a regulatory role for innate immunity in cns by regulation of microglial tnf- [ 22 , 23 ] although there are debatable views to the same . there are a variety of receptors expressed on microglia related to the different functions of these cells . tlr 19 receptors are known to be expressed by microglial cells ( discussed in detail later ) . nlr form complexes called inflammasomes ( for a detailed review see ) that have been shown to activate and recruit microglia in response to amyloid- ( a ) and prion peptide . some of the other innate immune receptors expressed on microglia surface are cd14 , cd18 , cd36 , cd68 , mannose , and lectin ( dendritic cell - specific intercellular adhesion molecule-3-grabbing nonintegrin or dc - sign ) receptors . morphologically , astrocytes are of two types : protoplasmic ( found in grey matter ) and fibrous ( found in white matter ) . however , recently they have been shown to play an active part in the modulation of neural activity , potentiation of synaptic transmission , sleep homeostasis , and even long - term memory formation . any insult to the cns is associated with changes in the structure , morphology , and hypertrophy of astrocytes , followed by cytokine and c1q secretion , leading to scar formation , collectively termed as reactive astrogliosis . like microglia , astrocytes have been shown to express innate immune prr like tlr , nlr , scavenger , complement , and mannose receptors . they have also been shown to release cytokines like tnf , il-6 , il-1 , interferon- , and chemokines when stimulated with lipopolysaccharide ( lps ) [ 36 , 37 ] . reactive astrogliosis is associated with a number of cns diseases such as ad [ 38 , 39 ] , pd , autism , and prion diseases [ 40 , 41 ] . tlrsare expressed on microglia , neurons , and astrocytes similar to dendritic cells , b cells , neutrophils , epithelia , and fibroblast . tlr is a type 1 membrane protein containing an extracellular leucine - rich repeat ( lrr ) domain and a toll / il-1 receptor ( tir ) domain in the cytoplasmic region ( figure 1 ) . lrr domain is involved in specific pathogen recognition and tir domain is involved in the signalling pathway . tlrs are considered to exist as dimers and bind to various ligands [ 44 , 45 ] . for example , tlr2 heterodimerises with tlr1 and also with tlr6 and recognises bacterial lipoproteins . upon sensing ligands , recruitment of adaptor proteins the adaptor proteins are ( i ) myeloid differentiating factor 88 ( myd88 ) ; ( ii ) myd88 adaptor - like protein ( mal ) ; ( iii ) tir domain - containing adaptor inducing interferon- ( trif ) ; ( iv ) trif - related adaptor molecule ; and ( v ) sterile- and armadillo - motif - containing protein . these adaptor proteins are recruited by tir domain leading to activation of nf-. nf- then induces production of proinflammatory cytokines such as tnf- , il-1 , and il-6 , and chemokines . all tlrs are activated by myd88 except tlr3 ; instead myd88 may be restricting tlr3 signalling . some of the other adaptors investigated in detail include major histocompatibility complex class ii molecules , small heterodimer partner , and dedicator of cytokinesis 8 ( dock8 ) . it has recently been shown that oligomerisation of tlr4 with myeloid differentiation protein-2 by morphine causes neuroinflammation . necrotic neurons have been shown to activate microglia via myd88 pathway leading to increased neuroinflammation . in mouse models , both myd88 and trif pathways have been implicated in regulation of il-6 and il-10 after cerebral ischaemia as well as regulation of il6 , tnf- , and il-1 following intracerebral haemorrhage . some of the exogenous and endogenous ligands of tlr are listed in table 2 [ 5962 ] . in vivo studies have shown that the administration of lps ( peripheral / intraperitoneal ) leads to expression of genes coding for proinflammatory cytokines in the microglial cells [ 63 , 64 ] . injection of lps directly into brain has been shown to produce an increased expression of genes of proinflammatory cytokines , chemokines , and complement proteins and receptors such as cd14 [ 66 , 67 ] . production of tnf by microglial cells upon lps stimulation has been found to cause death of dopaminergic neurons . tlr2 ligands stimulation of microglial and astrocytic cells leads to an increase in production of il-6 , chemokines , and ifn- . in mice studies , tlr9 ligand cpg has been found to be neuroprotective in cerebral ischaemia while similar findings have been reported in tlr4 knockout mice . tlr2 activation has been shown to be involved in neurogenesis while tlr8 induces apoptosis of neurons . tlr3 impairs plasticity and working memory while tlr7 and tlr9 have been found to be associated with the development of mouse brain . interestingly , increased peripheral responses of tlr2 , tlr4 , tlr8 , and tlr9 have been detected in psychosis while tlr9 is associated with posttraumatic anxiety . pneumococcal infection leads to innate immune response in brain and this depends on tlr2 and tlr4 . tlrs have been found to be involved in pneumococcal infection in hiv - associated neurocognitive disorders . tlr2 and tlr9 initiate immune response against herpes simplex virus ( hsv ) and also control hsv infection in the brain . tlr3 in astrocytes may be protective in hsv2 infection and has been reported to mediate entry of west nile virus ( wnv ) into the cns , causing encephalitis . tlrs have also been implicated in cns parasitic infections like toxoplasmosis , sleeping sickness , cerebral malaria , and neurocysticercosis . tlr2 is associated with protection from cerebral malaria and therapeutic targeting of tlrs has been shown to prevent experimental cerebral malaria [ 89 , 90 ] . in mouse model of ad , myd88 has been found to prevent memory and cognitive deficits while another study found myd88 deficiency to improve ad - related pathology . a loss - of - function mutation of tlr4 has been found to reduce microglial activation and increase a deposits with increased cognitive deficits . intracranial injection of lps ( a tlr4 ligand ) reduces a levels in brain . tlr9 may have a protective role in ad by improving cognitive functions , reducing a-toxicity , and clearing a . in amyotrophic lateral sclerosis ( als ) , myd88 has been shown to activate microglia due to mutant sod1 and in vitro studies show enhanced microglial activation and neurotoxicity when stimulated with tlr2 and tlr4 ligands [ 103 , 104 ] . myd88 pathway may also be involved in pd where -synuclein directly activates microglia and alters expression of tlrs . studies involving mice possessing mutant gene which prevents tlr4 signalling was found to have a shorter time for scrapie pathogenesis while administration of tlr9 agonist in prion - infected mice leads to delayed onset of the disease . however , myd88 knockout mice ( lacking tlr signalling ) were found to develop prion disease similar to wild - type mice both in terms of time and severity . like tlrs , nod - like receptors ( nlrs ) also detect pamps and damps . they consist of a central nucleotide - binding and oligomerisation ( nacht ) domain and a c - terminal lrrs . their n - terminal component may be variable based on which nlrs are further subdivided . it can be caspase activation and recruitment domain ( card ) ; a pyrin domain ( pyd ) , or baculovirus inhibitor of apoptosis protein repeat ( bir ) termed , respectively , as nlrc , nlrp , and nlrb . upon binding to agonists , nlr can lead to the activation of nf- or mapk signalling pathways and production of cytokines and chemokines . nlr binding to agonist also causes the activation of procaspase-1 leading to inflammasome formation ; pyroptosis ; autophagy ; and ifn-1 signalling [ 140145 ] ( figure 1 ) [ 141145 ] . inflammasomes are multiprotein complexes that activate caspase-1 , which in turn leads to processing and secretion of proinflammatory cytokines such as il-1 and il-18 . the members of nlr family that are capable of forming inflammasomes are pyd - containing nlrp1 , nlrp3 , nlrp6 , and card - containing nlrc4 . inflammasome complex formation occurs when a ligand binds to nlr and thereby induces a conformational change , leading to atp binding at nacht domain which causes receptor oligomerisation and recruitment of other complex members . inflammasomes have been implicated in various diseases such as gout , pseudogout , contact dermatitis , allergic dermatitis , vitiligo , hydatidiform mole , muckle - wells syndrome , atherosclerosis , type 2 diabetes mellitus , obesity , metabolic syndrome , acute myocardial infarction , coeliac disease , inflammatory bowel disease , asthma , pulmonary fibrosis , and viral and bacterial infections . nlrp3 inflammasome is involved in the innate immune response to a leading to ad pathology . in multiple sclerosis ( ms ) , nlrp3 knockout mice model of disease shows reduced demyelination , while another study shows nlrp3 involvement in migration of t - helper cells into cns . ifn- therapy is effective in treating inflammasome - dependent disease in mouse models of ms . nlrp1 has been found to be involved in tbi and neutralising its effect or formation was found to have beneficial effects . inflammasome complex inhibition has also been found to reduce inflammation and improve pathology in mouse models of stroke . nlrp3 inflammasome contributes to brain injury in pneumococcal meningitis and is associated with inflammation in japanese encephalitis . both nlrp1 and nlrp3 are increased in postmortem alcoholic human brains and inhibition of these inflammasomes was found to be beneficial in reversing ethanol - mediated neuroinflammation . srs are expressed on macrophages , dendritic cells , microglia , and endothelial cells [ 111 , 112 ] . the family of srs include class a ( macrophage receptors , marco ) , class b ( cd36 , sr - bi ) , cd68 and endothelial or lox-1 , cd163 , and receptor for advanced glycation end products ( rage ) [ 167 , 168 ] . some of the ligands that srs bind to are pathogen - specific : lps , lipoteichoic acid , streptococcus pneumoniae , staphylococcus aureus , mycoplasma pneumoniae , neisseriia meningitides , escherichia coli , apoptotic cells , and erythrocytes infected with plasmodium [ 171173 ] . srs have been implicated in atherosclerosis , lung inflammation , cystic fibrosis , sle , and ad . microglia express sr and thus bind to a fibrils which is associated with ad plaques . class a sr ( sr - a ) has also been shown to play an important role in cerebral injury due to ischemia . mice deficient in sr - a showed reduced expression levels of tnf- and il-1 as well as decreased infarct size . in experimental model of ms , sr - a knockout mice showed significantly reduced demyelination as well as reduced proinflammatory cytokines production . however , deficiency of sr - a in ad mouse models was not found to impact amyloid plaque deposition or clearance . in vitro studies have shown that astrocytes express sr - a and thus play a role in neuroinflammation . class b sr type i ( sr - bi ) has been shown to be produced in vivo in ad brains with increased expression being observed in cerebellum and cortex . in mice studies , sr - bi has also been shown to impair perivascular macrophages leading to ad pathology such as increased amyloid deposition , cerebral amyloid angiopathy ( deposition of a in cerebral arteries ) , and memory deficits . cd36 appears to be involved in neurovascular dysfunction due to a and promotes cerebral amyloid angiopathy leading to cognitive deficits . rage is a receptor for a and expressed on neurons , microglia , astrocytes , and endothelial cells . rage signalling in microglia due to p38 mapk signalling pathway leads to neuroinflammation and cognitive disturbances in ad as well as synaptic and neuronal dysfunction . the complement system comprises of more than 30 proteins in the serum as well as membrane - bound receptors and regulators . the complement system consists of 3 different initiating or activation pathways culminating into a final common lytic pathway , leading to the formation of membrane attack complex ( mac ) ( figure 2 ) . mac are pores that penetrate cell membrane ( lipid bilayers ) of pathogens or abnormal cells , thereby causing their lysis . the three initiating pathways are called ( i ) classical pathway which is mostly antibody mediated ( c1q being the first subcomponent ) and is activated by c1 complex ( c1q - c1r - c1s ) ; ( ii ) alternative pathway ( ap ) which is activated spontaneously involving low - level hydrolysis of c3 to c3 ( h20 ) ; and ( iii ) lectin pathway where activation occurs through binding of a carbohydrate pattern present on microorganisms called mannan , with mannan - binding lectin ( mbl ) and ficolins ( ficolin-1 , -2 and -3 ) . they circulate in the serum in combination with zymogen serine proteases called mbl - associated serine proteases ( masps ) [ 191196 ] . the c5b formed associates with c6 , c7 , c8 , and c9 to form mac . the functions of the complement system include opsonisation of pathogens , direct lysis of foreign cells , chemotaxis and activation of leukocytes , and clearance of apoptotic cells . the complement system also interacts with tlrs and plays a role in the regulation of humoral immunity . the complement system is kept in check by regulators in order to prevent overactivation leading to damage to tissues and autoimmune diseases . the regulators can be grouped into fluid - phase : factor h ( fh ) and properdin for alternative pathway , c1 inhibitor and c4b - binding protein ( c4bp ) for classical and mbl pathway ; host cell membrane - bound : cr1 , cr2 , cd55 , cd46 , cd59 ; cell surface - attached complement regulators : fh , factor h - like protein 1 ( fhl-1 ) , c4bp and clusterin [ 200 , 201 ] . for certain ligands , factor h can also regulate c1q - mediated classical pathway [ 202205 ] . complement is produced mainly in the liver and , over the years , it was thought that the brain was an immune - privileged organ due to the presence of blood - brain barrier . now , it is well known that components of innate immunity like complement are present and even produced within the cns . neuronal cells [ 206209 ] , astrocytes [ 210 , 211 ] , and microglia [ 212214 ] have been shown to produce complement and also express complement receptors . role of complement in cns is considered to be dual - neurotoxic and/or neuroprotective , depending on the level of its activation . complement receptors c3ar and c5ar are expressed on neural stem cells and reduced neurogenesis is observed in the absence of c3ar signalling . another complement receptor cr2 has been found to be expressed in neural progenitor cells and also negatively regulates hippocampal neurogenesis . an emerging area for complement involvement in cns is in relation to synapse ( reviewed in ) . c1q , initiating component of classical pathway and widely expressed by postnatal neurons and immature astrocytes , mediates the elimination of synapse [ 219 , 220 ] . show that c1q also promotes neuronal viability and survival . in vitro and in vivo studies many other in vitro and in vivo studies show neuroprotective functions for c3a and c5a that include protection against nmda - induced apoptosis and protection against glutamate - induced apoptosis via mapk - dependent inhibition of caspase 3 as well as regulation of glutamate receptor subunit 2 . meningococcus binds to factor h ( fh ) , a negative regulator of alternative pathway , and evades host innate immune system [ 229 , 230 ] . individuals with deficiency of properdin ( positive regulator of alternative pathway ) are susceptible to meningitis and individuals with combined properdin and mbl deficiency are at increased risk of infection with neisseria meningitidis . streptococcus pneumonia infection of cns is kept in check by complement system ( mainly c1q and c3 ) . c1q and c3 genetically deficient mice each showed considerably high bacterial titres in cns as compared to wild - type mice . escherichia coli , a cause for neonatal meningitis , crosses the blood - brain barrier by surviving in the serum where it binds to c4bp . complement controls antibody response in wnv infection with lectin pathway activation being found to be protective in wnv infection . fungal infection like cerebral aspergillosis leads to increased complement production seen in astrocytes , neurons , and oligodendrocytes , especially c1q production by infiltrating macrophages . some of the defence mechanisms developed by aspergillus fumigatus to avoid complement include secreting fungal proteases as well as production and recruitment of complement inhibitors . in cerebral malaria , c1q and c5 levels have been found to be increased in mice studies while another murine study also points to the requirement of mac in the pathogenesis of cerebral malaria . infectious particles called prions cause cns disorders like creutzfeldt - jakob disease and bovine spongiform encephalopathy . these prion particles which activate classical complement pathway are thought to bind to c1q and subsequently transported to the cns . c1q , c3b have been detected in postmortem brains of individuals with prion diseases , and mac deposition was found to co - relate with prion disease severity . complement activation occurs in tbi and act as mediators of secondary brain injury [ 250 , 251 ] . following injury , levels of mac corelate with blood - brain barrier ( bbb ) disruption . in mice studies , studies involving mice overexpressing complement inhibitor cr - related protein y ( crry ) show reduced neurological impairment following tbi . better outcome is seen in individuals with low levels of mbl activity and mice lacking mbl . immunohistochemistry on brains of stroke patients shows c1q deposition while complement regulator cd59 was found to be absent . studies involving c5- and c3-deficient mice as well as c1 inhibition have been successful in having beneficial effects in stroke therapy by targeting complement [ 256 , 265 ] . a major role for complement the neuropathology in ad includes loss of neurons , extracellular amyloid plaques , and intracellular neurofibrillary tangles consisting of abnormally phosphorylated tau protein . activated complement components c1q , c3d , and c4d have been detected in amyloid plaques [ 268 , 269 ] by immunohistochemistry . c1q binds to a [ 270 , 271 ] and modulates phagocytosis of a by microglia . upon exposure to a , c1q is expressed in neurons ( hippocampus ) , and it has been found that inhibiting the binding of c1q to a leads to protection of hippocampal cells . in mouse models of ad , complement regulators factor h , fhl-1 , and c4bp have also been localised in amyloid plaques and fh and c4bp have been shown to bind ain vitro [ 276278 ] . another interesting feature is the presence of microglia expressing complement receptors found in close proximity to plaques . microglia are found in and around plaques of ad brains and are found to express c1q and complement receptors c1qr , cr3 , cr4 , and c5ar , which help in the phagocytosis of a [ 281 , 282 ] . furthermore , inhibition of complement was found to be associated with an increased formation of plaque and neurodegeneration . amyloid precursor protein transgenic mouse models of ad that lack the ability to activate classical pathway ( appq ) ( i.e. , c1q phenotype ) show less neuropathology as compared to appq mice . however , appq mice also show increased c3 levels , providing evidence for alternative pathway activation in ad . in mice models , micro - rnas ( mirnas ) 9 , 125b , 146a , and 155 are found to be upregulated in ad and these mirnas target gene encoding fh . priming of microglia in ms has been found to be c3-dependent and , in the same study , it was found that in animal model of ms , crry - deficient mice show exacerbated and accelerated disease progression . pathological studies of ms lesions have found presence of complement components c3d , c4b , c1q , and mac on myelin sheath , surrounding vessel walls , microglia , and astrocytes [ 293296 ] . there is evidence for neuroinflammation in pd as well with the presence of reactive microglia and activated components of complement . elevated mrna levels of activated complement and markers of reactive microglia are also seen in pd . pathological studies show the presence of mac components intracellularly on the characteristic lewy bodies [ 299 , 300 ] . the cerebrospinal fluid levels of c3 and factor h have been observed to correlate with severity of pd . neuromelanin ( nm ) is a pigment that accumulates in dopaminergic neurons in normal aging process . in pd , these dopaminergic neurons are susceptible to degeneration which is thought to be caused by activation of microglia by nm . furthermore , this nm pigment is found to be opsonised by c1q and phagocytosed by c1q - positive microglia . huntington 's disease ( hd ) is another neurodegenerative disorder and a genetic cause of dementia . it is inherited as an autosomal - dominant trait characterised by abnormal ( at least 36 ) cag repeats on the coding sequence of huntingtin gene . neuropathological studies in hd brains show presence of complement components c1q , c4 , and c3 along with upregulation of complement regulators c1 inhibitor , clusterin , cd59 , and cd55 . in this study , schizophrenia is a psychiatric illness characterised by thought insertion , thought withdrawal , hallucinations , delusions , and negative symptoms such as apathy , speech problems , and slow cognition . there is an increase in serum levels of classical pathway complement proteins such as c1q , c1 , c3 , and c4 ; increased total complement activity ( ch50 ) , cr1 levels ; and decreased c4bp levels [ 307309 ] . the alternative pathway is also involved with increased factor b levels and increased activity in serum . mbl pathway shows increased activity as well ( increased mbl and masp-2 levels ) [ 311 , 312 ] . genetic studies have shown c1qb gene polymorphism , csmd1 and csmd2 ( code for complement regulatory proteins ) , c3 , mbl2 , and masp2 gene association [ 313316 ] . cells of the cns such as neurons , astrocytes , and microglia along with pattern recognition receptors , cytokines , chemokines , complement , peripheral immune cells , and signal pathways form the basis for neuroinflammation . local synthesis of a number of innate immune humoral factors within cns offers an opportunity for therapeutic intervention . furthermore , excessive activation of immune system is thought to be destructive to tissues whereas , simultaneously , it opens up possibilities to harness this activation in a controlled manner to obtain desired therapeutic or preventive strategies in cns diseases . a detailed understanding of the processes and mechanisms involved in the etiopathogenesis of cns diseases as well as normal functioning of cns immunity is essential and can pave the way for reducing excessive neuroinflammation and its effects . modulation of cellular processes , phenotypes , and functions looks increasingly likely to be a way forward in combating cns disorders .

inflammation of central nervous system ( cns ) is usually associated with trauma and infection . neuroinflammation occurs in close relation to trauma , infection , and neurodegenerative diseases . low - level neuroinflammation is considered to have beneficial effects whereas chronic neuroinflammation can be harmful . innate immune system consisting of pattern - recognition receptors , macrophages , and complement system plays a key role in cns homeostasis following injury and infection . here , we discuss how innate immune components can also contribute to neuroinflammation and neurodegeneration .

1. Introduction 2. Microglia 3. Astrocytes 4. Toll-Like Receptors (TLR) 5. NOD-Like Receptors 6. Scavenger Receptors 7. Complement 8. Conclusion

neuroinflammation is the mechanism of cns inflammation that occurs in response to trauma , infections , and/or neurodegenerative diseases . in neuroinflammation , cellular and molecular immune components such as specialised macrophages ( microglia ) , cytokines , complement , and pattern - recognition receptors are the contributing players . the effect of neuroinflammation is considered neuroprotective when the inflammatory activity is for a shorter period of time whereas chronic neuroinflammation is associated with harmful consequences for the cns . some of the components of first line of defence include epithelium ( skin , gut , and lungs ) that acts as a physical barrier and also produces several kinds of antimicrobial enzymes and peptides , namely , lysozyme , defensins , mucin , lectin . other components of innate immunity include the pattern - recognition receptors ( prrs ) such as toll - like receptors ( tlrs ) , nucleotide - binding , and oligomerisation domain , leucine - rich repeats containing ( nod)-like receptors ( nlrs ) ; and scavenger receptors ( srs ) . the innate immune system launches inflammatory and regulatory responses via prrs , phagocytes ( macrophages ) , complement system , cytokines , and chemokines in order to counteract infection , injury , and maintenance of tissue homeostasis . here the origin of these innate immune cells is debatable but it is now widely believed that they are of myeloid lineage . , bone marrow ) . these cells are found in brain , spinal cord , retina , and optic nerve . in this state , these cells constantly monitor and survey their area . activation of microglia by tlrs and nlrs is considered to be classical form of microglial activation where innate immune responses include production of proinflammatory cytokines like tumour necrosis factor ( tnf)- , interleukin ( il)-1 and il-6 , and chemokines . tnf- stimulation increases phagocytic activity of microglia , and deficiency of tnf receptors has been found to reduce microglial activation . tnf- is associated with activation of microglial cells involved in pathogenesis of neurodegenerative diseases like alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) . il-1 induces expression of tnf- and il-6 and is implicated in neuroinflammatory processes in traumatic brain injury ( tbi ) , ad , and pd . microglia also produce inhibitor of nuclear factor (nf- ) , mitogen - activated protein kinase ( mapk ) phosphatases , and suppressor of cytokine signalling proteins , which help in immune activation regulation . glucocorticoids have also been considered to play a regulatory role for innate immunity in cns by regulation of microglial tnf- [ 22 , 23 ] although there are debatable views to the same . some of the other innate immune receptors expressed on microglia surface are cd14 , cd18 , cd36 , cd68 , mannose , and lectin ( dendritic cell - specific intercellular adhesion molecule-3-grabbing nonintegrin or dc - sign ) receptors . however , recently they have been shown to play an active part in the modulation of neural activity , potentiation of synaptic transmission , sleep homeostasis , and even long - term memory formation . any insult to the cns is associated with changes in the structure , morphology , and hypertrophy of astrocytes , followed by cytokine and c1q secretion , leading to scar formation , collectively termed as reactive astrogliosis . like microglia , astrocytes have been shown to express innate immune prr like tlr , nlr , scavenger , complement , and mannose receptors . they have also been shown to release cytokines like tnf , il-6 , il-1 , interferon- , and chemokines when stimulated with lipopolysaccharide ( lps ) [ 36 , 37 ] . reactive astrogliosis is associated with a number of cns diseases such as ad [ 38 , 39 ] , pd , autism , and prion diseases [ 40 , 41 ] . tlrsare expressed on microglia , neurons , and astrocytes similar to dendritic cells , b cells , neutrophils , epithelia , and fibroblast . tlr is a type 1 membrane protein containing an extracellular leucine - rich repeat ( lrr ) domain and a toll / il-1 receptor ( tir ) domain in the cytoplasmic region ( figure 1 ) . tlrs are considered to exist as dimers and bind to various ligands [ 44 , 45 ] . for example , tlr2 heterodimerises with tlr1 and also with tlr6 and recognises bacterial lipoproteins . nf- then induces production of proinflammatory cytokines such as tnf- , il-1 , and il-6 , and chemokines . some of the other adaptors investigated in detail include major histocompatibility complex class ii molecules , small heterodimer partner , and dedicator of cytokinesis 8 ( dock8 ) . in mouse models , both myd88 and trif pathways have been implicated in regulation of il-6 and il-10 after cerebral ischaemia as well as regulation of il6 , tnf- , and il-1 following intracerebral haemorrhage . injection of lps directly into brain has been shown to produce an increased expression of genes of proinflammatory cytokines , chemokines , and complement proteins and receptors such as cd14 [ 66 , 67 ] . tlr2 ligands stimulation of microglial and astrocytic cells leads to an increase in production of il-6 , chemokines , and ifn- . in mice studies , tlr9 ligand cpg has been found to be neuroprotective in cerebral ischaemia while similar findings have been reported in tlr4 knockout mice . tlr3 impairs plasticity and working memory while tlr7 and tlr9 have been found to be associated with the development of mouse brain . interestingly , increased peripheral responses of tlr2 , tlr4 , tlr8 , and tlr9 have been detected in psychosis while tlr9 is associated with posttraumatic anxiety . pneumococcal infection leads to innate immune response in brain and this depends on tlr2 and tlr4 . tlr3 in astrocytes may be protective in hsv2 infection and has been reported to mediate entry of west nile virus ( wnv ) into the cns , causing encephalitis . tlrs have also been implicated in cns parasitic infections like toxoplasmosis , sleeping sickness , cerebral malaria , and neurocysticercosis . tlr2 is associated with protection from cerebral malaria and therapeutic targeting of tlrs has been shown to prevent experimental cerebral malaria [ 89 , 90 ] . intracranial injection of lps ( a tlr4 ligand ) reduces a levels in brain . tlr9 may have a protective role in ad by improving cognitive functions , reducing a-toxicity , and clearing a . studies involving mice possessing mutant gene which prevents tlr4 signalling was found to have a shorter time for scrapie pathogenesis while administration of tlr9 agonist in prion - infected mice leads to delayed onset of the disease . however , myd88 knockout mice ( lacking tlr signalling ) were found to develop prion disease similar to wild - type mice both in terms of time and severity . it can be caspase activation and recruitment domain ( card ) ; a pyrin domain ( pyd ) , or baculovirus inhibitor of apoptosis protein repeat ( bir ) termed , respectively , as nlrc , nlrp , and nlrb . inflammasomes are multiprotein complexes that activate caspase-1 , which in turn leads to processing and secretion of proinflammatory cytokines such as il-1 and il-18 . the members of nlr family that are capable of forming inflammasomes are pyd - containing nlrp1 , nlrp3 , nlrp6 , and card - containing nlrc4 . inflammasomes have been implicated in various diseases such as gout , pseudogout , contact dermatitis , allergic dermatitis , vitiligo , hydatidiform mole , muckle - wells syndrome , atherosclerosis , type 2 diabetes mellitus , obesity , metabolic syndrome , acute myocardial infarction , coeliac disease , inflammatory bowel disease , asthma , pulmonary fibrosis , and viral and bacterial infections . nlrp3 inflammasome is involved in the innate immune response to a leading to ad pathology . nlrp1 has been found to be involved in tbi and neutralising its effect or formation was found to have beneficial effects . nlrp3 inflammasome contributes to brain injury in pneumococcal meningitis and is associated with inflammation in japanese encephalitis . both nlrp1 and nlrp3 are increased in postmortem alcoholic human brains and inhibition of these inflammasomes was found to be beneficial in reversing ethanol - mediated neuroinflammation . srs are expressed on macrophages , dendritic cells , microglia , and endothelial cells [ 111 , 112 ] . the family of srs include class a ( macrophage receptors , marco ) , class b ( cd36 , sr - bi ) , cd68 and endothelial or lox-1 , cd163 , and receptor for advanced glycation end products ( rage ) [ 167 , 168 ] . some of the ligands that srs bind to are pathogen - specific : lps , lipoteichoic acid , streptococcus pneumoniae , staphylococcus aureus , mycoplasma pneumoniae , neisseriia meningitides , escherichia coli , apoptotic cells , and erythrocytes infected with plasmodium [ 171173 ] . srs have been implicated in atherosclerosis , lung inflammation , cystic fibrosis , sle , and ad . microglia express sr and thus bind to a fibrils which is associated with ad plaques . class a sr ( sr - a ) has also been shown to play an important role in cerebral injury due to ischemia . in experimental model of ms , sr - a knockout mice showed significantly reduced demyelination as well as reduced proinflammatory cytokines production . in vitro studies have shown that astrocytes express sr - a and thus play a role in neuroinflammation . class b sr type i ( sr - bi ) has been shown to be produced in vivo in ad brains with increased expression being observed in cerebellum and cortex . in mice studies , sr - bi has also been shown to impair perivascular macrophages leading to ad pathology such as increased amyloid deposition , cerebral amyloid angiopathy ( deposition of a in cerebral arteries ) , and memory deficits . rage is a receptor for a and expressed on neurons , microglia , astrocytes , and endothelial cells . rage signalling in microglia due to p38 mapk signalling pathway leads to neuroinflammation and cognitive disturbances in ad as well as synaptic and neuronal dysfunction . the complement system comprises of more than 30 proteins in the serum as well as membrane - bound receptors and regulators . the complement system consists of 3 different initiating or activation pathways culminating into a final common lytic pathway , leading to the formation of membrane attack complex ( mac ) ( figure 2 ) . the three initiating pathways are called ( i ) classical pathway which is mostly antibody mediated ( c1q being the first subcomponent ) and is activated by c1 complex ( c1q - c1r - c1s ) ; ( ii ) alternative pathway ( ap ) which is activated spontaneously involving low - level hydrolysis of c3 to c3 ( h20 ) ; and ( iii ) lectin pathway where activation occurs through binding of a carbohydrate pattern present on microorganisms called mannan , with mannan - binding lectin ( mbl ) and ficolins ( ficolin-1 , -2 and -3 ) . the c5b formed associates with c6 , c7 , c8 , and c9 to form mac . the functions of the complement system include opsonisation of pathogens , direct lysis of foreign cells , chemotaxis and activation of leukocytes , and clearance of apoptotic cells . the complement system also interacts with tlrs and plays a role in the regulation of humoral immunity . the complement system is kept in check by regulators in order to prevent overactivation leading to damage to tissues and autoimmune diseases . the regulators can be grouped into fluid - phase : factor h ( fh ) and properdin for alternative pathway , c1 inhibitor and c4b - binding protein ( c4bp ) for classical and mbl pathway ; host cell membrane - bound : cr1 , cr2 , cd55 , cd46 , cd59 ; cell surface - attached complement regulators : fh , factor h - like protein 1 ( fhl-1 ) , c4bp and clusterin [ 200 , 201 ] . for certain ligands , factor h can also regulate c1q - mediated classical pathway [ 202205 ] . neuronal cells [ 206209 ] , astrocytes [ 210 , 211 ] , and microglia [ 212214 ] have been shown to produce complement and also express complement receptors . role of complement in cns is considered to be dual - neurotoxic and/or neuroprotective , depending on the level of its activation . an emerging area for complement involvement in cns is in relation to synapse ( reviewed in ) . meningococcus binds to factor h ( fh ) , a negative regulator of alternative pathway , and evades host innate immune system [ 229 , 230 ] . streptococcus pneumonia infection of cns is kept in check by complement system ( mainly c1q and c3 ) . c1q and c3 genetically deficient mice each showed considerably high bacterial titres in cns as compared to wild - type mice . complement controls antibody response in wnv infection with lectin pathway activation being found to be protective in wnv infection . fungal infection like cerebral aspergillosis leads to increased complement production seen in astrocytes , neurons , and oligodendrocytes , especially c1q production by infiltrating macrophages . these prion particles which activate classical complement pathway are thought to bind to c1q and subsequently transported to the cns . c1q , c3b have been detected in postmortem brains of individuals with prion diseases , and mac deposition was found to co - relate with prion disease severity . complement activation occurs in tbi and act as mediators of secondary brain injury [ 250 , 251 ] . following injury , levels of mac corelate with blood - brain barrier ( bbb ) disruption . in mice studies , studies involving mice overexpressing complement inhibitor cr - related protein y ( crry ) show reduced neurological impairment following tbi . studies involving c5- and c3-deficient mice as well as c1 inhibition have been successful in having beneficial effects in stroke therapy by targeting complement [ 256 , 265 ] . a major role for complement the neuropathology in ad includes loss of neurons , extracellular amyloid plaques , and intracellular neurofibrillary tangles consisting of abnormally phosphorylated tau protein . activated complement components c1q , c3d , and c4d have been detected in amyloid plaques [ 268 , 269 ] by immunohistochemistry . upon exposure to a , c1q is expressed in neurons ( hippocampus ) , and it has been found that inhibiting the binding of c1q to a leads to protection of hippocampal cells . in mouse models of ad , complement regulators factor h , fhl-1 , and c4bp have also been localised in amyloid plaques and fh and c4bp have been shown to bind ain vitro [ 276278 ] . another interesting feature is the presence of microglia expressing complement receptors found in close proximity to plaques . microglia are found in and around plaques of ad brains and are found to express c1q and complement receptors c1qr , cr3 , cr4 , and c5ar , which help in the phagocytosis of a [ 281 , 282 ] . furthermore , inhibition of complement was found to be associated with an increased formation of plaque and neurodegeneration . , c1q phenotype ) show less neuropathology as compared to appq mice . in mice models , micro - rnas ( mirnas ) 9 , 125b , 146a , and 155 are found to be upregulated in ad and these mirnas target gene encoding fh . pathological studies of ms lesions have found presence of complement components c3d , c4b , c1q , and mac on myelin sheath , surrounding vessel walls , microglia , and astrocytes [ 293296 ] . the cerebrospinal fluid levels of c3 and factor h have been observed to correlate with severity of pd . neuromelanin ( nm ) is a pigment that accumulates in dopaminergic neurons in normal aging process . huntington 's disease ( hd ) is another neurodegenerative disorder and a genetic cause of dementia . neuropathological studies in hd brains show presence of complement components c1q , c4 , and c3 along with upregulation of complement regulators c1 inhibitor , clusterin , cd59 , and cd55 . in this study , schizophrenia is a psychiatric illness characterised by thought insertion , thought withdrawal , hallucinations , delusions , and negative symptoms such as apathy , speech problems , and slow cognition . there is an increase in serum levels of classical pathway complement proteins such as c1q , c1 , c3 , and c4 ; increased total complement activity ( ch50 ) , cr1 levels ; and decreased c4bp levels [ 307309 ] . genetic studies have shown c1qb gene polymorphism , csmd1 and csmd2 ( code for complement regulatory proteins ) , c3 , mbl2 , and masp2 gene association [ 313316 ] . cells of the cns such as neurons , astrocytes , and microglia along with pattern recognition receptors , cytokines , chemokines , complement , peripheral immune cells , and signal pathways form the basis for neuroinflammation . local synthesis of a number of innate immune humoral factors within cns offers an opportunity for therapeutic intervention . furthermore , excessive activation of immune system is thought to be destructive to tissues whereas , simultaneously , it opens up possibilities to harness this activation in a controlled manner to obtain desired therapeutic or preventive strategies in cns diseases . a detailed understanding of the processes and mechanisms involved in the etiopathogenesis of cns diseases as well as normal functioning of cns immunity is essential and can pave the way for reducing excessive neuroinflammation and its effects . modulation of cellular processes , phenotypes , and functions looks increasingly likely to be a way forward in combating cns disorders .

the alkali and alkaline - earth ternary oxides of uranium , neptunium , and plutonium have attracted interest since the 1960s because of their exciting and intriguing electronic and magnetic properties . in these systems with [ rn]6d5f ( uranium and neptunium ) and [ rn]6d5f ( plutonium ) electronic configurations , the 5f valence shell electrons have a large spatial extension and are close in energy to the 6d electrons , making them prone to chemical bonding , in contrast with the 4f electrons of the lanthanides , which are more core - like . this character leads to a wide range of oxidation states , between + 3 and + 7 , and the occurrence of magnetic ordering behavior . the theoretical description of these systems appears extremely challenging , however , as the crystal field interaction is usually of the same order of magnitude as the spin the crystal - field interaction can not be treated as a small perturbation of the electronic energy levels as is done for the [ xe]4f rare earths . in the case of [ rn]5f and [ rn]5f electronic configurations , however , the contribution from electronic repulsion is removed , which greatly simplifies the interpretation . a number of studies have recently been reported on several sodium actinide phases because of their relevance for the safety assessment of sodium - cooled fast reactors ( sfrs ) . those studies have revealed intriguing magnetic properties for the -na2npo4 and na4npo5 compositions and have stressed the need to re - evaluate the np(vi ) crystal - field ground state . the physical and chemical properties of k2npo4 have been investigated in the present work in an attempt to bring new insights into the complex behavior of np(vi ) phases . performed mssbauer spectroscopy and magnetic susceptibility measurements on this phase in 1981 and reported intriguing results . the authors suggested the occurrence of a first - order magnetic transition at 19.5(5 ) k as the mssbauer spectra showed hyperfine splitting below that temperature , with an associated magnetic hyperfine field of 122 t , corresponding to an ordered moment of about 0.6 b . however , their magnetic susceptibility data did not show any sign of an anomaly around 20 k , as could be expected from the mssbauer results . electronic structures can also be probed using x - ray absorption spectroscopy ( xas ) . coupling xas measurements in the high - energy - resolution fluorescence - detection ( herfd ) mode with theoretical calculations using the anderson impurity model , butorin et al . have recently estimated the crystal field parameters and 5f occupancy in pentavalent nauo3 ( [ rn]5f ) and hexavalent pb3uo6 ( [ rn]5f ) , revealing a significant covalent character of the chemical bond . the xas data available on solid actinide compounds with a valence state higher than iv is still very scarce , however , and the relationship between xas features and electronic density is not fully understood . in this work , we have synthesized k2npo4 and report for the first time a rietveld refinement of its crystal structure and xanes spectrum collected at the np - l3 edge . the relationship between local coordination environment and shape of the xanes spectrum is discussed , as well as the correlation between the edge absorption threshold of the neptunium xanes spectrum and the isomer shift value measured by np mssbauer spectroscopy . moreover , low - temperature heat capacity measurements have been performed to solve the discrepancy regarding the existence of magnetic ordering in this compound . k2npo4 and k2uo4 were synthesized under oxygen flow and air by reaction between accurately weighed samples of neptunium dioxide ( npo2 , ornl , oak ridge ) or uranium dioxide ( uo2.10 , jrc - karlsruhe stocks ) and potassium carbonate ( k2co3 , > 99% , baker ) . the stoichiometric mixtures were heated with intermediate regrinding steps at 1093 k for 22 h and 1073 k for 5 h for the neptunium ( green color ) and uranium ( orange color ) compounds , respectively . u is an emitter with a very long half - life ( 4.47 billion years ) , making it only weakly radioactive . np decays to pa by emission with a half - life of 2.14 million years . the pa daughter product is a emitter with a very short half - life ( 27 days ) and significant dose rate ( 1.335 10 ( msv / h)/mbq ) . the handling of those materials , requiring considerable safety precautions , was therefore done with limited quantities in gloveboxes . the x - ray diffraction measurements were carried out using a bruker d8 x - ray diffractometer mounted in the bragg brentano configuration with a copper tube ( 40 kv , 40 ma ) and a curved ge monochromator ( 111 ) , equipped with a linxeye position - sensitive detector . the data were collected by step scanning in the angle range 10 2 120 over a period of about 8 h. structural analysis at room temperature was performed by the rietveld method with the fullprof2k suite . xanes measurements were performed at the rossendorf beamline ( robl ) of the european synchrotron radiation facility ( esrf , grenoble , france ) on the k2npo4 material . small amounts ( 510 mg ) of powdered sample were mixed with boron nitride ( bn ) in an argon - filled glovebox and pressed into pellets for the measurements . the storage ring operating conditions were 6.0 gev and 170200 ma . a double - crystal monochromator mounted with a si(111 ) crystal coupled to collimating and focusing rh - coated mirrors was used . xanes spectra were collected at room temperature in transmission mode at the np - l3 edge . the energy e0 of the edge absorption threshold position was taken at the first inflection point of the spectrum by using the first node of the second derivative . the position of the white - line maximum was selected from the first node of the first derivative . several acquisitions were performed on the same sample and summed up to improve the signal to noise ratio . before the scans were averaged , each spectrum was aligned using the xanes spectrum of a metallic yttrium ( 17038 ev ) reference foil located between the second and the third ionization chambers and measured at the same time as the sample . the athena software ( version 0.9.20 ) was used to remove the background and to normalize the spectra . low - temperature heat capacity measurements were performed using thermal relaxation calorimetry with a ppms ( physical property measurement system , quantum design ) instrument at applied magnetic fields b = 0 and 9 t in the temperature ranges t = 2.1298.4 k for k2npo4 and t = 2.0312.4 k for k2uo4 , respectively . the measurements were carried out on 22.6(5 ) mg of k2npo4 material encapsulated in stycast 2850 ft , and the heat capacity contribution of the stycast was subtracted from the recorded data . a more detailed description of the experimental procedure , which is particularly well adapted to the study of radioactive materials , was given in ref ( 16 ) . the measurement of k2uo4 was done on 33.2(5 ) mg of material without additional encapsulation in stycast . the contributions of the sample platform , wires , and grease were deduced by a separate measurement of an addenda curve . considering the accuracy of the ppms instrument as estimated by lashley et al . , the reproducibility of the measurements , and the error introduced by the encapsulation procedure in stycast of the radioactive neptunium material , the final uncertainty was estimated to be about 12% in the middle range of acquisition ( 10100 k ) and reach about 3% at the lowest temperatures and near room temperature . self - heating effects coming from the radioactive decay of np were considered but appeared negligible . the use of stycast is the main contributor to the uncertainties on the heat capacity and entropy values quoted hereafter . the final uncertainty for the uranium material is estimated to be about 1% from 100 to 300 k and reach about 3% at the lowest temperatures . the structure of k2uo4 was refined recently on the basis of single - crystal data . the refined cell parameters obtained in the present study , a = 4.3322(3 ) and c = 13.1881(13 ) , are in good agreement with the literature . a rietveld refinement for the k2npo4 phase is reported for the first time in this work ( figure 1 ) . the cell parameters obtained are a = 4.2973(4 ) and c = 13.144(12 ) . the refined atomic positions are given in table 1 and selected bond lengths in table 2 . in this structure , the neptunium cations are 6-fold coordinated , and the octahedra are connected by their equatorial vertices , forming sheets in the ( ab ) plane ( figure 2a ) . the potassium cations , in 9-fold coordination , are located between the sheets , holding them together . the npo6 octahedra show a neptunyl type of coordination , with two short np o1 bonds at 1.84(1 ) in the axial direction and four long np o2 bonds at 2.15(1 ) in the equatorial plane ( figure 2b ) . the presence of a neptunyl configuration is quite common for hexavalent alkali - metal actinide oxide phases . it has been reported already for -na2npo4 ( np2oi = 1.762(5 ) , np4oii = 2.086(5 ) ) , -na2npo4 ( np2oi = 1.90 , np2oii = 2.16 , np2oiii = 2.17 ) ( orthorhombic in space groups pbam and pbca , respectively ) , and banpo4 ( np2oi = 1.89 , np2oii = 2.10 , np2oiii = 2.20 ) ( orthorhombic in space group pbcm ) . the unit cell volume in k2npo4 ( 242.7 ) is smaller than that for k2uo4 ( 247.5 ) , which can be related to the decreasing ionic radius along the series of the actinide elements . comparison between the observed ( yobs , in red ) and calculated ( ycalc , in black ) x - ray diffraction patterns of k2npo4 collected at room temperature ( t = 295 2 k ) . ycalc , in blue , is the difference between the experimental and calculated intensities . the inset shows an enlargement of the refinement in the angle range 2 = 70120. measurements were carried out with = cu k1 radiation . ( a ) crystal structure of k2npo4 ( k atoms in purple , o atoms in red , npo6 octahedra in gray ) showing the sheets of corner - sharing npo6 octahedra in the ( ab ) plane . the xanes spectrum of k2npo4 collected at the np - l3 edge is shown in figure 3 together with those of npo2 , na3npo4 , -na2npo4 , na4npo5 , and na5npo6 reference materials . the valence states of the sodium neptunates were confirmed by np mssbauer spectroscopy from the values of their isomer shifts , while the corresponding xanes spectra were reported in ref ( 10 ) . the values of the measured inflection points and white lines are reported in table 3 . the investigated series covered a wide range of oxidation states ( iv vii ) and a variety of local coordination geometries around the neptunium cation , i.e. , neptunyl ( na3npo4 and -na2npo4 ) , reverse neptunyl ( na4npo5 ) , and distorted npo6 octahedra ( na5npo6 ) , which has allowed us to correlate the shape of the xanes spectra with the local structural environments , as described later in this paper . normalized xanes spectrum of k2npo4 ( present work ) together with those of npo2 , na3npo4 , -na2npo4 , na4npo5 , and na5npo6 reference materials . the secondary white line , if present , is given in italics . the inflection point position of k2npo4 , corresponding to the absorption edge threshold e0 for the 2p 6d transitions , is well aligned with those of -na2npo4 and na4npo5 ( table 3 ) . these results confirm that neptunium is exclusively in the oxidation state vi in k2npo4 , corresponding to a [ rn]5f electronic configuration . the np ion in this structure is therefore a kramers ion with a f5/2 ground state manifold , and a f7/2 first excited state arising from spin orbit coupling . reported a linear correlation of the absorption edge threshold e0 determined by xanes versus the mssbauer isomer shift for the series of sodium neptunates . the value for k2npo4 fits very well with this trend ( figure 4 ) when the isomer shift value determined by nectoux et al . is used : i.e. , is = 56.9(6 ) mm s at 4.2 k relative to the standard npal2 absorber . the linear variation between e0 and is can be understood from the fact that both quantities result from the coulomb interaction with the surrounding electrons . the 5f shells produce a shielding effect on the electronic charge density of the s1/2 and p1/2 inner shells , which affects the isomer shift : is = [e(0 ) ] ( being a calibration constant and e(0 ) the difference in electronic charge density between the source material and the absorber at the nuclear origin ) . moreover , the increase in formal valence state produces a decrease in coulomb energy in the final state between the 5f and 6d electrons and the 2p3/2 core hole , which leads to a shift to higher energy of the absorption edge threshold e0 . absorption edge threshold e0 relative to npo2 versus isomer shift measured by mssbauer spectroscopy . in addition , the xanes spectrum of k2npo4 shows the typical double - peaked white lines ( wl ) of np(v ) , np(vi ) , and np(vii ) compounds , while the tetravalent npo2 compound exhibits a single wl peak . the double peak consists of the main white line at 17620.2(5 ) ev and a shoulder and reduced peak amplitude about 15 ev above the np edge . this feature has traditionally been attributed to localized multiple - scattering resonance of the neptunyl configuration . however , studies on the sodium uranates and neptunates have shown that the correlation between the shape of the xanes spectra and the local coordination geometries is probably more intricate . the isostructural compounds -na2uo4 and -na2npo4 , presenting a neptunyl type of configuration , do not show the expected secondary shoulder and reduced peak amplitude , whereas na4uo5 , presenting a reverse neptunyl type of configuration , does . other factors could play a role , and effects of the degree of localization of the 5f electrons and core - ionized final states with different 5f occupancies have been suggested . the absorption edge threshold e0 is finally slightly higher ( 0.3 ev ) for k2npo4 than for -na2np(vi)o4 , which could be related to the neptunyl bond distances ( table 2 ) , degree of covalency , or ( in other words ) degree of localization of the 5f electrons in both compounds . the shorter the np o bonds , the more localized the 5f electrons , and the greater the coulomb energy , making it easier to eject an electron from the 2p3/2 core shell . however , it is not possible to conclude definitively , given the experimental uncertainties ( 0.5 ev ) of the measurements at the np - l3 edge , and this would require performing high - energy - resolution fluorescence - detected ( herfd ) xanes measurements at the m4 edge . the heat capacity data of k2npo4 and k2uo4 measured at low temperatures in the absence of a magnetic field are shown in figure 5 and given in tables 5 and 6 of the appendix . the two curves cross above t = 150 k , although one would expect them to become equal , corresponding to the same lattice contribution at high temperatures for the uranium and neptunium compounds . however , this discrepancy can be related on the one hand to the uncertainty on our experimental results , which increases toward high temperatures using the ppms technique , and on the other hand to the fact that one compound was measured with stycast and the other without . the uncertainty on the neptunium data corrected for the stycast contribution is around 3% at room temperature , whereas that of the uranium data measured without stycast is around 1% . the heat capacities reach values that are about 1724 j k mol below the classical dulong petit limit ( clat = 21r 174.6 j k mol for the seven atoms in the formula unit ) as the temperature approaches 298.15 k. heat capacity of k2npo4 ( black ) and k2uo4 ( blue ) measured in zero magnetic field and the numerical fit to the neptunium ( red line ) and uranium ( blue dotted line ) data . the collected data for k2npo4 show a small anomaly at t = 25.9 k , which is almost unaffected by the application of a 9 t magnetic field apart from a small decrease in the amplitude . this feature could be interpreted at first as an indication of the presence of npo2 impurity within the investigated material . indeed , neptunium dioxide shows a sharp anomaly at t = 25.7 k due to rank 5 triacontadipolar order as described in the studies of santini et al . . however , the x - ray diffraction data did not reveal any secondary npo2 phase . moreover , the shape of the anomaly in k2npo4 does not match that of npo2 , although the critical temperatures are very close . the anomaly is very symmetrical in k2npo4 , in contrast with npo2 , showing an asymmetrical profile . on the basis of its amplitude , the amount of npo2 impurity would correspond to 26.9 1.0% , which should be detected easily by the x - rays . the corresponding magnetic contribution was derived as smag = 3.1 0.1 j k mol after subtraction of the lattice heat capacity contribution ( figure 7 ) . the latter was approximated with the heat capacity of k2uo4 ( which has electronic configuration [ rn]5f ) , as the two compounds are isostructural and have very similar atomic masses . it is worth pointing out that a low magnetic entropy , i.e. 0.19rln2 , has also been reported for -na2npo4 . interestingly , no anomaly was observed around 19.5(5 ) k , as could be expected from the mssbauer results of nectoux et al . an x - ray diffraction pattern collected after the low - temperature heat capacity measurement moreover confirmed that the sample had retained its integrity during the experiment . since the magnetic susceptibility measurements of the authors also did not show any anomaly around 20 k , the existence of a first - order magnetic phase transition at the latter temperature is unlikely . to explain the origin of the magnetic hyperfine splitting reported below 19.5(5 ) k , we could suggest the occurrence of a slow electron spin relaxation phenomenon in this paramagnetic system . however , this is doubtful , as it would require a sudden collapse of the relaxation time at the critical temperature . the cell parameters reported by for k2npo4 ( a = 4.26 and c = 13.01 ) are lower than those found in this study . a contamination of their sample with a magnetic impurity is possible although unlikely , as they reported a single - phase material , but they give very little detail on phase preparation and purity . the magnetic hyperfine splitting effect observed by the authors probably corresponds to the anomaly observed herein at 25.9 k , with a somewhat lower critical temperature . it should be pointed out that differences in critical temperatures of about 38 k have been reported in the literature between mssbauer and magnetic susceptibility results of the uranium for the magnetic susceptibilty results of nectoux et al . , a clear deviation from the curie weiss law is observed below about 40 k , which could suggest ferromagnetic ordering below the latter temperature . the hypothesis of a ferromagnetic transition is moreover in good agreement with the low - temperature heat capacity data , showing a slight decrease of the anomaly at t = 25.9 k upon application of a magnetic field . the negative value of the curie constant , i.e. , p = 150 k , derived from the curie it suggests a more complex order , possibly with a canting of the ferromagnetically coupled moments or with strong antiferromagnetic interactions . in the present work , the thermodynamic functions of k2npo4 and k2uo4 were derived at 298.15 k by fitting the experimental data to theoretical functions below t = 8.0 k and t = 20.0 k , respectively , and a combination of debye and einstein heat capacity functions at t = 7.8298.4 k and at t = 20.0312.4 k , respectively . the fitted data are shown with solid and dotted lines in figures 5 and 6 . the heat capacity values at 298.15 k were obtained by interpolation , yielding cp , m(k2npo4 , cr , 298.15 k ) = 152.7 4.5 j k mol and cp , m(k2uo4 , cr , 298.15 k ) = 156.5 1.6 j k mol ( in both cases , the quoted uncertainty corresponds to the standard uncertainty ) . the experimental standard entropies at 298.15 k were determined by numerical integration of cp , m / t = f(t ) using the aforementioned fitted functions and including the magnetic entropy contribution , yielding sm(k2npo4 , cr , 298.15 k ) = 209.3 4.9 j k mol and sm(k2uo4 , cr , 298.15 k ) = 210.1 2.7 j k mol , respectively . the values obtained for k2npo4 are slightly lower than that of k2uo4 , whereas the inverse behavior would be expected . however , this is due to the uncertainty introduced by the use of stycast as mentioned before and the crossing of the two curves . when adding the derived magnetic entropy to the lattice contribution of k2uo4 , one derives 213.2 j k mol for the standard entropy of k2npo4 , which remains within the uncertainty of the present measurement . cp / t for k2npo4 ( black ) and k2uo4 ( blue ) measured in zero magnetic field and the numerical fit to the neptunium ( red line ) and uranium ( blue dotted line ) data . at very low temperatures where the thermal expansion is negligible , the heat capacity at constant pressure can be approximated to the heat capacity at constant volume cp , m cv , m , which comprises lattice vibrations , electronic , and magnetic contributions the lattice contribution dominates at temperatures above about t = 820 k and can be modeled using a combination of debye and einstein functions , as shown in eq 1 . fitting with a single einstein function was attempted but could not reproduce accurately the high - temperature region:1where r is the universal gas constant equal to 8.3144621 j k mol , d(d ) , e(e1 ) , and e(e2 ) are the debye and einstein functions , respectively , as written in eqs 2 and 3 . nd , ne1 , and ne2 are adjustable parameters , whose sum nd + ne1 + ne2 should be approximately equal to the number of atoms in the formula unit ( i.e. , 7 in this case).23 the fitted parameters are listed in table 4 . the sum nd + ne1 + ne2 is very close to 7 . at very low temperatures ( t < 20 k ) , the phonon contribution is well - represented using a harmonic lattice model , as expressed by the polynomial function ( 4 ) , where the number of required terms augments the high - temperature limit of the fit:4the electronic contribution of the conduction electrons at the fermi surface are represented with the linear term t . for insulating materials such as k2npo4 and k2uo4 , however , a linear term was reported in materials such as -feooh , fe3(p2o7)2 , and sr2tisi2o8 , which was related to departure from stoichiometry , oxygen vacancies , or defects within the material . the heat capacity of k2npo4 was fitted with the harmonic model using four terms over the temperature range t = 2.18.0 k. that of k2uo4 was fitted with five terms over the temperature range t = 2.020.3 k. the corresponding coefficients are given in table 4 . in addition , the use of a linear t term appeared necessary to describe the experimental curve of k2npo4 . more recently , the occurrence of such a linear term was also reported in na4npo5 , which was related to the presence of defects within the material and an asymmetric peak profile shape in opposite directions for successive hkl reflections clearly visible on the x - ray diffraction pattern . the x - ray diffraction data of k2npo4 do not show such features , however . self - heating effects coming from the radioactive decay of np were considered but appeared negligible . moreover , departure from stoichiometry is unlikely according to the present np - l3 xanes results and mssbauer data of ref ( 8) . the appearance of a nuclear schottky effect arising from the magnetic hyperfine splitting interaction between the unpaired 5f electron and the magnetic moment at the np nucleus ( i = 5/2 ) was suggested for na2npo4 , as the corresponding data showed a reincrease below 3.7 k. k2npo4 might show similar behavior ( figure 7 ) , but we can not conclude in the absence of data below 2.0 k , which would require complementary measurements using a he refrigerator . electronic contribution to the heat capacity in k2npo4 obtained by subtracting the data for k2uo4 . a rietveld refinement of the crystal structure of k2npo4 , tetragonal in space group i4/mmm , is reported for the first time in the present work . the refined cell parameters and bond lengths are in good agreement with the trend of decreasing ionic radii along the actinide series . xanes data have also been collected at the np - l3 edge , which have confirmed the hexavalent state of neptunium in this compound and therefore the assigned stoichiometry . the measured absorption edge threshold e0 fits very well the linear correlation observed for the sodium neptunates between e0 and the isomer shift value is measured by mssbauer spectroscopy . moreover , double - peak white lines have been observed for k2npo4 , which are usually attributed to multiple scattering resonances of the actinyl compounds , but the interpretation could be more intricate . electronic density calculations are needed to obtain more insight into those complex features . low - temperature heat capacity data have been collected in the temperature range t = 2.1298.4 k for k2npo4 and t = 2.0312.4 k for k2uo4 , and the standard entropy and heat capacity of both compounds have been derived at 298.15 k. the latter data have revealed the presence of an anomaly at 25.9 k with an associated magnetic entropy smag = 3.1 0.1 j k mol , which most probably corresponds to the magnetic hyperfine splitting event observed in the literature by mssbauer spectroscopy at a slightly lower temperature : i.e. , t = 19.5(5 ) k. both the present low - temperature heat capacity data and the magnetic susceptibility measurements of nectoux et al . are consistent with the hypothesis of a ferromagnetic ordering transition around t = 25.9 k. complementary studies involving repeated mssbauer spectroscopy and magnetic susceptibility measurements , as well as neutron diffraction measurements on a well - characterized material , would allow confirmation of those results . finally , the amplitude of the anomaly at 25.9 k is smaller than expected for this kramers system ( smag = r ln 2 ) , but similar results have also been reported for -na2npo4 . the low values of the ordered moment derived from the mssbauer data ( 0.6 b ) , of the paramagnetic effective moment derived from the magnetic susceptibility data ( eff = 1.37 b ) , and of the magnetic entropy ( smag = 0.538r ln 2 ) , are not unusual for 5f systems . further investigations involving spectroscopy measurements at low energy and theoretical calculations are clearly required to get further insight into the np(vi ) crystal - field ground state and magnetic behavior of the alkali and alkaline - earth neptunates .

the physicochemical properties of the potassium neptunate k2npo4 have been investigated in this work using x - ray diffraction , x - ray absorption near edge structure ( xanes ) spectroscopy at the np - l3 edge , and low - temperature heat capacity measurements . a rietveld refinement of the crystal structure is reported for the first time . the np(vi ) valence state has been confirmed by the xanes data , and the absorption edge threshold of the xanes spectrum has been correlated to the mssbauer isomer shift value reported in the literature . the standard entropy and heat capacity of k2npo4 have been derived at 298.15 k from the low - temperature heat capacity data . the latter suggest the existence of a magnetic ordering transition around 25.9 k , most probably of the ferromagnetic type .

Introduction Experimental Methods Results and Discussion Conclusion

this character leads to a wide range of oxidation states , between + 3 and + 7 , and the occurrence of magnetic ordering behavior . the theoretical description of these systems appears extremely challenging , however , as the crystal field interaction is usually of the same order of magnitude as the spin the crystal - field interaction can not be treated as a small perturbation of the electronic energy levels as is done for the [ xe]4f rare earths . those studies have revealed intriguing magnetic properties for the -na2npo4 and na4npo5 compositions and have stressed the need to re - evaluate the np(vi ) crystal - field ground state . the physical and chemical properties of k2npo4 have been investigated in the present work in an attempt to bring new insights into the complex behavior of np(vi ) phases . however , their magnetic susceptibility data did not show any sign of an anomaly around 20 k , as could be expected from the mssbauer results . electronic structures can also be probed using x - ray absorption spectroscopy ( xas ) . the xas data available on solid actinide compounds with a valence state higher than iv is still very scarce , however , and the relationship between xas features and electronic density is not fully understood . in this work , we have synthesized k2npo4 and report for the first time a rietveld refinement of its crystal structure and xanes spectrum collected at the np - l3 edge . the relationship between local coordination environment and shape of the xanes spectrum is discussed , as well as the correlation between the edge absorption threshold of the neptunium xanes spectrum and the isomer shift value measured by np mssbauer spectroscopy . moreover , low - temperature heat capacity measurements have been performed to solve the discrepancy regarding the existence of magnetic ordering in this compound . the x - ray diffraction measurements were carried out using a bruker d8 x - ray diffractometer mounted in the bragg brentano configuration with a copper tube ( 40 kv , 40 ma ) and a curved ge monochromator ( 111 ) , equipped with a linxeye position - sensitive detector . xanes spectra were collected at room temperature in transmission mode at the np - l3 edge . the energy e0 of the edge absorption threshold position was taken at the first inflection point of the spectrum by using the first node of the second derivative . the position of the white - line maximum was selected from the first node of the first derivative . before the scans were averaged , each spectrum was aligned using the xanes spectrum of a metallic yttrium ( 17038 ev ) reference foil located between the second and the third ionization chambers and measured at the same time as the sample . low - temperature heat capacity measurements were performed using thermal relaxation calorimetry with a ppms ( physical property measurement system , quantum design ) instrument at applied magnetic fields b = 0 and 9 t in the temperature ranges t = 2.1298.4 k for k2npo4 and t = 2.0312.4 k for k2uo4 , respectively . the measurements were carried out on 22.6(5 ) mg of k2npo4 material encapsulated in stycast 2850 ft , and the heat capacity contribution of the stycast was subtracted from the recorded data . , the reproducibility of the measurements , and the error introduced by the encapsulation procedure in stycast of the radioactive neptunium material , the final uncertainty was estimated to be about 12% in the middle range of acquisition ( 10100 k ) and reach about 3% at the lowest temperatures and near room temperature . a rietveld refinement for the k2npo4 phase is reported for the first time in this work ( figure 1 ) . in this structure , the neptunium cations are 6-fold coordinated , and the octahedra are connected by their equatorial vertices , forming sheets in the ( ab ) plane ( figure 2a ) . comparison between the observed ( yobs , in red ) and calculated ( ycalc , in black ) x - ray diffraction patterns of k2npo4 collected at room temperature ( t = 295 2 k ) . ( a ) crystal structure of k2npo4 ( k atoms in purple , o atoms in red , npo6 octahedra in gray ) showing the sheets of corner - sharing npo6 octahedra in the ( ab ) plane . the xanes spectrum of k2npo4 collected at the np - l3 edge is shown in figure 3 together with those of npo2 , na3npo4 , -na2npo4 , na4npo5 , and na5npo6 reference materials . the valence states of the sodium neptunates were confirmed by np mssbauer spectroscopy from the values of their isomer shifts , while the corresponding xanes spectra were reported in ref ( 10 ) . , neptunyl ( na3npo4 and -na2npo4 ) , reverse neptunyl ( na4npo5 ) , and distorted npo6 octahedra ( na5npo6 ) , which has allowed us to correlate the shape of the xanes spectra with the local structural environments , as described later in this paper . normalized xanes spectrum of k2npo4 ( present work ) together with those of npo2 , na3npo4 , -na2npo4 , na4npo5 , and na5npo6 reference materials . the inflection point position of k2npo4 , corresponding to the absorption edge threshold e0 for the 2p 6d transitions , is well aligned with those of -na2npo4 and na4npo5 ( table 3 ) . the np ion in this structure is therefore a kramers ion with a f5/2 ground state manifold , and a f7/2 first excited state arising from spin orbit coupling . reported a linear correlation of the absorption edge threshold e0 determined by xanes versus the mssbauer isomer shift for the series of sodium neptunates . the 5f shells produce a shielding effect on the electronic charge density of the s1/2 and p1/2 inner shells , which affects the isomer shift : is = [e(0 ) ] ( being a calibration constant and e(0 ) the difference in electronic charge density between the source material and the absorber at the nuclear origin ) . moreover , the increase in formal valence state produces a decrease in coulomb energy in the final state between the 5f and 6d electrons and the 2p3/2 core hole , which leads to a shift to higher energy of the absorption edge threshold e0 . absorption edge threshold e0 relative to npo2 versus isomer shift measured by mssbauer spectroscopy . in addition , the xanes spectrum of k2npo4 shows the typical double - peaked white lines ( wl ) of np(v ) , np(vi ) , and np(vii ) compounds , while the tetravalent npo2 compound exhibits a single wl peak . however , studies on the sodium uranates and neptunates have shown that the correlation between the shape of the xanes spectra and the local coordination geometries is probably more intricate . other factors could play a role , and effects of the degree of localization of the 5f electrons and core - ionized final states with different 5f occupancies have been suggested . the absorption edge threshold e0 is finally slightly higher ( 0.3 ev ) for k2npo4 than for -na2np(vi)o4 , which could be related to the neptunyl bond distances ( table 2 ) , degree of covalency , or ( in other words ) degree of localization of the 5f electrons in both compounds . the shorter the np o bonds , the more localized the 5f electrons , and the greater the coulomb energy , making it easier to eject an electron from the 2p3/2 core shell . however , it is not possible to conclude definitively , given the experimental uncertainties ( 0.5 ev ) of the measurements at the np - l3 edge , and this would require performing high - energy - resolution fluorescence - detected ( herfd ) xanes measurements at the m4 edge . the heat capacity data of k2npo4 and k2uo4 measured at low temperatures in the absence of a magnetic field are shown in figure 5 and given in tables 5 and 6 of the appendix . the two curves cross above t = 150 k , although one would expect them to become equal , corresponding to the same lattice contribution at high temperatures for the uranium and neptunium compounds . however , this discrepancy can be related on the one hand to the uncertainty on our experimental results , which increases toward high temperatures using the ppms technique , and on the other hand to the fact that one compound was measured with stycast and the other without . the heat capacities reach values that are about 1724 j k mol below the classical dulong petit limit ( clat = 21r 174.6 j k mol for the seven atoms in the formula unit ) as the temperature approaches 298.15 k. heat capacity of k2npo4 ( black ) and k2uo4 ( blue ) measured in zero magnetic field and the numerical fit to the neptunium ( red line ) and uranium ( blue dotted line ) data . the collected data for k2npo4 show a small anomaly at t = 25.9 k , which is almost unaffected by the application of a 9 t magnetic field apart from a small decrease in the amplitude . however , the x - ray diffraction data did not reveal any secondary npo2 phase . interestingly , no anomaly was observed around 19.5(5 ) k , as could be expected from the mssbauer results of nectoux et al . an x - ray diffraction pattern collected after the low - temperature heat capacity measurement moreover confirmed that the sample had retained its integrity during the experiment . since the magnetic susceptibility measurements of the authors also did not show any anomaly around 20 k , the existence of a first - order magnetic phase transition at the latter temperature is unlikely . to explain the origin of the magnetic hyperfine splitting reported below 19.5(5 ) k , we could suggest the occurrence of a slow electron spin relaxation phenomenon in this paramagnetic system . the magnetic hyperfine splitting effect observed by the authors probably corresponds to the anomaly observed herein at 25.9 k , with a somewhat lower critical temperature . it should be pointed out that differences in critical temperatures of about 38 k have been reported in the literature between mssbauer and magnetic susceptibility results of the uranium for the magnetic susceptibilty results of nectoux et al . , a clear deviation from the curie weiss law is observed below about 40 k , which could suggest ferromagnetic ordering below the latter temperature . the hypothesis of a ferromagnetic transition is moreover in good agreement with the low - temperature heat capacity data , showing a slight decrease of the anomaly at t = 25.9 k upon application of a magnetic field . , p = 150 k , derived from the curie it suggests a more complex order , possibly with a canting of the ferromagnetically coupled moments or with strong antiferromagnetic interactions . in the present work , the thermodynamic functions of k2npo4 and k2uo4 were derived at 298.15 k by fitting the experimental data to theoretical functions below t = 8.0 k and t = 20.0 k , respectively , and a combination of debye and einstein heat capacity functions at t = 7.8298.4 k and at t = 20.0312.4 k , respectively . the heat capacity values at 298.15 k were obtained by interpolation , yielding cp , m(k2npo4 , cr , 298.15 k ) = 152.7 4.5 j k mol and cp , m(k2uo4 , cr , 298.15 k ) = 156.5 1.6 j k mol ( in both cases , the quoted uncertainty corresponds to the standard uncertainty ) . the experimental standard entropies at 298.15 k were determined by numerical integration of cp , m / t = f(t ) using the aforementioned fitted functions and including the magnetic entropy contribution , yielding sm(k2npo4 , cr , 298.15 k ) = 209.3 4.9 j k mol and sm(k2uo4 , cr , 298.15 k ) = 210.1 2.7 j k mol , respectively . however , this is due to the uncertainty introduced by the use of stycast as mentioned before and the crossing of the two curves . when adding the derived magnetic entropy to the lattice contribution of k2uo4 , one derives 213.2 j k mol for the standard entropy of k2npo4 , which remains within the uncertainty of the present measurement . at very low temperatures where the thermal expansion is negligible , the heat capacity at constant pressure can be approximated to the heat capacity at constant volume cp , m cv , m , which comprises lattice vibrations , electronic , and magnetic contributions the lattice contribution dominates at temperatures above about t = 820 k and can be modeled using a combination of debye and einstein functions , as shown in eq 1 . nd , ne1 , and ne2 are adjustable parameters , whose sum nd + ne1 + ne2 should be approximately equal to the number of atoms in the formula unit ( i.e. at very low temperatures ( t < 20 k ) , the phonon contribution is well - represented using a harmonic lattice model , as expressed by the polynomial function ( 4 ) , where the number of required terms augments the high - temperature limit of the fit:4the electronic contribution of the conduction electrons at the fermi surface are represented with the linear term t . the heat capacity of k2npo4 was fitted with the harmonic model using four terms over the temperature range t = 2.18.0 k. that of k2uo4 was fitted with five terms over the temperature range t = 2.020.3 k. the corresponding coefficients are given in table 4 . more recently , the occurrence of such a linear term was also reported in na4npo5 , which was related to the presence of defects within the material and an asymmetric peak profile shape in opposite directions for successive hkl reflections clearly visible on the x - ray diffraction pattern . the x - ray diffraction data of k2npo4 do not show such features , however . moreover , departure from stoichiometry is unlikely according to the present np - l3 xanes results and mssbauer data of ref ( 8) . the appearance of a nuclear schottky effect arising from the magnetic hyperfine splitting interaction between the unpaired 5f electron and the magnetic moment at the np nucleus ( i = 5/2 ) was suggested for na2npo4 , as the corresponding data showed a reincrease below 3.7 k. k2npo4 might show similar behavior ( figure 7 ) , but we can not conclude in the absence of data below 2.0 k , which would require complementary measurements using a he refrigerator . a rietveld refinement of the crystal structure of k2npo4 , tetragonal in space group i4/mmm , is reported for the first time in the present work . xanes data have also been collected at the np - l3 edge , which have confirmed the hexavalent state of neptunium in this compound and therefore the assigned stoichiometry . the measured absorption edge threshold e0 fits very well the linear correlation observed for the sodium neptunates between e0 and the isomer shift value is measured by mssbauer spectroscopy . low - temperature heat capacity data have been collected in the temperature range t = 2.1298.4 k for k2npo4 and t = 2.0312.4 k for k2uo4 , and the standard entropy and heat capacity of both compounds have been derived at 298.15 k. the latter data have revealed the presence of an anomaly at 25.9 k with an associated magnetic entropy smag = 3.1 0.1 j k mol , which most probably corresponds to the magnetic hyperfine splitting event observed in the literature by mssbauer spectroscopy at a slightly lower temperature : i.e. , t = 19.5(5 ) k. both the present low - temperature heat capacity data and the magnetic susceptibility measurements of nectoux et al . are consistent with the hypothesis of a ferromagnetic ordering transition around t = 25.9 k. complementary studies involving repeated mssbauer spectroscopy and magnetic susceptibility measurements , as well as neutron diffraction measurements on a well - characterized material , would allow confirmation of those results . finally , the amplitude of the anomaly at 25.9 k is smaller than expected for this kramers system ( smag = r ln 2 ) , but similar results have also been reported for -na2npo4 . the low values of the ordered moment derived from the mssbauer data ( 0.6 b ) , of the paramagnetic effective moment derived from the magnetic susceptibility data ( eff = 1.37 b ) , and of the magnetic entropy ( smag = 0.538r ln 2 ) , are not unusual for 5f systems . further investigations involving spectroscopy measurements at low energy and theoretical calculations are clearly required to get further insight into the np(vi ) crystal - field ground state and magnetic behavior of the alkali and alkaline - earth neptunates .

this study was approved by the institutional review boards of the participating centers , according to the tenets of the declaration of helsinki . informed consent was obtained from all patients at the initial visit in pakistan at the el shifa trust eye hospital , where two of our clinical collaborators saw the babies ( ak , ss ) and another oversaw the dna extractions ( rq ) ; the consent form contained a paragraph about genetic analyses of retinal genes , including whole exome sequencing . twenty - one ncrna families ( 28 affected members ) were recruited , phenotyped , and genotyped from pakistan and included in this study . entry criteria were as follows : the diagnosis of ncrna was given to patients with the history of congenital blindness because of partial to complete retinal detachment from infancy , shallow anterior chamber , leukocoria , and abnormal b - scan . all children included in this study were born full term , free from uncomplicated pregnancies , and did not have any known medical issues . all families were questioned for detailed , ocular , and visual histories , and pedigrees were drawn . blood samples from all available ncrna family members were collected at the al shifa trust eye hospital department of pediatric ophthalmology and strabismus , rawalpindi , pakistan . extraction of dna from the ncrna families and additional blood samples from 100 random unrelated healthy pakistani control individuals were provided by the comsats institute of information technology department of biosciences , rawalpindi , pakistan , for determination of allele frequency in the general pakistani population . genomic dna was extracted from peripheral blood leukocytes with an extraction kit ( flexigene ; qiagen , hilden , germany ) and a blood kit ( qiaamp dna ; qiagen ) , according to the instructions by the manufacturer . dna quantity and quality was verified by spectrophotometer ( nanodrop 1000 ; thermo fisher scientific , wilmington , de , usa ) . primers were designed by exon primer ( provided in the public domain by the institute for human genetics , technical university of munich , germany ( http://ihg.gsf.de/ihg/ exonprimer.html/ ) and by primer3 online program . to ensure the completeness and quality of the sequences and for detection of potential mutations located in splice sites , a minimal distance between primer and exon / the genomes of 11 out of our 17 consanguineous ncrna families were analyzed for homozygous chromosomal regions by using snp homozygosity mapping ( infinium hd 660k ; illumina , inc . , san diego , ca , usa ) , in accordance with the protocol provided by the manufacturer . homozygous regions were visualized and identified with a commercial software package ( bead studio plink ; illumina , inc . ) and a freeware spreadsheet , excludear . chromosomal segments were accepted as having significant homozygous regions if they contained 300 consecutive homozygous snps , as the likelihood that this would occur by chance is less than 1:100 with a particular focus to identified homozygous regions . all the known retinal genes ( the retinal genes listed in retinal network information website ) that resided in homozygous chromosomal segments were analyzed for mutations using sanger sequencing . fourteen patients diagnosed with ncrna were screened by our recently developed retnet known retinal disease gene panel , which contains all the currently known retinal disease genes ( 163 ) by targeted ngs . the chip helped us to screen all retinal genes listed in the retinal information network website and create a candidate retinal disease gene list for future study . we performed whole exome capture following the manufacturer 's protocol ( illumina , inc . ) . exome capture ( sureselect v4 ; agilent technologies , santa clara , ca , usa ) was performed according to the company 's standard protocol . the amplified capture product was sequenced with commercial software ( hiseq 2000 ; illumina , inc . ) to pinpoint the genetic variants responsible for the disease in those patients who did not carry mutations in known genes . sequence capture was performed in accordance with the protocol of the manufacturer ( illumina , inc . ) in the sequencing platform of the mcgill university and genome quebec innovation centre . known retinal genes within the homozygous regions , retinal detachment genes shown in retnet ( retinal information network , tables of genes and loci causing inherited retinal diseases ) , and all putative mutations found by retnet panel screening were sanger - sequenced . complete sequencing of the candidate genes within the homozygous regions was done to exclude all possible mutations . if a patient had several homozygous regions carrying different known genes for the given disease , the screening of the genes was started from a gene located in the biggest homozygous segment . the assays were performed by the sequencing platform of the mcgill university and genome quebec innovation centre using forward or reverse sequencing primers designed for pcr reactions that covered a designated exons and splice sites of the exons . the results were analyzed using an ngs software program ( sequencher ; gene codes corporation , ann arbor , mi , usa ) . for novel mutations found , we performed in silico analyses predicting the variants to be damaging by sift , blosum62 , and polyphen-2 . we then studied conservation of the residue in homologous proteins of lower animals and we verified that the variant has not been reported in the exome variant server ( nhlbi go exome sequencing project ) , dbsnp135 , or 1000 genome datasets . finally , segregation of the mutations between family members was then performed and we excluded the newly found variant from normal controls that are culturally matched to the original family . genomic dna was extracted from peripheral blood leukocytes with an extraction kit ( flexigene ; qiagen , hilden , germany ) and a blood kit ( qiaamp dna ; qiagen ) , according to the instructions by the manufacturer . dna quantity and quality was verified by spectrophotometer ( nanodrop 1000 ; thermo fisher scientific , wilmington , de , usa ) . primers were designed by exon primer ( provided in the public domain by the institute for human genetics , technical university of munich , germany ( http://ihg.gsf.de/ihg/ exonprimer.html/ ) and by primer3 online program . to ensure the completeness and quality of the sequences and for detection of potential mutations located in splice sites , a minimal distance between primer and exon / the genomes of 11 out of our 17 consanguineous ncrna families were analyzed for homozygous chromosomal regions by using snp homozygosity mapping ( infinium hd 660k ; illumina , inc . , san diego , ca , usa ) , in accordance with the protocol provided by the manufacturer . homozygous regions were visualized and identified with a commercial software package ( bead studio plink ; illumina , inc . ) and a freeware spreadsheet , excludear . chromosomal segments were accepted as having significant homozygous regions if they contained 300 consecutive homozygous snps , as the likelihood that this would occur by chance is less than 1:100 with a particular focus to identified homozygous regions . all the known retinal genes ( the retinal genes listed in retinal network information website ) that resided in homozygous chromosomal segments were analyzed for mutations using sanger sequencing . fourteen patients diagnosed with ncrna were screened by our recently developed retnet known retinal disease gene panel , which contains all the currently known retinal disease genes ( 163 ) by targeted ngs . the chip helped us to screen all retinal genes listed in the retinal information network website and create a candidate retinal disease gene list for future study . we performed whole exome capture following the manufacturer 's protocol ( illumina , inc . ) . exome capture ( sureselect v4 ; agilent technologies , santa clara , ca , usa ) was performed according to the company 's standard protocol . the amplified capture product was sequenced with commercial software ( hiseq 2000 ; illumina , inc . ) to pinpoint the genetic variants responsible for the disease in those patients who did not carry mutations in known genes . sequence capture was performed in accordance with the protocol of the manufacturer ( illumina , inc . ) in the sequencing platform of the mcgill university and genome quebec innovation centre . known retinal genes within the homozygous regions , retinal detachment genes shown in retnet ( retinal information network , tables of genes and loci causing inherited retinal diseases ) , and all putative mutations found by retnet panel screening were sanger - sequenced . complete sequencing of the candidate genes within the homozygous regions was done to exclude all possible mutations . if a patient had several homozygous regions carrying different known genes for the given disease , the screening of the genes was started from a gene located in the biggest homozygous segment . the assays were performed by the sequencing platform of the mcgill university and genome quebec innovation centre using forward or reverse sequencing primers designed for pcr reactions that covered a designated exons and splice sites of the exons . the results were analyzed using an ngs software program ( sequencher ; gene codes corporation , ann arbor , mi , usa ) . for novel mutations found , we performed in silico analyses predicting the variants to be damaging by sift , blosum62 , and polyphen-2 . we then studied conservation of the residue in homologous proteins of lower animals and we verified that the variant has not been reported in the exome variant server ( nhlbi go exome sequencing project ) , dbsnp135 , or 1000 genome datasets . finally , segregation of the mutations between family members was then performed and we excluded the newly found variant from normal controls that are culturally matched to the original family . in collaboration with a research group in islamabad , pakistan , we assembled 21 consanguineous pedigrees from pakistan with an ncrna diagnosis . consanguineous families have homozygous regions , one of which contains the causal gene with homozygous mutations . this fact allows for a genetic strategy that is well established , called homozygosity mapping and inheritance by descent ( ibd ) studies . our approach to finding the causal genes in ncrna patients was to first identify all the significant homozygous regions in the probands ( patients ) and then look for overlap of all these regions with all affected members of that family , followed by searching for overlap with other families . this three - step process allows us to narrow and decrease the number of homozygous regions . the remaining overlapping regions can then be probed for candidate genes that are expressed in retina , using available databases . in a preliminary study of our cohort , we screened families for known ncrna and retinal detachment genes ( including fevr genes ) which resided in one of the top five homozygous segments documented by the snp array . we hypothesized that the causal gene for ncrna resides in one of the top five largest intervals of our identified homozygous regions in consanguineous families and a new causal gene underlying the disease may locate in the overlapping homozygous regions of different family members with the same disease phenotype . analyzes of homozygous regions of the patients and direct sequencing of the candidate genes located in top five regions helped us to successfully genotype seven ncrna families with known genes located in the regions of interest ( table 1 ) . candidate genes for ncrna families in their ibd regions summary of analysis done by bioinformatics tools for novel mutations summary of successfully genotyped ncrna families the affected individuals presented with the history of congenital blindness and presented with searching roving eye movements . some patients had cloudy corneas due to iridocorneal adhesion , shallow anterior chamber due to forward displacement of lens iris diaphragm , and irregular pupils secondary to posterior synechiae and leukocoria ( white pupils ) ; but there was no consistent elevation of intraocular pressure . fundoscopy , when possible , revealed a dense fibrovasular mass behind the lens in each eye , originating from the optic nerve and attached behind the lens and peripheral fundus . in some cases , other cases showed total retinal detachments and fibrous bands originating from optic nerve posteriorly and attached anteriorly behind the lens . together , these clinical findings suggest bilateral persistence of the fetal blood vessels , with complete congenital detachment of the neural retina and intravitreal vascular proliferation during infancy . they have good vision , with no structural or functional eye pathology . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . surprisingly , two of these families harbored the reported deletion in atoh7 , which is the well - known , common mutation in the founder population of north khorasan , in iran . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . to be sure , we screened all our patients for atoh7 exonic and promoter mutations by sanger sequencing . two families ( ncrna5 and ncrna6 ) were found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 ( phenotypes of the patients are shown in fig . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue ( fig . family ncrna5 was found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 . anterior segment with leukocoria ( a ) and disorganized b scan showing retrolental mass with total vitreous condensation ( b ) caused by the variation in atoh7 gene in ncrna5 - 4 . polymerase chain reaction based genotyping of homozygous 6523-kb deletion in 5atoh7 in ncrna5 and ncrna6 families . ( a ) a polymerase chain reaction based deletion confirmation in the ncrna5 and ncrna6 families conducted as described by ghiasvand et al . letters ( c , d , e , f , g , h ) represent six adjacent amplicons in an ncrna subject that amplify conserved noncoding elements 20 kb upstream of atoh7 transcription start site . as it can be seen from the figure , c and h amplicons are present in both wild - type ( wt ) and mutant subjects , while the rest of the amplicons are missing in ncrna5 and ncrna6 family members . ( b ) cosegregation results between family members using primers designed by ghiasvand et al . in the first panel , primers for g and h amplicons are pooled , whereas in the second panel , only primers for h amplicons were used to verify if deletion of the area is present in the members of the family . we found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . in silico analyses predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . however , lrp5 was the only attractive retinal candidate gene in the region , and sanger sequencing identified the mutations in the gene . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . a member from a third family ( ncrna7 - 4 ) we then identified the reported homozygous p.gly1401asp mutation in three members of a single family ( ncrna19 - 3 , 19 - 4 , and 19 - 6 ) . panel screening with retnet then identified htz and hmz lrp5 mutations in an additional two families ( ncrna16 and ncrna20 , respectively ) ; ncrna16 has the previously reported p.arg142gln change and the ncrna20 family carries a novel p.leu541pro alteration . since the p.gly1401asp mutation in lrp5 found in the ncrna19 family members has previously been reported to cause osteoporosis - pseudoglioma , we checked bone mineral density ( bmd ) in patient ncrna19 - 4 and the result confirmed that the patient has reduced bmd and osteoporosis . the rest of the families were not able to come back for the given test . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . 4 ) . the altered residues are all evolutionarily conserved ( fig . 5 ) . we then documented delayed growth and skeletal abnormalities in some patients in these families ( fig . pedigrees for successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family positive for homozygous lrp5 mutations are shown in figure 6 . eye phenotype for ncrna2 , ncrna3 , ncrna7 and ncrna19 families and skeletal abnormalities found in a ncrna19 family member . ( a ) the fundus photo shows the fibrovascular fold arising from the optic nerve and extending to the retrolental area in the temporal region in ncrna2 - 5 ; family ncrna2 has been identified to carry a homozygous p. gly610arg alteration in lrp5 gene . ( b ) fundus photograph of ncrna3 - 4 show hemorrhage retinal fold with a fibrovascular mass behind the lens which is a typical presentation of ncrna caused by p.asp434asn mutation in lrp5 gene in ncrna3 - 4 proband . ( c , d ) anterior segment and b - scan for ncrna7 - 4 . ( e ) a member of ncrna19 family with p. gly1401asp mutation showing dense corneal opacity . ( f ) skeletal abnormalities ( ncrna19 - 4 ) . mutation conservation analysis for lrp5 . successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family pedigrees positive for homozygous lrp5 mutations . four families carrying mutation in lrp5 gene were consanguineous . to confirm the molecular diagnosis , we performed ngs in these patients and excluded other possible mutations . it is important to note that this is the first report relating lrp5 mutations with the ncrna phenotype . the gene tspan12 encodes a cell - surface protein that is characterized by the presence of four hydrophobic domains and is predicted to mediate signal transduction events that play a role in the regulation of cell development , activation , growth and motility ( tspan12 , omim#613138 ) . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . we confirmed a novel homozygous splice site mutation , ivs4 - 2a > g and a novel homozygous frameshift mutation , p. pro141fsx21 in tspan12 in two of our families ( ncrna9 and ncrna17 ; table 3 ) . the berkeley drosophila genome project splice site predictor analysis tool showed that the ivs4 - 2 a > g mutation found in ncrna9 deletes the acceptor site . cosegregation ( the tendency for closely linked genes and genetic markers to segregate together ) analysis showed perfect cosegregation of both mutations in the families . we revisited the phenotypes in those patients and found leukocoria , corneal opacities , a shallow ac , posterior synechiae , and total retinal detachment ( fig . 7 ) . six families had tspan12 as a candidate gene in their significant homozygous regions ( table 1 ) , but only ncrna9 and ncrna17 had mutations in this gene . ( a , c ) anterior segment photographs show shallow ac , posterior synechiae , white pupils . ( d ) retrolental mass and atrophic retina with thin atrophic band attachment to optic nerve , confirming severe disease ( red arrow shows site of the optic nerve ) . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . the gene ndp encodes a protein that plays a key role in retinal vascularization and activates the wnt signaling pathway with the help of its coreceptors , fzd4 and lrp5 . the gene is also thought to be implicated in neural cell differentiation and proliferation , as well as neuroectodermal cell - cell interaction ( ndp , omim#300658 ) . mutations in ndp are known to cause x - linked exudative vitreoretinopathy . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . a novel homozygous 244-bp deletion in ndp , which starts from the end of the third exon , deleting 67-bp from exon three and 177-bp for 3 utr , was identified in one of our families . this novel mutation is predicted to be probably damaging by an online polymorphism phenotyping tool , polyphen , with a score of 0.997 ( table 2 ) and the cys residue is evolutionally highly conserved ( fig . 9 ) . both the deletion and the missense mutations cosegregate within the families . this novel mutation is predicted to be probably damaging with a score of 0.997 ( polyphen , table 2 ) and the cys residue is evolutionally highly conserved . a novel homozygous 244-bp deletion in ndp gene ( gene deletion : 4380883943809101 ) was identified in ncrna14 family . family members with double bands ( correspond to the sizes 418 and 680 , wt and mutant bands , respectively ) are heterozygous female carriers in the pedigree . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . genetic causes of ncrna in the remaining eight families were not identified after extensive snp , wes , and retnet panel testing and are still under investigations for novel candidate ncrna genes . the affected individuals presented with the history of congenital blindness and presented with searching roving eye movements . some patients had cloudy corneas due to iridocorneal adhesion , shallow anterior chamber due to forward displacement of lens iris diaphragm , and irregular pupils secondary to posterior synechiae and leukocoria ( white pupils ) ; but there was no consistent elevation of intraocular pressure . fundoscopy , when possible , revealed a dense fibrovasular mass behind the lens in each eye , originating from the optic nerve and attached behind the lens and peripheral fundus . in some cases , other cases showed total retinal detachments and fibrous bands originating from optic nerve posteriorly and attached anteriorly behind the lens . together , these clinical findings suggest bilateral persistence of the fetal blood vessels , with complete congenital detachment of the neural retina and intravitreal vascular proliferation during infancy . they have good vision , with no structural or functional eye pathology . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . surprisingly , two of these families harbored the reported deletion in atoh7 , which is the well - known , common mutation in the founder population of north khorasan , in iran . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . to be sure , we screened all our patients for atoh7 exonic and promoter mutations by sanger sequencing . two families ( ncrna5 and ncrna6 ) were found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 ( phenotypes of the patients are shown in fig . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue ( fig . family ncrna5 was found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 . anterior segment with leukocoria ( a ) and disorganized b scan showing retrolental mass with total vitreous condensation ( b ) caused by the variation in atoh7 gene in ncrna5 - 4 . polymerase chain reaction based genotyping of homozygous 6523-kb deletion in 5atoh7 in ncrna5 and ncrna6 families . ( a ) a polymerase chain reaction based deletion confirmation in the ncrna5 and ncrna6 families conducted as described by ghiasvand et al . letters ( c , d , e , f , g , h ) represent six adjacent amplicons in an ncrna subject that amplify conserved noncoding elements 20 kb upstream of atoh7 transcription start site . as it can be seen from the figure , c and h amplicons are present in both wild - type ( wt ) and mutant subjects , while the rest of the amplicons are missing in ncrna5 and ncrna6 family members . ( b ) cosegregation results between family members using primers designed by ghiasvand et al . in the first panel , primers for g and h amplicons are pooled , whereas in the second panel , only primers for h amplicons were used to verify if deletion of the area is present in the members of the family . we found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . in silico analyses predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . however , lrp5 was the only attractive retinal candidate gene in the region , and sanger sequencing identified the mutations in the gene . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . a member from a third family ( ncrna7 - 4 ) had a novel homozygous p.trp79arg mutation . we then identified the reported homozygous p.gly1401asp mutation in three members of a single family ( ncrna19 - 3 , 19 - 4 , and 19 - 6 ) . panel screening with retnet then identified htz and hmz lrp5 mutations in an additional two families ( ncrna16 and ncrna20 , respectively ) ; ncrna16 has the previously reported p.arg142gln change and the ncrna20 family carries a novel p.leu541pro alteration . since the p.gly1401asp mutation in lrp5 found in the ncrna19 family members has previously been reported to cause osteoporosis - pseudoglioma , we checked bone mineral density ( bmd ) in patient ncrna19 - 4 and the result confirmed that the patient has reduced bmd and osteoporosis . the rest of the families were not able to come back for the given test . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . 4 ) . the altered residues are all evolutionarily conserved ( fig . 5 ) . we then documented delayed growth and skeletal abnormalities in some patients in these families ( fig . pedigrees for successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family positive for homozygous lrp5 mutations are shown in figure 6 . eye phenotype for ncrna2 , ncrna3 , ncrna7 and ncrna19 families and skeletal abnormalities found in a ncrna19 family member . ( a ) the fundus photo shows the fibrovascular fold arising from the optic nerve and extending to the retrolental area in the temporal region in ncrna2 - 5 ; family ncrna2 has been identified to carry a homozygous p. gly610arg alteration in lrp5 gene . ( b ) fundus photograph of ncrna3 - 4 show hemorrhage retinal fold with a fibrovascular mass behind the lens which is a typical presentation of ncrna caused by p.asp434asn mutation in lrp5 gene in ncrna3 - 4 proband . ( c , d ) anterior segment and b - scan for ncrna7 - 4 . ( e ) a member of ncrna19 family with p. gly1401asp mutation showing dense corneal opacity . successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family pedigrees positive for homozygous lrp5 mutations . four families carrying mutation in lrp5 gene were consanguineous . to confirm the molecular diagnosis , we performed ngs in these patients and excluded other possible mutations . it is important to note that this is the first report relating lrp5 mutations with the ncrna phenotype . the gene tspan12 encodes a cell - surface protein that is characterized by the presence of four hydrophobic domains and is predicted to mediate signal transduction events that play a role in the regulation of cell development , activation , growth and motility ( tspan12 , omim#613138 ) . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . we confirmed a novel homozygous splice site mutation , ivs4 - 2a > g and a novel homozygous frameshift mutation , p. pro141fsx21 in tspan12 in two of our families ( ncrna9 and ncrna17 ; table 3 ) . splice site predictor analysis tool showed that the ivs4 - 2 a > g mutation found in ncrna9 deletes the acceptor site . cosegregation ( the tendency for closely linked genes and genetic markers to segregate together ) analysis showed perfect cosegregation of both mutations in the families . we revisited the phenotypes in those patients and found leukocoria , corneal opacities , a shallow ac , posterior synechiae , and total retinal detachment ( fig . 7 ) . six families had tspan12 as a candidate gene in their significant homozygous regions ( table 1 ) , but only ncrna9 and ncrna17 had mutations in this gene . ( a , c ) anterior segment photographs show shallow ac , posterior synechiae , white pupils . ( d ) retrolental mass and atrophic retina with thin atrophic band attachment to optic nerve , confirming severe disease ( red arrow shows site of the optic nerve ) . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . the gene ndp encodes a protein that plays a key role in retinal vascularization and activates the wnt signaling pathway with the help of its coreceptors , fzd4 and lrp5 . the gene is also thought to be implicated in neural cell differentiation and proliferation , as well as neuroectodermal cell - cell interaction ( ndp , omim#300658 ) . mutations in ndp are known to cause x - linked exudative vitreoretinopathy . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . a novel homozygous 244-bp deletion in ndp , which starts from the end of the third exon , deleting 67-bp from exon three and 177-bp for 3 utr , was identified in one of our families . this novel mutation is predicted to be probably damaging by an online polymorphism phenotyping tool , polyphen , with a score of 0.997 ( table 2 ) and the cys residue is evolutionally highly conserved ( fig . this novel mutation is predicted to be probably damaging with a score of 0.997 ( polyphen , table 2 ) and the cys residue is evolutionally highly conserved . a novel homozygous 244-bp deletion in ndp gene ( gene deletion : 4380883943809101 ) was identified in ncrna14 family . family members with double bands ( correspond to the sizes 418 and 680 , wt and mutant bands , respectively ) are heterozygous female carriers in the pedigree . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . genetic causes of ncrna in the remaining eight families were not identified after extensive snp , wes , and retnet panel testing and are still under investigations for novel candidate ncrna genes . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . surprisingly , two of these families harbored the reported deletion in atoh7 , which is the well - known , common mutation in the founder population of north khorasan , in iran . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . to be sure , we screened all our patients for atoh7 exonic and promoter mutations by sanger sequencing . two families ( ncrna5 and ncrna6 ) were found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 ( phenotypes of the patients are shown in fig . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue ( fig . family ncrna5 was found to have the well - known 6523-bp deletion located 20 kb upstream from atoh7 . anterior segment with leukocoria ( a ) and disorganized b scan showing retrolental mass with total vitreous condensation ( b ) caused by the variation in atoh7 gene in ncrna5 - 4 . polymerase chain reaction based genotyping of homozygous 6523-kb deletion in 5atoh7 in ncrna5 and ncrna6 families . ( a ) a polymerase chain reaction based deletion confirmation in the ncrna5 and ncrna6 families conducted . letters ( c , d , e , f , g , h ) represent six adjacent amplicons in an ncrna subject that amplify conserved noncoding elements 20 kb upstream of atoh7 transcription start site . as it can be seen from the figure , c and h amplicons are present in both wild - type ( wt ) and mutant subjects , while the rest of the amplicons are missing in ncrna5 and ncrna6 family members . ( b ) cosegregation results between family members using primers designed by ghiasvand et al . in the first panel , primers for g and h amplicons are pooled , whereas in the second panel , only primers for h amplicons were used to verify if deletion of the area is present in the members of the family . arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . in silico analyses predicts that this mutation is probably damaging ( table 2 ) and conservation analysis shows the significance of the residue . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . however , lrp5 was the only attractive retinal candidate gene in the region , and sanger sequencing identified the mutations in the gene . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . a member from a third family ( ncrna7 - 4 ) had a novel homozygous p.trp79arg mutation . we then identified the reported homozygous p.gly1401asp mutation in three members of a single family ( ncrna19 - 3 , 19 - 4 , and 19 - 6 ) . panel screening with retnet then identified htz and hmz lrp5 mutations in an additional two families ( ncrna16 and ncrna20 , respectively ) ; ncrna16 has the previously reported p.arg142gln change and the ncrna20 family carries a novel p.leu541pro alteration . since the p.gly1401asp mutation in lrp5 found in the ncrna19 family members has previously been reported to cause osteoporosis - pseudoglioma , we checked bone mineral density ( bmd ) in patient ncrna19 - 4 and the result confirmed that the patient has reduced bmd and osteoporosis . the rest of the families were not able to come back for the given test . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . 4 ) . the altered residues are all evolutionarily conserved ( fig . 5 ) . we then documented delayed growth and skeletal abnormalities in some patients in these families ( fig . pedigrees for successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family positive for homozygous lrp5 mutations are shown in figure 6 . eye phenotype for ncrna2 , ncrna3 , ncrna7 and ncrna19 families and skeletal abnormalities found in a ncrna19 family member . ( a ) the fundus photo shows the fibrovascular fold arising from the optic nerve and extending to the retrolental area in the temporal region in ncrna2 - 5 ; family ncrna2 has been identified to carry a homozygous p. gly610arg alteration in lrp5 gene . ( b ) fundus photograph of ncrna3 - 4 show hemorrhage retinal fold with a fibrovascular mass behind the lens which is a typical presentation of ncrna caused by p.asp434asn mutation in lrp5 gene in ncrna3 - 4 proband . ( c , d ) anterior segment and b - scan for ncrna7 - 4 . ( e ) a member of ncrna19 family with p. gly1401asp mutation showing dense corneal opacity . ( f ) skeletal abnormalities ( ncrna19 - 4 ) . mutation conservation analysis for lrp5 . successfully genotyped ncrna2 , ncrna3 , ncrna7 and ncrna19 family pedigrees positive for homozygous lrp5 mutations . four families carrying mutation in lrp5 gene were consanguineous . to confirm the molecular diagnosis , we performed ngs in these patients and excluded other possible mutations . it is important to note that this is the first report relating lrp5 mutations with the ncrna phenotype . the gene tspan12 encodes a cell - surface protein that is characterized by the presence of four hydrophobic domains and is predicted to mediate signal transduction events that play a role in the regulation of cell development , activation , growth and motility ( tspan12 , omim#613138 ) . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . we confirmed a novel homozygous splice site mutation , ivs4 - 2a > g and a novel homozygous frameshift mutation , p. pro141fsx21 in tspan12 in two of our families ( ncrna9 and ncrna17 ; table 3 ) . the berkeley drosophila genome project splice site predictor analysis tool showed that the ivs4 - 2 a > g mutation found in ncrna9 deletes the acceptor site . cosegregation ( the tendency for closely linked genes and genetic markers to segregate together ) analysis showed perfect cosegregation of both mutations in the families . we revisited the phenotypes in those patients and found leukocoria , corneal opacities , a shallow ac , posterior synechiae , and total retinal detachment ( fig . six families had tspan12 as a candidate gene in their significant homozygous regions ( table 1 ) , but only ncrna9 and ncrna17 had mutations in this gene . ( a , c ) anterior segment photographs show shallow ac , posterior synechiae , white pupils . ( d ) retrolental mass and atrophic retina with thin atrophic band attachment to optic nerve , confirming severe disease ( red arrow shows site of the optic nerve ) . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . the gene ndp encodes a protein that plays a key role in retinal vascularization and activates the wnt signaling pathway with the help of its coreceptors , fzd4 and lrp5 . the gene is also thought to be implicated in neural cell differentiation and proliferation , as well as neuroectodermal cell - cell interaction ( ndp , omim#300658 ) . mutations in ndp are known to cause x - linked exudative vitreoretinopathy . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . a novel homozygous 244-bp deletion in ndp , which starts from the end of the third exon , deleting 67-bp from exon three and 177-bp for 3 utr , was identified in one of our families . another novel mutation , p. cys69gly , was identified in a second family ( ncrna11 , table 3 ) . this novel mutation is predicted to be probably damaging by an online polymorphism phenotyping tool , polyphen , with a score of 0.997 ( table 2 ) and the cys residue is evolutionally highly conserved ( fig . this novel mutation is predicted to be probably damaging with a score of 0.997 ( polyphen , table 2 ) and the cys residue is evolutionally highly conserved . a novel homozygous 244-bp deletion in ndp gene ( gene deletion : 4380883943809101 ) was identified in ncrna14 family . family members with double bands ( correspond to the sizes 418 and 680 , wt and mutant bands , respectively ) are heterozygous female carriers in the pedigree . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . genetic causes of ncrna in the remaining eight families were not identified after extensive snp , wes , and retnet panel testing and are still under investigations for novel candidate ncrna genes . nonsyndromic congenital retinal nonattachment ( omim # 221900 ) is a rare genetic disorder defined by congenital retinal detachments and children are completely blind at birth . often , the retinas are found to be fibrotic and appear as a white mass behind the crystalline lens . the goal of this study was to dissect an inbred cohort of clinically diagnosed ncrna patients , to determine the genetic causes of ncrna , in the hope of getting a molecular or genetic entry point into adult retinal detachments , a much more common condition , without any known molecular determinants . in this study , 28 pakistani blind children from 17 consanguineous and 4 nonconsanguineous families with the diagnosis of ncrna were analyzed with variety of gene - identifying techniques , including snp microarrays searching for homozygous regions . as expected , the majority of the patients in our cohort carried significant homozygous regions in their genomes as a result of consanguineous marriages commonly practiced in the region . in 7 ( 41% ) of 17 consanguineous families , homozygosity mapping successfully identified the region and subsequently a disease - causing mutation in the only currently known ncrna gene or other retinal disease genes . it is important to note that the mutated gene was in the largest or second largest homozygous region in 6 of the 11 families ( table 1 ) successfully genotyped using homozygosity mapping . in accordance with our previous study , the data obtained in this current study suggest that the ranking of the ibd regions ( continuous regions over which two haplotypes are identical by descent ) can be very helpful to identify which homozygous segment contains the possible underlying causal and mutant gene . in the rest of the families , the known ncrna and retinal disease genes located in significant homozygous regions were excluded from the study . the additional six successfully genotyped families were identified by retnet panel screening ( table 3 ) . although the clinical diagnosis and strict entry criteria of all children was ncrna , in eight families , the molecular diagnosis determined that the disease process was in fact a new and severe form of fevr . familial exudative vitreoretinopathy is a rare disease and usually characterized by a much milder , acquired phenotype characterized by retinal folds , inferotemporal dragging of the optic disc and macula , increased vessels in the equatorial region , and a peripheral avascular zone . familial exudative vitreoretinopathy was first described by criswick and schepens in 1969 ; it is currently not known to be associated with the severe developmental and congenital retinal detachment in babies and children . therefore , we have expanded the phenotypic spectrum of fevr to a severe retinal detachment phenotype that clinically overlaps with ncrna . in addition , we identified the identical large deletion ( 6523 bp ) in the atoh7 gene found in the kurdish founder population of northern iranian with a high incidence of ncrna , suggesting genetic overlap between the iranian and pakistani populations . therefore , we suggest that this founder mutation in atoh7 gene links the two populations . our molecular genetics study of clinically evaluated and diagnosed patients with ncrna revealed five important patterns . first , our work illustrates that ncrna and fevr overlap clinically ; fevr can be so severe that it can mimic ncrna . therefore , we show for the first time that the ncrna phenotype can be caused by fevr mutations . second , we found the atoh7 deletion links the pakistani population to the iranian population . possible explanations for this could be a common ancestor for both populations or substantial interbreeding by the patients ' ancestors who all lived in the same general large geographical area . third , we have found seven novel mutations : ( 1 ) a hmz c.125g > c , p.arg42pro mutation in atoh7 , ( 2 ) a hmz c.235t > c , p.trp79arg and ( 3 ) a hmz c.1622t > c , p.leu541pro mutation in lrp5 , ( 4 ) a hmz splice site mutation ivs4 - 2 a > g and ( 5 ) a hmz frameshift mutation c.423del t , p. pro141fsx21 in tspan12 , and ( 6 ) a hmz c.205t > g , p.cys69gly and ( 7 ) a hmz 244-bp c.336_402ivs4del deletion in ndp . these families were informed about their results and were genetically counseled about family planning and future treatment options . fourth , we confirmed that recessive mutations in tspan12 can cause much more severe fevr phenotype than the previously known dominant mutations related to the autosomal dominant fevr in accordance with a previous study . in addition , we used our newly developed retnet known retinal disease gene panel , which for the first time contains all the currently known retinal disease genes by targeted ngs which allowed us to screen all the genes listed in retinal information network website and create candidate retinal disease gene list for future study , in case if they are involved in new unknown mechanisms . although the clinical diagnosis of all children was ncrna , the molecular diagnosis determined that the disease process was in fact a severe new form of fevr in 13 families . however , the phenotype was severe congenital retinal detachment , previously not known to be associated with fevr mutations . therefore , we have expanded the phenotypic spectrum of fevr , and we also show that the disease spectrum is much wider than previously established . out of 21 pakistani families with clinically diagnosed ncrna , 10 families were discovered to have mutations in three fevr genes : lrp5 ( six families ) , tspan12 ( two families ) , and ndp ( two families ) , and three families were found to have mutations in atoh7 . thus , eight ncrna families in our cohort do not carry mutations in any known gene , which suggests that atoh7 is not the only gene to cause ncrna in children . we show that snp studies followed by retnet , wes , and sanger confirmation is a powerful protocol to identify the genetic cause of ncrna . finally , we established a genetic link between the iranian and pakistani populations , through a common founder deletion in atoh7 .

purposeto evaluate consanguineous pedigrees from pakistan with a clinical diagnosis of nonsyndromic congenital retinal nonattachment ( ncrna ) and identify genes responsible for the disease as currently only one ncrna gene is known ( atonal basic helix - loop - helix transcription factor 7 : atoh7).methodswe implemented a three - step genotyping platform : single nucleotide polymorphism genotyping to identify loss of heterozygosity regions in patients , retinal information network panel screening for mutations in currently known retinal genes . negative patients were then subjected to whole exome sequencing.resultswe evaluated 21 consanguineous ncrna pedigrees and identified the causal mutations in known retinal genes in 13 out of our 21 families . we found mutations in atoh7 in three families . surprisingly , we then found mutations in familial exudative vitreoretinopathy ( fevr ) genes ; low - density lipoprotein receptor - related protein 5 mutations ( six families ) , tetraspanin 12 mutations ( two families ) , and ndp mutations ( two families ) . thus , 62% of the patients were successfully genotyped in our study with seven novel and six previously reported mutations in known retinal genes.conclusionsalthough the clinical diagnosis of all children was ncrna with severe congenital fibrotic retinal detachments , the molecular diagnosis determined that the disease process was in fact a very severe form of fevr in 10 families . because severe congenital retinal detachment has not been previously associated with all the fevr genes , we have thus expanded the phenotypic spectrum of fevr , a highly variable retinal detachment phenotype that has clinical overlap with ncrna . we identified seven novel mutations . we also established for the first time genetic overlap between the iranian and pakistani populations . we identified eight ncrna families that do not harbor mutations in any known retinal genes , suggesting novel causal genes in these families .

Methods DNA Extraction and Primer Design Step 1: Single Nucleotide Polymorphism (SNP) Microarrays Step 2: RetNet Known Retinal Disease Gene Panel Screening by Targeted Next Generation Sequencing (NGS) Step 3: Whole Exome Sequencing Mutation Analysis Sanger Sequencing In Silico Mutational Analyses Results Clinical Findings Known Genes for Mutations in Mutations in Mutations in Discussion Conclusions

informed consent was obtained from all patients at the initial visit in pakistan at the el shifa trust eye hospital , where two of our clinical collaborators saw the babies ( ak , ss ) and another oversaw the dna extractions ( rq ) ; the consent form contained a paragraph about genetic analyses of retinal genes , including whole exome sequencing . twenty - one ncrna families ( 28 affected members ) were recruited , phenotyped , and genotyped from pakistan and included in this study . to ensure the completeness and quality of the sequences and for detection of potential mutations located in splice sites , a minimal distance between primer and exon / the genomes of 11 out of our 17 consanguineous ncrna families were analyzed for homozygous chromosomal regions by using snp homozygosity mapping ( infinium hd 660k ; illumina , inc . all the known retinal genes ( the retinal genes listed in retinal network information website ) that resided in homozygous chromosomal segments were analyzed for mutations using sanger sequencing . fourteen patients diagnosed with ncrna were screened by our recently developed retnet known retinal disease gene panel , which contains all the currently known retinal disease genes ( 163 ) by targeted ngs . to pinpoint the genetic variants responsible for the disease in those patients who did not carry mutations in known genes . known retinal genes within the homozygous regions , retinal detachment genes shown in retnet ( retinal information network , tables of genes and loci causing inherited retinal diseases ) , and all putative mutations found by retnet panel screening were sanger - sequenced . we then studied conservation of the residue in homologous proteins of lower animals and we verified that the variant has not been reported in the exome variant server ( nhlbi go exome sequencing project ) , dbsnp135 , or 1000 genome datasets . to ensure the completeness and quality of the sequences and for detection of potential mutations located in splice sites , a minimal distance between primer and exon / the genomes of 11 out of our 17 consanguineous ncrna families were analyzed for homozygous chromosomal regions by using snp homozygosity mapping ( infinium hd 660k ; illumina , inc . all the known retinal genes ( the retinal genes listed in retinal network information website ) that resided in homozygous chromosomal segments were analyzed for mutations using sanger sequencing . fourteen patients diagnosed with ncrna were screened by our recently developed retnet known retinal disease gene panel , which contains all the currently known retinal disease genes ( 163 ) by targeted ngs . to pinpoint the genetic variants responsible for the disease in those patients who did not carry mutations in known genes . known retinal genes within the homozygous regions , retinal detachment genes shown in retnet ( retinal information network , tables of genes and loci causing inherited retinal diseases ) , and all putative mutations found by retnet panel screening were sanger - sequenced . we then studied conservation of the residue in homologous proteins of lower animals and we verified that the variant has not been reported in the exome variant server ( nhlbi go exome sequencing project ) , dbsnp135 , or 1000 genome datasets . in collaboration with a research group in islamabad , pakistan , we assembled 21 consanguineous pedigrees from pakistan with an ncrna diagnosis . our approach to finding the causal genes in ncrna patients was to first identify all the significant homozygous regions in the probands ( patients ) and then look for overlap of all these regions with all affected members of that family , followed by searching for overlap with other families . in a preliminary study of our cohort , we screened families for known ncrna and retinal detachment genes ( including fevr genes ) which resided in one of the top five homozygous segments documented by the snp array . we hypothesized that the causal gene for ncrna resides in one of the top five largest intervals of our identified homozygous regions in consanguineous families and a new causal gene underlying the disease may locate in the overlapping homozygous regions of different family members with the same disease phenotype . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . we found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . a member from a third family ( ncrna7 - 4 ) we then identified the reported homozygous p.gly1401asp mutation in three members of a single family ( ncrna19 - 3 , 19 - 4 , and 19 - 6 ) . panel screening with retnet then identified htz and hmz lrp5 mutations in an additional two families ( ncrna16 and ncrna20 , respectively ) ; ncrna16 has the previously reported p.arg142gln change and the ncrna20 family carries a novel p.leu541pro alteration . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . to confirm the molecular diagnosis , we performed ngs in these patients and excluded other possible mutations . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . we found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . we then identified the reported homozygous p.gly1401asp mutation in three members of a single family ( ncrna19 - 3 , 19 - 4 , and 19 - 6 ) . panel screening with retnet then identified htz and hmz lrp5 mutations in an additional two families ( ncrna16 and ncrna20 , respectively ) ; ncrna16 has the previously reported p.arg142gln change and the ncrna20 family carries a novel p.leu541pro alteration . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . to confirm the molecular diagnosis , we performed ngs in these patients and excluded other possible mutations . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . in our 21 pakistani ncrna families , only three families had atoh7 in their largest ibd regions ( our mutation analysis confirmed this ) . eight out of our 21 ncrna families did not harbor mutations in atoh7 , nor in any other retinal detachment gene in their homozygous regions , suggesting that we have families with new gene / s for ncrna . we also found a novel homozygous missense mutation p. arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the third family ( ncrna4 ) . arg42pro , altering a highly conserved arginine for a proline residue in atoh7 in the ncrna4 family ; this is the first missense mutation reported in atoh7 . the gene lrp5 encodes a transmembrane low - density lipoprotein receptor and plays a key role in eye development , in skeletal homeostasis , and many bone density related diseases ( lrp5 , omim # 603506 ) . although the lrp5 gene is known for mild retinal detachments and folds and was located in ibd regions of the patients , fevr was not considered as the primary diagnosis since these patients presented with congenital blindness . we found five homozygous and one heterozygous ( htz ) mutations in the fevr gene lrp5 ; namely , two members of each ncrna2 ( ncrna2 - 5 and ncrna2 - 6 ) and ncrna3 ( ncrna3 - 4 and ncrna3 - 7 ) families were carrying the reported p.gly610arg and p.asp434asn homozygous mutations , respectively . the eye phenotype in our study consisted of congenital blindness with congenital retinal detachments and corneal opacity , posterior synechia , shallow ac , due to anteriorly displaced lens and iris , leukocoria , retrolenticular fibrovascular plaque , retinal folds ( in some patients ) , and complete retinal detachment , unlike the current phenotypes associated with lrp5 type fevr ( fig . tetraspanin-12 is currently known to cause autosomal dominant and recessive fevr , but not autosomal recessive ncrna , and it is not known to cause severe retinal detachments . thus , for the first time , we found recessive mutations in tspan12 in clinically diagnosed ncrna patients . with the help of the retnet panel and wes , we found ndp mutations in two families ( ncrna11 and ncrna14 ) . phenotypes of the patients with ndp mutations is consistent with the phenotype of previously described ncrna families in iran and also similar to the phenotypes of families carrying mutations in atoh7 , lrp5 , and tspan12 genes described in this study ; bilateral corneal opacities , shallow ac , posterior synechiae , retrolenticular fibrovascular plaque , and complete congenital retinal detachment . nonsyndromic congenital retinal nonattachment ( omim # 221900 ) is a rare genetic disorder defined by congenital retinal detachments and children are completely blind at birth . the goal of this study was to dissect an inbred cohort of clinically diagnosed ncrna patients , to determine the genetic causes of ncrna , in the hope of getting a molecular or genetic entry point into adult retinal detachments , a much more common condition , without any known molecular determinants . as expected , the majority of the patients in our cohort carried significant homozygous regions in their genomes as a result of consanguineous marriages commonly practiced in the region . it is important to note that the mutated gene was in the largest or second largest homozygous region in 6 of the 11 families ( table 1 ) successfully genotyped using homozygosity mapping . in accordance with our previous study , the data obtained in this current study suggest that the ranking of the ibd regions ( continuous regions over which two haplotypes are identical by descent ) can be very helpful to identify which homozygous segment contains the possible underlying causal and mutant gene . although the clinical diagnosis and strict entry criteria of all children was ncrna , in eight families , the molecular diagnosis determined that the disease process was in fact a new and severe form of fevr . familial exudative vitreoretinopathy is a rare disease and usually characterized by a much milder , acquired phenotype characterized by retinal folds , inferotemporal dragging of the optic disc and macula , increased vessels in the equatorial region , and a peripheral avascular zone . familial exudative vitreoretinopathy was first described by criswick and schepens in 1969 ; it is currently not known to be associated with the severe developmental and congenital retinal detachment in babies and children . therefore , we have expanded the phenotypic spectrum of fevr to a severe retinal detachment phenotype that clinically overlaps with ncrna . in addition , we identified the identical large deletion ( 6523 bp ) in the atoh7 gene found in the kurdish founder population of northern iranian with a high incidence of ncrna , suggesting genetic overlap between the iranian and pakistani populations . therefore , we show for the first time that the ncrna phenotype can be caused by fevr mutations . third , we have found seven novel mutations : ( 1 ) a hmz c.125g > c , p.arg42pro mutation in atoh7 , ( 2 ) a hmz c.235t > c , p.trp79arg and ( 3 ) a hmz c.1622t > c , p.leu541pro mutation in lrp5 , ( 4 ) a hmz splice site mutation ivs4 - 2 a > g and ( 5 ) a hmz frameshift mutation c.423del t , p. pro141fsx21 in tspan12 , and ( 6 ) a hmz c.205t > g , p.cys69gly and ( 7 ) a hmz 244-bp c.336_402ivs4del deletion in ndp . fourth , we confirmed that recessive mutations in tspan12 can cause much more severe fevr phenotype than the previously known dominant mutations related to the autosomal dominant fevr in accordance with a previous study . in addition , we used our newly developed retnet known retinal disease gene panel , which for the first time contains all the currently known retinal disease genes by targeted ngs which allowed us to screen all the genes listed in retinal information network website and create candidate retinal disease gene list for future study , in case if they are involved in new unknown mechanisms . although the clinical diagnosis of all children was ncrna , the molecular diagnosis determined that the disease process was in fact a severe new form of fevr in 13 families . however , the phenotype was severe congenital retinal detachment , previously not known to be associated with fevr mutations . therefore , we have expanded the phenotypic spectrum of fevr , and we also show that the disease spectrum is much wider than previously established . out of 21 pakistani families with clinically diagnosed ncrna , 10 families were discovered to have mutations in three fevr genes : lrp5 ( six families ) , tspan12 ( two families ) , and ndp ( two families ) , and three families were found to have mutations in atoh7 . thus , eight ncrna families in our cohort do not carry mutations in any known gene , which suggests that atoh7 is not the only gene to cause ncrna in children . finally , we established a genetic link between the iranian and pakistani populations , through a common founder deletion in atoh7 .

the steroid hormone estrogen and the estrogen receptor alpha ( er ) are necessary for the physiology of the female reproductive system ( musgrove and sutherland 2009 ) . these factors play an essential role in the breast , ovaries , and uterus , where they control cell division and differentiation , and the deregulation of er transcriptional activity may result in an increased proliferation and eventually in cancer onset . breast cancer is a heterogeneous disease with a different subgroup of patients showing distinct molecular profiles ( perou et al . 2000 ; sorlie et al . 2001 ; curtis et al . 2012 ; gray and druker 2012 ) . however , the most widespread type is the luminal group of tumors , and they share the common feature of being positive for the expression of er ( dowsett 2001 ; prat and baselga 2008 ) . er is a transcription factor that mediates the response to estrogens and to anticancer therapies , including the selective estrogen receptor modulator ( serm ) tamoxifen ( katzenellenbogen and frasor 2004 ; hurtado et al . the incorporation of new technologies such as high - throughput sequencing has been crucial for a deep understanding of er function . chromatin immunoprecipitation ( chip ) combined with sequencing studies in breast cancer cell lines and human tissue shows a dispersed occupancy pattern of er binding sites bearing heterogeneous recognition motifs ( carroll et al . 2006 ; lin et al . 2007 ; ross - innes et al . 2012 ) . estrogen and tamoxifen can affect the gene expression profile by inducing thousands of er binding events ( frasor et al . . moreover , er binds to chromatin with a multitude of transcription factors ( er - cooperating factors ) that influence transcriptional activity of er and ultimately affect the outcome of anti - estrogen therapies ( carroll et al . 2005a , b ; cheng et al . 2006 ; hurtado et al . 2011 ; kong et al . 2011 ) . a second group of breast cancer patients is characterized by an amplification of chromosome region 17q12 - 21 , leading to the overexpression of the epidermal growth factor receptor 2 , erbb2/her2/neu ( wolff et al . 2007 ) . moreover , about half of her2-positive patients are also positive for er ( dowsett 2001 ) , and the activation of other signaling pathways such as the pi3k pathway is critical for er / her-2-positive tumor development ( berns et al . yet , the molecular mechanism by which these signaling pathways modulate er and er - cooperating factors is not completely understood . in this review , we describe how cooperating factors influence the transcriptional activity of er , and we speculate how these signaling pathways may modulate the function of er and er - cooperating factors . er is a ligand - regulated transcription factor that recognizes a consensus sequence of nucleotides , establishing the binding to dna , and thereby triggering the recruitment of the transcription machinery . however , most of the genomic regions where er interacts are in a heterochromatic state ( hurtado et al . pioneer transcription factors interact with chromatin and expose dna for subsequent transcription factor binding and initiation of transcription ( liu et al . 1991 ; monaghan et al . 1993 ) . genomic analyses of er binding maps have shown that its union is accompanied with the binding of various transcription factors , which includes forkhead box a ( foxa ) ( carroll et al . 2005a , b ; eeckhoute et al . 2006 , 2007 ) , gata ( krum et al . 2011 ) , ap2 ( tan et al . 2011 ) , and pbx1 ( magnani et al . 2011 ) . in this section of the manuscript 1role of pioneering factors in regulation of er chromatin interactions . in the absence of pioneering factors , chromatin regions foxa1 , in cooperation with other transcription factors , opens chromatin regions and facilitates ligand er binding . pbx1 seems to have a foxa1-independent effect role of pioneering factors in regulation of er chromatin interactions . in the absence of pioneering factors , chromatin regions foxa1 , in cooperation with other transcription factors , opens chromatin regions and facilitates ligand er binding . pbx1 seems to have a foxa1-independent effect foxa proteins are the most studied pioneer transcription factors that bind to chromatin and enable gene activity . foxa1 ( also known as hnf3 ) recruitment to chromatin is mediated by the epigenetic signature consisting of mono- and dimethylated histone h3 on lysine 4 ( h3k4me1/me2 ) ( lupien et al . the pioneering properties of foxa1 reside on its protein structure , which contains a winged helix domain that can structurally mimic histone h1 and h5 , and thus permits its stable interaction with histone h3 and h4 with high affinity ( cirillo et al . the high chromatin affinity of foxa1 is a unique feature that allows its binding to the specific dna sequences on the nucleosome core and displaces the linker histones , leading to de - compaction of chromatin and facilitation of the binding of other transcription factors . in breast hormone - sensitive and resistant cancer cell lines , almost all er chromatin interactions and gene expression changes are dependent on the expression of foxa1 ( hurtado et al . moreover , foxa1 influences genome - wide chromatin accessibility of er ( hurtado et al . 2011 ) . recently , ross - innes et al . have established that hormone - resistant breast cancers still recruit er to the chromatin , and this binding is associated with foxa1 ( ross - innes et al . interestingly , er shows a distinct binding profile in patients with poor clinical outcome to anti - estrogen therapies . these newly identified regions are enriched toward the genes that previously were described to predict clinical outcome ( ross - innes et al . have shown that snps associated with breast cancer risk are located in a subset of the foxa1 binding regions , which influences the binding affinity for the pioneer factor foxa1 ( cowper - sal lari et al . therefore , data published to date suggest that foxa1 is a major determinant of estrogen er activity in breast cancer . six gata transcription factors have been identified in vertebrates ( gata-1 to gata-6 ) ( kouros - mehr et al . 2008 ) . in breast , gata-3 is expressed in luminal tumors ( sorlie et al . however , the mechanism of gata-3 action or its potential role as a pioneer factor of er has not been described yet . by contrast , gata-4 has been shown to have pioneering properties during early development ( bossard and zaret 1998 ) and for er binding in u2os osteosarcoma cell line ( krum et al . 2011 ) , which stably expresses exogenous er and very low levels of foxa1 ( hurtado et al . 2011 ) . dna interaction in mda - mb-231 breast cancer cell line ( stender et al . 2010 ) , which stably expresses exogenous er and is negative for the expression of foxa1 and her2 . these results support the idea that distinct pioneer proteins influence er binding in foxa1-negative tissues . the pre - b cell leukemia homeobox 1 factor ( pbx1 ) is a cofactor for homeobox ( hox ) transcription factors as it increases their affinity and specificity to chromatin ( moens and selleri 2006 ) . pbx1 has been described as a pioneer factor whose function is essential for the er - mediated transcriptional response ( magnani et al . magnani et al . demonstrated that estrogen - induced transcriptional response is preferentially associated with regulatory regions where er co - bounds with pbx1 or pbx1-foxa1 . indeed , the authors point out pbx1 , and not foxa1 , as a novel prognostic marker for recurrence in er - positive breast cancers ( magnani et al . genomic analyses of er binding sites from chip - sequencing experiments also identified enrichment for ap-2 motifs ( tan et al . the authors demonstrated that perturbations of the expression of the transcription factor ap-2 prevent er binding to dna and gene transcription . interestingly , the lack of this factor is even affecting er long - range chromatin interactions , which have been shown to be essential for er - mediated transcription ( fullwood et al . 2009 ) . further molecular studies indicate that both factors collaborate in er - mediated transcription ( tan et al . the groucho homologue transducin - like enhancer of split 1 ( tle1 ) is a multitasking transcriptional co - repressor . tle proteins can associate with condensed chromatin by binding to the histone tails of nucleosomes ( sekiya and zaret 2007 ) . the groucho / tle / grg family of co - repressors operates in many signaling pathways and distinct biological processes ( jennings et al . 1992 ) has critical transcription factor partners such as tcf / lef-1 ( daniels and weis 2005 ) , hairy / enhancer of split 1 ( dasen et al . 2010 ) , and the aml / cbfa runt domain transcription factor ( levanon et al . biologically , the loss of tle coincides with increased global protein synthesis and enhanced cell proliferation ( ali et al . moreover , recently , holmes et al . have published that tle1 positively assists some er chromatin interactions , a role that is distinct from its general role as a transcriptional repressor . the specific silencing of tle1 inhibits the ability of er to bind a subset of er binding sites within the genome , and this is accompanied by perturbations in phospho - rna pol ii recruitment ( holmes et al . interestingly , tle1 action occurs at regions where foxa1 binds more weakly ( holmes et al . foxa1 and gata3 proteins are expressed in er - positive luminal breast cancers ( sorlie et al . 2003 ) . in fact , foxa1 expression is associated with the expression of steroid hormone receptors ( er , progesterone receptor , and androgen receptor ) and other variables of good prognosis such as smaller tumor size , lower histological grade , and expression of luminal cytokeratins ( ck18 and ck7/8 ) , brca1 , and e - cadherin ( habashy et al . 2008 ) . these evidences imply that high foxa1 expression is linked with survival and a better outcome in breast cancer patients . accordingly , a recent publication suggests that foxa1 directly represses a subset of basal signature genes ( bernardo et al . the silencing of foxa1 causes a partial shift from luminal to basal gene expression signatures , which results in an increased migration and invasion of luminal cancer cells . this phenotype is representative of the basal subtype of tumors , which are negative for er and her2 expression . in breast , gata-3 plays an important role in mammary gland development and differentiation ( bossard and zaret 1998 ; ho and pai 2007 ) . moreover , the inactivation of gata-3 in mice results in contracted mammary epithelial structure , severely impaired lactogenesis , and disrupted differentiation of luminal progenitor cells into ductal and alveolar cells ( asselin - labat et al . , gata-3 has been positively implicated in mediating the estrogen er signaling ( eeckhoute et al . all in all , foxa1 and gata3 that are subsequently used by er to bind chromatin and regulate gene transcription , respectively ( carroll et al . 2005 ; eeckhoute et al . 2007 ; hurtado et al . 2011 ) , might be considered as biomarkers of luminal tumors . in fact , 83.1 % of foxa1-positive tumors are comprised in the luminal a subtype . similarly , 87.7 % of gata-3-positive tumors fall within this molecular subtype ( wilson and giguere 2008 ; albergaria et al . the pioneer factor foxa1 also plays an important role in androgen receptor ( ar ) signaling of molecular apocrine tumors , which have been recently identified as an additional subgroup of er - negative and ar - positive breast tumors ( ni et al . 2011 ) . on the one hand , ni et al . identified ar as a mediator of the ligand - dependent activation of wnt and her2 signaling pathways through direct transcriptional induction of wnt7b and her3 ( ni et al . 2011 ) . on the other hand , robinson et al . demonstrated that the specific silencing of foxa1 inhibits ar binding , expression of the majority of the molecular apocrine gene signature , and cell growth ( robinson et al . moreover , ni et al . proved that specific targeting of ar , wnt , or her2 signaling impairs androgen - stimulated tumor cell growth , suggesting potential therapeutic approaches for er/her2 + breast cancers ( ni et al . altogether , it seems that , in breast tumors , er and ar binding and their functionality is fully dependent on foxa1 . by contrast , in prostate cancer , the effect of foxa1 on ar binding is more complex . recently , two studies have reported a new paradigm for the forkhead protein foxa1 action in androgen signaling . besides the pioneering function on the ar pathway , foxa1 depletion elicited extensive redistribution of ar - binding sites ( sahu et al . interestingly , both groups identified three distinct classes of ar binding sites and androgen - responsive genes : some independent of foxa1 , others pioneered by foxa1 , and some others masked by foxa1 and functional upon foxa1 depletion . importantly , foxa1 protein level in primary prostate tumors has a significant association with the disease outcome ; high foxa1 level is associated with poor prognosis , whereas low foxa1 level , even in the presence of high ar expression , predicts good prognosis . the role of foxa1 in androgen signaling and prostate cancer ( gerhardt et al . 2012 ) is different from that in estrogen signaling and breast cancer ( sahu et al . by contrast , in breast cancer , there is a clear association between high foxa1 expression and a better survival ( habashy et al . 2008 ) . in fact , the oncotype dx test for breast cancer prognosis shows a negative and significant correlation between foxa1 expression and recurrence ( ademuyiwa et al . , studies focused on these tissue - specific properties of foxa1 will be instrumental for our understanding of hormone - regulated cancers . in endometrial cancer tumors , foxa1 is expressed in 37 % of the cases , and its expression is significantly and negatively associated with lymph node status ( abe et al . 2012 ) . interestingly , in er - positive endometrial cancer cells , foxa1 has been suggested to function as a tumor suppressor through modulation of proliferation and migration of endometrial cancer cells ( abe et al . 2012 ) . however , it is not clear whether foxa1 action occurs through er or progesterone receptor ( pr ) . very recently , clarke et al . have reported that foxa1 alters pr transcriptional response in normal breast ab32 cells , a pr - positive clone of the mcf-10a cell line ( clarke and graham 2012 ) . the conclusions of this study suggest that foxa1 is not absolutely required for progesterone response . however , when foxa1 is overexpressed in ab32 cells , it induces the expression of genes involved in negative regulation of apoptosis . yet , we do not know the role of foxa1 in progesterone- and estrogen - induced transcription in endometrial tissue . in summary , all the published data suggest that the idea of using foxa1 as a therapeutic target in breast and endometrial cancers could be an alternative for those patients with recurrence to current treatments . we need to know in what other functions , apart from that of pioneering , foxa1 is involved . furthermore , it is important to know how the function of foxa1 is regulated . finally , to decipher the specific weight of other pioneering functions that might compensate functionally the inactivation of foxa1 is critical for future therapies . estrogen may activate or repress transcription of er target genes potentially by recruiting distinct classes of co - regulators that have chromatin remodeling properties . structural and functional studies revealed that er co - activators are recruited to hormone - responsive genes through their interaction with activated receptors . in turn , the co - activator complex remodels the chromatin at this region through histone acetylation , facilitating rna polymerase ii - mediated transcription ( onate et al . 1995 ; anzick et al . 1997 ; torchia et al . 1997 ; chen et al . it has also been established that , in estrogen - repressed genes , estrogen er stimulates the selective association of co - repressors ( carroll et al . the interaction of these co - repressors prompts the binding of chromatin deacetylatases and therefore leads to transcriptional inhibition . some transcription factors have been shown to be responsible for er cofactor binding ( gata-3 , foxa1 , and rara ) , to function as cofactors by themselves ( xbp1 ) or to be mediators of er - repressive action ( pitx-1 ) . in the next paragraphs , we discuss the function of these transcription factors ( fig . cell lines , the complex created by foxa1 , gata3 , and er regulates estrogen ( red bold dot ) transcription . these three factors are necessary for the recruitment of the co - activator p300 and rna polymerase ii . rara , after binding its ligand atra ( blue bold dot ) , interacts and cooperates with er at er binding sites , where it stabilizes both er co - activator and co - repressor binding . pitx-1 represses transcription of a subset of er - regulated genes er - cooperating factors influence estrogen - mediated transcription . in breast carcinoma cell lines , the complex created by foxa1 , gata3 , and er regulates estrogen ( red bold dot ) transcription . these three factors are necessary for the recruitment of the co - activator p300 and rna polymerase ii . rara , after binding its ligand atra ( blue bold dot ) , interacts and cooperates with er at er binding sites , where it stabilizes both er co - activator and co - repressor binding . pitx-1 represses transcription of a subset of er - regulated genes recently , kong et al . reported in mcf-7 breast carcinoma cells that foxa1 , gata-3 , and er form a protein complex , which regulates er - mediated transcription ( kong et al . the chromatin regions occupied by all these three transcription factors were associated with the highest p300 co - activator recruitment ( histone acetylase enzyme ) , rna pol ii occupancy , and chromatin opening . interestingly , co - transfection of these three transcription factors was sufficient to restore the estrogen - responsive growth of er - negative mda - mb-231 and bt-549 cells . these findings are very significant and suggest that all three transcription factors are needed for co - activator recruitment and , ultimately , for er - mediated transcription in breast tissue . however , it is not clear yet whether the complex of er , foxa1 , and gata-3 is necessary for all er - regulated transcripts in breast tissue . retinoic acid receptor alpha ( rara ) is a nuclear receptor , which regulates gene expression by retinoic acid ( ra ) ( giguere et al . 2012 ) and antagonists of rara ( dawson et al . 1995 ; toma et al . moreover , rara is known to be an estrogen - induced target gene in breast cancer cells ( laganiere et al . yet , the mechanisms of action by which the rara agonists or antagonists carry out a repressive effect in breast cancer are not entirely clear . the white and carroll groups have tried to solve this conundrum . although both teams agree that rara binding throughout the genome is highly coincident with er binding ( hua et al . 2010 ) , they propose contradictory mechanisms of action . whereas white 's group supports a genomic antagonism between ra and estrogen signaling ( hua et al . 2009 ) , carroll 's group supports a cooperative interaction between rara and er ( ross - innes et al . altogether , it seems that rara might have two distinct roles in breast cancer cells : first , repressing estrogen transcription via the classic function of rara with its interacting partner retinoid x receptor and , second , interacting with er and maintaining er both rara agonist and antagonist actions show benefit on breast cancer , both mechanisms of action might be taking place . xbp1 is a transcription factor that belongs to the basic region / leucine zipper ( bzip ) family of proteins ( clauss et al . 1996 ) . regulation of transcription by xbp1 is a consequence of its binding to and activation of specific camp - responsive element . the xbp1 spliced form , xbp1(s ) , has the ability to bind to and activate er in a ligand - independent manner ( ding et al . furthermore , xbp1 is also rapidly induced in response to estrogen stimulation ( wang et al . 2009 ) , which suggests that transcription of er - regulated genes might be also modulated by the complex er xbp1 . recently , the pituitary homeobox 1 ( pitx-1 ) transcription factor has been related as a repressor for a subset of er target genes ( stender et al . all together , these evidences suggest that er - cooperating factors might be needed for estrogen er - mediated transcription . interestingly , the transcription of some of these cooperating factors is induced both by estrogen at early time points ( hurtado et al . 2011 ) as directly by foxa1 ( nakshatri and badve 2009 ) . this supports the established hypothesis and also suggests that these factors are needed for a sustained transcriptional activity of er . perhaps , foxa1 induces the transcription of these cooperating factors to allow the expression of early er - regulated genes . subsequent activation of the transcription of these cooperating factors by er would then allow the sustained expression of er targets . however , the mechanism of action of these factors for er - mediated transcription is not completely understood . it has been suggested that they might be important for recruitment of chromatin remodeling factors ( ross - innes et al . breast tumors positive for er expression represent around 70 % of these cancers ( dowsett 2001 ; prat and baselga 2008 ) . targeting estrogen action has been a therapeutic choice of breast cancer treatment so far ( harvey et al . , various endocrine treatments have been developed in order to block estrogen action in breast cancer cells . one of the most successful treatments is represented by the serm tamoxifen ( jensen and jordan 2003 ) . it antagonizes estrogen action by competing for the binding of er in breast cancer cells and is thought to repress er - mediated transcriptional activation by actively recruiting co - repressors ( katzenellenbogen and frasor 2004 ; malik et al . 2010 more recently , another class of anti - estrogen drug has been incorporated into clinical treatment , namely aromatase inhibitors ( ai ) . these inhibitors antagonize estrogen metabolism , and therefore , their use is restricted to postmenopausal women . unfortunately , one third of women treated with any of these treatments will relapse ( musgrove and sutherland 2009 ) . the molecular mechanisms by which tamoxifen induces repression on breast cancer cells are not completely understood ( harvey et al . 1999 ) , and diverse models of endocrine resistance have been hypothesized ( higgins and stearns 2009 ) . in this section of the review , we discuss which transcriptional cooperating factors can affect er genomic activity in response to endocrine treatment and how this process can affect cell proliferation and survival . the effectiveness of tamoxifen requires both the binding with er and the consequent interaction with dna . importantly , genomic maps of er binding induced with estrogen or tamoxifen are almost identical ( hurtado et al . 2011 ) , which evidences that tamoxifen er for its repressive action . from these evidences , one might assume that tamoxifen er and estrogen er use the same mechanisms to interact with dna ( hurtado et al . interestingly , ross - innes et al . observed that , in tumors resistant to endocrine therapy , er interactions were enriched with foxa1 motifs ( ross - innes et al . crosstalk between the er and her2 pathways has long been implicated in cancer onset and response to tamoxifen , but no direct connection at transcriptional level has been shown . tamoxifen - resistant breast tumors are characterized by elevated erbb2 levels ( osborne et al . 2003 ) , and er - positive cell line models overexpressing erbb2 acquire resistance to tamoxifen ( benz et al . 1992 ) . in 2008 , a new mechanism of resistance to tamoxifen treatment was suggested ( hurtado et al . 2008 ) , which revealed a novel interplay between er and erbb2 on a genomic level . this study proposes that the anti - proliferative effects of tamoxifen require repression of erbb2 and that breast cancer cells acquire resistance by deregulating the mechanisms that normally repress erbb2 transcription . this repression is mediated by the transcription factor pax2 , which cooperates with er at this locus . pax2 belongs to the pair box gene ( pax ) family , a group of transcription factors characterized by the presence of two dna - binding domains and are known for their role in terminal differentiation during organogenesis ( mansouri et al . pax2 is expressed in around 60 % of breast tumors ( muratovska et al . 2003 ) , and its nuclear localization is more frequent in luminal tumors than in nonluminal tumors ( silberstein et al . moreover , in luminal breast cancer cell lines , pax2 has been shown to be activated and confers a low invasive phenotype ( beauchemin et al . interestingly , pax2 silencing is able to abrogate the inhibition of erbb2 transcription and increases erbb2-dependent cell proliferation ( hurtado et al . moreover , the expression of pax2 is reduced in tamoxifen - resistant cells , and the overexpression of pax2 is able to restore the sensitivity to tamoxifen in these cells ( hurtado et al . nonetheless , another study showed that changes in erbb2 expression are not dependent on differences in pax2 expression among various populations of tamoxifen - resistant and estrogen - deprived mcf-7 cells ( leung et al . tamoxifen - resistant cells are also characterized by increased levels of the er co - activator aib-1 ( osborne et al . indeed , there is a competition between aib-1 and pax2 for the binding to the er binding region of the erbb2 gene , which might explain the differences between the two studies . this event has also been shown in tamoxifen - treated breast cancer samples , where the pax2-positive aib-1-negative tumors have the best prognosis and the lowest percentage of erbb2-positive cells ( hurtado et al . these results suggest that the critical event for erbb2 repression and tamoxifen resistance is not just explained by the loss of pax2 expression , but it supports the idea that the balance between pax2 and aib-1 recruitment at chromatin level is crucial for the determination of tamoxifen response and resistance . yet , the molecular mechanism underlying the competition between pax2 and aib-1 in er - mediated regulation of transcription is not completely understood . we have observed that tamoxifen mainly enhances the binding of pax2 at genome - wide level ( gilfillan et al . 2012 ) , which suggests that pax2 might be functioning as a general repressor for er tamoxifen action ( fig . 3 ) . however , all the genomic regions where pax2 interacts with dna after estrogen or tamoxifen treatment have not been identified yet , and therefore , it can not be established whether pax2 is required for all estrogen - repressed genes . furthermore , it is not clear yet if the competition between pax2 and aib-1 might be affecting the transcriptional regulation of many different er target genes . in summary , all these studies denote that the repressive action of tamoxifen is regulated by cooperating factors at least at two different levels : foxa1 , which orchestrates er binding on the chromatin , and pax2 , which dictates the transcriptional activity of er induced by tamoxifen . furthermore , all these findings highlight the complexity of er tamoxifen transcriptional regulation in human breast cancer.fig . 3the balance between aib-1 and pax2 governs er tamoxifen action . in breast cancer cells , after tamoxifen treatment ( in blue , bound to er ) , pax2 and aib-1 compete for the binding of er , and this competition determines tamoxifen response . high levels of pax2 may recruit co - repressors and other factors that promote chromatin compaction , er - mediated repression , and tamoxifen sensitivity ( on the left ) . on the contrary , high levels of aib-1 promote chromatin opening , transcription activation , and tamoxifen resistance ( on the right ) the balance between aib-1 and pax2 governs er tamoxifen action . in breast cancer cells , after tamoxifen treatment ( in blue , bound to er ) , pax2 and aib-1 compete for the binding of er , and this competition determines tamoxifen response . high levels of pax2 may recruit co - repressors and other factors that promote chromatin compaction , er - mediated repression , and tamoxifen sensitivity ( on the left ) . on the contrary , high levels of aib-1 promote chromatin opening , transcription activation , and tamoxifen resistance ( on the right ) although tamoxifen is a successful er antagonist in breast cancer therapy , it shows partial agonistic effects in other target tissues ( fisher et al . in particular , tamoxifen treatment has been associated with an increased incidence of endometrial carcinoma ( persson 2000 ; zeleniuch - jacquotte et al . gene expression analysis has shown that the genes targeted by tamoxifen are different from those targeted by estrogen , in endometrial epithelial cancer cells ( wu et al . 2005 ) . pax2 is a common target of estrogen- and tamoxifen - bound er and is a crucial effector for the proliferation of endometrial cancer cells . furthermore , pax2 is silenced in normal endometrium , and its expression is reactivated in endometrial cancer upon hypomethylation of its promoter . altogether , these evidences suggest that pax2 plays a crucial role in the determination of tamoxifen response both in breast and endometrial cancer cells , by repressing and promoting cell proliferation , respectively . for these reasons , further studies on the role of pax2 in cooperation with er may shed light on tamoxifen molecular mechanisms of action and resistance . in previous sections , we have discussed the role of xbp-1(s ) in ligand - independent activation of er . moreover , xbp-1(s ) is also a key mediator of er - independent growth ( gomez et al . 2007 ; davies et al . 2008 ) . gomez et al . showed that just the overexpression of xbp-1(s ) explained both phenotypes ( gomez et al . importantly , the study confirms xbp-1(s ) as an essential regulator of bcl2 transcription , which is a prosurvival / antiapoptotic factor and confers resistance to aromatase therapy in breast cancer patients ( ding et al . xbp-1(s ) may be considered an important diagnostic and prognostic biomarker of breast cancer samples and may be also a useful tool in the identification of er - positive breast tumors with a relatively poor response.fig . 4xbp-1(s ) has a role in ligand - independent er activation and anti - estrogen drug resistance . xbp-1(s ) overexpression plays a dual role in estrogen independence and anti - estrogen resistance . xbp-1(s ) can bind and activate er in a ligand - independent manner ( upper panel ) and induces transcription of bcl-2 gene ( lower panel ) , which might have implications in anti - estrogen drug resistance xbp-1(s ) has a role in ligand - independent er activation and anti - estrogen drug resistance . xbp-1(s ) overexpression plays a dual role in estrogen independence and anti - estrogen resistance . xbp-1(s ) can bind and activate er in a ligand - independent manner ( upper panel ) and induces transcription of bcl-2 gene ( lower panel ) , which might have implications in anti - estrogen drug resistance in addition to ligand binding , posttranslational modifications ( acetylation and phosphorylation ) of er and its associated co - activators ( e.g. , src1 , src2 , aib1 , p300 ) and co - repressors ( e.g. , mta1 , ncor , and smrt ) play a role in er action . histone - modifying enzymes interact with er and influence its activity and that of its cooperating factors . moreover , in response to estrogen treatment , er can activate a variety of kinases ( e.g. , map kinases , erk , and akt ) and phosphatases ( e.g. , pp1 , pp2a , and pdxp ) , which can regulate histone proteins ( e.g. , msk1 , msk2 , and histone h1 ) and er co - regulators . in this section , we review and discuss the importance of these enzymes in modulating er , er cooperating partners , and their relevance in hormone - resistant tumors ( fig . receptor tyrosine kinases egfr , her2 , and igfr1 activate downstream signaling pathways including pi3k / akt , map kinases , and erk . these kinases may phosphorylate er , which can be activated in a ligand - independent manner . igfr-1 represses pax2 transcription factor by inducing specific phosphorylation the crosstalk between growth factor signaling pathways and er - cooperating factors . receptor tyrosine kinases egfr , her2 , and igfr1 activate downstream signaling pathways including pi3k / akt , map kinases , and erk . these kinases may phosphorylate er , which can be activated in a ligand - independent manner . igfr-1 represses pax2 transcription factor by inducing specific phosphorylation er is modulated by membrane receptor tyrosine kinases / growth factor signaling , including epidermal growth factor receptor ( egfr ) , her2 , and insulin - like growth factor receptor ( nicholson et al . 2003 ) , which contributes to endocrine resistance ( drury et al . 2011 ; fagan et al . the overexpression of these receptor kinases can activate the downstream mapk / erk and pi3k / akt pathways ( kato et al . 2011 ) , which results in phosphorylation of er at multiple serine residues ( e.g. , 104 , 106 , 118 , 167 , and 305 ) , and can influence er signaling ( arnold et al . importantly , phosphorylation of er at serine 305 is associated with endocrine resistance and poor prognosis ( kok et al . the phosphorylation of er by cytoplasmic kinases also regulates its function via interaction with other transcription factors such as ap-1 , sp1 , and creb , which mediate er interaction with chromatin ( porter et al . her2 signaling has also connection with the er - cooperating factors foxa1 , pax2 , and aib-1 . in molecular apocrine however , the precise mechanism behind the crosstalk between her2 and foxa1 signaling is not completely understood yet . her2 signaling also induces aib-1 phosphorylation ( osborne and schiff 2003 ) , which contributes to tamoxifen resistance . moreover , igf-1 negatively regulates pax2 activity in breast by inducing its phosphorylation ( beauchemin et al . perhaps , regulation of protein phosphorylation might be a mechanism of control of the activity of pax2 and aib-1 and may ultimately dictate the outcome to anti - estrogen therapies . in summary , er signaling and its crosstalk with various signaling pathways have been clinically associated with poor clinical outcome and resistance to anti - estrogen therapies . therefore , affecting either kinases or phosphatases regulating er might help in treating patients with resistance to these therapies . importantly , pp1 phosphatase is known to dephosphorylate aib-1 , and this results in suppression of its degradation ( li et al . the other phosphatases pdxp and pp2a inhibit src3 interaction with er in the absence of ligand ( li et al . future studies identifying how these phosphatases and kinases regulate er and its cooperating factors might improve the anti - estrogen therapies .

estrogen receptor ( er ) is a hormone - regulated transcription factor that controls cell division and differentiation in the ovary , breast , and uterus . the expression of er is a common feature of the majority of breast cancers , which is used as a therapeutic target . recent genetic studies have shown that er binding occurs in regions distant to the promoters of estrogen target genes . these studies have also demonstrated that er binding is accompanied with the binding of other transcription factors , which regulate the function of er and response to anti - estrogen therapies . in this review , we explain how these factors influence the interaction of er to chromatin and their cooperation for er transcriptional activity . moreover , we describe how the expression of these factors dictates the response to anti - estrogen therapies . finally , we discuss how cytoplasmatic signaling pathways may modulate the function of er and its cooperating transcription factors .

Introduction Pioneer transcription factors mediate ER binding Function of ER-cooperating factors in hormone-regulated cancers ER-cooperating factors influence estrogen-mediated transcription ER-cooperating transcription factors and anti-estrogen drug response Cell signaling pathways modulating ER and ER-cooperating factors

the steroid hormone estrogen and the estrogen receptor alpha ( er ) are necessary for the physiology of the female reproductive system ( musgrove and sutherland 2009 ) . these factors play an essential role in the breast , ovaries , and uterus , where they control cell division and differentiation , and the deregulation of er transcriptional activity may result in an increased proliferation and eventually in cancer onset . however , the most widespread type is the luminal group of tumors , and they share the common feature of being positive for the expression of er ( dowsett 2001 ; prat and baselga 2008 ) . er is a transcription factor that mediates the response to estrogens and to anticancer therapies , including the selective estrogen receptor modulator ( serm ) tamoxifen ( katzenellenbogen and frasor 2004 ; hurtado et al . chromatin immunoprecipitation ( chip ) combined with sequencing studies in breast cancer cell lines and human tissue shows a dispersed occupancy pattern of er binding sites bearing heterogeneous recognition motifs ( carroll et al . moreover , er binds to chromatin with a multitude of transcription factors ( er - cooperating factors ) that influence transcriptional activity of er and ultimately affect the outcome of anti - estrogen therapies ( carroll et al . a second group of breast cancer patients is characterized by an amplification of chromosome region 17q12 - 21 , leading to the overexpression of the epidermal growth factor receptor 2 , erbb2/her2/neu ( wolff et al . moreover , about half of her2-positive patients are also positive for er ( dowsett 2001 ) , and the activation of other signaling pathways such as the pi3k pathway is critical for er / her-2-positive tumor development ( berns et al . yet , the molecular mechanism by which these signaling pathways modulate er and er - cooperating factors is not completely understood . in this review , we describe how cooperating factors influence the transcriptional activity of er , and we speculate how these signaling pathways may modulate the function of er and er - cooperating factors . er is a ligand - regulated transcription factor that recognizes a consensus sequence of nucleotides , establishing the binding to dna , and thereby triggering the recruitment of the transcription machinery . pioneer transcription factors interact with chromatin and expose dna for subsequent transcription factor binding and initiation of transcription ( liu et al . genomic analyses of er binding maps have shown that its union is accompanied with the binding of various transcription factors , which includes forkhead box a ( foxa ) ( carroll et al . in this section of the manuscript 1role of pioneering factors in regulation of er chromatin interactions . in the absence of pioneering factors , chromatin regions foxa1 , in cooperation with other transcription factors , opens chromatin regions and facilitates ligand er binding . in the absence of pioneering factors , chromatin regions foxa1 , in cooperation with other transcription factors , opens chromatin regions and facilitates ligand er binding . pbx1 seems to have a foxa1-independent effect foxa proteins are the most studied pioneer transcription factors that bind to chromatin and enable gene activity . the pioneering properties of foxa1 reside on its protein structure , which contains a winged helix domain that can structurally mimic histone h1 and h5 , and thus permits its stable interaction with histone h3 and h4 with high affinity ( cirillo et al . the high chromatin affinity of foxa1 is a unique feature that allows its binding to the specific dna sequences on the nucleosome core and displaces the linker histones , leading to de - compaction of chromatin and facilitation of the binding of other transcription factors . in breast hormone - sensitive and resistant cancer cell lines , almost all er chromatin interactions and gene expression changes are dependent on the expression of foxa1 ( hurtado et al . moreover , foxa1 influences genome - wide chromatin accessibility of er ( hurtado et al . have established that hormone - resistant breast cancers still recruit er to the chromatin , and this binding is associated with foxa1 ( ross - innes et al . interestingly , er shows a distinct binding profile in patients with poor clinical outcome to anti - estrogen therapies . have shown that snps associated with breast cancer risk are located in a subset of the foxa1 binding regions , which influences the binding affinity for the pioneer factor foxa1 ( cowper - sal lari et al . therefore , data published to date suggest that foxa1 is a major determinant of estrogen er activity in breast cancer . however , the mechanism of gata-3 action or its potential role as a pioneer factor of er has not been described yet . 2011 ) , which stably expresses exogenous er and very low levels of foxa1 ( hurtado et al . 2010 ) , which stably expresses exogenous er and is negative for the expression of foxa1 and her2 . the pre - b cell leukemia homeobox 1 factor ( pbx1 ) is a cofactor for homeobox ( hox ) transcription factors as it increases their affinity and specificity to chromatin ( moens and selleri 2006 ) . indeed , the authors point out pbx1 , and not foxa1 , as a novel prognostic marker for recurrence in er - positive breast cancers ( magnani et al . the authors demonstrated that perturbations of the expression of the transcription factor ap-2 prevent er binding to dna and gene transcription . interestingly , the lack of this factor is even affecting er long - range chromatin interactions , which have been shown to be essential for er - mediated transcription ( fullwood et al . the groucho homologue transducin - like enhancer of split 1 ( tle1 ) is a multitasking transcriptional co - repressor . 2010 ) , and the aml / cbfa runt domain transcription factor ( levanon et al . the specific silencing of tle1 inhibits the ability of er to bind a subset of er binding sites within the genome , and this is accompanied by perturbations in phospho - rna pol ii recruitment ( holmes et al . in fact , foxa1 expression is associated with the expression of steroid hormone receptors ( er , progesterone receptor , and androgen receptor ) and other variables of good prognosis such as smaller tumor size , lower histological grade , and expression of luminal cytokeratins ( ck18 and ck7/8 ) , brca1 , and e - cadherin ( habashy et al . this phenotype is representative of the basal subtype of tumors , which are negative for er and her2 expression . in breast , gata-3 plays an important role in mammary gland development and differentiation ( bossard and zaret 1998 ; ho and pai 2007 ) . moreover , the inactivation of gata-3 in mice results in contracted mammary epithelial structure , severely impaired lactogenesis , and disrupted differentiation of luminal progenitor cells into ductal and alveolar cells ( asselin - labat et al . all in all , foxa1 and gata3 that are subsequently used by er to bind chromatin and regulate gene transcription , respectively ( carroll et al . the pioneer factor foxa1 also plays an important role in androgen receptor ( ar ) signaling of molecular apocrine tumors , which have been recently identified as an additional subgroup of er - negative and ar - positive breast tumors ( ni et al . identified ar as a mediator of the ligand - dependent activation of wnt and her2 signaling pathways through direct transcriptional induction of wnt7b and her3 ( ni et al . demonstrated that the specific silencing of foxa1 inhibits ar binding , expression of the majority of the molecular apocrine gene signature , and cell growth ( robinson et al . altogether , it seems that , in breast tumors , er and ar binding and their functionality is fully dependent on foxa1 . importantly , foxa1 protein level in primary prostate tumors has a significant association with the disease outcome ; high foxa1 level is associated with poor prognosis , whereas low foxa1 level , even in the presence of high ar expression , predicts good prognosis . , studies focused on these tissue - specific properties of foxa1 will be instrumental for our understanding of hormone - regulated cancers . in endometrial cancer tumors , foxa1 is expressed in 37 % of the cases , and its expression is significantly and negatively associated with lymph node status ( abe et al . however , when foxa1 is overexpressed in ab32 cells , it induces the expression of genes involved in negative regulation of apoptosis . in summary , all the published data suggest that the idea of using foxa1 as a therapeutic target in breast and endometrial cancers could be an alternative for those patients with recurrence to current treatments . furthermore , it is important to know how the function of foxa1 is regulated . estrogen may activate or repress transcription of er target genes potentially by recruiting distinct classes of co - regulators that have chromatin remodeling properties . structural and functional studies revealed that er co - activators are recruited to hormone - responsive genes through their interaction with activated receptors . the interaction of these co - repressors prompts the binding of chromatin deacetylatases and therefore leads to transcriptional inhibition . some transcription factors have been shown to be responsible for er cofactor binding ( gata-3 , foxa1 , and rara ) , to function as cofactors by themselves ( xbp1 ) or to be mediators of er - repressive action ( pitx-1 ) . in the next paragraphs , we discuss the function of these transcription factors ( fig . pitx-1 represses transcription of a subset of er - regulated genes er - cooperating factors influence estrogen - mediated transcription . pitx-1 represses transcription of a subset of er - regulated genes recently , kong et al . reported in mcf-7 breast carcinoma cells that foxa1 , gata-3 , and er form a protein complex , which regulates er - mediated transcription ( kong et al . the chromatin regions occupied by all these three transcription factors were associated with the highest p300 co - activator recruitment ( histone acetylase enzyme ) , rna pol ii occupancy , and chromatin opening . interestingly , co - transfection of these three transcription factors was sufficient to restore the estrogen - responsive growth of er - negative mda - mb-231 and bt-549 cells . these findings are very significant and suggest that all three transcription factors are needed for co - activator recruitment and , ultimately , for er - mediated transcription in breast tissue . however , it is not clear yet whether the complex of er , foxa1 , and gata-3 is necessary for all er - regulated transcripts in breast tissue . retinoic acid receptor alpha ( rara ) is a nuclear receptor , which regulates gene expression by retinoic acid ( ra ) ( giguere et al . altogether , it seems that rara might have two distinct roles in breast cancer cells : first , repressing estrogen transcription via the classic function of rara with its interacting partner retinoid x receptor and , second , interacting with er and maintaining er both rara agonist and antagonist actions show benefit on breast cancer , both mechanisms of action might be taking place . xbp1 is a transcription factor that belongs to the basic region / leucine zipper ( bzip ) family of proteins ( clauss et al . 2009 ) , which suggests that transcription of er - regulated genes might be also modulated by the complex er xbp1 . recently , the pituitary homeobox 1 ( pitx-1 ) transcription factor has been related as a repressor for a subset of er target genes ( stender et al . this supports the established hypothesis and also suggests that these factors are needed for a sustained transcriptional activity of er . perhaps , foxa1 induces the transcription of these cooperating factors to allow the expression of early er - regulated genes . subsequent activation of the transcription of these cooperating factors by er would then allow the sustained expression of er targets . however , the mechanism of action of these factors for er - mediated transcription is not completely understood . breast tumors positive for er expression represent around 70 % of these cancers ( dowsett 2001 ; prat and baselga 2008 ) . it antagonizes estrogen action by competing for the binding of er in breast cancer cells and is thought to repress er - mediated transcriptional activation by actively recruiting co - repressors ( katzenellenbogen and frasor 2004 ; malik et al . 2010 more recently , another class of anti - estrogen drug has been incorporated into clinical treatment , namely aromatase inhibitors ( ai ) . in this section of the review , we discuss which transcriptional cooperating factors can affect er genomic activity in response to endocrine treatment and how this process can affect cell proliferation and survival . the effectiveness of tamoxifen requires both the binding with er and the consequent interaction with dna . importantly , genomic maps of er binding induced with estrogen or tamoxifen are almost identical ( hurtado et al . crosstalk between the er and her2 pathways has long been implicated in cancer onset and response to tamoxifen , but no direct connection at transcriptional level has been shown . 2008 ) , which revealed a novel interplay between er and erbb2 on a genomic level . this repression is mediated by the transcription factor pax2 , which cooperates with er at this locus . pax2 belongs to the pair box gene ( pax ) family , a group of transcription factors characterized by the presence of two dna - binding domains and are known for their role in terminal differentiation during organogenesis ( mansouri et al . 2003 ) , and its nuclear localization is more frequent in luminal tumors than in nonluminal tumors ( silberstein et al . moreover , the expression of pax2 is reduced in tamoxifen - resistant cells , and the overexpression of pax2 is able to restore the sensitivity to tamoxifen in these cells ( hurtado et al . indeed , there is a competition between aib-1 and pax2 for the binding to the er binding region of the erbb2 gene , which might explain the differences between the two studies . 2012 ) , which suggests that pax2 might be functioning as a general repressor for er tamoxifen action ( fig . in summary , all these studies denote that the repressive action of tamoxifen is regulated by cooperating factors at least at two different levels : foxa1 , which orchestrates er binding on the chromatin , and pax2 , which dictates the transcriptional activity of er induced by tamoxifen . in breast cancer cells , after tamoxifen treatment ( in blue , bound to er ) , pax2 and aib-1 compete for the binding of er , and this competition determines tamoxifen response . in breast cancer cells , after tamoxifen treatment ( in blue , bound to er ) , pax2 and aib-1 compete for the binding of er , and this competition determines tamoxifen response . on the contrary , high levels of aib-1 promote chromatin opening , transcription activation , and tamoxifen resistance ( on the right ) although tamoxifen is a successful er antagonist in breast cancer therapy , it shows partial agonistic effects in other target tissues ( fisher et al . pax2 is a common target of estrogen- and tamoxifen - bound er and is a crucial effector for the proliferation of endometrial cancer cells . furthermore , pax2 is silenced in normal endometrium , and its expression is reactivated in endometrial cancer upon hypomethylation of its promoter . in previous sections , we have discussed the role of xbp-1(s ) in ligand - independent activation of er . moreover , xbp-1(s ) is also a key mediator of er - independent growth ( gomez et al . importantly , the study confirms xbp-1(s ) as an essential regulator of bcl2 transcription , which is a prosurvival / antiapoptotic factor and confers resistance to aromatase therapy in breast cancer patients ( ding et al . xbp-1(s ) may be considered an important diagnostic and prognostic biomarker of breast cancer samples and may be also a useful tool in the identification of er - positive breast tumors with a relatively poor response.fig . 4xbp-1(s ) has a role in ligand - independent er activation and anti - estrogen drug resistance . xbp-1(s ) can bind and activate er in a ligand - independent manner ( upper panel ) and induces transcription of bcl-2 gene ( lower panel ) , which might have implications in anti - estrogen drug resistance xbp-1(s ) has a role in ligand - independent er activation and anti - estrogen drug resistance . xbp-1(s ) overexpression plays a dual role in estrogen independence and anti - estrogen resistance . xbp-1(s ) can bind and activate er in a ligand - independent manner ( upper panel ) and induces transcription of bcl-2 gene ( lower panel ) , which might have implications in anti - estrogen drug resistance in addition to ligand binding , posttranslational modifications ( acetylation and phosphorylation ) of er and its associated co - activators ( e.g. histone - modifying enzymes interact with er and influence its activity and that of its cooperating factors . moreover , in response to estrogen treatment , er can activate a variety of kinases ( e.g. , pp1 , pp2a , and pdxp ) , which can regulate histone proteins ( e.g. in this section , we review and discuss the importance of these enzymes in modulating er , er cooperating partners , and their relevance in hormone - resistant tumors ( fig . receptor tyrosine kinases egfr , her2 , and igfr1 activate downstream signaling pathways including pi3k / akt , map kinases , and erk . igfr-1 represses pax2 transcription factor by inducing specific phosphorylation the crosstalk between growth factor signaling pathways and er - cooperating factors . receptor tyrosine kinases egfr , her2 , and igfr1 activate downstream signaling pathways including pi3k / akt , map kinases , and erk . igfr-1 represses pax2 transcription factor by inducing specific phosphorylation er is modulated by membrane receptor tyrosine kinases / growth factor signaling , including epidermal growth factor receptor ( egfr ) , her2 , and insulin - like growth factor receptor ( nicholson et al . 2011 ) , which results in phosphorylation of er at multiple serine residues ( e.g. the phosphorylation of er by cytoplasmic kinases also regulates its function via interaction with other transcription factors such as ap-1 , sp1 , and creb , which mediate er interaction with chromatin ( porter et al . her2 signaling has also connection with the er - cooperating factors foxa1 , pax2 , and aib-1 . perhaps , regulation of protein phosphorylation might be a mechanism of control of the activity of pax2 and aib-1 and may ultimately dictate the outcome to anti - estrogen therapies . in summary , er signaling and its crosstalk with various signaling pathways have been clinically associated with poor clinical outcome and resistance to anti - estrogen therapies . future studies identifying how these phosphatases and kinases regulate er and its cooperating factors might improve the anti - estrogen therapies .

wound healing after tooth extraction or dental implantation is an abstruse process involving a series of biological events which consist of repair and remodeling of soft and hard tissues in response to injury . the focus of research in bone biology and healing is now centered on molecular events that regulate the repair of injured tissue . identification of cellular and molecular biology and many signaling molecules associated with formation and repair of skeletal tissues , like members of the transforming growth factor- superfamily ( including the bone morphogenetic proteins ) and several additional signaling molecules such as fibroblast growth factors , insulin - like growth factors and platelet derived growth factors have resulted in rapid progress of our knowledge of the complex process of wound healing . many types of cells involve in the wound healing process , such as immune cells , endothelial cells , fibroblasts , progenitors , and stem cells , whose proliferation , differentiation and migration are a prerequisite for this phenomenon . one of the mechanisms which plays an important role in the remodeling of bone is the relationship between the elements osteoprotegerin ( opg ) , receptor activator of nuclear factor b ( rank ) , and rank ligand ( rankl ) . the manner in which opg interacts with the target cells is binding to rankl ; a transmembrane cytokine expressed on the surface of the preosteoclastic / stromal cells ; this binds to rank and rankl , triggers a series of mechanisms that result in differentiation , maturation and activation of osteoclasts . tartrate - resistant acid phosphatase ( trap or trapase ) , also called acid phosphatase 5 , tartrate resistant , is a glycosylated monomeric metalloprotein enzyme expressed in mammals . under normal circumstances , trap is highly expressed by osteoclasts , activated macrophages , neurons , and by the porcine endometrium during pregnancy . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . it has been shown that osteopontin and bone sialoprotein , and bone matrix phosphoproteins , are highly efficient in vitro trap substrates , which bind to osteoclasts when phosphorylated . on partial dephosphorylation , both osteopontin and bone sialoprotein are incapable of binding to osteoclasts . from this effect , it has been hypothesized that trap is secreted from the ruffled border of osteoclasts dephosphorylates osteopontin and allows osteoclast migration , and further resorption to occur . a number of drugs such as bisphosphontes , esteriods , nonesteroidal anti - inflammatory drugs , and chemically modified teracyclines are recognized for interaction with these complicated mechanisms in different stages , although the exact mechanisms of these agents have not been exactly described . chemically modified tetracyclines ( cmts ) are tetracycline compounds which have substantially no antibacterial activity but have been found to possess a number of interesting properties , such as the inhibition of excessive collagenolytic activity in vivo . they have been used for their anticancer potential in a variety of cancers : melanoma , lung , breast , and prostate cancers . bone resorption is also suppressed due to their combined antiproteinase and apoptotic effects on osteoblasts and osteoclasts , respectively . development of resistant bacteria and gastrointestinal toxicity seen with parent tetracyclines is not produced by cmts . in 1984 , gomes et al . compared four antibiotics ; penicillin , streptomycin , ampicillin , and tetracycline for their inhibitory effect on bone resorption . after the introduction of nonantibacterial tetracycline formulations in dentistry , a low - dose of minocycline was initially tested but was soon replaced by low - dose doxycycline ( dox ) . showed more linear bone fill using a membrane loaded with 25% dox paste in the treatment of human periodontal infrabony defects . macrolides such as erythromycin ( em ) are reported to reduce exacerbation of chronic inflammatory respiratory disease and chronic obstructive pulmonary disease ; their anti - inflammatory effects in vitro and in vivo are shown . ren et al . concluded that em can inhibit wear debris - induced osteoclastogenesis by modulation of murine macrophage nf-b activity . they also reported that when em was applied to the peri - apatite layer of the titanium pins in the tibial bone of rats , the bone volume percentage increased significantly around the pin area . to the best of our knowledge , no in vivo study has yet been conducted to report the effect of dox on the expression of opg and rankl genes following tooth extraction . the objective of this research is to evaluate the rankl and opg gene expressions using real - time polymerase chain reaction ( rt - pcr ) and the number of osteoclasts by trap staining , in the presence of subantimicrobial concentrations of dox and em following tooth extraction in rats . in this animal study , forty five ( 810 ) week - old male wistar rats had their maxillary right molar extracted after immobilization and using general anesthesia with ketamine 10% ( alfasan international , woerden , holland , 80 mg / kg ) and xylazine ( neurotranq , alfasan , woerden , holand , 8 mg / kg ) . teeth were loosed using a hemostat with modified beaks ( two cavities were made in each beak ) ( day 1 ) . finally , they were extracted by a cotton plier . the percedures of this study were approved by the animal research ethics committee of the isfahan university of medical sciences , isfahan , iran . then animals were divided into three groups of fifteen . in group 1 ( control ) , groups 2 and 3 the rats received normal saline ( 5 ml / kg / day ) , dox ( subantimicrobial dose , 5 mg / kg / day by gavage ) and em ( subantimicrobial dose , 2 mg / kg / day intraperitoneally [ i.p . ] ) , respectively at day 1 and daily for 1 week . each group was evaluated at three different times : in 7 , 14 , and 21 days following tooth extraction . the animals were kept in an artificially controlled environment with temperature ranging from 20c to 24c , on a 12:12 h light / dark cycle and were fed food and water . then samples were euthanized in 7 , 14 , and 21 days after surgery in a chamber saturated with halothane vapor . after the administration of subantimicrobial dose of dox , immobilization of the rats , an aqueous solution of dox hyclate 10% was released into their stomachs using a gavage tube . in group 3 , em dissolved in distilled water and was injected i.p . from the day of tooth extraction and maintained daily injection until the 7 day . the right sections of upper jaws of animals were cut and maintained in a 4% paraformaldehyde solution . the study was approved by the ethics committee of the by the ethical committee for animal experiment in isfahan university of medical sciences . real - time quantitative polymerase chain reaction ( taqman pcr ) using an abi step one real - time sequence detection system and a taqman pcr core reagent kit ( perkin elmer corp . ) was performed according to the manufacturer 's protocol . the copy number of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) mrna was used as an internal control . gapdh : forward 5'-gcattgatggtgaggtgagcaaa-3 ' , reverse 5'-tcgctcctggaagatggtga-3 ' , taqman probe 5 ' ( fam)-ccacggcaagttcaacggcacagt-(tamra ) 3 ' ; opg : forward , 5'-agagggcgcatagtcagtagaca-3 ' , reverse 5'-atattgcccccaacgttcaac-3 ' , taqman probe 5 ' ( fam)-tgtgcactcctggtgttcttggaca-(tamra ) 3 ' ; rankl : forward 5'-cttggcccagcctcgat-3 ' , reverse 5-accatcaatgctgccgacat-3 ' , taqman probe 5 ' ( fam)-aaggttcgtggctcgatgtggcc-(tamra ) 3 ' . the conditions for the opg gene were as follows : 95c for 5 min , followed by 48 cycles of 95c for 30 s , 62c for 45 s , and 72c for 28 s. for rankl gene the setting was 95c for 5 min , followed by 28 cycles of 95c for 25 s , 58c for 30 s , and 72c for 20 s and an extra cycle of 62c for 10 s. the negative controls for each target showed an absence of carryover . to detect the specific antigens of trap , the tissues were immunohistochemically stained by biotin streptavidin method . briefly , the main procedure included serial sectioning ( in 34 m sections ) , deparaffinization , rehydration , and antigen retrieval . all specimens were placed in phosphate - buffered saline , treated with protein block ( re 1102 ) for 5 min to prevent any false staining . next , the specimens were incubated for 30 min with primary antibody of trap ( ncl - trap ) clone 514h12 ( novocastra ) . ( the sections were then exposed to novolink polymer [ re7112 ] or secondary antibody for 30 min and washed in phosphate buffer saline , after that they were incubated with 3,3'-diaminobenzidine for 5 min for visualization . sections through tonsils served as positive controls and the breast tissue were used as negative control . to quantities the number of cells within the lesions positive for trap marker in the middle third of the rat alveolus , sections were observed by a single examiner who was also blinded to the identity of samples using a 400 magnification of olympus light microscope ( olympus corporation , tokyo , japan ) . the amount of stained cells ( multinuclear trap - positive cells that were considered as osteoclasts ) in each field was measured . all data of three groups were presented as the mean standard error of the mean in table 1 and were analyzed by spss version 20 software ( statistical package for the social sciences , ibm spss , inc . in chicago , if a significant difference between them was found , post hoc ( tukey multiple comparisons ) test was used for comparison between groups . a significance level of p the mean expression of receptor activator of nuclear factor b ligand , osteoprotegerin , receptor activator of nuclear factor b ligand / osteoprotegerin ratio and the mean number of osteoclasts in dental socket of rat in test and control groups real - time quantitative polymerase chain reaction ( taqman pcr ) using an abi step one real - time sequence detection system and a taqman pcr core reagent kit ( perkin elmer corp . ) was performed according to the manufacturer 's protocol . one microliter of the first strand cdna was used in the following assay . the copy number of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) mrna was used as an internal control . gapdh : forward 5'-gcattgatggtgaggtgagcaaa-3 ' , reverse 5'-tcgctcctggaagatggtga-3 ' , taqman probe 5 ' ( fam)-ccacggcaagttcaacggcacagt-(tamra ) 3 ' ; opg : forward , 5'-agagggcgcatagtcagtagaca-3 ' , reverse 5'-atattgcccccaacgttcaac-3 ' , taqman probe 5 ' ( fam)-tgtgcactcctggtgttcttggaca-(tamra ) 3 ' ; rankl : forward 5'-cttggcccagcctcgat-3 ' , reverse 5-accatcaatgctgccgacat-3 ' , taqman probe 5 ' ( fam)-aaggttcgtggctcgatgtggcc-(tamra ) 3 ' . the conditions for the opg gene were as follows : 95c for 5 min , followed by 48 cycles of 95c for 30 s , 62c for 45 s , and 72c for 28 s. for rankl gene the setting was 95c for 5 min , followed by 28 cycles of 95c for 25 s , 58c for 30 s , and 72c for 20 s and an extra cycle of 62c for 10 s. the negative controls for each target showed an absence of carryover . to detect the specific antigens of trap , the tissues were immunohistochemically stained by biotin streptavidin method . briefly , the main procedure included serial sectioning ( in 34 m sections ) , deparaffinization , rehydration , and antigen retrieval . all specimens were placed in phosphate - buffered saline , treated with protein block ( re 1102 ) for 5 min to prevent any false staining . next , the specimens were incubated for 30 min with primary antibody of trap ( ncl - trap ) clone 514h12 ( novocastra ) . ( the sections were then exposed to novolink polymer [ re7112 ] or secondary antibody for 30 min and washed in phosphate buffer saline , after that they were incubated with 3,3'-diaminobenzidine for 5 min for visualization . sections through tonsils served as positive controls and the breast tissue were used as negative control . to quantities the number of cells within the lesions positive for trap marker in the middle third of the rat alveolus , sections were observed by a single examiner who was also blinded to the identity of samples using a 400 magnification of olympus light microscope ( olympus corporation , tokyo , japan ) . the amount of stained cells ( multinuclear trap - positive cells that were considered as osteoclasts ) in each field was measured . all data of three groups were presented as the mean standard error of the mean in table 1 and were analyzed by spss version 20 software ( statistical package for the social sciences , ibm spss , inc . in chicago , if a significant difference between them was found , post hoc ( tukey multiple comparisons ) test was used for comparison between groups . a significance level of p the mean expression of receptor activator of nuclear factor b ligand , osteoprotegerin , receptor activator of nuclear factor b ligand / osteoprotegerin ratio and the mean number of osteoclasts in dental socket of rat in test and control groups one - way anova showed that differences in the mean of rankl in em , dox , and control groups were statistically significant with regard to evaluated time points [ table 1 ] . in all of the studied intervals in em group compared to control group , a significant rankl reduction on gene expression level was observed . in the dox group , the expression of rankl in three evaluated time points diminished significantly rather than in the control group ( p < 0.05 ) . based on post hoc analysis ( tukey 's multiple comparisons test ) , no meaningful difference in the expression of rankl gene was observed between dox and em groups [ p > 0.05 , table 1 and chart 1a ] . ( a ) receptor activator of nuclear factor b ligand expression in the dental socket of rat , ( b ) osteoprotegerin expression in the dental socket of rat , ( c ) receptor activator of nuclear factor b ligand / osteoprotegerin ratio . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points . p values derived from by tukey 's post hoc test . in all of the studied intervals in dox and em groups , the expression of opg was higher when compared to control group in same periods [ table 1 ] , although this enhancement was not statistically significant ( p > 0.05 ) . when two intervened groups were compared in similar evaluated time points , despite higher levels of opg expression in em group , no significant difference was observed [ p > 0.05 , table 1 and chart 1b ] . as shown in table 1 , the ratio of rankl / opg in em and dox groups in the days 7 and 14 , was significantly lower than the control group ( p < 0.05 ) , but on day 21 it was not . in the comparison of two drugs , there was no meaningful difference between them [ p > 0.05 , table 1 , chart 1c ] . this permits characterization of osteoclasts by their staining for high expression of trap , and cathepsin k. osteoclast rough endoplasmic reticulum is sparse , and the golgi complex is extensive . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . on day 7 , the number of trap + cells in control group was significantly lower than dox and em groups , without any meaningful difference between two tested groups [ p < 0.05 , table 1 and chart 2 ] . on days 14 and 21 , based on one - way anova , there was no significant difference in the studied groups [ p > 0.05 , table 1 and figure 1 ] . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points , y - axis : mean + standard error of mean of variables , p derived from by tukey 's post hoc test . rat alveolar socket 1 week after tooth extraction ( trap , 400 ) . ( a ) control group , ( b ) erythromycin , ( c ) doxycyclin . one - way anova showed that differences in the mean of rankl in em , dox , and control groups were statistically significant with regard to evaluated time points [ table 1 ] . in all of the studied intervals in em group compared to control group , a significant rankl reduction on gene expression level was observed . in the dox group , the expression of rankl in three evaluated time points diminished significantly rather than in the control group ( p < 0.05 ) . based on post hoc analysis ( tukey 's multiple comparisons test ) , no meaningful difference in the expression of rankl gene was observed between dox and em groups [ p > 0.05 , table 1 and chart 1a ] . ( a ) receptor activator of nuclear factor b ligand expression in the dental socket of rat , ( b ) osteoprotegerin expression in the dental socket of rat , ( c ) receptor activator of nuclear factor b ligand / osteoprotegerin ratio . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points . in all of the studied intervals in dox and em groups , the expression of opg was higher when compared to control group in same periods [ table 1 ] , although this enhancement was not statistically significant ( p > 0.05 ) . when two intervened groups were compared in similar evaluated time points , despite higher levels of opg expression in em group , no significant difference was observed [ p > 0.05 , table 1 and chart 1b ] . as shown in table 1 , the ratio of rankl / opg in em and dox groups in the days 7 and 14 , was significantly lower than the control group ( p < 0.05 ) , but on day 21 it was not . in the comparison of two drugs , there was no meaningful difference between them [ p > 0.05 , table 1 , chart 1c ] . this permits characterization of osteoclasts by their staining for high expression of trap , and cathepsin k. osteoclast rough endoplasmic reticulum is sparse , and the golgi complex is extensive . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . on day 7 , the number of trap + cells in control group was significantly lower than dox and em groups , without any meaningful difference between two tested groups [ p < 0.05 , table 1 and chart 2 ] . on days 14 and 21 , based on one - way anova , there was no significant difference in the studied groups [ p > 0.05 , table 1 and figure 1 ] . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points , y - axis : mean + standard error of mean of variables , p derived from by tukey 's post hoc test . rat alveolar socket 1 week after tooth extraction ( trap , 400 ) . ( a ) control group , ( b ) erythromycin , ( c ) doxycyclin . to the best of our knowledge , no in vivo study has yet been conducted to report the effect of dox on the expression of opg and rankl genes following tooth extraction . in fact , we reported the short - term sequential expression of rankl and opg from week 1 to week 3 ( every week in the dental socket of rat ) , using the advanced method of rt - pcr to register the delicate alterations as far as possible . the rankl / opg ratio illustrates the importance of the balance between these molecules in bone remodeling , in health , and in disease states . both studied genes , opg and rankl , are expressed by osteoblasts and play a major role at early stages of alveolar bone healing . opg is the natural decoy receptor for rankl that is known as osteoclastogenesis inhibitory factor since these molecules are known as key factors for osteoclastogenesis and primary regulators of bone remodeling , we focused our attention on them . glucocorticoids have been demonstrated to upregulate rankl mrna levels and decrease opg mrna level in human osteoblasts . a similar pattern of these changes has been indicated following the administration of immunosuppressant ( cyclosporine a , rapanyein , tacrolimus ) , by contrast bisphosphonates have been shown a reverse pattern in the expression of these genes . in control group , on days 7 and 14 , the rankl / opg ratio was significantly higher than other two groups that indicate the anti - osteoclastogenesis of dox and em , again . the increase in opg ( marker of bone formation ) and reduction in rankl ( marker of bone resorption ) expressions observed in both dox and em groups , ( than the control group , ) indicates more activation or increase of the osteoblast cells . in em - treated group , a significant down - regulation of rankl gene expression was observed at 3 weeks , suggesting low differentiation of osteoclasts , and show the low level of bone resorption than control group . this finding is confirmed the decreased number of osteoclasts by trap staining ( a genetic marker of osteoclast differentiation ) with the first week in control group and increased with the amount of new bone formation . in the dox - treated group , there was a gradual up - regulation of opg gene expression over time ; while the pattern of rankl gene expression had a gradually decreasing behavior . the rankl expression peak , which occurred during the bone resorption phase ( 7 day ) , preceded the expression of opg that occurred during the bone formation phase ( 21 day ) . it meant that bone formation was stimulated by dox and bone resorption was inhibited ; comparing the control group in which rankl gene expression or induction of bone resorption was enhanced gradually . the rankl / opg ratio peaked in the first week , rather than other periods , and then gradually decreased . this might be related to the initial increase in bone resorption that preceded normal bone formation following tooth extraction which has been noted by guglielmotti and cabrini and iizuka et al . it has been demonstrated that 14 days after tooth extraction in rats is a time in which maximal bone formation and alveolar volume occurs . the effect of tetracyclines on osteoblastic cells was addressed in few previous studies , by different cell systems and situations . gomes and fernandes reported that therapeutic concentrations of doxycycline and minocycline can stimulate the proliferation of osteoblastic - induced bone marrow cells . they caused an increase in the cell growth , in the maintenance of alkaline phosphatase activity and higher abundance of mineral deposition . . used nanohydroxyapatite microspheres as a delivery system for em , amoxicillin , and augmentin to evaluate their interaction with osteoblasts . he concluded that em - induced osteoblastic cells proliferation higher than others , and also suggested the use of microsphere and em as a good alternative carrier to enhance bone regeneration while treating periodontal defects . reduction of resorbing activity in our investigation was not in agreement of folwarczna et al . 's study which indicated administration of dox in rats with normal process of bone remodeling , increased bone resorption , and this effect may be dose - dependent . in another study with the aim of comparison of new bone formation following use of dox and vehicle in the bony defects , no meaningful difference was observed . on the other hand , many articles showed the inhibitory effect of dox in bone resorption . in an in vivo investigation , it was reported that the dox - treated rats ' ( 7 days , i.p . , 3.0 mg / kg ) skin , muscle and bone healing clearly improved compared with the saline - treated animals . in another investigation by use of dox after periradicular surgeries or following applied orthodontic forces , bone loss reduced significantly . reported that a bioabsorbable polymer that delivers doxycycline ( atridox ) can be used for the treatment of severe peri - implant bone loss when is combined with autogenous bone . in another study in 2008 , metzger et al . concluded that low - dose doxycycline reduced the area of bone resorption associated with apical periodontitis in the mandibular first molar teeth of rats . we know that there is a biologic balance between inflammatory cytokines and growth factors so that upregulation of one results in the down - regulation of the other one . both studied drugs have an anti - inflammatory effect which is independent of their antimicrobial one , so a decrease in the inflammatory cytokines caused enhancement of the growth factors and increased regeneration and rate of wound healing . since the principles of osseointegration of dental implants are on the basis of bone healing , understanding its mechanism , rate and the factors which may accelerate this phenomenon is very important . other routes of administration of these drugs such as local delivery as surface coating have not yet been investigated in conjunction with endosseous dental implants , which is strongly suggested for the future studies . according to these findings , it seems that dox and em improve healing of alveolar bony socket by decreasing rankl / opg ratio and the number of osteoclasts that suggests their anti - osteoclastogenesis activity . it is reasonable to propose that the rankl / opg system plays an important role in bone healing . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .

background : the aim of this study was to evaluate the effects of bone resorption inhibitors , doxycycline ( dox ) and erythromycin ( em ) , on osseous wound healing in rat alveolar socket.materials and methods : in this randomized controlled trial , 45 810-week - old male wistar rats had their maxillary right molar extracted . they were divided into three groups of 15 . in group 1 normal saline , group 2 dox , and group 3 em were administered at the doses of 5 ml / kg / day , 5 mg / kg / day , and 2 mg / kg / day , respectively , for 7 consecutive days . the rats were sacrificed 7 , 14 , and 21 days after surgery . real - time polymerase chain reaction was employed to evaluate the mrna expression of receptor activator of nuclear factor b ligand ( rankl ) and osteoprotegerin ( opg ) and immunohistochemical staining for tartrate - resistant acid phosphatase ( trap ) to determine osteoclasts . the data were analyzed by one - way analysis of variance followed by tukey 's post hoc test using spss version 20 . significant level was set at 0.05.results:the results showed that when drug - treated groups compared to control groups , rankl gene expression significantly decreased , trap+ cells decreased on day 7 . the rankl / opg ratios in the first two weeks in the test groups were significantly lower than the control group . there was no significant difference in the studied indices between dox and em groups.conclusion:following administration of dox and em , the number of osteoclasts and rankl / opg ratio decreased suggesting their anti - osteoclastogenesis activity . these two drugs have no advantage over each other in increasing the bone formation .

INTRODUCTION MATERIALS AND METHODS Real-time quantitative polymerase chain reaction Immunohistochemistry Statistical analysis RESULTS Receptor activator of nuclear factor B ligand gene expression Osteoprotegerin gene expression Receptor activator of nuclear factor B ligand/osteoprotegerin ratio Immunohistochemical observation (tartrate-resistant acid phosphatase + cells) DISCUSSION CONCLUSION Financial support and sponsorship Conflicts of interest

identification of cellular and molecular biology and many signaling molecules associated with formation and repair of skeletal tissues , like members of the transforming growth factor- superfamily ( including the bone morphogenetic proteins ) and several additional signaling molecules such as fibroblast growth factors , insulin - like growth factors and platelet derived growth factors have resulted in rapid progress of our knowledge of the complex process of wound healing . many types of cells involve in the wound healing process , such as immune cells , endothelial cells , fibroblasts , progenitors , and stem cells , whose proliferation , differentiation and migration are a prerequisite for this phenomenon . one of the mechanisms which plays an important role in the remodeling of bone is the relationship between the elements osteoprotegerin ( opg ) , receptor activator of nuclear factor b ( rank ) , and rank ligand ( rankl ) . tartrate - resistant acid phosphatase ( trap or trapase ) , also called acid phosphatase 5 , tartrate resistant , is a glycosylated monomeric metalloprotein enzyme expressed in mammals . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . from this effect , it has been hypothesized that trap is secreted from the ruffled border of osteoclasts dephosphorylates osteopontin and allows osteoclast migration , and further resorption to occur . a number of drugs such as bisphosphontes , esteriods , nonesteroidal anti - inflammatory drugs , and chemically modified teracyclines are recognized for interaction with these complicated mechanisms in different stages , although the exact mechanisms of these agents have not been exactly described . bone resorption is also suppressed due to their combined antiproteinase and apoptotic effects on osteoblasts and osteoclasts , respectively . compared four antibiotics ; penicillin , streptomycin , ampicillin , and tetracycline for their inhibitory effect on bone resorption . after the introduction of nonantibacterial tetracycline formulations in dentistry , a low - dose of minocycline was initially tested but was soon replaced by low - dose doxycycline ( dox ) . macrolides such as erythromycin ( em ) are reported to reduce exacerbation of chronic inflammatory respiratory disease and chronic obstructive pulmonary disease ; their anti - inflammatory effects in vitro and in vivo are shown . they also reported that when em was applied to the peri - apatite layer of the titanium pins in the tibial bone of rats , the bone volume percentage increased significantly around the pin area . to the best of our knowledge , no in vivo study has yet been conducted to report the effect of dox on the expression of opg and rankl genes following tooth extraction . the objective of this research is to evaluate the rankl and opg gene expressions using real - time polymerase chain reaction ( rt - pcr ) and the number of osteoclasts by trap staining , in the presence of subantimicrobial concentrations of dox and em following tooth extraction in rats . in this animal study , forty five ( 810 ) week - old male wistar rats had their maxillary right molar extracted after immobilization and using general anesthesia with ketamine 10% ( alfasan international , woerden , holland , 80 mg / kg ) and xylazine ( neurotranq , alfasan , woerden , holand , 8 mg / kg ) . the percedures of this study were approved by the animal research ethics committee of the isfahan university of medical sciences , isfahan , iran . then animals were divided into three groups of fifteen . in group 1 ( control ) , groups 2 and 3 the rats received normal saline ( 5 ml / kg / day ) , dox ( subantimicrobial dose , 5 mg / kg / day by gavage ) and em ( subantimicrobial dose , 2 mg / kg / day intraperitoneally [ i.p . ] each group was evaluated at three different times : in 7 , 14 , and 21 days following tooth extraction . then samples were euthanized in 7 , 14 , and 21 days after surgery in a chamber saturated with halothane vapor . after the administration of subantimicrobial dose of dox , immobilization of the rats , an aqueous solution of dox hyclate 10% was released into their stomachs using a gavage tube . in group 3 , em dissolved in distilled water and was injected i.p . real - time quantitative polymerase chain reaction ( taqman pcr ) using an abi step one real - time sequence detection system and a taqman pcr core reagent kit ( perkin elmer corp . ) the conditions for the opg gene were as follows : 95c for 5 min , followed by 48 cycles of 95c for 30 s , 62c for 45 s , and 72c for 28 s. for rankl gene the setting was 95c for 5 min , followed by 28 cycles of 95c for 25 s , 58c for 30 s , and 72c for 20 s and an extra cycle of 62c for 10 s. the negative controls for each target showed an absence of carryover . briefly , the main procedure included serial sectioning ( in 34 m sections ) , deparaffinization , rehydration , and antigen retrieval . next , the specimens were incubated for 30 min with primary antibody of trap ( ncl - trap ) clone 514h12 ( novocastra ) . ( the sections were then exposed to novolink polymer [ re7112 ] or secondary antibody for 30 min and washed in phosphate buffer saline , after that they were incubated with 3,3'-diaminobenzidine for 5 min for visualization . to quantities the number of cells within the lesions positive for trap marker in the middle third of the rat alveolus , sections were observed by a single examiner who was also blinded to the identity of samples using a 400 magnification of olympus light microscope ( olympus corporation , tokyo , japan ) . all data of three groups were presented as the mean standard error of the mean in table 1 and were analyzed by spss version 20 software ( statistical package for the social sciences , ibm spss , inc . in chicago , if a significant difference between them was found , post hoc ( tukey multiple comparisons ) test was used for comparison between groups . a significance level of p the mean expression of receptor activator of nuclear factor b ligand , osteoprotegerin , receptor activator of nuclear factor b ligand / osteoprotegerin ratio and the mean number of osteoclasts in dental socket of rat in test and control groups real - time quantitative polymerase chain reaction ( taqman pcr ) using an abi step one real - time sequence detection system and a taqman pcr core reagent kit ( perkin elmer corp . ) one microliter of the first strand cdna was used in the following assay . the conditions for the opg gene were as follows : 95c for 5 min , followed by 48 cycles of 95c for 30 s , 62c for 45 s , and 72c for 28 s. for rankl gene the setting was 95c for 5 min , followed by 28 cycles of 95c for 25 s , 58c for 30 s , and 72c for 20 s and an extra cycle of 62c for 10 s. the negative controls for each target showed an absence of carryover . briefly , the main procedure included serial sectioning ( in 34 m sections ) , deparaffinization , rehydration , and antigen retrieval . next , the specimens were incubated for 30 min with primary antibody of trap ( ncl - trap ) clone 514h12 ( novocastra ) . ( the sections were then exposed to novolink polymer [ re7112 ] or secondary antibody for 30 min and washed in phosphate buffer saline , after that they were incubated with 3,3'-diaminobenzidine for 5 min for visualization . to quantities the number of cells within the lesions positive for trap marker in the middle third of the rat alveolus , sections were observed by a single examiner who was also blinded to the identity of samples using a 400 magnification of olympus light microscope ( olympus corporation , tokyo , japan ) . all data of three groups were presented as the mean standard error of the mean in table 1 and were analyzed by spss version 20 software ( statistical package for the social sciences , ibm spss , inc . in chicago , if a significant difference between them was found , post hoc ( tukey multiple comparisons ) test was used for comparison between groups . a significance level of p the mean expression of receptor activator of nuclear factor b ligand , osteoprotegerin , receptor activator of nuclear factor b ligand / osteoprotegerin ratio and the mean number of osteoclasts in dental socket of rat in test and control groups one - way anova showed that differences in the mean of rankl in em , dox , and control groups were statistically significant with regard to evaluated time points [ table 1 ] . in all of the studied intervals in em group compared to control group , a significant rankl reduction on gene expression level was observed . in the dox group , the expression of rankl in three evaluated time points diminished significantly rather than in the control group ( p < 0.05 ) . based on post hoc analysis ( tukey 's multiple comparisons test ) , no meaningful difference in the expression of rankl gene was observed between dox and em groups [ p > 0.05 , table 1 and chart 1a ] . ( a ) receptor activator of nuclear factor b ligand expression in the dental socket of rat , ( b ) osteoprotegerin expression in the dental socket of rat , ( c ) receptor activator of nuclear factor b ligand / osteoprotegerin ratio . * significant difference was found between intervention groups with control group , p < 0.05 . p values derived from by tukey 's post hoc test . in all of the studied intervals in dox and em groups , the expression of opg was higher when compared to control group in same periods [ table 1 ] , although this enhancement was not statistically significant ( p > 0.05 ) . when two intervened groups were compared in similar evaluated time points , despite higher levels of opg expression in em group , no significant difference was observed [ p > 0.05 , table 1 and chart 1b ] . as shown in table 1 , the ratio of rankl / opg in em and dox groups in the days 7 and 14 , was significantly lower than the control group ( p < 0.05 ) , but on day 21 it was not . in the comparison of two drugs , there was no meaningful difference between them [ p > 0.05 , table 1 , chart 1c ] . this permits characterization of osteoclasts by their staining for high expression of trap , and cathepsin k. osteoclast rough endoplasmic reticulum is sparse , and the golgi complex is extensive . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . on day 7 , the number of trap + cells in control group was significantly lower than dox and em groups , without any meaningful difference between two tested groups [ p < 0.05 , table 1 and chart 2 ] . on days 14 and 21 , based on one - way anova , there was no significant difference in the studied groups [ p > 0.05 , table 1 and figure 1 ] . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points , y - axis : mean + standard error of mean of variables , p derived from by tukey 's post hoc test . rat alveolar socket 1 week after tooth extraction ( trap , 400 ) . one - way anova showed that differences in the mean of rankl in em , dox , and control groups were statistically significant with regard to evaluated time points [ table 1 ] . in all of the studied intervals in em group compared to control group , a significant rankl reduction on gene expression level was observed . in the dox group , the expression of rankl in three evaluated time points diminished significantly rather than in the control group ( p < 0.05 ) . based on post hoc analysis ( tukey 's multiple comparisons test ) , no meaningful difference in the expression of rankl gene was observed between dox and em groups [ p > 0.05 , table 1 and chart 1a ] . ( a ) receptor activator of nuclear factor b ligand expression in the dental socket of rat , ( b ) osteoprotegerin expression in the dental socket of rat , ( c ) receptor activator of nuclear factor b ligand / osteoprotegerin ratio . * significant difference was found between intervention groups with control group , p < 0.05 . in all of the studied intervals in dox and em groups , the expression of opg was higher when compared to control group in same periods [ table 1 ] , although this enhancement was not statistically significant ( p > 0.05 ) . when two intervened groups were compared in similar evaluated time points , despite higher levels of opg expression in em group , no significant difference was observed [ p > 0.05 , table 1 and chart 1b ] . as shown in table 1 , the ratio of rankl / opg in em and dox groups in the days 7 and 14 , was significantly lower than the control group ( p < 0.05 ) , but on day 21 it was not . in the comparison of two drugs , there was no meaningful difference between them [ p > 0.05 , table 1 , chart 1c ] . this permits characterization of osteoclasts by their staining for high expression of trap , and cathepsin k. osteoclast rough endoplasmic reticulum is sparse , and the golgi complex is extensive . in osteoclasts , trap is localized within the ruffled border area , the lysosomes , the golgi cisternae , and vesicles . on day 7 , the number of trap + cells in control group was significantly lower than dox and em groups , without any meaningful difference between two tested groups [ p < 0.05 , table 1 and chart 2 ] . on days 14 and 21 , based on one - way anova , there was no significant difference in the studied groups [ p > 0.05 , table 1 and figure 1 ] . * significant difference was found between intervention groups with control group , p < 0.05 . x - axis : time points , y - axis : mean + standard error of mean of variables , p derived from by tukey 's post hoc test . rat alveolar socket 1 week after tooth extraction ( trap , 400 ) . to the best of our knowledge , no in vivo study has yet been conducted to report the effect of dox on the expression of opg and rankl genes following tooth extraction . in fact , we reported the short - term sequential expression of rankl and opg from week 1 to week 3 ( every week in the dental socket of rat ) , using the advanced method of rt - pcr to register the delicate alterations as far as possible . the rankl / opg ratio illustrates the importance of the balance between these molecules in bone remodeling , in health , and in disease states . a similar pattern of these changes has been indicated following the administration of immunosuppressant ( cyclosporine a , rapanyein , tacrolimus ) , by contrast bisphosphonates have been shown a reverse pattern in the expression of these genes . in control group , on days 7 and 14 , the rankl / opg ratio was significantly higher than other two groups that indicate the anti - osteoclastogenesis of dox and em , again . the increase in opg ( marker of bone formation ) and reduction in rankl ( marker of bone resorption ) expressions observed in both dox and em groups , ( than the control group , ) indicates more activation or increase of the osteoblast cells . in em - treated group , a significant down - regulation of rankl gene expression was observed at 3 weeks , suggesting low differentiation of osteoclasts , and show the low level of bone resorption than control group . this finding is confirmed the decreased number of osteoclasts by trap staining ( a genetic marker of osteoclast differentiation ) with the first week in control group and increased with the amount of new bone formation . in the dox - treated group , there was a gradual up - regulation of opg gene expression over time ; while the pattern of rankl gene expression had a gradually decreasing behavior . the rankl expression peak , which occurred during the bone resorption phase ( 7 day ) , preceded the expression of opg that occurred during the bone formation phase ( 21 day ) . it meant that bone formation was stimulated by dox and bone resorption was inhibited ; comparing the control group in which rankl gene expression or induction of bone resorption was enhanced gradually . the rankl / opg ratio peaked in the first week , rather than other periods , and then gradually decreased . this might be related to the initial increase in bone resorption that preceded normal bone formation following tooth extraction which has been noted by guglielmotti and cabrini and iizuka et al . it has been demonstrated that 14 days after tooth extraction in rats is a time in which maximal bone formation and alveolar volume occurs . used nanohydroxyapatite microspheres as a delivery system for em , amoxicillin , and augmentin to evaluate their interaction with osteoblasts . he concluded that em - induced osteoblastic cells proliferation higher than others , and also suggested the use of microsphere and em as a good alternative carrier to enhance bone regeneration while treating periodontal defects . 's study which indicated administration of dox in rats with normal process of bone remodeling , increased bone resorption , and this effect may be dose - dependent . in another study with the aim of comparison of new bone formation following use of dox and vehicle in the bony defects , no meaningful difference was observed . on the other hand , many articles showed the inhibitory effect of dox in bone resorption . , 3.0 mg / kg ) skin , muscle and bone healing clearly improved compared with the saline - treated animals . concluded that low - dose doxycycline reduced the area of bone resorption associated with apical periodontitis in the mandibular first molar teeth of rats . both studied drugs have an anti - inflammatory effect which is independent of their antimicrobial one , so a decrease in the inflammatory cytokines caused enhancement of the growth factors and increased regeneration and rate of wound healing . according to these findings , it seems that dox and em improve healing of alveolar bony socket by decreasing rankl / opg ratio and the number of osteoclasts that suggests their anti - osteoclastogenesis activity . it is reasonable to propose that the rankl / opg system plays an important role in bone healing . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article . the authors of this manuscript declare that they have no conflicts of interest , real or perceived , financial or non - financial in this article .

a severe gonadotropin deficiency together with chronic estradiol deficiency leading to amenorrhea characterizes hypogonadotropic hypogonadism ( hh ) . deficiency in luteinizing hormone ( lh ) and follicle - stimulating hormone ( fsh ) levels may result from either hypothalamic or pituitary causes . hh is most frequently acquired and caused by a number of hypothalamic or pituitary pathological processes . when secondary causes of hh can apparently be excluded , the diagnosis of idiopathic or isolated hh factors may be recognized . exogenous administration of both fsh and lh in women with hh has been shown to be essential in achieving successful stimulation of follicular development , ovulation , and fertility restoration.13 in women with hh and normal pituitary function , pulsatile gonadotropin - releasing hormone ( gnrh ) therapy can be used to induce the periodic release of fsh and lh , leading to proper ovulation.48 the use of a portable pump injecting gnrh either intravenously or subcutaneously for several weeks can cause practical and clinical problems . additionally , the use of daily injections of exogenous gonadotropins has proven to be as effective as gnrh therapy in inducing ovulation in all hh women , including those with hh of pituitary origin , when gnrh therapy is clearly not effective . for many years , human menopausal gonadotropin , which provides both fsh and lh activities , has been applied as the drug of choice in hh patients needing restoration of ovulation.3,5,7,9 in recent years , the availability of both recombinant fsh ( rfsh ) and recombinant lh ( rlh ) has provided a new therapeutic option for hh patients . in 1997 , agrawal et al10 reported the first birth in a hypopituitary hypogonadotropic woman ( world health organization [ who ] type i ) following stimulation of follicular growth with rfsh and rlh . the aim of this article is to review the data reported in the literature on the efficacy of the administration of both rfsh and rlh in the treatment of women with severe lh / fsh deficiency . the two cell two gonadotropin theory was established to understand the roles of lh and fsh , as well as their correlation with the physiological hormonal milieu , which lead to follicular growth , maturation , and ovulation in the woman . in the antral follicle , lh receptors are present only in the theca cells , whereas granulosa cells express only fsh receptors . androgen production and release during folliculogenesis is dependent on the stimulation of the theca cells by lh . during follicular growth , the theca cells produce androgens in response to lh , which are then converted into estrogen by fsh - induced aromatase in the neighboring granulosa cells in the selected growing follicles . in the midfollicular phase , lh receptors begin to appear on the granulosa cells and are involved in the induction and maintenance of a complex paracrine system ( which involves inhibin b and insulin - like growth factor [ igf-1 ] ) necessary for granulosa cell growth and regulation of oocyte maturation . according to the so - called spare receptor hypothesis,11 at a given time , when inhibin b and igf-1 are adequately secreted , androgen synthesis and release are optimal even with < 1% of lh receptors occupied . the decline in fsh level , which occurs during follicular growth , has a key role in the selection of the dominant follicle , as evinced by the fact that on the selected follicle , fsh receptors appear with a lower threshold than on the other growing follicles . on the other hand , according to the lh ceiling theory , each follicle would have an upper limit of stimulation , which is higher in larger follicles and lower in smaller ones . lh would promote leading follicle progression when its concentration is less than its ceiling , and it would cause the degeneration of secondary follicles by overcoming their ceiling.12 at midcycle , the lh surge triggers the final oocyte maturation and ovulation and , at the same time , prevents further growth of granulosa cells , leading to absence of atresia of dominant follicles.13 the two cell two gonadotropin theory was established to understand the roles of lh and fsh , as well as their correlation with the physiological hormonal milieu , which lead to follicular growth , maturation , and ovulation in the woman . in the antral follicle , lh receptors are present only in the theca cells , whereas granulosa cells express only fsh receptors . androgen production and release during folliculogenesis is dependent on the stimulation of the theca cells by lh . during follicular growth , the theca cells produce androgens in response to lh , which are then converted into estrogen by fsh - induced aromatase in the neighboring granulosa cells in the selected growing follicles . in the midfollicular phase , lh receptors begin to appear on the granulosa cells and are involved in the induction and maintenance of a complex paracrine system ( which involves inhibin b and insulin - like growth factor [ igf-1 ] ) necessary for granulosa cell growth and regulation of oocyte maturation . according to the so - called spare receptor hypothesis,11 at a given time , when inhibin b and igf-1 are adequately secreted , androgen synthesis and release are optimal even with < 1% of lh receptors occupied . the decline in fsh level , which occurs during follicular growth , has a key role in the selection of the dominant follicle , as evinced by the fact that on the selected follicle , fsh receptors appear with a lower threshold than on the other growing follicles . on the other hand , according to the lh ceiling theory , each follicle would have an upper limit of stimulation , which is higher in larger follicles and lower in smaller ones . lh would promote leading follicle progression when its concentration is less than its ceiling , and it would cause the degeneration of secondary follicles by overcoming their ceiling.12 at midcycle , the lh surge triggers the final oocyte maturation and ovulation and , at the same time , prevents further growth of granulosa cells , leading to absence of atresia of dominant follicles.13 hh may result either from absent or inadequate hypothalamic gnrh secretion or from failure of pituitary gonadotropin secretion . several congenital and acquired causes , including functional and organic forms , congenital isolated forms of hh are characterized by partial or complete lack of pubertal development due to a deficiency in gnrh - induced gonadotropin secretion , in the absence of anatomical abnormalities in the hypothalamic and pituitary region , although baseline and reserve testing of the remaining pituitary hormones are normal . this could also be associated with olfactory dysfunction , as demonstrated in ~50%60% of patients ( kallmann syndrome).14 from a genetic point of view , congenital isolated forms of hh constitute a very heterogeneous condition , and many genes are implicated in the mechanism of hh.1416 acquired causes of hh are mainly invasive disorders of the hypothalamic pituitary tract , such as pituitary adenomas sarcoidosis , craniopharyngiomas , histiocytosis , and other central nervous system tumors . in these cases , associated pituitary hormone deficiencies are the common result.16 adult onset of isolated pituitary gonadotropin deficiency can be related to systemic disorders , drugs ( glucocorticoid , opiates , and psychotropic agents ) , functional abnormalities due to excessive exercise , hyperprolactinemia , nutritional deficits , psychological distress , or even idiopathic condition.17 actually , the discussion of the various causes of hypogonadism is not inherent to our article as the clinical presentations of the congenital forms are still variable and debatable . from a clinical point of view , amenorrhea , infertility , decreased libido , and osteoporosis are the main symptoms caused by anovulation and chronic estradiol deficiency , which in turn are related to the severe gonadotropin deficiency determined by hh.15 fsh and lh are glycoproteins composed of two noncovalently linked protein subunits , the alpha and beta subunits.18,19 the alpha subunit contains 92 amino acids and is identical in fsh , lh , and human chorionic gonadotropin ( hcg ) , whereas the beta subunit varies between the different glycoproteins and confers unique receptor specificity and specific biological properties.20 its biological activity is provided by the attachment of carbohydrate moieties , forming heterodimers ; the extent and pattern of glycosylation convey the spectrum of different charges , bioactivities , and half - lives for each glycoprotein.21,22 endogenous gonadotropins exist in a number of different isoforms , which have similar amino acid sequences but differ in their terminal sialic acid content . although gonadotropin isoforms influence a variety of biological activities , including cellular growth and development , steroidogenesis , and protein synthesis , their clinical roles are still to be determined.2327 recently , the use of genetic engineering and recombinant dna technology has led to the development of the recombinant human gonadotropin preparations follitropin alpha and follitropin beta.28,29 follitropin alpha was the first recombinant human fsh ( hfsh ) preparation successfully implemented in ovarian stimulation protocols . more recently , lutropin alpha , the first rlh has also been produced and became commercially available for clinical use.30,31 recombinant dna technology used to produce follitropin alpha and lutropin alpha implements the incorporation of the gene encoded for the bioformation of each hormone into a genetically engineered chinese hamster cell line . subsequently , the extraction and purification of rfsh and rlh are carried out by the use of immunochromatographic techniques.28,32 follitropin alpha and lutropin alpha are glycoproteins structurally similar to endogenous fsh and lh . they contain similar alpha subunit and different beta subunits with specific bioactivities.25,33 a different rfsh , follitropin beta , has been later produced and has become commercially available for clinical use.34,35 follitropin alpha resembles the natural fsh isoform detected at midcycle , whereas follitropin beta is similar to that detected in the early follicular phase.36 rfsh preparations have been introduced successfully in the treatment of couples with infertility problems . some clinical trials have shown that rfsh is highly effective in terms of oocyte yield , embryo quality , and dose of fsh needed , with less risk of causing ovarian hyperstimulation syndrome ( ohss).37,38 other studies , however , have demonstrated that the efficacy of rfsh in terms of oocyte and embryo quality is not superior to that of urinary hfsh . some authors have argued that the difference between the two types of fsh may be due to the presence of lh activity in hfsh preparations , which has a positive effect on oocyte maturation and embryo quality,39,40 while other investigators have postulated that such differences may reside in the nature of fsh isoform activities.39,41 fsh isoforms differ in their ability to bind to the target cell receptors surviving in the circulation and to induce biological responses in vivo and in vitro.42,43 a significant difference exists between human - derived fsh and rfsh in terms of their glycosylation patterns and sialic acid content : hfsh contains a higher proportion of acidic isoforms , whereas rfsh contains a higher proportion of less acidic isoforms.27,4446 this difference in the glycosylation pattern of fsh is reflected in its bioactivity , its clearance rate , and its biological function.4749 it has been suggested that the less acidic isoforms have a faster circulatory clearance and , thus , a shorter circulatory half - life48 than the acidic isoforms.50,51 however , a more recent study has shown that the slow clearance of the acidic isoform results in better follicular maturation and estradiol secretion compared with the less acidic isoform.41 a growing body of evidence shows that follicular development patterns and oocyte quality are strongly affected by the fsh glycoform range , and that the requirements of the growing follicle may change during its progress through different stages of follicular development.52,53 indeed , the glycosylation patterns of the two types of fsh implemented for ovarian stimulation have an important role in oocyte maturation competence and clinical outcomes.54 on the other hand , human menopausal gonadotropin ( hmg ) , widely used in a variety of infertility treatment protocols , has both fsh and lh , and sometimes hcg , activities . it contains 75 iu of fsh and 75 iu of lh , as measured by standard bioassays . recently , more purified forms of hmg with specific activity of > 2,500 iu mg for both fsh and lh activities are produced and they also contain some hcg activity . a highly purified form of hmg ( hmg - hp ) has become available , which contains more hcg ( 10 iu ) and less lh ( 5 iu ) than the other forms of hmg preparations , and nowadays , it is successfully used in ovarian stimulation regimens . currently , the rlh lutropin alpha has become commercially available,32 and it has been successfully used in combination with rfsh for infertility treatment , as an alternative to hmg , and the results achieved are as expected . although the role of lh in sensitizing antral follicles to fsh still remains to be elucidated , it has been argued that lh is required for normal hormone production and normal oocyte and embryo development . nevertheless , follicular responses to lh may depend upon the stage of follicular development , it is clearly evident that lh has an important role in the final oocyte maturation and ovulation trigger . additionally , a new combination of rfsh and rlh ( follitropin alpha + lutropin alpha ) at a 2:1 ratio was first introduced for infertility treatment in 2007 . among the advantages of this combination , used for women requiring lh supplementation , is that both fsh and lh can be administered in a single injection , rather than two . however , it has been shown that a similar bioequivalence of rfsh and rlh was observed when they were administered either alone or in combination.19,55,56 because both recombinant gonadotropins ( rfsh and rlh ) have become available , they have also been applied in the treatment of who type i anovulatory patients , ie , patients with oligomenorrhea / amenorrhea caused by hh . due to the rarity of this condition ( ~1% of infertile patients ) , most of the initial studies described some isolated case reports on the administration of both rfsh and rlh to treat hh patients.10,5762 in 1998 , the european recombinant human lh study group63 published the first study on the use of recombinant gonadotropins in a large group of hh women with the aim to determine the minimal effective dose of rlh for supporting rfsh - induced follicular development in lh- and fsh - deficient anovulatory women ( hh ) . patients were randomly assigned to receive daily 0 iu , 25 iu , 75 iu , or 225 iu rlh , in addition to 150 iu rfsh daily , administered for up to 20 days . as expected , patients receiving rfsh without rlh supplement did not show successful follicular growth , whereas patients receiving rlh supplement exhibited improved ovarian response , as measured by the presence of at least one follicle > 17 mm , a level of estradiol 400 pmol / l , and midluteal phase progesterone 4 nmol / l . the study also showed that the efficacy of stimulation increased with increasing dose of rlh administered . the authors suggested that the presence of a ceiling effect is related to the rlh dose : the group of patients who received 225 iu / d of rlh had a smaller number of growing follicles than the group who received 75 iu of rlh / d . this could reflect an lh ceiling effect , whereby some secondary follicles underwent atresia due to their high sensitivity to lh . pregnancy rates ( prs ) achieved were comparable to those in a previous study , wherein hmg had been administered to induce ovulation and pregnancy in hh women , ranging from a pr of 29% per cycle with a 75 iu rlh dose to a 20% pr per cycle with a dose of 225 iu of rlh.63 these data were confirmed by loumaye et al,64 studying 24 who type i patients and 36 who type ii patients ; they showed that rlh alone can trigger arrest of follicular growth in a significant number of patients , suggesting the existence of an initially , burgus et al65 conducted another study on a large group of 38 who type i women , who were stimulated with an initial fixed dose of 150 iu of rfsh and 75 iu of rlh . eighty - four cycles underwent ovarian stimulation , of which 79 ( 94% ) achieved sufficient follicular growth . clinical pregnancies were established in 15 ( 39.5% ) out of 38 patients , with a pr of 18% per started cycle and a pr of 22.4% per given hcg . six years later , kaufmann et al66 evaluated the efficacy of rfsh and rlh in hh women in a wide prospective , randomized , placebo - controlled , double - blind , multicenter study , involving 23 centers in three countries . the stimulation doses were flexible , with a starting dose of 75 iu of rlh and 150 iu of rfsh daily in all the patients . in the case of patients with suboptimal response , the rfsh dose was increased to a maximum of 225 iu , or decreased if necessary . in 27 out of 31 patients , follicular development was achieved in a maximum of three cycles , and 20 ( 74.1% ) of those patients became pregnant . in a similar double - blind , randomized , placebo - controlled trial conducted in 25 medical centers in four countries , shoham et al67 investigated the safety and efficacy of administration of 75 iu of lutropin alpha in combination with follitropin alpha for follicular development induction in women with profound gonadotropin deficiency . they administered a fixed dose of 75 iu rlh and 150 iu rfsh to 27 hh patients and a fixed dose of 150 iu of rfsh and a placebo without rlh to 12 hh patients . they observed a significant improvement of follicular growth in patients treated with lutropin alpha compared to those patients in the placebo group ( p=0.023 ) : 66.7% ( 16 of 24 ) vs 20.0% ( two out of ten ) . two patients of the study group had a positive hcg with a pr of 15% per given hcg and the response rate of combined lutropin alpha and follitropin alpha treatment was similar to that ( 66.7% ) reported by the european study63 that used the same entry criteria . further study was conducted by odea et al,68 in order to evaluate the need for lh supplementation in women with gonadotropin deficiency and to assess the requirement of rlh supplementation to support rfsh for ovulation induction in anovulatory , amenorrhoeic women . a group of 43 patients underwent a total of 61 treatment cycles with rlh and rfsh . iu / d , 25 iu / d , 75 iu / d , or 225 iu / d of rlh and a fixed dose of 150 iu / d of rfsh . only 15 out of 43 patients had a typical hh with lh level 1.2 iu / l and really needed rlh supplementation to achieve adequate follicular growth and maturation , while in the remaining amenorrhoeic women , with higher basal level of lh , proper ovulation was obtained by the administration of rfsh alone . more recently , carone et al69 were the first to compare the efficacy of recombinant vs urinary gonadotropins in women with severe hh . seventeen hh women were administered recombinant gonadotropins ( 150 iu rfsh + 75 iu rlh daily ) for 27 cycles of stimulation , and 18 hh women received urinary gonadotropin ( 150 iu hmg - hp daily , which is equal to 150 iu fsh + 150 iu lh - like activity ) for 43 cycles of stimulation . their results showed that rlh is highly superior compared to hcg in supporting fsh - induced follicular development in who type i women in terms of pr : 55% per cycle in rfsh / rlh patients vs 23.2% per cycle in hmg - hp patients ( p<0.05 ) . interestingly , no statistical differences were observed between hmg - hp and rfsh + rlh patients when considering only ovulation induction . ovulation was achieved in 88% of hmg - hp cycles compared to 70% of recombinant cycles , suggesting that rfsh / rlh is equally efficient as hmg - hp in inducing ovulation . according to the authors , the difference in terms of pregnancies among the two groups of patients may be due to the different sources of lh activity present in the two pharmaceutical preparations . in fact , in hmg - hp , the lh activity is derived from urinary hcg.70 despite the fact that lh and hcg have 90% amino acid homology , an evident difference exists between them : hcg is characterized by the presence of a carboxyl terminal peptide and a high glycosylation pattern , whereas lh contains three n - linked residues . although they act on the same receptor , they stimulate different bioactivity mechanisms.71 moreover , hcg has a longer half - life and capability of accumulation , which may contribute to lh receptors desensitization and downregulation , when compared to lh.7276 additionally , the differences between the two molecules may reside in their different interactions with the same receptor , and this could partially explain the significant differences in the clinical outcome.69 in a recent study , papaleo et al77 have analyzed the study reported by carone et al69 from a pharmacoeconomic point of view in order to develop a cost - effectiveness model , comparing between rfsh + rlh and hmg - hp . their study indicates that the average cost per pregnancy is lower for patients treated with rfsh + rlh than for those treated with hmg - hp ; this may be due to the strong impact of the efficacy of the recombinant gonadotropin therapy with respect to the urinary gonadotropin therapy . they found that rfsh + rlh is associated with a higher total cost ( 3,453.50 ) and higher efficacy ( 0.87 ) compared with hmg - hp ( 2,719.70 ) and lower efficacy ( 0.50 ) , but the average cost estimated per pregnancy is around 3,990.00 for the recombinant strategy and 5,439.80 for the urinary strategy . the authors concluded that despite the higher acquisition cost in comparison to hmg - hp , using rfsh + rlh resulted in a higher pregnancy rate , which makes it the recommended choice of treatment when considering the cost - effectiveness of rlh used in supporting fsh - induced follicular growth in hh women . additionally , awwad et al78 investigated whether split daily doses of recombinant human lh is more effective than the single daily dose in supporting follicular development and ovulation in primary hh . in their study , 27 women with hh received a 150 iu fixed dose of recombinant hfsh daily , subcutaneously administered , supplemented with 75 iu dose daily of rlh . the patients received the therapy either as a single dose ( n=9 ; single - dose group ) or four equally divided doses ( n=18 ; split - dose group ) . although no statistical significance was observed between the two groups , the proportion of women in the rlh split - dose group who achieved proper ovulation ( at least one follicle 17 mm in diameter , preovulatory serum estradiol 400 pmol / l , and a midluteal progesterone 25 nmol / l ) was higher than in the single - dose group ( 72.2% vs 55.6% ) . although no data were reported on pregnancy outcomes , the authors concluded that administering rlh in split daily doses appears to be superior in terms of ovulation induction compared with the traditional single daily dose . the availability of pure recombinant human gonadotropin preparations ( rfsh and rlh ) helped to achieve more insight to confirm the two cell theory of ovarian steroidogenesis , which predicts that both fsh and lh are required to ensure adequate follicular growth and maturation . fsh alone is insufficient for full follicular maturation and oocyte competence in the case of severe gonadotropin deficiency ; nevertheless , using fsh alone has been demonstrated to be sufficient for ovarian stimulation in normogonadotropic women.62,63,79 concerns about the minimal effective dose of rlh needed to induce appropriate ovulation in association with rfsh have been investigated only in two clinical trials.63,68 these studies have tested the capability of different doses of rlh ( 0 iu , 25 iu , 75 iu , and 225 iu daily ) to stimulate adequate follicular growth and maturation . as shown in table 1 , no follicular growth was seen in hh women when no rlh was administered , and this seems to be in accordance with the two cell theory . the minimal dose of rlh necessary to achieve ovulation induction seems to be 25 iu , in association with a fixed dose of rfsh of 150 iu / d ; nevertheless , no pregnancy was achieved in this group of patients . on the other hand , the proportion of patients achieving appropriate ovulation increased with increasing doses of rlh to 75 iu and 225 iu , and six pregnancies were achieved out of 26 treated patients , with a pr of 23% . this could be the consequence of a better hormonal milieu in these groups of patients.63,68 interestingly , an improvement in clinical results could be obtained when both rlh and rfsh doses were tailored to the patient s response to stimulation , and adjusted where necessary by increasing the doses of recombinant gonadotropins , according to the results of ovarian monitoring . in such groups of patients , personalizing the stimulation doses , the clinical outcome results achieved fluctuated from 39.5% to 88.5% pr per patient10,58,61,66,69 ( table 2 ) . on the other hand , although the efficacy of using combined rfsh and rlh to stimulate hh patients has been proven , a few studies have reported on the possible side effects , such as local reaction , tolerability , and the risk of developing ohss , when using recombinant rfsh / rlh , compared to conventional hmg , in the treatment of such a group of hh patients . a study published by burgus et al65 evaluated the safety of using rfsh / rlh combined protocol in the treatment of who type 1 anovulation patients , and they found that the risk of ohss occurred in three out of 38 treated patients ( one mild and two moderate ohss cases ) . they also assessed 984 injections for local tolerance and observed that 9% ( 88/984 ) of injections were associated with local reactions ; only 1.1% of injections were associated with severe pain and 0.5% with severe itching . despite the lack of a control hmg group in this study , the authors concluded that the combined protocol of rfsh / rlh is well tolerated . moreover , carone et al69 reported no adverse events , and no mild or moderate ohss was observed in 35 patients , treated for a total of 70 cycles , following ovarian stimulation either with hmg - hp or rfsh / rlh . other studies , however , reported a significantly increased incidence of ohss risk in normogonadotropic patients stimulated with rfsh + rlh compared to patients treated with hmg.80,81 indeed , further comparative studies are warranted to investigate the tolerability , acceptability , and other adverse events , such as the risk of ohss , on using rfsh / rlh to stimulate hypogonadotropic patients compared to the same using conventional hmg regimens . a peculiar form of hh could be relieved following controlled ovarian hyperstimulation treatment during assisted reproductive technology ( art ) cycles . ovarian stimulation protocols implement the administration of a gnrh agonist ( gnrh - a ) for pituitary desensitization , known as cycle downregulation phase . for many years , the gnrh - a long protocol has been considered the stimulation protocol of choice because of its ability to reversibly block pituitary function , thus preventing premature lh surge , by depletion of pituitary gonadotropin after an initial stimulatory phase . gonad axis is achieved after administration of gnrh - a.82 the degree of pituitary suppression depends also on the gnrh - a formulation as well as the mode and dose of administration.83,84 the administration of gnrh - a produces a sort of iatrogenic hh state.12 as a consequence of pituitary postsuppression , a decline occurs in either fsh concentrations ( ranged between 1.5 iu / l and 3.5 iu / l ) or lh concentrations ( ranged between 0.5 in some cases , the lh concentration often decreases to < 0.5 iu / l during the intermediate - to - late stages of stimulation , which is even less than the value observed in true hh.12 however , according to the previously reported , androgen synthesis and release are optimal even with < 1% of lh receptors occupied , allowing adequate multifollicular growth and maturation with the administration of fsh alone.11 moreover , in some normogonadotropic women , with normal ovarian reserve , using rfsh alone is not efficient in inducing appropriate multifollicular growth following pituitary gnrh downregulation.84,85 some authors attributed this phenomenon to the profound suppression of lh level after long downregulation gnrh - a protocol.83,84 several studies have been conducted with the aim of establishing a valuable cutoff of circulating lh level after pituitary desensitization in order to identify those patients , but the results obtained are conflicting.84,85 conversely , a meta - analysis published by kolibianakis et al86 showed no evidence that low endogenous lh levels , which may occur during long protocols of ovarian stimulation for in vitro fertilization , require exogenous lh supplementation to improve the probability of ongoing pregnancy . additionally , other studies have demonstrated that a subgroup of normogonadotropic women , who are hyporesponsive to rfsh monotherapy following gnrh - a down - regulation , may benefit from rlh administration , irrespective of the basal lh levels , to achieve proper multifollicular growth.8790 in another study , lisi et al91 reported that using rlh and rfsh to stimulate patients who had a hyporesponse to rfsh alone in a previous downregulated cycle improves the fertilization rate ( from 60.9% to 86% ) and the clinical pr ( from 5.9% to 50% ) . after these preliminary reports , it clearly appears that lh levels after downregulation may not have predictive value in identifying hyporesponsive patients . this has induced other researchers to attempt other strategies to identify hyporesponsive patients during the early stimulation phase and to evaluate the possible use of rlh in the same cycle of stimulation , thus trying a sort of rescue of stimulation treatment.8790 the results were compared with those obtained in the controlled group of hyporesponsive patients , in which the rescue strategy was based on an increase of rfsh daily doses , and/or in normoresponsive patients.8790 in all these studies , different protocols were used to evaluate rlh doses , timing of rlh administration , and starting doses of rfsh . primary and secondary end points evaluated differed in each of the reported studies ; nevertheless , all observed results showed a beneficial effect of the administration of rlh in these iatrogenic hh subgroup patients . ferraretti et al88 report a statistically significant difference in terms of implantation rate ( 36.8% in rlh + rfsh group vs 14.1% in rfsh - only group ) and pr ( 54% in rlh + rfsh group vs 24% in rfsh - only group ) with a dose of 75 iu or 150 iu rlh daily . conversely , de placido et al87 found an increase in the number of oocytes retrieved and in the percentage of mature oocytes when 150 iu / d of rlh was added to stimulate the steady responder patients , as well as a statistically significant decrease in both variables when adding 75 iu / d of rlh . in another study , de placido et al89 evaluated the difference in the number and maturity of retrieved oocytes in a group of steady responders when 150 iu / d of rlh was added to rfsh compared to increased doses of rfsh without rlh supplementation . as expected , in the group that received rlh in association with rfsh , the stimulation outcomes were comparable with those obtained in normal responders , whereas in the group of steady responders , who received only increased doses of rfsh , reductions in the total number of retrieved oocytes and in the total number of mature oocytes were observed . more recently , yazici yilmaz et al90 conducted a similar study , using 75 iu / d of rlh instead of 150 iu / d ; they found no differences between the two subgroups of steady responders , while the total number of retrieved oocytes and the total number of mature oocytes were reduced as compared to those in normoresponsive patients . interestingly , when considering implantation rate and pr , they observed similar results in the subgroup of patients supplemented with rlh compared to those observed in normoresponsive patients , whereas a statistically significant decrease was observed in terms of implantation rate and pr in the subgroup of steady responders who received only increasing doses of rfsh alone daily . these findings may be related to a discrepancy between the bioactive and immunoreactive forms of lh.9294 in some of these patients , the presence of a polymorphism in the lh beta subunit gene ( the variant being termed v - betalh ) , which affects ~10% of the population , may explain the hyporesponse to rfsh monotherapy.95 alternatively , ovarian resistance to rfsh has also been advocated as a possible cause for this hyporesponsivness.96 from the data in literature , it appears that the subgroup of hyporesponder patients needs lh activity ( rlh or hmg ) supplementation , instead of increased doses of rfsh , in order to achieve appropriate follicular growth , ovulation , and oocyte competence , as well as results comparable to those of the normoresponder patients in terms of pr.12 although studies on this issue are limited and the experiences available are few due to the small number of such patients , it is clearly evident that the recombinant gonadotropins rfsh and rlh are efficient in treating patients affected by hh . the results observed in the studies reported in this review suggest that recombinant gonadotropins are able to induce appropriate follicular growth , oocyte maturation , and eventually pregnancy in this group of women . moreover , the clinical use of recombinant gonadotropins in this type of patient has elucidated some endocrinological aspects of ovarian function , which have not yet been fully understood by using urinary gonadotropins . however , additional studies should seek to address the safety and efficiency of recombinant gonadotropins in ovarian stimulation and aim to heighten our knowledge on the physiological hormonal mechanism governing follicular growth and ovulation and , consequently , to improve the clinical applications of recombinant gonadotropins in fertility restoration .

a severe gonadotropin deficiency together with chronic estradiol deficiency leading to amenorrhea characterizes patients suffering from hypogonadotropic hypogonadism . administration of both follicle - stimulating hormone ( fsh ) and luteinizing hormone ( lh ) to these patients has been shown to be essential in achieving successful stimulation of follicular development , ovulation , and rescue of fertility . in recent years , the availability of both recombinant fsh ( rfsh ) and recombinant lh ( rlh ) has provided a new therapeutic option for the stimulation of follicular growth in hypopituitary hypogonadotropic women ( world health organization group i ) . in this article , we review the data reported in the literature to highlight the role and the efficacy of using recombinant gonadotropins , rfsh and rlh , in the treatment of women with severe lh / fsh deficiency . although the studies on this issue are limited and the experiences available in the literature are few due to the small number of such patients , it is clearly evident that the recombinant gonadotropins rfsh and rlh are efficient in treating patients affected by hypogonadotropic hypogonadism . the results observed in the studies reported in this review suggest that recombinant gonadotropins are able to induce proper follicular growth , oocyte maturation , and eventually pregnancy in this group of women . moreover , the clinical use of recombinant gonadotropins in this type of patients has given more insight into some endocrinological aspects of ovarian function that have not yet been fully understood .

Introduction Role of gonadotropins in follicular growth and ovulation The two celltwo gonadotropin theory Clinical and endocrinological features of HH Follitropin alpha and lutropin alpha Stimulation of follicular development with rFSH and rLH in women with profound FSH and LH deficiency: clinical results Iatrogenic severe LH and FSH deficiency during assisted reproductive technology cycles Conclusion

a severe gonadotropin deficiency together with chronic estradiol deficiency leading to amenorrhea characterizes hypogonadotropic hypogonadism ( hh ) . deficiency in luteinizing hormone ( lh ) and follicle - stimulating hormone ( fsh ) levels may result from either hypothalamic or pituitary causes . exogenous administration of both fsh and lh in women with hh has been shown to be essential in achieving successful stimulation of follicular development , ovulation , and fertility restoration.13 in women with hh and normal pituitary function , pulsatile gonadotropin - releasing hormone ( gnrh ) therapy can be used to induce the periodic release of fsh and lh , leading to proper ovulation.48 the use of a portable pump injecting gnrh either intravenously or subcutaneously for several weeks can cause practical and clinical problems . for many years , human menopausal gonadotropin , which provides both fsh and lh activities , has been applied as the drug of choice in hh patients needing restoration of ovulation.3,5,7,9 in recent years , the availability of both recombinant fsh ( rfsh ) and recombinant lh ( rlh ) has provided a new therapeutic option for hh patients . in 1997 , agrawal et al10 reported the first birth in a hypopituitary hypogonadotropic woman ( world health organization [ who ] type i ) following stimulation of follicular growth with rfsh and rlh . the aim of this article is to review the data reported in the literature on the efficacy of the administration of both rfsh and rlh in the treatment of women with severe lh / fsh deficiency . the two cell two gonadotropin theory was established to understand the roles of lh and fsh , as well as their correlation with the physiological hormonal milieu , which lead to follicular growth , maturation , and ovulation in the woman . lh would promote leading follicle progression when its concentration is less than its ceiling , and it would cause the degeneration of secondary follicles by overcoming their ceiling.12 at midcycle , the lh surge triggers the final oocyte maturation and ovulation and , at the same time , prevents further growth of granulosa cells , leading to absence of atresia of dominant follicles.13 the two cell two gonadotropin theory was established to understand the roles of lh and fsh , as well as their correlation with the physiological hormonal milieu , which lead to follicular growth , maturation , and ovulation in the woman . during follicular growth , the theca cells produce androgens in response to lh , which are then converted into estrogen by fsh - induced aromatase in the neighboring granulosa cells in the selected growing follicles . in these cases , associated pituitary hormone deficiencies are the common result.16 adult onset of isolated pituitary gonadotropin deficiency can be related to systemic disorders , drugs ( glucocorticoid , opiates , and psychotropic agents ) , functional abnormalities due to excessive exercise , hyperprolactinemia , nutritional deficits , psychological distress , or even idiopathic condition.17 actually , the discussion of the various causes of hypogonadism is not inherent to our article as the clinical presentations of the congenital forms are still variable and debatable . from a clinical point of view , amenorrhea , infertility , decreased libido , and osteoporosis are the main symptoms caused by anovulation and chronic estradiol deficiency , which in turn are related to the severe gonadotropin deficiency determined by hh.15 fsh and lh are glycoproteins composed of two noncovalently linked protein subunits , the alpha and beta subunits.18,19 the alpha subunit contains 92 amino acids and is identical in fsh , lh , and human chorionic gonadotropin ( hcg ) , whereas the beta subunit varies between the different glycoproteins and confers unique receptor specificity and specific biological properties.20 its biological activity is provided by the attachment of carbohydrate moieties , forming heterodimers ; the extent and pattern of glycosylation convey the spectrum of different charges , bioactivities , and half - lives for each glycoprotein.21,22 endogenous gonadotropins exist in a number of different isoforms , which have similar amino acid sequences but differ in their terminal sialic acid content . although gonadotropin isoforms influence a variety of biological activities , including cellular growth and development , steroidogenesis , and protein synthesis , their clinical roles are still to be determined.2327 recently , the use of genetic engineering and recombinant dna technology has led to the development of the recombinant human gonadotropin preparations follitropin alpha and follitropin beta.28,29 follitropin alpha was the first recombinant human fsh ( hfsh ) preparation successfully implemented in ovarian stimulation protocols . subsequently , the extraction and purification of rfsh and rlh are carried out by the use of immunochromatographic techniques.28,32 follitropin alpha and lutropin alpha are glycoproteins structurally similar to endogenous fsh and lh . they contain similar alpha subunit and different beta subunits with specific bioactivities.25,33 a different rfsh , follitropin beta , has been later produced and has become commercially available for clinical use.34,35 follitropin alpha resembles the natural fsh isoform detected at midcycle , whereas follitropin beta is similar to that detected in the early follicular phase.36 rfsh preparations have been introduced successfully in the treatment of couples with infertility problems . some clinical trials have shown that rfsh is highly effective in terms of oocyte yield , embryo quality , and dose of fsh needed , with less risk of causing ovarian hyperstimulation syndrome ( ohss).37,38 other studies , however , have demonstrated that the efficacy of rfsh in terms of oocyte and embryo quality is not superior to that of urinary hfsh . some authors have argued that the difference between the two types of fsh may be due to the presence of lh activity in hfsh preparations , which has a positive effect on oocyte maturation and embryo quality,39,40 while other investigators have postulated that such differences may reside in the nature of fsh isoform activities.39,41 fsh isoforms differ in their ability to bind to the target cell receptors surviving in the circulation and to induce biological responses in vivo and in vitro.42,43 a significant difference exists between human - derived fsh and rfsh in terms of their glycosylation patterns and sialic acid content : hfsh contains a higher proportion of acidic isoforms , whereas rfsh contains a higher proportion of less acidic isoforms.27,4446 this difference in the glycosylation pattern of fsh is reflected in its bioactivity , its clearance rate , and its biological function.4749 it has been suggested that the less acidic isoforms have a faster circulatory clearance and , thus , a shorter circulatory half - life48 than the acidic isoforms.50,51 however , a more recent study has shown that the slow clearance of the acidic isoform results in better follicular maturation and estradiol secretion compared with the less acidic isoform.41 a growing body of evidence shows that follicular development patterns and oocyte quality are strongly affected by the fsh glycoform range , and that the requirements of the growing follicle may change during its progress through different stages of follicular development.52,53 indeed , the glycosylation patterns of the two types of fsh implemented for ovarian stimulation have an important role in oocyte maturation competence and clinical outcomes.54 on the other hand , human menopausal gonadotropin ( hmg ) , widely used in a variety of infertility treatment protocols , has both fsh and lh , and sometimes hcg , activities . a highly purified form of hmg ( hmg - hp ) has become available , which contains more hcg ( 10 iu ) and less lh ( 5 iu ) than the other forms of hmg preparations , and nowadays , it is successfully used in ovarian stimulation regimens . currently , the rlh lutropin alpha has become commercially available,32 and it has been successfully used in combination with rfsh for infertility treatment , as an alternative to hmg , and the results achieved are as expected . although the role of lh in sensitizing antral follicles to fsh still remains to be elucidated , it has been argued that lh is required for normal hormone production and normal oocyte and embryo development . nevertheless , follicular responses to lh may depend upon the stage of follicular development , it is clearly evident that lh has an important role in the final oocyte maturation and ovulation trigger . however , it has been shown that a similar bioequivalence of rfsh and rlh was observed when they were administered either alone or in combination.19,55,56 because both recombinant gonadotropins ( rfsh and rlh ) have become available , they have also been applied in the treatment of who type i anovulatory patients , ie , patients with oligomenorrhea / amenorrhea caused by hh . due to the rarity of this condition ( ~1% of infertile patients ) , most of the initial studies described some isolated case reports on the administration of both rfsh and rlh to treat hh patients.10,5762 in 1998 , the european recombinant human lh study group63 published the first study on the use of recombinant gonadotropins in a large group of hh women with the aim to determine the minimal effective dose of rlh for supporting rfsh - induced follicular development in lh- and fsh - deficient anovulatory women ( hh ) . the authors suggested that the presence of a ceiling effect is related to the rlh dose : the group of patients who received 225 iu / d of rlh had a smaller number of growing follicles than the group who received 75 iu of rlh / d . pregnancy rates ( prs ) achieved were comparable to those in a previous study , wherein hmg had been administered to induce ovulation and pregnancy in hh women , ranging from a pr of 29% per cycle with a 75 iu rlh dose to a 20% pr per cycle with a dose of 225 iu of rlh.63 these data were confirmed by loumaye et al,64 studying 24 who type i patients and 36 who type ii patients ; they showed that rlh alone can trigger arrest of follicular growth in a significant number of patients , suggesting the existence of an initially , burgus et al65 conducted another study on a large group of 38 who type i women , who were stimulated with an initial fixed dose of 150 iu of rfsh and 75 iu of rlh . in a similar double - blind , randomized , placebo - controlled trial conducted in 25 medical centers in four countries , shoham et al67 investigated the safety and efficacy of administration of 75 iu of lutropin alpha in combination with follitropin alpha for follicular development induction in women with profound gonadotropin deficiency . more recently , carone et al69 were the first to compare the efficacy of recombinant vs urinary gonadotropins in women with severe hh . according to the authors , the difference in terms of pregnancies among the two groups of patients may be due to the different sources of lh activity present in the two pharmaceutical preparations . although they act on the same receptor , they stimulate different bioactivity mechanisms.71 moreover , hcg has a longer half - life and capability of accumulation , which may contribute to lh receptors desensitization and downregulation , when compared to lh.7276 additionally , the differences between the two molecules may reside in their different interactions with the same receptor , and this could partially explain the significant differences in the clinical outcome.69 in a recent study , papaleo et al77 have analyzed the study reported by carone et al69 from a pharmacoeconomic point of view in order to develop a cost - effectiveness model , comparing between rfsh + rlh and hmg - hp . their study indicates that the average cost per pregnancy is lower for patients treated with rfsh + rlh than for those treated with hmg - hp ; this may be due to the strong impact of the efficacy of the recombinant gonadotropin therapy with respect to the urinary gonadotropin therapy . the availability of pure recombinant human gonadotropin preparations ( rfsh and rlh ) helped to achieve more insight to confirm the two cell theory of ovarian steroidogenesis , which predicts that both fsh and lh are required to ensure adequate follicular growth and maturation . fsh alone is insufficient for full follicular maturation and oocyte competence in the case of severe gonadotropin deficiency ; nevertheless , using fsh alone has been demonstrated to be sufficient for ovarian stimulation in normogonadotropic women.62,63,79 concerns about the minimal effective dose of rlh needed to induce appropriate ovulation in association with rfsh have been investigated only in two clinical trials.63,68 these studies have tested the capability of different doses of rlh ( 0 iu , 25 iu , 75 iu , and 225 iu daily ) to stimulate adequate follicular growth and maturation . the minimal dose of rlh necessary to achieve ovulation induction seems to be 25 iu , in association with a fixed dose of rfsh of 150 iu / d ; nevertheless , no pregnancy was achieved in this group of patients . this could be the consequence of a better hormonal milieu in these groups of patients.63,68 interestingly , an improvement in clinical results could be obtained when both rlh and rfsh doses were tailored to the patient s response to stimulation , and adjusted where necessary by increasing the doses of recombinant gonadotropins , according to the results of ovarian monitoring . in such groups of patients , personalizing the stimulation doses , the clinical outcome results achieved fluctuated from 39.5% to 88.5% pr per patient10,58,61,66,69 ( table 2 ) . on the other hand , although the efficacy of using combined rfsh and rlh to stimulate hh patients has been proven , a few studies have reported on the possible side effects , such as local reaction , tolerability , and the risk of developing ohss , when using recombinant rfsh / rlh , compared to conventional hmg , in the treatment of such a group of hh patients . a study published by burgus et al65 evaluated the safety of using rfsh / rlh combined protocol in the treatment of who type 1 anovulation patients , and they found that the risk of ohss occurred in three out of 38 treated patients ( one mild and two moderate ohss cases ) . gonad axis is achieved after administration of gnrh - a.82 the degree of pituitary suppression depends also on the gnrh - a formulation as well as the mode and dose of administration.83,84 the administration of gnrh - a produces a sort of iatrogenic hh state.12 as a consequence of pituitary postsuppression , a decline occurs in either fsh concentrations ( ranged between 1.5 iu / l and 3.5 iu / l ) or lh concentrations ( ranged between 0.5 in some cases , the lh concentration often decreases to < 0.5 iu / l during the intermediate - to - late stages of stimulation , which is even less than the value observed in true hh.12 however , according to the previously reported , androgen synthesis and release are optimal even with < 1% of lh receptors occupied , allowing adequate multifollicular growth and maturation with the administration of fsh alone.11 moreover , in some normogonadotropic women , with normal ovarian reserve , using rfsh alone is not efficient in inducing appropriate multifollicular growth following pituitary gnrh downregulation.84,85 some authors attributed this phenomenon to the profound suppression of lh level after long downregulation gnrh - a protocol.83,84 several studies have been conducted with the aim of establishing a valuable cutoff of circulating lh level after pituitary desensitization in order to identify those patients , but the results obtained are conflicting.84,85 conversely , a meta - analysis published by kolibianakis et al86 showed no evidence that low endogenous lh levels , which may occur during long protocols of ovarian stimulation for in vitro fertilization , require exogenous lh supplementation to improve the probability of ongoing pregnancy . this has induced other researchers to attempt other strategies to identify hyporesponsive patients during the early stimulation phase and to evaluate the possible use of rlh in the same cycle of stimulation , thus trying a sort of rescue of stimulation treatment.8790 the results were compared with those obtained in the controlled group of hyporesponsive patients , in which the rescue strategy was based on an increase of rfsh daily doses , and/or in normoresponsive patients.8790 in all these studies , different protocols were used to evaluate rlh doses , timing of rlh administration , and starting doses of rfsh . as expected , in the group that received rlh in association with rfsh , the stimulation outcomes were comparable with those obtained in normal responders , whereas in the group of steady responders , who received only increased doses of rfsh , reductions in the total number of retrieved oocytes and in the total number of mature oocytes were observed . these findings may be related to a discrepancy between the bioactive and immunoreactive forms of lh.9294 in some of these patients , the presence of a polymorphism in the lh beta subunit gene ( the variant being termed v - betalh ) , which affects ~10% of the population , may explain the hyporesponse to rfsh monotherapy.95 alternatively , ovarian resistance to rfsh has also been advocated as a possible cause for this hyporesponsivness.96 from the data in literature , it appears that the subgroup of hyporesponder patients needs lh activity ( rlh or hmg ) supplementation , instead of increased doses of rfsh , in order to achieve appropriate follicular growth , ovulation , and oocyte competence , as well as results comparable to those of the normoresponder patients in terms of pr.12 although studies on this issue are limited and the experiences available are few due to the small number of such patients , it is clearly evident that the recombinant gonadotropins rfsh and rlh are efficient in treating patients affected by hh . the results observed in the studies reported in this review suggest that recombinant gonadotropins are able to induce appropriate follicular growth , oocyte maturation , and eventually pregnancy in this group of women . moreover , the clinical use of recombinant gonadotropins in this type of patient has elucidated some endocrinological aspects of ovarian function , which have not yet been fully understood by using urinary gonadotropins . however , additional studies should seek to address the safety and efficiency of recombinant gonadotropins in ovarian stimulation and aim to heighten our knowledge on the physiological hormonal mechanism governing follicular growth and ovulation and , consequently , to improve the clinical applications of recombinant gonadotropins in fertility restoration .

the limited capacity of adult mammalian central nervous system axons to regenerate following spinal cord injury ( sci ) leads to substantial functional defects . one likely cause of this diminished growth ability is that the expression levels of proteins necessary for robust growth is dramatically lower in mature neurons than in younger , developing neurons . one tool that can modify gene expression in the mature central nervous system , potentially allowing for the overcoming of this significant hurdle , is recombinant viral vectors . adeno - associated virus ( aav ) is one type of viral vector that is often used for gene therapy in the central nervous system because of its non - pathogenic nature , replication deficiency and high neuronal transduction efficiency . this helper - dependent , single - stranded dna parvovirus has the ability to effectively transduce post - mitotic cells producing prolonged , stable gene expression without triggering an inflammatory response and toxicity . several serotypes of aav exist , each with distinct cellular tropisms determined by the surface features of the capsid . the ability of aav to transduce fully differentiated neurons makes it an ideal candidate to manipulate gene expression in adult , supraspinal neurons to promote axon regeneration , synaptic plasticity and neuronal survival following the injury of their axons that project to spinal cord . in various sci models , application of aav in the vicinity of targeted cell bodies results in their successful transduction . because sci interrupts many different axon tracts projecting from the brain to the spinal cord , promoting regeneration of several injured , descending tracts would likely restore more interrupted circuitry than regeneration of a specific tract , thereby possibly improving functional recovery . therefore , it seems reasonable to imagine that it would be advantageous to simultaneously modify a variety of supraspinal neuronal pools affected by the injury . recently , widespread transduction was achieved by injecting aav - green fluorescent protein ( gfp ) into multiple sites throughout the brainstem . while this injection paradigm effectively transduces many neurons within the brainstem , it is also very invasive . interestingly , another means to transduce neurons using aav is via retrograde transport . injecting aav into a target muscle or peripheral nerve results in the transduction of motoneurons and dorsal root ganglion neurons that innervate the targeted muscle or that send projections via the injected nerve . whether aav is taken up by central axons within the spinal cord after injury and is then retrogradely transported to transduce neurons within the brain has not been determined . in these experiments , we sought to assess the effectiveness of a novel , less invasive approach to transduce multiple descending populations following sci . we hypothesized that aav injected into spinal cord tissue immediately rostral to an injury site would not only transduce local spinal neurons but would also be taken up by severed , descending tract axons and retrogradely transported to transduce diverse brain neurons that project to spinal cord . successful retrograde transport would allow manipulation of gene expression in several remote neuronal populations upon administration into one location . in addition , because different aav serotypes have dissimilar tropisms and potential for retrograde transport , we sought to identify which aav serotype most efficiently retrogradely transduces neurons in brain after sci . immediately following a complete transection of the spinal cord at thoracic level 3 ( t3 ) , equivalent titers of aav1- , aav2- , aav5- , aav8- , or aav9-cba - gfp ( gfp transgene under a chicken -actin promoter ; n = 3 per serotype ) were injected into four different locations rostral to the transection site ( figure 1a ) at two different depths per location ( figure 1b ) . this was done to flood with viral vectors tissue encompassing the intermediate gray matter and the lateral and ventral funiculi just rostral to the injury . intraspinal injection of all tested aav serotypes resulted in transduction of spinal cord neurons rostral to a sci site . in animals from all groups , numerous gfp neurons were observed in gray matter ( figure 2a , c ) above the injury . moreover , many gfp axons ( visualized as more punctate than linear staining in figure 2a , b because the axons are seen in cross - section ) were observed in the lateral and ventral white matter rostral to the transection site . most gfp cells within the spinal cord had a neuronal phenotype ( figure 2a , c , d ) but a few gfp cells colocalized with the astrocytic marker glial fibrillary acidic protein ( figure 2e , arrows ) . nonetheless , the vast majority of glial fibrillary acidic protein - positive cells did not coexpress gfp ( figure 2e , arrowheads ) , even in regions containing many gfp neurons ( figure 2f , arrowheads ) . these data indicate that each serotype transduced more neurons than astrocytes , agreeing with literature that aav with a non - cell specific promoter preferentially transduces neurons and not glia . to determine if different aav serotypes transduce spinal cord neurons to disparate degrees , all gfp neurons in a subset of spinal cord sections rostral to the spinal transection site ( every 400 m ; figure 3a ) were counted . there were 2,700 212.3 gfp spinal cord neurons in animals injected with aav1 and 3,439 338.3 neurons in animals injected with aav5 . both aav1 and aav5 were significantly better at transducing spinal cord neurons than the other tested serotypes ( p < 0.05 ) . there were 548 71.3 gfp spinal neurons in animals injected with aav2 , 848 193.3 in animals injected with aav8 and 1,572 104.5 in animals injected with aav9 . all serotypes were able to transduce spinal cord neurons as far as 56 mm rostral to the injury site ( figure 3b ) . moreover , neurons as far as 10 mm rostral to the transection site were transduced following aav5 injection . to determine if aav injected immediately rostral to a sci site is taken up by injured axons and retrogradely transported to transduce neurons within the brain , gfp neurons in brain sections every 400 m apart were counted . we found many gfp neurons in the reticular formation ( figure 4a , arrows ) , vestibular nucleus ( figure 4b , arrows ) and red nucleus ( figure 4c , arrows ) . moreover , we found many gfp processes in these same areas ( figure 4 , arrowheads ) , some appearing to emerge from gfp somas . gfp neurons were observed in all brains , indicating that each of the tested aav serotypes is capable of being retrogradely transported by injured axons to transduce brain neurons ( figure 4d ) . however , there was a significantly greater number ( p < 0.05 ) of gfp neurons in the brains of animals intraspinally injected with aav5 ( 2,713 186.0 ) than aav1 ( 1,700 229.3 ) , aav2 ( 943.3 93.8 ) , aav8 ( 1,471.7 359.3 ) or aav9 ( 890.7 262.1 ) . we did not observe any transduced neurons within cortex ( data not shown ) . to determine if the tested serotypes had different tropisms for various supraspinal neuronal pools , we assessed where each gfp neuron in the brain was located . significantly more neurons within the reticular formation ( figure 5a ) that project via the reticulospinal tract were transduced with aav5 ( 1,873 168.7 ) than aav1 ( 743 130.4 ) , aav2 ( 441.7 55.9 ) , aav8 ( 844.7 207.8 ) or aav9 ( 378.7 174.5 ; p all aav serotypes resulted in statistically equal numbers of gfp neurons in the vestibular nuclei ( figure 5b ) that form the vestibulospinal tract . aav1 transduced 436.7 132 neurons , aav2 transduced 228.3 51.1 , aav5 transduced 523.7 70.1 , aav8 transduced 276.7 129.1 , and aav9 transduced 239 127.5 . injecting aav1 into the spinal cord transduced significantly more neurons in the raphe nuclei ( 18.4 2.2 ) that form the raphespinal tract than aav5 ( 9 5.5 ; p < 0.05 ; figure 5c ) . we did not observe any gfp+ neurons in the raphe after injecting aav2 , aav8 or aav9 rostral to the spinal transection . all aav serotypes were transported by injured rubrospinal tract axons to result in gfp expression in neurons within the red nucleus ( figure 5d ) . in the subset of sections analyzed , there were 211 14 gfp neurons in the red nucleus of animals injected with aav1 , 273.3 16.5 injected with aav2 , 307.3 53.5 injected with aav5 , 350.5 61.2 injected with aav8 and 273 48.1 injected with aav9 . these data demonstrate that multiple supraspinal neuronal pools that project to spinal cord are effectively retrogradely transduced when aav is intraspinally injected after sci . moreover , these data indicate all aav serotypes tested are capable of being retrogradely transported by injured axons . intraspinal injection of all tested aav serotypes resulted in transduction of spinal cord neurons rostral to a sci site . in animals from all groups , numerous gfp neurons were observed in gray matter ( figure 2a , c ) above the injury . moreover , many gfp axons ( visualized as more punctate than linear staining in figure 2a , b because the axons are seen in cross - section ) were observed in the lateral and ventral white matter rostral to the transection site . most gfp cells within the spinal cord had a neuronal phenotype ( figure 2a , c , d ) but a few gfp cells colocalized with the astrocytic marker glial fibrillary acidic protein ( figure 2e , arrows ) . nonetheless , the vast majority of glial fibrillary acidic protein - positive cells did not coexpress gfp ( figure 2e , arrowheads ) , even in regions containing many gfp neurons ( figure 2f , arrowheads ) . these data indicate that each serotype transduced more neurons than astrocytes , agreeing with literature that aav with a non - cell specific promoter preferentially transduces neurons and not glia . to determine if different aav serotypes transduce spinal cord neurons to disparate degrees , all gfp neurons in a subset of spinal cord sections rostral to the spinal transection site ( every 400 m ; figure 3a ) were counted . there were 2,700 212.3 gfp spinal cord neurons in animals injected with aav1 and 3,439 338.3 neurons in animals injected with aav5 . both aav1 and aav5 were significantly better at transducing spinal cord neurons than the other tested serotypes ( p < 0.05 ) . there were 548 71.3 gfp spinal neurons in animals injected with aav2 , 848 193.3 in animals injected with aav8 and 1,572 104.5 in animals injected with aav9 . all serotypes were able to transduce spinal cord neurons as far as 56 mm rostral to the injury site ( figure 3b ) . moreover , neurons as far as 10 mm rostral to the transection site were transduced following aav5 injection . to determine if aav injected immediately rostral to a sci site is taken up by injured axons and retrogradely transported to transduce neurons within the brain , gfp neurons in brain sections every 400 m apart were counted . we found many gfp neurons in the reticular formation ( figure 4a , arrows ) , vestibular nucleus ( figure 4b , arrows ) and red nucleus ( figure 4c , arrows ) . moreover , we found many gfp processes in these same areas ( figure 4 , arrowheads ) , some appearing to emerge from gfp somas . gfp neurons were observed in all brains , indicating that each of the tested aav serotypes is capable of being retrogradely transported by injured axons to transduce brain neurons ( figure 4d ) . however , there was a significantly greater number ( p < 0.05 ) of gfp neurons in the brains of animals intraspinally injected with aav5 ( 2,713 186.0 ) than aav1 ( 1,700 229.3 ) , aav2 ( 943.3 93.8 ) , aav8 ( 1,471.7 359.3 ) or aav9 ( 890.7 262.1 ) . we did not observe any transduced neurons within cortex ( data not shown ) . to determine if the tested serotypes had different tropisms for various supraspinal neuronal pools , we assessed where each gfp neuron in the brain was located . significantly more neurons within the reticular formation ( figure 5a ) that project via the reticulospinal tract were transduced with aav5 ( 1,873 168.7 ) than aav1 ( 743 130.4 ) , aav2 ( 441.7 55.9 ) , aav8 ( 844.7 207.8 ) or aav9 ( 378.7 174.5 ; p all aav serotypes resulted in statistically equal numbers of gfp neurons in the vestibular nuclei ( figure 5b ) that form the vestibulospinal tract . aav1 transduced 436.7 132 neurons , aav2 transduced 228.3 51.1 , aav5 transduced 523.7 70.1 , aav8 transduced 276.7 129.1 , and aav9 transduced 239 127.5 . injecting aav1 into the spinal cord transduced significantly more neurons in the raphe nuclei ( 18.4 2.2 ) that form the raphespinal tract than aav5 ( 9 5.5 ; p < 0.05 ; figure 5c ) . we did not observe any gfp+ neurons in the raphe after injecting aav2 , aav8 or aav9 rostral to the spinal transection . all aav serotypes were transported by injured rubrospinal tract axons to result in gfp expression in neurons within the red nucleus ( figure 5d ) . in the subset of sections analyzed , there were 211 14 gfp neurons in the red nucleus of animals injected with aav1 , 273.3 16.5 injected with aav2 , 307.3 53.5 injected with aav5 , 350.5 61.2 injected with aav8 and 273 48.1 injected with aav9 . these data demonstrate that multiple supraspinal neuronal pools that project to spinal cord are effectively retrogradely transduced when aav is intraspinally injected after sci . moreover , these data indicate all aav serotypes tested are capable of being retrogradely transported by injured axons . while various treatments have successfully promoted some axonal regeneration after sci , gene therapy may help surmount the decreased expression of regeneration - associated genes in adult neurons to significantly enhance their intrinsic growth potential . we chose to focus on aav because it is non - pathogenic , able to transduce post - mitotic cells , preferentially transduces neurons rather than glia and does not trigger an immune or gliotic response . because multiple descending populations are affected by sci , it is plausible that increasing the expression of growth - promoting genes in a wide population of supraspinal neurons that project to spinal cord could further improve functionally relevant axonal regeneration . one means of accomplishing an extensive transduction of brain neurons in a sci model is to inject aav into multiple locations throughout the brainstem , which is home to many neuronal pools that project to spinal cord . while this method is effective , it is very invasive and involves 19 separate injections . while aav effectively transduces neurons when applied to the cell body , it also can be retrogradely transported following injection into peripheral nerve or muscle ( after uptake at synaptic terminals ) to transduce neurons remote from the injection site . retrograde transport of aav appears to be dependent upon interaction with the motor protein dynein that , in turn , binds to and travels along microtubules . indeed , colchicine , which inhibits microtubule polymerization and induces microtubule disassembly , prevents the transduction of spinal motoneurons following aav injection into peripheral nerve . in this study , we sought to determine if we could take advantage of the ability of aav to be retrogradely transported to transduce a large and diverse population of neurons whose axons are injured by sci while concurrently minimizing additional trauma due to delivery of viral vectors . to this end as one would expect , thousands of gfp neurons were found within the spinal cord itself . while this is likely primarily due to the transduction of spinal neurons by virus that diffused within the parenchyma away from the injection sites , the fact that some gfp neurons were located 10 mm away ( figure 3b ) suggests that some propriospinal neurons were also retrogradely transduced . in addition , we found thousands of gfp neurons in brain regions far distant from the injury and injection sites in upper thoracic spinal cord . these data indicate that acutely injured , descending axons are capable of retrogradely transporting intraspinally - delivered aav to effectively transduce neurons within the brainstem that project to spinal cord . this process is likely due ( at least partly ) to active endocytosis because axonal membranes typically seal up within 30 minutes after injury and the injections took about an hour to complete . if uptake of virus into injured axons was merely passive , it seems likely that we would have noticed more gfp neurons in tissue on the side that was injected into first , before membrane sealing , which we did not observe ( data not shown ) . it is important to note that only a fraction of all brain sections were analyzed ( one section every 400 m ) . thus , it is possible and likely that our gfp neuron counts underestimate the absolute number of neurons that were transduced in this fashion . in addition , using higher titers of virus may also increase the number of transduced neurons . we are confident that the expression of the reporter gene gfp in the brain resulted from transport of aav and not from uptake by cell bodies of excess viral vector circulating in cerebral spinal fluid because we found no evidence of gfp neurons surrounding the ventricles ( data not shown ) . we only observed gfp neurons within the spinal cord or in brain regions that project down to spinal cord . in the latter case , gfp neurons were found in the reticular formation , the vestibular nuclei , the red nucleus and , to a lesser extent , the raphe nuclei . this indicates that aav was taken up and transported by axons forming the reticulospinal , vestibulospinal , rubrospinal and raphespinal tracts present in the lateral and ventral funiculi . while other groups have found that transport of several different aav serotypes within the brain is possible , to our knowledge , these data are the first to demonstrate that retrograde transport of aav after sci can remotely transduce neurons . while the direction of transport ( retrograde versus anterograde ) is not always clear , we are confident that in this study , transduction in the brainstem is due to retrograde transport . neurons residing within the spinal cord do not project to the red nucleus or the raphe , so furthermore , while some spinal neurons project via the spinoreticular tract to the reticular formation and via the spinovestibular tract to the vestibular nuclei , the vast majority of these neurons reside in cervical ( for spinoreticular and spinovestibular tracts ) and lumbar spinal cord ( for spinoreticular tract only ) , far from the injury and injection site within thoracic spinal cord . furthermore , we did not observe gfp , transduced neurons within the dorsal column nuclei ( data not shown ) , which is where ascending dorsal column axons terminate , providing additional support that transduction within the brain is due to retrograde and not anterograde transport . while we observed many gfp neurons within brainstem , we did not find any evidence that neurons within primary motor cortex that project to spinal cord via the corticospinal tract were transduced . this is a bit surprising given that these neurons are well - transduced following intracortical injections of aav1 and aav8 , two of the serotypes tested in this study . while it is not entirely clear if the lack of transduction in this study is due to a poor ability of these axons to take up and retrogradely transport the vectors or virus not diffusing to the dorsal funiculus , figure 2a suggests that there was sufficient vector present in the dorsal funiculus , where the primary component of the corticospinal tract is present in the rat . furthermore , when a mixture of horseradish peroxidase and adenovirus encoding for lacz was injected into lumbar spinal cord of naive mice , there were many more horseradish peroxidase - positive than -galactosidase neurons in motor cortex , indicating that corticospinal tract axons either endocytosed or transported horseradish peroxidase and the adenovirus at different rates . these data along with the lack of transduction of primary motor cortical neurons in our study suggest that corticospinal tract axons may inherently be difficult to transduce via retrograde transport of viral vectors , be it aav or adenovirus . it also indicates that transduction efficiencies following delivering virus to the cell body can be quite different from after delivering virus to the axon . because our protocol entails injecting aav into the spinal cord , we are able to more specifically target descending tracts than if we were to inject virus throughout brainstem , which will transduce neurons that project to spinal cord as well as other areas within the brain . interestingly , retrograde transport of aav has been reported in nonhuman primate , suggesting that retrograde transport of aav is not specific to small animal models and is likely possible in the human . another potential advantage of this paradigm for future clinical application is that as surgical intervention in the lesion vicinity is already likely in people who sustained sci , it is foreseeable that aav could be injected into tissue immediately rostral to the injury site during this surgical procedure , negating the need for an additional surgery . although it is beyond the scope of the present study , which was focused on ascertaining whether aav was transported by injured axons , at all , it would be interesting to determine if injury itself affects transport or transduction efficiency . furthermore , while we found that our injection paradigm results in robust neuronal transduction of brainstem neurons in an acute sci model , it would be very important to assess if aav is also taken up and retrogradely transported by chronically injured axons , as studies have demonstrated retrograde transport in long - injured axons is impaired . this information would allow us to better understand if this aav injection paradigm could possibly be used to treat the millions of people within the united states alone already living with some form of sci . as alluded to above , one potential application of this aav injection paradigm is to enhance axonal regeneration after sci . our labuses a well - established grafting model in which segments of peripheral nerve are transplanted to fill the lesion cavity with a growth - supportive environment . some axonal tracts , including the populations that we found to be well - transduced in this study ( e.g. , reticulospinal , vestibulospinal , rubrospinal , and propriospinal pathways ) regenerate fairly well into these peripheral nerve grafts . a significant challenge has proven to be getting axons to emerge from the graft to reinnervate spinal cord tissue . this step is necessary if these axons are to reform functionally relevant synapses upon target neurons . injecting aav rostral to a sci site could be one means to overexpress regeneration - associated genes or knockdown genes that negatively regulate growth specifically in neurons that regenerate into the graft ( e.g. , those originating from the reticular formation , vestibular nuclei , and red nucleus ) to enhance their axons ' ability to traverse the distal graft host interface . we also found that there were transduction efficiency differences between the tested serotypes , with aav1 and aav5 transducing the most neurons within spinal cord and aav5 transducing the most neurons within brain . it is not yet known if this is due to distinct , capsid - dependent neurotropisms of the various aav serotypes or differential ability of aav serotypes to be retrogradely transported . thus , for sci , aav5 seems like the best viral vector to use since it results inefficient transduction of a diverse population of both descending neurons and propriospinal neurons , whose regeneration and/or plasticity can also potentially mediate functional recovery . however , with continued improvements in our understanding of aav capsids , it is possible that another serotype will be discovered or developed that will surpass aav5 in usefulness for our very specific application ( i.e. , retrograde transduction after sci ) . in addition , the further refinement of inducible promoter systems to better control transgene expression in specific neuronal populations will greatly affect the potential of using aav to promote axonal regeneration in the clinic . overall , we have identified a novel , minimally invasive method to effectively transduce a variety of neuronal populations within both the spinal cord and the brain following sci . while the field is still in the nascent stages of using gene therapy as a means to help repair the injured spinal cord , our findings demonstrate the ability to simultaneously and specifically affect multiple neurons after injecting aav5 into one location , i.e. , intraspinally into tissue just rostral to a sci site . this paradigm to broadly distribute viral vectors has the potential to be an important component of an eventual bench - to - bedside combinatorial strategy to promote functional axonal regeneration . all single - stranded aav vectors were obtained from the university of north carolina 's gene therapy center ( chapel hill , nc ) and encoded for the reporter gene gfp under the control of a chicken -actin promoter . aav serotypes used were aav1 , aav2 , aav5 , aav8 , and aav9 . all viral vectors used were hybrids in that the replication gene was from aav2 and the capsid gene was specific to the particular serotype . surgical procedures . all procedures complied with drexel university 's institutional animal care and use committee and national institutes of health guidelines for experimentation with laboratory animals . adult female sprague dawley rats ( 225250 g ; charles river , wilmington , ma ) were anesthetized with isoflurane inhalation . the dorsal surface of thoracic level 3 ( t3 ) spinal cord was exposed by laminectomy . the dura was incised and one vertebral body length ( 23 mm ) of spinal cord was removed via aspiration . the dura was closed using a 10 - 0 suture . to target lateral and ventral funiculi and gray matter 1 l of aav1- , aav2- , aav 5- , aav8- , or aav9-gfp ( n = 3 per group , 10 transducing unit ) was slowly injected into four different site locations at two different depths ~1 mm rostral to the transection using a glass micropipette attached to a hamilton syringe ( figure 1a ) . specifically , aav was bilaterally injected just medial to the lateral edge of the spinal cord at a depth of 1 and 2 mm and 1 mm lateral to midline , and 2.5 and 1.5 mm deep ( figure 1b ) . the laminectomy site was covered with a silastic membrane ( biobrane ; udl laboratories , rockford , il ) . the overlying musculature was closed using 4 - 0 sutures , and the skin was closed using wound clips . mg / kg ) for postoperative pain management , and cephazolin ( 160 mg / kg ) for 7 days to prevent infection . in addition , bladders were expressed manually two- to three - times a day until the voiding reflex returned ( ~14 days ) . the brain and spinal cord rostral to the transection were dissected and post - fixed overnight in 4% paraformaldehyde at 4 c . every eighth section ( every 400 m ) was mounted onto glass slides and coverslipped using fluorsave ( emd millipore , billerica , ma ) . in addition , some sections were blocked in 5% normal goat serum , 1% bovine serum albumin , 0.1% triton x-100 in phosphate - buffered saline for 1 hour at room temperature before incubation in the appropriate primary antibody overnight at room temperate . the primary antibodies used were against gfp ( 1:1,000 ; millipore , billerica , ma ) and glial fibrillary acidic protein ( 1:1,000 ; millipore ) . sections were rinsed in phosphate - buffered saline and incubated in the appropriate secondary antibody for 2 hours at room temperature . secondary antibodies used were conjugated to alexa 488 or 594 ( 1:500 ; life technologies , grand island , ny ) . sections were rinsed in phosphate - buffered saline , mounted onto glass sides , and coverslipped . slides were imaged using an olympus bx51 and a leica dm5500b microscope ( leica microsystems , wetzlar , germany ) . quantification of gfp transduced neurons . to quantify transduced spinal cord neurons , all gfp neurons in sections differences in the number of gfp neurons amongst aav serotype groups was assessed for significance using a one - way analysis of variance and post - hoc tukey 's tests . to quantify transduced neurons within the brain , all gfp neurons in sections 200 m apart were counted and their location noted . statistical significance was determined using one - way analysis of variance and post - hoc tukey 's tests ( graphpad , la jolla , ca ) .

in the vast majority of studies utilizing adeno - associated virus ( aav ) in central nervous system applications , including those published with spinal cord injury ( sci ) models , aav has been administered at the level of the cell body of neurons targeted for genetic modification , resulting in transduction of neurons in the vicinity of the injection site . however , as sci interrupts many axon tracts , it may be more beneficial to transduce a diverse pool of supraspinal neurons . we determined if descending axons severed by sci are capable of retrogradely transporting aav to remotely transduce a variety of brain regions . different aav serotypes encoding the reporter green fluorescent protein ( gfp ) were injected into gray and white matter immediately rostral to a spinal transection site . this resulted in the transduction of thousands of neurons within the spinal cord and in multiple regions within the brainstem that project to spinal cord . in addition , we established that different serotypes had disparate regional specificity and that aav5 transduced the most brain and spinal cord neurons . this is the first demonstration that retrograde transport of aav by axons severed by sci is an effective means to transduce a collection of supraspinal neurons . thus , we identify a novel , minimally invasive means to transduce a variety of neuronal populations within both the spinal cord and the brain following sci . this paradigm to broadly distribute viral vectors has the potential to be an important component of a combinatorial strategy to promote functional axonal regeneration .

Introduction Results Intraspinal injection of AAV rostral to a SCI site transduces many spinal cord neurons AAV is retrogradely transported by axons injured by SCI and robustly transduces various supraspinal brain neurons Discussion Materials and methods

the limited capacity of adult mammalian central nervous system axons to regenerate following spinal cord injury ( sci ) leads to substantial functional defects . one tool that can modify gene expression in the mature central nervous system , potentially allowing for the overcoming of this significant hurdle , is recombinant viral vectors . adeno - associated virus ( aav ) is one type of viral vector that is often used for gene therapy in the central nervous system because of its non - pathogenic nature , replication deficiency and high neuronal transduction efficiency . the ability of aav to transduce fully differentiated neurons makes it an ideal candidate to manipulate gene expression in adult , supraspinal neurons to promote axon regeneration , synaptic plasticity and neuronal survival following the injury of their axons that project to spinal cord . in various sci models , application of aav in the vicinity of targeted cell bodies results in their successful transduction . because sci interrupts many different axon tracts projecting from the brain to the spinal cord , promoting regeneration of several injured , descending tracts would likely restore more interrupted circuitry than regeneration of a specific tract , thereby possibly improving functional recovery . therefore , it seems reasonable to imagine that it would be advantageous to simultaneously modify a variety of supraspinal neuronal pools affected by the injury . recently , widespread transduction was achieved by injecting aav - green fluorescent protein ( gfp ) into multiple sites throughout the brainstem . while this injection paradigm effectively transduces many neurons within the brainstem , it is also very invasive . interestingly , another means to transduce neurons using aav is via retrograde transport . injecting aav into a target muscle or peripheral nerve results in the transduction of motoneurons and dorsal root ganglion neurons that innervate the targeted muscle or that send projections via the injected nerve . whether aav is taken up by central axons within the spinal cord after injury and is then retrogradely transported to transduce neurons within the brain has not been determined . in these experiments , we sought to assess the effectiveness of a novel , less invasive approach to transduce multiple descending populations following sci . we hypothesized that aav injected into spinal cord tissue immediately rostral to an injury site would not only transduce local spinal neurons but would also be taken up by severed , descending tract axons and retrogradely transported to transduce diverse brain neurons that project to spinal cord . in addition , because different aav serotypes have dissimilar tropisms and potential for retrograde transport , we sought to identify which aav serotype most efficiently retrogradely transduces neurons in brain after sci . immediately following a complete transection of the spinal cord at thoracic level 3 ( t3 ) , equivalent titers of aav1- , aav2- , aav5- , aav8- , or aav9-cba - gfp ( gfp transgene under a chicken -actin promoter ; n = 3 per serotype ) were injected into four different locations rostral to the transection site ( figure 1a ) at two different depths per location ( figure 1b ) . this was done to flood with viral vectors tissue encompassing the intermediate gray matter and the lateral and ventral funiculi just rostral to the injury . intraspinal injection of all tested aav serotypes resulted in transduction of spinal cord neurons rostral to a sci site . moreover , many gfp axons ( visualized as more punctate than linear staining in figure 2a , b because the axons are seen in cross - section ) were observed in the lateral and ventral white matter rostral to the transection site . most gfp cells within the spinal cord had a neuronal phenotype ( figure 2a , c , d ) but a few gfp cells colocalized with the astrocytic marker glial fibrillary acidic protein ( figure 2e , arrows ) . to determine if different aav serotypes transduce spinal cord neurons to disparate degrees , all gfp neurons in a subset of spinal cord sections rostral to the spinal transection site ( every 400 m ; figure 3a ) were counted . all serotypes were able to transduce spinal cord neurons as far as 56 mm rostral to the injury site ( figure 3b ) . to determine if aav injected immediately rostral to a sci site is taken up by injured axons and retrogradely transported to transduce neurons within the brain , gfp neurons in brain sections every 400 m apart were counted . gfp neurons were observed in all brains , indicating that each of the tested aav serotypes is capable of being retrogradely transported by injured axons to transduce brain neurons ( figure 4d ) . however , there was a significantly greater number ( p < 0.05 ) of gfp neurons in the brains of animals intraspinally injected with aav5 ( 2,713 186.0 ) than aav1 ( 1,700 229.3 ) , aav2 ( 943.3 93.8 ) , aav8 ( 1,471.7 359.3 ) or aav9 ( 890.7 262.1 ) . to determine if the tested serotypes had different tropisms for various supraspinal neuronal pools , we assessed where each gfp neuron in the brain was located . significantly more neurons within the reticular formation ( figure 5a ) that project via the reticulospinal tract were transduced with aav5 ( 1,873 168.7 ) than aav1 ( 743 130.4 ) , aav2 ( 441.7 55.9 ) , aav8 ( 844.7 207.8 ) or aav9 ( 378.7 174.5 ; p all aav serotypes resulted in statistically equal numbers of gfp neurons in the vestibular nuclei ( figure 5b ) that form the vestibulospinal tract . injecting aav1 into the spinal cord transduced significantly more neurons in the raphe nuclei ( 18.4 2.2 ) that form the raphespinal tract than aav5 ( 9 5.5 ; p < 0.05 ; figure 5c ) . we did not observe any gfp+ neurons in the raphe after injecting aav2 , aav8 or aav9 rostral to the spinal transection . all aav serotypes were transported by injured rubrospinal tract axons to result in gfp expression in neurons within the red nucleus ( figure 5d ) . these data demonstrate that multiple supraspinal neuronal pools that project to spinal cord are effectively retrogradely transduced when aav is intraspinally injected after sci . moreover , these data indicate all aav serotypes tested are capable of being retrogradely transported by injured axons . intraspinal injection of all tested aav serotypes resulted in transduction of spinal cord neurons rostral to a sci site . moreover , many gfp axons ( visualized as more punctate than linear staining in figure 2a , b because the axons are seen in cross - section ) were observed in the lateral and ventral white matter rostral to the transection site . most gfp cells within the spinal cord had a neuronal phenotype ( figure 2a , c , d ) but a few gfp cells colocalized with the astrocytic marker glial fibrillary acidic protein ( figure 2e , arrows ) . nonetheless , the vast majority of glial fibrillary acidic protein - positive cells did not coexpress gfp ( figure 2e , arrowheads ) , even in regions containing many gfp neurons ( figure 2f , arrowheads ) . to determine if different aav serotypes transduce spinal cord neurons to disparate degrees , all gfp neurons in a subset of spinal cord sections rostral to the spinal transection site ( every 400 m ; figure 3a ) were counted . all serotypes were able to transduce spinal cord neurons as far as 56 mm rostral to the injury site ( figure 3b ) . to determine if aav injected immediately rostral to a sci site is taken up by injured axons and retrogradely transported to transduce neurons within the brain , gfp neurons in brain sections every 400 m apart were counted . gfp neurons were observed in all brains , indicating that each of the tested aav serotypes is capable of being retrogradely transported by injured axons to transduce brain neurons ( figure 4d ) . to determine if the tested serotypes had different tropisms for various supraspinal neuronal pools , we assessed where each gfp neuron in the brain was located . significantly more neurons within the reticular formation ( figure 5a ) that project via the reticulospinal tract were transduced with aav5 ( 1,873 168.7 ) than aav1 ( 743 130.4 ) , aav2 ( 441.7 55.9 ) , aav8 ( 844.7 207.8 ) or aav9 ( 378.7 174.5 ; p all aav serotypes resulted in statistically equal numbers of gfp neurons in the vestibular nuclei ( figure 5b ) that form the vestibulospinal tract . injecting aav1 into the spinal cord transduced significantly more neurons in the raphe nuclei ( 18.4 2.2 ) that form the raphespinal tract than aav5 ( 9 5.5 ; p < 0.05 ; figure 5c ) . we did not observe any gfp+ neurons in the raphe after injecting aav2 , aav8 or aav9 rostral to the spinal transection . all aav serotypes were transported by injured rubrospinal tract axons to result in gfp expression in neurons within the red nucleus ( figure 5d ) . these data demonstrate that multiple supraspinal neuronal pools that project to spinal cord are effectively retrogradely transduced when aav is intraspinally injected after sci . moreover , these data indicate all aav serotypes tested are capable of being retrogradely transported by injured axons . because multiple descending populations are affected by sci , it is plausible that increasing the expression of growth - promoting genes in a wide population of supraspinal neurons that project to spinal cord could further improve functionally relevant axonal regeneration . one means of accomplishing an extensive transduction of brain neurons in a sci model is to inject aav into multiple locations throughout the brainstem , which is home to many neuronal pools that project to spinal cord . while aav effectively transduces neurons when applied to the cell body , it also can be retrogradely transported following injection into peripheral nerve or muscle ( after uptake at synaptic terminals ) to transduce neurons remote from the injection site . retrograde transport of aav appears to be dependent upon interaction with the motor protein dynein that , in turn , binds to and travels along microtubules . in this study , we sought to determine if we could take advantage of the ability of aav to be retrogradely transported to transduce a large and diverse population of neurons whose axons are injured by sci while concurrently minimizing additional trauma due to delivery of viral vectors . to this end as one would expect , thousands of gfp neurons were found within the spinal cord itself . while this is likely primarily due to the transduction of spinal neurons by virus that diffused within the parenchyma away from the injection sites , the fact that some gfp neurons were located 10 mm away ( figure 3b ) suggests that some propriospinal neurons were also retrogradely transduced . in addition , we found thousands of gfp neurons in brain regions far distant from the injury and injection sites in upper thoracic spinal cord . these data indicate that acutely injured , descending axons are capable of retrogradely transporting intraspinally - delivered aav to effectively transduce neurons within the brainstem that project to spinal cord . thus , it is possible and likely that our gfp neuron counts underestimate the absolute number of neurons that were transduced in this fashion . we are confident that the expression of the reporter gene gfp in the brain resulted from transport of aav and not from uptake by cell bodies of excess viral vector circulating in cerebral spinal fluid because we found no evidence of gfp neurons surrounding the ventricles ( data not shown ) . we only observed gfp neurons within the spinal cord or in brain regions that project down to spinal cord . while other groups have found that transport of several different aav serotypes within the brain is possible , to our knowledge , these data are the first to demonstrate that retrograde transport of aav after sci can remotely transduce neurons . while the direction of transport ( retrograde versus anterograde ) is not always clear , we are confident that in this study , transduction in the brainstem is due to retrograde transport . neurons residing within the spinal cord do not project to the red nucleus or the raphe , so furthermore , while some spinal neurons project via the spinoreticular tract to the reticular formation and via the spinovestibular tract to the vestibular nuclei , the vast majority of these neurons reside in cervical ( for spinoreticular and spinovestibular tracts ) and lumbar spinal cord ( for spinoreticular tract only ) , far from the injury and injection site within thoracic spinal cord . furthermore , we did not observe gfp , transduced neurons within the dorsal column nuclei ( data not shown ) , which is where ascending dorsal column axons terminate , providing additional support that transduction within the brain is due to retrograde and not anterograde transport . while we observed many gfp neurons within brainstem , we did not find any evidence that neurons within primary motor cortex that project to spinal cord via the corticospinal tract were transduced . while it is not entirely clear if the lack of transduction in this study is due to a poor ability of these axons to take up and retrogradely transport the vectors or virus not diffusing to the dorsal funiculus , figure 2a suggests that there was sufficient vector present in the dorsal funiculus , where the primary component of the corticospinal tract is present in the rat . furthermore , when a mixture of horseradish peroxidase and adenovirus encoding for lacz was injected into lumbar spinal cord of naive mice , there were many more horseradish peroxidase - positive than -galactosidase neurons in motor cortex , indicating that corticospinal tract axons either endocytosed or transported horseradish peroxidase and the adenovirus at different rates . these data along with the lack of transduction of primary motor cortical neurons in our study suggest that corticospinal tract axons may inherently be difficult to transduce via retrograde transport of viral vectors , be it aav or adenovirus . because our protocol entails injecting aav into the spinal cord , we are able to more specifically target descending tracts than if we were to inject virus throughout brainstem , which will transduce neurons that project to spinal cord as well as other areas within the brain . interestingly , retrograde transport of aav has been reported in nonhuman primate , suggesting that retrograde transport of aav is not specific to small animal models and is likely possible in the human . another potential advantage of this paradigm for future clinical application is that as surgical intervention in the lesion vicinity is already likely in people who sustained sci , it is foreseeable that aav could be injected into tissue immediately rostral to the injury site during this surgical procedure , negating the need for an additional surgery . furthermore , while we found that our injection paradigm results in robust neuronal transduction of brainstem neurons in an acute sci model , it would be very important to assess if aav is also taken up and retrogradely transported by chronically injured axons , as studies have demonstrated retrograde transport in long - injured axons is impaired . some axonal tracts , including the populations that we found to be well - transduced in this study ( e.g. injecting aav rostral to a sci site could be one means to overexpress regeneration - associated genes or knockdown genes that negatively regulate growth specifically in neurons that regenerate into the graft ( e.g. we also found that there were transduction efficiency differences between the tested serotypes , with aav1 and aav5 transducing the most neurons within spinal cord and aav5 transducing the most neurons within brain . it is not yet known if this is due to distinct , capsid - dependent neurotropisms of the various aav serotypes or differential ability of aav serotypes to be retrogradely transported . thus , for sci , aav5 seems like the best viral vector to use since it results inefficient transduction of a diverse population of both descending neurons and propriospinal neurons , whose regeneration and/or plasticity can also potentially mediate functional recovery . in addition , the further refinement of inducible promoter systems to better control transgene expression in specific neuronal populations will greatly affect the potential of using aav to promote axonal regeneration in the clinic . overall , we have identified a novel , minimally invasive method to effectively transduce a variety of neuronal populations within both the spinal cord and the brain following sci . while the field is still in the nascent stages of using gene therapy as a means to help repair the injured spinal cord , our findings demonstrate the ability to simultaneously and specifically affect multiple neurons after injecting aav5 into one location , i.e. this paradigm to broadly distribute viral vectors has the potential to be an important component of an eventual bench - to - bedside combinatorial strategy to promote functional axonal regeneration . to target lateral and ventral funiculi and gray matter 1 l of aav1- , aav2- , aav 5- , aav8- , or aav9-gfp ( n = 3 per group , 10 transducing unit ) was slowly injected into four different site locations at two different depths ~1 mm rostral to the transection using a glass micropipette attached to a hamilton syringe ( figure 1a ) . specifically , aav was bilaterally injected just medial to the lateral edge of the spinal cord at a depth of 1 and 2 mm and 1 mm lateral to midline , and 2.5 and 1.5 mm deep ( figure 1b ) . the brain and spinal cord rostral to the transection were dissected and post - fixed overnight in 4% paraformaldehyde at 4 c . in addition , some sections were blocked in 5% normal goat serum , 1% bovine serum albumin , 0.1% triton x-100 in phosphate - buffered saline for 1 hour at room temperature before incubation in the appropriate primary antibody overnight at room temperate . to quantify transduced spinal cord neurons , all gfp neurons in sections differences in the number of gfp neurons amongst aav serotype groups was assessed for significance using a one - way analysis of variance and post - hoc tukey 's tests . to quantify transduced neurons within the brain , all gfp neurons in sections 200 m apart were counted and their location noted .

chronic low - grade inflammation and aberrant regulation of the stress response system are two prominent hallmarks of obesity , a major risk factor for the development of insulin resistance , type 2 diabetes , metabolic syndrome , and cardiovascular diseases [ 1 , 2 ] . sedentary lifestyles and excessive food intake are considered as key contributors to this chronic condition . the white adipose tissue has been identified as the predominant site of obesity - associated inflammatory reactions due to its infiltration by immune inflammatory cells such as monocytes , macrophages , th1 t cells , and dendritic cells [ 24 ] . these immune cells , together with adipocytes and stromal vascular cells , constitute a cellular network that produces various inflammatory mediators . obesity - induced inflammatory response impairs insulin signaling in insulin - responsive organs and causes systemic insulin resistance , which leads to a perturbation of glucose homeostasis and ultimately type-2 diabetes [ 5 , 6 ] . studies on mice indicated that obesity also alters the balance between pro- and anti - inflammatory activities in adipose tissue by promoting the phenotypic switch from m2 anti - inflammatory macrophages to m1 proinflammatory macrophages and thereby perpetuating further the inflammatory response and insulin resistance [ 5 , 7 ] . regulated upon activation normal t cells expressed and secreted ( rantes or ccl5 ) is a powerful proinflammatory mediator of the cc chemokine family that regulates the mobilization and , in certain cases , promotes survival of immune inflammatory cells from the bloodstream into tissues and other areas of injury and infection [ 3 , 8 , 9 ] . although the chemotactic activity of rantes on immune cells to injured and infected areas is beneficial , sustained production of rantes is associated with several detrimental effects such as atherosclerosis [ 10 , 11 ] , arthritis rheumatoid , liver disease [ 13 , 14 ] , and viral infection that share in common chronic inflammatory response . consistent with its critical role in the pathophysiology of these chronic inflammatory - related diseases , treatments that interfere with rantes signaling such as neutralizing antibody [ 16 , 17 ] , peptide mimetics [ 18 , 19 ] , and pharmacological inhibitors [ 20 , 21 ] are associated with improved outcomes . rantes orchestrates its effects through binding to one of its cognate receptors including ccr1 , ccr3 , and ccr5 [ 22 , 23 ] . the crucial role of ccr5 in mediating the inflammatory response in adipose tissue was demonstrated recently by using ccr5 knockout mice that showed a dominant shift from m1-phenotype to m2-phenotype , which contributed to attenuated inflammatory response and improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity . physical exercise is an important component of healthy lifestyle that is widely advocated as a first line for the treatment and management of obesity , insulin resistance , diabetes , and cardiovascular diseases [ 2527 ] . previous studies linked the protective effect of physical exercise to the improvement of the inflammatory and stress responses [ 2830 ] . the mechanisms by which physical exercise mediates its anti - inflammatory effect are multiples and they include a reduction of visceral fat mass with subsequent decrease in the production of proinflammatory adipokines and a reduction in the circulating number of proinflammatory monocytes in favor of an increase in the circulating number of regulatory t cells [ 31 , 32 ] . other studies revealed also that physical exercise prevents monocytes and macrophages from infiltrating into adipose tissue and induces a phenotypic switching from m1-phenotype macrophages to m2-macrophages within the adipose tissue . other beneficial effects of physical exercise include attenuated activation of c - jun nh2-terminal kinase together with improvement of lipid , glucose , and oxidative homeostasis [ 3537 ] . in the current study , we investigated the effect of a 3-month physical exercise on the expression and release of rantes in obese subjects . given the critical role of rantes and its ccr5 receptor in promoting obesity - induced metabolic inflammation , we hypothesized that physical exercise may mediates its anti - inflammatory effect in part , by impairing rantes signaling pathway via downregulation of rantes and/or its ccr5 receptor . we report here for the first time a decrease in the expression of both rantes and ccr5 receptor by physical exercise in the adipose tissue of obese humans . the study was conducted on adult male and female nondiabetic subjects consisting of 17 lean ( bmi = 2024.9 kg / m ) and 40 obese ( bmi = 3040 kg / m ) . all subjects gave written informed consent and the study was approved by the ethical review board of dasman diabetes institute ( reference number : ra-2010 - 003 ) . participants that followed any physical exercise within the last 6 months prior to this study as well as those with prior major illness or intake of medications or supplements known to influence the body composition or bone mass were excluded from the study . none of the participants that were enrolled in this study reported any cerebrovascular complication , kidney disease , asthma , and food and drug allergies and chronic viral infection . according to the medical questionnaire , furthermore , no specific diet or nutritional change was prescribed to our subject during the 3 months of physical exercise protocol as our aim was mainly to investigate the sole effect of physical exercise on those subjects . all eligible subjects were enrolled to a supervised exercise program at the fitness and rehabilitation center ( frc ) of dasman diabetes institute . prior to exercise prescription , each individual underwent a symptom - limited maximal incremental cardiopulmonary exercise test as well as a muscle strength and endurance test . it consisted of a combination of both moderate intensity of aerobic exercise and resistance training using either treadmill or cycling . each exercise session included 10 minutes of warming - up and cooling down steps at 5060% of max hr , along with 40 minutes of the prescribed exercise program at 6580% of max hr . for the duration of the 3-month period , participants exercised 3 to 5 times per week and they were instructed to reach and maintain the recommended heart rate range . strength training was performed 2 to 3 times a week according to the program plan . all trainings were supervised by qualified fitness professionals from frc . to assess the effectiveness of the exercise , the same physical stress and fitness tests were performed for all subjects at the end of the exercise program . venous peripheral blood and subcutaneous adipose tissue biopsies were obtained prior to the start of the 3-month protocol and within 2 to 3 days after its completion . plasma and serum were prepared using vacutainer tubes and then aliquoted and stored at 80c . subcutaneous superficial adipose tissue biopsies ( ~0.5 g ) were obtained from the periumbilical area by surgical biopsy after a local anesthesia . once removed , the biopsy was rinsed in cold pbs , divided into 4 pieces , and stored appropriately until assayed . glucose and lipid profiles were measured on the siemens dimension rxl chemistry analyzer ( diamond diagnostics , holliston , ma ) . hemoglobin a1c ( hba1c ) was determined using the variant device ( biorad , hercules , ca ) . plasma levels of inflammatory and metabolic markers were measured using the bio - plex 27-plex human cytokine and 12 bio - plex human diabetes kits , respectively ( biorad , hercules , ca ) . data were analyzed with bio - plex manager software version 6 ( biorad , hercules , ca ) . lipid peroxidation was assessed by measuring plasma levels of malonaldehyde , using tbars assay kit ( cayman chemical company , ann arbor , mi ) . serum levels of ros were determined using the oxiselect ros assay kit ( cell biolabs inc , san diego , ca ) . total rna was extracted from frozen adipose tissue and pbmcs using rneasy lipid tissue mini kit and allprep rna / protein kit , respectively ( qiagen , inc . , the cdna was synthesized from total rna sample using high capacity cdna reverse transcription kits ( applied biosystems , foster city , ca ) . qrt - pcr was performed on rotor - disc 100 system using sybr green normalized to gapdh ( qiagen , inc . , cell lysates were prepared from pbmcs by the addition of ripa buffer ( 50 mm tris - hcl ph 7.5 , 150 mm nacl , 1% triton x100 , 1 mm edta , 0.5% sodium deoxycholate , and 0.1% sds ) . protein concentration was determined by bradford method using globulin as a standard and 20 g of proteins was resolved on 10% sds - page gels . proteins were transferred onto pvdf membranes and probed with primary and secondary antibodies using standard protocols . protein bands were visualized by chemiluminescence and the images were captured by using the versadoc 5000 system ( biorad , hercules , ca ) . the primary antibodies used in this study are raised against rantes ( abcam , cambridge , ma ) and actin ( santa cruz biotechnology , santa cruz , ca ) . for densitometric analysis , the intensity of the bands was determined using quantity one software ( biorad , hercules , ca ) . formalin - fixed and paraffin - embedded adipose tissue sections were deparaffinized and rehydrated prior to antigen retrieval by boiling in the unmasking solution ( dako , denmark ) . sections were blocked with 5% fat - free milk for 60 min at rt followed by 1% bsa for another 60 min and then incubated at 4c for overnight with primary antibodies against rantes ( abcam , cambridge , ma ) , tnf - a ( abcam , cambridge , ma ) , il-6 ( novus biologicals , littleton , co ) , and phospho - jnk ( staining with horseradish conjugated secondary antibody ( dako , denmark ) was done for 60 minutes at rt . colors were developed using dab kit ( dako , denmark ) and sections were counterstained with hematoxylin ( sigma aldrich , st . quantification of the immunohistochemical data was done using aperio software version 6.3 ( molecular devices , downingtown , pa ) with an established arbitrary threshold . approximately 5 10 of frozen pbmcs were thawed and washed with rpmi medium supplemented with 10% fbs , antibiotics , and l - glutamine ( complete media ) . for rantes staining , cells were cultured overnight in complete media at 37c and then washed with pbs containing 2% fbs followed by facs buffer ( bd biosciences , san jose , ca ) and then costained with anti - human apc - conjugated rantes ( r&d systems ) , fitc - conjugated cd3 ( mca463f , abd serotec , raleigh , nc ) , and pe - conjugated cd14 ( fab3832p , r&d systems , minneapolis , mn ) antibodies for 45 minutes on ice . cells were then washed twice with permeabilization buffer ( ebioscience , san diego , ca ) and resuspended in 300 l facs buffer for analysis . regarding ccr5 staining , cells were allowed to recover for 1 - 2 hours in complete media . the cells were then cultured in complete media in presence of 2 m monensin ( ebioscience , san diego , ca ) at 37c and 5% co2 for 16 hours . pbmcs were then washed with fb and surface stained simultaneously with fitc - conjugated cd3 ( mca463f ) and pe - conjugated cd14 ( fab3832p ) antibodies . after 45 minutes of incubation on ice in the dark , cells were washed twice and fixed with fixation buffer ( ebioscience , san diego , ca ) for 20 minutes . cells were then washed twice with permeabilization buffer ( ebioscience , san diego , ca ) according to manufacturer 's recommendation followed by staining with ccr5-apc antibody ( fab1802a , r&d systems , minneapolis , mn ) for 20 minutes in the dark . acquisition and analysis were carried out on a facs canto ii flow cytometer using facsdiva software ( bd biosciences , san jose , ca ) . after forward scatter ( fsc ) and side scatter ( ssc ) gating on either lymphocytes or monocytes the rantes expression level was determined in the cd3 as well as in cd14 subsets . statistical analyses were performed with sas version 9.2 ( sas institute inc , cary , nc ) . unless otherwise stated , all descriptive statistics for the variables in the study were reported as means standard error . nonparametric mann - whitney test was used to determine significance of difference in means between the two groups as indicated in the figure legends . the physical characteristics of the two groups , namely , lean ( n = 17 ) and obese ( n = 40 ) , enrolled in this study are displayed in table 1 . as expected , body mass index , percent body fat , and waist and hip circumferences were significantly higher in obese group compared to lean group ( p < 0.0001 ) . obese subjects had also higher systolic blood pressure and higher levels of triglycerides ( p < 0.05 ) ; however , there was no significant difference in the cardiopulmonary parameters between the two groups . obese subjects had higher levels of glucose and hba1c ( p < 0.05 ) in spite of not being diabetic . compared to lean group , the metabolic and inflammatory profiles are dysregulated in obese group as indicated by increased levels of leptin ( p = 0.0006 ) , resistin ( p = 0.04 ) , ip-10 ( p = 0.0008 ) , and rantes chemokines ( p = 0.023 ) . no significant difference was observed in the other metabolic and inflammatory mediators as well as the oxidative stress markers ( table 1 and data not shown ) . in agreement with our recent investigation , obese subjects had higher levels of rantes in the circulation that correlated negatively with il-1ra ( r = 0.42 ; p = 0.001 ) and positively with ip-10 ( r = 0.31 ; p = 0.02 ) and tbars levels ( r = 0.29 ; p = 0.03 ) ( figure 1(a ) ) . to investigate the endogenous expression of rantes between the two groups at the protein level , we determined its expression in pbmcs and adipose tissue by western blot , flow cytometry , and immunohistochemistry ( ihc ) . as shown in figure 2(a ) , there was no difference in the expression of rantes protein in pbmcs between lean and obese subjects ( n = 3 for each group ) . consistent with this finding , flow cytometry indicated that rantes levels were comparable between lean and obese subjects ( n = 3 for each group ) in both monocytes and lymphocytes ( figure 2(b ) ) . by contrast to pbmcs , the expression of rantes protein in the adipose tissue as monitored by ihc studies was significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.01 ; figure 2(c ) ) . the differential expression of rantes between pbmcs and adipose tissue in lean and obese subjects was also confirmed at the mrna level by using quantitative real - time pcr ( qrt - pcr ) and the results are displayed in figures 2(d ) and 2(e ) . accordingly , there was no change in rantes mrna levels in pbmcs from lean and obese subjects ( n = 10 for both lean and obese , figure 2(d ) ) , whereas the levels of rantes mrna in the adipose tissue were significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.02 , figure 2(e ) ) . under the same conditions , the endogenous expression of the two key inflammatory markers tnf- and il-6 in the adipose tissue were significantly increased in obese subjects both at the protein level ( p < 0.05 , figure 2(f ) ) and mrna level ( p < 0.05 , figure 2(g ) ) . we next examined the expression profile of ccr5 receptor between lean and obese subjects using pbmcs and adipose tissue . indeed , there was a clear upregulation of ccr5 mrna in the adipose tissue of obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.03 , figure 3(b ) ) . by contrast , ccr5 mrna was significantly downregulated in pbmcs of obese subjects compared to lean subjects ( n = 10 for both lean and obese ) ( p = 0.04 , figure 3(a ) ) . on the other hand , flow cytometry analysis carried out on pbmcs from lean and obese ( n = 5 for each group ) revealed a significant and unique increase of ccr5 in cd14 + monocyte subset from obese subjects ( p = 0.02 , figure 3(c ) ) . the specific mean fluorescence intensity for ccr5 in obese individuals revealed a higher signal for cd14 + monocytes compared to cd14++ monocytes ( p = 0.001 , figure 3(c ) ) . we previously reported the effectiveness of our physical exercise protocol on improving the physical , clinical , and metabolic parameters on obese subjects . accordingly , there was a significant reduction of pbf and sbp and increase in vo2max along with a decrease in tbars levels and a reduction of inflammatory markers tnf- and il-6 in the circulation . however , physical exercise did not reduce the levels of rantes in the circulation . to investigate whether physical exercise has an impact on the endogenous expression of rantes and ccr5 , qrt - pcr and ihc were carried out on adipose tissue from obese subjects before and after the exercise program . as shown in figure 4(a ) , ihc carried out on adipose tissue from obese subjects before ( n = 11 ) and after ( n = 7 ) the exercise program indicated a significant decrease in the expression of rantes by physical exercise ( p = 0.003 ) . qrt - pcr performed on obese before and after the exercise program ( n = 10 for each group ) confirmed the reduction of rantes mrna expression by physical exercise ( p = 0.01 , figure 4(b ) ) . likewise , ccr5 mrna was significantly reduced by physical exercise in the adipose tissue ( p = 0.02 , figure 4(b ) ) . using the same samples , we observed a decrease in the endogenous expression of tnf- and il-6 both at the mrna level ( p < 0.05 , figure 4(b ) ) and protein level ( p < 0.05 , figure 4(c ) ) . since obesity is known to induce the phosphorylation of jnk protein , we measured the levels of phosphorylated jnk in adipose tissue by ihc before ( n = 11 ) and after ( n = 7 ) the physical exercise program . data shown on figure 4(c ) indicated that physical exercise decreases significantly the levels of phosphorylated jnk ( p = 0.002 ) in obese in a manner that was concomitant with a decrease of ccr5 and its ligand rantes . no effect was observed for total jnk before and after exercise in obese subjects ( data not shown ) . taken together , these data suggest that exercise is interfering with obesity - mediated expression of rantes and ccr5 . chronic low - grade inflammation state is a key feature of obesity and it is caused mainly by infiltration of macrophages and other inflammatory cells into the adipose tissue . the present study explored the effect of physical exercise on the expression of rantes and its main receptor ccr5 in obese humans . our focus on ccr5 receptor was based on a recent study in which ccr5 knockout mice were protected from obesity - induced adipose tissue inflammation and insulin resistance . in addition , it has been previously shown that obesity triggers a modest change in the expression of ccr3 and no change in that of ccr1 . the main findings of our current investigation are as follows : ( 1 ) the expression of both rantes and ccr5 was significantly higher in the subcutaneous adipose tissue of obese subjects , ( 2 ) by contrast to the adipose tissue , ccr5 was downregulated in pbmcs of obese subjects compared to lean subjects but there was no significant difference in the expression of rantes between the two groups , and ( 3 ) physical exercise corrected the dysregulated expression of both rantes and ccr5 in the subcutaneous adipose tissue but has a marginal effect on circulating levels of rantes . while our findings showing increased expression of rantes and ccr5 in the adipose tissue are consistent with previous clinical and animal studies [ 17 , 40 ] , the downregulation of their expression by physical exercise is novel . based on the crucial role of rantes / ccr5 signaling pathway in the pathology of obesity and its associated complications , our findings add further evidence that physical exercise might be one of nonpharmacologic approaches that can attenuate rantes / ccr5 signaling pathway and thereby mitigating the inflammatory and metabolic stress triggered by obesity . rantes is a powerful proinflammatory chemokine that controls the trafficking of immune inflammatory cells such as monocytes , macrophages , th1 t cells , and dendritic cells from the circulation into various tissues including adipose tissue [ 3 , 9 ] . previous studies carried out on obese humans and rodents reported high levels of rantes as well as increased frequency of macrophages in the adipose tissue that were concomitant with increased inflammatory response , insulin resistance , and type 2 diabetes [ 5 , 17 , 4144 ] . our findings showing high levels of rantes in the adipose tissue of obese subjects at both mrna and protein levels corroborate these pioneer studies that associated rantes with obesity . unlike the adipose tissue , the levels of rantes were however comparable between lean and obese subjects as monitored by western blotting , flow cytometry , and qrt - pcr . the mechanism underlying this differential expression of rantes between pbmcs and adipose tissue remains to be investigated but there is evidence for depot - specific differences that dictate the levels of rantes released by various adipose tissues in humans . one of the key characteristics of macrophages that infiltrate the adipose tissue is the heterogeneity of their phenotype and depending on their polarization status ; they can be proinflammatory macrophages ( m1-phenotype ) secreting various inflammatory mediators such as tnf- , il-6 , and inos [ 45 , 46 ] or anti - inflammatory macrophages ( m2-phenotype ) that secrete anti - inflammatory cytokines such as il-10 [ 41 , 47 ] . in obesity , the differentiation of m2 into m1 macrophages is considered as a major event that sustains chronic inflammation . indeed , studies on mice indicated that obesity induces a shift in macrophage balance towards the m1-phenotype macrophages that perpetuate further the inflammatory response and insulin resistance [ 5 , 46 ] . in our study population , we did not evaluate the status of m1- and m2-phenotypes , which may represent a limitation to this investigation . however , in our study the high levels of circulating rantes correlated negatively with the anti - inflammatory il-1ra and positively with the levels of proinflammatory ip-10 chemokine and tbars supporting its role in mediating inflammation . in the current study , we have also investigated the expression pattern of ccr5 in obese humans and similar to our findings with rantes , both mrna and protein levels of ccr5 were increased in the adipose tissue of obese compared to lean group . the observed increase is consistent with recent studies both on humans and rodents [ 24 , 40 ] . the direct role of ccr5 in the regulation of obesity - induced adipose tissue inflammation and development of insulin resistance was recently demonstrated using ccr5 knockout mice . a dominant shift occurred from proinflammatory m1 to anti - inflammatory m2 macrophage phenotypes , which contributed to an improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity in these animals . unexpectedly , our data demonstrated that , unlike the adipose tissue , the expression of ccr5 mrna was significantly downregulated in pbmcs from obese individuals . to the best of our knowledge , our data is the first to report the downregulation of ccr5 mrna in the pbmcs of obese individuals . this observed downregulation is intriguing and might suggest a complex regulation process of its transcription . the altered expression of ccr5 in pbmcs has been described in rheumatoid arthritis and in women with systemic lupus erythematosus . it is possible that other receptors for rantes such as ccr1 and ccr3 may be compensating for the reduced expression of ccr5 in obese subjects . interestingly , ccr5 expression on cd14 + monocyte subset appears to be upregulation in comparison with cd14++ subset in obese individuals . cd14 + cells are known to exhibit a macrophage - like phenotype with enhanced antigen - presenting capacities , higher endothelial affinity , and they are potent producers of proinflammatory cytokines ; thus , they may contribute to the development of chronic inflammation . of particular interest , our study emphasized more on the role of physical exercise in regulating the expression of rantes and its receptor ccr5 . it is well established that exercise reduces the risk of chronic metabolic and cardiorespiratory diseases by modulating the inflammatory and stress responses [ 51 , 52 ] . our data showed for the first time that physical exercise significantly reduced the expression of both rantes and ccr5 receptor in the adipose tissue in a manner that was concomitant with decreased pbf and improved cardiopulmonary performance as monitored by decreased sbp and increased vo2max. the reduced expression of rantes and ccr5 by physical exercise was also consistent with attenuated inflammatory response as indicated by decreased tnf- and il-6 in the circulation and reduced levels of tbars . our finding revealed also that exercise - mediated decrease in the expression of rantes and ccr5 in the adipose tissue was not translated to a decrease in the circulating levels of rantes . the lack of parallel changes between systemic and endogenous levels of rantes after exercise raises the question regarding the role of rantes in the circulation . earlier study indicated that systemic levels of rantes are 100-fold higher than those being released from any of the adipose tissue depots . therefore , it is sensible to suggest that while adipose tissue is able to produce this chemokine , its contribution to systemic levels may be less significant . physical exercise is also known to inhibit metabolic stress in part by dephosphorylating and thus inactivating jnk stress kinase [ 34 , 53 , 54 ] . in agreement with these findings , our data indicate that there was a significant increase in the levels of phosphorylated jnk along with high levels of rantes and ccr5 in obese subjects that were all reduced by physical exercise ( figure 4(c ) ) . whether there is a direct crosstalk between rantes / ccr5 signaling pathway and jnk signaling or not remains to be investigated , despite the fact that ccr5 knockout mice displayed attenuated signaling via nf-b and jnk . in conclusion , the dysregulation of rantes and its negative correlation with the metabolic and stress responses are supporting the pathological role of rantes in obesity . our results also provide strong evidence that physical exercise downregulates the expression of both rantes and ccr5 in the adipose tissue . these findings give strong support to the concept that physical exercise can be used as a nonpharmacologic approach to mitigate the complications associated with obesity , particularly inflammation , in part by attenuating rantes / ccr5 signaling in the adipose tissue .

rantes and its ccr5 receptor trigger inflammation and its progression to insulin resistance in obese . in the present study , we investigated for the first time the effect of physical exercise on the expression of rantes and ccr5 in obese humans . fifty - seven adult nondiabetic subjects ( 17 lean and 40 obese ) were enrolled in a 3-month supervised physical exercise . rantes and ccr5 expressions were measured in pbmcs and subcutaneous adipose tissue before and after exercise . circulating plasma levels of rantes were also investigated . there was a significant increase in rantes and ccr5 expression in the subcutaneous adipose tissue of obese compared to lean . in pbmcs , however , while the levels of rantes mrna and protein were comparable between both groups , ccr5 mrna was downregulated in obese subjects ( p < 0.05 ) . physical exercise significantly reduced the expression of both rantes and ccr5 ( p < 0.05 ) in the adipose tissue of obese individuals with a concomitant decrease in the levels of the inflammatory markers tnf- , il-6 , and p - jnk . circulating rantes correlated negatively with anti - inflammatory il-1ra ( p = 0.001 ) and positively with proinflammatory ip-10 and tbars levels ( p < 0.05 ) . therefore , physical exercise may provide an effective approach for combating the deleterious effects associated with obesity through rantes signaling in the adipose tissue .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

chronic low - grade inflammation and aberrant regulation of the stress response system are two prominent hallmarks of obesity , a major risk factor for the development of insulin resistance , type 2 diabetes , metabolic syndrome , and cardiovascular diseases [ 1 , 2 ] . studies on mice indicated that obesity also alters the balance between pro- and anti - inflammatory activities in adipose tissue by promoting the phenotypic switch from m2 anti - inflammatory macrophages to m1 proinflammatory macrophages and thereby perpetuating further the inflammatory response and insulin resistance [ 5 , 7 ] . although the chemotactic activity of rantes on immune cells to injured and infected areas is beneficial , sustained production of rantes is associated with several detrimental effects such as atherosclerosis [ 10 , 11 ] , arthritis rheumatoid , liver disease [ 13 , 14 ] , and viral infection that share in common chronic inflammatory response . consistent with its critical role in the pathophysiology of these chronic inflammatory - related diseases , treatments that interfere with rantes signaling such as neutralizing antibody [ 16 , 17 ] , peptide mimetics [ 18 , 19 ] , and pharmacological inhibitors [ 20 , 21 ] are associated with improved outcomes . rantes orchestrates its effects through binding to one of its cognate receptors including ccr1 , ccr3 , and ccr5 [ 22 , 23 ] . the crucial role of ccr5 in mediating the inflammatory response in adipose tissue was demonstrated recently by using ccr5 knockout mice that showed a dominant shift from m1-phenotype to m2-phenotype , which contributed to attenuated inflammatory response and improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity . physical exercise is an important component of healthy lifestyle that is widely advocated as a first line for the treatment and management of obesity , insulin resistance , diabetes , and cardiovascular diseases [ 2527 ] . previous studies linked the protective effect of physical exercise to the improvement of the inflammatory and stress responses [ 2830 ] . the mechanisms by which physical exercise mediates its anti - inflammatory effect are multiples and they include a reduction of visceral fat mass with subsequent decrease in the production of proinflammatory adipokines and a reduction in the circulating number of proinflammatory monocytes in favor of an increase in the circulating number of regulatory t cells [ 31 , 32 ] . other studies revealed also that physical exercise prevents monocytes and macrophages from infiltrating into adipose tissue and induces a phenotypic switching from m1-phenotype macrophages to m2-macrophages within the adipose tissue . other beneficial effects of physical exercise include attenuated activation of c - jun nh2-terminal kinase together with improvement of lipid , glucose , and oxidative homeostasis [ 3537 ] . in the current study , we investigated the effect of a 3-month physical exercise on the expression and release of rantes in obese subjects . given the critical role of rantes and its ccr5 receptor in promoting obesity - induced metabolic inflammation , we hypothesized that physical exercise may mediates its anti - inflammatory effect in part , by impairing rantes signaling pathway via downregulation of rantes and/or its ccr5 receptor . we report here for the first time a decrease in the expression of both rantes and ccr5 receptor by physical exercise in the adipose tissue of obese humans . the study was conducted on adult male and female nondiabetic subjects consisting of 17 lean ( bmi = 2024.9 kg / m ) and 40 obese ( bmi = 3040 kg / m ) . none of the participants that were enrolled in this study reported any cerebrovascular complication , kidney disease , asthma , and food and drug allergies and chronic viral infection . according to the medical questionnaire , furthermore , no specific diet or nutritional change was prescribed to our subject during the 3 months of physical exercise protocol as our aim was mainly to investigate the sole effect of physical exercise on those subjects . venous peripheral blood and subcutaneous adipose tissue biopsies were obtained prior to the start of the 3-month protocol and within 2 to 3 days after its completion . subcutaneous superficial adipose tissue biopsies ( ~0.5 g ) were obtained from the periumbilical area by surgical biopsy after a local anesthesia . glucose and lipid profiles were measured on the siemens dimension rxl chemistry analyzer ( diamond diagnostics , holliston , ma ) . plasma levels of inflammatory and metabolic markers were measured using the bio - plex 27-plex human cytokine and 12 bio - plex human diabetes kits , respectively ( biorad , hercules , ca ) . sections were blocked with 5% fat - free milk for 60 min at rt followed by 1% bsa for another 60 min and then incubated at 4c for overnight with primary antibodies against rantes ( abcam , cambridge , ma ) , tnf - a ( abcam , cambridge , ma ) , il-6 ( novus biologicals , littleton , co ) , and phospho - jnk ( staining with horseradish conjugated secondary antibody ( dako , denmark ) was done for 60 minutes at rt . for rantes staining , cells were cultured overnight in complete media at 37c and then washed with pbs containing 2% fbs followed by facs buffer ( bd biosciences , san jose , ca ) and then costained with anti - human apc - conjugated rantes ( r&d systems ) , fitc - conjugated cd3 ( mca463f , abd serotec , raleigh , nc ) , and pe - conjugated cd14 ( fab3832p , r&d systems , minneapolis , mn ) antibodies for 45 minutes on ice . unless otherwise stated , all descriptive statistics for the variables in the study were reported as means standard error . the physical characteristics of the two groups , namely , lean ( n = 17 ) and obese ( n = 40 ) , enrolled in this study are displayed in table 1 . as expected , body mass index , percent body fat , and waist and hip circumferences were significantly higher in obese group compared to lean group ( p < 0.0001 ) . obese subjects had also higher systolic blood pressure and higher levels of triglycerides ( p < 0.05 ) ; however , there was no significant difference in the cardiopulmonary parameters between the two groups . obese subjects had higher levels of glucose and hba1c ( p < 0.05 ) in spite of not being diabetic . compared to lean group , the metabolic and inflammatory profiles are dysregulated in obese group as indicated by increased levels of leptin ( p = 0.0006 ) , resistin ( p = 0.04 ) , ip-10 ( p = 0.0008 ) , and rantes chemokines ( p = 0.023 ) . in agreement with our recent investigation , obese subjects had higher levels of rantes in the circulation that correlated negatively with il-1ra ( r = 0.42 ; p = 0.001 ) and positively with ip-10 ( r = 0.31 ; p = 0.02 ) and tbars levels ( r = 0.29 ; p = 0.03 ) ( figure 1(a ) ) . to investigate the endogenous expression of rantes between the two groups at the protein level , we determined its expression in pbmcs and adipose tissue by western blot , flow cytometry , and immunohistochemistry ( ihc ) . as shown in figure 2(a ) , there was no difference in the expression of rantes protein in pbmcs between lean and obese subjects ( n = 3 for each group ) . consistent with this finding , flow cytometry indicated that rantes levels were comparable between lean and obese subjects ( n = 3 for each group ) in both monocytes and lymphocytes ( figure 2(b ) ) . by contrast to pbmcs , the expression of rantes protein in the adipose tissue as monitored by ihc studies was significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.01 ; figure 2(c ) ) . the differential expression of rantes between pbmcs and adipose tissue in lean and obese subjects was also confirmed at the mrna level by using quantitative real - time pcr ( qrt - pcr ) and the results are displayed in figures 2(d ) and 2(e ) . accordingly , there was no change in rantes mrna levels in pbmcs from lean and obese subjects ( n = 10 for both lean and obese , figure 2(d ) ) , whereas the levels of rantes mrna in the adipose tissue were significantly higher in obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.02 , figure 2(e ) ) . under the same conditions , the endogenous expression of the two key inflammatory markers tnf- and il-6 in the adipose tissue were significantly increased in obese subjects both at the protein level ( p < 0.05 , figure 2(f ) ) and mrna level ( p < 0.05 , figure 2(g ) ) . we next examined the expression profile of ccr5 receptor between lean and obese subjects using pbmcs and adipose tissue . indeed , there was a clear upregulation of ccr5 mrna in the adipose tissue of obese ( n = 11 ) compared to lean ( n = 7 ) subjects ( p = 0.03 , figure 3(b ) ) . by contrast , ccr5 mrna was significantly downregulated in pbmcs of obese subjects compared to lean subjects ( n = 10 for both lean and obese ) ( p = 0.04 , figure 3(a ) ) . on the other hand , flow cytometry analysis carried out on pbmcs from lean and obese ( n = 5 for each group ) revealed a significant and unique increase of ccr5 in cd14 + monocyte subset from obese subjects ( p = 0.02 , figure 3(c ) ) . the specific mean fluorescence intensity for ccr5 in obese individuals revealed a higher signal for cd14 + monocytes compared to cd14++ monocytes ( p = 0.001 , figure 3(c ) ) . we previously reported the effectiveness of our physical exercise protocol on improving the physical , clinical , and metabolic parameters on obese subjects . accordingly , there was a significant reduction of pbf and sbp and increase in vo2max along with a decrease in tbars levels and a reduction of inflammatory markers tnf- and il-6 in the circulation . however , physical exercise did not reduce the levels of rantes in the circulation . to investigate whether physical exercise has an impact on the endogenous expression of rantes and ccr5 , qrt - pcr and ihc were carried out on adipose tissue from obese subjects before and after the exercise program . as shown in figure 4(a ) , ihc carried out on adipose tissue from obese subjects before ( n = 11 ) and after ( n = 7 ) the exercise program indicated a significant decrease in the expression of rantes by physical exercise ( p = 0.003 ) . qrt - pcr performed on obese before and after the exercise program ( n = 10 for each group ) confirmed the reduction of rantes mrna expression by physical exercise ( p = 0.01 , figure 4(b ) ) . likewise , ccr5 mrna was significantly reduced by physical exercise in the adipose tissue ( p = 0.02 , figure 4(b ) ) . using the same samples , we observed a decrease in the endogenous expression of tnf- and il-6 both at the mrna level ( p < 0.05 , figure 4(b ) ) and protein level ( p < 0.05 , figure 4(c ) ) . since obesity is known to induce the phosphorylation of jnk protein , we measured the levels of phosphorylated jnk in adipose tissue by ihc before ( n = 11 ) and after ( n = 7 ) the physical exercise program . data shown on figure 4(c ) indicated that physical exercise decreases significantly the levels of phosphorylated jnk ( p = 0.002 ) in obese in a manner that was concomitant with a decrease of ccr5 and its ligand rantes . no effect was observed for total jnk before and after exercise in obese subjects ( data not shown ) . taken together , these data suggest that exercise is interfering with obesity - mediated expression of rantes and ccr5 . chronic low - grade inflammation state is a key feature of obesity and it is caused mainly by infiltration of macrophages and other inflammatory cells into the adipose tissue . the present study explored the effect of physical exercise on the expression of rantes and its main receptor ccr5 in obese humans . our focus on ccr5 receptor was based on a recent study in which ccr5 knockout mice were protected from obesity - induced adipose tissue inflammation and insulin resistance . in addition , it has been previously shown that obesity triggers a modest change in the expression of ccr3 and no change in that of ccr1 . the main findings of our current investigation are as follows : ( 1 ) the expression of both rantes and ccr5 was significantly higher in the subcutaneous adipose tissue of obese subjects , ( 2 ) by contrast to the adipose tissue , ccr5 was downregulated in pbmcs of obese subjects compared to lean subjects but there was no significant difference in the expression of rantes between the two groups , and ( 3 ) physical exercise corrected the dysregulated expression of both rantes and ccr5 in the subcutaneous adipose tissue but has a marginal effect on circulating levels of rantes . while our findings showing increased expression of rantes and ccr5 in the adipose tissue are consistent with previous clinical and animal studies [ 17 , 40 ] , the downregulation of their expression by physical exercise is novel . based on the crucial role of rantes / ccr5 signaling pathway in the pathology of obesity and its associated complications , our findings add further evidence that physical exercise might be one of nonpharmacologic approaches that can attenuate rantes / ccr5 signaling pathway and thereby mitigating the inflammatory and metabolic stress triggered by obesity . rantes is a powerful proinflammatory chemokine that controls the trafficking of immune inflammatory cells such as monocytes , macrophages , th1 t cells , and dendritic cells from the circulation into various tissues including adipose tissue [ 3 , 9 ] . previous studies carried out on obese humans and rodents reported high levels of rantes as well as increased frequency of macrophages in the adipose tissue that were concomitant with increased inflammatory response , insulin resistance , and type 2 diabetes [ 5 , 17 , 4144 ] . our findings showing high levels of rantes in the adipose tissue of obese subjects at both mrna and protein levels corroborate these pioneer studies that associated rantes with obesity . unlike the adipose tissue , the levels of rantes were however comparable between lean and obese subjects as monitored by western blotting , flow cytometry , and qrt - pcr . the mechanism underlying this differential expression of rantes between pbmcs and adipose tissue remains to be investigated but there is evidence for depot - specific differences that dictate the levels of rantes released by various adipose tissues in humans . one of the key characteristics of macrophages that infiltrate the adipose tissue is the heterogeneity of their phenotype and depending on their polarization status ; they can be proinflammatory macrophages ( m1-phenotype ) secreting various inflammatory mediators such as tnf- , il-6 , and inos [ 45 , 46 ] or anti - inflammatory macrophages ( m2-phenotype ) that secrete anti - inflammatory cytokines such as il-10 [ 41 , 47 ] . however , in our study the high levels of circulating rantes correlated negatively with the anti - inflammatory il-1ra and positively with the levels of proinflammatory ip-10 chemokine and tbars supporting its role in mediating inflammation . in the current study , we have also investigated the expression pattern of ccr5 in obese humans and similar to our findings with rantes , both mrna and protein levels of ccr5 were increased in the adipose tissue of obese compared to lean group . the direct role of ccr5 in the regulation of obesity - induced adipose tissue inflammation and development of insulin resistance was recently demonstrated using ccr5 knockout mice . a dominant shift occurred from proinflammatory m1 to anti - inflammatory m2 macrophage phenotypes , which contributed to an improved impaired glucose tolerance and insulin sensitivity in response to diet - induced obesity in these animals . unexpectedly , our data demonstrated that , unlike the adipose tissue , the expression of ccr5 mrna was significantly downregulated in pbmcs from obese individuals . to the best of our knowledge , our data is the first to report the downregulation of ccr5 mrna in the pbmcs of obese individuals . the altered expression of ccr5 in pbmcs has been described in rheumatoid arthritis and in women with systemic lupus erythematosus . it is possible that other receptors for rantes such as ccr1 and ccr3 may be compensating for the reduced expression of ccr5 in obese subjects . interestingly , ccr5 expression on cd14 + monocyte subset appears to be upregulation in comparison with cd14++ subset in obese individuals . of particular interest , our study emphasized more on the role of physical exercise in regulating the expression of rantes and its receptor ccr5 . our data showed for the first time that physical exercise significantly reduced the expression of both rantes and ccr5 receptor in the adipose tissue in a manner that was concomitant with decreased pbf and improved cardiopulmonary performance as monitored by decreased sbp and increased vo2max. the reduced expression of rantes and ccr5 by physical exercise was also consistent with attenuated inflammatory response as indicated by decreased tnf- and il-6 in the circulation and reduced levels of tbars . our finding revealed also that exercise - mediated decrease in the expression of rantes and ccr5 in the adipose tissue was not translated to a decrease in the circulating levels of rantes . the lack of parallel changes between systemic and endogenous levels of rantes after exercise raises the question regarding the role of rantes in the circulation . earlier study indicated that systemic levels of rantes are 100-fold higher than those being released from any of the adipose tissue depots . therefore , it is sensible to suggest that while adipose tissue is able to produce this chemokine , its contribution to systemic levels may be less significant . in agreement with these findings , our data indicate that there was a significant increase in the levels of phosphorylated jnk along with high levels of rantes and ccr5 in obese subjects that were all reduced by physical exercise ( figure 4(c ) ) . in conclusion , the dysregulation of rantes and its negative correlation with the metabolic and stress responses are supporting the pathological role of rantes in obesity . our results also provide strong evidence that physical exercise downregulates the expression of both rantes and ccr5 in the adipose tissue . these findings give strong support to the concept that physical exercise can be used as a nonpharmacologic approach to mitigate the complications associated with obesity , particularly inflammation , in part by attenuating rantes / ccr5 signaling in the adipose tissue .

perspective taking is a capability that we all learn as infants , at least in principle ( e.g. , baillargeon et al . , 2010 ) , and yet even as adults , we still have our difficulties accomplishing it ( galinsky et al . , 2006 ; wu and keysar , 2007 ) : in our attempts to find orientation in the outside world , in social relations and interactions , or in our inner life , we often presume that we are adopting the only possible perspective , and hence find it difficult to view things from a different angle . in this regard , cognitive scientists barely differ from anybody else . for good reasons ( in fact , better reasons than most other people ) they assume to understand cognitive processes , and for similar good reasons , they believe to have achieved this insight by well - suited methods . when visiting a far - away country , most of us are hit by realizing that the people we meet show opinions , values , and behaviors that do not at all match our expectations . suddenly , we have no difficulties imagining and might even find it inevitable that the others will perceive and most likely reason about the world differently from us . but how great are these differences really , and on which level do they arise ? when we are in contact with cultures , do we overestimate that , which we tend to underestimate in daily life ? or does what ( and maybe even how ) we perceive , think , and feel actually depend on the culture in which we grew up ? in this regard , a substantial proportion of cognitive scientists seem to differ considerably from anybody else by assuming that cognition is largely independent from culture . whereas anthropologists and ethnolinguists explore the specific cultural context and content of cognition , cognitive psychologists and psycholinguists prefer to work on what they assume to be universal aspects of cognition . more importantly , these two groups tend to ignore each other 's perspectives of the subject they both work on . research on human cognition has all too often ignored cultural diversity , and anthropology has all too often remained mute to discussions in cognitive science . as a consequence , research in cognitive science may fall prey to certain biases even in their approach to research questions and design ( e.g. , white , 2006 ) . in this paper , we aim at elaborating the advantages of taking different perspectives , and we do so in at least two different senses : taking a cross - cultural perspective on cognition , and taking a cross - disciplinary perspective in cognitive research . although we hope to contribute to a more general discussion in the cognitive sciences , we will basically focus on the value of anthropology for questions of cognitive psychology , as these are the two disciplines we are most familiar with . we argue that combining these perspectives will give rise to synergistic effects and will allow for a more comprehensive understanding of human cognition the opinion that cognitions are constituted by vs. independent of culture mark two endpoints between which not only lay - psychology explanations , but also scientific perspectives on cognition , seem to have oscillated over the last decades . while during colonialism , europeans increasingly came into contact with people from other cultures , one leading school in the social sciences assumed that lvy - bruhl ( 1910 , 1922 ) , for instance , defined this primitive mentality as a mystic - prelogic mode of thinking that largely consists of religious and supernatural elements . it was therefore impossible to describe this mode of thinking with logic - based rules or to make it compatible with the cartesian mode of thinking , which he believed to be characteristic of europeans . in his later years however , a modified version , according to which two modes of thinking are still distinguished sometimes termed as rule - based vs. associative , reflective vs. intuitive , or abstract vs. content - specific yet assumed to co - exist in all cultures , is still discussed in research on thinking and reasoning , albeit controversially ( e.g. , sloman , 1996 ; sperber , 1997 ; beller and spada , 2003 ; evans , 2003 ; for a related discussion , see also nisbett et al . the conception of the psychic unity of humankind , in contrast , holds that , irrespective of their cultural background , all humans have at their disposal the same cognitive toolkit . dating back at least to the enlightenment , this position experienced a huge uplift in the 1950s and 1960s , when the cognitive revolution laid the foundation for a new field ( miller , 2003 ) . several disciplines among them psychology and cultural anthropology joined forces to explore the foundations of the human mind and its achievements ( for overviews , see gardner , 1985 ; boden , 2006 ) . not only did they share a common goal , but they did so from a perspective that focused on mental states , which are generated and altered by way of information input , processing , storage , and transmission . according to a popular metaphor , information processing in a human mind is analogous to information processing that takes place in a computer , consists of algorithms and data and varies with regard to input and output , but not with regard to processing itself . we assume that it was this computer metaphor that gave rise to the following three basic although often implicit assumptions ( see also block , 1995 ; norenzayan and heine , 2005 ) : cognition can be split into processing and content ( i.e. , the information being processed ) . cognition takes place in people 's heads , which implies that processing is largely independent of context . only content varies across cultures , whereas the processor itself , and hence the processing , are universal , that is , independent of people 's cultural background . for research on human cognition , these assumptions entail far - reaching consequences . first , the assumed separability of process and content seemed to justify a division of labor according to which anthropology would account for the concrete cultural content of mental representations , and psychology would account for their cognitive processing ( d'andrade , 1981 , p. 182 ) . in recent years , though , this distinction turned out to be neither reasonable nor tenable ( medin and atran , 2004 ; bang et al . , 2007 ) , and this insight , however , should not have come as a surprise , if the computer metaphor had been taken more seriously . computer science teaches us that the format of the data determines which algorithms can be performed smoothly and which can not ( e.g. , mehlhorn and sanders , 2008 ) , and so do specific formats of representing content affect the cognitive processes operating on them . a thorough examination of psychological processes must therefore always take content and its representation into account . second , the focus in early cognitive science on mental phenomena is comprehensible if one considers that it originated as counter - movement to behaviorism ( cf . whereas behaviorism categorically rejected the usability of mental constructs due to their inaccessibility for direct observation , the new discipline realized that behavior could only be accounted for if one understands what people are attending to and what information they are processing . for cognitive scientists and anthropologists alike this set the path : we must get inside our subjects heads ( frake , 1964 , p. 133 ) . the downside of this concentration on mental phenomena was a disregard for other factors with potential influence on cognitive processing . a range of cognitive activities are not exclusively performed internally , but rather in interaction with one 's environment and with artifacts , which often serve precisely the purpose of facilitating cognitive processing ( e.g. , norman , 1993 ) . instructive examples of this interaction are the usage of digits for calculations ( nickerson , 1988 ; zhang and norman , 1995 ) or of maps and technical instruments in navigation ( hutchins , 1995 ) . for the incorporation of cognitive artifacts into cognitive processing , hutchins ( 2006 ) coined the term distributed cognition . even beyond concrete tools , the context in which we process information may affect how we process it , particularly with regard to whether ( and which ) other people are present . one simple example of this effect is social facilitation ( i.e. , increasing individual performance on simple tasks when others are present ) , which has actually been known for at least a century ( bond and titus , 1983 ; guerin , 1993 ) . to ensure their external validity , the common practice of examining cognitive processes under controlled conditions in labs is therefore rather critical . yet , this preference for controlled experiments on cognition has been reinforced by the third assumption , according to which cognitive processes are universal , that is , independent of people 's cultural background . support for this assumption even seemed to come from cognitive anthropology , which , in its early days , had found evidence for universal principles of organizing cognitive domains ( e.g. , berlin et al . hence , up to the end of the last century , the potential of culture to affect cognitive processes has been widely ignored ( cf . , 2007 ; henrich et al . , 2010b ) , and this seemed to justify the widespread habit of exploring cognitive processes with selective samples , namely graduate students of one 's own university . after all , if cognitive processes are universal , each student should be as good a subject for their exploration as anybody else . harley , 2008 ) , which were largely based on the assumption that language has no effect on cognition and hence were all too often content with the english language and its speakers . and again , if one adopts the chomskyan idea of a universal grammar , english should be as good a testing bed for its exploration as any other language . but how can we ascertain that these processes are indeed universal if we do not test for universality ? criticism in this regard is far from new , but has gained increasing attention in recent years . as arnett ( 2008 ) complains , the studies reported in six leading apa journals from 2003 to 2007 were almost exclusively conducted not only by researchers from english speaking and european countries , but also with samples from these same countries . in this respect , researchers neglected roughly 95% of the world population , about which from a psychological and cognitive science perspective we know practically nothing ( see also henry , 2008 ) . the heavily researched 5% , on the other hand , belong to the weirdest people in the world western , educated , industrialized , rich , and democratic societies , and , in global comparison , they must be considered a psychological outlier . with numerous examples , ranging from visual perception through spatial cognition , ethnobiological concepts , and economic decision - making , to self - concept and various social phenomena , henrich et al . ( 2010b ) demonstrate that many of the effects previously assumed to be robust and universal occur only weakly or not at all when sampling beyond weird people ( similar observations hold for linguistic universals ; cf . , evans and levinson , 2009 ) . this is not to say that such universals do not exist . given the current research practice , however , only few of the assumed universals can be regarded as sufficiently established . exploring cognitive diversity thus remains a fundamental goal for all cognitive sciences , and has become one of the hot topics in the field ( cohen , 2001 ; norenzayan and heine , 2005 ; lloyd , 2007 ; gentner , 2010 ) . meanwhile , conclusive evidence for deep cultural impacts not only on cognition , but on the very architecture of the brain , is even provided by neuroscience ( ambady and bharucha , 2009 ; kitayama and uskul , 2011 ) , which of all disciplines , is the one that might have been associated the most with universal claims from the outset , as it is perceived by many non - experts as not only reducing cognition to algorithms but to the very hardware of the processor : the neurons ( cf . , neuroscience increasingly provides evidence for the assumption that the brain is altered by learning and experience , which itself is organized by culture . however , while cross - cultural research is indispensable for scrutinizing cognitive diversity , it is a delicate thing to do . the first home - field disadvantage is a tendency to leave one of the cultures under comparison unmarked , thereby taking it as the standard from which others deviate ; as one consequence of this , peculiarities of the unmarked culture fall prey to cultural blind spots . the second home - field disadvantage is a tendency to consider other cultures ( and occasionally even whole hosts of cultures ) as more homogeneous than one 's own culture , and definitely as more homogeneous than they actually are . the indians , or chinese , japanese , and south koreans are referred to as the east asians . the third home - field disadvantage is an excessive trust in the equivalence of tasks across cultures . this raises at least two concerns : a concern with how these tasks are understood in different cultural contexts ( e.g. , astuti and bloch , 2010 ) and how the response scales have to be interpreted ( e.g. , heine et al . , 2002 ) , and a concern with what the obtained data would be able to reveal . as most tasks are specifically tailored to bring about a specific effect in the culture for which they were developed , regression toward the mean demands that in other conditions and this implies in other cultures this effect will be less likely to show up with the same task ( medin et al . , 2010 ) . the home - field disadvantages result from presumptions that researchers hold about their field and the rules of the game . such presumptions are nurtured by several handicaps , which increase psychological distance between the researchers and those they are researching ( trope and liberman , 2003 ) , and thereby hamper perspective taking ( galinsky et al . however , this requires constant efforts in marking the unmarked cultural group , collaborating with the group researched , conducting research on the terms of the respective culture , and taking multiple perspectives ( medin et al . , 2010 ) . it is exactly here , in the efforts to combat the home - field disadvantages , where cross - cultural research in cognitive science could profit most from taking an anthropological perspective . for almost a century now , anthropology has been cultivating the ideal of perspective taking . the conditio sine qua non of being a good anthropologist instead , the ultimate goal , as malinowski ( 1922 ) once put it , is to grasp the native 's point of view , his relation to life , to realize his vision of his world to put it more technically : anthropologists prefer an emic analysis , based on the categories from inside the system , over the etic analysis ( pike , 1967 ; and see headland et al . anthropology , however , once a pioneer discipline and a founding member of the cognitive sciences ( gardner , 1985 ; d'andrade , 1995 ) , has increasingly withdrawn from the mutual endeavor during the last decades and is meanwhile considered the missing discipline in cognitive science ( boden , 2006 ) . several reasons have been identified for this alienation , and diverging methodological preferences are among the most important ( bender et al . nevertheless , in addition to the distinct perspective just mentioned , there are at least three more reasons why anthropology would still and indeed , more so than ever be an invaluable partner for cognitive sciences , and why efforts to re - integrate the former into the latter need to be intensified . first , as we have seen , the division of labor between the disciplines was never really conducive in the first place ; separating cognitive processes and cultural content does not do justice to the topic . if we assume that cognitive processes are affected by content , information on cultural variation in content is important for a more comprehensive understanding of processing ( for an example , see atran and medin , 2008 ) . for a broad range of cultures , in addition , anthropology brings to the table its expertise on culture in general as a heuristic concept . this entails not only a heightened awareness of home - field disadvantages and continuous efforts for overcoming them as outlined above ( cf . , , 1990 ; agar , 1996 ; ross , 2004 ; bernard , 2006 ) , but also a broader perspective on what culture is in the first place . the increasing interest in cognitive science for culture 's constitutive role in human cognition has spurred an increasing number of cross - cultural and cross - linguistic studies . all too often , however , these studies tend to reduce culture to simple , dichotomous variables , such as individualism vs. collectivism ( for overview and critical assessments , cf . , still largely neglected are interactions between cognition and culture in the sense of a cognitive ecology ( cole , 1996 ; shweder , 2007 ; hutchins , 2010 ) . and finally , the diverging methodological approaches of the disciplines could provide valuable complements provided that they manage to overcome the reservations they hold against each other 's preferences . despite the fact that cognitive science and anthropology share a common goal , they nevertheless take diverging perspectives , with regard to both focus and methods ( for an extensive treatment , see boster , 2011 ; mishra and dasen , 2007 ) . most cognitive sciences , and particularly so psychology , are primarily interested in how cognitive processes operate and how they are related to each other . the preferred method is the experiment , as it allows potentially interfering factors to be eliminated to a large extent , thus enabling the pure process to be scrutinized , which is essential in order to uncover causal mechanisms of cognitive phenomena . cognitive anthropology , on the other hand , is primarily interested in cultural meaning in a more holistic manner , thus focusing on how cultural knowledge of groups of persons is organized and described , transmitted and modified . as most anthropologists conceive of culture not only as the origin ( tomasello , 1999 ) , but also as an integral part of cognition ( d'andrade , 1981 ; hutchins , 1995 ; shore , 1996 ) , they embed systematic data collection in field studies with participant observation ( agar , 1996 ) . at first glance as boster ( 2011 ) elegantly puts it , cognitive psychologists examine trees and cognitive anthropologists contemplate forests . a more thorough examination , however , reveals that in taking different angles , they are indeed complementary . used together , they compensate for each other 's weaknesses with their distinct strengths , thus paving the way for synergies beyond what could be achieved by each discipline alone ( beller and bender , 2010 ) . in order to obtain a comprehensive understanding of human cognition ( including its cultural constitution ) , combining these approaches will be inevitable . one critical prerequisite for making use of this complementarity , however , is knowledge of and respect for the methodological principles of the respective other discipline . collaboration across disciplines is encumbered with a host of difficulties , including different backgrounds in terms of knowledge , distinct theoretical conceptualizations and vocabulary , diverging research paradigms and methodological approaches , and different practical habits . for instance , psychologists are used to conducting research in teams and to publishing their results jointly , whereas anthropologists typically work in their field all by themselves , which is then reflected in their publications . establishing respective cooperation is hence no simple endeavor . and only if they mutually acknowledge and respect each other 's research traditions will they be prepared to invest the required effort . inducing researchers from different traditions to collaborate will require more than simply making an appeal to do so , and anybody with experience in interdisciplinary work will be well aware of the pitfalls . however , these difficulties do not justify refraining from collaboration . according to our own experience , there is little that is so supportive of a constructive exchange across disciplines as the willingness to jointly address common research questions and goals . in the case of cognitive research one promising starting point might be to place onto the shared research agenda the very topics upon which the various disciplines appear to disagree : from a serious discussion of the conceptualization of culture and its implications , through methodological issues , to a critical examination of universals . crucially , however , this endeavor should not be undertaken on an abstract level , but needs to be grounded in a concrete cognitive domain and coupled to precise questions ( even if these questions may have to be rephrased in the course of collaboration ) . it will also require identifying the level at which each discipline will discuss the phenomenon under scrutiny , and again , the different disciplines can complement each other in this respect , with cognitive psychology contributing a perspective on individual processing and tendencies of populations , and anthropologists contributing a perspective on the content and context of cognition in interactions . a few cases of good practice provide evidence that cross - disciplinary collaboration can not only work well , but may be prosperous in giving rise to new and innovative research . among the most convincing examples are the work on ethnobiological and ecological cognition and behavior conducted by the team of douglas medin and scott atran at northwestern university ( e.g. , medin and atran , 2004 ; medin et al . , 2006 ; atran and medin , 2008 ) and the work on interactions of language , culture and cognition in stephen levinson 's research group at the max planck institute for psycholinguistics in nijmegen ( e.g. , majid et al . , 2004 , 2007 ; other instances include research on numerical cognition ( wassmann and dasen , 1994 ; pica et al . beller and bender , 2008 ) , on causal and temporal reasoning ( beller et al . , 2009 ; bender et al . , 2010a ) , on the cognitive foundations of religion and concepts of an afterlife ( bloch et al . , 2004 ; whitehouse and mccauley , 2005 ; astuti and harris , 2008 ) , on theory of mind ( wassmann et al . , 2010 ) , and on decision - making and rules of fairness in social dilemma situations ( henrich et al . , 2005 , 2010a ) . institutional attempts to support cross - disciplinary collaboration include the international cognition and culture institute ( icci ) , which provides a web - based platform for exchange , the culture and the mind project at the university of sheffield , which investigates the cognitive and evolutionary foundations of culture and its impact on the mind , and the center for interdisciplinary research ( zif ) of bielefeld university , which provides funding for interdisciplinary residential groups . one of these groups ( taking residence in 2011/2012 ) is specifically designed as a platform for re - integrating anthropology into the cognitive sciences and will focus on the cultural constitution of causal cognition . these instances for communication and even cooperation between anthropology and the wider cognitive sciences are still the exception rather than the rule , and a lot of work remains to be done . nevertheless , the potential for synergistic effects is remarkable , and the need for enhanced cross - disciplinary collaboration is increasingly acknowledged across disciplines . if cognitive science strives for a comprehensive understanding of human cognition , it needs to consider and integrate the perspective offered by anthropology on the cultural constitution of cognition . the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

to what extent is cognition affected by culture ? and how might cognitive science profit from an intensified collaboration with anthropology in exploring this issue ? in order to answer these questions , we will first give a brief description of different perspectives on cognition , one that prevails in most cognitive sciences particularly in cognitive psychology and one in anthropology . three basic assumptions of cognitive science regarding the separability of content and process , the context - independence of processing , and the culture - independence of processing will then be discussed . we argue that these assumptions need to be questioned and scrutinized cross - culturally . a thorough examination of these issues would profit considerably from collaboration with anthropologists , not only by enabling deeper insight into the cultures under scrutiny , but also by synergistic effects that would allow for a more comprehensive understanding of human cognition .

Introduction Perspectives on Cognition Cultural Variance and Challenges for Research The Gift of Anthropology: Taking Multiple Perspectives The Case for Collaboration and Cases of Good Practice Conflict of Interest Statement

perspective taking is a capability that we all learn as infants , at least in principle ( e.g. , 2010 ) , and yet even as adults , we still have our difficulties accomplishing it ( galinsky et al . , 2006 ; wu and keysar , 2007 ) : in our attempts to find orientation in the outside world , in social relations and interactions , or in our inner life , we often presume that we are adopting the only possible perspective , and hence find it difficult to view things from a different angle . in this regard , cognitive scientists barely differ from anybody else . for good reasons ( in fact , better reasons than most other people ) they assume to understand cognitive processes , and for similar good reasons , they believe to have achieved this insight by well - suited methods . when visiting a far - away country , most of us are hit by realizing that the people we meet show opinions , values , and behaviors that do not at all match our expectations . suddenly , we have no difficulties imagining and might even find it inevitable that the others will perceive and most likely reason about the world differently from us . but how great are these differences really , and on which level do they arise ? when we are in contact with cultures , do we overestimate that , which we tend to underestimate in daily life ? or does what ( and maybe even how ) we perceive , think , and feel actually depend on the culture in which we grew up ? in this regard , a substantial proportion of cognitive scientists seem to differ considerably from anybody else by assuming that cognition is largely independent from culture . whereas anthropologists and ethnolinguists explore the specific cultural context and content of cognition , cognitive psychologists and psycholinguists prefer to work on what they assume to be universal aspects of cognition . more importantly , these two groups tend to ignore each other 's perspectives of the subject they both work on . research on human cognition has all too often ignored cultural diversity , and anthropology has all too often remained mute to discussions in cognitive science . as a consequence , research in cognitive science may fall prey to certain biases even in their approach to research questions and design ( e.g. in this paper , we aim at elaborating the advantages of taking different perspectives , and we do so in at least two different senses : taking a cross - cultural perspective on cognition , and taking a cross - disciplinary perspective in cognitive research . although we hope to contribute to a more general discussion in the cognitive sciences , we will basically focus on the value of anthropology for questions of cognitive psychology , as these are the two disciplines we are most familiar with . we argue that combining these perspectives will give rise to synergistic effects and will allow for a more comprehensive understanding of human cognition the opinion that cognitions are constituted by vs. independent of culture mark two endpoints between which not only lay - psychology explanations , but also scientific perspectives on cognition , seem to have oscillated over the last decades . while during colonialism , europeans increasingly came into contact with people from other cultures , one leading school in the social sciences assumed that lvy - bruhl ( 1910 , 1922 ) , for instance , defined this primitive mentality as a mystic - prelogic mode of thinking that largely consists of religious and supernatural elements . it was therefore impossible to describe this mode of thinking with logic - based rules or to make it compatible with the cartesian mode of thinking , which he believed to be characteristic of europeans . in his later years however , a modified version , according to which two modes of thinking are still distinguished sometimes termed as rule - based vs. associative , reflective vs. intuitive , or abstract vs. content - specific yet assumed to co - exist in all cultures , is still discussed in research on thinking and reasoning , albeit controversially ( e.g. , sloman , 1996 ; sperber , 1997 ; beller and spada , 2003 ; evans , 2003 ; for a related discussion , see also nisbett et al . the conception of the psychic unity of humankind , in contrast , holds that , irrespective of their cultural background , all humans have at their disposal the same cognitive toolkit . dating back at least to the enlightenment , this position experienced a huge uplift in the 1950s and 1960s , when the cognitive revolution laid the foundation for a new field ( miller , 2003 ) . several disciplines among them psychology and cultural anthropology joined forces to explore the foundations of the human mind and its achievements ( for overviews , see gardner , 1985 ; boden , 2006 ) . not only did they share a common goal , but they did so from a perspective that focused on mental states , which are generated and altered by way of information input , processing , storage , and transmission . according to a popular metaphor , information processing in a human mind is analogous to information processing that takes place in a computer , consists of algorithms and data and varies with regard to input and output , but not with regard to processing itself . we assume that it was this computer metaphor that gave rise to the following three basic although often implicit assumptions ( see also block , 1995 ; norenzayan and heine , 2005 ) : cognition can be split into processing and content ( i.e. , the information being processed ) . only content varies across cultures , whereas the processor itself , and hence the processing , are universal , that is , independent of people 's cultural background . for research on human cognition , these assumptions entail far - reaching consequences . first , the assumed separability of process and content seemed to justify a division of labor according to which anthropology would account for the concrete cultural content of mental representations , and psychology would account for their cognitive processing ( d'andrade , 1981 , p. 182 ) . in recent years , though , this distinction turned out to be neither reasonable nor tenable ( medin and atran , 2004 ; bang et al . , 2007 ) , and this insight , however , should not have come as a surprise , if the computer metaphor had been taken more seriously . computer science teaches us that the format of the data determines which algorithms can be performed smoothly and which can not ( e.g. , mehlhorn and sanders , 2008 ) , and so do specific formats of representing content affect the cognitive processes operating on them . a thorough examination of psychological processes must therefore always take content and its representation into account . second , the focus in early cognitive science on mental phenomena is comprehensible if one considers that it originated as counter - movement to behaviorism ( cf . whereas behaviorism categorically rejected the usability of mental constructs due to their inaccessibility for direct observation , the new discipline realized that behavior could only be accounted for if one understands what people are attending to and what information they are processing . a range of cognitive activities are not exclusively performed internally , but rather in interaction with one 's environment and with artifacts , which often serve precisely the purpose of facilitating cognitive processing ( e.g. , norman , 1993 ) . instructive examples of this interaction are the usage of digits for calculations ( nickerson , 1988 ; zhang and norman , 1995 ) or of maps and technical instruments in navigation ( hutchins , 1995 ) . for the incorporation of cognitive artifacts into cognitive processing , hutchins ( 2006 ) coined the term distributed cognition . even beyond concrete tools , the context in which we process information may affect how we process it , particularly with regard to whether ( and which ) other people are present . one simple example of this effect is social facilitation ( i.e. to ensure their external validity , the common practice of examining cognitive processes under controlled conditions in labs is therefore rather critical . yet , this preference for controlled experiments on cognition has been reinforced by the third assumption , according to which cognitive processes are universal , that is , independent of people 's cultural background . hence , up to the end of the last century , the potential of culture to affect cognitive processes has been widely ignored ( cf . , 2010b ) , and this seemed to justify the widespread habit of exploring cognitive processes with selective samples , namely graduate students of one 's own university . after all , if cognitive processes are universal , each student should be as good a subject for their exploration as anybody else . harley , 2008 ) , which were largely based on the assumption that language has no effect on cognition and hence were all too often content with the english language and its speakers . but how can we ascertain that these processes are indeed universal if we do not test for universality ? criticism in this regard is far from new , but has gained increasing attention in recent years . as arnett ( 2008 ) complains , the studies reported in six leading apa journals from 2003 to 2007 were almost exclusively conducted not only by researchers from english speaking and european countries , but also with samples from these same countries . in this respect , researchers neglected roughly 95% of the world population , about which from a psychological and cognitive science perspective we know practically nothing ( see also henry , 2008 ) . the heavily researched 5% , on the other hand , belong to the weirdest people in the world western , educated , industrialized , rich , and democratic societies , and , in global comparison , they must be considered a psychological outlier . with numerous examples , ranging from visual perception through spatial cognition , ethnobiological concepts , and economic decision - making , to self - concept and various social phenomena , henrich et al . ( 2010b ) demonstrate that many of the effects previously assumed to be robust and universal occur only weakly or not at all when sampling beyond weird people ( similar observations hold for linguistic universals ; cf . , evans and levinson , 2009 ) . this is not to say that such universals do not exist . given the current research practice , however , only few of the assumed universals can be regarded as sufficiently established . exploring cognitive diversity thus remains a fundamental goal for all cognitive sciences , and has become one of the hot topics in the field ( cohen , 2001 ; norenzayan and heine , 2005 ; lloyd , 2007 ; gentner , 2010 ) . meanwhile , conclusive evidence for deep cultural impacts not only on cognition , but on the very architecture of the brain , is even provided by neuroscience ( ambady and bharucha , 2009 ; kitayama and uskul , 2011 ) , which of all disciplines , is the one that might have been associated the most with universal claims from the outset , as it is perceived by many non - experts as not only reducing cognition to algorithms but to the very hardware of the processor : the neurons ( cf . , neuroscience increasingly provides evidence for the assumption that the brain is altered by learning and experience , which itself is organized by culture . however , while cross - cultural research is indispensable for scrutinizing cognitive diversity , it is a delicate thing to do . the first home - field disadvantage is a tendency to leave one of the cultures under comparison unmarked , thereby taking it as the standard from which others deviate ; as one consequence of this , peculiarities of the unmarked culture fall prey to cultural blind spots . the second home - field disadvantage is a tendency to consider other cultures ( and occasionally even whole hosts of cultures ) as more homogeneous than one 's own culture , and definitely as more homogeneous than they actually are . the indians , or chinese , japanese , and south koreans are referred to as the east asians . this raises at least two concerns : a concern with how these tasks are understood in different cultural contexts ( e.g. , astuti and bloch , 2010 ) and how the response scales have to be interpreted ( e.g. , heine et al . , 2002 ) , and a concern with what the obtained data would be able to reveal . as most tasks are specifically tailored to bring about a specific effect in the culture for which they were developed , regression toward the mean demands that in other conditions and this implies in other cultures this effect will be less likely to show up with the same task ( medin et al . the home - field disadvantages result from presumptions that researchers hold about their field and the rules of the game . such presumptions are nurtured by several handicaps , which increase psychological distance between the researchers and those they are researching ( trope and liberman , 2003 ) , and thereby hamper perspective taking ( galinsky et al . however , this requires constant efforts in marking the unmarked cultural group , collaborating with the group researched , conducting research on the terms of the respective culture , and taking multiple perspectives ( medin et al . it is exactly here , in the efforts to combat the home - field disadvantages , where cross - cultural research in cognitive science could profit most from taking an anthropological perspective . the conditio sine qua non of being a good anthropologist instead , the ultimate goal , as malinowski ( 1922 ) once put it , is to grasp the native 's point of view , his relation to life , to realize his vision of his world to put it more technically : anthropologists prefer an emic analysis , based on the categories from inside the system , over the etic analysis ( pike , 1967 ; and see headland et al . anthropology , however , once a pioneer discipline and a founding member of the cognitive sciences ( gardner , 1985 ; d'andrade , 1995 ) , has increasingly withdrawn from the mutual endeavor during the last decades and is meanwhile considered the missing discipline in cognitive science ( boden , 2006 ) . several reasons have been identified for this alienation , and diverging methodological preferences are among the most important ( bender et al . nevertheless , in addition to the distinct perspective just mentioned , there are at least three more reasons why anthropology would still and indeed , more so than ever be an invaluable partner for cognitive sciences , and why efforts to re - integrate the former into the latter need to be intensified . first , as we have seen , the division of labor between the disciplines was never really conducive in the first place ; separating cognitive processes and cultural content does not do justice to the topic . if we assume that cognitive processes are affected by content , information on cultural variation in content is important for a more comprehensive understanding of processing ( for an example , see atran and medin , 2008 ) . for a broad range of cultures , in addition , anthropology brings to the table its expertise on culture in general as a heuristic concept . this entails not only a heightened awareness of home - field disadvantages and continuous efforts for overcoming them as outlined above ( cf . , , 1990 ; agar , 1996 ; ross , 2004 ; bernard , 2006 ) , but also a broader perspective on what culture is in the first place . the increasing interest in cognitive science for culture 's constitutive role in human cognition has spurred an increasing number of cross - cultural and cross - linguistic studies . all too often , however , these studies tend to reduce culture to simple , dichotomous variables , such as individualism vs. collectivism ( for overview and critical assessments , cf . and finally , the diverging methodological approaches of the disciplines could provide valuable complements provided that they manage to overcome the reservations they hold against each other 's preferences . despite the fact that cognitive science and anthropology share a common goal , they nevertheless take diverging perspectives , with regard to both focus and methods ( for an extensive treatment , see boster , 2011 ; mishra and dasen , 2007 ) . most cognitive sciences , and particularly so psychology , are primarily interested in how cognitive processes operate and how they are related to each other . the preferred method is the experiment , as it allows potentially interfering factors to be eliminated to a large extent , thus enabling the pure process to be scrutinized , which is essential in order to uncover causal mechanisms of cognitive phenomena . cognitive anthropology , on the other hand , is primarily interested in cultural meaning in a more holistic manner , thus focusing on how cultural knowledge of groups of persons is organized and described , transmitted and modified . as most anthropologists conceive of culture not only as the origin ( tomasello , 1999 ) , but also as an integral part of cognition ( d'andrade , 1981 ; hutchins , 1995 ; shore , 1996 ) , they embed systematic data collection in field studies with participant observation ( agar , 1996 ) . at first glance as boster ( 2011 ) elegantly puts it , cognitive psychologists examine trees and cognitive anthropologists contemplate forests . a more thorough examination , however , reveals that in taking different angles , they are indeed complementary . used together , they compensate for each other 's weaknesses with their distinct strengths , thus paving the way for synergies beyond what could be achieved by each discipline alone ( beller and bender , 2010 ) . in order to obtain a comprehensive understanding of human cognition ( including its cultural constitution ) , combining these approaches will be inevitable . collaboration across disciplines is encumbered with a host of difficulties , including different backgrounds in terms of knowledge , distinct theoretical conceptualizations and vocabulary , diverging research paradigms and methodological approaches , and different practical habits . for instance , psychologists are used to conducting research in teams and to publishing their results jointly , whereas anthropologists typically work in their field all by themselves , which is then reflected in their publications . inducing researchers from different traditions to collaborate will require more than simply making an appeal to do so , and anybody with experience in interdisciplinary work will be well aware of the pitfalls . however , these difficulties do not justify refraining from collaboration . in the case of cognitive research one promising starting point might be to place onto the shared research agenda the very topics upon which the various disciplines appear to disagree : from a serious discussion of the conceptualization of culture and its implications , through methodological issues , to a critical examination of universals . crucially , however , this endeavor should not be undertaken on an abstract level , but needs to be grounded in a concrete cognitive domain and coupled to precise questions ( even if these questions may have to be rephrased in the course of collaboration ) . it will also require identifying the level at which each discipline will discuss the phenomenon under scrutiny , and again , the different disciplines can complement each other in this respect , with cognitive psychology contributing a perspective on individual processing and tendencies of populations , and anthropologists contributing a perspective on the content and context of cognition in interactions . a few cases of good practice provide evidence that cross - disciplinary collaboration can not only work well , but may be prosperous in giving rise to new and innovative research . , medin and atran , 2004 ; medin et al . , 2006 ; atran and medin , 2008 ) and the work on interactions of language , culture and cognition in stephen levinson 's research group at the max planck institute for psycholinguistics in nijmegen ( e.g. beller and bender , 2008 ) , on causal and temporal reasoning ( beller et al . , 2010a ) , on the cognitive foundations of religion and concepts of an afterlife ( bloch et al . , 2010 ) , and on decision - making and rules of fairness in social dilemma situations ( henrich et al . institutional attempts to support cross - disciplinary collaboration include the international cognition and culture institute ( icci ) , which provides a web - based platform for exchange , the culture and the mind project at the university of sheffield , which investigates the cognitive and evolutionary foundations of culture and its impact on the mind , and the center for interdisciplinary research ( zif ) of bielefeld university , which provides funding for interdisciplinary residential groups . one of these groups ( taking residence in 2011/2012 ) is specifically designed as a platform for re - integrating anthropology into the cognitive sciences and will focus on the cultural constitution of causal cognition . these instances for communication and even cooperation between anthropology and the wider cognitive sciences are still the exception rather than the rule , and a lot of work remains to be done . nevertheless , the potential for synergistic effects is remarkable , and the need for enhanced cross - disciplinary collaboration is increasingly acknowledged across disciplines . if cognitive science strives for a comprehensive understanding of human cognition , it needs to consider and integrate the perspective offered by anthropology on the cultural constitution of cognition .

the old 1960s tularemia focus was in the srebarna lake nature reserve and its surroundings ( fig . 1 and supplementary file 1 ) . the focus covered an area of about 7 km where the marsh lake abuts with the danube river . the new 1990s focus covered an area of 1,0001,500 km situated on 500800 m altitude adjacent to mountains and the capital of sofia ( fig . 1 and supplementary file 1 ) . the genetic distance between strains l2 and 81 ( orange dots ) comprised five snps in 33 years . b. the genetic distance between strain 94 from ussr and strain 19c from srebarna ( yellow dots ) comprised two snps in 5 years . c. there was an accumulation of > 20 snps between the srebarna 19 from the old focus and l1 , mn , and mmm in the new focus ( red dots ) . d. the first bulgarian genome in the b.4 basal clade was represented by strain b1 isolated in breznik in 1999 ( black dot ) . three strains from the 1960s outbreak in srebarna lake reserve in northeast bulgaria and seven strains from the new 1990s focus in west bulgaria were genome sequenced ( table 1 and fig . 1 ) . in addition , two strains recovered in the old focus in the 1960s were cansnp typed . the 1960s strains had been lyophilized after their isolation and kept at room temperature until culturing for genome sequencing in 2011 . the isolation and storage of the seven strains from the 1990s outbreaks have been previously described ( 10 ) . one archive strain isolated 1956 in ussr was sequenced for comparison purposes . for a more detailed epidemiology description , francisella tularensis strains investigated in the study the strains are from the strain collection of the military medical academy ( mma ) , sofia , bulgaria . the strains srebarna 5 , srebarna 19 , and srebarna 56 were recovered by an mma team ( gotev / kupenov ) and originate from the bulgarian type culture collection ( btcc ) in sofia note that the strain l1 is not identical to a strain also named l1 in the reference ( 16 ) b.34/35 should be read as that the strain showed the derived snp state for b.34 and the ancestral state for b.35 no further subtyping with b.77 was performed on these two strains due to insufficient amount of dna ( fig . 3 ) the strains 19c , 81 , and 94 are part of a strain collection maintained at the military institute of hygiene and epidemiology ( mihe ) in warsaw , poland , and originate from a strain collection at the former institute of marine medicine in gdansk , poland ( 21 ) . strains 19c and 94 originated from the medical academy , moscow , ussr and strain 81 originated from centre of epidemiology and microbiology in prague , former czechoslovakia the original documentation reads bulgaria ( 21 ) . no other outbreaks than the srebarna outbreak nor other areas of strains isolation are described in the literature , institutional records , or known by personal communication to our knowledge ( marinov , personal communication ) . the strains srebarna 5 , srebarna 19 , srebarna 56 , l1 , l2 , dm1 , dm2 , b1 , mmm , and mn were cultured as previously described at the military medical academy ( mma ) in sofia , bulgaria ( 10 ) . lysis of the bacteria at mma was achieved by suspending the samples in 1.0 ml 0.9% nacl and heat - treated at 65c for 2 h. the strains 19c , 81 , and 94 were re - cultured at military institute of hygiene and epidemiology ( mihe ) in pulawy , poland , and sent to the swedish defence research agency ( foi ) in ume . prior to dna preparation , strains 19c , 81 , and 94 were re - cultured at foi on gc ii agar with 1% hemoglobin and 1% isovitalex at 37c ( 17 ) dna from l1 , l2 , dm1 , b1 , mn , 19c , 81 , and 94 was prepared at foi using phenol - chloroform extraction , as previously described ( 18 ) , while dna from dm2 , mmm , srebarna 5 , srebarna 19 , and srebarna 56 was prepared using the ez1 robot preparation method , as previously described ( 19 ) . dna concentrations were measured using the nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and the qubit ( life technologies , carlsbad , ca , usa ) . the amount of dna prepared from srebarna 5 and srebarna 56 stock samples was insufficient for dna sequencing and therefore only genotyped with the cansnp markers b.12 and b.23 . genome sequencing of isolates was performed at scilifelab , uppsala , sweden , using the illumina hiseq 2000 platform with truseq dna library prep kits ( strain 91 with truseq nano dna ht sample prep kit ) according to standard protocols to produce 100 bp pair - end data . the resulting sequence data were assembled using abyss versions 1.3.2 and 1.3.3 using standard parameters ( table 2 ) . nucleotide distances and phylogeny were inferred using mega 5.2 ( complete deletion option and the number of differences method , maximum parsimony ) . genome data for 10 bulgarian strains and one archive strain sequenced in this study n50 is the length of the smallest contig in the set that contains the fewest ( largest ) contigs whose combined length represents at least 50% of the assembly ncbi bioproject prjna285143 mma = military medical academy , sofia , bulgaria ; mihe = military institute of hygiene and epidemiology , pulawy , poland . in silico typing of genomes the strains were snp genotyped with an sybr green - based pcr assay adopted from the protocol developed by svensson et al . changes were made from the original protocol to simplify the procedure by reducing the number of components and to increase the discriminatory power of the assays . ( 6 ) was exchanged for sybr green pcr mix ( applied biosystems , life technologies corporation , carlsbad , ca , usa ) that contains all the necessary components except the primers and template . snp - typing was performed in two separate reactions , differing by the two allele specific forward primers , specifically designed for each marker . each 10 l reaction contained 1sybr green pcr mix ( applied biosystems , life technologies corporation , carlsbad , ca , usa ) , 300 nm of each forward and reverse primer , and 1 ng template . the pcr was initiated by an activation and denaturation step for 10 min at 95c , followed by 35 cycles of 15 s at 95c and 60 s at 55c , and plate reading . finally , product dissociation ( melt analysis ) was performed at 6595c at 0.2c increments . the snp state was determined by comparing the cq values between the product generated by the ancestor and derived primers , respectively ( supplementary file 2 ) . the primers and markers used in these assays were as far as possible adopted from previous publications ( 5 , 6 , 20 ) ( table 3 ) . however , intentional mismatches at the penultimate or antepenultimate nucleotide ( already present in the primers published by vogler et al . ( 5 ) and gyuranecz et al . existing assays using modified primer sequences from previous publications schu s4 has genbank accession no . aj749949.2 d = derived snp state primer , a = ancestral snp state primer , c = common primer svensson et al . phylogeography of francisella tularensis : global expansion of a highly fit clone gyuranecz et al . the old 1960s tularemia focus was in the srebarna lake nature reserve and its surroundings ( fig . 1 and supplementary file 1 ) . the focus covered an area of about 7 km where the marsh lake abuts with the danube river . the new 1990s focus covered an area of 1,0001,500 km situated on 500800 m altitude adjacent to mountains and the capital of sofia ( fig . 1 and supplementary file 1 ) . the genetic distance between strains l2 and 81 ( orange dots ) comprised five snps in 33 years . b. the genetic distance between strain 94 from ussr and strain 19c from srebarna ( yellow dots ) comprised two snps in 5 years . c. there was an accumulation of > 20 snps between the srebarna 19 from the old focus and l1 , mn , and mmm in the new focus ( red dots ) . d. the first bulgarian genome in the b.4 basal clade was represented by strain b1 isolated in breznik in 1999 ( black dot ) . three strains from the 1960s outbreak in srebarna lake reserve in northeast bulgaria and seven strains from the new 1990s focus in west bulgaria were genome sequenced ( table 1 and fig . 1 ) . in addition , two strains recovered in the old focus in the 1960s were cansnp typed . the 1960s strains had been lyophilized after their isolation and kept at room temperature until culturing for genome sequencing in 2011 . the isolation and storage of the seven strains from the 1990s outbreaks have been previously described ( 10 ) . one archive strain isolated 1956 in ussr was sequenced for comparison purposes . for a more detailed epidemiology description , francisella tularensis strains investigated in the study the strains are from the strain collection of the military medical academy ( mma ) , sofia , bulgaria . the strains srebarna 5 , srebarna 19 , and srebarna 56 were recovered by an mma team ( gotev / kupenov ) and originate from the bulgarian type culture collection ( btcc ) in sofia note that the strain l1 is not identical to a strain also named l1 in the reference ( 16 ) b.34/35 should be read as that the strain showed the derived snp state for b.34 and the ancestral state for b.35 no further subtyping with b.77 was performed on these two strains due to insufficient amount of dna ( fig . 3 ) the strains 19c , 81 , and 94 are part of a strain collection maintained at the military institute of hygiene and epidemiology ( mihe ) in warsaw , poland , and originate from a strain collection at the former institute of marine medicine in gdansk , poland ( 21 ) . strains 19c and 94 originated from the medical academy , moscow , ussr and strain 81 originated from centre of epidemiology and microbiology in prague , former czechoslovakia the original documentation reads bulgaria ( 21 ) . no other outbreaks than the srebarna outbreak nor other areas of strains isolation are described in the literature , institutional records , or known by personal communication to our knowledge ( marinov , personal communication ) . the strains srebarna 5 , srebarna 19 , srebarna 56 , l1 , l2 , dm1 , dm2 , b1 , mmm , and mn were cultured as previously described at the military medical academy ( mma ) in sofia , bulgaria ( 10 ) . lysis of the bacteria at mma was achieved by suspending the samples in 1.0 ml 0.9% nacl and heat - treated at 65c for 2 h. the strains 19c , 81 , and 94 were re - cultured at military institute of hygiene and epidemiology ( mihe ) in pulawy , poland , and sent to the swedish defence research agency ( foi ) in ume . prior to dna preparation , strains 19c , 81 , and 94 were re - cultured at foi on gc ii agar with 1% hemoglobin and 1% isovitalex at 37c ( 17 ) dna from l1 , l2 , dm1 , b1 , mn , 19c , 81 , and 94 was prepared at foi using phenol - chloroform extraction , as previously described ( 18 ) , while dna from dm2 , mmm , srebarna 5 , srebarna 19 , and srebarna 56 was prepared using the ez1 robot preparation method , as previously described ( 19 ) . dna concentrations were measured using the nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and the qubit ( life technologies , carlsbad , ca , usa ) . the amount of dna prepared from srebarna 5 and srebarna 56 stock samples was insufficient for dna sequencing and therefore only genotyped with the cansnp markers b.12 and b.23 . genome sequencing of isolates was performed at scilifelab , uppsala , sweden , using the illumina hiseq 2000 platform with truseq dna library prep kits ( strain 91 with truseq nano dna ht sample prep kit ) according to standard protocols to produce 100 bp pair - end data . the resulting sequence data were assembled using abyss versions 1.3.2 and 1.3.3 using standard parameters ( table 2 ) . nucleotide distances and phylogeny were inferred using mega 5.2 ( complete deletion option and the number of differences method , maximum parsimony ) . genome data for 10 bulgarian strains and one archive strain sequenced in this study n50 is the length of the smallest contig in the set that contains the fewest ( largest ) contigs whose combined length represents at least 50% of the assembly ncbi bioproject prjna285143 mma = military medical academy , sofia , bulgaria ; mihe = military institute of hygiene and epidemiology , pulawy , poland . the strains were snp genotyped with an sybr green - based pcr assay adopted from the protocol developed by svensson et al . changes were made from the original protocol to simplify the procedure by reducing the number of components and to increase the discriminatory power of the assays . the pcr mix used by svensson et al . ( 6 ) was exchanged for sybr green pcr mix ( applied biosystems , life technologies corporation , carlsbad , ca , usa ) that contains all the necessary components except the primers and template . snp - typing was performed in two separate reactions , differing by the two allele specific forward primers , specifically designed for each marker . each 10 l reaction contained 1sybr green pcr mix ( applied biosystems , life technologies corporation , carlsbad , ca , usa ) , 300 nm of each forward and reverse primer , and 1 ng template . the pcr was initiated by an activation and denaturation step for 10 min at 95c , followed by 35 cycles of 15 s at 95c and 60 s at 55c , and plate reading . finally , product dissociation ( melt analysis ) was performed at 6595c at 0.2c increments . the snp state was determined by comparing the cq values between the product generated by the ancestor and derived primers , respectively ( supplementary file 2 ) . the primers and markers used in these assays were as far as possible adopted from previous publications ( 5 , 6 , 20 ) ( table 3 ) . however , intentional mismatches at the penultimate or antepenultimate nucleotide ( already present in the primers published by vogler et al . ( 5 ) and gyuranecz et al . ( 20 ) ) were introduced in the primers published by svensson et al . existing assays using modified primer sequences from previous publications schu s4 has genbank accession no . aj749949.2 d = derived snp state primer , a = ancestral snp state primer , c = common primer svensson et al . phylogeography of francisella tularensis : global expansion of a highly fit clone gyuranecz et al . ( 20 ) phylogeography of francisella tularensis subsp . following a three - decade epidemiological silence , an outbreak of tularemia occurred in 1997 in west bulgaria , where tularemia had never been registered previously , 300 km from the old outbreak area in northeast bulgaria ( fig . , there were also some sporadic cases close to the old focus . by genome sequencing of 10 strains from bulgaria , three from the old outbreaks in 1960s and seven strains from the new outbreaks in 1990s , we were able to assign the strains to genetic groups using cansnps and compare them with reference genomes ( table 1 ) . the two strains srebarna 5 and srebarna 56 were subtyped by cansnp typing assays only and were assigned to the genetic subclade b.12 ( b.23 ) . the genetic analysis of the 12 strains showed that the 1960s as well as the 1990s outbreaks of tularemia in bulgaria involved multiple clones of f. tularensis with five and six members of the two b.12 subclades b.20 and b.23 , respectively , and one member of the basal clade b.4 isolated in 1999 ( table 1 and fig . the relatively large genetic distances between the clones isolated in the old and new foci ( fig . 2 and fig . 3 ) argue against the fact that the same clone caused the epidemics in the new focus . they are divided by the balkan mountain , a long and high mountain chain that limits natural movement of vectors , rodents , and hares that could transfer f. tularensis . thus , neither common ecological factors nor close genetic kinships suggest that there was a transfer of f. tularensis from the old focus to the new focus . holarctica based on snps in an alignment of nine published genomes ( strain names are indicated ) showing the placement of the bulgarian strains in a global phylogenetic framework . bulgarian outbreak strains and the non - outbreak strain 94 sequenced in this study are indicated in bold text . b.4 , b.6 , b.12 , and b.16 indicate the four major cansnp clades of the subspecies . b.20 and b.23 indicate two b.12 subclades that included bulgarian strains the tree was mid - point rooted in fsc022 . the new bulgarian genomes placed in the phylogenetic framework of cansnps . four assays b.76 b.79 were developed in this study to target subclades , where strains from bulgaria were members . b.4 , b.12 , b.23 , b.24 , b.34 , and b.35 have been previously published ( 5 , 6 , 20 ) . alternative cansnps ( b.17 , b.19 , and b.42 ) are indicated in gray below the first published cansnp in indicated branch ( 6 , 19 ) . a local persistence of f. tularensis is suggested in the old focus by the close kinship of strain 81 and strain l2 . 3a , subclade b.34/35 ) and the l2 strain was isolated in close proximity to srebarna in 2005 ( fig . 3a , subclade b.34/35 ) , and the genomes of the strains differed by only five snps . this finding may suggest that f. tularensis was preserved in the srebarna area over 43 years and thus would lend support to pavlovsky 's inclusion of tularemia as one of the diseases that adhere to the nidality concept ( 3 ) . however , the close genetic relationships of strains from bulgaria with strains from hungary , sweden , the former soviet union , and turkey caution for making firm conclusions on persistence ( see fig . there was similarly a close genetic relationship but little epidemiological connection between the bulgarian strain b1 and isolates from sweden , norway , and austria ( see basal clade b.4 , fig . the 19c strain isolated in 1961 in the old focus and member of the subclade b.79 showed a very close relationship to the reference strain 94 isolated from a water rat in 1956 in ussr , with only one unique snp each ( fig . this corroborates previous findings on the possible long - distance movement of f. tularensis clones ( 8 , 9 ) . in this case , a ussr origin is also supported by historical data on movement of 24 muskrats ( ondatra zibethica ) from the ussr into the srebarna reserve in 1956 for the purpose of hunting and production of fur ( 11 ) . five years later , in 1961 , the muskrat population had reached approximately 20,000 individuals and an epizootic of tularemia occurred . the same year , f. tularensis strain 19c was isolated from a dead muskrat in the reserve ( 11 ) . potentially , a parental clone to strain 19c and the ussr strain 94 of 1956 may have been transferred along with muskrats into the reserve ( 13 ) . the genetic analysis showed that the 1960s as well as the 1990s outbreaks involved new genetic diversity . the new diversity was characterized by four new cansnps denoted b.76 , b.77 , b.78 , and b.79 in the basal clades b.12 ( b.76 , b.77 , and b.79 ) and b.4 ( b.78 ) ( fig . we developed cansnp assays for these new genetic branches of f. tularensis for use in future epidemiological investigations in bulgaria ( table 3 ) . the cansnp system is a generic taxonomic system for detailed intra - subspecies division that has been developed for f. tularensis ( 5 , 6 ) and other strictly clonal bacteria such as bacillus anthracis ( 22 ) , yersinia pestis ( 23 ) , and coxiella burnetii ( 24 ) . the system is based on identification of selected representative ( canonical ) snps for each branch in a phylogenetic tree making it possible to use cansnp assays for rapidly placing samples into the taxonomic system , for example , during a suspected outbreak . the genetic analysis of the 12 strains showed that the 1960s as well as the 1990s outbreaks of tularemia in bulgaria involved multiple clones of f. tularensis with five and six members of the two b.12 subclades b.20 and b.23 , respectively , and one member of the basal clade b.4 isolated in 1999 ( table 1 and fig . the relatively large genetic distances between the clones isolated in the old and new foci ( fig . 2 and fig . 3 ) argue against the fact that the same clone caused the epidemics in the new focus . they are divided by the balkan mountain , a long and high mountain chain that limits natural movement of vectors , rodents , and hares that could transfer f. tularensis . thus , neither common ecological factors nor close genetic kinships suggest that there was a transfer of f. tularensis from the old focus to the new focus . holarctica based on snps in an alignment of nine published genomes ( strain names are indicated ) showing the placement of the bulgarian strains in a global phylogenetic framework . bulgarian outbreak strains and the non - outbreak strain 94 sequenced in this study are indicated in bold text . b.4 , b.6 , b.12 , and b.16 indicate the four major cansnp clades of the subspecies . b.20 and b.23 indicate two b.12 subclades that included bulgarian strains the tree was mid - point rooted in fsc022 . four assays b.76 b.79 were developed in this study to target subclades , where strains from bulgaria were members . b.4 , b.12 , b.23 , b.24 , b.34 , and b.35 have been previously published ( 5 , 6 , 20 ) . alternative cansnps ( b.17 , b.19 , and b.42 ) are indicated in gray below the first published cansnp in indicated branch ( 6 , 19 ) . a local persistence of f. tularensis is suggested in the old focus by the close kinship of strain 81 and strain l2 . 3a , subclade b.34/35 ) and the l2 strain was isolated in close proximity to srebarna in 2005 ( fig . 3a , subclade b.34/35 ) , and the genomes of the strains differed by only five snps . this finding may suggest that f. tularensis was preserved in the srebarna area over 43 years and thus would lend support to pavlovsky 's inclusion of tularemia as one of the diseases that adhere to the nidality concept ( 3 ) . however , the close genetic relationships of strains from bulgaria with strains from hungary , sweden , the former soviet union , and turkey caution for making firm conclusions on persistence ( see fig . there was similarly a close genetic relationship but little epidemiological connection between the bulgarian strain b1 and isolates from sweden , norway , and austria ( see basal clade b.4 , fig . 3d ) . the 19c strain isolated in 1961 in the old focus and member of the subclade b.79 showed a very close relationship to the reference strain 94 isolated from a water rat in 1956 in ussr , with only one unique snp each ( fig . this corroborates previous findings on the possible long - distance movement of f. tularensis clones ( 8 , 9 ) . in this case , a ussr origin is also supported by historical data on movement of 24 muskrats ( ondatra zibethica ) from the ussr into the srebarna reserve in 1956 for the purpose of hunting and production of fur ( 11 ) . five years later , in 1961 , the muskrat population had reached approximately 20,000 individuals and an epizootic of tularemia occurred . the same year , f. tularensis strain 19c was isolated from a dead muskrat in the reserve ( 11 ) . potentially , a parental clone to strain 19c and the ussr strain 94 of 1956 may have been transferred along with muskrats into the reserve ( 13 ) . the genetic analysis showed that the 1960s as well as the 1990s outbreaks involved new genetic diversity . the new diversity was characterized by four new cansnps denoted b.76 , b.77 , b.78 , and b.79 in the basal clades b.12 ( b.76 , b.77 , and b.79 ) and b.4 ( b.78 ) ( fig . we developed cansnp assays for these new genetic branches of f. tularensis for use in future epidemiological investigations in bulgaria ( table 3 ) . the cansnp system is a generic taxonomic system for detailed intra - subspecies division that has been developed for f. tularensis ( 5 , 6 ) and other strictly clonal bacteria such as bacillus anthracis ( 22 ) , yersinia pestis ( 23 ) , and coxiella burnetii ( 24 ) . the system is based on identification of selected representative ( canonical ) snps for each branch in a phylogenetic tree making it possible to use cansnp assays for rapidly placing samples into the taxonomic system , for example , during a suspected outbreak . the existence of strain - pairs isolated in the same year and same area ( dm1 and dm2 ; mmm and mn ) , and different hosts ( mmm and mn ) , supports an expansion of individual f. tularensis clones in local areas during outbreaks among wildlife and humans . the data were inconclusive to infer a common origin of f. tularensis strains that caused the bulgarian tularemia outbreaks in the 1960s and the 1990s . the finding of very few snp differences ( n=5 ) across the whole genome in two strains isolated in the srebarna region in 1961 and 2005 , respectively , indicates local persistence and/or slow natural mutation rate in this bacterial species . a modified low - cost cansnp detection system was developed that can be used in future outbreak investigations to place f. tularensis strains into the global phylogenetic context . taken together , this study provides information on the genetic diversity of f. tularensis in bulgaria and suggests that muskrat implantation from ussr contributed to this diversity . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .

introductionoutbreaks of the zoonotic disease tularemia occurred in north - east bulgaria in the 1960s . then came 30 years of epidemiological silence until new outbreaks occurred in west bulgaria in the 1990s . to investigate how bacterial strains of francisella tularensis causing tularemia in wildlife and humans in the 1960s and the 1990s were related , we explored their genetic diversity.material and methodsten f. tularensis genomes from the 1960s ( n=3 ) and the 1990s ( n=7 ) were sequenced , assigned to canonical single - nucleotide polymorphism ( cansnp ) clades , and compared to reference genomes . we developed four new cansnp polymerase chain reaction ( pcr ) assays based on the genome sequence information.results and discussionthe genetic analysis showed that the outbreaks in the 1960s as well as in the 1990s involved multiple clones and new genetic diversity . the smallest genetic difference found between any of the bulgarian strains was five snps between the strains l2 and 81 isolated 43 years apart , indicating that f. tularensis may persist locally over long time periods without causing outbreaks . the existence of genetically highly similar strain - pairs isolated the same year in the same area from different hosts supports a hypothesis of local expansion of clones during outbreaks . close relationship ( two snps ) was found between one strain isolated 1961 in northeast bulgaria and one strain isolated 5 years before in ussr . historical data coinciding with the actual time point describe the introduction of water rats from ussr into the bulgarian outbreak area , which may explain the close genetic relationship and the origin of the outbreak.conclusiongenome analysis of strains from two outbreaks in the 1960s and the 1990s provided valuable information on the genetic diversity and persistence of f. tularensis in bulgaria .

Materials and methods Tularemia outbreaks Tularemia outbreak strains DNA preparation Genome sequencing and phylogenetic analysis Typing and design of four new SNP assays for clades that include Bulgarian isolates Results and discussion Genetic diversity and persistence of tularemia in Bulgaria Assays for Bulgarian specific clades Conclusions Supplementary Material Conflict of interest and funding

the genetic distance between strains l2 and 81 ( orange dots ) comprised five snps in 33 years . b. the genetic distance between strain 94 from ussr and strain 19c from srebarna ( yellow dots ) comprised two snps in 5 years . c. there was an accumulation of > 20 snps between the srebarna 19 from the old focus and l1 , mn , and mmm in the new focus ( red dots ) . three strains from the 1960s outbreak in srebarna lake reserve in northeast bulgaria and seven strains from the new 1990s focus in west bulgaria were genome sequenced ( table 1 and fig . in addition , two strains recovered in the old focus in the 1960s were cansnp typed . the isolation and storage of the seven strains from the 1990s outbreaks have been previously described ( 10 ) . one archive strain isolated 1956 in ussr was sequenced for comparison purposes . for a more detailed epidemiology description , francisella tularensis strains investigated in the study the strains are from the strain collection of the military medical academy ( mma ) , sofia , bulgaria . the strains srebarna 5 , srebarna 19 , and srebarna 56 were recovered by an mma team ( gotev / kupenov ) and originate from the bulgarian type culture collection ( btcc ) in sofia note that the strain l1 is not identical to a strain also named l1 in the reference ( 16 ) b.34/35 should be read as that the strain showed the derived snp state for b.34 and the ancestral state for b.35 no further subtyping with b.77 was performed on these two strains due to insufficient amount of dna ( fig . 3 ) the strains 19c , 81 , and 94 are part of a strain collection maintained at the military institute of hygiene and epidemiology ( mihe ) in warsaw , poland , and originate from a strain collection at the former institute of marine medicine in gdansk , poland ( 21 ) . no other outbreaks than the srebarna outbreak nor other areas of strains isolation are described in the literature , institutional records , or known by personal communication to our knowledge ( marinov , personal communication ) . the strains srebarna 5 , srebarna 19 , srebarna 56 , l1 , l2 , dm1 , dm2 , b1 , mmm , and mn were cultured as previously described at the military medical academy ( mma ) in sofia , bulgaria ( 10 ) . lysis of the bacteria at mma was achieved by suspending the samples in 1.0 ml 0.9% nacl and heat - treated at 65c for 2 h. the strains 19c , 81 , and 94 were re - cultured at military institute of hygiene and epidemiology ( mihe ) in pulawy , poland , and sent to the swedish defence research agency ( foi ) in ume . dna concentrations were measured using the nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and the qubit ( life technologies , carlsbad , ca , usa ) . genome data for 10 bulgarian strains and one archive strain sequenced in this study n50 is the length of the smallest contig in the set that contains the fewest ( largest ) contigs whose combined length represents at least 50% of the assembly ncbi bioproject prjna285143 mma = military medical academy , sofia , bulgaria ; mihe = military institute of hygiene and epidemiology , pulawy , poland . in silico typing of genomes the strains were snp genotyped with an sybr green - based pcr assay adopted from the protocol developed by svensson et al . changes were made from the original protocol to simplify the procedure by reducing the number of components and to increase the discriminatory power of the assays . phylogeography of francisella tularensis : global expansion of a highly fit clone gyuranecz et al . the old 1960s tularemia focus was in the srebarna lake nature reserve and its surroundings ( fig . the genetic distance between strains l2 and 81 ( orange dots ) comprised five snps in 33 years . b. the genetic distance between strain 94 from ussr and strain 19c from srebarna ( yellow dots ) comprised two snps in 5 years . c. there was an accumulation of > 20 snps between the srebarna 19 from the old focus and l1 , mn , and mmm in the new focus ( red dots ) . three strains from the 1960s outbreak in srebarna lake reserve in northeast bulgaria and seven strains from the new 1990s focus in west bulgaria were genome sequenced ( table 1 and fig . in addition , two strains recovered in the old focus in the 1960s were cansnp typed . the isolation and storage of the seven strains from the 1990s outbreaks have been previously described ( 10 ) . one archive strain isolated 1956 in ussr was sequenced for comparison purposes . for a more detailed epidemiology description , francisella tularensis strains investigated in the study the strains are from the strain collection of the military medical academy ( mma ) , sofia , bulgaria . the strains srebarna 5 , srebarna 19 , and srebarna 56 were recovered by an mma team ( gotev / kupenov ) and originate from the bulgarian type culture collection ( btcc ) in sofia note that the strain l1 is not identical to a strain also named l1 in the reference ( 16 ) b.34/35 should be read as that the strain showed the derived snp state for b.34 and the ancestral state for b.35 no further subtyping with b.77 was performed on these two strains due to insufficient amount of dna ( fig . 3 ) the strains 19c , 81 , and 94 are part of a strain collection maintained at the military institute of hygiene and epidemiology ( mihe ) in warsaw , poland , and originate from a strain collection at the former institute of marine medicine in gdansk , poland ( 21 ) . no other outbreaks than the srebarna outbreak nor other areas of strains isolation are described in the literature , institutional records , or known by personal communication to our knowledge ( marinov , personal communication ) . the strains srebarna 5 , srebarna 19 , srebarna 56 , l1 , l2 , dm1 , dm2 , b1 , mmm , and mn were cultured as previously described at the military medical academy ( mma ) in sofia , bulgaria ( 10 ) . lysis of the bacteria at mma was achieved by suspending the samples in 1.0 ml 0.9% nacl and heat - treated at 65c for 2 h. the strains 19c , 81 , and 94 were re - cultured at military institute of hygiene and epidemiology ( mihe ) in pulawy , poland , and sent to the swedish defence research agency ( foi ) in ume . dna concentrations were measured using the nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) and the qubit ( life technologies , carlsbad , ca , usa ) . nucleotide distances and phylogeny were inferred using mega 5.2 ( complete deletion option and the number of differences method , maximum parsimony ) . genome data for 10 bulgarian strains and one archive strain sequenced in this study n50 is the length of the smallest contig in the set that contains the fewest ( largest ) contigs whose combined length represents at least 50% of the assembly ncbi bioproject prjna285143 mma = military medical academy , sofia , bulgaria ; mihe = military institute of hygiene and epidemiology , pulawy , poland . the strains were snp genotyped with an sybr green - based pcr assay adopted from the protocol developed by svensson et al . changes were made from the original protocol to simplify the procedure by reducing the number of components and to increase the discriminatory power of the assays . the pcr was initiated by an activation and denaturation step for 10 min at 95c , followed by 35 cycles of 15 s at 95c and 60 s at 55c , and plate reading . ( 20 ) ) were introduced in the primers published by svensson et al . phylogeography of francisella tularensis : global expansion of a highly fit clone gyuranecz et al . ( 20 ) phylogeography of francisella tularensis subsp . following a three - decade epidemiological silence , an outbreak of tularemia occurred in 1997 in west bulgaria , where tularemia had never been registered previously , 300 km from the old outbreak area in northeast bulgaria ( fig . by genome sequencing of 10 strains from bulgaria , three from the old outbreaks in 1960s and seven strains from the new outbreaks in 1990s , we were able to assign the strains to genetic groups using cansnps and compare them with reference genomes ( table 1 ) . the two strains srebarna 5 and srebarna 56 were subtyped by cansnp typing assays only and were assigned to the genetic subclade b.12 ( b.23 ) . the genetic analysis of the 12 strains showed that the 1960s as well as the 1990s outbreaks of tularemia in bulgaria involved multiple clones of f. tularensis with five and six members of the two b.12 subclades b.20 and b.23 , respectively , and one member of the basal clade b.4 isolated in 1999 ( table 1 and fig . the relatively large genetic distances between the clones isolated in the old and new foci ( fig . 3 ) argue against the fact that the same clone caused the epidemics in the new focus . they are divided by the balkan mountain , a long and high mountain chain that limits natural movement of vectors , rodents , and hares that could transfer f. tularensis . thus , neither common ecological factors nor close genetic kinships suggest that there was a transfer of f. tularensis from the old focus to the new focus . holarctica based on snps in an alignment of nine published genomes ( strain names are indicated ) showing the placement of the bulgarian strains in a global phylogenetic framework . bulgarian outbreak strains and the non - outbreak strain 94 sequenced in this study are indicated in bold text . b.4 , b.6 , b.12 , and b.16 indicate the four major cansnp clades of the subspecies . b.4 , b.12 , b.23 , b.24 , b.34 , and b.35 have been previously published ( 5 , 6 , 20 ) . a local persistence of f. tularensis is suggested in the old focus by the close kinship of strain 81 and strain l2 . 3a , subclade b.34/35 ) and the l2 strain was isolated in close proximity to srebarna in 2005 ( fig . 3a , subclade b.34/35 ) , and the genomes of the strains differed by only five snps . this finding may suggest that f. tularensis was preserved in the srebarna area over 43 years and thus would lend support to pavlovsky 's inclusion of tularemia as one of the diseases that adhere to the nidality concept ( 3 ) . however , the close genetic relationships of strains from bulgaria with strains from hungary , sweden , the former soviet union , and turkey caution for making firm conclusions on persistence ( see fig . there was similarly a close genetic relationship but little epidemiological connection between the bulgarian strain b1 and isolates from sweden , norway , and austria ( see basal clade b.4 , fig . the 19c strain isolated in 1961 in the old focus and member of the subclade b.79 showed a very close relationship to the reference strain 94 isolated from a water rat in 1956 in ussr , with only one unique snp each ( fig . this corroborates previous findings on the possible long - distance movement of f. tularensis clones ( 8 , 9 ) . in this case , a ussr origin is also supported by historical data on movement of 24 muskrats ( ondatra zibethica ) from the ussr into the srebarna reserve in 1956 for the purpose of hunting and production of fur ( 11 ) . the same year , f. tularensis strain 19c was isolated from a dead muskrat in the reserve ( 11 ) . potentially , a parental clone to strain 19c and the ussr strain 94 of 1956 may have been transferred along with muskrats into the reserve ( 13 ) . the genetic analysis showed that the 1960s as well as the 1990s outbreaks involved new genetic diversity . the new diversity was characterized by four new cansnps denoted b.76 , b.77 , b.78 , and b.79 in the basal clades b.12 ( b.76 , b.77 , and b.79 ) and b.4 ( b.78 ) ( fig . we developed cansnp assays for these new genetic branches of f. tularensis for use in future epidemiological investigations in bulgaria ( table 3 ) . the cansnp system is a generic taxonomic system for detailed intra - subspecies division that has been developed for f. tularensis ( 5 , 6 ) and other strictly clonal bacteria such as bacillus anthracis ( 22 ) , yersinia pestis ( 23 ) , and coxiella burnetii ( 24 ) . the system is based on identification of selected representative ( canonical ) snps for each branch in a phylogenetic tree making it possible to use cansnp assays for rapidly placing samples into the taxonomic system , for example , during a suspected outbreak . the genetic analysis of the 12 strains showed that the 1960s as well as the 1990s outbreaks of tularemia in bulgaria involved multiple clones of f. tularensis with five and six members of the two b.12 subclades b.20 and b.23 , respectively , and one member of the basal clade b.4 isolated in 1999 ( table 1 and fig . the relatively large genetic distances between the clones isolated in the old and new foci ( fig . 3 ) argue against the fact that the same clone caused the epidemics in the new focus . they are divided by the balkan mountain , a long and high mountain chain that limits natural movement of vectors , rodents , and hares that could transfer f. tularensis . thus , neither common ecological factors nor close genetic kinships suggest that there was a transfer of f. tularensis from the old focus to the new focus . holarctica based on snps in an alignment of nine published genomes ( strain names are indicated ) showing the placement of the bulgarian strains in a global phylogenetic framework . bulgarian outbreak strains and the non - outbreak strain 94 sequenced in this study are indicated in bold text . b.4 , b.6 , b.12 , and b.16 indicate the four major cansnp clades of the subspecies . a local persistence of f. tularensis is suggested in the old focus by the close kinship of strain 81 and strain l2 . 3a , subclade b.34/35 ) and the l2 strain was isolated in close proximity to srebarna in 2005 ( fig . 3a , subclade b.34/35 ) , and the genomes of the strains differed by only five snps . this finding may suggest that f. tularensis was preserved in the srebarna area over 43 years and thus would lend support to pavlovsky 's inclusion of tularemia as one of the diseases that adhere to the nidality concept ( 3 ) . however , the close genetic relationships of strains from bulgaria with strains from hungary , sweden , the former soviet union , and turkey caution for making firm conclusions on persistence ( see fig . there was similarly a close genetic relationship but little epidemiological connection between the bulgarian strain b1 and isolates from sweden , norway , and austria ( see basal clade b.4 , fig . the 19c strain isolated in 1961 in the old focus and member of the subclade b.79 showed a very close relationship to the reference strain 94 isolated from a water rat in 1956 in ussr , with only one unique snp each ( fig . this corroborates previous findings on the possible long - distance movement of f. tularensis clones ( 8 , 9 ) . in this case , a ussr origin is also supported by historical data on movement of 24 muskrats ( ondatra zibethica ) from the ussr into the srebarna reserve in 1956 for the purpose of hunting and production of fur ( 11 ) . five years later , in 1961 , the muskrat population had reached approximately 20,000 individuals and an epizootic of tularemia occurred . the same year , f. tularensis strain 19c was isolated from a dead muskrat in the reserve ( 11 ) . potentially , a parental clone to strain 19c and the ussr strain 94 of 1956 may have been transferred along with muskrats into the reserve ( 13 ) . the genetic analysis showed that the 1960s as well as the 1990s outbreaks involved new genetic diversity . the new diversity was characterized by four new cansnps denoted b.76 , b.77 , b.78 , and b.79 in the basal clades b.12 ( b.76 , b.77 , and b.79 ) and b.4 ( b.78 ) ( fig . we developed cansnp assays for these new genetic branches of f. tularensis for use in future epidemiological investigations in bulgaria ( table 3 ) . the cansnp system is a generic taxonomic system for detailed intra - subspecies division that has been developed for f. tularensis ( 5 , 6 ) and other strictly clonal bacteria such as bacillus anthracis ( 22 ) , yersinia pestis ( 23 ) , and coxiella burnetii ( 24 ) . the system is based on identification of selected representative ( canonical ) snps for each branch in a phylogenetic tree making it possible to use cansnp assays for rapidly placing samples into the taxonomic system , for example , during a suspected outbreak . the existence of strain - pairs isolated in the same year and same area ( dm1 and dm2 ; mmm and mn ) , and different hosts ( mmm and mn ) , supports an expansion of individual f. tularensis clones in local areas during outbreaks among wildlife and humans . the data were inconclusive to infer a common origin of f. tularensis strains that caused the bulgarian tularemia outbreaks in the 1960s and the 1990s . a modified low - cost cansnp detection system was developed that can be used in future outbreak investigations to place f. tularensis strains into the global phylogenetic context . taken together , this study provides information on the genetic diversity of f. tularensis in bulgaria and suggests that muskrat implantation from ussr contributed to this diversity .

why are some human genetic diseases so common ? we expect natural selection to favour the alleles of genes that confer health and hinder alleles of genes that confer disease . yet , there are a glut of genetically based human diseases , behavioural syndromes , and birth defects that occur at relatively high frequencies . the classic explanation is that diseases are maintained in balance between mutation , which creates the disease , and purifying selection , which removes it . though this theory is roughly supported by diseases with simple genetic inheritance , it is not designed to apply for diseases with complex patterns of inheritance [ 3 , 4 ] . one form of complex inheritance occurs when the genetic makeup of mothers influences the disease status of their offspring . this can either happen through maternal genetic effects , whereby the genotype of mothers influences trait expression in offspring , or through maternal - zygotic epistasis , whereby the phenotype of an offspring is dependent on the interaction between genes in the maternal genome and genes in the offspring genome [ 7 , 8 ] . it can be viewed as kind of genotype x environment interaction , in which the maternal genome provides the environment for the offspring genotype . however , unlike standard discussions of genotype x environment interaction where the environment is fixed , the maternal environment is genetic and can evolve . the most detailed evidence for this type of inheritance comes from work in plant and animal systems [ 815 ] , but there is also direct evidence from humans . we do not know the full extent to which maternal genetic effects and maternal - zygotic epistasis contribute to human diseases . our ability to detect complex inheritance is hampered by a range of factors , particularly sampling limitations , population structure , and environmental influences on disease [ 1621 ] . our ability to detect maternal genetic sources of inheritance is further hampered by the multigenerational complexity of the data sets required to test for it [ 8 , 2224 ] . despite these limitations , there is emerging evidence from association studies that maternal genetic effects and maternal - zygotic epistasis might be common mechanisms of inheritance of human diseases and birth defects [ 16 , 2531 ] . the mechanism of inheritance is important to understand because it may affect the prevalence of disease . for simple genetic diseases , prevalence is determined by the mutation - selection balance . however , in maternally expressed diseases half of the alleles are hidden from selection , as the genotype of fathers has no effect on offspring phenotype [ 3235 ] . this means that purifying selection might only be half as effective at removing diseases that result from maternal gene expression . thus , given the same mutation rate , we might expect maternal genetic diseases to have a substantially higher prevalence than zygotic genetic diseases ( diseases caused by gene expression in offspring ) . the relaxed selective constraint on maternal genetic effects is supported by studies of population variation and diversification of maternal effect genes [ 3436 ] . epistasis is known to confound disease gene mapping [ 5 , 33 , 37 ] because alleles at one locus can mask the effects of expression of alleles at another locus [ 3841 ] . thus , under specific forms of gene interaction , even genes that confer death in one genetic background might be maintained at higher frequencies than we would expect under traditional , mutation / selection balance , theories of disease evolution . previous theories have described general models of maternal genetic effects and maternal - zygotic epistasis in trait expression [ 7 , 8 , 34 , 36 ] . however , there are many unresolved questions about their roles in genetic diseases . do maternal genetic effects increase the incidence of genetic diseases ? how do epistatic interactions between maternal and offspring genomes affect disease susceptibility ? and , can models of the evolution of complex diseases assist us in identifying the genes which underlie complex diseases ? here , we address these questions from the perspective of evolutionary population genetics . we present results from population genetic simulations of the evolution of genes affecting viability , whether owing to birth defects or adult diseases . these results are the first to demonstrate that genes that reduce fitness are more likely to be maintained in populations over time and at higher equilibrium frequencies when they are expressed in mothers than when they are expressed in offspring . the results also reveal the specific forms of maternal - fetal epistasis which increase the incidence of alleles with direct and deleterious effects on fetal survival . we show that a gene with a deleterious main effect can become fixed and mean fitness can increase because fixing a genetic background with a strong positive interaction effect may add more to mean fitness than the deleterious main effect removes . we discuss the implications of these findings for how genetic diseases evolve and for detecting genes associated with disease . before we can model the evolution of genetic diseases , we need to develop an appropriate decomposition of genetic effects . from the perspective of population genetics , a diseased individual has a genetic makeup that encodes lower offspring production ( lower fitness ) than the average individual in the population . simple genetic diseases can be described by the additive and dominance effects that their alleles encode . the additive effect of a disease allele is half the fitness difference between the homozygote classes . the dominance effect of an allele is the deviation between the observed fitness of heterozygous individuals and the fitness midpoint between the homozygous classes . for example , the following are the genotypic values of a hypothetical gene o , with alleles o and o , which causes lethal birth defects in all homozygous recessive offspring : oo fitness = 1 , oo fitness = 1 , oo fitness = 0 . the additive and dominance values for the genotypes in this scenario are as follows : additive value for o allele = 0.5 so that the fitness of the oo genotype equals 1 + 2(0.5 ) ; dominance value for the oo genotype = + 0.5 , so that its fitness equals 1 + ( + .50 ) + 1(0.5 ) . in our simulations the following are the genotypic values when the hypothetical gene o causes lethal birth defects in 20% of homozygous recessive offspring : oo fitness = 1 , oo fitness = 1 , and oo fitness = 0.8 . the additive and dominance values for this scenario are as follows : additive value = 0.1 ; dominance value = + 0.1 . for simple genetic diseases , we can calculate the probability that a given genotype will express the disease from the additive and dominance effects . however , when there are gene - by - gene interactions then the fitness of a given genotype is dependent on the sum of the additive and dominance values plus the effect of the interaction . we chose to focus on diploid statistical epistasis , rather than other kinds of gene interactions , because they can be estimated from genetically based epidemiological data and they describe the statistical effect of gene interactions on phenotypes in a similar manner to simple genetic effects . our goal is to illuminate the evolutionary process in terms of such statistically detectable gene effects . complex genetic diseases can be described by additive and dominance effects at one locus , additive and dominance effects at another locus , and all possible two - way interactions among genetic effects . in total , the additive and epistatic effects of a pair of genes ( hypothetical genes a and b ) on phenotypes can be decomposed into eight separate orthogonal components ( see table 1 ) . for the sake of clarity , means additive genetics at the a locus , acting on genotypes aa and aa ; da means dominance at the a locus , acting on the genotype aa ; means additive genetics at the b locus , acting on genotypes bb and bb ; db means dominance at the b locus , acting on the genotype bb ; iab means interaction between additive alleles at a and b loci , acting on aabb , aabb , aabb , and aabb genotypes ; kaab means interaction between the aa genotype with bb and bb genotypes ; kabb means interaction between the bb genotype with aa and aa genotypes ; jaabb means interaction between aa and bb genotypes . for example , in table 1 , the fitness of an individual of genotype aabb is the sum of the additive effects of the a and b loci ( and ) and their additive by additive interaction ( iab ) . traditionally , epistasis has been considered for gene combinations within the same individual or genome ( g g epistasis ) . however , genes expressed in one individual can also interact with genes expressed in another [ 8 , 32 ] . the best described form of such between - genome epistasis ( often described as g g epistasis ; here we use the term maternal - zygotic epistasis to describe offspring genotype x mother genotype epistasis ) is maternal - zygotic epistasis , which occurs when trait expression in offspring is determined by interactions between genes expressed in a mother and genes expressed in her offspring . maternal genetic effects and maternal - zygotic epistasis can be described by maternally expressed additive and dominance effects at one locus , additive and dominance effects expressed in offspring , and all possible two - way interactions among genetic effects . in total , the additive and epistatic effects of a pair of hypothetical gene expressed in mothers and offspring ( m and o ) that determine diseases in offspring can be decomposed into eight separate components ( table 2 ) . for the sake of clarity , means additive genetics at the m locus , acting on the offspring of mm and mm mothers ; dm means dominance at the m locus , acting on the offspring of mm mothers ; means additive genetic effect at the o locus , acting on the oo and oo offspring ; do means dominance at the o locus , acting on the oo offspring . loci , acting on oo and oo offspring of mm mothers , and oo and oo offspring of mm mothers ; kmmb means interaction between the oo and oo offspring of mm mothers ; kmoo means interaction between the oo offspring of mm and mm mothers ; jmmoo means interaction between oo offspring of mm mothers . although they look similar , the fitness calculations are more complicated in transgenerational genetic models ( table 2 ) than physiological epistatic models ( table 1 ) . in transgenerational genetic models thus , two offspring of the same genotype but with different mothers can have different viabilities . for example , in table 2 , the fitness of oomm offspring from oomm and oomm mothers is the sum of the additive effects of the o locus ( ) , additive effects of the m locus ( ) and their zygotic additive by maternal additive interaction ( iom ) . in contrast , the fitness of oomm offspring from oomm and oomm mothers is the sum of the additive effects of the o locus ( ) , dominance effects of the m locus ( dm ) and the zygotic additive by maternal dominance interaction ( komm ) . it is this conditioning on maternal genotype that gives maternal - zygotic gene interaction its unique evolutionary properties [ 7 , 8 ] . although the interplay between the maternal and zygotic genomes probably involves complex interactions between thousands of genes and gene products , we can begin to understand the fundamental nature of these interactions by simulating maternal - zygotic interactions under greatly simplified conditions . m , with two alleles , m and m , is expressed in mothers and influences the fitness of offspring . a second locus , o , with two alleles , o and o , is expressed in offspring and influences the fitness of offspring . in our simulations , we also assume for simplicity standard population genetic assumptions , which include that there are diploid males and females in a population of infinite size that mate randomly and produce offspring via sexual reproduction . we assumed that there was no inbreeding depression and that fitnesses were equivalent for males and females . the model also assumes that fitness is strictly determined by the expression of alleles and by interactions between alleles at the m and o loci . fitness is assigned to offspring after they are born , but before they reproduce . in every simulation , we set the initial starting frequency of the m and o alleles at 0.25 at two - locus hardy - weinberg equilibrium . because the strength and direction of epistatic effects depends on allele frequency [ 37 , 43 ] restricting the analysis to an initial allele frequency to 0.25 for both alleles limits general inferences from our simulations ; however , previous models have shown that over a large range of initial allele frequencies ( between 0 and 0.5 ) the evolutionary dynamics of maternal - zygotic interactions are determined by the relative strengths of direct selection and epistasis . our goal was to uncover some of the dynamics of how deleterious genes are maintained in populations under reasonable values of selection and epistasis , not to characterize all dynamics across all allele frequencies , selection coefficients and epistatic interactions . we chose our initial allele frequency because at a frequency of 0.25 it is easy to visualize both increases and decreases in allele frequency . we undertook simulations to understand the evolution of genes and gene combinations which reduce fitness , our definition of genes that encode birth defects and genetic diseases . the simulations operate by measuring how the frequency of each genotype changes over 100 generations when we assign values of fitness to the sets of the parameters described in table 2 , which are the genetic effects of and epistatic interactions between the m and o genes . for each generation we also calculated the average fitness of the population and the linkage disequilibrium between the m and o loci . linkage disequilibrium ( ld ) , also referred to as gametic phase disequilibrium , is the nonrandom association of alleles at different loci into gametes [ 44 , 45 ] . in our simulations , a positive ld means that there are a greater proportion of mo and mo gametes in the population than mo and mo gametes . conversely , a negative ld indicates that there are a greater proportion of mo and mo gametes in the population than mo and mo gametes . we conducted a total of 56 simulations of the evolution of genetic diseases under simple and complex patterns of inheritance . purifying selection : = 0.1 , = 0.1 , dm = 0.1 , and do = 0.1 , each in separate simulations ( figure 1 ) . maternal additive by zygotic additive epistasis with and without purifying selection : imo = 0.4 , 0 , and 0.4 with = 0 and = 0 ; imo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0.1 and = 0 ; imo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0 and = 0.1 ( figure 2 ) . maternal dominant by zygotic additive epistasis with and without purifying selection : kmmo = 0.4 , 0 , and 0.4 with = 0 and = 0 ; kmmo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0.1 and = 0 ; kmmo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0 and = 0.1 ( figure 3 ) . maternal additive by zygotic dominant epistasis with and without purifying selection : kmoo = 0.4 , 0 , and 0.4 with = 0 and = 0 ; kmoo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0.1 and = 0 ; kmoo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0 and = 0.1 ( figure 4 ) . maternal dominant by zygotic dominant epistasis with and without purifying selection : jmmoo = 0.4 , 0 , and 0.4 with = 0 and = 0 ; jmmoo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0.1 and = 0 ; jmmoo = 0.4 , 0.2 , 0 , 0.2 , and 0.4 with = 0 and = 0.1 ( figure 5 ) . in each case we found , as we would expect , that genes that encode alleles which reduce fitness are selectively disadvantageous . we found that additive deleterious alleles are lost more quickly than alleles with some level of dominance . dominance , in effect , hides copies of the deleterious allele away from some of the action of purifying natural selection and retards the overall evolutionary process . complete dominance hides alleles more effectively than the partial dominance we illustrate here . by comparing the maternal and zygotic patterns of gene expression , we found that maternally expressed alleles are always lost more slowly than offspring alleles with the same additive and dominance effects ( figure 1 ) . maternal gene expression , like dominance , effectively hides copies of the deleterious alleles in fathers , where they are not expressed and hence are hidden from the action of purifying natural selection . note that average population fitness smoothly increases in all cases as expected when deleterious genotypes are removed by selection and their place is taken by genotypes of higher fitness . these results indicate that genetic diseases caused by gene expression in mothers will have a higher incidence than diseases exclusively caused by gene expression in offspring . we see the same basic findings when purifying selection acts in conjunction with maternal - zygotic epistasis . note that , in all cases , purifying selection acting on maternal effect genes is slower than it is on similarly acting zygotic effect genes . this reinforces and extends our general finding for single genes with only additive and dominance effects to more complex genetic architectures : maternal effect disease - causing genes will be more common in populations than similar zygotically expressed genes . because selection on genes with only maternal effects is weakened , we note that , in some cases , the evolutionary outcome differs for maternal and zygotic effect genes . in particular , with additive - by - dominance epistasis , strong interaction outweighs the additive , deleterious effect and produces permanent polymorphism of the maternal effect gene but not for the zygotic effect gene ( compare the left panels of figures 3(b ) and 4(b ) ) . this is the most dramatic case where the evolutionary equilibrium frequency of a maternal effect gene ( pm ~ 0.36 ) exceeds that of a zygotic gene ( po ~ 0.0 ) of comparable effect . our results show that epistasis between a maternally expressed allele and a zygotically expressed allele can alter the evolution of genetic diseases . in the absence of maternal - zygotic epistasis , purifying selection rapidly removes disease genes with additive or dominant effects ( figure 1 ) . however , in the presence of maternal - zygotic epistasis purifying selection can have a variety of different consequences for the evolution of disease - encoding alleles . it can hasten the loss of the allele ( figures 2(a ) and 2(b ) ) . it can slow the loss of the allele ( figures 3(c ) , 4(b ) , 4(c ) , 5(b ) and 5(c ) ) . it can increase the frequency of the allele before eventual loss ( figures 2(b ) , 2(c ) , 4(b ) and 4(c ) ) ; and it can result in permanent maintenance of disease - causing alleles ( figures 3(b ) , 3(c ) , 5(b ) and 5(c ) ) . for additive - by - additive epistasis , m gene , its deleterious effect on fitness is intensified , while on others it is diminished . in order to determine the overall effect of the interaction , the fitness of the o allele has to be averaged over all genetic backgrounds at the m this averaging produces some counter intuitive results with additive - by - additive epistasis . for example , when iom = + 0.4 ( iom in table 2 and heavy solid line in ( figure 2(a ) left panel ) , the fitness of the mmoo genotype is substantially greater than 1.0 when produced by mm mothers ( fitness = 1.3 [ 1 0.1 + 0.4 ] ) , and close to 1.0 when produced by mm mothers ( fitness = 0.9 [ 1 0.1 ] ) . because the fitness of the mmoo genotype offspring from mm mothers is high , we might expect selection to favor the spread of m and o alleles ; however , the mm genotypes also have lowered fitness in the absence of the o allele , so it is not clear , a priori , how the frequencies of o and m will change each generation . the simulations indicate that under positive iom epistasis both the m and o alleles are quickly removed from the population because the fitness of mmoo and mmoo intermediate genotypes is low and because oo genotypes have a fitness advantage when they are produced by mm mothers . in fact , the m and o alleles are actually lost more quickly with positive iom epistasis than with no epistasis ( figure 2(a ) left panel ) . as the bad gene combinations are selected against , the mean fitness of the population is increased ( figure 2(a ) top middle panel ) . during this process , mo and mo gametes are more common than random expectation so that ld is negative ( figure 2(a ) top right panel ) . with fixation of the o allele , ld returns to zero . when we change the sign of iom , mmoo , and mmoo genotypes have the highest fitness , and the mmoo and mmoo genotypes have the lowest fitness . here selection against the o disease susceptibility allele is weaker than with no epistasis because the mmoo and mmoo genotypes have higher fitness which causes the m allele to fix , after which in the mm genetic background , the o allele is no longer favored . note that changes in mean population fitness under negative iom do not increase monotonically and that the rate of change in mean population fitness shifts as genotypes are sorted by selection ( figure 2(b ) middle panel ) . during this process , om and om gametes are more common than random expectation so that ld is positive ( figure 2(b ) middle right panel ) . these findings show that additive - by - additive epistasis opposite in sign of an allele 's direct effect on fitness will mitigate purifying selection . intuitively , by synergistically making a bad allele worse , selection will act to remove the allele more quickly , while in the opposite case , selection will be reduced . negative maternal dominance by zygotic additive epistasis appears to increase selection against additive zygotically expressed disease genes ( figure 4 left panels ) . without epistasis , the fitnesses of the o locus genotypes , oo , oo , and oo are 0.9 , 1 , and 1.1 , respectively ; however , with kmmo equal to 0.4 the fitnesses are 0.5 and 0.9 for oo , 1 for oo , and 1.1 and 1.5 , for oo , depending on maternal m locus genotype . for the m locus , on the most common o locus genotypic background , oo , the fitnesses are 1.1 when they are derived from mm or mm mothers and 1.5 when they are derived from mm mothers . locus ( i.e. , marginal overdominance or balancing selection ) . as a result , for negative values of koom , the zygotic effect disease gene is rapidly lost and the maternal effect gene with no additive effect on its own evolves to a frequency of 0.5 ( figure 4(a ) top left panel ) . mean population fitness quickly increases for negative values of epistasis ( figure 4 middle panels ) . for positive values of koom on the most common o locus background there is marginal under - dominance of the fitnesses of the three locus genotypes , mm , mm , and mm , 1.1 , 0.7 , 1.1 , respectively . though the o disease causing allele has a substantial fitness advantage in an mm background , because the m allele is lost over time as a result of the marginal under - dominance , the o allele is concurrently lost , though more slowly and with greater levels of negative ld than without epistasis ( figure 4(c ) bottom right panel ) . with the maternal additive by zygotic additive epistasis equal to 0.4 for mm , the most common m locus background , the fitnesses of the three o locus genotypes , oo , oo , and oo are 0.90 , 1.4 , and 1.1 , respectively . this is the fitness pattern of balancing selection at the o locus . as a result , for negative values of koom , the frequency of the deleterious o allele evolves to a stable intermediate frequency ( figure 5 left panels ) . mean population fitness is not as high as in previous cases , because at the polymorphic equilibrium , recombination and segregation produce deleterious genotypes in the population every generation ( figure 5 middle panels ) . for positive values of koom , there is marginal under - dominance of the fitnesses of the three o locus genotypes , oo , oo , and oo : 0.90 , 0.6 , and 1.1 , respectively when in the mm maternal background . as a result , the o allele is lost more quickly with epistasis than without epistasis . overall , only moderate levels of ld are generated by positive and negative dominance - by - additive epistasis ( figure 5 left panels ) . with dominance - by - dominance epistasis and joomm equal to + 0.4 ( joomm in table 1 ) , again there is marginal overdominance and heterozygote advantage at the o locus and the population achieves a stable intermediate polymorphism . mean population fitness is lowest at this equilibrium because recombination and segregation continue to produce deleterious offspring genotypes . it is also important to note that negative ld is generated by strong positive dominance - by - dominance epistasis for nearly 200 generations although it is absent at equilibrium ( figure 5(b ) right panel ) . when only maternal - zygotic interaction determines disease risk , the variety of possible evolutionary outcomes is greater for both genes relative to all cases that we discussed above . in particular , stable polymorphism is the most common outcome with any kind of dominance epistasis ( figures 3 , 4 , and 5 left panels ) . at the polymorphic equilibria , no detectable ld exists in a population ( figures 3 , 4 , and 5 right panels ) . as a result of the polymorphism , equilibrium disease risk and incidence will be high and , owing to the absence of ld , nonrandom associations across the population between maternal and zygotic alleles , that might point us toward a causal interaction , will be lacking . this might well represent the current state of genetic risk for maternal - zygotic diseases in the us population . maternal - zygotic epistasis is known to produce half the levels of ld as traditional epistasis . our results indicate that this finding also applies to maternal genetic disease involved in maternal - zygotic interactions . by comparing the middle and lower panels of figures 2 , 3 , 4 , and 5 , it is clear that zygotic genetic diseases generate lower levels of ld than maternal genetic diseases when they exhibit maternal - zygotic epistasis . the implication of this finding is that studies designed to identify maternal - zygotic epistasis up to a particular effect size will need substantially larger sample sizes than studies designed to identify epistasis with effects of the same magnitude . second , epistasis generates patterns that , without knowledge of the exact mechanism of inheritance , appear to be similar to simple genetic effects on disease . our results illustrate cases where allele frequencies appear be selected for or against , or can be maintained by balancing selection . without considering the fate and frequency of the interacting alleles , but , in each of these cases the pattern is influenced by the fate and frequency of interacting loci ( see ) . this finding indicates that characterizations of simple genetic diseases might need to be qualified with the possibility that gene interactions drive the disease expression . this finding also indicates that larger , more genetically detailed data sets may provide a deeper understanding of the evolution of genetic disease . all genetic diseases are affected by the same constellation of evolutionary forces : natural selection , mutation , migration , and random genetic drift ; however , the complexity of the pattern of gene expression changes the way these forces work together . the evolutionary equilibrium of simple genetic diseases is thought to be primarily determined by the mutation - selection balance . however , the results of our simulations show that the pattern of gene expression ( maternal versus offspring ) and the form and level of gene interaction can greatly affect the incidence of genetic diseases . diseases genes persist longer and at higher frequencies in a population when they are expressed maternally than when they are expressed zygotically . we have shown that genes which reduce fitness evolve differently under each of the four statistical forms of epistasis . we found that genes with additive main effects on increased risk of disease can be selected for and even brought to fixation by epistasis between maternally and zygotically expressed alleles . this was particularly true when the form of epistasis involves dominance . by tracking gene frequency changes in conjunction with the mean fitness of the population we found that as the frequencies of particular genotypes change in the population , the mean population fitness can shift in nonintuitive ways . we also found that epistasis temporarily generates ld between maternal- and zygotic - effect genes . thus , even with the simplifying assumptions of infinite population size and random mating , maternal / zygotic epistasis had a large confounding effect on the detection of single genes underlying disease etiology . maternal genetic effects and maternal - zygotic epistatic interactions on offspring phenotypes arise because maternal genotype and condition guide early embryonic development [ 7 , 12 , 13 , 46 , 47 ] . for example , it is now well established that birth weight is determined by interactions between the maternal uterine genotype and offspring genotype [ 8 , 10 , 11 , 26 ] . mothers contribute prenatal nutrients , mrna , and antibodies to offspring as well as postnatal effects , most prominently during lactation [ 10 , 11 , 48 ] . in vertebrate embryos , maternal gene expression establishes the formation of axes and induces developmental genes in the embryo . in mammals , zygotic gene expression becomes the predominant controlling factor in development between the 2 and 8-cell stage , which is called the maternal - zygotic transition ( mzt ) [ 5052 ] . perhaps the best known maternal - zygotic interaction in human disease is the rhesus ( rh ) blood factor in pregnancy . maternally expressed alleles of the rhd gene negatively interact with zygotically expressed alleles of the rhag gene . if mothers are serologically rh , then exposure to serologically rh+ molecules in fetal blood can result in the formation of antibodies against the rh+ factor , which can result in rejection of the red blood cells of the baby and subsequent anemia , brain damage and even fetal death . similarly , maternal , zygotic and maternal - zygotic interaction effects in several genes in the folate - dependent homocysteine pathway are associated with neural tube defects . though we emphasize maternal - zygotic epistasis in disease etiology , it should be noted that maternal condition also influences the health of offspring . this type of effect results from genotype - by - environment interactions . in utero exposure to smoking and to alcohol are probably the best knows causes of fetal syndromes , but maternal age [ 53 , 54 ] and in utero exposure to disease may have long - term negative impacts on offspring health . interactions between the maternal environment and genotype can also affect susceptibility to disease and birth defects . for example , the strength of effect of maternal age on the incidence of obsessive - compulsive behaviours in offspring appears to depend on interactions with the dopamine d1 gene . of course , the natural word is much more complex than our simulations , although digenic interaction models should capture much of the phenotypic variation owing to epistasis . conditions where epistasis and maternal effect would be more important [ 7 , 21 , 33 ] . in the last few centuries , the development of a global economy has increased migration and as a consequence has brought genotypes together that have independently evolved for thousands of years and created populations that are unlikely to be at evolutionary genetic equilibrium . such admixture of populations also creates ld and segregation among large blocks of genes . how can we ever hope to uncover the genetic basis of disease given this historical complexity ? as one can clearly see from our simulations , complex genetic disorders have a confusing pattern of inheritance and a nonintuitive evolutionary trajectory . in the last two decades , there has been increasing interest in uncovering the genetic basis of complex diseases . though association and linkage disequilibrium studies have identified many putative disease genes , they have often been difficult to confirm in independent population samples . some investigators have argued that this irreproducibility is largely a consequence of weak statistical power [ 17 , 18 , 56 ] . however , the lack of replication of the association between genes and traits is also the signature of epistasis . in some populations the genetic background will facilitate detection of disease genes , while in others , the genetic background masks detection [ 19 , 33 ] . these complexities do not mean that we can not map genetic basis of complex genetic disorders . we need to use and develop methods that identify the contexts that lead to increased risk of disease . our simulations also show that each of the four forms of epistasis affects levels of linkage disequilibrium and , at equilibrium , ld tends toward zero . this implies that linkage association mapping of disease genes may not be that useful when the disease gene involves epistasis . single allele - disease associations might occasionally point us in the right direction , but for most complex diseases we need approaches that embrace population subdivision and epistasis . for example , templeton 's nested cladistic analysis of phenotypic associations with haplotypes identifies the specific genetic backgrounds that generate the strongest signal between alleles and disease ( or any other trait ) in nonexperimental populations . for particular diseases it is now possible to estimate an individual 's risk of aquiring disease based on a genetic profile which classifies the individual 's ethnicity . there are also experimental methods that are specifically designed to detect gene interactions and maternal - zygotic gene interactions [ 2224 , 35 ] . by stratifying the population and searching for genetic sources of disease in each distinct biologically relevant clade by embracing population subdivision and epistasis we will be more likely to determine the genetic basis of birth defects and disease .

many birth defects and genetic diseases are expressed in individuals that do not carry the disease causing alleles . genetic diseases observed in offspring can be caused by gene expression in mothers and by interactions between gene expression in mothers and offspring . it is not clear whether the underlying pattern of gene expression ( maternal versus offspring ) affects the incidence of genetic disease . here we develop a 2-locus population genetic model with epistatic interactions between a maternal gene and a zygotic gene to address this question . we show that maternal effect genes that affect disease susceptibility in offspring persist longer and at higher frequencies in a population than offspring genes with the same effects . we find that specific forms of maternal - zygotic epistasis can maintain disease causing alleles at high frequencies over a range of plausible values . our findings suggest that the strength and form of epistasis and the underlying pattern of gene expression may greatly influence the prevalence of human genetic diseases .

1. Introduction 2. Materials and Methods 3. Results 4. Discussion

why are some human genetic diseases so common ? we expect natural selection to favour the alleles of genes that confer health and hinder alleles of genes that confer disease . yet , there are a glut of genetically based human diseases , behavioural syndromes , and birth defects that occur at relatively high frequencies . the classic explanation is that diseases are maintained in balance between mutation , which creates the disease , and purifying selection , which removes it . though this theory is roughly supported by diseases with simple genetic inheritance , it is not designed to apply for diseases with complex patterns of inheritance [ 3 , 4 ] . one form of complex inheritance occurs when the genetic makeup of mothers influences the disease status of their offspring . this can either happen through maternal genetic effects , whereby the genotype of mothers influences trait expression in offspring , or through maternal - zygotic epistasis , whereby the phenotype of an offspring is dependent on the interaction between genes in the maternal genome and genes in the offspring genome [ 7 , 8 ] . it can be viewed as kind of genotype x environment interaction , in which the maternal genome provides the environment for the offspring genotype . we do not know the full extent to which maternal genetic effects and maternal - zygotic epistasis contribute to human diseases . our ability to detect complex inheritance is hampered by a range of factors , particularly sampling limitations , population structure , and environmental influences on disease [ 1621 ] . despite these limitations , there is emerging evidence from association studies that maternal genetic effects and maternal - zygotic epistasis might be common mechanisms of inheritance of human diseases and birth defects [ 16 , 2531 ] . the mechanism of inheritance is important to understand because it may affect the prevalence of disease . for simple genetic diseases , prevalence is determined by the mutation - selection balance . this means that purifying selection might only be half as effective at removing diseases that result from maternal gene expression . thus , given the same mutation rate , we might expect maternal genetic diseases to have a substantially higher prevalence than zygotic genetic diseases ( diseases caused by gene expression in offspring ) . the relaxed selective constraint on maternal genetic effects is supported by studies of population variation and diversification of maternal effect genes [ 3436 ] . epistasis is known to confound disease gene mapping [ 5 , 33 , 37 ] because alleles at one locus can mask the effects of expression of alleles at another locus [ 3841 ] . thus , under specific forms of gene interaction , even genes that confer death in one genetic background might be maintained at higher frequencies than we would expect under traditional , mutation / selection balance , theories of disease evolution . previous theories have described general models of maternal genetic effects and maternal - zygotic epistasis in trait expression [ 7 , 8 , 34 , 36 ] . however , there are many unresolved questions about their roles in genetic diseases . do maternal genetic effects increase the incidence of genetic diseases ? how do epistatic interactions between maternal and offspring genomes affect disease susceptibility ? we present results from population genetic simulations of the evolution of genes affecting viability , whether owing to birth defects or adult diseases . these results are the first to demonstrate that genes that reduce fitness are more likely to be maintained in populations over time and at higher equilibrium frequencies when they are expressed in mothers than when they are expressed in offspring . the results also reveal the specific forms of maternal - fetal epistasis which increase the incidence of alleles with direct and deleterious effects on fetal survival . we show that a gene with a deleterious main effect can become fixed and mean fitness can increase because fixing a genetic background with a strong positive interaction effect may add more to mean fitness than the deleterious main effect removes . we discuss the implications of these findings for how genetic diseases evolve and for detecting genes associated with disease . before we can model the evolution of genetic diseases , we need to develop an appropriate decomposition of genetic effects . simple genetic diseases can be described by the additive and dominance effects that their alleles encode . the dominance effect of an allele is the deviation between the observed fitness of heterozygous individuals and the fitness midpoint between the homozygous classes . for example , the following are the genotypic values of a hypothetical gene o , with alleles o and o , which causes lethal birth defects in all homozygous recessive offspring : oo fitness = 1 , oo fitness = 1 , oo fitness = 0 . the additive and dominance values for the genotypes in this scenario are as follows : additive value for o allele = 0.5 so that the fitness of the oo genotype equals 1 + 2(0.5 ) ; dominance value for the oo genotype = + 0.5 , so that its fitness equals 1 + ( + .50 ) + 1(0.5 ) . in our simulations the following are the genotypic values when the hypothetical gene o causes lethal birth defects in 20% of homozygous recessive offspring : oo fitness = 1 , oo fitness = 1 , and oo fitness = 0.8 . for simple genetic diseases , we can calculate the probability that a given genotype will express the disease from the additive and dominance effects . we chose to focus on diploid statistical epistasis , rather than other kinds of gene interactions , because they can be estimated from genetically based epidemiological data and they describe the statistical effect of gene interactions on phenotypes in a similar manner to simple genetic effects . complex genetic diseases can be described by additive and dominance effects at one locus , additive and dominance effects at another locus , and all possible two - way interactions among genetic effects . in total , the additive and epistatic effects of a pair of genes ( hypothetical genes a and b ) on phenotypes can be decomposed into eight separate orthogonal components ( see table 1 ) . traditionally , epistasis has been considered for gene combinations within the same individual or genome ( g g epistasis ) . however , genes expressed in one individual can also interact with genes expressed in another [ 8 , 32 ] . the best described form of such between - genome epistasis ( often described as g g epistasis ; here we use the term maternal - zygotic epistasis to describe offspring genotype x mother genotype epistasis ) is maternal - zygotic epistasis , which occurs when trait expression in offspring is determined by interactions between genes expressed in a mother and genes expressed in her offspring . maternal genetic effects and maternal - zygotic epistasis can be described by maternally expressed additive and dominance effects at one locus , additive and dominance effects expressed in offspring , and all possible two - way interactions among genetic effects . in total , the additive and epistatic effects of a pair of hypothetical gene expressed in mothers and offspring ( m and o ) that determine diseases in offspring can be decomposed into eight separate components ( table 2 ) . in transgenerational genetic models thus , two offspring of the same genotype but with different mothers can have different viabilities . it is this conditioning on maternal genotype that gives maternal - zygotic gene interaction its unique evolutionary properties [ 7 , 8 ] . although the interplay between the maternal and zygotic genomes probably involves complex interactions between thousands of genes and gene products , we can begin to understand the fundamental nature of these interactions by simulating maternal - zygotic interactions under greatly simplified conditions . m , with two alleles , m and m , is expressed in mothers and influences the fitness of offspring . a second locus , o , with two alleles , o and o , is expressed in offspring and influences the fitness of offspring . in our simulations , we also assume for simplicity standard population genetic assumptions , which include that there are diploid males and females in a population of infinite size that mate randomly and produce offspring via sexual reproduction . the model also assumes that fitness is strictly determined by the expression of alleles and by interactions between alleles at the m and o loci . in every simulation , we set the initial starting frequency of the m and o alleles at 0.25 at two - locus hardy - weinberg equilibrium . because the strength and direction of epistatic effects depends on allele frequency [ 37 , 43 ] restricting the analysis to an initial allele frequency to 0.25 for both alleles limits general inferences from our simulations ; however , previous models have shown that over a large range of initial allele frequencies ( between 0 and 0.5 ) the evolutionary dynamics of maternal - zygotic interactions are determined by the relative strengths of direct selection and epistasis . our goal was to uncover some of the dynamics of how deleterious genes are maintained in populations under reasonable values of selection and epistasis , not to characterize all dynamics across all allele frequencies , selection coefficients and epistatic interactions . we chose our initial allele frequency because at a frequency of 0.25 it is easy to visualize both increases and decreases in allele frequency . we undertook simulations to understand the evolution of genes and gene combinations which reduce fitness , our definition of genes that encode birth defects and genetic diseases . the simulations operate by measuring how the frequency of each genotype changes over 100 generations when we assign values of fitness to the sets of the parameters described in table 2 , which are the genetic effects of and epistatic interactions between the m and o genes . for each generation we also calculated the average fitness of the population and the linkage disequilibrium between the m and o loci . linkage disequilibrium ( ld ) , also referred to as gametic phase disequilibrium , is the nonrandom association of alleles at different loci into gametes [ 44 , 45 ] . we conducted a total of 56 simulations of the evolution of genetic diseases under simple and complex patterns of inheritance . in each case we found , as we would expect , that genes that encode alleles which reduce fitness are selectively disadvantageous . by comparing the maternal and zygotic patterns of gene expression , we found that maternally expressed alleles are always lost more slowly than offspring alleles with the same additive and dominance effects ( figure 1 ) . maternal gene expression , like dominance , effectively hides copies of the deleterious alleles in fathers , where they are not expressed and hence are hidden from the action of purifying natural selection . these results indicate that genetic diseases caused by gene expression in mothers will have a higher incidence than diseases exclusively caused by gene expression in offspring . we see the same basic findings when purifying selection acts in conjunction with maternal - zygotic epistasis . note that , in all cases , purifying selection acting on maternal effect genes is slower than it is on similarly acting zygotic effect genes . this reinforces and extends our general finding for single genes with only additive and dominance effects to more complex genetic architectures : maternal effect disease - causing genes will be more common in populations than similar zygotically expressed genes . because selection on genes with only maternal effects is weakened , we note that , in some cases , the evolutionary outcome differs for maternal and zygotic effect genes . in particular , with additive - by - dominance epistasis , strong interaction outweighs the additive , deleterious effect and produces permanent polymorphism of the maternal effect gene but not for the zygotic effect gene ( compare the left panels of figures 3(b ) and 4(b ) ) . this is the most dramatic case where the evolutionary equilibrium frequency of a maternal effect gene ( pm ~ 0.36 ) exceeds that of a zygotic gene ( po ~ 0.0 ) of comparable effect . our results show that epistasis between a maternally expressed allele and a zygotically expressed allele can alter the evolution of genetic diseases . in the absence of maternal - zygotic epistasis , purifying selection rapidly removes disease genes with additive or dominant effects ( figure 1 ) . however , in the presence of maternal - zygotic epistasis purifying selection can have a variety of different consequences for the evolution of disease - encoding alleles . for additive - by - additive epistasis , m gene , its deleterious effect on fitness is intensified , while on others it is diminished . because the fitness of the mmoo genotype offspring from mm mothers is high , we might expect selection to favor the spread of m and o alleles ; however , the mm genotypes also have lowered fitness in the absence of the o allele , so it is not clear , a priori , how the frequencies of o and m will change each generation . when we change the sign of iom , mmoo , and mmoo genotypes have the highest fitness , and the mmoo and mmoo genotypes have the lowest fitness . here selection against the o disease susceptibility allele is weaker than with no epistasis because the mmoo and mmoo genotypes have higher fitness which causes the m allele to fix , after which in the mm genetic background , the o allele is no longer favored . note that changes in mean population fitness under negative iom do not increase monotonically and that the rate of change in mean population fitness shifts as genotypes are sorted by selection ( figure 2(b ) middle panel ) . for the m locus , on the most common o locus genotypic background , oo , the fitnesses are 1.1 when they are derived from mm or mm mothers and 1.5 when they are derived from mm mothers . as a result , for negative values of koom , the zygotic effect disease gene is rapidly lost and the maternal effect gene with no additive effect on its own evolves to a frequency of 0.5 ( figure 4(a ) top left panel ) . mean population fitness quickly increases for negative values of epistasis ( figure 4 middle panels ) . though the o disease causing allele has a substantial fitness advantage in an mm background , because the m allele is lost over time as a result of the marginal under - dominance , the o allele is concurrently lost , though more slowly and with greater levels of negative ld than without epistasis ( figure 4(c ) bottom right panel ) . this is the fitness pattern of balancing selection at the o locus . mean population fitness is not as high as in previous cases , because at the polymorphic equilibrium , recombination and segregation produce deleterious genotypes in the population every generation ( figure 5 middle panels ) . with dominance - by - dominance epistasis and joomm equal to + 0.4 ( joomm in table 1 ) , again there is marginal overdominance and heterozygote advantage at the o locus and the population achieves a stable intermediate polymorphism . when only maternal - zygotic interaction determines disease risk , the variety of possible evolutionary outcomes is greater for both genes relative to all cases that we discussed above . at the polymorphic equilibria , no detectable ld exists in a population ( figures 3 , 4 , and 5 right panels ) . this might well represent the current state of genetic risk for maternal - zygotic diseases in the us population . maternal - zygotic epistasis is known to produce half the levels of ld as traditional epistasis . our results indicate that this finding also applies to maternal genetic disease involved in maternal - zygotic interactions . by comparing the middle and lower panels of figures 2 , 3 , 4 , and 5 , it is clear that zygotic genetic diseases generate lower levels of ld than maternal genetic diseases when they exhibit maternal - zygotic epistasis . the implication of this finding is that studies designed to identify maternal - zygotic epistasis up to a particular effect size will need substantially larger sample sizes than studies designed to identify epistasis with effects of the same magnitude . this finding indicates that characterizations of simple genetic diseases might need to be qualified with the possibility that gene interactions drive the disease expression . this finding also indicates that larger , more genetically detailed data sets may provide a deeper understanding of the evolution of genetic disease . all genetic diseases are affected by the same constellation of evolutionary forces : natural selection , mutation , migration , and random genetic drift ; however , the complexity of the pattern of gene expression changes the way these forces work together . the evolutionary equilibrium of simple genetic diseases is thought to be primarily determined by the mutation - selection balance . however , the results of our simulations show that the pattern of gene expression ( maternal versus offspring ) and the form and level of gene interaction can greatly affect the incidence of genetic diseases . diseases genes persist longer and at higher frequencies in a population when they are expressed maternally than when they are expressed zygotically . we have shown that genes which reduce fitness evolve differently under each of the four statistical forms of epistasis . we found that genes with additive main effects on increased risk of disease can be selected for and even brought to fixation by epistasis between maternally and zygotically expressed alleles . this was particularly true when the form of epistasis involves dominance . by tracking gene frequency changes in conjunction with the mean fitness of the population we found that as the frequencies of particular genotypes change in the population , the mean population fitness can shift in nonintuitive ways . we also found that epistasis temporarily generates ld between maternal- and zygotic - effect genes . thus , even with the simplifying assumptions of infinite population size and random mating , maternal / zygotic epistasis had a large confounding effect on the detection of single genes underlying disease etiology . maternal genetic effects and maternal - zygotic epistatic interactions on offspring phenotypes arise because maternal genotype and condition guide early embryonic development [ 7 , 12 , 13 , 46 , 47 ] . for example , it is now well established that birth weight is determined by interactions between the maternal uterine genotype and offspring genotype [ 8 , 10 , 11 , 26 ] . in vertebrate embryos , maternal gene expression establishes the formation of axes and induces developmental genes in the embryo . in mammals , zygotic gene expression becomes the predominant controlling factor in development between the 2 and 8-cell stage , which is called the maternal - zygotic transition ( mzt ) [ 5052 ] . perhaps the best known maternal - zygotic interaction in human disease is the rhesus ( rh ) blood factor in pregnancy . similarly , maternal , zygotic and maternal - zygotic interaction effects in several genes in the folate - dependent homocysteine pathway are associated with neural tube defects . though we emphasize maternal - zygotic epistasis in disease etiology , it should be noted that maternal condition also influences the health of offspring . interactions between the maternal environment and genotype can also affect susceptibility to disease and birth defects . for example , the strength of effect of maternal age on the incidence of obsessive - compulsive behaviours in offspring appears to depend on interactions with the dopamine d1 gene . conditions where epistasis and maternal effect would be more important [ 7 , 21 , 33 ] . as one can clearly see from our simulations , complex genetic disorders have a confusing pattern of inheritance and a nonintuitive evolutionary trajectory . however , the lack of replication of the association between genes and traits is also the signature of epistasis . these complexities do not mean that we can not map genetic basis of complex genetic disorders . our simulations also show that each of the four forms of epistasis affects levels of linkage disequilibrium and , at equilibrium , ld tends toward zero . for particular diseases it is now possible to estimate an individual 's risk of aquiring disease based on a genetic profile which classifies the individual 's ethnicity . there are also experimental methods that are specifically designed to detect gene interactions and maternal - zygotic gene interactions [ 2224 , 35 ] . by stratifying the population and searching for genetic sources of disease in each distinct biologically relevant clade by embracing population subdivision and epistasis we will be more likely to determine the genetic basis of birth defects and disease .

rna interference ( rnai ) achieved by double - stranded rna ( dsrna ) mediated sequence - specific breakdown of the mrna ( fire et al , 1998 ; montgomery and fire , 1998 ; elbashir et al , 2001a ) . it is an evolutionarily conserved phenomenon initially characterized in c. elegans and drosophila , but also occurs in plants , fungi and mammals ( boshare and labouesse , 2000 ) . mechanistic studies have indicated that the effector molecules of the rnai process is not the long dsrna itself , but a family of short double - stranded rna fragments ( 19 - 21 base pairs in length ) derived through cleavage of the long dsrna by rnase iii enzyme(s ) ( elbashir et al , 2001b ) . the short double - stranded rna fragments generated as such , as well as through chemical synthesis or vector - based expression , are collectively referred to as small interfering rna ( sirna ) . the long dsrna can not be used in mammalian cells for gene silencing due to the fact that introduction of long dsrna elicits a strong antiviral response that obscures any gene - specific silencing effect . much of this response is caused by activation of the dsrna - dependent protein kinase pkr , which phosphorylates and inactivates the translation initiation factor eif2a ( proud , 1995 ) . introduction of sirna into mammalian cells was found not to elicit the antiviral response to such an extreme level , but still can result in efficient gene knockdown ( sledz et al , 2003 ; elbashir et al , 2001b ) . short sirna molecules can be prepared by direct chemical synthesis or transcription driven by rna polymerase promoters . in term of high - throughput applications , vector - based strategies are favoured because such strategies enjoy the advantages of much lower cost and ability to regenerate . the only large - scale synthetic sirna library that was produced so far is the genome - wide sirna collection made for novartis by qiagen ( hilden , germany ) and dhamarcon ( lafayette , usa ) . transfection efficiency of sirna into cells depends on cell types and the rnai effect of synthetic sirna only sustain for a period of time . the advantage of the vector - based sirna is the capability of removing those cells that are not transfected with the plasmids by selecting the transfected cells with antibiotic resistant genes . virus vectors also enable the delivery of sirna expression cassettes into cells with higher transfection efficiency , and in case of lentivirus and retrovirus , it is easy to make stable knockdown cells by integration into the genome . if one wants to use sirna expression vectors as a combinatorial library , the viral vectors would be most suitable . in the current review , we will focus on the vector - based sirna libraries and the methods to produce them . the use of pol iii promoters in vectors for encoding sirna was pioneered by a number of groups in early 2002 ( brummelkamp et al , 2002 ; miyagishi and taira , 2002 ; sui et al , 2002 ; xia et al , 2002 ; and yu et al , 2002 ) . the vectors contain a single pol iii promoter followed by a segment of dna that encodes a short hairpin rna ( shrna ) ( figure 1a ) . the shrna closely resembles the structure of micrornas and will be integrated into the risc complex in the same way as the normal sirna does . the backbones of these constructs were initially plasmids , but the grafting the shrna expressing cassettes into different viral vectors such as lentivirus , retrovirus and even adenovirus also explored successfully to facilitate the transfection of cells that were hard to penetrate , such as primary cells and suspension cells ( li et al , 2003 ; hosono et al , 2004 ; devroe and silver , 2004 ) diagrams of three general ways of encoding sirna in a plasmid or viral vector . a , a hairpin - like sirna ( shrna ) is generated by transcription from a segment of dna ( 50 - 60 bp in length ) driven by a single promoter for pol iii rna polymerases . b , expression of a sirna from a single 19-mer dna fragment through transcription driven two opposing promoters for pol iii rna polymerases . c , expression of a sirna from a two separate 19-mer dna fragments through transcription driven two tandem promoters for pol iii rna polymerases . another strategy of encoding sirna that has gain more popularity is a vector that contains dual pol iii promoters arranged in a convergent manner that drive the expression of two short complementary rna strand from a single 19 bp dna region ( figure 1b ) ( chen et al , 2005 ; zheng et al 2004 ) . while being as effective as the shrna encoding vectors , these dual - promoter ( genebuster ) vectors are first of all more robust for cloning both because of the lack of secondary structure in the sirna coding region , and much lower level of dna synthesis errors due to the shorter sequence , and they have tremendous advantages in constructing large scale sirna libraries in a combinatorial way , as will be discussed below . a third method is a technique using two tandem pol iii promoters to drive the expression of sirna from separate 19 mer dna fragments ( figure 1c ) ( lee et al , 2002 ) . this method might not be widely used due to the fact that construction of such a plasmid is considerably more cumbersome than the other two methods . this method as well as the shrna method , however , offers the possibility to integrate mismatches into the resulting sirna , thus possibly enhance the efficacy of the sirna or increase the frequency of effective sirna . h1 and u6 promoters from human and mouse are the most widely used pol iii promoters in the sirna vector construction . other promoters that have been used include promoters of trna and trna ( oshima et al , 2003 ; boden et al , 2003 ) . all these pol iii promoters are active in a large variety of cells , and appear to perform similarly in different species . one interesting feature of the pol iii promoters is that they can be easily transformed into inducible promoters ( van de wetering et al , 2003 ) . in addition to plasmid - based systems , pcr - derived sirna expression cassettes based on the single - promoter system have been shown to efficiently suppress gene activities in transfected cells ( castanotto et al , 2002 ) . other promoters that have been used for sirna production include t7 promoter and cmv promoter ( xia et al , 2002 ; holle et al , 2004 ) . since t7 promoter does not work in mammalian cells , strategies employing t7 or similar promoters normally can only support sirna production in test tubes , but not within cells . people have overcome this shortcoming by either pre - load the promoter with cmv promoter is a much stronger promoter compared to other pol iii promoters , and this can allow more sirna molecules to be transcribed from a given amount of dna vector . but since cmv promoter is an rna polymerase ii ( pol ii ) promoter , the resulting transcripts are normally capped at the 5-end and tailed at the 3-end with a long poly ( a ) sequence . due to the potentially higher degree of flexibility in enabling different spatial and temporal expression patterns , these types of vector - based sirna strategies might be much more useful for in vivo research purposes and for sirna based gene - therapy at a later stage although pol iii based vectors seem to be able to satisfy most of the gene knock - down needs in cultured cells . one of the critical initial steps in the high throughput sirna application is the formation of a physical sirna library , which many people also refer to as a sirna library . to generate a sirna library , one critical factor to consider is the design of sirna sequence . since the first guideline for sirna design was published by tuschl and colleagues ( elbashir et al , 2001b ) several studies of currently available information for effective and non - effective sirna have been carried out , and these have resulted in different sirna design tools that based on only partially overlapping criteria ( henschel et al , 2004 ; wang and mu , 2004 ; chalk et al , 2005 ) . one common drawback of all currently existing sirna design software is that they are all based on limited data sets about sirna efficacy . it is anticipated that , with the development of high throughput sirna construction and validation methods , algorithms based on randomly chosen sirna will emerge to provide better predictions for functional sirna . although a sirna library can be constructed by chemical synthesis , as done by qiagen for novartis and more recently by dharmacon , most of the sirna libraries covering more than a couple of thousand of genes are done by vector - based approaches . this is because , i ) a vector - based sirna library can be regenerated with minimal effort , ii ) the constructions can be carried out in a standard molecular biology laboratory , and iii ) a vector - based sirna library can be much more affordable than a chemically synthesized library . three large - scale sirna libraries have recently been constructed by academic researchers ( paddison et al , 2004 ; berns et al 2004 , michiels et al , 2002 ) and galapagos has generated the first large scale adenovirus based sirna library in the industrial sector . multiple efforts have been initiated since then by commercial companies , and notably among them , genordia ab , a sweden - based company will most likely be able to deliver the first sirna library that covers a whole mammalian genome in 2005 . hannon and colleagues have constructed a large - scale sirna library targeting a selected panel of human and mouse genes using a hairpin sirna encoding cassette ( berns et al , 2004 ) . the sirna - encoding cassette was first cloned into a pshag - magic donor vector . although in most sirna applications people has chosen to use 19 - 21mer sirna , this particular sirna library encoded sirna expression unit that contained 29 nucleotides , simple loop structure and u6 promoter . it was found that 29-nucleotide hairpins were more effective than shorter hairpin sirna in this study . since the efficacy of short sirna is well established , this interesting finding might need more independent confirmation before being adopted as a guideline for sirna libraries construction . also concerns have been raised for possibly higher level of off - target hits as well as induction of antiviral responses . totally 28,659 shrnas targeting 9,610 human genes and 9,119 shrnas targeting 5,563 mouse genes were included in the hannon sirna library . the extensive sequence verification was warranted because 2575% of cloned shrnas were found to contain significant mutations that arose during chemical synthesis . 2 ) only coding sequences were targeted , and each shrna was chosen such that it contained not more than 26 bp matches to any other gene . it should be cautioned that recent studies have demonstrated that for processed sirna sequence identity up to 16 - 17 might already allow the sirna to have off - target effects . 3 ) where possible , shrnas were designed to have sequence identity to the mouse orthologue of the targeted gene . in terms of gene family representation , this library covers about 85% of all known kinases and phosphatases by three or more sirnas per gene . most other functional classes contain between 3060% coverage with three or more hairpins , and 80% of genes by at least one sequence - verified hairpin . a unique 60-nuclotide dna bar code has been integrated to each vector to allow identification of shrnas in populations of virally transduced cells through hybridization methods . the human rnai library ( the nki library ) constructed by bernards and colleagues contains 23,742 sirna vectors that cover 7,914 human genes ( michiels et al , 2002 ) . gene within this collection include components of major cellular pathways , such as the cell cycle , transcription regulation , stress signaling , signal transduction and important biological processes such as biosynthesis , proteolysis and metabolism . in addition , genes implicated in cancer and other diseases are included in the library . the sirna encoding dna fragments were cloned in a high - throughput fashion into pretrosuper ( prs ) , a previously reported retroviral vector containing the shrna expression cassette . this is one of the first large scale sirna collection in the academic sector and in the validation of this library , researchers have achieved on average 70% expression inhibition for approximately 70% of the genes in the library using a pool of three knockdown vectors against a single gene . compared to sirna library made in plasmid vectors , sirna librarys constructed in viral vectors has to be packaged and mass produced before being used in any knock - down experiments . these might comprise a major limitation in the application of viral vector based sirna libraries in very large - scale screening . schultz and his colleagues have developed a pcr based system to generate sirna for about 8000 genes at 2-sirna / gene coverage using an opposing promoter approach . the sirna has been used to discover novel members of the nf - kb pathway ( zheng et al , 2004 ) . a single - step pcr protocol was used to produce sirna expression cassettes in a high - throughput fashion and showed that these sirna expression cassettes can also specifically and efficiently suppress the expression of both transfected and endogenous genes . this single - step pcr approach is efficient and cost - effective and makes high - throughput production of sirna expression cassette libraries for genome - wide functional gene annotation practical . the arrayed library was used to screen for genes involved in the nf - b signalling pathway that controls many diverse cellular processes including growth , development , inflammation , immune response , apoptosis , and oncogenesis . notably , one of the first viral based sirna libraries was generated by an industrial player , galapagos genomics . this adenoviral sirna library contains knock - down reagents for over 4,900 human druggable transcripts , including g - protein coupled receptors ( gpcrs ) , ion channels , nuclear hormone receptors , kinases , phosphodiesterases and other druggable transcripts . this three - fold redundancy in sirna sequences gives a 90% probability that the mrna for any given gene will be knocked - down by at least 75% . such a loss - of - function gene collection provides an ideal complement to the arrayed adenoviral gain - of - function libraries from the same source . other characteristics of this sirna library include : 1 ) the sirna expression cassette is based on the human u6 promoter , a strong , ubiquitously active promoter that lacks essential promoter elements within the transcribed region . 2 ) the u6 promoter - based expression cassette was cloned in an adenoviral adapter plasmid at the position of the e1 region . the adenoviral vectors were based on adenovirus serotype 5 with deleted e1 and e2a regions . 3 ) the viral vectors were found to be able to express processed hairpin rnas for at least 10 d. 4 ) the adenoviral vectors used have a broad tropism and very efficiently infect many different primary cell types including primary keratinocytes and primary synoviocytes , two cell types that are notoriously difficult to transfect . the use of sirna tools in primary cells has been hampered by inefficient transfection protocols . advantages in the transfection aspect are of critical importance for some applications , and thus counter - balance the burden imposed by the cumbersome viral handling . in a conventional sirna application , a target gene is chosen first , and then sirna sequences are designed against the transcript of this gene . in this approach , the choice of gene , and choice of targeting sites are inevitably biased due to the limited number of genes and sites that can be included in a particular study . the bias also arise inevitably from the fact that we have just started to get a relatively complete picture of the part of the mouse transcriptome that has a poly ( a ) tail , and knowing little about transcripts that do not have a such a tail . the development of sirna expressing libraries that encode substantially all permutations of 19-mer sirna thus become an appealing approach to allow the use of it as a single affordable tool for un - biased genome - wide screening . towards this direction , sirna libraries have been synthesized by integration of 19-mer fully randomized dna sequences into different sirna encoding vectors ( chen et al , 2005 ) . such vectors should have theoretical complexity of 2.7510 in order to encode all the permutations of sirna . this is a complexity that , albeit being reachable , will impose significant burden on the manipulation of the library and use of it for screening . lucky or not , the sirna was actually found to be tolerant to mismatches towards its terminus . this makes it reasonable to assume that a library will about 1 10 complexity will cover all possible sirna . such a complexity then comes in a range that is practical for plasmid library construction and maintenance . the library was used for phenotype - driven screening using cell proliferation as a model , and multiple sirna that can induce significant enhancement of cell growth were identified . another alternative approach is to generate semi - random sirna library from an mrna source . in this case , a specific mrna population is converted into double stranded cdna , which will be cut into short dna fragments that can then inserted into the vectors for encoding sirna . the advantage of this type of libraries is that it will only have a complexity of in the neighborhood of 10 but already be able to cover a substantial part of the genome . the limiting aspect of the approach is that a library made in this way will only be useful for the same organism , or even only for the same cell type , whereas other fully randomized libraries can be used in all cell types and all relevant organisms . there have been several reports ( luo et al , 2004 ; sen et al , 2004 ; shirane et al , 2004 ) for the construction of such semi - random sirna libraries and one of these is briefly described here as an example ( sen et al , 2004 ) . in this method , mrna is converted into double - stranded cdna mixture that was further cleaved with a mixture of five restriction endonucleases , each of which leaves a 5-cg overhang but has a different 4-bp recognition sequence . this combination covers 6 of 256 possible 4-bp sequences , resulting in one cleavage every 43 bp . this hairpin has a recognition site for mmei , a type ii restriction endonuclease that cleaves 2021 bp away from its recognition sequence and leaves a two - base 5 overhang . a hairpin linker is then ligated to either the 5 or 3 end of the double - stranded cdna fragment . the 5 and 3 ends of the extended hairpin become ligated to the extension sense oligonucleotide ( green ) and the extension antisense the extension priming oligonucleotide serves as primer for dna elongation by the highly processive bst dna polymerase large fragment , which lacks a 53 exonuclease domain . the process has been used to construct a sirna library from a mouse embryo cdna library . twenty - seven clones were chosen at random for sequencing , all of which revealed matches against known mouse genes . northern blotting with probes corresponding to their mrna targets demonstrated that each of the cdna - derived inserts produced the expected processed 21-base sirnas and these clones caused specific reductions in their corresponding target mrna . genome - wide rnai screens were explored in c. elegans using libraries of in vitro - transcribed long dsrnas or simply the bacteria that express dsrna . for example , an rnai feeding library consisting of 16,757 bacterial clones covering majority of the worm genome was constructed ( fraser et al , 2000 ; kamath et al , 2003 ) . upon feeding the worms , these clones generated transient loss - of - function phenotypes for many genes by inactivating the target genes via rnai . genome - wide rnai approaches have been used successfully for phenotype - based screens also in drosophila melanogaster . in part , these successes derived from the availability of convenient and inexpensive methods for producing and introducing dsrna . to date , the use of rnai libraries in mammalian systems is becoming well accepted but most of the work has been limited to specific protein families because of technical and practical issues associated with generating large synthetic sirna and/or vector - based short hairpin rna ( shrna ) expression libraries , but the situation is rapidly improving with the technology development mentioned above . screening schemes using large - scale sirna libraries are understandably diverse due to differences in aims , vector systems , and read - out methods . the five case studies presented below could give us a hint about how such libraries can be deployed in hts . retroviral vectors can be used to transfect a large variety of cell types , and its short - coming on the handling side can be well balanced by using it in a combinatorial manner , e.g. , in libraries . work done by berns et al ( 2004 ) has provided a typical example of such applications in a medium scale . in this study , retroviral vectors were generated in pools and then the pools were used to transfect bj - tert - tslt cells at 32 c . after two days ' incubation at 32 c , the incubation temperature was increased to 39 c for proliferating colony screening . in the first - round screening , six pools of infected cells were found able to form colonies at 39c . these shrna inserts were then re - cloned from these colonies , and their identity was established by dna sequence analysis . only those shrna inserts that were presented in multiple independently derived colonies were further analyzed in the second - round screening in bj - tert - tslt fibroblast . using this approach , shrnas against six genes were identified that could suppress the temperature - shift - induced proliferation arrest in the bj - tert - tslt cells . remarkably one identified gene was p53 , which underscores the quality of the nki library . knockdown of other five identified genes also mediated escape from proliferation arrest in bj - tert - tslt cells when tested in the absence of retroviral vector , suggesting that inhibition of these genes allowed bypass of the p53 response . in screening as such , it would always be a concern whether the change in read - out is a consequence of on - target hit or off - target hits . in this case , it was found that two of the three shrnas targeting a single gene could mediate escape from growth arrest for three of the five newly identified genes . this could be considered as a confirmation that the effects of the sirnas were on - target. for genes that only have a single sirna hit , normally additional sirna need to be designed to confirm any phenotypical changes observed with the initial sirna clone . in a similar screening , the 26s proteasome is the major non - lysosomal protease in eukaryotic cells . to search the library for shrnas that compromise proteasome function , a reporter assay was used in which a fluorescent protein ( zoanthus green fluorescent , zsgreen ) was coupled to a well characterized degradation signal , the mouse ornithine decarboxylase ( modc ) gene . another plasmid encoded discosoma red fluorescent protein ( dsred ) was used as a normalization control for transfection . individual transfection of 6,712 shrnas revealed approximately 100 rnai constructs that increased the accumulation of zsgreen modc , of which 22 corresponded to 15 known proteasome subunits . as mentioned above , a sirna expression cassette library targeting 8,000 human genes from the public unigene library were generated in schultz lab in scripps institute in a vector containing dual pol iii promoters , with two targeting sequences per gene zheng et al , 2004 ) . to screen for regulators of nf - kb transcriptional activation by tnfa , the pnf - b - luc reporter plasmid was cotransfected with individual sirna expression cassettes into hek293 t cells . of 94 genes identified in the initial screening , 20 hits were further assessed by introducing additional sirnas , and 17 of them seemed to be genuine on - target hits . among them , eight are known to be involved in the nf - kb signalling . of the remaining genes , some might be involved in cellular processes that indirectly affect the nf - b pathway , whereas others might have previously unrecognized roles in the nf - b signalling pathway . all screening work mentioned above were done in microtitre plates , which will remain a major screening platform in the future . cell microarrays were created to provide much higher throughput than microtitre plates for sirna based screening . cell microarray was first described by ziauddin and sabatini ( 2001 ) who demonstrated that cells grown on a glass substrate could take up dna lipid complexes that had been deposited on the slide before cells were plated . cells became transfected in situ , with defined spots of transfected cells localized over the printed dnas . cell microarrays represent a novel alternative to classical approaches to phenotype - based assays in mammalian cells , at least for some of the easy - to - transfect cells such as hek 293 t , imr90/e1a , nih 3t3 , and hela cells . in this screening format , sirna ( plasmids or pcr cassettes ) are spotted onto glass slides together with transfection reagents ( normally with gelatin too for immobilization purpose ) . when cells are overlaid on top of a spot on the slides , corresponding sirna vector will be taken into the cells so that sirna will be expressed , and the effect of the sirna can be read out in high throughput according to the phenotypes or the reporter systems used . the initial proof - of principle was done by sirnas corresponding to the enhanced green fluorescent protein ( gfp ) gene ( egfp ) on microscope slides , and plated hela cells permanently expressing a destabilized version of egfp on top of the slides . the result showed the cellular uptake , reverse transfection , of rhodamine - tagged sirna ( rh - egfp ) , and silencing of gfp expression in cells . an experimental comparison was provided by carrying out , side by side , the array - based and microtitre plate screening of sirna that can perturb proteosome functions . the result showed that the array method does work in screening for sirna that can manipulate endogenous genes , and likely to provide data that is even better than the classic microtitre plate based screening process . advantage of the method is that large number of sirna can be rapidly screening through a given phenotype or reporter system . the limitation of the method is , in additional to the need of complex instrumentation , that many phenotypes ca n't be fit into such an array - based read - out format . short sirna molecules can be prepared by direct chemical synthesis or transcription driven by rna polymerase promoters . in term of high - throughput applications , vector - based strategies are favoured because such strategies enjoy the advantages of much lower cost and ability to regenerate . the only large - scale synthetic sirna library that was produced so far is the genome - wide sirna collection made for novartis by qiagen ( hilden , germany ) and dhamarcon ( lafayette , usa ) . transfection efficiency of sirna into cells depends on cell types and the rnai effect of synthetic sirna only sustain for a period of time . the advantage of the vector - based sirna is the capability of removing those cells that are not transfected with the plasmids by selecting the transfected cells with antibiotic resistant genes . virus vectors also enable the delivery of sirna expression cassettes into cells with higher transfection efficiency , and in case of lentivirus and retrovirus , it is easy to make stable knockdown cells by integration into the genome . if one wants to use sirna expression vectors as a combinatorial library , the viral vectors would be most suitable . in the current review , we will focus on the vector - based sirna libraries and the methods to produce them . the use of pol iii promoters in vectors for encoding sirna was pioneered by a number of groups in early 2002 ( brummelkamp et al , 2002 ; miyagishi and taira , 2002 ; sui et al , 2002 ; xia et al , 2002 ; and yu et al , 2002 ) . the vectors contain a single pol iii promoter followed by a segment of dna that encodes a short hairpin rna ( shrna ) ( figure 1a ) . the shrna closely resembles the structure of micrornas and will be integrated into the risc complex in the same way as the normal sirna does . the backbones of these constructs were initially plasmids , but the grafting the shrna expressing cassettes into different viral vectors such as lentivirus , retrovirus and even adenovirus also explored successfully to facilitate the transfection of cells that were hard to penetrate , such as primary cells and suspension cells ( li et al , 2003 ; hosono et al , 2004 ; devroe and silver , 2004 ) diagrams of three general ways of encoding sirna in a plasmid or viral vector . a , a hairpin - like sirna ( shrna ) is generated by transcription from a segment of dna ( 50 - 60 bp in length ) driven by a single promoter for pol iii rna polymerases . b , expression of a sirna from a single 19-mer dna fragment through transcription driven two opposing promoters for pol iii rna polymerases . c , expression of a sirna from a two separate 19-mer dna fragments through transcription driven two tandem another strategy of encoding sirna that has gain more popularity is a vector that contains dual pol iii promoters arranged in a convergent manner that drive the expression of two short complementary rna strand from a single 19 bp dna region ( figure 1b ) ( chen et al , 2005 ; zheng et al 2004 ) . while being as effective as the shrna encoding vectors , these dual - promoter ( genebuster ) vectors are first of all more robust for cloning both because of the lack of secondary structure in the sirna coding region , and much lower level of dna synthesis errors due to the shorter sequence , and they have tremendous advantages in constructing large scale sirna libraries in a combinatorial way , as will be discussed below . a third method is a technique using two tandem pol iii promoters to drive the expression of sirna from separate 19 mer dna fragments ( figure 1c ) ( lee et al , 2002 ) . this method might not be widely used due to the fact that construction of such a plasmid is considerably more cumbersome than the other two methods . this method as well as the shrna method , however , offers the possibility to integrate mismatches into the resulting sirna , thus possibly enhance the efficacy of the sirna or increase the frequency of effective sirna . h1 and u6 promoters from human and mouse are the most widely used pol iii promoters in the sirna vector construction . other promoters that have been used include promoters of trna and trna ( oshima et al , 2003 ; boden et al , 2003 ) . all these pol iii promoters are active in a large variety of cells , and appear to perform similarly in different species . one interesting feature of the pol iii promoters is that they can be easily transformed into inducible promoters ( van de wetering et al , 2003 ) . this feature is very important for some functional studies and screening . in addition to plasmid - based systems , pcr - derived sirna expression cassettes based on the single - promoter system have been shown to efficiently suppress gene activities in transfected cells ( castanotto et al , 2002 ) . other promoters that have been used for sirna production include t7 promoter and cmv promoter ( xia et al , 2002 ; holle et al , 2004 ) . since t7 promoter does not work in mammalian cells , strategies employing t7 or similar promoters normally can only support sirna production in test tubes , but not within cells . people have overcome this shortcoming by either pre - load the promoter with cmv promoter is a much stronger promoter compared to other pol iii promoters , and this can allow more sirna molecules to be transcribed from a given amount of dna vector . but since cmv promoter is an rna polymerase ii ( pol ii ) promoter , the resulting transcripts are normally capped at the 5-end and tailed at the 3-end with a long poly ( a ) sequence . due to the potentially higher degree of flexibility in enabling different spatial and temporal expression patterns , these types of vector - based sirna strategies might be much more useful for in vivo research purposes and for sirna based gene - therapy at a later stage although pol iii based vectors seem to be able to satisfy most of the gene knock - down needs in cultured cells . one of the critical initial steps in the high throughput sirna application is the formation of a physical sirna library , which many people also refer to as a sirna library . to generate a sirna library , one critical factor to consider is the design of sirna sequence . since the first guideline for sirna design was published by tuschl and colleagues ( elbashir et al , 2001b ) several studies of currently available information for effective and non - effective sirna have been carried out , and these have resulted in different sirna design tools that based on only partially overlapping criteria ( henschel et al , 2004 ; wang and mu , 2004 ; chalk et al , 2005 ) . one common drawback of all currently existing sirna design software is that they are all based on limited data sets about sirna efficacy . it is anticipated that , with the development of high throughput sirna construction and validation methods , algorithms based on randomly chosen sirna will emerge to provide better predictions for functional sirna . although a sirna library can be constructed by chemical synthesis , as done by qiagen for novartis and more recently by dharmacon , most of the sirna libraries covering more than a couple of thousand of genes are done by vector - based approaches . this is because , i ) a vector - based sirna library can be regenerated with minimal effort , ii ) the constructions can be carried out in a standard molecular biology laboratory , and iii ) a vector - based sirna library can be much more affordable than a chemically synthesized library . three large - scale sirna libraries have recently been constructed by academic researchers ( paddison et al , 2004 ; berns et al 2004 , michiels et al , 2002 ) and galapagos has generated the first large scale adenovirus based sirna library in the industrial sector . multiple efforts have been initiated since then by commercial companies , and notably among them , genordia ab , a sweden - based company will most likely be able to deliver the first sirna library that covers a whole mammalian genome in 2005 . hannon and colleagues have constructed a large - scale sirna library targeting a selected panel of human and mouse genes using a hairpin sirna encoding cassette ( berns et al , 2004 ) . the sirna - encoding cassette was first cloned into a pshag - magic donor vector . although in most sirna applications people has chosen to use 19 - 21mer sirna , this particular sirna library encoded sirna expression unit that contained 29 nucleotides , simple loop structure and u6 promoter . it was found that 29-nucleotide hairpins were more effective than shorter hairpin sirna in this study . since the efficacy of short sirna is well established , this interesting finding might need more independent confirmation before being adopted as a guideline for sirna libraries construction . also concerns have been raised for possibly higher level of off - target hits as well as induction of antiviral responses . totally 28,659 shrnas targeting 9,610 human genes and 9,119 shrnas targeting 5,563 mouse genes were included in the hannon sirna library . the extensive sequence verification was warranted because 2575% of cloned shrnas were found to contain significant mutations that arose during chemical synthesis . 2 ) only coding sequences were targeted , and each shrna was chosen such that it contained not more than 26 bp matches to any other gene . it should be cautioned that recent studies have demonstrated that for processed sirna sequence identity up to 16 - 17 might already allow the sirna to have off - target effects . 3 ) where possible , shrnas were designed to have sequence identity to the mouse orthologue of the targeted gene . in terms of gene family representation , this library covers about 85% of all known kinases and phosphatases by three or more sirnas per gene . most other functional classes contain between 3060% coverage with three or more hairpins , and 80% of genes by at least one sequence - verified hairpin . a unique 60-nuclotide dna bar code has been integrated to each vector to allow identification of shrnas in populations of virally transduced cells through hybridization methods . the human rnai library ( the nki library ) constructed by bernards and colleagues contains 23,742 sirna vectors that cover 7,914 human genes ( michiels et al , 2002 ) . gene within this collection include components of major cellular pathways , such as the cell cycle , transcription regulation , stress signaling , signal transduction and important biological processes such as biosynthesis , proteolysis and metabolism . in addition , genes implicated in cancer and other diseases are included in the library . the sirna encoding dna fragments were cloned in a high - throughput fashion into pretrosuper ( prs ) , a previously reported retroviral vector containing the shrna expression cassette . this is one of the first large scale sirna collection in the academic sector and in the validation of this library , researchers have achieved on average 70% expression inhibition for approximately 70% of the genes in the library using a pool of three knockdown vectors against a single gene . compared to sirna library made in plasmid vectors , sirna librarys constructed in viral vectors has to be packaged and mass produced before being used in any knock - down experiments . these might comprise a major limitation in the application of viral vector based sirna libraries in very large - scale screening . schultz and his colleagues have developed a pcr based system to generate sirna for about 8000 genes at 2-sirna / gene coverage using an opposing promoter approach . the sirna has been used to discover novel members of the nf - kb pathway ( zheng et al , 2004 ) . a single - step pcr protocol was used to produce sirna expression cassettes in a high - throughput fashion and showed that these sirna expression cassettes can also specifically and efficiently suppress the expression of both transfected and endogenous genes . this single - step pcr approach is efficient and cost - effective and makes high - throughput production of sirna expression cassette libraries for genome - wide functional gene annotation practical . the arrayed library was used to screen for genes involved in the nf - b signalling pathway that controls many diverse cellular processes including growth , development , inflammation , immune response , apoptosis , and oncogenesis . notably , one of the first viral based sirna libraries was generated by an industrial player , galapagos genomics . this adenoviral sirna library contains knock - down reagents for over 4,900 human druggable transcripts , including g - protein coupled receptors ( gpcrs ) , ion channels , nuclear hormone receptors , kinases , phosphodiesterases and other druggable transcripts . this three - fold redundancy in sirna sequences gives a 90% probability that the mrna for any given gene will be knocked - down by at least 75% . such a loss - of - function gene collection provides an ideal complement to the arrayed adenoviral gain - of - function libraries from the same source . other characteristics of this sirna library include : 1 ) the sirna expression cassette is based on the human u6 promoter , a strong , ubiquitously active promoter that lacks essential promoter elements within the transcribed region . 2 ) the u6 promoter - based expression cassette was cloned in an adenoviral adapter plasmid at the position of the e1 region . the adenoviral vectors were based on adenovirus serotype 5 with deleted e1 and e2a regions . 3 ) the viral vectors were found to be able to express processed hairpin rnas for at least 10 d. 4 ) the adenoviral vectors used have a broad tropism and very efficiently infect many different primary cell types including primary keratinocytes and primary synoviocytes , two cell types that are notoriously difficult to transfect . the use of sirna tools in primary cells has been hampered by inefficient transfection protocols . advantages in the transfection aspect are of critical importance for some applications , and thus counter - balance the burden imposed by the cumbersome viral handling . in a conventional sirna application , a target gene is chosen first , and then sirna sequences are designed against the transcript of this gene . in this approach , the choice of gene , and choice of targeting sites are inevitably biased due to the limited number of genes and sites that can be included in a particular study . the bias also arise inevitably from the fact that we have just started to get a relatively complete picture of the part of the mouse transcriptome that has a poly ( a ) tail , and knowing little about transcripts that do not have a such a tail . the development of sirna expressing libraries that encode substantially all permutations of 19-mer sirna thus become an appealing approach to allow the use of it as a single affordable tool for un - biased genome - wide screening . towards this direction , sirna libraries have been synthesized by integration of 19-mer fully randomized dna sequences into different sirna encoding vectors ( chen et al , 2005 ) . such vectors should have theoretical complexity of 2.7510 in order to encode all the permutations of sirna . this is a complexity that , albeit being reachable , will impose significant burden on the manipulation of the library and use of it for screening . lucky or not , the sirna was actually found to be tolerant to mismatches towards its terminus . this makes it reasonable to assume that a library will about 1 10 complexity will cover all possible sirna . such a complexity then comes in a range that is practical for plasmid library construction and maintenance . the library was used for phenotype - driven screening using cell proliferation as a model , and multiple sirna that can induce significant enhancement of cell growth were identified . another alternative approach is to generate semi - random sirna library from an mrna source . in this case , a specific mrna population is converted into double stranded cdna , which will be cut into short dna fragments that can then inserted into the vectors for encoding sirna . the advantage of this type of libraries is that it will only have a complexity of in the neighborhood of 10 but already be able to cover a substantial part of the genome . the limiting aspect of the approach is that a library made in this way will only be useful for the same organism , or even only for the same cell type , whereas other fully randomized libraries can be used in all cell types and all relevant organisms . there have been several reports ( luo et al , 2004 ; sen et al , 2004 ; shirane et al , 2004 ) for the construction of such semi - random sirna libraries and one of these is briefly described here as an example ( sen et al , 2004 ) . in this method , mrna is converted into double - stranded cdna mixture that was further cleaved with a mixture of five restriction endonucleases , each of which leaves a 5-cg overhang but has a different 4-bp recognition sequence . this combination covers 6 of 256 possible 4-bp sequences , resulting in one cleavage every 43 bp . this hairpin has a recognition site for mmei , a type ii restriction endonuclease that cleaves 2021 bp away from its recognition sequence and leaves a two - base 5 overhang . a hairpin linker is then ligated to either the 5 or 3 end of the double - stranded cdna fragment . the 5 and 3 ends of the extended hairpin become ligated to the extension sense oligonucleotide ( green ) and the extension antisense the extension priming oligonucleotide serves as primer for dna elongation by the highly processive bst dna polymerase large fragment , which lacks a 53 exonuclease domain . the process has been used to construct a sirna library from a mouse embryo cdna library . twenty - seven clones were chosen at random for sequencing , all of which revealed matches against known mouse genes . northern blotting with probes corresponding to their mrna targets demonstrated that each of the cdna - derived inserts produced the expected processed 21-base sirnas and these clones caused specific reductions in their corresponding target mrna . genome - wide rnai screens were explored in c. elegans using libraries of in vitro - transcribed long dsrnas or simply the bacteria that express dsrna . for example , an rnai feeding library consisting of 16,757 bacterial clones covering majority of the worm genome was constructed ( fraser et al , 2000 ; kamath et al , 2003 ) . upon feeding the worms , these clones generated transient loss - of - function phenotypes for many genes by inactivating the target genes via rnai . genome - wide rnai approaches have been used successfully for phenotype - based screens also in drosophila melanogaster . in part , these successes derived from the availability of convenient and inexpensive methods for producing and introducing dsrna . to date , the use of rnai libraries in mammalian systems is becoming well accepted but most of the work has been limited to specific protein families because of technical and practical issues associated with generating large synthetic sirna and/or vector - based short hairpin rna ( shrna ) expression libraries , but the situation is rapidly improving with the technology development mentioned above . screening schemes using large - scale sirna libraries are understandably diverse due to differences in aims , vector systems , and read - out methods . the five case studies presented below could give us a hint about how such libraries can be deployed in hts . retroviral vectors can be used to transfect a large variety of cell types , and its short - coming on the handling side can be well balanced by using it in a combinatorial manner , e.g. , in libraries . work done by berns et al ( 2004 ) has provided a typical example of such applications in a medium scale . in this study , retroviral vectors were generated in pools and then the pools were used to transfect bj - tert - tslt cells at 32 c . after two days ' incubation at 32 c , the incubation temperature was increased to 39 c for proliferating colony screening . in the first - round screening , six pools of infected cells were found able to form colonies at 39c . these shrna inserts were then re - cloned from these colonies , and their identity was established by dna sequence analysis . only those shrna inserts that were presented in multiple independently derived colonies were further analyzed in the second - round screening in bj - tert - tslt fibroblast . using this approach , shrnas against six genes were identified that could suppress the temperature - shift - induced proliferation arrest in the bj - tert - tslt cells . remarkably one identified gene was p53 , which underscores the quality of the nki library . knockdown of other five identified genes also mediated escape from proliferation arrest in bj - tert - tslt cells when tested in the absence of retroviral vector , suggesting that inhibition of these genes allowed bypass of the p53 response . in screening as such , it would always be a concern whether the change in read - out is a consequence of on - target hit or off - target hits . in this case , it was found that two of the three shrnas targeting a single gene could mediate escape from growth arrest for three of the five newly identified genes . this could be considered as a confirmation that the effects of the sirnas were on - target. for genes that only have a single sirna hit , normally additional sirna need to be designed to confirm any phenotypical changes observed with the initial sirna clone . in a similar screening , the 26s proteasome is the major non - lysosomal protease in eukaryotic cells . to search the library for shrnas that compromise proteasome function , a reporter assay was used in which a fluorescent protein ( zoanthus green fluorescent , zsgreen ) was coupled to a well characterized degradation signal , the mouse ornithine decarboxylase ( modc ) gene . another plasmid encoded discosoma red fluorescent protein ( dsred ) was used as a normalization control for transfection . individual transfection of 6,712 shrnas revealed approximately 100 rnai constructs that increased the accumulation of zsgreen modc , of which 22 corresponded to 15 known proteasome subunits . as mentioned above , a sirna expression cassette library targeting 8,000 human genes from the public unigene library were generated in schultz lab in scripps institute in a vector containing dual pol iii promoters , with two targeting sequences per gene zheng et al , 2004 ) . to screen for regulators of nf - kb transcriptional activation by tnfa , the pnf - b - luc reporter plasmid was cotransfected with individual sirna expression cassettes into hek293 t cells . of 94 genes identified in the initial screening , 20 hits were further assessed by introducing additional sirnas , and 17 of them seemed to be genuine on - target hits . among them , eight are known to be involved in the nf - kb signalling . of the remaining genes , some might be involved in cellular processes that indirectly affect the nf - b pathway , whereas others might have previously unrecognized roles in the nf - b signalling pathway . all screening work mentioned above were done in microtitre plates , which will remain a major screening platform in the future . cell microarrays were created to provide much higher throughput than microtitre plates for sirna based screening . cell microarray was first described by ziauddin and sabatini ( 2001 ) who demonstrated that cells grown on a glass substrate could take up dna lipid complexes that had been deposited on the slide before cells were plated . cells became transfected in situ , with defined spots of transfected cells localized over the printed dnas . cell microarrays represent a novel alternative to classical approaches to phenotype - based assays in mammalian cells , at least for some of the easy - to - transfect cells such as hek 293 t , imr90/e1a , nih 3t3 , and hela cells . in this screening format , sirna ( plasmids or pcr cassettes ) are spotted onto glass slides together with transfection reagents ( normally with gelatin too for immobilization purpose ) . when cells are overlaid on top of a spot on the slides , corresponding sirna vector will be taken into the cells so that sirna will be expressed , and the effect of the sirna can be read out in high throughput according to the phenotypes or the reporter systems used . the initial proof - of principle was done by sirnas corresponding to the enhanced green fluorescent protein ( gfp ) gene ( egfp ) on microscope slides , and plated hela cells permanently expressing a destabilized version of egfp on top of the slides . the result showed the cellular uptake , reverse transfection , of rhodamine - tagged sirna ( rh - egfp ) , and silencing of gfp expression in cells . an experimental comparison was provided by carrying out , side by side , the array - based and microtitre plate screening of sirna that can perturb proteosome functions . the result showed that the array method does work in screening for sirna that can manipulate endogenous genes , and likely to provide data that is even better than the classic microtitre plate based screening process . advantage of the method is that large number of sirna can be rapidly screening through a given phenotype or reporter system . the limitation of the method is , in additional to the need of complex instrumentation , that many phenotypes ca n't be fit into such an array - based read - out format . rnai provides a perfect example of how a natural occurring process can be hijacked by researchers and used as a research tool . the penetration speed of this technology into the labs in both academic and industrial settings has been astonishing even though the mechanism of rnai has not been fully understood . in this reveal , we tried to focus on the development and application of vector based sirna libraries due to that fact that although synthetic sirna has become much more affordable now than two years ago due to stiff competition and improvement in the throughput of rna synthesis , vector - based sirna will still be the reagent of choice for large scale sirna based screening in most academic institutions and companies . it is worth to stress that synthetic sirna has also certain advantages over vector - based sirna approaches in dosing , integration of modified nucleotides , and delivery , in just name a few aspects for example . it is anticipated that plasmid libraries that have a genome - wide coverage will become available in 2005 for screening using both transient transfection as well as stable transfections . while lentivirus and other viral vectors provide obvious advantages in the transfection of hard - to - transfect cells , they are bound to be significantly cumbersome to work with in a high throughput screening . it is more likely that they will serve as second line of verification tools instead of being used as the first line screening tools in many occasions other than combinatorial screening .

application of sirna in high - throughput fashion is still in its early phase although the principle has been established for three years . in this review , we outline the different vector - based sirna delivery platforms as well as resources that are becoming available for high - throughput applications , and some initial outcomes of vector sirna high - throughput screening efforts using vector encoded sirna . it is expected that further improvement of the sirna technology and availability of the sirna resources will help to materialize the potential of sirna for functional genomics and drug target validation .

INTRODUCTION Methods for generating siRNAs siRNA production by pol III promoters siRNA production by other promoters Construction of siRNA libraries siRNA libraries with degenerated sequences Functional screening using different siRNA libraries CONCLUSIONS

mechanistic studies have indicated that the effector molecules of the rnai process is not the long dsrna itself , but a family of short double - stranded rna fragments ( 19 - 21 base pairs in length ) derived through cleavage of the long dsrna by rnase iii enzyme(s ) ( elbashir et al , 2001b ) . the short double - stranded rna fragments generated as such , as well as through chemical synthesis or vector - based expression , are collectively referred to as small interfering rna ( sirna ) . much of this response is caused by activation of the dsrna - dependent protein kinase pkr , which phosphorylates and inactivates the translation initiation factor eif2a ( proud , 1995 ) . in term of high - throughput applications , vector - based strategies are favoured because such strategies enjoy the advantages of much lower cost and ability to regenerate . transfection efficiency of sirna into cells depends on cell types and the rnai effect of synthetic sirna only sustain for a period of time . the advantage of the vector - based sirna is the capability of removing those cells that are not transfected with the plasmids by selecting the transfected cells with antibiotic resistant genes . virus vectors also enable the delivery of sirna expression cassettes into cells with higher transfection efficiency , and in case of lentivirus and retrovirus , it is easy to make stable knockdown cells by integration into the genome . in the current review , we will focus on the vector - based sirna libraries and the methods to produce them . while being as effective as the shrna encoding vectors , these dual - promoter ( genebuster ) vectors are first of all more robust for cloning both because of the lack of secondary structure in the sirna coding region , and much lower level of dna synthesis errors due to the shorter sequence , and they have tremendous advantages in constructing large scale sirna libraries in a combinatorial way , as will be discussed below . this method as well as the shrna method , however , offers the possibility to integrate mismatches into the resulting sirna , thus possibly enhance the efficacy of the sirna or increase the frequency of effective sirna . one interesting feature of the pol iii promoters is that they can be easily transformed into inducible promoters ( van de wetering et al , 2003 ) . in addition to plasmid - based systems , pcr - derived sirna expression cassettes based on the single - promoter system have been shown to efficiently suppress gene activities in transfected cells ( castanotto et al , 2002 ) . due to the potentially higher degree of flexibility in enabling different spatial and temporal expression patterns , these types of vector - based sirna strategies might be much more useful for in vivo research purposes and for sirna based gene - therapy at a later stage although pol iii based vectors seem to be able to satisfy most of the gene knock - down needs in cultured cells . one of the critical initial steps in the high throughput sirna application is the formation of a physical sirna library , which many people also refer to as a sirna library . it is anticipated that , with the development of high throughput sirna construction and validation methods , algorithms based on randomly chosen sirna will emerge to provide better predictions for functional sirna . although a sirna library can be constructed by chemical synthesis , as done by qiagen for novartis and more recently by dharmacon , most of the sirna libraries covering more than a couple of thousand of genes are done by vector - based approaches . this is because , i ) a vector - based sirna library can be regenerated with minimal effort , ii ) the constructions can be carried out in a standard molecular biology laboratory , and iii ) a vector - based sirna library can be much more affordable than a chemically synthesized library . multiple efforts have been initiated since then by commercial companies , and notably among them , genordia ab , a sweden - based company will most likely be able to deliver the first sirna library that covers a whole mammalian genome in 2005 . it was found that 29-nucleotide hairpins were more effective than shorter hairpin sirna in this study . also concerns have been raised for possibly higher level of off - target hits as well as induction of antiviral responses . it should be cautioned that recent studies have demonstrated that for processed sirna sequence identity up to 16 - 17 might already allow the sirna to have off - target effects . most other functional classes contain between 3060% coverage with three or more hairpins , and 80% of genes by at least one sequence - verified hairpin . a unique 60-nuclotide dna bar code has been integrated to each vector to allow identification of shrnas in populations of virally transduced cells through hybridization methods . the sirna encoding dna fragments were cloned in a high - throughput fashion into pretrosuper ( prs ) , a previously reported retroviral vector containing the shrna expression cassette . these might comprise a major limitation in the application of viral vector based sirna libraries in very large - scale screening . schultz and his colleagues have developed a pcr based system to generate sirna for about 8000 genes at 2-sirna / gene coverage using an opposing promoter approach . the sirna has been used to discover novel members of the nf - kb pathway ( zheng et al , 2004 ) . a single - step pcr protocol was used to produce sirna expression cassettes in a high - throughput fashion and showed that these sirna expression cassettes can also specifically and efficiently suppress the expression of both transfected and endogenous genes . this single - step pcr approach is efficient and cost - effective and makes high - throughput production of sirna expression cassette libraries for genome - wide functional gene annotation practical . notably , one of the first viral based sirna libraries was generated by an industrial player , galapagos genomics . other characteristics of this sirna library include : 1 ) the sirna expression cassette is based on the human u6 promoter , a strong , ubiquitously active promoter that lacks essential promoter elements within the transcribed region . 2 ) the u6 promoter - based expression cassette was cloned in an adenoviral adapter plasmid at the position of the e1 region . the use of sirna tools in primary cells has been hampered by inefficient transfection protocols . advantages in the transfection aspect are of critical importance for some applications , and thus counter - balance the burden imposed by the cumbersome viral handling . in this approach , the choice of gene , and choice of targeting sites are inevitably biased due to the limited number of genes and sites that can be included in a particular study . the bias also arise inevitably from the fact that we have just started to get a relatively complete picture of the part of the mouse transcriptome that has a poly ( a ) tail , and knowing little about transcripts that do not have a such a tail . the development of sirna expressing libraries that encode substantially all permutations of 19-mer sirna thus become an appealing approach to allow the use of it as a single affordable tool for un - biased genome - wide screening . in this case , a specific mrna population is converted into double stranded cdna , which will be cut into short dna fragments that can then inserted into the vectors for encoding sirna . the advantage of this type of libraries is that it will only have a complexity of in the neighborhood of 10 but already be able to cover a substantial part of the genome . the limiting aspect of the approach is that a library made in this way will only be useful for the same organism , or even only for the same cell type , whereas other fully randomized libraries can be used in all cell types and all relevant organisms . a hairpin linker is then ligated to either the 5 or 3 end of the double - stranded cdna fragment . in part , these successes derived from the availability of convenient and inexpensive methods for producing and introducing dsrna . to date , the use of rnai libraries in mammalian systems is becoming well accepted but most of the work has been limited to specific protein families because of technical and practical issues associated with generating large synthetic sirna and/or vector - based short hairpin rna ( shrna ) expression libraries , but the situation is rapidly improving with the technology development mentioned above . retroviral vectors can be used to transfect a large variety of cell types , and its short - coming on the handling side can be well balanced by using it in a combinatorial manner , e.g. in this case , it was found that two of the three shrnas targeting a single gene could mediate escape from growth arrest for three of the five newly identified genes . cell microarrays represent a novel alternative to classical approaches to phenotype - based assays in mammalian cells , at least for some of the easy - to - transfect cells such as hek 293 t , imr90/e1a , nih 3t3 , and hela cells . when cells are overlaid on top of a spot on the slides , corresponding sirna vector will be taken into the cells so that sirna will be expressed , and the effect of the sirna can be read out in high throughput according to the phenotypes or the reporter systems used . the initial proof - of principle was done by sirnas corresponding to the enhanced green fluorescent protein ( gfp ) gene ( egfp ) on microscope slides , and plated hela cells permanently expressing a destabilized version of egfp on top of the slides . the result showed the cellular uptake , reverse transfection , of rhodamine - tagged sirna ( rh - egfp ) , and silencing of gfp expression in cells . an experimental comparison was provided by carrying out , side by side , the array - based and microtitre plate screening of sirna that can perturb proteosome functions . advantage of the method is that large number of sirna can be rapidly screening through a given phenotype or reporter system . the limitation of the method is , in additional to the need of complex instrumentation , that many phenotypes ca n't be fit into such an array - based read - out format . in term of high - throughput applications , vector - based strategies are favoured because such strategies enjoy the advantages of much lower cost and ability to regenerate . the advantage of the vector - based sirna is the capability of removing those cells that are not transfected with the plasmids by selecting the transfected cells with antibiotic resistant genes . virus vectors also enable the delivery of sirna expression cassettes into cells with higher transfection efficiency , and in case of lentivirus and retrovirus , it is easy to make stable knockdown cells by integration into the genome . in the current review , we will focus on the vector - based sirna libraries and the methods to produce them . the backbones of these constructs were initially plasmids , but the grafting the shrna expressing cassettes into different viral vectors such as lentivirus , retrovirus and even adenovirus also explored successfully to facilitate the transfection of cells that were hard to penetrate , such as primary cells and suspension cells ( li et al , 2003 ; hosono et al , 2004 ; devroe and silver , 2004 ) diagrams of three general ways of encoding sirna in a plasmid or viral vector . while being as effective as the shrna encoding vectors , these dual - promoter ( genebuster ) vectors are first of all more robust for cloning both because of the lack of secondary structure in the sirna coding region , and much lower level of dna synthesis errors due to the shorter sequence , and they have tremendous advantages in constructing large scale sirna libraries in a combinatorial way , as will be discussed below . this method as well as the shrna method , however , offers the possibility to integrate mismatches into the resulting sirna , thus possibly enhance the efficacy of the sirna or increase the frequency of effective sirna . due to the potentially higher degree of flexibility in enabling different spatial and temporal expression patterns , these types of vector - based sirna strategies might be much more useful for in vivo research purposes and for sirna based gene - therapy at a later stage although pol iii based vectors seem to be able to satisfy most of the gene knock - down needs in cultured cells . it is anticipated that , with the development of high throughput sirna construction and validation methods , algorithms based on randomly chosen sirna will emerge to provide better predictions for functional sirna . although a sirna library can be constructed by chemical synthesis , as done by qiagen for novartis and more recently by dharmacon , most of the sirna libraries covering more than a couple of thousand of genes are done by vector - based approaches . this is because , i ) a vector - based sirna library can be regenerated with minimal effort , ii ) the constructions can be carried out in a standard molecular biology laboratory , and iii ) a vector - based sirna library can be much more affordable than a chemically synthesized library . three large - scale sirna libraries have recently been constructed by academic researchers ( paddison et al , 2004 ; berns et al 2004 , michiels et al , 2002 ) and galapagos has generated the first large scale adenovirus based sirna library in the industrial sector . multiple efforts have been initiated since then by commercial companies , and notably among them , genordia ab , a sweden - based company will most likely be able to deliver the first sirna library that covers a whole mammalian genome in 2005 . it was found that 29-nucleotide hairpins were more effective than shorter hairpin sirna in this study . also concerns have been raised for possibly higher level of off - target hits as well as induction of antiviral responses . 3 ) where possible , shrnas were designed to have sequence identity to the mouse orthologue of the targeted gene . the sirna encoding dna fragments were cloned in a high - throughput fashion into pretrosuper ( prs ) , a previously reported retroviral vector containing the shrna expression cassette . these might comprise a major limitation in the application of viral vector based sirna libraries in very large - scale screening . the sirna has been used to discover novel members of the nf - kb pathway ( zheng et al , 2004 ) . a single - step pcr protocol was used to produce sirna expression cassettes in a high - throughput fashion and showed that these sirna expression cassettes can also specifically and efficiently suppress the expression of both transfected and endogenous genes . this single - step pcr approach is efficient and cost - effective and makes high - throughput production of sirna expression cassette libraries for genome - wide functional gene annotation practical . the arrayed library was used to screen for genes involved in the nf - b signalling pathway that controls many diverse cellular processes including growth , development , inflammation , immune response , apoptosis , and oncogenesis . notably , one of the first viral based sirna libraries was generated by an industrial player , galapagos genomics . 2 ) the u6 promoter - based expression cassette was cloned in an adenoviral adapter plasmid at the position of the e1 region . the use of sirna tools in primary cells has been hampered by inefficient transfection protocols . advantages in the transfection aspect are of critical importance for some applications , and thus counter - balance the burden imposed by the cumbersome viral handling . in this approach , the choice of gene , and choice of targeting sites are inevitably biased due to the limited number of genes and sites that can be included in a particular study . the bias also arise inevitably from the fact that we have just started to get a relatively complete picture of the part of the mouse transcriptome that has a poly ( a ) tail , and knowing little about transcripts that do not have a such a tail . the library was used for phenotype - driven screening using cell proliferation as a model , and multiple sirna that can induce significant enhancement of cell growth were identified . in this case , a specific mrna population is converted into double stranded cdna , which will be cut into short dna fragments that can then inserted into the vectors for encoding sirna . the advantage of this type of libraries is that it will only have a complexity of in the neighborhood of 10 but already be able to cover a substantial part of the genome . the limiting aspect of the approach is that a library made in this way will only be useful for the same organism , or even only for the same cell type , whereas other fully randomized libraries can be used in all cell types and all relevant organisms . in part , these successes derived from the availability of convenient and inexpensive methods for producing and introducing dsrna . to date , the use of rnai libraries in mammalian systems is becoming well accepted but most of the work has been limited to specific protein families because of technical and practical issues associated with generating large synthetic sirna and/or vector - based short hairpin rna ( shrna ) expression libraries , but the situation is rapidly improving with the technology development mentioned above . these shrna inserts were then re - cloned from these colonies , and their identity was established by dna sequence analysis . in this case , it was found that two of the three shrnas targeting a single gene could mediate escape from growth arrest for three of the five newly identified genes . of 94 genes identified in the initial screening , 20 hits were further assessed by introducing additional sirnas , and 17 of them seemed to be genuine on - target hits . of the remaining genes , some might be involved in cellular processes that indirectly affect the nf - b pathway , whereas others might have previously unrecognized roles in the nf - b signalling pathway . cell microarrays represent a novel alternative to classical approaches to phenotype - based assays in mammalian cells , at least for some of the easy - to - transfect cells such as hek 293 t , imr90/e1a , nih 3t3 , and hela cells . in this screening format , sirna ( plasmids or pcr cassettes ) are spotted onto glass slides together with transfection reagents ( normally with gelatin too for immobilization purpose ) . when cells are overlaid on top of a spot on the slides , corresponding sirna vector will be taken into the cells so that sirna will be expressed , and the effect of the sirna can be read out in high throughput according to the phenotypes or the reporter systems used . the initial proof - of principle was done by sirnas corresponding to the enhanced green fluorescent protein ( gfp ) gene ( egfp ) on microscope slides , and plated hela cells permanently expressing a destabilized version of egfp on top of the slides . an experimental comparison was provided by carrying out , side by side , the array - based and microtitre plate screening of sirna that can perturb proteosome functions . advantage of the method is that large number of sirna can be rapidly screening through a given phenotype or reporter system . the limitation of the method is , in additional to the need of complex instrumentation , that many phenotypes ca n't be fit into such an array - based read - out format . in this reveal , we tried to focus on the development and application of vector based sirna libraries due to that fact that although synthetic sirna has become much more affordable now than two years ago due to stiff competition and improvement in the throughput of rna synthesis , vector - based sirna will still be the reagent of choice for large scale sirna based screening in most academic institutions and companies . it is worth to stress that synthetic sirna has also certain advantages over vector - based sirna approaches in dosing , integration of modified nucleotides , and delivery , in just name a few aspects for example . it is anticipated that plasmid libraries that have a genome - wide coverage will become available in 2005 for screening using both transient transfection as well as stable transfections .

epidemic keratoconjunctivitis ( ekc ) is an acute infection of the eye caused by adenovirus , of which several different types have been implicated . common manifestations of this infection include inflammation of the conjunctivae , edema of the eyelid , pain , photophobia , and blurred vision . transmission may occur through direct contact with eye secretions of an infected person , or indirectly through contact with contaminated surfaces , instruments or solutions . ekc is highly contagious and frequently occurs in epidemic fashion , as its name implies . the incubation period can range from 2 days to 2 weeks , with an average of 8 - 10 days , and infected people remain contagious for up to 2 weeks . viral particles can remain infectious on surfaces for up to a month . in march 2009 , the florida department of health ( fldoh ) was contacted by a regional academic ophthalmology referral center , regarding a local outpatient ophthalmology practice ( practice a ) that had requested assistance with an outbreak of ekc at the practice . practice a was contacted and a site visit was arranged . at the same time , anecdotal reports were noted of increased ekc activity throughout the county . to assess whether the problems experienced at practice a were isolated or part of a more wide - spread increase in ekc activity , a brief survey was developed of outpatient ophthalmology clinics in the county . this report describes the investigation of the ekc outbreak at practice a and the survey results of other out - patient ophthalmology clinics in the county . the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators . office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months . confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens . suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation . the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited . based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms . specific information regarding medical procedures performed during previous visits or past visits to other providers was not obtained . during the site visit , a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] . specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day . viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl . viral culture was conducted by inoculation of a549 and mrc cells ( viromed laboratories , minnetonka , mn , usa ) and incubation for up to 21 days , followed by immunoflourescent staining with monoclonal antibody ( pathodx , remel , lenexa , ks , usa ) . viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes . fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records . a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire . the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available . analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed . the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators . office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months . confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens . suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation . the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited . based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms . specific information regarding medical procedures performed during previous visits or past visits to other providers was not obtained . during the site visit , a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] . specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day . viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl . viral culture was conducted by inoculation of a549 and mrc cells ( viromed laboratories , minnetonka , mn , usa ) and incubation for up to 21 days , followed by immunoflourescent staining with monoclonal antibody ( pathodx , remel , lenexa , ks , usa ) . viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes . fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records . a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire . the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available . analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed . practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week . the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . at office a1 , there are 4 exam rooms and 2 ophthalmology technicians who assist with patient care ; 2 other staff assist with administrative duties . office a2 is a smaller satellite facility where physician a and the technicians work 2 half - days per week . approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice . interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 . a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 . twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 . two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test . all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset . interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms . no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) . nineteen ekc patients ( 51% ) had been seen for an unrelated condition by physician a , within 2 weeks of the onset of their symptoms ( figure 2 ) . four additional ekc cases were seen at practice a between 15 and 17 days prior to symptom onset . of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 , the day the health department was notified of the outbreak , practice a was closed for terminal cleaning . all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned . when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . for patient encounters , office staff implemented aggressive use of gloves and wiping of surfaces with germicidal wipes following each patient encounter . these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures , 2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection . no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak . approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates . medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns . eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers , the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . none of these 84 suspect case - patients were laboratory confirmed . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem . of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient . none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff . finally , 7 of 18 ( 39% ) clinics that responded indicated future interest in submitting specimens to reference laboratories for diagnostic testing . practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week . the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . at office a1 , there are 4 exam rooms and 2 ophthalmology technicians who assist with patient care ; 2 other staff assist with administrative duties . office a2 is a smaller satellite facility where physician a and the technicians work 2 half - days per week . approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice . interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 . a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 . twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 . two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test . all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset . interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms . no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) . nineteen ekc patients ( 51% ) had been seen for an unrelated condition by physician a , within 2 weeks of the onset of their symptoms ( figure 2 ) . four additional ekc cases were seen at practice a between 15 and 17 days prior to symptom onset . of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 , the day the health department was notified of the outbreak , practice a was closed for terminal cleaning . all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned . when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . for patient encounters , office staff implemented aggressive use of gloves and wiping of surfaces with germicidal wipes following each patient encounter . these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures , 2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection . no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak . approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates . medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns . eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers , the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . none of these 84 suspect case - patients were laboratory confirmed . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem . of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient . none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff . finally , 7 of 18 ( 39% ) clinics that responded indicated future interest in submitting specimens to reference laboratories for diagnostic testing . the outbreak at practice a described in this report involved 37 patients diagnosed with ekc over a 5 month period . approximately one - half of case - patients had recently been seen by physician a for an unrelated condition , and previous visits occurred at both offices of the practice . one additional suspect case involved the ophthalmologist , raising the strong possibility of iatrogenic transmission within the practice . at least 2 other ophthalmology clinics in the county reported a similar number of case - patients during the same time period and at least 1 of these clinics may also have had nosocomial transmission , based on the large number of ekc patients who were recently seen for unrelated conditions . patients diagnosed with ekc were also seen in several other clinics throughout the county during this time frame . taken together , these observations suggest broad distribution of ekc - like illness in the community with focal amplification in practice a , likely due to nosocomial transmission . the etiologic agent identified in 4 ekc patients at practice a was adenovirus , serotype 8 ( ad8 ) . ad8 is a common cause of severe ekc , along with adenoviral serotypes 19 and 37 . other serotypes , such as ad3 , ad4 , ad7 , and ad11 , generally cause milder conjunctivitis with systemic involvement . diagnosis of severe ekc is usually only clinical , but milder forms of the illness due to these other serotypes may require laboratory confirmation . among those infected with ad8 , eyelid edema may be significantly more common , and serotype ad8 may persist longer in ocular tissue and secretions than other serotypes . the fact that ad8 was found to be the etiologic agent in this outbreak suggests that those patients diagnosed clinically at practice a , without laboratory confirmation , were also likely infected with ad8 . there is no population based disease surveillance for ekc in florida or other states , making estimates of disease incidence difficult in the usa . this episode was classified as an outbreak based on the experienced clinician reporting it as such , rather than by comparing disease incidence in practice a to background rates , which are not reliably available for florida . in japan , where nationwide surveillance has occurred over several years , an estimated 1 million cases of adenoviral conjunctivitis occur each year . in the usa and elsewhere , outbreaks of ekc are believed to be common , and several outbreak investigations have been described in the literature . risk factors for ekc transmission identified in healthcare settings include use of inadequately disinfected tonometers to measure intraocular pressure , use of multi - dose drops , or exposure to specific care givers . adenoviruses have been shown to persist in the environment and remain viable in a desiccated state for lengthy periods and to be recoverable from the hands of infected individuals , even following hand washing . guidelines for the disinfection and sterilization of ophthalmology equipment that may contribute to ekc transmission have been published by the healthcare infection control practices advisory committee . firstly , we only became aware of the outbreak in practice a late in its course , after strict control measures had been implemented . thus , sampling to identify an environmental source of infection was unlikely to yield useful information and no environmental sampling was conducted . in addition , more than one month had elapsed since physician a became symptomatic , making diagnostic testing of physician a impractical . since the outbreak began to come under control at the time the health department investigation was beginning , fewer patients with active symptoms were available for laboratory confirmation to support an epidemiologic investigation of risk factors . since physician a was the only ophthalmologist in the practice , all patients were exposed to physician a , making measures of association with this exposure difficult to estimate . therefore , a specific common source of transmission could not be conclusively identified . when practitioners are confronted with a suspected outbreak of ekc at their facility , there are some recognized best practices , many of which were followed by practice a. they sought laboratory assistance to identify the pathogen , conducted thorough cleaning of the facility and equipment with appropriate disinfectants , segregated symptomatic and non - symptomatic patients , and cooperated fully with the health department in the investigation . however , healthcare providers with known or suspected ekc should also avoid direct patient contact for 14 days following onset of symptoms in the most recently involved eye , which practice a did not follow . under florida administrative code 64d-3 , any disease outbreak in a community , hospital or other institution is reportable to public health authorities . our survey of ophthalmologists in the county found that at least 2 other outpatient facilities witnessed a similar number of ekc patients during the same time period , yet neither facility reported these as outbreaks . without laboratory confirmation and serotyping , it is difficult to determine if the patients diagnosed with ekc seen at these other facilities were somehow linked to patients at practice a. our findings suggest that passive surveillance based on administrative rule results in poor surveillance sensitivity to detect ekc outbreaks in florida . the same is likely true in many other states where disease outbreaks are reportable , but individual cases of the same disease are not . ophthalmologists and other providers experiencing a perceived increase in ekc activity among patients in their facility are encouraged to contact their local health department . laboratory diagnostic support to confirm certain infectious causes may be available through state health department laboratories .

epidemic keratoconjunctivitis ( ekc ) is an acute eye infection caused by adenovirus . we investigated an outbreak of ekc at an outpatient ophthalmology practice in the context of a suspected community wide increase in ekc activity . a site visit was made to the facility reporting the outbreak . a line list was created of patients clinically diagnosed with ekc at the practice during the previous 5 months . a questionnaire was faxed to all other licensed ophthalmologists in the county regarding recent ekc activity in their facility . descriptive data analyses were conducted . the outbreak facility reported 37 patients clinically diagnosed with ekc during the previous 5 months . in addition , the single ophthalmologist at the practice also had symptoms compatible with ekc during the outbreak period . specimens were collected on 4 patients and all were positive for adenovirus serotype 8 . forty percent of ophthalmologists surveyed in the county saw at least one ekc patient in the previous 3 months , and 20% reported a perceived increase in ekc activity in recent months over normal seasonal patterns . the outbreak at the facility likely began as part of a widespread community increase in ekc that may have been amplified at the facility through nosocomial transmission . medical providers experiencing increases in ekc activity above seasonally expected norms should contact their public health department for assistance with etiologic diagnoses and outbreak control .

Introduction Materials and Methods Investigation of practice A Survey of outpatient ophthalmology clinics Results Investigation of practice A Survey of outpatient clinics in the community Discussion

epidemic keratoconjunctivitis ( ekc ) is an acute infection of the eye caused by adenovirus , of which several different types have been implicated . common manifestations of this infection include inflammation of the conjunctivae , edema of the eyelid , pain , photophobia , and blurred vision . the incubation period can range from 2 days to 2 weeks , with an average of 8 - 10 days , and infected people remain contagious for up to 2 weeks . in march 2009 , the florida department of health ( fldoh ) was contacted by a regional academic ophthalmology referral center , regarding a local outpatient ophthalmology practice ( practice a ) that had requested assistance with an outbreak of ekc at the practice . practice a was contacted and a site visit was arranged . at the same time , anecdotal reports were noted of increased ekc activity throughout the county . to assess whether the problems experienced at practice a were isolated or part of a more wide - spread increase in ekc activity , a brief survey was developed of outpatient ophthalmology clinics in the county . this report describes the investigation of the ekc outbreak at practice a and the survey results of other out - patient ophthalmology clinics in the county . the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators . office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months . confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens . suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation . the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited . based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms . during the site visit , a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] . specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day . viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl . viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes . fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records . a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire . the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available . analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed . the practice consists of a main office ( office a1 ) and an auxiliary office ( office a2 ) , located in different parts of the county , and only office a1 was visited by investigators . office staff assembled a line list from patient charts of all persons diagnosed with ekc at practice a during the previous 5 months . confirmed cases were defined as those with compatible clinical symptoms of ekc and laboratory confirmed adenovirus infection through viral culture of ocular swab specimens . suspect cases were defined as those with a clinical diagnosis of ekc , without laboratory confirmation . the line list included the onset date of symptoms , dates previously seen in the practice prior to symptoms , and which office visited . based on the estimated outside incubation period for ekc of 2 weeks , cases were further classified as possibly healthcare associated if they were seen at practice a for an unrelated condition within 2-weeks of the onset of ekc symptoms . during the site visit , a rapid test was performed on 2 patients with compatible symptoms , using the adeno detector test kit [ www.rpt-tests.com/products_ad.html ] . specimens for viral culture were obtained from the same 2 patients and from 2 additional patients seen the next day . viral cultures were performed at the bascom palmer eye institute , microbiology laboratory , in miami , fl . viral culture isolates positive for adenovirus were forwarded to the centers for disease control and prevention ( cdc ) for viral serotyping by polymerase chain reaction , using primers specific for adenovirus serotypes . fldoh developed a list of all licensed ophthalmologists in the county from medical licensing records . a cover letter and brief 1-page questionnaire were faxed to all ophthalmology practices in the county alerting them to the possible ekc problem , and requesting the completion and return of the questionnaire . the questionnaire included items regarding their perceptions of recent ekc incidence , number of ekc cases diagnosed in their clinic during the last three months , similar illness among office staff , and any laboratory test results available . analyses of data collected from both practice a and the county wide survey were descriptive and no statistical tests of significance were performed . practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week . the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice . interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 . a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 . twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 . two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test . all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset . interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms . no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) . of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 , the day the health department was notified of the outbreak , practice a was closed for terminal cleaning . all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned . when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures , 2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection . no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak . approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates . medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns . eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers , the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . none of these 84 suspect case - patients were laboratory confirmed . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem . of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient . none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff . practice a is a busy , outpatient ophthalmology practice that serves a predominantly elderly patient population and sees approximately 150 - 200 patients per week . the practice has 1 general ophthalmologist ( physician a ) , specializing in cataract and corneal surgery , who founded the practice and has worked for several years in the community . approximately two - thirds of patient visits occur at office a1 , where all patient records are maintained for the practice . interviews with staff revealed that the first ekc patient believed to be part of this outbreak was seen in office a1 in late november 2008 . a total of 37 patients were clinically diagnosed with ekc at practice a from november 2008 through march 2009 . twenty - three ( 62% ) ekc patients were seen at office a1 , 12 ( 32% ) at office a2 , and 2 patients were seen at both offices ( figure 1 ) . of the 4 patients seen at office a1 for whom specimens were collected for viral culture , all 4 were positive for adenovirus , serotype 8 . two of the four laboratory confirmed patients had rapid tests performed during the clinical visit , and both of these patients were negative for adenovirus by rapid test . all of the laboratory confirmed patients had a previous visit at office a1 within the previous 2 weeks , prior to their symptom onset . interviews revealed that physician a had symptoms compatible with ekc , with onset of these symptoms on approximately february 5 , 2009 and lasting approximately 4 - 5 weeks before resolution of symptoms . no other staff members had similar symptoms and no diagnostic specimens were collected from any staff members . among the other 37 confirmed or suspected ekc patients , the median age was 79 years ( table 1 ) . of the 19 ekc patients seen at practice a within 2 weeks of symptom onset , 13 had been seen at office a1 , 5 at office a2 , and 1 at both offices . on march 18 , 2009 , the day the health department was notified of the outbreak , practice a was closed for terminal cleaning . all surfaces were wiped by office staff with a bleach solution , reusable vials of drops were discarded , and tonometers and other medical equipment were thoroughly cleaned . when practice a opened the following day , one exam room in office a1 was dedicated exclusively for ekc patients , and ekc and non - ekc patients were separated on different sides of the waiting room . these control measures were initiated independently by practice a , prior to the health department becoming involved in the investigation . following implementation of these control measures , 2 additional cases were diagnosed with ekc at practice a , 2 and 6 days respectively following the terminal cleaning . the patient with onset 2 days after implementation of control measures had a previous office visit 15 days earlier ; the other patient had not been seen in practice a during the past year . since prevention measures had already been implemented and the outbreak had begun to dissipate by the time the epidemiologic investigation was under way , no environmental samples were collected and extensive analyses were not conducted to identify additional risk factors associated with infection . no additional ekc patients were seen at practice a in the two months following this outbreak , however , physician a reported seeing 4 additional ekc patients within 6 months following the outbreak . approximately 8 ekc case patients from the outbreak , including physician a , developed post - viral keratitis , requiring long - term topical steroid treatment to control corneal infiltrates . medical licensing records revealed 29 ophthalmologists in general practice in the county , including physician a. in april 2009 , questionnaires were faxed to all 28 providers , other than physician a , and responses were received from 20 ( 71% ) . of those who responded , 4 ( 20% ) reported an increase in conjunctivitis during the previous 6 months above normal seasonal patterns . eight of twenty ( 40% ) respondents believed they had seen patients from january through march 2009 with symptoms compatible with ekc or acute hemorrhagic conjunctivitis ( ahc ) . among these providers , the median number of ekc / ahc patients seen per clinic during this period was 10 ( range 1 to 25 ) with a total of 84 patients with compatible symptoms seen at these 8 clinics . for comparison , during the same 3-month period , physician a saw 28 ekc patients , making 112 total ekc patients for all 21 providers . because denominator data were not collected on the total number of patients seen at each clinic , attack rates at each clinic two of the eight clinics ( 25% ) reported seeing symptoms compatible with ekc in patients who had visited their clinic in the previous 2 weeks for an unrelated problem . of these 2 clinics , one reported seeing ekc symptoms in 10 return patients , and the other clinic reported 1 ekc case in a return patient . none of the 20 clinics who responded to the survey reported similar symptoms among clinic staff . the outbreak at practice a described in this report involved 37 patients diagnosed with ekc over a 5 month period . approximately one - half of case - patients had recently been seen by physician a for an unrelated condition , and previous visits occurred at both offices of the practice . one additional suspect case involved the ophthalmologist , raising the strong possibility of iatrogenic transmission within the practice . at least 2 other ophthalmology clinics in the county reported a similar number of case - patients during the same time period and at least 1 of these clinics may also have had nosocomial transmission , based on the large number of ekc patients who were recently seen for unrelated conditions . patients diagnosed with ekc were also seen in several other clinics throughout the county during this time frame . taken together , these observations suggest broad distribution of ekc - like illness in the community with focal amplification in practice a , likely due to nosocomial transmission . the etiologic agent identified in 4 ekc patients at practice a was adenovirus , serotype 8 ( ad8 ) . other serotypes , such as ad3 , ad4 , ad7 , and ad11 , generally cause milder conjunctivitis with systemic involvement . among those infected with ad8 , eyelid edema may be significantly more common , and serotype ad8 may persist longer in ocular tissue and secretions than other serotypes . there is no population based disease surveillance for ekc in florida or other states , making estimates of disease incidence difficult in the usa . this episode was classified as an outbreak based on the experienced clinician reporting it as such , rather than by comparing disease incidence in practice a to background rates , which are not reliably available for florida . in the usa and elsewhere , outbreaks of ekc are believed to be common , and several outbreak investigations have been described in the literature . adenoviruses have been shown to persist in the environment and remain viable in a desiccated state for lengthy periods and to be recoverable from the hands of infected individuals , even following hand washing . guidelines for the disinfection and sterilization of ophthalmology equipment that may contribute to ekc transmission have been published by the healthcare infection control practices advisory committee . firstly , we only became aware of the outbreak in practice a late in its course , after strict control measures had been implemented . in addition , more than one month had elapsed since physician a became symptomatic , making diagnostic testing of physician a impractical . since the outbreak began to come under control at the time the health department investigation was beginning , fewer patients with active symptoms were available for laboratory confirmation to support an epidemiologic investigation of risk factors . since physician a was the only ophthalmologist in the practice , all patients were exposed to physician a , making measures of association with this exposure difficult to estimate . when practitioners are confronted with a suspected outbreak of ekc at their facility , there are some recognized best practices , many of which were followed by practice a. they sought laboratory assistance to identify the pathogen , conducted thorough cleaning of the facility and equipment with appropriate disinfectants , segregated symptomatic and non - symptomatic patients , and cooperated fully with the health department in the investigation . however , healthcare providers with known or suspected ekc should also avoid direct patient contact for 14 days following onset of symptoms in the most recently involved eye , which practice a did not follow . under florida administrative code 64d-3 , any disease outbreak in a community , hospital or other institution is reportable to public health authorities . our survey of ophthalmologists in the county found that at least 2 other outpatient facilities witnessed a similar number of ekc patients during the same time period , yet neither facility reported these as outbreaks . without laboratory confirmation and serotyping , it is difficult to determine if the patients diagnosed with ekc seen at these other facilities were somehow linked to patients at practice a. our findings suggest that passive surveillance based on administrative rule results in poor surveillance sensitivity to detect ekc outbreaks in florida . ophthalmologists and other providers experiencing a perceived increase in ekc activity among patients in their facility are encouraged to contact their local health department . laboratory diagnostic support to confirm certain infectious causes may be available through state health department laboratories .

the study was performed between 27 september 2004 and 13 september 2007 at three study sites , in sweden , finland , and the netherlands . in total , 69 patients were randomly assigned using a permutated block randomization scheme stratified by site and screening a1c to receive exenatide or insulin glargine , in addition to ongoing metformin treatment ( fig . inclusion criteria were age 3075 years , a1c 6.59.5% , bmi 2540 kg / m , and metformin treatment at a stable dose for at least 2 months . no other blood glucose lowering agents were allowed within 3 months before screening . no changes in other agents known to affect -cell function ( such as ace inhibitors and angiotensin receptor blockers ) were allowed during the study . the study protocol was approved by the ethics review committee at each site and was in accordance with the principles described in the declaration of helsinki . patients randomly assigned to exenatide ( n = 36 ) initiated treatment at a dose of 5 g b.i.d . , injected 15 min before breakfast and dinner , for a period of 4 weeks , followed by a dose increase to 10 g b.i.d . exenatide was titrated to a maximum dose of 20 g t.i.d . , or the maximum tolerated dose , when a1c ranged from 7.1 to 7.5% at two consecutive visits or when a1c was 7.6% at any given visit . patients randomly assigned to insulin glargine ( n = 33 ) started at an initial dose of 10 iu q.d . , injected at bedtime . patients were instructed to increase the daily dose based on their fasting self - monitored blood glucose ( smbg ) levels , according to a prespecified algorithm ( 14 ) . mmol / l on 3 consecutive days , the insulin dose was increased by 2 units until finally fasting smbg would range between 4.5 and 5.5 mmol / l . if a hypoglycemic event ( < 3.3 mmol / l ) occurred , patients were instructed to refrain from increasing the insulin glargine dose for 7 days and to contact the study physician . insulin secretion and sensitivity were measured during a combined euglycemic - hyperinsulinemic and hyperglycemic clamp procedure ( supplementary fig . first- and second - phase c - peptide secretion was calculated as area under the curve ( auc)180190 min and auc190260 min . arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc260270 min above the fasting c - peptide concentration . arginine was administered during a hyperglycemic clamp to measure maximum insulin secretory capacity at a steady - state glucose concentration of 15 mmol / l ( 17 ) . clamps were performed before randomization , after 52 weeks of treatment , and after a 4-week off - drug period . after an overnight fast , an indwelling cannula was inserted into an antecubital vein for infusion of glucose and insulin . to obtain arterialized venous blood samples , an cannula was inserted in a retrograde fashion into a dorsal hand or wrist vein and maintained in a heated box at 50c . during the clamp at week 52 , patients randomly assigned to exenatide , were given the study drug 15 min before the onset of the hyperglycemic clamp , and patients randomly assigned to insulin glargine received their last insulin dose the night before at bedtime . a1c ( normal range : 4.36.1% , diabetes control and complications trial standardized bio - rad assay ) was measured using the fasting plasma glucose , and safety parameters were measured before randomization and during each follow - up visit until the end of the 12-week off - drug period by a central laboratory ( quintiles , livingston , u.k . ) . patients were instructed to record seven - point ( fasting , 2 h after breakfast , before lunch , 2 h after lunch , before dinner , 2 h after dinner , and at bedtime ) smbg profiles using an onetouch ultra blood glucose meter ( lifescan , milpitas , ca ) before each visit . plasma glucose concentrations during the clamp were measured using an ysi 2300 stat plus analyzer ( ysi , yellow springs , oh ) in sweden and the netherlands and using a beckman coulter glucose analyzer ii ( beckman coulter , fullerton , ca ) in finland . c - peptide samples were analyzed at the vu university medical center using an immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , netherlands ) . the primary efficacy end point of this study is the treatment effect on -cell function as measured by the ratio of week 52 combined glucose- and arginine - stimulated insulin secretion during a hyperglycemic clamp . a sample size of 26 patients per group was required to provide 90% power to detect a between - group significant difference in arginine - stimulated insulin secretion between the two treatment groups , assuming that the mean incremental auc value at baseline is 200 pmol min l for both groups and values at week 52 are 1,100 and 300 pmol min the dependent variable used in the model is the loge - transformed ratio to pretreatment for the -cell function parameters ( airarg , first phase , and second phase ) . for all other end points the dependent value used is the mean at the corresponding visit . the model includes factors for treatment group ( exenatide / glargine ) , site ( netherlands / sweden / finland ) , and baseline a1c stratum ( 8.5%/>8.5% ) , and the pretreatment variable of the corresponding dependent variable as a covariate . statistical analysis was done using sas software ( sas institute , cary , nc ) . all inferential statistical tests were conducted at a significance level of 0.05 ( two - sided ) . insulin secretion and sensitivity were measured during a combined euglycemic - hyperinsulinemic and hyperglycemic clamp procedure ( supplementary fig . 1a , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1797/dc1 ) first- and second - phase c - peptide secretion was calculated as area under the curve ( auc)180190 min and auc190260 min . arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc260270 min above the fasting c - peptide concentration . arginine was administered during a hyperglycemic clamp to measure maximum insulin secretory capacity at a steady - state glucose concentration of 15 mmol / l ( 17 ) . clamps were performed before randomization , after 52 weeks of treatment , and after a 4-week off - drug period . after an overnight fast , an indwelling cannula was inserted into an antecubital vein for infusion of glucose and insulin . to obtain arterialized venous blood samples , an cannula was inserted in a retrograde fashion into a dorsal hand or wrist vein and maintained in a heated box at 50c . during the clamp at week 52 , patients randomly assigned to exenatide , were given the study drug 15 min before the onset of the hyperglycemic clamp , and patients randomly assigned to insulin glargine received their last insulin dose the night before at bedtime . a1c ( normal range : 4.36.1% , diabetes control and complications trial standardized bio - rad assay ) was measured using the fasting plasma glucose , and safety parameters were measured before randomization and during each follow - up visit until the end of the 12-week off - drug period by a central laboratory ( quintiles , livingston , u.k . ) . patients were instructed to record seven - point ( fasting , 2 h after breakfast , before lunch , 2 h after lunch , before dinner , 2 h after dinner , and at bedtime ) smbg profiles using an onetouch ultra blood glucose meter ( lifescan , milpitas , ca ) before each visit . plasma glucose concentrations during the clamp were measured using an ysi 2300 stat plus analyzer ( ysi , yellow springs , oh ) in sweden and the netherlands and using a beckman coulter glucose analyzer ii ( beckman coulter , fullerton , ca ) in finland . c - peptide samples were analyzed at the vu university medical center using an immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , netherlands ) . the primary efficacy end point of this study is the treatment effect on -cell function as measured by the ratio of week 52 combined glucose- and arginine - stimulated insulin secretion during a hyperglycemic clamp . a sample size of 26 patients per group was required to provide 90% power to detect a between - group significant difference in arginine - stimulated insulin secretion between the two treatment groups , assuming that the mean incremental auc value at baseline is 200 pmol l for both groups and values at week 52 are 1,100 and 300 pmol min the dependent variable used in the model is the loge - transformed ratio to pretreatment for the -cell function parameters ( airarg , first phase , and second phase ) . for all other end points the dependent value used is the mean at the corresponding visit . the model includes factors for treatment group ( exenatide / glargine ) , site ( netherlands / sweden / finland ) , and baseline a1c stratum ( 8.5%/>8.5% ) , and the pretreatment variable of the corresponding dependent variable as a covariate . statistical analysis was done using sas software ( sas institute , cary , nc ) . all inferential statistical tests were conducted at a significance level of 0.05 ( two - sided ) . of the patients randomly assigned to exenatide , 62.1% ( n = 18 ) were treated with exenatide 10 g b.i.d . at 52 weeks of treatment . five ( 17.2% ) patients were using 20 g t.i.d . , two ( 6.9% ) were using 10 g t.i.d . , one ( 3.4% ) was using 15 g b.i.d . , and one ( 3.4% ) was using 15 g of t.i.d . the daily exenatide dose was reduced to 5 g b.i.d . in two patients ( 6.9% ) . despite this increase in daily exenatide dose , none of these patients reached the a1c target of < 7.1% . at 52 weeks , the mean the corresponding fasting smbg in the insulin glargine treated group was 5.6 0.2 mmol / l . exenatide and insulin glargine treatment resulted in similar reductions in a1c ( 0.8 0.1 and 0.7 0.2% , respectively ; p = 0.55 ) , with both groups achieving a mean a1c of 6.8% at 52 weeks . the insulin glargine group showed a significantly greater reduction in fasting plasma glucose compared with the exenatide group ( 2.9 0.4 vs. 1.6 0.3 mmol / l , respectively ; p < 0.0001 ) , whereas smbg profiles demonstrated significantly greater reductions in postprandial glucose excursions in the exenatide - treated patients ( fig . 2c and d ) . during the off - drug period , both a1c and fasting plasma glucose increased in both groups and were not significantly different compared with pretreatment values after 12 weeks off - drug ( fig . . changes in body weight ( e ) and insulin sensitivity were measured as the m value ( f ) . , exenatide ; , insulin glargine ; , pretreatment ; , 52 weeks on - drug ; , 4 weeks off - drug . bb , before breakfast ; ab , after breakfast ; bl , before lunch ; al , after lunch ; bd , before dinner ; ad , after dinner ; bt , bedtime . fifty - two weeks of exenatide treatment resulted in a lowering of body weight of 3.6 0.6 kg , whereas treatment with insulin glargine resulted in a body weight increase of + 1.0 0.8 kg ( between - group difference , 4.6 1.1 kg ; p < 0.0001 ) ( fig . body weight trended toward baseline values with both therapies ( between - group difference 2.4 1.1 kg ; p = 0.03 ) . at baseline , insulin - mediated glucose uptake did not differ between the two treatment groups ( fig . treatment with exenatide and insulin glargine improved insulin sensitivity to the same extent by 0.9 0.3 and 1.1 0.3 mg min kg , respectively ( p = 0.49 ) . after a 4-week discontinuation of study medication , the m value was not significantly different from pretreatment values in the insulin glargine treated group , whereas it remained significantly higher in the exenatide - treated group ( between - group difference 0.8 0.4 mg / min / kg ; p = 0.03 ) . at baseline , both glucose- and arginine - stimulated c - peptide secretion did not differ between the two treatment groups ( table 1 ; fig . accordingly , exenatide treatment significantly increased first- and second - phase glucose - stimulated c - peptide secretion by 1.53 0.11- and 2.85 0.22-fold , respectively ( p the c - peptide response to arginine during hyperglycemia increased 3.19 0.24-fold from pretreatment in the exenatide group compared with a 1.31 0.07-fold increase in the insulin glargine group ( between - group difference 2.46 0.20-fold ; p < 0.0001 ) . after 4 weeks discontinuation of the study medication , measures of -cell function returned to pretreatment values in both groups . measures of -cell secretory function during hyperglycemic clamp and ratio to pretreatment in the exenatide- and insulin glargine treated groups data represent mean sem . airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ( nmol min l ) ; first - phase , first - phase c - peptide response to glucose ( nmol min l ; second - phase , second - phase c - peptide response to glucose ( nmol min l ) . c - peptide concentrations during hyperglycemic clamp and ratio to pretreatment in the exenatide ( a and c)- and insulin glargine ( b and d)-treated group . data represent mean sem in a and b and geometric mean sem in c and d. airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose . , , pretreatment ; , , 52-weeks on - drug ; , , 4 weeks off - drug . the most frequently observed adverse event in exenatide - treated patients was mild - to - moderate nausea ( 50% ) . other gastrointestinal adverse events were reported more commonly in exenatide - treated patients , including vomiting , diarrhea , and abdominal distension . mmol / l ) was observed more frequently in the insulin glargine group ( 24.2% ) than in the exenatide - treated patients ( 8.3% ) . other adverse events observed more frequently in the insulin glargine group included influenza and gastroenteritis . one patient randomly assigned to exenatide developed pancreatitis , which resolved after withdrawal of the study medication . of the patients randomly assigned to exenatide , 62.1% ( n = 18 ) were treated with exenatide 10 g b.i.d . at 52 weeks of treatment . five ( 17.2% ) patients were using 20 g t.i.d . , two ( 6.9% ) were using 10 g t.i.d . , one ( 3.4% ) was using 15 g b.i.d . , and one ( 3.4% ) was using 15 g of t.i.d . the daily exenatide dose was reduced to 5 g b.i.d . in two patients ( 6.9% ) . despite this increase in daily exenatide dose , none of these patients reached the a1c target of < 7.1% . at 52 weeks , the mean the corresponding fasting smbg in the insulin glargine treated group was 5.6 0.2 mmol / l . exenatide and insulin glargine treatment resulted in similar reductions in a1c ( 0.8 0.1 and 0.7 0.2% , respectively ; p = 0.55 ) , with both groups achieving a mean a1c of 6.8% at 52 weeks . the insulin glargine group showed a significantly greater reduction in fasting plasma glucose compared with the exenatide group ( 2.9 0.4 vs. 1.6 0.3 mmol / l , respectively ; p < 0.0001 ) , whereas smbg profiles demonstrated significantly greater reductions in postprandial glucose excursions in the exenatide - treated patients ( fig . 2c and d ) . during the off - drug period , both a1c and fasting plasma glucose increased in both groups and were not significantly different compared with pretreatment values after 12 weeks off - drug ( fig . smbg concentrations before ( c ) and after ( d ) 52 weeks of treatment . changes in body weight ( e ) and insulin sensitivity were measured as the m value ( f ) . , exenatide ; , insulin glargine ; , pretreatment ; , 52 weeks on - drug ; , 4 weeks off - drug . bb , before breakfast ; ab , after breakfast ; bl , before lunch ; al , after lunch ; bd , before dinner ; ad , after dinner ; bt , bedtime . fifty - two weeks of exenatide treatment resulted in a lowering of body weight of 3.6 0.6 kg , whereas treatment with insulin glargine resulted in a body weight increase of + 1.0 0.8 kg ( between - group difference , 4.6 1.1 kg ; p < 0.0001 ) ( fig . body weight trended toward baseline values with both therapies ( between - group difference 2.4 1.1 kg ; p = 0.03 ) . at baseline , insulin - mediated glucose uptake did not differ between the two treatment groups ( fig . treatment with exenatide and insulin glargine improved insulin sensitivity to the same extent by 0.9 0.3 and 1.1 0.3 mg min kg , respectively ( p = 0.49 ) . after a 4-week discontinuation of study medication , the m value was not significantly different from pretreatment values in the insulin glargine treated group , whereas it remained significantly higher in the exenatide - treated group ( between - group difference 0.8 0.4 mg / min / kg ; p = 0.03 ) . at baseline , both glucose- and arginine - stimulated c - peptide secretion did not differ between the two treatment groups ( table 1 ; fig . accordingly , exenatide treatment significantly increased first- and second - phase glucose - stimulated c - peptide secretion by 1.53 0.11- and 2.85 0.22-fold , respectively ( p the c - peptide response to arginine during hyperglycemia increased 3.19 0.24-fold from pretreatment in the exenatide group compared with a 1.31 0.07-fold increase in the insulin glargine group ( between - group difference 2.46 0.20-fold ; p < 0.0001 ) . after 4 weeks discontinuation of the study medication , measures of -cell function returned to pretreatment values in both groups . measures of -cell secretory function during hyperglycemic clamp and ratio to pretreatment in the exenatide- and insulin glargine treated groups data represent mean sem . airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ( nmol min l ) ; first - phase , first - phase c - peptide response to glucose ( nmol min l ; second - phase , second - phase c - peptide response to glucose ( nmol min l ) . see research design and methods for calculations of -cell function measures . c - peptide concentrations during hyperglycemic clamp and ratio to pretreatment in the exenatide ( a and c)- and insulin glargine ( b and d)-treated group . data represent mean sem in a and b and geometric mean sem in c and d. airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose . , , pretreatment ; , , 52-weeks on - drug ; , , 4 weeks off - drug . the most frequently observed adverse event in exenatide - treated patients was mild - to - moderate nausea ( 50% ) . other gastrointestinal adverse events were reported more commonly in exenatide - treated patients , including vomiting , diarrhea , and abdominal distension . mmol / l ) was observed more frequently in the insulin glargine group ( 24.2% ) than in the exenatide - treated patients ( 8.3% ) . other adverse events observed more frequently in the insulin glargine group included influenza and gastroenteritis . one patient randomly assigned to exenatide developed pancreatitis , which resolved after withdrawal of the study medication . this study demonstrates that 52 weeks of treatment with exenatide significantly improves -cell function compared with insulin glargine in metformin - treated type 2 diabetic patients . in addition , exenatide treatment achieved similar improvements in glycemic control , reduced body weight , and resulted in fewer hypoglycemic events . both exenatide and insulin glargine resulted in similar improvements in whole - body insulin sensitivity . after cessation of both treatments , end point measures returned to pretreatment values . in type 2 diabetes , defects in -cell function include an absent first - phase insulin response and a gradually diminishing second - phase response to glucose ( 1 ) . this progressive loss of -cell function is considered to be the main factor responsible for the gradual increase of glycemia over time , regardless of the therapy used ( 3 ) . acute and chronic exposure to exenatide has been shown to improve -cell function ( 46,8 ) . however , there have been no studies comparing -cell function after long - term exposure to exenatide or other glucose - lowering therapies . in the current study , exenatide therapy for 1 year significantly improved -cell function compared with titrated insulin glargine therapy , in the presence of comparable improvements in glycemic control . a number of prior studies have demonstrated that exenatide is not inferior to insulin regimens as a glucose - lowering treatment option in type 2 diabetic patients with inadequate glucose control ( 810 ) . these previous insulin comparator trials have been criticized for not achieving optimal insulin doses in the comparator arm of the study ( 19 ) despite mean reductions in a1c levels that were within the range of reductions observed in comparable insulin trials . in the current study , insulin titration resulted in a mean daily insulin dose of 34 19 units . although this insulin glargine dose is lower than that used in other studies of type 2 diabetes ( 20,21 ) , smbg targets were achieved in the current study , suggesting that insulin doses were appropriately titrated in the majority of patients . in addition , the intensive treatment with both therapies reduced mean a1c values after 52 weeks to 6.8% . in the lanmet study ( 22 ) , in which , comparable to our study , insulin glargine was added to metformin monotherapy in patients with type 2 diabetes , a higher dose of insulin glargine ( 68 units / day ) was used . these apparent differences in insulin glargine dose may be attributed to the relatively good baseline a1c in our population compared with that of the lanmet participants ( a1c 9.5% ) . one limitation of the current study should be highlighted : exenatide was given 15 min before the start of the hyperglycemic clamp study . we therefore can not discriminate between acute and long - term effects of exenatide on -cell function . the current study does , however , support the observation that longer - term treatment with exenatide does not attenuate the known acute effects of this therapy on -cell function , whereas active insulin therapy did not improve -cell function to the same degree . because the primary study objective was to determine the effects of both active exenatide and insulin therapy on -cell function in type 2 diabetic patients , we decided to perform the tests on therapy , in accordance with previous studies ( 18,23,24 ) . these findings support the idea that longer - term exenatide treatment could have an enduring effect on -cell function . in the current study , treatment with both exenatide and insulin glargine resulted in a similar reduction in a1c . of interest , the two therapies achieved this result through different ways : exenatide primarily affected postprandial glucose excursions , with a modest effect on fasting glucose , whereas insulin glargine predominantly reduced fasting plasma glucose , without influencing postprandial glucose elevations . the resultant average glucose concentrations are similar in both treatment groups , as reflected by the a1c concentration . albeit modest , the improved glycemic control in either group may have lowered the hyperglycemia - associated oxidative stress burden , which in part may explain the modest improvement in -cell function in the insulin glargine group ( 25 ) . obviously , patients treated with exenatide showed a greater improvement in acute -cell function , which is considered to be the result of binding to the -cell glp-1 receptor ( 13 ) . these findings are consistent with work in a number of animal models , in which exenatide or glp-1 has been shown to increase -cell mass and to reduce apoptosis ( 13 ) . collectively , these observations lead to the question whether long - term exenatide administration to patients with type 2 diabetes may restore some of the -cell functionality that is lost as part of the natural history of the disease . in our study the improved -cell function at 52 weeks was lost 4 weeks after cessation of either study treatment , and this was accompanied by an increase in plasma glucose and a1c to pretreatment values in both study arms . interestingly , insulin sensitivity remained significantly improved after 4 weeks cessation of treatment in the exenatide - treated group only . this finding may suggest additional effects of exenatide , possibly mediated by weight loss , which may be of longer duration . whether longer - term exposure to exenatide can alter functional -cell mass in the absence of active exenatide treatment will require further study . these effects may be dependent on other factors including diabetes duration , the amount of functional -cells present at the initiation of therapy and overall metabolic control achieved . to study a possible preserving effect on -cell function , both titrated exenatide and insulin glargine were generally well tolerated with > 80% of patients in both groups completing 1 year of therapy . these therapeutic approaches differed in that the most common side effects with exenatide were of gastrointestinal origin , occurring in a proportion of patients similar to that reported in previous studies ( 410 ) . mild to moderate nausea ( 47% ) was the most commonly reported adverse event with exenatide . in contrast , hypoglycemia was the most common adverse event reported in 25% of the insulin glargine treated patients . in summary , this study uniquely demonstrates that 1 year of treatment with exenatide significantly improved -cell function and reduced body weight in the presence of similar improvements in glycemic control compared with insulin glargine treatment . after cessation of therapy , the beneficial effects on -cell function , glycemic control , and body weight were not sustained , suggesting that active treatment is necessary to maintain these beneficial effects of exenatide in patients in whom oral blood glucose lowering therapy has failed .

objectivetraditional blood glucose lowering agents do not sustain adequate glycemic control in most type 2 diabetic patients . preclinical studies with exenatide have suggested sustained improvements in -cell function . we investigated the effects of 52 weeks of treatment with exenatide or insulin glargine followed by an off - drug period on hyperglycemic clamp derived measures of -cell function , glycemic control , and body weight.research design and methodssixty - nine metformin - treated patients with type 2 diabetes were randomly assigned to exenatide ( n = 36 ) or insulin glargine ( n = 33 ) . -cell function was measured during an arginine - stimulated hyperglycemic clamp at week 0 , at week 52 , and after a 4-week off - drug period . additional end points included effects on glycemic control , body weight , and safety.resultstreatment-induced change in combined glucose- and arginine - stimulated c - peptide secretion was 2.46-fold ( 95% ci 2.092.90 , p < 0.0001 ) greater after a 52-week exenatide treatment compared with insulin glargine treatment . both exenatide and insulin glargine reduced a1c similarly : 0.8 0.1 and 0.7 0.2% , respectively ( p = 0.55 ) . exenatide reduced body weight compared with insulin glargine ( difference 4.6 kg , p < 0.0001 ) . -cell function measures returned to pretreatment values in both groups after a 4-week off - drug period . a1c and body weight rose to pretreatment values 12 weeks after discontinuation of either exenatide or insulin glargine therapy.conclusionsexenatide significantly improves -cell function during 1 year of treatment compared with titrated insulin glargine . after cessation of both exenatide and insulin glargine therapy , -cell function and glycemic control returned to pretreatment values , suggesting that ongoing treatment is necessary to maintain the beneficial effects of either therapy .

RESEARCH DESIGN AND METHODS Study end points Statistical analysis RESULTS Patient disposition and baseline clinical characteristics A1C and fasting plasma glucose Body weight and insulin sensitivity Hyperglycemic clampderived measures of -cell function Adverse effects and tolerability CONCLUSIONS Supplementary Material

in total , 69 patients were randomly assigned using a permutated block randomization scheme stratified by site and screening a1c to receive exenatide or insulin glargine , in addition to ongoing metformin treatment ( fig . no other blood glucose lowering agents were allowed within 3 months before screening . patients randomly assigned to exenatide ( n = 36 ) initiated treatment at a dose of 5 g b.i.d . patients randomly assigned to insulin glargine ( n = 33 ) started at an initial dose of 10 iu q.d . first- and second - phase c - peptide secretion was calculated as area under the curve ( auc)180190 min and auc190260 min . arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc260270 min above the fasting c - peptide concentration . clamps were performed before randomization , after 52 weeks of treatment , and after a 4-week off - drug period . during the clamp at week 52 , patients randomly assigned to exenatide , were given the study drug 15 min before the onset of the hyperglycemic clamp , and patients randomly assigned to insulin glargine received their last insulin dose the night before at bedtime . a1c ( normal range : 4.36.1% , diabetes control and complications trial standardized bio - rad assay ) was measured using the fasting plasma glucose , and safety parameters were measured before randomization and during each follow - up visit until the end of the 12-week off - drug period by a central laboratory ( quintiles , livingston , u.k . ) patients were instructed to record seven - point ( fasting , 2 h after breakfast , before lunch , 2 h after lunch , before dinner , 2 h after dinner , and at bedtime ) smbg profiles using an onetouch ultra blood glucose meter ( lifescan , milpitas , ca ) before each visit . c - peptide samples were analyzed at the vu university medical center using an immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , netherlands ) . the primary efficacy end point of this study is the treatment effect on -cell function as measured by the ratio of week 52 combined glucose- and arginine - stimulated insulin secretion during a hyperglycemic clamp . a sample size of 26 patients per group was required to provide 90% power to detect a between - group significant difference in arginine - stimulated insulin secretion between the two treatment groups , assuming that the mean incremental auc value at baseline is 200 pmol min l for both groups and values at week 52 are 1,100 and 300 pmol min the dependent variable used in the model is the loge - transformed ratio to pretreatment for the -cell function parameters ( airarg , first phase , and second phase ) . 1a , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc08-1797/dc1 ) first- and second - phase c - peptide secretion was calculated as area under the curve ( auc)180190 min and auc190260 min . arginine - stimulated c - peptide secretion ( airarg ) was calculated as the incremental auc260270 min above the fasting c - peptide concentration . clamps were performed before randomization , after 52 weeks of treatment , and after a 4-week off - drug period . during the clamp at week 52 , patients randomly assigned to exenatide , were given the study drug 15 min before the onset of the hyperglycemic clamp , and patients randomly assigned to insulin glargine received their last insulin dose the night before at bedtime . a1c ( normal range : 4.36.1% , diabetes control and complications trial standardized bio - rad assay ) was measured using the fasting plasma glucose , and safety parameters were measured before randomization and during each follow - up visit until the end of the 12-week off - drug period by a central laboratory ( quintiles , livingston , u.k . ) patients were instructed to record seven - point ( fasting , 2 h after breakfast , before lunch , 2 h after lunch , before dinner , 2 h after dinner , and at bedtime ) smbg profiles using an onetouch ultra blood glucose meter ( lifescan , milpitas , ca ) before each visit . c - peptide samples were analyzed at the vu university medical center using an immunoradiometric assay ( centaur ; bayer diagnostics , mijdrecht , netherlands ) . the primary efficacy end point of this study is the treatment effect on -cell function as measured by the ratio of week 52 combined glucose- and arginine - stimulated insulin secretion during a hyperglycemic clamp . a sample size of 26 patients per group was required to provide 90% power to detect a between - group significant difference in arginine - stimulated insulin secretion between the two treatment groups , assuming that the mean incremental auc value at baseline is 200 pmol l for both groups and values at week 52 are 1,100 and 300 pmol min the dependent variable used in the model is the loge - transformed ratio to pretreatment for the -cell function parameters ( airarg , first phase , and second phase ) . of the patients randomly assigned to exenatide , 62.1% ( n = 18 ) were treated with exenatide 10 g b.i.d . at 52 weeks of treatment . at 52 weeks , the mean the corresponding fasting smbg in the insulin glargine treated group was 5.6 0.2 mmol / l . exenatide and insulin glargine treatment resulted in similar reductions in a1c ( 0.8 0.1 and 0.7 0.2% , respectively ; p = 0.55 ) , with both groups achieving a mean a1c of 6.8% at 52 weeks . the insulin glargine group showed a significantly greater reduction in fasting plasma glucose compared with the exenatide group ( 2.9 0.4 vs. 1.6 0.3 mmol / l , respectively ; p < 0.0001 ) , whereas smbg profiles demonstrated significantly greater reductions in postprandial glucose excursions in the exenatide - treated patients ( fig . during the off - drug period , both a1c and fasting plasma glucose increased in both groups and were not significantly different compared with pretreatment values after 12 weeks off - drug ( fig . changes in body weight ( e ) and insulin sensitivity were measured as the m value ( f ) . , exenatide ; , insulin glargine ; , pretreatment ; , 52 weeks on - drug ; , 4 weeks off - drug . fifty - two weeks of exenatide treatment resulted in a lowering of body weight of 3.6 0.6 kg , whereas treatment with insulin glargine resulted in a body weight increase of + 1.0 0.8 kg ( between - group difference , 4.6 1.1 kg ; p < 0.0001 ) ( fig . body weight trended toward baseline values with both therapies ( between - group difference 2.4 1.1 kg ; p = 0.03 ) . treatment with exenatide and insulin glargine improved insulin sensitivity to the same extent by 0.9 0.3 and 1.1 0.3 mg min kg , respectively ( p = 0.49 ) . after a 4-week discontinuation of study medication , the m value was not significantly different from pretreatment values in the insulin glargine treated group , whereas it remained significantly higher in the exenatide - treated group ( between - group difference 0.8 0.4 mg / min / kg ; p = 0.03 ) . at baseline , both glucose- and arginine - stimulated c - peptide secretion did not differ between the two treatment groups ( table 1 ; fig . accordingly , exenatide treatment significantly increased first- and second - phase glucose - stimulated c - peptide secretion by 1.53 0.11- and 2.85 0.22-fold , respectively ( p the c - peptide response to arginine during hyperglycemia increased 3.19 0.24-fold from pretreatment in the exenatide group compared with a 1.31 0.07-fold increase in the insulin glargine group ( between - group difference 2.46 0.20-fold ; p < 0.0001 ) . after 4 weeks discontinuation of the study medication , measures of -cell function returned to pretreatment values in both groups . measures of -cell secretory function during hyperglycemic clamp and ratio to pretreatment in the exenatide- and insulin glargine treated groups data represent mean sem . airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ( nmol min l ) ; first - phase , first - phase c - peptide response to glucose ( nmol min l ; second - phase , second - phase c - peptide response to glucose ( nmol min l ) . c - peptide concentrations during hyperglycemic clamp and ratio to pretreatment in the exenatide ( a and c)- and insulin glargine ( b and d)-treated group . data represent mean sem in a and b and geometric mean sem in c and d. airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose . , , pretreatment ; , , 52-weeks on - drug ; , , 4 weeks off - drug . the most frequently observed adverse event in exenatide - treated patients was mild - to - moderate nausea ( 50% ) . other gastrointestinal adverse events were reported more commonly in exenatide - treated patients , including vomiting , diarrhea , and abdominal distension . mmol / l ) was observed more frequently in the insulin glargine group ( 24.2% ) than in the exenatide - treated patients ( 8.3% ) . one patient randomly assigned to exenatide developed pancreatitis , which resolved after withdrawal of the study medication . of the patients randomly assigned to exenatide , 62.1% ( n = 18 ) were treated with exenatide 10 g b.i.d . at 52 weeks of treatment . exenatide and insulin glargine treatment resulted in similar reductions in a1c ( 0.8 0.1 and 0.7 0.2% , respectively ; p = 0.55 ) , with both groups achieving a mean a1c of 6.8% at 52 weeks . the insulin glargine group showed a significantly greater reduction in fasting plasma glucose compared with the exenatide group ( 2.9 0.4 vs. 1.6 0.3 mmol / l , respectively ; p < 0.0001 ) , whereas smbg profiles demonstrated significantly greater reductions in postprandial glucose excursions in the exenatide - treated patients ( fig . during the off - drug period , both a1c and fasting plasma glucose increased in both groups and were not significantly different compared with pretreatment values after 12 weeks off - drug ( fig . smbg concentrations before ( c ) and after ( d ) 52 weeks of treatment . changes in body weight ( e ) and insulin sensitivity were measured as the m value ( f ) . , exenatide ; , insulin glargine ; , pretreatment ; , 52 weeks on - drug ; , 4 weeks off - drug . fifty - two weeks of exenatide treatment resulted in a lowering of body weight of 3.6 0.6 kg , whereas treatment with insulin glargine resulted in a body weight increase of + 1.0 0.8 kg ( between - group difference , 4.6 1.1 kg ; p < 0.0001 ) ( fig . body weight trended toward baseline values with both therapies ( between - group difference 2.4 1.1 kg ; p = 0.03 ) . treatment with exenatide and insulin glargine improved insulin sensitivity to the same extent by 0.9 0.3 and 1.1 0.3 mg min kg , respectively ( p = 0.49 ) . after a 4-week discontinuation of study medication , the m value was not significantly different from pretreatment values in the insulin glargine treated group , whereas it remained significantly higher in the exenatide - treated group ( between - group difference 0.8 0.4 mg / min / kg ; p = 0.03 ) . at baseline , both glucose- and arginine - stimulated c - peptide secretion did not differ between the two treatment groups ( table 1 ; fig . accordingly , exenatide treatment significantly increased first- and second - phase glucose - stimulated c - peptide secretion by 1.53 0.11- and 2.85 0.22-fold , respectively ( p the c - peptide response to arginine during hyperglycemia increased 3.19 0.24-fold from pretreatment in the exenatide group compared with a 1.31 0.07-fold increase in the insulin glargine group ( between - group difference 2.46 0.20-fold ; p < 0.0001 ) . after 4 weeks discontinuation of the study medication , measures of -cell function returned to pretreatment values in both groups . measures of -cell secretory function during hyperglycemic clamp and ratio to pretreatment in the exenatide- and insulin glargine treated groups data represent mean sem . airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ( nmol min l ) ; first - phase , first - phase c - peptide response to glucose ( nmol min l ; second - phase , second - phase c - peptide response to glucose ( nmol min l ) . see research design and methods for calculations of -cell function measures . c - peptide concentrations during hyperglycemic clamp and ratio to pretreatment in the exenatide ( a and c)- and insulin glargine ( b and d)-treated group . data represent mean sem in a and b and geometric mean sem in c and d. airarg , c - peptide response to arginine at 15 mmol / l glucose concentration ; 1st phase , first - phase c - peptide response to glucose ; 2nd phase , second - phase c - peptide response to glucose . , , pretreatment ; , , 52-weeks on - drug ; , , 4 weeks off - drug . other gastrointestinal adverse events were reported more commonly in exenatide - treated patients , including vomiting , diarrhea , and abdominal distension . mmol / l ) was observed more frequently in the insulin glargine group ( 24.2% ) than in the exenatide - treated patients ( 8.3% ) . one patient randomly assigned to exenatide developed pancreatitis , which resolved after withdrawal of the study medication . this study demonstrates that 52 weeks of treatment with exenatide significantly improves -cell function compared with insulin glargine in metformin - treated type 2 diabetic patients . in addition , exenatide treatment achieved similar improvements in glycemic control , reduced body weight , and resulted in fewer hypoglycemic events . both exenatide and insulin glargine resulted in similar improvements in whole - body insulin sensitivity . after cessation of both treatments , end point measures returned to pretreatment values . in type 2 diabetes , defects in -cell function include an absent first - phase insulin response and a gradually diminishing second - phase response to glucose ( 1 ) . acute and chronic exposure to exenatide has been shown to improve -cell function ( 46,8 ) . however , there have been no studies comparing -cell function after long - term exposure to exenatide or other glucose - lowering therapies . in the current study , exenatide therapy for 1 year significantly improved -cell function compared with titrated insulin glargine therapy , in the presence of comparable improvements in glycemic control . a number of prior studies have demonstrated that exenatide is not inferior to insulin regimens as a glucose - lowering treatment option in type 2 diabetic patients with inadequate glucose control ( 810 ) . although this insulin glargine dose is lower than that used in other studies of type 2 diabetes ( 20,21 ) , smbg targets were achieved in the current study , suggesting that insulin doses were appropriately titrated in the majority of patients . in the lanmet study ( 22 ) , in which , comparable to our study , insulin glargine was added to metformin monotherapy in patients with type 2 diabetes , a higher dose of insulin glargine ( 68 units / day ) was used . the current study does , however , support the observation that longer - term treatment with exenatide does not attenuate the known acute effects of this therapy on -cell function , whereas active insulin therapy did not improve -cell function to the same degree . because the primary study objective was to determine the effects of both active exenatide and insulin therapy on -cell function in type 2 diabetic patients , we decided to perform the tests on therapy , in accordance with previous studies ( 18,23,24 ) . these findings support the idea that longer - term exenatide treatment could have an enduring effect on -cell function . in the current study , treatment with both exenatide and insulin glargine resulted in a similar reduction in a1c . albeit modest , the improved glycemic control in either group may have lowered the hyperglycemia - associated oxidative stress burden , which in part may explain the modest improvement in -cell function in the insulin glargine group ( 25 ) . obviously , patients treated with exenatide showed a greater improvement in acute -cell function , which is considered to be the result of binding to the -cell glp-1 receptor ( 13 ) . collectively , these observations lead to the question whether long - term exenatide administration to patients with type 2 diabetes may restore some of the -cell functionality that is lost as part of the natural history of the disease . in our study the improved -cell function at 52 weeks was lost 4 weeks after cessation of either study treatment , and this was accompanied by an increase in plasma glucose and a1c to pretreatment values in both study arms . interestingly , insulin sensitivity remained significantly improved after 4 weeks cessation of treatment in the exenatide - treated group only . to study a possible preserving effect on -cell function , both titrated exenatide and insulin glargine were generally well tolerated with > 80% of patients in both groups completing 1 year of therapy . in summary , this study uniquely demonstrates that 1 year of treatment with exenatide significantly improved -cell function and reduced body weight in the presence of similar improvements in glycemic control compared with insulin glargine treatment . after cessation of therapy , the beneficial effects on -cell function , glycemic control , and body weight were not sustained , suggesting that active treatment is necessary to maintain these beneficial effects of exenatide in patients in whom oral blood glucose lowering therapy has failed .

amongst adult patients with medically intractable epilepsy , focal epilepsy from a temporal lobe focus represents the largest single etiology [ 14 ] . while only a small percentage ( < 25% ) among patients operated for temporal lobe epilepsy ( tle ) are in the pediatric age group , it is well documented that most adults in surgical series have had a childhood onset of epilepsy . it has been suggested that a significant proportion of children with refractory epilepsy on the other hand may have tle and , more specifically , may have hippocampal sclerosis ( hs ) on mri ( magnetic resonance imaging ) . there exist gaps in our understanding of the natural history of tle and its progression , from its antecedents in fetal life and childhood , eventually culminating in a stage of medical refractoriness that is reached in adult life . current understanding of the natural history of tle identifies the need for well - designed prospective studies to elucidate risk factors and triggers [ 7 , 8 ] . clinical information of this nature might provide additional avenues for interventional strategies to be investigated and developed . in this paper we present a brief outline and review of prior published data on tle and identify critical areas where additional information on the evolution of tle can be prospectively derived . for the purpose of clarification , the term tle used is inclusive of more common mesial temporal lobe epilepsy ( mtle ) , as well as other forms of neocortical temporal lobe epilepsy ( ntle ) , unless otherwise specified . a pubmed search strategy using the following terms tle and natural history ; tle and antecedents ; tle and benign ; tle and clinical and outcome was adopted for the purpose of this review . the search yielded a total of 678 publications when the limits of human and english were applied . studies were selected on the basis of ( 1 ) whether patient selection criteria clearly defined tle . we ascertained that each of the definitions used clearly either carried mention of detailed semiological features of habitual seizures and latent period or resection of the temporal lobe for refractory epilepsy with outcome details and ( 2 ) if the study mentioned any clinical details of antecedents or precipitating events that includes fs or fse , age at onset , and time to refractoriness of epilepsy . after reviewing 89 abstracts and 23 full text articles reporting large series of patients with tle , many of which were outcome studies of surgery for tle we identified nine studies that documented details of past history obtained preoperatively . a summary of key findings in the studies critical to this review is introduced in table 1 . the participants of group 1 ( for definition and natural history of mtle ) of the ilae ( international league against epilepsy ) commission on neurosurgery observed that most surgical series reported a high incidence of ipes in the form of fs , trauma , hypoxia or intracranial infection . , since the detailed and exhaustive case histories that are required to determine frequency of the ipes ( antecedents ) and time to onset as well as time to intractability of epilepsy might not be available from these retrospective series . in an elegant study , french et al . presented a comparative analysis of data obtained from 193 patients with predefined clinical and eeg criteria to define tle and that obtained from 164 normal individuals . etiological factors in the past histories of 77.2% patients with tle , compared with only 7.3% among the control population reporting birth abnormalities or other significant past history . clinical data regarding ipes in childhood have been identified in surgical series that include patients with hs on mri ( magnetic resonance imaging ) and underwent temporal lobectomy ( tly ) for the same . the data presented by harvey et al . however evaluated patients who did not undergo surgery . in this group nearly 40% of patients with tle confirmed the occurrence of an ipe , in the form of simple or complicated fs . a history of perinatal problems was reported in only a small proportion of patients ( < 1% ) by several investigators [ 10 , 12 , 13 , 17 ] with the exception of the study by ozkara et al . . in the latter , the authors reported an antecedent history suggestive of birth asphyxia in 21 out of 62 patients ( 33.9% ) , a significantly higher proportion . a history of fs was also noted in a majority of patients ( 70% ) among their group of postoperative patients with mtle and mri evidence of hs . in a study by rathore et al . , a similar proportion , that is , ( 68.5% ) of postoperative patients were found to have a documented history of fs , while only 2 out of 124 patients ( 1.6% ) had history of perinatal hypoxia . in the largest series among the selected studies that were reviewed , uijl et al . found history of fs in 37% of the patients , while details of other ipes were not described . in a study of factors determining the development of treatment resistance among patients undergoing epilepsy surgery , berg et al . are only able to provide data on history of febrile seizures , while information on other ipes is lacking . an analysis of these selected as well as other studies on surgical outcomes in tle leads one to the conclusion that there is little prospective data or agreement regarding ipes amongst most published series . it is clear from these studies that there is a considerable variation encountered when comparing results from different studies even when similar clinical variables are considered . differences in the documentation of historical details , different selection criteria for surgery , and the lack of prospectively collected information surrounding ipes may serve as plausible reasons for the variation in information available . thus , while perinatal critical events and febrile seizures appear to be known variables , no definitive conclusions other than a speculative inference regarding the potential role for ipe 's can be drawn from these published studies . studied the age at onset of a homogenous group of 118 patients with mtle and history of childhood febrile convulsions ( cfcs ) and reported the age at epilepsy onset to be a trimodal pattern with peaks at ages of 5.5 , 15.3 , and 26.7 years . these authors observed that the latent / silent period between occurrence of cfcs and epilepsy onset was significantly longer in patients with childhood onset , when compared to patients with an adolescent onset of mtle . harvey et al . reported a bimodal pattern of the age at onset in a much smaller group of tle patients with hs on mri . the mean age observed was 5.1 3.8 years with two peaks identified , the first occurring early ( second year ) and the second , late ( ninth year ) during childhood years . a younger age of onset of epilepsy also appears to bear a strong correlation with mri findings of hs , even controlling for disease duration and history of fs [ 8 , 20 ] . in a study aimed at evaluating predictive prognostic factors for a cohort of patients with sporadic tle associated with either hs or a nonlesional tle aguglia et al . observed an older average age at onset ( among 100 seizure free ) patients to be 33.5 19.9 years in comparison to an average age of 17.2 14 . 4 years ( in 90 non - seizure - free ) patients on univariate analysis . furthermore , an older age at onset was found to be the single independent prognostic factor for good seizure outcome , even on multivariate analysis . heuser and colleagues propose the occurrence of mtle among patients with history of fs ( mtle - fs ) as a separate entity based on the observation that the age at epilepsy onset is significantly lower in this group as compared to those mtle patients who do not have prior history of fs . it is evident from these studies that tle , dominantly represented by patients with hs on mri , has a variable age at onset . despite suggestions that a prior cerebral insult predisposes an individual to development of tle , at least among a subgroup of patients , the latency to the onset of epilepsy remains variable . in addition , the latency to the development of the more commonly encountered refractory form of epilepsy remains largely unpredictable in the individual setting . thus , the evidence from published case series suggests a role for the duration of epilepsy from the time of occurrence of the ipe in determining development of hs and prospects for a remission . prospective cohort studies among subgroups of patients with perinatal brain injury and those with fs and fse will be necessary to determine whether distinct subtypes of tle can be delineated based on the age of onset of epilepsy and the occurrence of fs / fse . it is well known that among patients with tle as well as other focal epilepsies , treatment resistance or refractoriness to antiepileptic drugs ( aeds ) appears after a significant time lapse or latency from onset of first seizure , at least in a subset of patients . the mechanisms underlying this latent period remain largely unidentified . in a study analyzing information obtained from 333 patients with medically refractory partial epilepsy , berg et al . could identify the average time to intractability as 9.1 years ( median 5 and range 046 years ) in 282 patients . the authors defined time to intractability as time between occurrence of second seizure and failure of second aed . on multivariate logistic regression analysis , the authors found that the age at onset provided the most significant correlation of this latency period ; that is , earlier the age at onset , longer the latency period to intractability . in another retrospective study of 162 patients fulfilling the inclusion criteria of tle followed up for at least 2 years , the best model to predict refractoriness to medication included the variable failure of first aed trial , with a positive predictive value of 0.89 ( 95% ci 0.76 , 0.96 ) and negative predictive value of 0.95 ( 95% ci 0.87 , 0.99 ) . it has been suggested that the time to establish failure to respond to an adequate trial of a second aed ( or even the first aed ) does not necessarily imply latency to intractability , as intractability may have been present long before the current standard of proof is attained . any direct study of human tle , suggesting that it is a progressive condition , is not yet available . confounding factors like the genetic profile of the individual ( susceptibility genes , genetic polymorphisms ) , epigenetic modifiers , underlying systemic disease , effect of epileptic discharges on human brain , neuropathological effects of seizure - related systemic perturbations , trauma , aed effects ( including inappropriate drugs ) , and multiple drug - resistant transporters will no doubt influence any estimate . fs have long been known to be associated with the future development of epilepsy , especially mtle [ 25 , 26 ] . while retrospective studies have favored this association , prospective studies thus far have failed to confirm a causal association between the two [ 2729 ] . in the retrospective study by abou - khalil et al . , amongst 47 patients operated for temporal resection , a subgroup of 19 patients with history of fs showed excellent surgical outcome ( 85% seizure - free ) in comparison to the remaining 28 patients ( 32% ) who did not have prior history of fs . importantly , almost all patients had prolonged fs ( mean duration 4 hours , range 20 minutes to 24 hours ) . a major selection bias could potentially have contributed to this difference , as the history of fs may have well influenced the decision to take up those patients with hs for tly , resulting in an expectedly good surgical outcome . it is then interesting to note the findings of a large prospective study on patients with a history of fs obtained through questionnaires administered to parents of infants born over a week in the uk . the questionnaires were administered when the children were 5 years of age ( 82% responses obtained ) and when they were 10 years of age ( 93% responses obtained ) . thirteen of these 382 ( 3.4% ) experienced one or more afebrile seizures , nine of whom developed epilepsy . a higher proportion of children with complex fs ( 6/95 ) rather than simple fs ( 3/287 ) developed epilepsy , the risk being highest for those who had had focal fs ( 5/17 ; x2 = 399 , p < 0 001 ) . the authors concluded that epilepsy developing after fs in childhood is not as common as expected from hospital studies , rather rare . a systematic review of 63 studies on the outcome of pediatric status epilepticus found that the risk of sequelae in unprovoked and febrile convulsive status epilepticus ( cse ) is low . the same review finds evidence that cse , especially febrile cse , might cause hippocampal injury , although its role in the development of mts is unknown . additional lines of evidence favor a role for human herpes virus 6 ( hhv 6 ) in the causation of mtle based on the detection of the viral antigen in resected temporal lobe tissues at surgery , and exposure to the virus in the early childhood is exceedingly common . detection of the virus or viral antigen in body fluids of children with febrile seizures has ranged from 8 to 40% in different studies . the rate of hhv6 detection in resected temporal lobe tissue from surgical specimens in patients undergoing temporal lobectomy has varied greatly [ 33 , 34 ] . recently , niehusmann et al . carried out molecular and histochemical analysis of excised pathological specimens in patients undergoing temporal lobectomy for pharmacoresistant epilepsy . they reported a 55% positivity for hhv 6 on nested pcr experiments in surgically excised temporal lobe tissue in a group of 9 tle patients who met strict criteria of a well - defined history of encephalitis in childhood , compared to none among 26 others with tle without ipes , tle with history of complex fs , and lesional tle with 10 autopsy controls . the authors suggest that while their findings argue against a causative role for hhv6 in tle with a history of complex fs , the viral infection may play an important role in tle with a history of encephalitis , despite the obvious heterogeneity of postencephalitic patients in their series . they suggest that , in patients with encephalitis of different cause , the presence of hhv6 may facilitate the development of tle ; however , they caution that it may neither be sufficient or necessary factor . it is also interesting to note that the presence of hhv6 was associated with significant gliosis in the temporal lobe tissue being examined , a feature which points to a role for inflammation in tle [ 35 , 36 ] . interval data analysis from the febstat study , a multi - center prospective study on children 1 month to 5 year in age , designed to study the consequences of fse , indicates that primary hhv-6b may be an important cause of fse . the evidence thus far offers weak associations between the occurrence of fs , fse , and viral infections ( in particular hhv-6 ) in childhood and subsequent development of tle . it is also suggested that age and coexistent neuropathology may be added as contributory factors in the variable rates of detection of herpesvirus dna in surgically resected tissue . an older age at onset is the only independent predictor of 2-year seizure freedom or remission among patients with nonlesional tle . studying 190 patients with clinically diagnosed tle , with a mean followup of 11 years , the authors found that more than half of the patients underwent spontaneous remissions . although occurrence of fs and hs on mri was found to be predictors of poor prognosis on univariate analysis , these did not attain significance on multivariate analysis . spooner et al . , in a prospective study on 64 patients with tle followed up for a median of 13 years , found 19 ( 31% ) to have attained 515-year remission and successfully remained off treatment while the remainder ( 69% ) continued to have seizures and had to be considered for epilepsy surgery evaluation , the only predictor of significance for continued seizures , being a lesion on mri . both these studies published nearly 15 years apart suggest similar facts about remissions in tle that a sizeable proportion of patients with an older age of onset do remit , and that the presence of lesions on mri and a younger age at onset predict lower chance of remission . a review of the literature surrounding tle clearly suggests that the etiology varies depending on age of onset . the maximal information regarding etiology has been obtained from pathological studies on surgically resected tissue in individuals undergoing anterior temporal lobectomy ( atl ) . the existence of other forms of tle where the outcome is better than the more common form of pharmacoresistant tle is also being recognized ( benign tle and familial tle ) . the findings of a recently published study suggest that the most frequently reported pathology in childhood onset tle who develop medically refractory epilepsy consists of dual pathology in the form of a neocortical lesion associated with hs ( 80% of cases accounted for by cortical dysplasia or tumors such as gangliogliomas with hs ) . isolated malformations of cortical development ( mcd ) , gangliogliomas without hippocampal pathology , accounted for the remainder . in infants , hs tends to be even less frequent , with the majority of the pathology reported consisting of nonprogressive lesions such as mcd , hamartomas , and low grade tumors such as dysembryoplastic neuroepithelial tumors ( dnet ) . the etiologies in adult onset tle differ from those of childhood onset . while the epidemiological studies of late onset tle point to infective , vascular ( arteriovenous malformation , cavernous hemangioma , meningioangiomatosis ) , tumoral , traumatic , and in older individuals ( > 64 years of age ) neurodegenerative etiologies , the most frequently observed and reported pathology is hs . the causes leading to adult tle have been less frequently reported [ 42 , 43 ] . it is of interest that the etiologies in those individuals who developed tle with an age of onset beyond 20 years differ further from those individuals developing tle in late adulthood ( often > 6065 years ) . in a recent study , soeder et al . identified limbic encephalitis ( le ) associated with paraneoplastic and nonparaneoplastic ( autoimmune ) conditions ( 25% ) as constituting the largest group , followed by hs that was preceded by an ipe ( 22% ) . tumors ( dnet , ganglioglioma , and other low - grade tumors ) and amygdalar lesions made up constituted ( 14% and 135 ) , respectively . finally , a miscellaneous group ( 24% ) was made of patients with posttraumatic lesions , cavernomas , chronic herpes simplex encephalitis , and progressive tumors ( anaplastic oligodendroglioma , glioblastoma multiforme , and astrocytomas ) . the italian group of labate , gambardella , aguglia , and colleagues have addressed various issues related to benign mtle in great detail [ 4446 ] . they described this entity for patients , who were seizure free or had auras or not more than 2 disabling ( cpss and/or secondarily generalized ) seizures in 24 months , with or without appropriate medications . in a study of 101 patients diagnosed as benign tle , they found mri evidence of unilateral mts in 39 ( 38.6% ) patients . the antecedent history of febrile seizures alone predicted the association with mts in this group of patients with benign tle . it has , thus , been clearly demonstrated that neither clinical presentation with features of tle nor mri evidence of mts is determinant of future refractoriness of epilepsy in a large proportion of patients with tle . first described by berkovic and colleagues in 38 patients from 13 families in australia , familial tle has been recognized as a benign syndrome with typical temporal lobe seizures and good prognosis . in a recent report by the same group , the epilepsies in these families were found to be generally benign , and history of febrile seizures was infrequent ( 9.8% ) . a single individual underwent atl , with subsequent seizure freedom and histopathological evidence of hs was not found . santos and colleagues reported 15 families with tle , 14 of which manifested with features of mtle and one family with ntle . this study is published in abstract format and no data on course of the epilepsy has been presented . within this group of patients with a relatively benign outcome , pathogenic mutations in the lgi 1 ( leucine - rich glioma - inactivated 1 ) gene have been identified in patients with a partial epilepsy syndrome with auditory features ( both autosomal dominant and sporadic forms ) . the existence of genetic heterogeneity in this group is well documented [ 50 , 51 ] . while no definitive causal associations can be established , there does not appear strong evidence to support the existence of a single common pathway to the development of tle and medical intractability . the most important conclusion that one can draw is that tle is not a homogeneous entity . rather , there may be several subsets that could be characterized on the basis of the following features : ( 1 ) age of onset of epilepsy ( early versus late ) ( 2 ) the presence or absence of a lesion on imaging ( lesional versus nonlesional ) , ( 3 ) the nature of the ipe , ( 4 ) the occurrence of fs and fse , ( 5 ) genetic and familial predisposition , and finally ( 6 ) response to treatment and outcome . any combination and interaction within different factors probably confer susceptibility as well as development and progression to tle ( figure 1 ) . the current body of literature on the subject is vast and confusing at times . the limitations of most studies include the retrospective nature , the lack of longitudinal and observational studies , and weaknesses in study design . single - center studies with poorly defined criteria for selection and inherent biases in patient selection further add to the challenges in interpretation of results . several important questions that have been raised need to be addressed in carefully designed studies . these questions could be addressed through careful assessment of patients from the index event of the first seizure by epileptologists . in this context longitudinal observation of selected cohorts of patients sharing ipe 's etiologies will form the groups where specialized high - resolution imaging and genetic studies should be considered . tracking the patients to emergence of treatment resistance and eventual surgery in some will be important in identifying benign versus poor outcomes . postsurgical evaluation of pathological material and specialized studies will further clarify role of infection and inflammation . animal models could be developed to clarify questions pertaining to epileptogenesis , establish , and explore the usefulness of treatment interventions . future prospective studies will need to be multicenter and prospective in nature , perhaps undertaken by teams of investigators , with attention to study design . such studies should take into account not only the factors discussed , but additionally be prepared to take a multipronged approach in investigating genetic and epigenetic factors that influence the development of an epileptogenic network that eventually leads to the development of different forms of tle .

temporal lobe epilepsy represents the largest group of patients with treatment resistant / medically intractable epilepsy undergoing epilepsy surgery . the underpinnings of common forms of tle in many instances begin in early life with the occurrence of an initial precipitating event . the first epileptic seizure often occurs after a variable latency period following this event . the precise natural history and progression following the first seizure to the development of tle , its subsequent resolution through spontaneous remission or the development of treatment resistant epilepsy remain poorly understood . our present understanding of the role played by these initial events , the subsequent latency to development of temporal lobe epilepsy , and the emergence of treatment resistance remains incomplete . a critical analysis of published data suggest that tle is a heterogeneous condition , where the age of onset , presence or absence of a lesion on neuroimaging , the initial precipitating event , association with febrile seizures , febrile status epilepticus , and neurotropic viral infections influence the natural history and outcome . the pathways and processes through which these variables coalesce into a framework will provide the basis for an understanding of the natural history of tle . the questions raised need to be addressed in future prospective and longitudinal observational studies .

1. Introduction 2. Methods 3. Results and Discussion 4. Conclusions and Future Directions

amongst adult patients with medically intractable epilepsy , focal epilepsy from a temporal lobe focus represents the largest single etiology [ 14 ] . while only a small percentage ( < 25% ) among patients operated for temporal lobe epilepsy ( tle ) are in the pediatric age group , it is well documented that most adults in surgical series have had a childhood onset of epilepsy . there exist gaps in our understanding of the natural history of tle and its progression , from its antecedents in fetal life and childhood , eventually culminating in a stage of medical refractoriness that is reached in adult life . current understanding of the natural history of tle identifies the need for well - designed prospective studies to elucidate risk factors and triggers [ 7 , 8 ] . clinical information of this nature might provide additional avenues for interventional strategies to be investigated and developed . in this paper we present a brief outline and review of prior published data on tle and identify critical areas where additional information on the evolution of tle can be prospectively derived . for the purpose of clarification , the term tle used is inclusive of more common mesial temporal lobe epilepsy ( mtle ) , as well as other forms of neocortical temporal lobe epilepsy ( ntle ) , unless otherwise specified . a pubmed search strategy using the following terms tle and natural history ; tle and antecedents ; tle and benign ; tle and clinical and outcome was adopted for the purpose of this review . studies were selected on the basis of ( 1 ) whether patient selection criteria clearly defined tle . we ascertained that each of the definitions used clearly either carried mention of detailed semiological features of habitual seizures and latent period or resection of the temporal lobe for refractory epilepsy with outcome details and ( 2 ) if the study mentioned any clinical details of antecedents or precipitating events that includes fs or fse , age at onset , and time to refractoriness of epilepsy . after reviewing 89 abstracts and 23 full text articles reporting large series of patients with tle , many of which were outcome studies of surgery for tle we identified nine studies that documented details of past history obtained preoperatively . the participants of group 1 ( for definition and natural history of mtle ) of the ilae ( international league against epilepsy ) commission on neurosurgery observed that most surgical series reported a high incidence of ipes in the form of fs , trauma , hypoxia or intracranial infection . , since the detailed and exhaustive case histories that are required to determine frequency of the ipes ( antecedents ) and time to onset as well as time to intractability of epilepsy might not be available from these retrospective series . presented a comparative analysis of data obtained from 193 patients with predefined clinical and eeg criteria to define tle and that obtained from 164 normal individuals . etiological factors in the past histories of 77.2% patients with tle , compared with only 7.3% among the control population reporting birth abnormalities or other significant past history . clinical data regarding ipes in childhood have been identified in surgical series that include patients with hs on mri ( magnetic resonance imaging ) and underwent temporal lobectomy ( tly ) for the same . in this group nearly 40% of patients with tle confirmed the occurrence of an ipe , in the form of simple or complicated fs . a history of perinatal problems was reported in only a small proportion of patients ( < 1% ) by several investigators [ 10 , 12 , 13 , 17 ] with the exception of the study by ozkara et al . in the latter , the authors reported an antecedent history suggestive of birth asphyxia in 21 out of 62 patients ( 33.9% ) , a significantly higher proportion . a history of fs was also noted in a majority of patients ( 70% ) among their group of postoperative patients with mtle and mri evidence of hs . , a similar proportion , that is , ( 68.5% ) of postoperative patients were found to have a documented history of fs , while only 2 out of 124 patients ( 1.6% ) had history of perinatal hypoxia . in the largest series among the selected studies that were reviewed , uijl et al . found history of fs in 37% of the patients , while details of other ipes were not described . in a study of factors determining the development of treatment resistance among patients undergoing epilepsy surgery , berg et al . are only able to provide data on history of febrile seizures , while information on other ipes is lacking . an analysis of these selected as well as other studies on surgical outcomes in tle leads one to the conclusion that there is little prospective data or agreement regarding ipes amongst most published series . differences in the documentation of historical details , different selection criteria for surgery , and the lack of prospectively collected information surrounding ipes may serve as plausible reasons for the variation in information available . thus , while perinatal critical events and febrile seizures appear to be known variables , no definitive conclusions other than a speculative inference regarding the potential role for ipe 's can be drawn from these published studies . studied the age at onset of a homogenous group of 118 patients with mtle and history of childhood febrile convulsions ( cfcs ) and reported the age at epilepsy onset to be a trimodal pattern with peaks at ages of 5.5 , 15.3 , and 26.7 years . these authors observed that the latent / silent period between occurrence of cfcs and epilepsy onset was significantly longer in patients with childhood onset , when compared to patients with an adolescent onset of mtle . reported a bimodal pattern of the age at onset in a much smaller group of tle patients with hs on mri . the mean age observed was 5.1 3.8 years with two peaks identified , the first occurring early ( second year ) and the second , late ( ninth year ) during childhood years . a younger age of onset of epilepsy also appears to bear a strong correlation with mri findings of hs , even controlling for disease duration and history of fs [ 8 , 20 ] . in a study aimed at evaluating predictive prognostic factors for a cohort of patients with sporadic tle associated with either hs or a nonlesional tle aguglia et al . observed an older average age at onset ( among 100 seizure free ) patients to be 33.5 19.9 years in comparison to an average age of 17.2 14 . furthermore , an older age at onset was found to be the single independent prognostic factor for good seizure outcome , even on multivariate analysis . heuser and colleagues propose the occurrence of mtle among patients with history of fs ( mtle - fs ) as a separate entity based on the observation that the age at epilepsy onset is significantly lower in this group as compared to those mtle patients who do not have prior history of fs . it is evident from these studies that tle , dominantly represented by patients with hs on mri , has a variable age at onset . despite suggestions that a prior cerebral insult predisposes an individual to development of tle , at least among a subgroup of patients , the latency to the onset of epilepsy remains variable . in addition , the latency to the development of the more commonly encountered refractory form of epilepsy remains largely unpredictable in the individual setting . thus , the evidence from published case series suggests a role for the duration of epilepsy from the time of occurrence of the ipe in determining development of hs and prospects for a remission . prospective cohort studies among subgroups of patients with perinatal brain injury and those with fs and fse will be necessary to determine whether distinct subtypes of tle can be delineated based on the age of onset of epilepsy and the occurrence of fs / fse . it is well known that among patients with tle as well as other focal epilepsies , treatment resistance or refractoriness to antiepileptic drugs ( aeds ) appears after a significant time lapse or latency from onset of first seizure , at least in a subset of patients . in a study analyzing information obtained from 333 patients with medically refractory partial epilepsy , berg et al . the authors defined time to intractability as time between occurrence of second seizure and failure of second aed . on multivariate logistic regression analysis , the authors found that the age at onset provided the most significant correlation of this latency period ; that is , earlier the age at onset , longer the latency period to intractability . in another retrospective study of 162 patients fulfilling the inclusion criteria of tle followed up for at least 2 years , the best model to predict refractoriness to medication included the variable failure of first aed trial , with a positive predictive value of 0.89 ( 95% ci 0.76 , 0.96 ) and negative predictive value of 0.95 ( 95% ci 0.87 , 0.99 ) . it has been suggested that the time to establish failure to respond to an adequate trial of a second aed ( or even the first aed ) does not necessarily imply latency to intractability , as intractability may have been present long before the current standard of proof is attained . any direct study of human tle , suggesting that it is a progressive condition , is not yet available . confounding factors like the genetic profile of the individual ( susceptibility genes , genetic polymorphisms ) , epigenetic modifiers , underlying systemic disease , effect of epileptic discharges on human brain , neuropathological effects of seizure - related systemic perturbations , trauma , aed effects ( including inappropriate drugs ) , and multiple drug - resistant transporters will no doubt influence any estimate . fs have long been known to be associated with the future development of epilepsy , especially mtle [ 25 , 26 ] . , amongst 47 patients operated for temporal resection , a subgroup of 19 patients with history of fs showed excellent surgical outcome ( 85% seizure - free ) in comparison to the remaining 28 patients ( 32% ) who did not have prior history of fs . a major selection bias could potentially have contributed to this difference , as the history of fs may have well influenced the decision to take up those patients with hs for tly , resulting in an expectedly good surgical outcome . it is then interesting to note the findings of a large prospective study on patients with a history of fs obtained through questionnaires administered to parents of infants born over a week in the uk . thirteen of these 382 ( 3.4% ) experienced one or more afebrile seizures , nine of whom developed epilepsy . a higher proportion of children with complex fs ( 6/95 ) rather than simple fs ( 3/287 ) developed epilepsy , the risk being highest for those who had had focal fs ( 5/17 ; x2 = 399 , p < 0 001 ) . a systematic review of 63 studies on the outcome of pediatric status epilepticus found that the risk of sequelae in unprovoked and febrile convulsive status epilepticus ( cse ) is low . the same review finds evidence that cse , especially febrile cse , might cause hippocampal injury , although its role in the development of mts is unknown . additional lines of evidence favor a role for human herpes virus 6 ( hhv 6 ) in the causation of mtle based on the detection of the viral antigen in resected temporal lobe tissues at surgery , and exposure to the virus in the early childhood is exceedingly common . detection of the virus or viral antigen in body fluids of children with febrile seizures has ranged from 8 to 40% in different studies . the rate of hhv6 detection in resected temporal lobe tissue from surgical specimens in patients undergoing temporal lobectomy has varied greatly [ 33 , 34 ] . carried out molecular and histochemical analysis of excised pathological specimens in patients undergoing temporal lobectomy for pharmacoresistant epilepsy . they reported a 55% positivity for hhv 6 on nested pcr experiments in surgically excised temporal lobe tissue in a group of 9 tle patients who met strict criteria of a well - defined history of encephalitis in childhood , compared to none among 26 others with tle without ipes , tle with history of complex fs , and lesional tle with 10 autopsy controls . the authors suggest that while their findings argue against a causative role for hhv6 in tle with a history of complex fs , the viral infection may play an important role in tle with a history of encephalitis , despite the obvious heterogeneity of postencephalitic patients in their series . they suggest that , in patients with encephalitis of different cause , the presence of hhv6 may facilitate the development of tle ; however , they caution that it may neither be sufficient or necessary factor . it is also interesting to note that the presence of hhv6 was associated with significant gliosis in the temporal lobe tissue being examined , a feature which points to a role for inflammation in tle [ 35 , 36 ] . the evidence thus far offers weak associations between the occurrence of fs , fse , and viral infections ( in particular hhv-6 ) in childhood and subsequent development of tle . an older age at onset is the only independent predictor of 2-year seizure freedom or remission among patients with nonlesional tle . studying 190 patients with clinically diagnosed tle , with a mean followup of 11 years , the authors found that more than half of the patients underwent spontaneous remissions . although occurrence of fs and hs on mri was found to be predictors of poor prognosis on univariate analysis , these did not attain significance on multivariate analysis . , in a prospective study on 64 patients with tle followed up for a median of 13 years , found 19 ( 31% ) to have attained 515-year remission and successfully remained off treatment while the remainder ( 69% ) continued to have seizures and had to be considered for epilepsy surgery evaluation , the only predictor of significance for continued seizures , being a lesion on mri . both these studies published nearly 15 years apart suggest similar facts about remissions in tle that a sizeable proportion of patients with an older age of onset do remit , and that the presence of lesions on mri and a younger age at onset predict lower chance of remission . a review of the literature surrounding tle clearly suggests that the etiology varies depending on age of onset . the existence of other forms of tle where the outcome is better than the more common form of pharmacoresistant tle is also being recognized ( benign tle and familial tle ) . the findings of a recently published study suggest that the most frequently reported pathology in childhood onset tle who develop medically refractory epilepsy consists of dual pathology in the form of a neocortical lesion associated with hs ( 80% of cases accounted for by cortical dysplasia or tumors such as gangliogliomas with hs ) . in infants , hs tends to be even less frequent , with the majority of the pathology reported consisting of nonprogressive lesions such as mcd , hamartomas , and low grade tumors such as dysembryoplastic neuroepithelial tumors ( dnet ) . while the epidemiological studies of late onset tle point to infective , vascular ( arteriovenous malformation , cavernous hemangioma , meningioangiomatosis ) , tumoral , traumatic , and in older individuals ( > 64 years of age ) neurodegenerative etiologies , the most frequently observed and reported pathology is hs . it is of interest that the etiologies in those individuals who developed tle with an age of onset beyond 20 years differ further from those individuals developing tle in late adulthood ( often > 6065 years ) . identified limbic encephalitis ( le ) associated with paraneoplastic and nonparaneoplastic ( autoimmune ) conditions ( 25% ) as constituting the largest group , followed by hs that was preceded by an ipe ( 22% ) . tumors ( dnet , ganglioglioma , and other low - grade tumors ) and amygdalar lesions made up constituted ( 14% and 135 ) , respectively . finally , a miscellaneous group ( 24% ) was made of patients with posttraumatic lesions , cavernomas , chronic herpes simplex encephalitis , and progressive tumors ( anaplastic oligodendroglioma , glioblastoma multiforme , and astrocytomas ) . the italian group of labate , gambardella , aguglia , and colleagues have addressed various issues related to benign mtle in great detail [ 4446 ] . the antecedent history of febrile seizures alone predicted the association with mts in this group of patients with benign tle . it has , thus , been clearly demonstrated that neither clinical presentation with features of tle nor mri evidence of mts is determinant of future refractoriness of epilepsy in a large proportion of patients with tle . first described by berkovic and colleagues in 38 patients from 13 families in australia , familial tle has been recognized as a benign syndrome with typical temporal lobe seizures and good prognosis . in a recent report by the same group , the epilepsies in these families were found to be generally benign , and history of febrile seizures was infrequent ( 9.8% ) . within this group of patients with a relatively benign outcome , pathogenic mutations in the lgi 1 ( leucine - rich glioma - inactivated 1 ) gene have been identified in patients with a partial epilepsy syndrome with auditory features ( both autosomal dominant and sporadic forms ) . while no definitive causal associations can be established , there does not appear strong evidence to support the existence of a single common pathway to the development of tle and medical intractability . the most important conclusion that one can draw is that tle is not a homogeneous entity . rather , there may be several subsets that could be characterized on the basis of the following features : ( 1 ) age of onset of epilepsy ( early versus late ) ( 2 ) the presence or absence of a lesion on imaging ( lesional versus nonlesional ) , ( 3 ) the nature of the ipe , ( 4 ) the occurrence of fs and fse , ( 5 ) genetic and familial predisposition , and finally ( 6 ) response to treatment and outcome . the limitations of most studies include the retrospective nature , the lack of longitudinal and observational studies , and weaknesses in study design . single - center studies with poorly defined criteria for selection and inherent biases in patient selection further add to the challenges in interpretation of results . several important questions that have been raised need to be addressed in carefully designed studies . these questions could be addressed through careful assessment of patients from the index event of the first seizure by epileptologists . in this context longitudinal observation of selected cohorts of patients sharing ipe 's etiologies will form the groups where specialized high - resolution imaging and genetic studies should be considered . tracking the patients to emergence of treatment resistance and eventual surgery in some will be important in identifying benign versus poor outcomes . animal models could be developed to clarify questions pertaining to epileptogenesis , establish , and explore the usefulness of treatment interventions . future prospective studies will need to be multicenter and prospective in nature , perhaps undertaken by teams of investigators , with attention to study design . such studies should take into account not only the factors discussed , but additionally be prepared to take a multipronged approach in investigating genetic and epigenetic factors that influence the development of an epileptogenic network that eventually leads to the development of different forms of tle .

there are several histological types of thyroid cancer , including papillary thyroid carcinoma ( ptc ) , follicular thyroid carcinoma ( ftc ) , medullary thyroid carcinoma ( mtc ) , and anaplastic thyroid carcinoma ( atc ) . determining the type of thyroid cancer is crucial for the assessment of prognosis and treatment selection . most patients with thyroid carcinoma are initially diagnosed based on the result of fine needle aspiration cytology . refined diagnosis and classification / staging of this cancer unfortunately , in some cases , proper classification could be problematic when histopathological patterns are inconclusive . therefore , classical histopathological approach in the diagnosis of thyroid cancer could be potentially supported by molecular biomarkers . nowadays , a number of molecular tests to confirm the diagnosis of thyroid nodules have been proposed , which included panel of somatic mutations ( e.g. , ret - ptc , ras ) and immunocytochemistry tests ( e.g. , braf v600e ihc ) [ 24 ] . this approach , although not implemented widely in clinical practice yet , could change the attitude towards classification of thyroid cancers in near future . further application studies and clinical trials such applications are likely to become a component of the standard diagnostic approach for patients with thyroid cancer . therefore , there is an urgent need to determine the most cost - effective protocols to utilize these molecular - based diagnostic tools . metabolomics is one of the high - throughput omics techniques , which beside genomics , transcriptomics , and proteomics play an important role in systems biology . metabolome is the final downstream product of gene expression and therefore reflects changes in the transcriptome ( mrna ) and the proteome ( proteins ) . additionally , metabolomics reflects phenotypic changes and alterations in pathophysiological states of biological systems and therefore represents the most downstream level of molecular life of a cell . metabolomics targets different classes of low molecular weight ( mw < 1500 da ) metabolites . in contrast to plants , human metabolomes are relatively well known , defined , and catalogued . the estimated number of components of human metabolomes ranges from thousands to tens of thousands , depending on type of a cell or tissue . changes in metabolome composition reflect alterations in enzymes concentration , cellular regulation , control of signalling pathways , genetic variations , and catabolic and anabolic reactions . therefore , metabolome most directly reflects the phenotype , physiology , and molecular state of an organism . the main drawback of metabolomic studies , reflecting chemical variability of this cellular component , is the fact that there is no single analytical method allowing simultaneous measurement of such broad spectrum of bioactive compounds . hence , different combinations of liquid ( lc ) and gas ( gc ) chromatography coupled with mass spectrometry ( ms ) or nuclear magnetic resonance ( nmr ) are the most accepted and widely used analytical approaches in this field . metabolomics provides valuable information about metabolism of malignant cells and has a great potential in cancer research as well as in identification of novel diagnostic and prognostic markers . several studies proved that metabolomics approaches allowed for classification of different types of malignancies and identification of potential biomarkers in the case of brain , breast , kidney , and prostate cancers . although studies regarding metabolome of thyroid cancer are not very common , there are several recent works showing that metabolomics approach could help to discriminate different types of thyroid lesions and provide significant information about their progression . here , we aim to review recent progress in the field of thyroid metabolomics and discuss its contribution to understanding thyroid tumorigenesis and potential refinement of molecular classification of thyroid cancers . palpable thyroid nodules occur in 47% of the population ; however , lesions found incidentally during ultrasonographic examination suggest a prevalence of 1967% . the classification of such nodules includes numerous entities , both nonneoplastic and neoplastic , benign and malignant . although thyroid tumors occur in roughly 510% of palpable nodules , they represent the most common endocrine malignancy and pose a significant challenge to pathologists , surgeons , and oncologists . fine - needle aspiration ( fna ) cytology is a simple , rapid , inexpensive , and minimally invasive procedure which plays an important role in decision - making regarding clinical management of patients with thyroid nodules . it has six diagnostic categories , which correlate with risk of malignancy , and provides clear management guidelines to clinicians to go for follow - up fna or surgery . in general , many thyroid cancers can be diagnosed with certainty using fna , yet the method has some limitations . for example , the nuclear and architectural changes of some ptcs are subtle and focal . this is particularly true of the follicular variant of ptc , which can be difficult to distinguish from a benign follicular nodule . other ptcs may be incompletely sampled and yield only a small number of abnormal cells . some rare benign lesions like hyalinizing trabecular tumors are frequently misdiagnosed by fna cytology as ptc [ 17 , 18 ] . histological distinguishing of follicular carcinoma from follicular adenoma is based on the presence of capsular or / and vascular invasion , whose features could not be analyzed in cytological material . moreover , the cytological features alone can not reliably separate the detected thyroid tumors into benign or malignant ; hence , patients with lesions are advised to undergo a diagnostic surgery . in fact , less than 30% of such lesions were diagnosed after surgery as malignant . therefore , several molecular tests for adjustment of cytological diagnosis of thyroid nodules have been proposed , which included panel of somatic mutations ( e.g. , braf and nras mutation and/or ret / ptc translocation ) and immunocytochemistry tests based on gene expression signatures . brafv600e is very specific for ptc , yet the failure to detect this mutation does not rule out ptc . another approach proposed recently to improve diagnostics based on fna material included commercially available tests : afirma gene expression classifier for thyroid fna analysis and mirinform thyroid test by asuragen [ 22 , 23 ] . when concerning postoperative material , a reliable histopathological diagnosis can be reached by an experienced pathologist by sole morphologic assessment for most cases of thyroid tumors . however , several types of morphologic features ( e.g. , the presence of nuclear atypia ) are not sufficient for reliable diagnosis of malignancy . for example , in hashimoto 's thyroiditis and dyshormonogenesis , isolated cells with pleomorphic nuclei are very common . furthermore , in the case of tumors exhibiting unusual morphologic patterns or in order to confirm the diagnosis of medullary carcinoma , additional molecular tests ( e.g. , immunohistochemistry ) are required . nevertheless , the currently used molecular tests have only limited application in routine diagnosis of thyroid neoplasia . although several ihc tests ( e.g. , for cytokeratin-19 and galectin-3 ) have been proposed for distinguishing malignant from benign thyroid lesions , they frequently show high level of false - positive and false - negative results . also molecular markers suggested for differential diagnosis of ptc versus benign thyroid lesions or other thyroid tumors have been widely tested in clinical practice . in fact , certain normal thyroid follicles , nonneoplastic thyroid lesions ( in particular thyroiditis ) , and benign thyroid tumors can exhibit focal or extensive staining for many of putative thyroid cancer markers . for instance , 3155% of adenomatous hyperplasia could be positive for cytokeratin-19 or galectin-3 . however , even application of a panel of markers for ihc studies ( e.g. , ck-19 , gal-3 , and hbme-1 ) did not improve significantly the diagnostic performance of the test . the apparent role of interactions between the tumor and its niche in the tumor - thyroid interface represents additional diagnostic challenge . recently , promising results have been reported for the usage of cd56 for differentiation of benign lesions and ptc [ 2628 ] . additionally , in the case of a follicular patterned neoplasm lacking the cytoarchitectural features of papillary carcinoma , the only feature that distinguishes carcinoma from adenoma is the presence of unequivocal vascular and/or capsular invasion in the former . in conclusion , histopathological evaluation remains the gold standard in the distinction between different types of thyroid cancer , for example , between follicular carcinoma and follicular adenoma . various molecular tests have been tested beside histological analysis , including immunohistochemical assessment of proteins and genetic tests for gene mutations , yet none of them proved its actual clinical applicability . therefore , there is a constant need for development of unambiguous molecular markers which would bring a real improvement in diagnosis and classification of thyroid cancers . the term omics defines high - throughput approaches to complex molecular composition of tissues enabling simultaneous analysis of thousands of genes / proteins . the main assumption of systems biology is integration of different omics datasets in order to obtain a broad perspective on the complex processes occurring in a living organism . for this purpose , platforms integrating genomics , transcriptomics , proteomics , and metabolomics data are constructed , enabling better understanding of mechanisms involved in natural history of cancer . therefore , there is a generally expressed expectation that such approach would also deliver new biomarkers to be applied in clinical practice . microarray and next - generation - sequencing approaches to the analysis of gene expression as well as mass spectrometry techniques used for the analysis of proteins and/or peptides have delivered valuable information on various types of human malignancies . the best example of how the achievements of the genomics era could alter the clinicopathologic paradigm in classification of cancer types and affect the decision making process in selection of a treatment is breast cancer [ 3134 ] . similar approaches have been tested during the last decade for many other malignancies , including thyroid cancer . the first global microarray - based gene expression profile of thyroid cancer was reported in 2001 , when the gene signature characteristic for papillary thyroid cancer was identified . another microarray - based study allowed for identification of the gene expression signatures associated with mutations in braf , ras , and ret / ptc genes , as well as distinguishing the classic ptc from the tall cell and follicular variants . comparative analysis of the expression profiles in ptc and ftc , the two most common forms of thyroid carcinoma , enabled identification of the gene signatures characteristic for these cancers ; the differentiating signature includes five genes ( cited1 , cav1 , cav2 , igfbp6 , and cldn10 ) . since then many other works have been published describing the differences between thyroid cancer and normal thyroid tissue , as well as differences between types of thyroid neoplasia . these works revealed the significance of hundreds of genes , including the key genes involved in thyroid hormone biosynthesis [ 38 , 39 ] . an important issue in the diagnostics of thyroid cancer is differentiation between follicular adenoma , follicular carcinoma , and the follicular variant of papillary carcinoma . currently , several biomarkers have proved their applicability in solving this problem , including lgals3 , hemoglobin , epsilon 1 ( hbe1 ) , keratin 19 ( ck-19 ) , and tpo ( thyroid peroxidase ) . another powerful and promising approach in the search of cancer biomarkers is proteomics based on mass spectrometry tools [ 4145 ] . proteins can be extracted , identified , and quantified from different cell and tissue sources . it should be stressed that proteomics studies of thyroid represent a real challenge due to high heterogeneity observed in this tissue and very broad range between the most abundant ( e.g. , thyroglobulin ) and the least abundant proteins . important disease - related proteins are likely to be discovered in the subpicomolar range , which means that thyroid proteome profiling methods must cover the dynamic range substantially wider than 10 . the comparative analysis of ptc specimens matched with the normal thyroid tissue from the same patients and benign follicular adenomas allowed identification of three proteins , namely , s100a6 ( an isoform of s100 protein ) , peroxiredoxin 2 , and heat shock protein 70 ( hsp70 ) , whose expression levels were markedly higher in ptc tissue . furthermore , the study confirmed overexpression of several ptc markers identified earlier by mrna study ( e.g. , galectin-3 , cytokeratin-19 , and cathepsin - b ) . another study compared proteome profiles between thyroid follicular adenomas and follicular carcinomas and revealed statistically significant difference in abundance of 43 proteins detected in thyroid tissue . genomics and proteomics studies have a significant impact on general understanding of cancer - related processes and have delivered several promising candidates for cancer biomarkers . however , some limitations in the prognostic and prediction power of gene / protein expression data have been recognized in recent years . the challenge is that correlation between the established mrna and protein expression levels and their real influence on phenotype of cancer has appeared elusive in many cases . therefore , other approaches directly addressing phenotypic features of a cancer tissue , represented by metabolomics , have a large potential in cancer studies . cancer progression is a complex process which involves proliferation , hypoxia , angiogenesis , apoptosis , metastasis , inflammation , and increased tolerance to reactive oxygen species . these tumor associated processes significantly affect the primary metabolic pathways ; hence , cancer cells are characterized by altered metabolism in comparison with the normal differentiated cells , whose pathways are depicted schematically in figure 1 . the major difference between cancerous and normal cells concerns the pathways involved in production of energy . in healthy tissues , glucose is used for the production of nadh and atp during the tricarboxylic acid cycle ( also known as the krebs cycle or the citric acid cycle ) and oxidative phosphorylation . in marked contrast , most cancer cells use aerobic glycolysis , known as the warburg effect , to produce both energy and building blocks ( amino acids , nucleotides , and fatty acids ) needed for extensive growth and proliferation . otto warburg was the first to observe that tumor cells take up large amounts of glucose which is converted to lactic acid . since then numerous studies have shown the increased level of lactate and other glycolytic products in cancer tissues [ 5355 ] , which is a phenomenon that can be used in clinical practice for detection of tumors . cancer metabolome is also characterized by elevated amounts of fatty acids and lipids , which are essential for cell membrane building . fatty acids are synthesized de novo during cancer progression [ 5658 ] which requires nadph and acetyl - coa . in cancer cells , ppp , which is upregulated by glycolysis , provides pentose phosphates which are essential also in the synthesis of nucleotides . moreover , increased glutaminolysis provides acetyl - coa in the reverse reaction of citrate synthase . in addition to nadph and acetyl - coa , choline is the next essential substrate required for lipids biosynthesis , and accumulation of this compound in cancer cells was observed in different studies . choline - containing compounds , including phosphocholine , phosphatidylcholine , and glycerophosphocholine , are the key components of a cell membrane . thus , alterations in membrane phospholipids may influence key aspects of cancer phenotype like invasiveness and metastatic potential . changes in the levels of lipids and their derivatives were observed in patients with different type of malignancies , including breast , prostate , brain , and thyroid cancers . saturated and unsaturated lipids forming a cell membrane could move through the cytosol as mobile droplets and accumulate in cytosolic vesicles . currently , it becomes widely accepted that lipids associated with proliferation and inflammation have apparent prognostic value in cancer diagnostics [ 69 , 70 ] . in addition to lipids , there is a large group of small metabolites , including amino acids , nucleotides , sugars , and organic acids , important for cancer development . as a consequence of the warburg effect , the decreased level of glucose and simultaneously increased levels of lactic acid and alanine are observed , especially during hypoxia and ischemia [ 7173 ] . levels of taurine and myo - inositol , other small molecules involved in osmoregulation , are also affected in cancer , which was reported in thyroid , prostate , colon , breast , and ovarian cancers [ 11 , 55 ] . although elevated levels of phospholipids and products of glycolysis characterize cancer cell in general , it should be emphasised that specific levels of various metabolites , including glycine , alanine , lactate , citrate , nucleotides , and lipids , may depend on the type of a cancer . consequently , metabolomics approach allows distinguishing not only between the normal and cancer tissue but also between different types and stages of malignancy . breast cancer is generally characterized by elevated level of total choline - containing compounds ( tcho ) , low glycerophosphocholine , and low glucose , when compared to healthy tissues and benign tumors [ 64 , 7477 ] . furthermore , specific differences in features of metabolome were observed between different histotypes of breast cancer . another example of well - described malignancies is brain tumors , whose different histological types showed distinct metabolic profiles reflecting the levels of alanine , threonine , creatine , glutamate , and phosphocholine [ 7880 ] . also the metabolome of prostate cancer was characterized , where high levels of tcho and phosphocholine , along with increased amounts of alanine and lactate , were observed . therefore , metabolomics has an apparent potential in the studies focused on the discovery of cancer biomarkers . current research efforts are focused on the use of metabolomics screening in preclinical and clinical studies to improve diagnosis and support therapy . however , there is still a significant need to establish the rigorous and effective analytical protocols which could be widely accepted and find their appropriate place in clinical trials . the number of metabolites present in a human organism is currently estimated as approximately 17,000 ( according to the human metabolome database - hmdb version 3.6 ) , yet this number is still expanding ; hence , the exact figure remains unknown . due to extremely diverse physicochemical properties of different metabolites and highly dynamic changes in the composition of the metabolomes , there is no single analytical method allowing examination of the entire metabolome . metabolomic fingerprinting is the least precise approach which enables rapid monitoring of the composition of low molecular weight compounds , without the need for detailed identification . the most commonly used method is metabolite profiling , allowing qualitative and quantitative analysis of a given group of metabolites . targeted analysis enables the most detailed study of a selected class of compounds [ 10 , 82 ] . regardless of the chosen analytical approach , there are several general steps in the metabolomics studies , including sample collection and preparation , data acquisition and processing , biostatistical analysis , and data interpretation , which have been schematically depicted in figure 2 . in order to obtain reproducible results that could be compared between laboratories , strict compliance with the standardized procedures of metabolomic analysis metabolomics standard initiative ( msi ) ( http://www.msi-workgroups.sourceforge.net/ ) published standard reporting requirements for each step of metabolomics experiments [ 8387 ] . the first and also the most critical steps in metabolomics study are sample collection , storage , and preparation for instrumental analysis . blood ( and its derivatives like serum ) and urine are the most commonly used specimens for biomarker studies because of easy repetitive access and high yield . moreover , saliva , breath condensate , bronchial washes , pancreatic juices , prostatic secretions , faeces , and other types of physiological liquids or surrogate tissues can be also used for metabolomics studies . for different biofluids , the standard sample volume required for most types of analyses is within the range of 0.1 to 0.5 ml . most obviously , tissue specimens are also widely examined in metabolomics studies using mass spectrometry ( ms ) , imaging mass spectrometry ( ims ) , and nuclear magnetic resonance ( nmr ) spectroscopy techniques . however , tissue samples are usually characterized by large heterogeneity and therefore require more complex preparation procedures before analysis , including the use of laser - capture microdissection ( lcm ) techniques . the collected material can be fresh - frozen and stored in the temperature below 80c , which is a gold standard in the analysis of metabolites . however , tissue specimens fixed in formalin and stored as formalin - fixed paraffin - embedded ( ffpe ) samples ( normally stored in room temperature ) are the most accessible source of clinical material for molecular studies . this type of tissue material is also suitable for certain metabolomic analyses when properly collected and stored . each type of specimen is characterized by different features , for example , volatility , extraction efficiency , and storage requirement . nevertheless , due to high sensibility of metabolites to exogenous environment , maintaining low temperature and selection of the appropriate method of extraction are essential in most of the cases . metabolites present in biological samples differ in terms of molecular weight , thermostability , volatility , and polarity . therefore , it is impossible to isolate all of them simultaneously , using the same method of purification . consequently , the choice of the sample preparation method depends on the character of the studied compound class and the type of the analytical technique . for isolation of a relatively large group of metabolites , organic solvents of different polarity ( i.e. , methanol , acetonitrile , chloroform , or hexane ) should be used for analyte extraction . additionally , separation techniques based on gas chromatography require further derivatization of analytes , which enhances their volatility . the techniques based on nmr require less complicated procedures for sample processing , yet these analytical methods are less sensitive when compared to ms . the most commonly applied analytical methods in cancer metabolomics studies are liquid or gas chromatography coupled with mass spectrometry ( lc / gc - ms ) and nuclear magnetic resonance ( nmr ) spectroscopy . ms is a highly sensitive technique which allows identification and quantification of multiple metabolites , even at very low concentrations , based on the mass to charge ratio of the analyte ions generated in the spectrometer . high - resolution ms experiments with sequential fragmentation of the analyte ions permit obtaining structural information about the studied compounds , yet not all metabolites can be ionized using either positive or negative ionization mode . chromatographic techniques coupled with ms allow separation of complex mixture of analysed compounds , which is a typical problem in the case of biological materials . gc / ms approach requires more time - consuming sample processing , yet it is a reproducible method allowing for access to large databases for automatic identification of metabolites . nmr spectroscopy also enables identification of small biomolecules based on the resonance spectra of the atomic nuclei h , c , and p , which are commonly present in metabolites . methods based on nmr are generally less sensitive in comparison to ms techniques ; however , they can be used in analyses of liquid and solid samples with minimal preparation stage . high - resolution magic angel spinning ( hr - mas ) nmr spectroscopy is widely tested for metabolite - based cancer tissue classification [ 55 , 100 ] . more recently , direct analysis of spatial distribution of metabolites in a tissue is an emerging approach in metabolomics . spatial distribution of metabolic markers can be analyzed both in vitro and in vivo using localized magnetic resonance spectroscopy imaging ( mrsi ) . another method for in vitro differentiation of tissue regions based on their molecular content is imaging mass spectrometry ( ims ) . ims allows for high - resolution determination of spatial distribution of metabolites and lipids within the tissue , which was utilized in several studies oriented on cancer metabolome [ 102104 ] . the combination of different analytical methods , including ms , nmr , and imaging techniques , apparently provides the most powerful approach to metabolomics studies in current and future applications . the last stage of a metabolomics study is data analysis and interpretation , including several different computation and bioinformatics steps . the collected ms or nmr spectral data requires multistep processing and normalization for further identification of components and their quantification [ 105 , 106 ] . subsequently , the registered molecules are identified by annotation at metabolomics databases , such as human metabolome database ( hmdb ) , metlin , golm database , massbank , or lipid maps . next , multivariate statistics , including supervised and/or unsupervised methods , is required for pattern - recognition and identification of multicomponent signatures or classifiers characteristic for different biological states ( e.g. , tumor versus normal tissue ) . supervised methods ( e.g. , pls - da , opls - da ) use the information of class membership to classify a given dataset . unsupervised approaches ( e.g. , pca , hca ) have been applied to investigate the innate variation in dataset . nevertheless , biomarker candidates obtained in existing studies apparently require further testing and validation using independent material and ( most preferably ) cross - validation studies involving interlaboratory cooperation [ 113 , 114 ] . there are numerous works proving successful application of genomics , transcriptomics , and proteomics in the field of thyroid cancer research [ 2 , 46 , 115 ] . in contrast , metabolomic studies concerning this malignancy are currently limited to relatively few papers . however , the metabolomics approach implemented in identification of biomarkers for diagnosis and classification of thyroid tumors has been dynamically expanding in recent years [ 55 , 58 , 67 , 116118 ] . the state - of - the - art technologies including maldi - ims ( matrix assisted laser desorption ionization - imaging mass spectrometry ) and hr - mas nmr ( high - resolution magic angle spinning nuclear magnetic resonance ) have been used for the identification of a large group of metabolites present in thyroid tissues , which are potential diagnostic biomarkers [ 58 , 116 ] . in general , significant differences were observed between the metabolomes of normal thyroid tissues and neoplastic lesions , as well as between benign and malignant nodules . major groups of metabolites , whose different abundance in thyroid tissue and/or blood was observed between different types of thyroid lesions , are listed there . discrimination of different thyroid lesions , such as nonneoplastic nodules , follicular adenoma , and malignant tumors , based on metabolite profiles constitutes a technically challenging but clinically relevant problem . the first studies demonstrating the potential of h nmr to differentiate between normal and malignant thyroid metabolites were conducted during the last decade of 20th century [ 119122 ] . shortly afterwards , the potential of nmr techniques in the analysis of lipid component of thyroid cancer was reported [ 123 , 124 ] . more recently , hr mas hmr and mri methods have been applied to distinguish between benign and malignant thyroid nodules [ 125 , 126 ] . in fact , nmr metabolomic data processed with multivariate statistical approaches allowed separation between different types of thyroid lesions and several multicomponent metabolic signatures / classifiers were proposed . . took advantage of hr mas hmr and noted elevated levels of several amino acids ( mainly phenylalanine and taurine ) and lactate combined with decreased levels of choline ( and its derivatives ) and scyllo-/myo - inositol in malignant lesions ( ptc , fvptc , and ftc ) in comparison to the benign ones , yet significant differences between the different cancer histotypes were not detected . the observed changes in the levels of lactates and inositols were in agreement with the changes generally observed in many types of tumors . however , the decrease in the content of choline and its derivatives was not generally observed in cancer tissue [ 57 , 127 , 128 ] , also in other works on thyroid cancer [ 58 , 67 , 118 , 129 , 130 ] . deja et al . implemented h nmr technique in analysis of aqueous tissue extracts of healthy thyroid tissue , nonneoplastic nodules , follicular adenomas , and malignant cancer . the authors subjected acquired nmr data to multivariate analysis including both unsupervised method ( pca ) and supervised modelling ( opls - da ) and identified metabolites characteristic for different types of thyroid lesions . potential biomarkers common to all thyroid lesions included alanine , methionine , glutamate , glycine , tyrosine , phenylalanine , hypoxanthine , acetone , and lactate . the reduced levels of scyllo- and myo - inositol , which act as osmoregulators , were found to be specific for thyroid cancers . moreover , a decrease of lipids content in malignant lesions was detected when compared to the healthy tissue . the increased level of lactate was found not only in cancer tissue but also in other thyroid lesions . in general , the observed features of the cancer metabolome reflected dysregulation of the tricarboxylic acid cycle , ammonia recycling , amino acids metabolism , and osmotic regulation . based on the specific metabolite profile of follicular adenomas and significant differences between healthy control , nonneoplastic nodules , and thyroid cancer , the authors classified this lesion as an intermediate step in thyroid cancer progression . this observation gives a promise of new potential metabolomic biomarkers of different thyroid cancer stages . imaging mass spectrometry ( ims ) has already been used for simultaneous detection and spatial localization of lipids in differentiated thyroid cancer tissues . ishikawa et al . focused on distribution of phospholipids in papillary carcinoma in comparison to normal thyroid tissue . they identified phosphatidylcholines pc ( 16:0/18:1 ) , pc ( 16:0/18:2 ) , and sphingomyelin sm ( 18:0/16:1 ) , whose levels in ptc were significantly higher than in nontumor tissue . more recently , guo et al . presented the results of their work where molecular tissue imaging using maldi - fticr - ims was combined with serum lipidome profiling . the study was focused on phosphatidylcholines ( pc ) , phosphatidic acids ( pa ) , and sphingomyelins ( sm ) in three types of tissues : normal thyroid , benign tumors ( thyroid adenoma and multinodular goiter ) , and malignant tumors ( papillary and follicular thyroid carcinoma ) . based on ims analysis performed on 36 tissue samples and lc / ms profiling of almost 300 sera from three different groups of patients ( healthy , with malignant thyroid tumor , or with benign thyroid tumor ) , the authors identified ten differentiating lipid species : phosphatidylcholines pc ( 34:1 ) , pc ( 36:1 ) , pc ( 38:6 ) , phosphatidic acids pa ( 36:2 ) , pa ( 36:3 ) , pa ( 38:3 ) , pa ( 38:4 ) , pa ( 38:5 ) , pa ( 40:5 ) , and sphingomyelin sm ( 34:1 ) . these potential biomarkers ( or their subpanels ; see table 1 ) detected in serum showed potential diagnostic power to differentiate between cancer patients and healthy individuals as well as between patients with malignant and benign thyroid lesions . moreover , the authors validated the previous findings [ 56 , 57 ] that de novo synthesis of fatty acids was associated with tumorigenesis . for this purpose , expression of key enzymes , scd1 and fasn , involved in de novo fatty acids synthesis [ 131 , 132 ] was correlated with the levels of monosaturated pc in malignant , benign , and normal thyroid tissues . immunohistochemical detection of scd1 and fasn confirmed their association with high level of monosaturated pc in thyroid cancer tissues . the impact of lipid metabolism on thyroid tumorigenesis has also been shown by yao et al . . the authors conducted serum metabolic profiling of ptc , nodular goiter cases , and healthy control using lc / ms technique . significant changes in the levels of amino acids , free fatty acids ( ffa ) , and phospholipids were observed between different groups of donors . moreover , the most significant differences between benign and malignant nodules were observed in lipid metabolism . the deregulation of lipid metabolism in a group of patients with ptc was mirrored by an increased level of sphingosine , ffa , 3-hydroxybutyric acid , and carnitine . particularly , the level of 3-hydroxybutyric acid , which is intermediate product of fatty acid metabolism , was significantly higher in patients with ptc than in benign tumors and healthy control cases . metabolomics has an apparent potential to expand our knowledge on molecular factors involved in thyroid cancer . recent works have indeed shown significant differences in metabolomes of normal and neoplastic thyroid tissues , as well as between various stages of neoplasia . however , the example of breast cancer studies clearly indicates the need for combining the metabolomics information with other systems biology datasets to provide the holistic view of the processes ongoing in this endocrine malignancy . such metabolomics network has already been created for thyroid hormone secretion pathway , which helped to identify the potential drug targets essential in treatment of thyroid disorders such as hypo- or hyperthyroidism [ 133 , 134 ] . along with the technological development of analytical tools ( first of all ms- and nmr - based spectral techniques ) , metabolomics becomes an emerging research approach also in the field of thyroid cancer . however , despite the promising value of initial studies , there is a need for rigorous standardization of analytical methods and validation of preliminary results using large independent datasets . moreover , there is an obvious necessity for integration of metabolomic data to genomics , transcriptomics , and proteomics results to bring better insights into the cellular mechanisms of thyroid cancer progression . nevertheless , one should expect that metabolomics studies may deliver in the next years the basic knowledge not only on cancer - related processes but also on novel biomarkers to be implemented in diagnosis and classification of thyroid cancer .

thyroid cancer is the most common endocrine malignancy with four major types distinguished on the basis of histopathological features : papillary , follicular , medullary , and anaplastic . classification of thyroid cancer is the primary step in the assessment of prognosis and selection of the treatment . however , in some cases , cytological and histological patterns are inconclusive ; hence , classification based on histopathology could be supported by molecular biomarkers , including markers identified with the use of high - throughput omics techniques . beside genomics , transcriptomics , and proteomics , metabolomic approach emerges as the most downstream attitude reflecting phenotypic changes and alterations in pathophysiological states of biological systems . metabolomics using mass spectrometry and magnetic resonance spectroscopy techniques allows qualitative and quantitative profiling of small molecules present in biological systems . this approach can be applied to reveal metabolic differences between different types of thyroid cancer and to identify new potential candidates for molecular biomarkers . in this review , we consider current results concerning application of metabolomics in the field of thyroid cancer research . recent studies show that metabolomics can provide significant information about the discrimination between different types of thyroid lesions . in the near future , one could expect a further progress in thyroid cancer metabolomics leading to development of molecular markers and improvement of the tumor types classification and diagnosis .

1. Introduction 2. Dilemmas in Thyroid Cancers Diagnosis 3. 4. Cancer Metabolism 5. Methods of Metabolomics 6. Metabolomics in the Studies on Thyroid Cancer 7. Conclusions

there are several histological types of thyroid cancer , including papillary thyroid carcinoma ( ptc ) , follicular thyroid carcinoma ( ftc ) , medullary thyroid carcinoma ( mtc ) , and anaplastic thyroid carcinoma ( atc ) . determining the type of thyroid cancer is crucial for the assessment of prognosis and treatment selection . most patients with thyroid carcinoma are initially diagnosed based on the result of fine needle aspiration cytology . refined diagnosis and classification / staging of this cancer unfortunately , in some cases , proper classification could be problematic when histopathological patterns are inconclusive . therefore , classical histopathological approach in the diagnosis of thyroid cancer could be potentially supported by molecular biomarkers . nowadays , a number of molecular tests to confirm the diagnosis of thyroid nodules have been proposed , which included panel of somatic mutations ( e.g. this approach , although not implemented widely in clinical practice yet , could change the attitude towards classification of thyroid cancers in near future . further application studies and clinical trials such applications are likely to become a component of the standard diagnostic approach for patients with thyroid cancer . metabolomics is one of the high - throughput omics techniques , which beside genomics , transcriptomics , and proteomics play an important role in systems biology . metabolome is the final downstream product of gene expression and therefore reflects changes in the transcriptome ( mrna ) and the proteome ( proteins ) . additionally , metabolomics reflects phenotypic changes and alterations in pathophysiological states of biological systems and therefore represents the most downstream level of molecular life of a cell . changes in metabolome composition reflect alterations in enzymes concentration , cellular regulation , control of signalling pathways , genetic variations , and catabolic and anabolic reactions . hence , different combinations of liquid ( lc ) and gas ( gc ) chromatography coupled with mass spectrometry ( ms ) or nuclear magnetic resonance ( nmr ) are the most accepted and widely used analytical approaches in this field . metabolomics provides valuable information about metabolism of malignant cells and has a great potential in cancer research as well as in identification of novel diagnostic and prognostic markers . several studies proved that metabolomics approaches allowed for classification of different types of malignancies and identification of potential biomarkers in the case of brain , breast , kidney , and prostate cancers . although studies regarding metabolome of thyroid cancer are not very common , there are several recent works showing that metabolomics approach could help to discriminate different types of thyroid lesions and provide significant information about their progression . here , we aim to review recent progress in the field of thyroid metabolomics and discuss its contribution to understanding thyroid tumorigenesis and potential refinement of molecular classification of thyroid cancers . palpable thyroid nodules occur in 47% of the population ; however , lesions found incidentally during ultrasonographic examination suggest a prevalence of 1967% . although thyroid tumors occur in roughly 510% of palpable nodules , they represent the most common endocrine malignancy and pose a significant challenge to pathologists , surgeons , and oncologists . this is particularly true of the follicular variant of ptc , which can be difficult to distinguish from a benign follicular nodule . when concerning postoperative material , a reliable histopathological diagnosis can be reached by an experienced pathologist by sole morphologic assessment for most cases of thyroid tumors . however , several types of morphologic features ( e.g. furthermore , in the case of tumors exhibiting unusual morphologic patterns or in order to confirm the diagnosis of medullary carcinoma , additional molecular tests ( e.g. also molecular markers suggested for differential diagnosis of ptc versus benign thyroid lesions or other thyroid tumors have been widely tested in clinical practice . in fact , certain normal thyroid follicles , nonneoplastic thyroid lesions ( in particular thyroiditis ) , and benign thyroid tumors can exhibit focal or extensive staining for many of putative thyroid cancer markers . however , even application of a panel of markers for ihc studies ( e.g. , ck-19 , gal-3 , and hbme-1 ) did not improve significantly the diagnostic performance of the test . the apparent role of interactions between the tumor and its niche in the tumor - thyroid interface represents additional diagnostic challenge . additionally , in the case of a follicular patterned neoplasm lacking the cytoarchitectural features of papillary carcinoma , the only feature that distinguishes carcinoma from adenoma is the presence of unequivocal vascular and/or capsular invasion in the former . in conclusion , histopathological evaluation remains the gold standard in the distinction between different types of thyroid cancer , for example , between follicular carcinoma and follicular adenoma . various molecular tests have been tested beside histological analysis , including immunohistochemical assessment of proteins and genetic tests for gene mutations , yet none of them proved its actual clinical applicability . therefore , there is a constant need for development of unambiguous molecular markers which would bring a real improvement in diagnosis and classification of thyroid cancers . the term omics defines high - throughput approaches to complex molecular composition of tissues enabling simultaneous analysis of thousands of genes / proteins . for this purpose , platforms integrating genomics , transcriptomics , proteomics , and metabolomics data are constructed , enabling better understanding of mechanisms involved in natural history of cancer . microarray and next - generation - sequencing approaches to the analysis of gene expression as well as mass spectrometry techniques used for the analysis of proteins and/or peptides have delivered valuable information on various types of human malignancies . the best example of how the achievements of the genomics era could alter the clinicopathologic paradigm in classification of cancer types and affect the decision making process in selection of a treatment is breast cancer [ 3134 ] . similar approaches have been tested during the last decade for many other malignancies , including thyroid cancer . another microarray - based study allowed for identification of the gene expression signatures associated with mutations in braf , ras , and ret / ptc genes , as well as distinguishing the classic ptc from the tall cell and follicular variants . comparative analysis of the expression profiles in ptc and ftc , the two most common forms of thyroid carcinoma , enabled identification of the gene signatures characteristic for these cancers ; the differentiating signature includes five genes ( cited1 , cav1 , cav2 , igfbp6 , and cldn10 ) . since then many other works have been published describing the differences between thyroid cancer and normal thyroid tissue , as well as differences between types of thyroid neoplasia . these works revealed the significance of hundreds of genes , including the key genes involved in thyroid hormone biosynthesis [ 38 , 39 ] . an important issue in the diagnostics of thyroid cancer is differentiation between follicular adenoma , follicular carcinoma , and the follicular variant of papillary carcinoma . currently , several biomarkers have proved their applicability in solving this problem , including lgals3 , hemoglobin , epsilon 1 ( hbe1 ) , keratin 19 ( ck-19 ) , and tpo ( thyroid peroxidase ) . another powerful and promising approach in the search of cancer biomarkers is proteomics based on mass spectrometry tools [ 4145 ] . proteins can be extracted , identified , and quantified from different cell and tissue sources . it should be stressed that proteomics studies of thyroid represent a real challenge due to high heterogeneity observed in this tissue and very broad range between the most abundant ( e.g. the comparative analysis of ptc specimens matched with the normal thyroid tissue from the same patients and benign follicular adenomas allowed identification of three proteins , namely , s100a6 ( an isoform of s100 protein ) , peroxiredoxin 2 , and heat shock protein 70 ( hsp70 ) , whose expression levels were markedly higher in ptc tissue . genomics and proteomics studies have a significant impact on general understanding of cancer - related processes and have delivered several promising candidates for cancer biomarkers . however , some limitations in the prognostic and prediction power of gene / protein expression data have been recognized in recent years . these tumor associated processes significantly affect the primary metabolic pathways ; hence , cancer cells are characterized by altered metabolism in comparison with the normal differentiated cells , whose pathways are depicted schematically in figure 1 . in marked contrast , most cancer cells use aerobic glycolysis , known as the warburg effect , to produce both energy and building blocks ( amino acids , nucleotides , and fatty acids ) needed for extensive growth and proliferation . in addition to nadph and acetyl - coa , choline is the next essential substrate required for lipids biosynthesis , and accumulation of this compound in cancer cells was observed in different studies . choline - containing compounds , including phosphocholine , phosphatidylcholine , and glycerophosphocholine , are the key components of a cell membrane . changes in the levels of lipids and their derivatives were observed in patients with different type of malignancies , including breast , prostate , brain , and thyroid cancers . in addition to lipids , there is a large group of small metabolites , including amino acids , nucleotides , sugars , and organic acids , important for cancer development . levels of taurine and myo - inositol , other small molecules involved in osmoregulation , are also affected in cancer , which was reported in thyroid , prostate , colon , breast , and ovarian cancers [ 11 , 55 ] . although elevated levels of phospholipids and products of glycolysis characterize cancer cell in general , it should be emphasised that specific levels of various metabolites , including glycine , alanine , lactate , citrate , nucleotides , and lipids , may depend on the type of a cancer . breast cancer is generally characterized by elevated level of total choline - containing compounds ( tcho ) , low glycerophosphocholine , and low glucose , when compared to healthy tissues and benign tumors [ 64 , 7477 ] . therefore , metabolomics has an apparent potential in the studies focused on the discovery of cancer biomarkers . current research efforts are focused on the use of metabolomics screening in preclinical and clinical studies to improve diagnosis and support therapy . however , there is still a significant need to establish the rigorous and effective analytical protocols which could be widely accepted and find their appropriate place in clinical trials . the number of metabolites present in a human organism is currently estimated as approximately 17,000 ( according to the human metabolome database - hmdb version 3.6 ) , yet this number is still expanding ; hence , the exact figure remains unknown . metabolomic fingerprinting is the least precise approach which enables rapid monitoring of the composition of low molecular weight compounds , without the need for detailed identification . the most commonly used method is metabolite profiling , allowing qualitative and quantitative analysis of a given group of metabolites . regardless of the chosen analytical approach , there are several general steps in the metabolomics studies , including sample collection and preparation , data acquisition and processing , biostatistical analysis , and data interpretation , which have been schematically depicted in figure 2 . in order to obtain reproducible results that could be compared between laboratories , strict compliance with the standardized procedures of metabolomic analysis metabolomics standard initiative ( msi ) ( http://www.msi-workgroups.sourceforge.net/ ) published standard reporting requirements for each step of metabolomics experiments [ 8387 ] . the first and also the most critical steps in metabolomics study are sample collection , storage , and preparation for instrumental analysis . moreover , saliva , breath condensate , bronchial washes , pancreatic juices , prostatic secretions , faeces , and other types of physiological liquids or surrogate tissues can be also used for metabolomics studies . most obviously , tissue specimens are also widely examined in metabolomics studies using mass spectrometry ( ms ) , imaging mass spectrometry ( ims ) , and nuclear magnetic resonance ( nmr ) spectroscopy techniques . however , tissue samples are usually characterized by large heterogeneity and therefore require more complex preparation procedures before analysis , including the use of laser - capture microdissection ( lcm ) techniques . the collected material can be fresh - frozen and stored in the temperature below 80c , which is a gold standard in the analysis of metabolites . however , tissue specimens fixed in formalin and stored as formalin - fixed paraffin - embedded ( ffpe ) samples ( normally stored in room temperature ) are the most accessible source of clinical material for molecular studies . nevertheless , due to high sensibility of metabolites to exogenous environment , maintaining low temperature and selection of the appropriate method of extraction are essential in most of the cases . metabolites present in biological samples differ in terms of molecular weight , thermostability , volatility , and polarity . consequently , the choice of the sample preparation method depends on the character of the studied compound class and the type of the analytical technique . the most commonly applied analytical methods in cancer metabolomics studies are liquid or gas chromatography coupled with mass spectrometry ( lc / gc - ms ) and nuclear magnetic resonance ( nmr ) spectroscopy . ms is a highly sensitive technique which allows identification and quantification of multiple metabolites , even at very low concentrations , based on the mass to charge ratio of the analyte ions generated in the spectrometer . high - resolution ms experiments with sequential fragmentation of the analyte ions permit obtaining structural information about the studied compounds , yet not all metabolites can be ionized using either positive or negative ionization mode . nmr spectroscopy also enables identification of small biomolecules based on the resonance spectra of the atomic nuclei h , c , and p , which are commonly present in metabolites . methods based on nmr are generally less sensitive in comparison to ms techniques ; however , they can be used in analyses of liquid and solid samples with minimal preparation stage . spatial distribution of metabolic markers can be analyzed both in vitro and in vivo using localized magnetic resonance spectroscopy imaging ( mrsi ) . another method for in vitro differentiation of tissue regions based on their molecular content is imaging mass spectrometry ( ims ) . the combination of different analytical methods , including ms , nmr , and imaging techniques , apparently provides the most powerful approach to metabolomics studies in current and future applications . there are numerous works proving successful application of genomics , transcriptomics , and proteomics in the field of thyroid cancer research [ 2 , 46 , 115 ] . however , the metabolomics approach implemented in identification of biomarkers for diagnosis and classification of thyroid tumors has been dynamically expanding in recent years [ 55 , 58 , 67 , 116118 ] . the state - of - the - art technologies including maldi - ims ( matrix assisted laser desorption ionization - imaging mass spectrometry ) and hr - mas nmr ( high - resolution magic angle spinning nuclear magnetic resonance ) have been used for the identification of a large group of metabolites present in thyroid tissues , which are potential diagnostic biomarkers [ 58 , 116 ] . major groups of metabolites , whose different abundance in thyroid tissue and/or blood was observed between different types of thyroid lesions , are listed there . discrimination of different thyroid lesions , such as nonneoplastic nodules , follicular adenoma , and malignant tumors , based on metabolite profiles constitutes a technically challenging but clinically relevant problem . shortly afterwards , the potential of nmr techniques in the analysis of lipid component of thyroid cancer was reported [ 123 , 124 ] . in fact , nmr metabolomic data processed with multivariate statistical approaches allowed separation between different types of thyroid lesions and several multicomponent metabolic signatures / classifiers were proposed . the observed changes in the levels of lactates and inositols were in agreement with the changes generally observed in many types of tumors . however , the decrease in the content of choline and its derivatives was not generally observed in cancer tissue [ 57 , 127 , 128 ] , also in other works on thyroid cancer [ 58 , 67 , 118 , 129 , 130 ] . the authors subjected acquired nmr data to multivariate analysis including both unsupervised method ( pca ) and supervised modelling ( opls - da ) and identified metabolites characteristic for different types of thyroid lesions . potential biomarkers common to all thyroid lesions included alanine , methionine , glutamate , glycine , tyrosine , phenylalanine , hypoxanthine , acetone , and lactate . based on the specific metabolite profile of follicular adenomas and significant differences between healthy control , nonneoplastic nodules , and thyroid cancer , the authors classified this lesion as an intermediate step in thyroid cancer progression . this observation gives a promise of new potential metabolomic biomarkers of different thyroid cancer stages . imaging mass spectrometry ( ims ) has already been used for simultaneous detection and spatial localization of lipids in differentiated thyroid cancer tissues . the study was focused on phosphatidylcholines ( pc ) , phosphatidic acids ( pa ) , and sphingomyelins ( sm ) in three types of tissues : normal thyroid , benign tumors ( thyroid adenoma and multinodular goiter ) , and malignant tumors ( papillary and follicular thyroid carcinoma ) . based on ims analysis performed on 36 tissue samples and lc / ms profiling of almost 300 sera from three different groups of patients ( healthy , with malignant thyroid tumor , or with benign thyroid tumor ) , the authors identified ten differentiating lipid species : phosphatidylcholines pc ( 34:1 ) , pc ( 36:1 ) , pc ( 38:6 ) , phosphatidic acids pa ( 36:2 ) , pa ( 36:3 ) , pa ( 38:3 ) , pa ( 38:4 ) , pa ( 38:5 ) , pa ( 40:5 ) , and sphingomyelin sm ( 34:1 ) . for this purpose , expression of key enzymes , scd1 and fasn , involved in de novo fatty acids synthesis [ 131 , 132 ] was correlated with the levels of monosaturated pc in malignant , benign , and normal thyroid tissues . immunohistochemical detection of scd1 and fasn confirmed their association with high level of monosaturated pc in thyroid cancer tissues . the authors conducted serum metabolic profiling of ptc , nodular goiter cases , and healthy control using lc / ms technique . significant changes in the levels of amino acids , free fatty acids ( ffa ) , and phospholipids were observed between different groups of donors . moreover , the most significant differences between benign and malignant nodules were observed in lipid metabolism . metabolomics has an apparent potential to expand our knowledge on molecular factors involved in thyroid cancer . however , the example of breast cancer studies clearly indicates the need for combining the metabolomics information with other systems biology datasets to provide the holistic view of the processes ongoing in this endocrine malignancy . such metabolomics network has already been created for thyroid hormone secretion pathway , which helped to identify the potential drug targets essential in treatment of thyroid disorders such as hypo- or hyperthyroidism [ 133 , 134 ] . along with the technological development of analytical tools ( first of all ms- and nmr - based spectral techniques ) , metabolomics becomes an emerging research approach also in the field of thyroid cancer . moreover , there is an obvious necessity for integration of metabolomic data to genomics , transcriptomics , and proteomics results to bring better insights into the cellular mechanisms of thyroid cancer progression . nevertheless , one should expect that metabolomics studies may deliver in the next years the basic knowledge not only on cancer - related processes but also on novel biomarkers to be implemented in diagnosis and classification of thyroid cancer .

considerable improvements in health care during the course of the last century have led to unprecedented increases in current life expectancies . however , longevity continues to favor women and as a consequence chinese and australian national health policies recognize the need to address male health in aging . while increased life expectancy is a broad consequence of quality health care , it also produces an aging population whose health care burden suggests wellbeing has not equally paralleled longevity . several areas of male health decline with age with increasing incidence of impaired spatial ability and depression being significant barriers to mental health . a sex difference in longevity intuitively points to a role of sex - hormones in maintaining health during aging . the sex - hormone testosterone is the primary androgen produced in men and circulating levels progressively decrease from 30 years onward so that by 60 years of age , up to 1 in 4 men have developed androgen deficiency . with increasing longevity the proportion of androgen deficient or hypogonadal men is likely to grow , along with predicted adverse consequences for male health . although clinically diagnosed hypogonadism is remediable with testosterone replacement therapy , understanding a causative relationship between age - related physiological decline and reduced androgen levels will enable better defined interventional strategies . the hippocampus is a key component of the temporal lobe and is notable for retaining ongoing adult neurogenesis within the dentate gyrus subregion . impaired hippocampal function resulting from neurogenesis deficits is hypothesised to contribute to the etiology of both depression and spatial memory impairment . in considering the collective clinical and rodent model data , a coincident age - related decline in circulating testosterone , hippocampal neurogenesis and hippocampal function becomes evident . however , a causal relationship between the age - related decline of these factors has yet to be established . this review raises a hypothesis that testosterone contributes to preserving hippocampal function in aging men by maintaining neurogenesis levels . herein , the intention is to briefly discuss the available evidence and highlight the necessity of further studies to establish , or refute , clinically relevant causal relationships . production of the primary male androgen testosterone is reduced as men age ( 1 ) . circulating testosterone targets the hippocampus , which has been functionally demarcated into dorsal ( 2 ) and ventral segments ( 3 ) . testosterone can support spatial memory ( 2 ) and provide anti - depressant efficacy ( 3 ) . intrinsic hippocampal circuitry comprises a well described tri - synaptic connection between ca1 , ca3 and dentate gyrus ( dg ) subregions ( 4 ) . ongoing adult neurogenesis proceeds in the sub - granular zone ( sgz ) of the dg through sequential proliferative and post - mitotic steps to produce functionally integrated neurons in the granule cell layer ( gcl ) of the dg ( 5 ) . androgen receptors are expressed in the dg but whether testosterone can directly or indirectly affect neurogenesis remains unresolved ( 6 ) . dosage and duration of testosterone and aromatasemediated metabolism into 17-estradiol may be important determinants of efficacy ( 7 ) . declines in circulating testosterone levels , hippocampal neurogenesis and hippocampal function are coincident in aging ( 8) . testosterone is the primary male androgen and is produced through the co - ordinated actions of the hypothalamic - pituitary - testes ( hpt ) axis . while there is normal variation among men , serum total testosterone in men aged under 35 years generally exceeds 20 nm . according to several endocrinology health bodies serum concentrations of total testosterone below 12 nm constitute a clinical diagnosis of hypogonadism , which can be further classified according to a taxonomy that directs remedial strategies . for example , testicular dysfunction , or primary hypogonadism , manifests in the presence of high luteinizing hormone / follicle stimulating hormone ( lh / fsh ) and is potentially indicated for testosterone replacement therapy . secondary hypogonadism occurs when hypothalamic / pituitary pathology fails to produce lh / fsh in sufficient levels to stimulate testicular androgen production . in this case , age - related low circulating testosterone can be the result of either primary or secondary hypogonadism . otherwise normal aging is associated with reduced total circulating testosterone levels ( below 20 nm ) concomitant with increased sex - hormone binding globulin ( shbg ) . testosterone bound to shbg is not bioactive and can affect the clearance rate of testosterone by the liver . however , the age - related increase in shbg levels has been shown to be independent of the age - related decline in bioactive testosterone levels . testosterone replacement therapy is advised based on symptomatic low circulating testosterone levels . however , applying this criterion to aging men presents potential limitations . firstly the age at which testosterone levels become low is an important determinant of resulting symptoms . age - related , or late - onset hypogonadism , may therefore elicit a unique spectrum of symptoms that are yet to be fully established . secondly , controversy surrounds the reliability of widely used measures of total and free serum testosterone to detect low circulating levels . as symptoms other than sexual dysfunction and frailty are not readily attributed to low circulating testosterone , symptomatic testosterone deficiency could be more common than generally recognised . testosterone therapy in aging men has hitherto been directed toward treating sexual and cardiovascular dysfunction , muscle mass loss and immobility . hence , the symptom profile of late - onset hypogonadism may well include declining mental health . although there are cross - sectional variations in measures of cognitive ability , a more robust decline is observed in longitudinal analyses . notwithstanding the view held by some that age - related cognitive decline is normal , maintaining cognition in longevity at young levels would have an undeniable impact on wellbeing . procedural memory remains largely unaffected , whereas declarative memory , particularly episodic memory shows demonstrable decline with increasing age . declarative memory as a whole is closely associated with temporal lobe function and abilities related to spatial memory are intimately coupled with the hippocampus . the decline in spatial ability of aging men is a domain of cognition particularly studied in relation to testosterone levels . this interest may be attributed to the fact that aspects of spatial ability are superior in men compared to women . a sex bias in spatial ability is also reflected in rodent studies that have been largely demonstrated using maze navigation tasks . men and women employ the same brain regions , including the hippocampus , when undertaking spatial navigation tasks . these observations suggest that testosterone may target the hippocampus in a manner that enhances spatial ability in men . notions of sex differences in brain anatomy and function have centred largely on the effects of sex hormones in development . evidence supports a possible organisational role of androgen signalling during brain development that imparts superior spatial cognitive ability in adulthood . longitudinal studies showing a correlation between age - related cognitive decline and reduced testosterone , raise the possibility that testosterone may perform a similar , but activational role in the mature brain . this idea led to studies demonstrating that testosterone therapy promotes spatial ability in aged men . additionally , the substantial evidence demonstrating pro - cognitive effects of oestrogen replacement therapy in postmenopausal women , including enhanced hippocampal plasticity and function , underscores the possibility of a similar mechanistic role of testosterone in aging men . oestrogen is a major metabolite of testosterone in the male brain further suggesting that low circulating testosterone may be a readily modifiable factor predisposing aging men to cognitive decline . androgens protect the adult brain from degenerative insults such as stroke and traumatic brain injury . although neurodegenerative diseases increase with advancing age , their consideration needs to be distinguished from normal age - related cognitive decline . along with the dentate gyrus the ca1 and ca3 subregions form anatomical demarcations of the hippocampus . androgen receptors are expressed in the hippocampus of humans , non - human primates , mice and rats with higher levels in ca1 compared to the ca3 or dentate gyrus subregions . evidence derived from young rodents shows that androgen withdrawal in development will reversibly reduce ca1 and ca3 synaptogenesis and subsequent adult spatial ability . synaptic loss during aging may underlie functional decline of the hippocampus , ( however also see ) . studies from rodents and non - human primates have provided evidence that serum androgens support ca1 synaptic density in young adulthood . these data provide a mechanism by which androgens could participate in the long - term preservation of hippocampal function . considered in reverse , the data suggests that age - related androgen deficiency may lead to ca1 synaptic loss and hippocampal dysfunction . as acknowledged above however , longitudinal studies indicate that the dentate gyrus subregion is more closely associated with age - related hippocampal impairment . evidence that sex - hormones increase dentate gyrus synaptogenesis has been extensively derived from examining the effects of oestrogen . in contrast , there is a relative paucity in studies reporting the effects of testosterone on dendritic structure and synaptic density of dentate gyrus granule neurons . it remains plausible that circulating androgens also target the aging dentate gyrus to preserve mechanisms of neuronal plasticity and hippocampal function . however , these theories were immersed in the basic tenet that the mature brain lacks ongoing neuronal production . this dogma was overturned with the widespread acceptance that adult neurogenesis occurs in mammalian brains . although spatially restricted , neurons generated in the adult dentate gyrus provide a functional form of neuronal plasticity . despite ongoing debate regarding the function of adult hippocampal neurogenesis , the weight of associative evidence points to a role of adult - generated granule neurons in spatial ability and perhaps emotional behaviour related to depression . in contrast to the ca1 subregion , androgens could preserve functional plasticity in the dentate gyrus through the addition of new granule neurons . levels of adult hippocampal neurogenesis decline with age , which has been hypothesised as a contributing factor to age - related decline in spatial ability . importantly , age - related declining adult hippocampal neurogenesis and spatial ability are reversible with factors such as physical exercise . if androgens play a critical role in maintaining adult hippocampal neurogenesis levels then late - onset hypogonadism may be a remediable cause of hippocampal dysfunction in aging men . adult hippocampal neurogenesis proceeds through a series of sequential steps : asymmetric division of neural stem cells , proliferative expansion of neural progenitors , neuronal differentiation and finally survival of mature neurons including both efferent and afferent synaptogenesis . intrinsic proliferation and differentiation markers together with exogenous markers such as 5-bromo-2deoxyuridine ( brdu ; which is incorporated into dividing cells and can assess proliferation and survival at short and longer time - points , respectively ) are widely used to measure adult hippocampal neurogenesis . mechanisms of age - induced reductions in adult hippocampal neurogenesis include decreased progenitor proliferation and neuronal survival , but factors mediating these mechanisms remain unknown . notions of sex - specific regulation of adult hippocampal neurogenesis were a significant impetus for exploring the effects of sex hormones on proliferation , differentiation and survival of adult - generated hippocampal neurons ( reviewed in ) . there is more hippocampal neurogenesis in neonatal male rats than females and several studies have demonstrated sex - specific rates of hippocampal neurogenesis in adult rodents . whether there is a sex - specific decline in adult hippocampal neurogenesis with advancing age remains hitherto unestablished . early studies withdrawing androgen through gonadectomy in young male rats showed no effect on proliferation but reduced neuronal survival as demonstrated by a reduction in the number of 30-day - old brdu - labelled cells in the dentate gyrus . repletion of testosterone at high but not low levels in these animals restored adult hippocampal neurogenesis in an androgen receptor - dependent manner . in contrast , another study showed that exogenous injections of nandrolone , a synthetic derivative of testosterone and androgen receptor agonist , reduced adult hippocampal neurogenesis by decreasing neuronal precursor proliferation in young adult male rats . our own recent work demonstrated that chronic testosterone therapy in young male adult mice had no effect on precursor proliferation as measured by the intrinsic marker ki67 , but did promote adult hippocampal neurogenesis through increased neuronal survival and differentiation 28-days after brdu administration . we measured hippocampal neurogenesis during the last 4 weeks of 3 months of supraphysiological doses of testosterone through sustained - release pellets , whereas a similar dosage limited to 3 days via repeated injections had no effect on neurogenesis . another study of young male mice demonstrated that precursor proliferation also measured by ki67 expression was unchanged , while the generation of maturing neurons measured by another intrinsic marker doublecortin , was decreased in the absence of androgen through gonadectomy . single administration of brdu to measure precursor proliferation in gonadectomised young adult male rats untreated or made replete with physiological levels of testosterone showed no effect of androgen withdrawal . however , the same study showed that testosterone exacerbated the up - regulated proliferation in response to the anti - depressant imipramine . in a contrasting finding , ki67 was used to demonstrate a decrease in precursor proliferation in gonadectomised young male rats compared to sham - surgery controls . this study also employed brdu to further examine survival and neuronal differentiation , showing that androgen withdrawal through gonadectomy decreased surviving 24-day - old brdu - labelled cells in the dentate gyrus without change in neuronal differentiation . furthermore , polysialic acid - neural cell adhesion molecule ( psa - ncam ) was used to demonstrate androgen withdrawal decreases maturing / migrating neurons . using a different approach , intact male rats given placebo or testosterone and brdu to measure proliferation , survival and differentiation showed no effect of exogenous androgen on any of these hippocampal neurogenesis parameters . manipulating testosterone levels in young male rats by gonadectomy or exogenous administration to intact the authors of this study make a pertinent argument that testosterone 's effects on hippocampal neurogenesis may be dose and duration - dependent and specific to the developmental stage of newly - generated neurons . in addition , the mode of hormone delivery , repeated injections versus constant release implants likely impacts on serum testosterone levels . hence differences in delivery mode could confound direct comparisons between studies in which serum testosterone levels are reported . this notion is reflected clinically , where testosterone therapy through long - term skin patches could provide better efficacy compared to repeated surges that accompany periodic injections . further potential confounds reside in the methodology used to measure hippocampal neurogenesis , for example intrinsic markers such as ki67 , psa - ncam and doublecortin versus the timing and dose of exogenous markers like brdu . more importantly , the manipulations of testosterone levels in young male rodents in these studies do not recapitulate the late - adult onset and gradual decrease in circulating androgen manifest in normal aging . hence , extrapolating these data to explain a possible causal relationship between low testosterone and declining adult hippocampal neurogenesis in age is limited and specific longitudinal studies examining models of normal aging are required . androgen receptor signal transduction incorporates both genomic mechanisms and non - genomic pathways including ; camp response element - binding protein ( creb ) , extracellular signal - regulated kinase ( erk ) and akt signalling . these non - genomic signalling pathways in particular could represent intrinsic cellular mechanisms by which testosterone directly effects hippocampal neurogenesis . however , despite androgen receptor expression in the dentate gyrus region , whether neural stem cells , transient precursor intermediates or maturing neurons exhibit differential expression is unclear . cultured neural stem cells have been found to express androgen receptor , though these in vitro studies used cells derived from the sub - ventricular zone of adult female rats . hence it remains to be determined if hippocampal neurogenesis can be directly affected by testosterone or whether effects are transmitted by cellular and/or molecular intermediates . the local microenvironment , or neurogenic niche , is considered to play a significant role in regulating neurogenesis levels in the adult hippocampus . astrocytes are an integral cellular component of the neurogenic niche that support hippocampal neurogenesis through mechanisms not fully understood but includes promoting neuronal differentiation and maturation . one mechanism by which astrocytes stimulate hippocampal neurogenesis is through the secretion of soluble neurotrophic factors such as fibroblast growth factor ( fgf)-2 . expression of fgf-2 by astrocytes residing in the hippocampal neurogenic microenvironment decreases during aging and may contribute to the age - related decline in hippocampal neurogenesis . hippocampal astrocytes in male rats express androgen receptor and hence testosterone could influence hippocampal neurogenesis indirectly . testosterone triggers fgf-2 production in germ - line stem cells of the adult male testis that may play a role in spermatogenesis . whether such signalling also occurs in neural stem cells to promote hippocampal neurogenesis and whether reductions in this signalling precipitate age - related decline in hippocampal neurogenesis remains untested . brain derived neurotrophic factor ( bdnf ) is another compound that both maintains and mediates up - regulation of hippocampal neurogenesis in rodents and is decreased in the aged hippocampus . the ability of testosterone to promote survival of adult - generated neurons in adult canaries is mediated by bdnf . however , fluctuations in circulating testosterone during the early development of male mice are independent of hippocampal bdnf levels . studies using aging rats have shown no sex - specific changes in declining hippocampal bdnf levels , nor any correlations between levels of serum oestrogen or testosterone with hippocampal bdnf in aging male rats . the collective evidence would suggest that age - related declining serum testosterone and hippocampal bdnf levels are independent and do not interact to cause falling hippocampal neurogenesis . this association could be important in aging men given the recent findings that circulating bdnf levels are ; higher in aging women compared to men , negatively correlated with age in men and positively correlated with bioavailable circulating testosterone levels in men . these observations raise a possibility that bdnf could be a systemic factor mediating the cognitive efficacy of testosterone therapy . moreover , age - related decline in cellular and molecular elements including signal transduction pathways necessary to mediate testosterone 's effects could significantly impact on the effectiveness of androgen therapy and underscores the importance of their elucidation . the physiological metabolism of testosterone is another important consideration in elucidating the mechanisms of cognitive efficacy . testosterone is metabolised by 5-reductase into the high - affinity androgen receptor ligand dihydrotestosterone and by aromatase into the oestrogen receptors and ligand 17-estradiol . thus , whether it be intrinsic production or the result of exogenous therapy , effects of circulating testosterone can be mediated through either androgen or oestrogen receptors . however , while both androgen and oestrogen mediated signalling contribute to preserving ca1 synaptic density in females , the same effect in males is androgen receptor dependent . although one study using rats showed that testosterone therapy tended to increase circulating levels of 17-estradiol ( albeit a non - statistically significant increase ) , oestrogen activity was not responsible for the increased adult hippocampal neurogenesis . the ability of testosterone therapy to restore declining spatial ability in aging rats was tested in advance of the general acceptance of adult hippocampal neurogenesis . more recent studies administering testosterone to aged rats however , have shown restorative effects on declining spatial ability . moreover , studies examining the effects of androgens on spatial abilities in rodents across life - span have yielded complex results that more generally reflect the collective clinical data examining aging men . the relationship between testosterone levels and different parameters of cognition in aging men has been recently reviewed in detail . decreased circulating testosterone in aging men is linked to poorer performances on tasks requiring spatial ability . several clinical studies show that spatial ability is improved in aging men receiving testosterone therapy . this effect of testosterone is also seen in men suffering mild cognitive impairment and even alzheimer 's disease . however , others have shown no effect of testosterone therapy on cognitive parameters in healthy and mildly cognitive impaired aged men . the magnitude of demonstrated spatial ability improvements have varied and may dependent on the dose of testosterone and the length of therapy . spatial ability varies with endogenous circulating testosterone levels in aging men and appears to respond better to intermediate doses of exogenous testosterone compared low and supraphysiological doses that show no efficacy . circulating 17-estradiol levels could change in elderly men receiving testosterone therapy as a consequence of peripheral aromatisation . however , the efficacy of testosterone therapy on spatial ability in aging men appears to be independent of oestrogen activity , though 17-estradiol may mediate concomitant improvements in verbal memory . prospective clinical studies of prostate health support the idea that both testosterone and 17-estradiol levels impact on male aging . both rodent and clinical studies suggest spatial ability in aged males is enhanced when high testosterone is coincident with low 17-estradiol levels . thus , in parallel with the notion of optimal testosterone levels , is the view that the balance of testosterone and 17-estradiol plays a significant role in spatial ability . debate over the function of adult hippocampal neurogenesis has evolved to include a specific role in pattern separation , which is to encode and retrieve similar events and is linked to dentate gyrus function . the strongest evidence from rodent studies also suggest an association between age - related decline in adult hippocampal neurogenesis and impaired pattern separation ability . given the relative infancy of interest in age - related pattern separation impairment , there is hitherto no definitive evidence for sex differences in the decline of this domain of spatial cognition . it therefore remains possible that testosterone therapy in aging men may preserve pattern separation ability , which could be mediated through maintaining neurogenesis levels in the aging dentate gyrus . the notion that androgens subserve specific domains of mental health includes effects on emotion - based behaviours . furthermore , along with spatial ability , adult hippocampal neurogenesis has also been linked with emotion - based behaviour . a hypothesis based on several associative links posits that impaired adult hippocampal neurogenesis elicits depression and that efficacy of antidepressant interventions is mediated through adult hippocampal neurogenesis . how does a link between depression and adult hippocampal neurogenesis relate to testosterone levels and advancing age ? as with memory loss , age is a risk factor for depression . gender difference in the incidence of depression disappears with advancing age and the occurrence of depression in elderly men is associated with low testosterone . to explore these observations in the context of declining adult hippocampal neurogenesis requires a consideration of a functional distinction in hippocampal structure . the discussion has hitherto observed the classic tri - synaptic circuitry intrinsic to the hippocampus mediated through the interconnectiveness of the dentate gyrus , ca1 and ca3 subregions . however , on a more global level the rodent hippocampus has been regionalised into dorsal ( septal pole or posterior in humans ) and ventral ( temporal pole or anterior in humans ) functional subregions . this regionalisation has partly been based on functional assessments , differential gene expression and connectivity , with spatial ability and emotion - based behaviour preferential to the dorsal and ventral hippocampal regions , respectively . neurogenesis rates may be higher in the dorsal compared to the ventral region of young adult hippocampus , but this regional difference attenuates with advancing age , indicating a relatively higher impact of age on dorsal hippocampal neurogenesis . clinical efficacy of antidepressant treatment is associated with specific increases in adult neurogenesis in the anterior hippocampus in depressed patients . whether androgen deficiency with advancing age differentially affects neurogenesis rates along the septal - temporal axis in the adult hippocampus in parallel with functional deficits such as depression and impaired spatial ability is not known . the question of whether antidepressant and spatial ability efficacy of testosterone therapy for aging men is mediated by region - specific hippocampal neurogenesis remains intriguing . intriguing in part , because addressing this cause - effect relationship will help refine the neurogenesis - depression and neurogenesis - cognition hypotheses and also elucidate links between impaired neural substrates , hypogonadism and symptoms of cognitive dysfunction . this latter point will in turn help define position statements that advocate testosterone therapy based on low testosterone in conjunction with manifest symptoms . clinical studies using testosterone therapy in elderly men showing either benefit or no effect rather than deterioration in cognition have employed inconsistent and even inadequate measures of mental health . a significant point to consider in evaluating this literature is the notion that androgens likely affect mental health in a domain - specific manner . moreover , conclusions drawn from clinical studies will depend significantly on the cognitive measures used . uncertainty as to what biochemically measured level of circulating free testosterone constitutes late - onset hypogonadism and associated symptoms in aging men is a significant impediment to standardised testosterone replacement therapy . the prevailing view that age - related mental health decline is not an indicator for testosterone therapy is because of inconclusive rather than negative evidence . ethical and technical limitations in clinical studies , including varied cognitive assessment methodologies , study inclusion criteria and population size and age range of participants have played a large part in generating this equivocal evidence . this review premises that reduced testosterone is a mediating factor of impaired spatial ability and depression incidence in aging males because of reduced dorsal and ventral hippocampal neurogenesis , respectively . in discussing the known effects of both androgen withdrawal and therapy on hippocampal function and plasticity , it is clear new understanding will be needed to establish a causal relationship between these factors in the context of normal male aging . there remain several key questions concerning the role androgens play in regulating adult hippocampal neurogenesis , including age - related decline and mediation of stimulatory factors such as exercise . an active lifestyle predicts higher circulating testosterone levels in aging men ; could testosterone mediate the ability of physical activity to promote neurogenesis and function in the aged hippocampus ? if so , given that intensity could determine the effects of exercise on testosterone and neurogenesis levels , is aerobic or anaerobic exercise best ? emerging studies showing the cognitive benefit of resistance training in the elderly could provide a paradigm shift in the way physical activity is prescribed to preserve mental health . testosterone promotes synaptogenesis in the ca1 subregion ; does testosterone support synaptogenesis in neurons generated in the aging dentate gyrus ? . menopause - mediated oestrogen withdrawal is posited play a role in cognitive decline and depressive symptoms in women that manifest early than in males . because menopause is a far more precipitous event compared to the gradual decline in male androgen production , what role in maintaining hippocampal neurogenesis and function in aging men does testosterone aromatisation into 17-estradiol play ? the hippocampus expresses receptors for lh and gonadotropin - releasing hormone ( gnrh ) and their role , directly or indirectly through promoting local steriodogenesis , in age - related decline is uncertain . studies examining these questions will address the paucity in knowledge regarding the areas of impaired mental health to which low testosterone predisposes and also elucidate ongoing debate surrounding the clinical relevance of adult hippocampal neurogenesis . new clinical and basic research endeavours that encompass measures of hippocampal - dependent spatial memory , pattern separation and depression in conjunction with serum measures of hpt axis hormones will shed further light on physiological roles of testosterone in maintaining mental health in aging men .

interest surrounds the role of sex - hormones in regulating brain function outside of reproductive behaviour . declining androgen production in aging males has been associated with cognitive impairment , depression and increased risk of developing alzheimer 's disease . indication for testosterone replacement therapy is based on biochemically determined low circulating testosterone combined with manifest symptoms . however , which aspects of age - related cognitive decline are attributable to low circulating testosterone remain ambiguous . studies examining cognition in aging men receiving testosterone replacement therapy have yielded equivocal results . the exact role of testosterone in maintaining cognitive function and the underlying neural mechanisms are largely unknown , though it would appear to be domain specific . clarity in this area will provide clinical direction toward addressing an increasing healthcare burden of mental health decline coincident with increasing longevity . the premise that androgens contribute to maintaining aspects of mental health in aging men by preserving hippocampal neurogenesis will be used as a forum in this review to discuss current knowledge and the need for further studies to better define testosterone replacement strategies for aging male health .

INTRODUCTION ANDROGEN PRODUCTION DURING MALE AGING COGNITIVE IMPAIRMENT WITH ADVANCING AGE ANDROGENS AND HIPPOCAMPAL PLASTICITY AND FUNCTION ANDROGENS AND THE REGULATION OF ADULT HIPPOCAMPAL NEUROGENESIS ANDROGEN RECEPTOR-MEDIATED EFFECTS ON ADULT HIPPOCAMPAL NEUROGENESIS EFFECTS OF TESTOSTERONE THERAPY ON COGNITION IN ELDERLY MEN CONCLUDING REMARKS

however , longevity continues to favor women and as a consequence chinese and australian national health policies recognize the need to address male health in aging . several areas of male health decline with age with increasing incidence of impaired spatial ability and depression being significant barriers to mental health . a sex difference in longevity intuitively points to a role of sex - hormones in maintaining health during aging . the sex - hormone testosterone is the primary androgen produced in men and circulating levels progressively decrease from 30 years onward so that by 60 years of age , up to 1 in 4 men have developed androgen deficiency . with increasing longevity the proportion of androgen deficient or hypogonadal men is likely to grow , along with predicted adverse consequences for male health . although clinically diagnosed hypogonadism is remediable with testosterone replacement therapy , understanding a causative relationship between age - related physiological decline and reduced androgen levels will enable better defined interventional strategies . impaired hippocampal function resulting from neurogenesis deficits is hypothesised to contribute to the etiology of both depression and spatial memory impairment . in considering the collective clinical and rodent model data , a coincident age - related decline in circulating testosterone , hippocampal neurogenesis and hippocampal function becomes evident . however , a causal relationship between the age - related decline of these factors has yet to be established . this review raises a hypothesis that testosterone contributes to preserving hippocampal function in aging men by maintaining neurogenesis levels . herein , the intention is to briefly discuss the available evidence and highlight the necessity of further studies to establish , or refute , clinically relevant causal relationships . circulating testosterone targets the hippocampus , which has been functionally demarcated into dorsal ( 2 ) and ventral segments ( 3 ) . dosage and duration of testosterone and aromatasemediated metabolism into 17-estradiol may be important determinants of efficacy ( 7 ) . declines in circulating testosterone levels , hippocampal neurogenesis and hippocampal function are coincident in aging ( 8) . according to several endocrinology health bodies serum concentrations of total testosterone below 12 nm constitute a clinical diagnosis of hypogonadism , which can be further classified according to a taxonomy that directs remedial strategies . for example , testicular dysfunction , or primary hypogonadism , manifests in the presence of high luteinizing hormone / follicle stimulating hormone ( lh / fsh ) and is potentially indicated for testosterone replacement therapy . in this case , age - related low circulating testosterone can be the result of either primary or secondary hypogonadism . otherwise normal aging is associated with reduced total circulating testosterone levels ( below 20 nm ) concomitant with increased sex - hormone binding globulin ( shbg ) . testosterone bound to shbg is not bioactive and can affect the clearance rate of testosterone by the liver . however , the age - related increase in shbg levels has been shown to be independent of the age - related decline in bioactive testosterone levels . testosterone replacement therapy is advised based on symptomatic low circulating testosterone levels . however , applying this criterion to aging men presents potential limitations . age - related , or late - onset hypogonadism , may therefore elicit a unique spectrum of symptoms that are yet to be fully established . secondly , controversy surrounds the reliability of widely used measures of total and free serum testosterone to detect low circulating levels . as symptoms other than sexual dysfunction and frailty are not readily attributed to low circulating testosterone , symptomatic testosterone deficiency could be more common than generally recognised . testosterone therapy in aging men has hitherto been directed toward treating sexual and cardiovascular dysfunction , muscle mass loss and immobility . hence , the symptom profile of late - onset hypogonadism may well include declining mental health . notwithstanding the view held by some that age - related cognitive decline is normal , maintaining cognition in longevity at young levels would have an undeniable impact on wellbeing . declarative memory as a whole is closely associated with temporal lobe function and abilities related to spatial memory are intimately coupled with the hippocampus . notions of sex differences in brain anatomy and function have centred largely on the effects of sex hormones in development . evidence supports a possible organisational role of androgen signalling during brain development that imparts superior spatial cognitive ability in adulthood . longitudinal studies showing a correlation between age - related cognitive decline and reduced testosterone , raise the possibility that testosterone may perform a similar , but activational role in the mature brain . additionally , the substantial evidence demonstrating pro - cognitive effects of oestrogen replacement therapy in postmenopausal women , including enhanced hippocampal plasticity and function , underscores the possibility of a similar mechanistic role of testosterone in aging men . oestrogen is a major metabolite of testosterone in the male brain further suggesting that low circulating testosterone may be a readily modifiable factor predisposing aging men to cognitive decline . although neurodegenerative diseases increase with advancing age , their consideration needs to be distinguished from normal age - related cognitive decline . considered in reverse , the data suggests that age - related androgen deficiency may lead to ca1 synaptic loss and hippocampal dysfunction . as acknowledged above however , longitudinal studies indicate that the dentate gyrus subregion is more closely associated with age - related hippocampal impairment . evidence that sex - hormones increase dentate gyrus synaptogenesis has been extensively derived from examining the effects of oestrogen . however , these theories were immersed in the basic tenet that the mature brain lacks ongoing neuronal production . despite ongoing debate regarding the function of adult hippocampal neurogenesis , the weight of associative evidence points to a role of adult - generated granule neurons in spatial ability and perhaps emotional behaviour related to depression . levels of adult hippocampal neurogenesis decline with age , which has been hypothesised as a contributing factor to age - related decline in spatial ability . importantly , age - related declining adult hippocampal neurogenesis and spatial ability are reversible with factors such as physical exercise . if androgens play a critical role in maintaining adult hippocampal neurogenesis levels then late - onset hypogonadism may be a remediable cause of hippocampal dysfunction in aging men . adult hippocampal neurogenesis proceeds through a series of sequential steps : asymmetric division of neural stem cells , proliferative expansion of neural progenitors , neuronal differentiation and finally survival of mature neurons including both efferent and afferent synaptogenesis . intrinsic proliferation and differentiation markers together with exogenous markers such as 5-bromo-2deoxyuridine ( brdu ; which is incorporated into dividing cells and can assess proliferation and survival at short and longer time - points , respectively ) are widely used to measure adult hippocampal neurogenesis . mechanisms of age - induced reductions in adult hippocampal neurogenesis include decreased progenitor proliferation and neuronal survival , but factors mediating these mechanisms remain unknown . notions of sex - specific regulation of adult hippocampal neurogenesis were a significant impetus for exploring the effects of sex hormones on proliferation , differentiation and survival of adult - generated hippocampal neurons ( reviewed in ) . there is more hippocampal neurogenesis in neonatal male rats than females and several studies have demonstrated sex - specific rates of hippocampal neurogenesis in adult rodents . whether there is a sex - specific decline in adult hippocampal neurogenesis with advancing age remains hitherto unestablished . repletion of testosterone at high but not low levels in these animals restored adult hippocampal neurogenesis in an androgen receptor - dependent manner . in contrast , another study showed that exogenous injections of nandrolone , a synthetic derivative of testosterone and androgen receptor agonist , reduced adult hippocampal neurogenesis by decreasing neuronal precursor proliferation in young adult male rats . our own recent work demonstrated that chronic testosterone therapy in young male adult mice had no effect on precursor proliferation as measured by the intrinsic marker ki67 , but did promote adult hippocampal neurogenesis through increased neuronal survival and differentiation 28-days after brdu administration . we measured hippocampal neurogenesis during the last 4 weeks of 3 months of supraphysiological doses of testosterone through sustained - release pellets , whereas a similar dosage limited to 3 days via repeated injections had no effect on neurogenesis . single administration of brdu to measure precursor proliferation in gonadectomised young adult male rats untreated or made replete with physiological levels of testosterone showed no effect of androgen withdrawal . however , the same study showed that testosterone exacerbated the up - regulated proliferation in response to the anti - depressant imipramine . using a different approach , intact male rats given placebo or testosterone and brdu to measure proliferation , survival and differentiation showed no effect of exogenous androgen on any of these hippocampal neurogenesis parameters . manipulating testosterone levels in young male rats by gonadectomy or exogenous administration to intact the authors of this study make a pertinent argument that testosterone 's effects on hippocampal neurogenesis may be dose and duration - dependent and specific to the developmental stage of newly - generated neurons . further potential confounds reside in the methodology used to measure hippocampal neurogenesis , for example intrinsic markers such as ki67 , psa - ncam and doublecortin versus the timing and dose of exogenous markers like brdu . hence , extrapolating these data to explain a possible causal relationship between low testosterone and declining adult hippocampal neurogenesis in age is limited and specific longitudinal studies examining models of normal aging are required . these non - genomic signalling pathways in particular could represent intrinsic cellular mechanisms by which testosterone directly effects hippocampal neurogenesis . however , despite androgen receptor expression in the dentate gyrus region , whether neural stem cells , transient precursor intermediates or maturing neurons exhibit differential expression is unclear . cultured neural stem cells have been found to express androgen receptor , though these in vitro studies used cells derived from the sub - ventricular zone of adult female rats . hence it remains to be determined if hippocampal neurogenesis can be directly affected by testosterone or whether effects are transmitted by cellular and/or molecular intermediates . the local microenvironment , or neurogenic niche , is considered to play a significant role in regulating neurogenesis levels in the adult hippocampus . one mechanism by which astrocytes stimulate hippocampal neurogenesis is through the secretion of soluble neurotrophic factors such as fibroblast growth factor ( fgf)-2 . expression of fgf-2 by astrocytes residing in the hippocampal neurogenic microenvironment decreases during aging and may contribute to the age - related decline in hippocampal neurogenesis . hippocampal astrocytes in male rats express androgen receptor and hence testosterone could influence hippocampal neurogenesis indirectly . testosterone triggers fgf-2 production in germ - line stem cells of the adult male testis that may play a role in spermatogenesis . whether such signalling also occurs in neural stem cells to promote hippocampal neurogenesis and whether reductions in this signalling precipitate age - related decline in hippocampal neurogenesis remains untested . brain derived neurotrophic factor ( bdnf ) is another compound that both maintains and mediates up - regulation of hippocampal neurogenesis in rodents and is decreased in the aged hippocampus . the ability of testosterone to promote survival of adult - generated neurons in adult canaries is mediated by bdnf . however , fluctuations in circulating testosterone during the early development of male mice are independent of hippocampal bdnf levels . studies using aging rats have shown no sex - specific changes in declining hippocampal bdnf levels , nor any correlations between levels of serum oestrogen or testosterone with hippocampal bdnf in aging male rats . the collective evidence would suggest that age - related declining serum testosterone and hippocampal bdnf levels are independent and do not interact to cause falling hippocampal neurogenesis . this association could be important in aging men given the recent findings that circulating bdnf levels are ; higher in aging women compared to men , negatively correlated with age in men and positively correlated with bioavailable circulating testosterone levels in men . moreover , age - related decline in cellular and molecular elements including signal transduction pathways necessary to mediate testosterone 's effects could significantly impact on the effectiveness of androgen therapy and underscores the importance of their elucidation . the physiological metabolism of testosterone is another important consideration in elucidating the mechanisms of cognitive efficacy . thus , whether it be intrinsic production or the result of exogenous therapy , effects of circulating testosterone can be mediated through either androgen or oestrogen receptors . however , while both androgen and oestrogen mediated signalling contribute to preserving ca1 synaptic density in females , the same effect in males is androgen receptor dependent . although one study using rats showed that testosterone therapy tended to increase circulating levels of 17-estradiol ( albeit a non - statistically significant increase ) , oestrogen activity was not responsible for the increased adult hippocampal neurogenesis . the ability of testosterone therapy to restore declining spatial ability in aging rats was tested in advance of the general acceptance of adult hippocampal neurogenesis . more recent studies administering testosterone to aged rats however , have shown restorative effects on declining spatial ability . moreover , studies examining the effects of androgens on spatial abilities in rodents across life - span have yielded complex results that more generally reflect the collective clinical data examining aging men . the relationship between testosterone levels and different parameters of cognition in aging men has been recently reviewed in detail . decreased circulating testosterone in aging men is linked to poorer performances on tasks requiring spatial ability . several clinical studies show that spatial ability is improved in aging men receiving testosterone therapy . this effect of testosterone is also seen in men suffering mild cognitive impairment and even alzheimer 's disease . however , others have shown no effect of testosterone therapy on cognitive parameters in healthy and mildly cognitive impaired aged men . the magnitude of demonstrated spatial ability improvements have varied and may dependent on the dose of testosterone and the length of therapy . spatial ability varies with endogenous circulating testosterone levels in aging men and appears to respond better to intermediate doses of exogenous testosterone compared low and supraphysiological doses that show no efficacy . circulating 17-estradiol levels could change in elderly men receiving testosterone therapy as a consequence of peripheral aromatisation . however , the efficacy of testosterone therapy on spatial ability in aging men appears to be independent of oestrogen activity , though 17-estradiol may mediate concomitant improvements in verbal memory . both rodent and clinical studies suggest spatial ability in aged males is enhanced when high testosterone is coincident with low 17-estradiol levels . thus , in parallel with the notion of optimal testosterone levels , is the view that the balance of testosterone and 17-estradiol plays a significant role in spatial ability . debate over the function of adult hippocampal neurogenesis has evolved to include a specific role in pattern separation , which is to encode and retrieve similar events and is linked to dentate gyrus function . the strongest evidence from rodent studies also suggest an association between age - related decline in adult hippocampal neurogenesis and impaired pattern separation ability . given the relative infancy of interest in age - related pattern separation impairment , there is hitherto no definitive evidence for sex differences in the decline of this domain of spatial cognition . it therefore remains possible that testosterone therapy in aging men may preserve pattern separation ability , which could be mediated through maintaining neurogenesis levels in the aging dentate gyrus . the notion that androgens subserve specific domains of mental health includes effects on emotion - based behaviours . a hypothesis based on several associative links posits that impaired adult hippocampal neurogenesis elicits depression and that efficacy of antidepressant interventions is mediated through adult hippocampal neurogenesis . how does a link between depression and adult hippocampal neurogenesis relate to testosterone levels and advancing age ? gender difference in the incidence of depression disappears with advancing age and the occurrence of depression in elderly men is associated with low testosterone . to explore these observations in the context of declining adult hippocampal neurogenesis requires a consideration of a functional distinction in hippocampal structure . however , on a more global level the rodent hippocampus has been regionalised into dorsal ( septal pole or posterior in humans ) and ventral ( temporal pole or anterior in humans ) functional subregions . this regionalisation has partly been based on functional assessments , differential gene expression and connectivity , with spatial ability and emotion - based behaviour preferential to the dorsal and ventral hippocampal regions , respectively . neurogenesis rates may be higher in the dorsal compared to the ventral region of young adult hippocampus , but this regional difference attenuates with advancing age , indicating a relatively higher impact of age on dorsal hippocampal neurogenesis . clinical efficacy of antidepressant treatment is associated with specific increases in adult neurogenesis in the anterior hippocampus in depressed patients . whether androgen deficiency with advancing age differentially affects neurogenesis rates along the septal - temporal axis in the adult hippocampus in parallel with functional deficits such as depression and impaired spatial ability is not known . the question of whether antidepressant and spatial ability efficacy of testosterone therapy for aging men is mediated by region - specific hippocampal neurogenesis remains intriguing . intriguing in part , because addressing this cause - effect relationship will help refine the neurogenesis - depression and neurogenesis - cognition hypotheses and also elucidate links between impaired neural substrates , hypogonadism and symptoms of cognitive dysfunction . this latter point will in turn help define position statements that advocate testosterone therapy based on low testosterone in conjunction with manifest symptoms . clinical studies using testosterone therapy in elderly men showing either benefit or no effect rather than deterioration in cognition have employed inconsistent and even inadequate measures of mental health . a significant point to consider in evaluating this literature is the notion that androgens likely affect mental health in a domain - specific manner . uncertainty as to what biochemically measured level of circulating free testosterone constitutes late - onset hypogonadism and associated symptoms in aging men is a significant impediment to standardised testosterone replacement therapy . the prevailing view that age - related mental health decline is not an indicator for testosterone therapy is because of inconclusive rather than negative evidence . this review premises that reduced testosterone is a mediating factor of impaired spatial ability and depression incidence in aging males because of reduced dorsal and ventral hippocampal neurogenesis , respectively . in discussing the known effects of both androgen withdrawal and therapy on hippocampal function and plasticity , it is clear new understanding will be needed to establish a causal relationship between these factors in the context of normal male aging . there remain several key questions concerning the role androgens play in regulating adult hippocampal neurogenesis , including age - related decline and mediation of stimulatory factors such as exercise . an active lifestyle predicts higher circulating testosterone levels in aging men ; could testosterone mediate the ability of physical activity to promote neurogenesis and function in the aged hippocampus ? emerging studies showing the cognitive benefit of resistance training in the elderly could provide a paradigm shift in the way physical activity is prescribed to preserve mental health . menopause - mediated oestrogen withdrawal is posited play a role in cognitive decline and depressive symptoms in women that manifest early than in males . because menopause is a far more precipitous event compared to the gradual decline in male androgen production , what role in maintaining hippocampal neurogenesis and function in aging men does testosterone aromatisation into 17-estradiol play ? the hippocampus expresses receptors for lh and gonadotropin - releasing hormone ( gnrh ) and their role , directly or indirectly through promoting local steriodogenesis , in age - related decline is uncertain . studies examining these questions will address the paucity in knowledge regarding the areas of impaired mental health to which low testosterone predisposes and also elucidate ongoing debate surrounding the clinical relevance of adult hippocampal neurogenesis . new clinical and basic research endeavours that encompass measures of hippocampal - dependent spatial memory , pattern separation and depression in conjunction with serum measures of hpt axis hormones will shed further light on physiological roles of testosterone in maintaining mental health in aging men .

pneumonia more frequently afflicts patients aged 65 years , with an incidence that increases proportionally with increasing age . it was estimated that 1 in 20 persons aged 85 years experiences a new community - acquired pneumonia ( cap ) episode each year . several factors such as comorbidities , nutritional status , cognitive impairment may contribute to the frailty and the increased susceptibility of these patients to pneumonia . additionally , hospitalization , required in approximately 40% of episodes among elderly persons , is associated with a high risk of readmission within the following year and with high mortality rates ( 40% ) ( janssens 2005 ; schmidt - ioanas and lode 2006 ) . appropriate and timely antibiotic treatment is required for elderly patients in order to enhance the likelihood of a good clinical outcome . patients requiring hospital admission for treatment of cap should be promptly treated with potent broad spectrum intravenous antimicrobials in order to cover the major respiratory pathogens involved . beta - lactam antibiotics have been commonly prescribed over the past decades for the treatment of outpatients and patients hospitalized with cap ; however , the emergence in recent years in several countries in the world of penicillin - resistant streptococcus pneumoniae isolates ( resistance rates ranging from less than 5% to over 50% ) has forced a reassessment of the approach to treating cap ( heffelfinger et al 2000 ) . in this context , recent fluoroquinolones with antipneumococcal activity have been suggested as monotherapy in several international guidelines for the management of both outpatients and inpatients on a general medical ward and as a part of combination therapy for intensive care patients ( bts 2001 ; niederman et al 2001 ; mandell et al 2003 ) . among the newest generation of fluoroquinolones , moxifloxacin has been shown to display excellent activity against the most important respiratory pathogens including multi - drug resistant pneumococcal strains , and to possess satisfactory pharmacokinetic and pharmacodynamic characteristics at respiratory level ( balfour et al 2000 ; zhanel et al 2002 ) . this review will focus on the efficacy of moxifloxacin in the treatment of elderly patients with cap and on the safety and tolerability aspects of the drug in this population . pneumonia represents the leading infection - related cause of death and the fifth cause of overall mortality in the geriatric population ( schmidt - ioanas and lode 2006 ) . several factors such as alcoholism , asthma , immunosuppression , lung and heart diseases , institutionalization , and increasing age have been found to be associated with an increased risk of pneumonia in the elderly . the clinical presentation of cap in elderly patients is frequently characterized by a reduced prevalence of nonrespiratory symptoms and by the absence of the typical acute symptoms observed in young adults ( torres et al 1999 ; loeb 2003 ; niederman and ahmed 2003 ; schmidt - ioanas and lode 2006 ) . elderly people sometimes show only atypical clinical manifestations ( eg , weakness , urinary incontinence , and changes in mental status ) . these atypical findings could be responsible for a delay in diagnosis and treatment contributing to increased morbidity and mortality . the term cap should be reserved , in the elderly population , for pneumonia acquired outside the nursing home setting , since nursing home - acquired pneumonia ( nhap ) differs from cap in terms of its etiology and clinical manifestations ( lieberman and lieberman 2000 ) . although in most of the elderly the etiological agent of cap remains undetermined because of the difficulty in attaining adequate bronchial specimens and/or contamination of sputum by oral colonizing gram - negative bacilli , s. pneumoniae represents the most frequently isolated pathogen in this population , accounting for up to 50% of the causative agents ( marrie 2000 ; niederman et al 2001 ) . moreover , increasing age , per se , represents a risk factor for drug - resistant s. pneumoniae . relatively high frequency is also reported in the elderly population for haemophilus influenzae and moraxella catarrhalis , while staphylococcus aureus and gram - negative bacteria occur less frequently in post - viral influenza and in high - risk patient groups , respectively ( cunha 2001 ) . atypical pathogens , mainly chlamydia pneumoniae , have been emphasized recently in old people as the cause of cap and nhap outbreaks with a high mortality rate in nursing homes . outbreaks of pneumonia owing to respiratory viruses ( influenza and respiratory syncytial viruses ) may also occur in this population . etiology of aspiration pneumonia , which represents 5%15% of cap in elderly patients , in outpatients is associated with s. aureus , s. pneumoniae , and h. influenzae , while in hospitalized or nursing home residents more likely results in infection by gram - negative rods . in addition , anerobes may also contribute to the pathogenesis of aspiration pneumonia , although evidence for their major role has not been confirmed in recent studies ( schmidt - ioanas and lode 2006 ) . despite advances in diagnosis and therapy , the management of pneumonia still represents a challenge to the physicians mainly in old patients . when respiratory infections occur , rapid diagnosis and prompt administration of appropriate antibacterial therapy that ensures adequate coverage of the major pathogens causing cap in old people is likely to increase the probability of a successful outcome , reducing the risk of hospitalization and death and preventing the spread of antimicrobial resistance ( rajagopalan and yoshikawa 2001 ; neralla and meyer 2004 ) . the initial antibiotic therapy for elderly patients with cap should be empirical and the selection of antibacterials should be based upon local resistance patterns of chosen drugs . the algorithm for therapy suggested by international guidelines recommends the use of either a selected beta - lactam combined with a macrolide or monotherapy with a new antipneumococcal quinolone for both adult outpatients and inpatients ( not in an intensive care unit ) ( niederman et al 2001 ; bts guidelines 2001 ; mandell et al 2003 ) . no difference in antimicrobial selection for elderly with cap has been suggested by the recent infectious diseases society of america update containing a new chapter on pneumonia in the elderly ( mandell et al 2003 ) . moxifloxacin , like other recent fluoroquinolones , has a broad antibacterial spectrum that provides excellent coverage of the major respiratory tract pathogens . it displays excellent activity against gram - negative bacilli ( enterobacteriaceae , h. influenzae , m. catarrhalis ) and improved gram - positive activity against s. pneumoniae ( both penicillin - susceptible and penicillin - resistant strains ) , and s. aureus compared with ciprofloxacin . moreover , it retains good activity against atypical pathogens with a significantly better antibacterial effect against legionella pneumophila compared with erythromycin , and displays improved activity against anerobes compared with ciprofloxacin ( zhanel et al 2002 ; tano et al 2005 ) . moxifloxacin may inhibit both dna gyrase and topoisomeras iv , but its mechanism of action is slightly different from that of other fluoroquinolones . for s. pneumoniae the preferential target of fluoroquinolone action appears to vary depending on the chosen antibacterial agent . moxifloxacin primarily targets the gyra subunit of dna gyrase , whereas levofloxacin and other less recent derivatives such as ofloxacin and ciprofloxacin preferentially target the subunits parc or pare of the topoisomerase iv . owing to this difference , moxifloxacin may retain high activity against increasingly common pneumococcal strains bearing substitutions in topoisomerase iv due to the wide use of old derivatives . in addition , due to modification of the substituent at the c-7 position of the fluoroquinolone structure , moxifloxacin is a poor substrate for active efflux in s. pneumoniae ( pestova et al 2000 ) . moxifloxacin may be administered as oral and/or intravenous formulations with excellent bioavailability ; the drug is well absorbed after oral administration and achieves good tissue penetration at respiratory level , reaching higher concentrations in alveolar macrophages ( 56.7 g / ml ) and epithelial lining fluid ( 20.7 g / ml ) than in serum ( 3.2 g / ml ) after a single 400 mg oral dose ( soman et al 1999 ) . recently , high intrapulmonary concentrations have also been confirmed in older adults ( capitano et al 2004 ) . moreover , moxifloxacin both intravenously and orally exhibts high penetration in lung tissue with maximal lung concentrations of 12.37 g / g and 16.21 g / g for iv and oral administration respectively ( breilh et al 2003 ) . this is consistent with moxifloxacin being metabolized mainly by means of phase ii hepatic reactions , the activity of which was shown not to decline with age ( pea et al 2006 ) . dose adjustment does not appear to be necessary in patients of advanced age or those with mild to moderate renal or hepatic impairment ( balfour and lamb 2000 ; ball et al 2004 ) . like other fluoroquinolones , moxifloxacin can cause qt interval prolongation ; therefore it should be avoided in patients with known prolongation of the qt interval , patients with uncorrected hypokalemia or hypomagnesemia , and patients receiving class ia ( eg , quinidine , procainamide ) or class iii ( eg , amiodarone , sotalol ) antiarrhytmic agents or other drugs such as certain antimicrobials ( eg , erythromycin , halofantrine , pentamidine ) , certain antihistaminics ( eg , astemizole , mizolastine , terfenadine ) , neuroleptics , and tricyclic antidepressive agents ( stahlmann and lode 2003 ; miravitlles 2005 ) . moxifloxacin has a low propensity for causing phototoxic reactions and a low potential for causing excitatory effects and , like other agents of the group , can potentially cause tendon disorders mainly in aged patients in presence of concomitant use of corticosteroids and chronic renal diseases ( balfour and lamb 2000 ; zhanel et al 2002 ; stahlmann and lode 2003 ) . moxifloxacin lacks other significant drug interactions with a number of commonly prescribed drugs , although its absorption is decreased by concomitant administration of iron and cationic antacids ( ball et al 2004 ) . clinical efficacy of moxifloxacin in cap has been compared in some trials with different comparator agents both in adult and old patients ( table 1 ) . all studies excluding those of patel et al ( 2000 ) , and fogarty et al ( 2005 ) are comparative trials of moxifloxacin versus standard therapies including beta - lactams ( amoxicillin , amoxicillin - clavulanate , ceftriaxone ) alone or in combination with a macrolide ( erythromycin , roxithromycin , clarithromycin ) , a macrolide alone ( clarithromycin ) , or another fluoroquinolone , levofloxacin ( fogarty et al 1999 ; hoeffken et al 2001 ; petitpretz 2001 ; finch et al 2002 ; torres et al 2003 ; jardim et al 2003 ; katz et al 2004 ; portier et al 2005 ; welte et al 2006 ; anzueto et al 2006 ) . all these trials have confirmed that moxifloxacin is at least as effective as comparator regimens , since the overall clinical and bacteriological success rates for moxifloxacin ( range , 83%97% and 77%97% , respectively ) are comparable to those obtained for comparators ( range , 80%95% and 62%93% , respectively ) . aged subjects as a proportion of total number of patients included in these studies are not always available ; clinical trials providing such demographic data ( petitpretz et al 2001 ; torres et al 2003 ; jardim et al 2003 ; portier et al 2005 ; welte et al 2005 ) , and the only study entirely carried out on a population of elderly patients ( anzueto et al 2006 ) will be briefly summarized . welte et al ( 2005 ) compared the efficacy , safety , and speed and quality of defervescence of sequential intravenous or oral moxifloxacin ( 400 mg od ) and high dose ceftriaxone ( 2 g intravenously od ) with or without erythromycin ( 1 g intravenously every 68 hours ) for 714 days in patient with cap requiring parenteral therapy . in both arms of the study the validated per - protocol ( pp ) population aged 65 years achieved rates of 43.5% for moxifloxacin and 41.0% for comparators respectively . clinical response rates at the test of cure ( toc ) visit in the validated pp population ( 317 patients ) were 85.7% in the moxifloxacin group ( 138 of 161 patients ) and 86.5% in the ceftriaxone erythromycin group ( 135 of 156 patients ) ; at the end of treatment , resolution was reported for 87.6% and 88.5% of patients for moxifloxacin and for the other group respectively . in subgroups of patients matched for age , sex , and pneumonia severity index score , no significant differences were observed at the toc visit in both treatment groups . compared with the ceftriaxone erythromycin , the moxifloxacin regimen was observed to shorten the duration of hospitalization by a mean of 1.3 days in the validated pp population . in addition , defervescence and relief of signs and symptoms associated with cap such as chest pain and weakness , occurred significantly earlier in the moxifloxacin - treated group than in the comparator - treated group . no relevant differences between treatment groups in the incidence for drug - related adverse events were observed and the vast majority of them were mild to moderate . in a recent multicenter randomized open - label study , portier et al ( 2005 ) compared 10 days oral treatment with moxifloxacin ( 400 mg od ) with amoxicillin clavulanate ( 1000/125 mg tid ) plus roxithromycin ( 150 mg bid ) for non - severe cap in adults with risk factors . patients aged > 65 years included in the intention - to - treat ( itt ) analysis were 46.8% ( 80/171 ) and 53.1% ( 93/175 ) , respectively , and half of the entire population had at least one comorbid condition . respective per - protocol clinical success rates at the toc visit for moxifloxacin and comparator were 131 of 151 ( 86.8% ) and 120 of 138 ( 87.0% ) , with a 95% confidence interval ( ci ) of 8.0 to 7.6 for the difference . similar success rates were reached and maintained for patients with bacteriologically proven cap and for those in whom the causative pathogen was s. pneumoniae . the bacteriological success rates at the toc visit were also comparable for moxifloxacin- and comparator - treated patients with respective success rates of 23 of 30 ( 76.7% ) and 23 of 31 ( 74.2% ) . persistent clinical success rates at follow - up were 118 of 120 ( 98.3% ) and 102 of 106 ( 96.2% ) with a 95% ci of 2.2 to 6.4 for the difference . the clinical success rates were also maintained when subgroups were analyzed according to risk factors such as age ( > 65 years ) , comorbidities , prior hospitalization , and alcohol consumption . the numbers of drug - related adverse events ( predominantly digestive disorders ) in the itt population were comparable for both treatment arms : 24.6% ( 42 patients ) for moxifloxacin and 28.6% ( 50 patients ) for comparator group . age 70 years was reported for 22% of moxifloxacin - treated patients ( 52/233 ) and for 18% of comparator - treated patients ( 44/244 ) of the itt population , in the study of torres et al ( 2003 ) . in this study , 564 patients were randomized to either oral moxifloxacin ( 400 mg od ) or to standard oral therapy ( amoxicillin 1 g tid or clarithromycin 500 mg bid alone or in combination ) for up to 14 days using a double - blind procedure . in the pp population ( 446 patients ) , clinical success was reported for 201 of 215 ( 93.5% ) and 217 of 231 ( 93.9% ) in the moxifloxacin and standard groups , respectively , at 710 days post - therapy . at 2835 days follow - up , continued clinical cure was observed in 183 of 192 ( 95.3% ) moxifloxacin and 207 of 221 ( 93.7% ) standard groups . moxifloxacin treatment was significantly better tolerated than standard regimens with fewer adverse events and premature discontinuation . drug - related adverse events were reported in 55 of 274 ( 20% ) moxifloxacin and 86 of 279 ( 31% ) standard patients with a predominance of mild gastrointestinal upsets . the study of jardim et al ( 2003 ) evaluated the efficacy and safety of treatment with either moxifloxacin or amoxicillin administered for 10 days to patients suspected of having cap caused by a pneumococcal infection . over a total of 84 patients ( itt population ) included in the study and enrolled from 5 latin american countries , 70 patients ( pp population , 34 patients for moxifloxacin and 36 for amoxicillin ) were evaluated at the end of the trial . in the itt population patients aged > 65 years comprised 28.2% ( 11/39 ) and 31.1% ( 14/45 ) of the moxifloxacin and amoxicillin arm , respectively . the clinical success rate in the pp population at the final visit after treatment was 94.1% ( 32/34 ) for moxifloxacin and 91.7% ( 33/36 ) for amoxicillin , while the bacteriological success rate in microbiologically valid patients was 88.2% ( 15/17 ) and 87.5% ( 14/16 ) , respectively . in terms of pneumococcal etiology 15 of 34 and 13 of 36 patients evaluated and treated with moxifloxacin and amoxicillin , respectively , had proven pneumococcal pneumonia with prevalence of isolates with reduced susceptibility to penicillin . drug - related adverse events in both treatment groups were mainly mild to moderate in intensity and were subsequently resolved . petitpretz et al ( 2001 ) compared the efficacy and safety of moxifloxacin with amoxicillin for the treatment of mild - to - moderate , suspected pneumococcal cap in adult patients ; 25% and 22% respectively of the pp population ( 362 patients ) were aged 70 years . clinical success rate at the toc visit in the pp population was 91.5% for moxifloxacin and 89.7% for amoxicillin , while the clinical cure rate in patients with proven pneumococcal pneumonia was similar in both treatment groups ( 87.8% ) . the bacteriological success rate in 136 bacteriologically evaluable patients at the toc visit was 89.7% for moxifloxacin and 82.4% for amoxicillin . the bacteriological success rate against s. pneumoniae was 89.6% for moxifloxacin and 84.8% for amoxicillin . finally , the study of anzueto et al ( 2006 ) was the first comparative trial entirely carried out in hospitalized elderly patients ( mean age 77.4 7.7 years ) . in this double - blind , randomized trial , eligible patients for clinical efficacy were stratified by cap severity to receive treatment with either intravenous / oral moxifloxacin ( 400 mg od ) or intravenous / oral levofloxacin ( 500 mg od ) for 714 days . cure rates at the toc visit for the clinically valid ( pp ) population ( 141 and 140 patients in the moxifloxacin and levofloxacin group , respectively ) were 92.9% in the moxifloxacin arm and 87.9% in the levofloxacin arm ( 95% ci , 1.9 to 11.9 ; p = 0.2 ) . moreover , in subgroup analyses based on cap severity and patient age , clinical cure rates in the moxifloxacin arm were consistently higher than , although not statistically significantly different from , those for levofloxacin . in the moxifloxacin group , cure rates were 92.6% for patients with mild or moderate cap and 94.7% for patients with severe cap , compared with cure rates of 88.6% and 84.6% , respectively , in the levofloxacin group . cure rates in the moxifloxacin arm were 90% for patients aged 6574 years and 94.5% for patients aged 75 years , compared with 85.0% and 90.0% , respectively , in the levofloxacin arm . bacteriological success at the toc visit in the microbiologically valid population was 81.0% in the moxifloxacin arm ( 17 of 21 patients ) and 75.0% in the levofloxacin arm ( 21 of 28 patients ) ( p = 0.9 ) . the bacteriological response was in agreement with the clinical response : clinical cure rates for the microbiologically valid population were 81.0% in the moxifloxacin arm ( 17 of 21 patients ) vs 76.7% in the levofloxacin arm ( 23 of 30 patients ) ( 95% ci , 0.22 to 0.31 ; p = 0.98 ) . although no differences emerged in duration of stay or duration of intravenous therapy between the two regimens , sequential intravenous / oral moxifloxacin therapy provided significantly higher clinical recovery rates by day 35 after initiation of treatment . no statistically significant differences were observed between the treatment groups in drug - related adverse events . aging is well known to be associated with physiological changes and a higher risk of drug interactions ; for these reasons special attention needs to be directed towards the safety of medications in elderly people . cumulative safety data from the most recent clinical trials and post - marketing surveillance studies including a large number of patients have shown that gastrointestinal complaints such as nausea , diarrhea , and dizziness were the most commonly reported drug - related adverse events ( 7.1 , 5.2 , and 2.6% , respectively ) following administration of an oral dosage of 400 mg od ( ball et al 2004 ) . gastrointestinal and central nervous system ( cns ) disturbances are in line with those of other fluoroquinolones ; however , as adverse cns reactions are of particular concern for the elderly , old patients with impairments of the cns ( eg , epilepsy , pronounced arteriosclerosis ) should be treated with a quinolone only under close supervision ( stahlmann and lode 2003 ) . the safety of oral moxifloxacin in adult and elderly patients pooled by age group ( 4939 patients aged < 65 years , 842 patients aged 6574 years , 489 patients aged 75 years ) has been evaluated in a retrospective analysis versus comparator ( cefuroxime and clarithromycin , the most frequently used comparators ) ( andriole et al 2005 ) . drug - related adverse event rates associated with oral moxifloxacin or the comparator therapy used in these studies showed no significant increase with advancing age . no arrhythmias related to corrected qt interval prolongation were reported in this large group of young and elderly patients and a similar number of deaths was observed between the treatment groups ( 17 moxifloxacin , 19 comparator ) . the cardiac rhythm safety of moxifloxacin versus levofloxacin in high - risk elderly patients with cap ( eg , comorbid conditions and multiple medications ) was recently evaluated in a randomized , double - blind trial ( morganroth et al 2005 ) . in the studied population ( 394 patients ; two - third of the patients were > 75 years old , and 74.1% had a history of cardiac disease ) , iv / oral moxifloxacin demonstrated a comparable cardiac rhythm safety profile to iv / oral levofloxacin ; moreover , no deaths clearly related to study drugs were reported to occur during the observation period . to date , sporadic moxifloxacin - related adverse events observed in the elderly population may be limited to very few cases : these include tendinitis , in a 65-year - old female ( burkhardt et al 2004 ) , nephrotoxicity in a 68-year - old woman who experienced acute tubulointerstitial nephritis developed approximately 10 days after the end of moxifloxacin therapy for a non - specific bronchial infection ( argirov et al 2005 ) , and cholestasis in a 69-year - old man treated with moxifloxacin because of a respiratory infection ( soto et al 2002 ) . of major concern , several case reports have recently documented , mainly in old patients , a clinically relevant interaction between moxifloxacin and warfarin with significantly elevated international normalized ratio ( inr ) in patients receiving concomitant therapy ( arnold et al 2005 ; elbe and chang 2005 ) . routine , frequent inr monitoring and a suitable warfarin dosage adjustment should be recommended mainly in old patients receiving this combination of drugs . the treatment of cap is often based on an empirical approach ; therefore , antibiotic choice must cover key pathogens and take into account , at the same time , the steady increase in resistance observed worldwide during recent years in important respiratory pathogens . basically , the antibiotic therapy of cap in the elderly should mainly cover s. pneumoniae , including the penicillin - resistant strains in countries with a high prevalence ; when previous antimicrobial treatment , prior hospitalization , or structural lung disease are present , pseudomonas aeruginosa should be suspected and should guide the antibiotic choice ; and enteric gram - negative rods and anerobes should be considered in residents of nursing homes and in suspicion of aspiration pneumonia . isolates of penicillin- or macrolide - resistant pneumococci are now a consideration in elderly patients , as an age of > 65 years represents a recognized risk factor for infection with these organisms . given the increasing resistance of s. pneumoniae to the traditional first - line agents employed in cap treatment , recent fluoroquinolones with improved antipneumococcal activity are emerging as important therapeutic options . moxifloxacin , thanks to its excellent microbiological , pharmacokinetic , and pharmacodynamic profile , fulfils all the requirements of an optimal antimicrobial agent useful for lower respiratory tract infections . results of clinical trials available so far have shown that generally moxifloxacin may achieve more than 90% cure in all patients irrespective of severity of pneumonia , patient s age , and underlying comorbidities , and may retain bacteriological and clinical efficacy also in presence of penicillin- , macrolide- and multidrug - resistant pneumococcal isolates ( petitpretz et al 2001 ; finch et al 2002 ; jardim et al 2003 ; fogarty et al 2005 ) . in addition , moxifloxacin therapy has been observed to be often associated with faster clinical recovery than comparator therapies . finally , based on pharmacoeconomic considerations , the option of moxifloxacin sequential therapy , leading to early discharge from the hospital , may save costs compared with standard therapy ( drummond et al 2003 ) . proven clinical and bacteriological efficacy and safety profile in line with established fluoroquinolones and comparator agents suggest that moxifloxacin should prove a successful agent for the treatment of elderly patients with cap . the growing use of the new - generation fluoroquinolones could , however , lead to emergence of resistant pneumococcal isolates ; although resistance is currently low , levofloxacin clinical failures in the management of pneumococcal pneumonia have been reported recently , requiring a re - evaluation of the newer agents ( ferrara 2005 ) . no reports have shown the same phenomenon in patients treated with the more active moxifloxacin , suggesting that the selection of the most potent fluoroquinolone , which may assure the best coverage against s. pneumoniae , will reduce the opportunity for resistance to develop . however , to preserve the activity of moxifloxacin for the future , it seems prudent to avoid the massive use of this new agent as long as effective standard antibiotics for the treatment of cap are still available . targeted and judicious use of newer fluoroquinolones in selected cap patients , including those with infection owing to highly - resistant pneumococci , or following treatment failure with first - line regimen , will minimize the emergence of bacterial resistance to the entire class and maintain class efficacy ( heffelfnger et al 2000 ) .

community - acquired pneumonia ( cap ) remains a common cause of morbidity and a potentially life - threatening illness throughout the world mainly in elderly patients . initial antibacterial treatment , usually empirical , should be as effective as possible in order to assure rapid clinical resolution and reduce high rates of hospitalization and mortality especially affecting aged patients . new fluoroquinolones with potent activity against the most important respiratory pathogens including streptococcus pneumoniae , a key pathogen mainly in old patients with cap , have been recently suggested by several international guidelines as monotherapy for the treatment of most cap patient categories . among newer derivatives , moxifloxacin , an advanced generation 8-methoxy quinolone , has demonstrated good clinical and bacteriological efficacy in large , well designed clinical trials both in adults and old patients with cap , achieving also in aged people efficacy comparable with that of standard treatments . good pharmacokinetic characteristics such as excellent penetration into respiratory tract tissues and fluids , optimal bioavailability , simplicity of once - daily dosing , and good tolerability , represent potential advantages of moxifloxacin over other therapies . in addition , primarily due to a shorter length of hospital stay , moxifloxacin has been shown to save costs compared with standard therapy .

Introduction CAP in the elderly Moxifloxacin: a respiratory quinolone Clinical efficacy of moxifloxacin in CAP Safety considerations Conclusion

it was estimated that 1 in 20 persons aged 85 years experiences a new community - acquired pneumonia ( cap ) episode each year . appropriate and timely antibiotic treatment is required for elderly patients in order to enhance the likelihood of a good clinical outcome . patients requiring hospital admission for treatment of cap should be promptly treated with potent broad spectrum intravenous antimicrobials in order to cover the major respiratory pathogens involved . beta - lactam antibiotics have been commonly prescribed over the past decades for the treatment of outpatients and patients hospitalized with cap ; however , the emergence in recent years in several countries in the world of penicillin - resistant streptococcus pneumoniae isolates ( resistance rates ranging from less than 5% to over 50% ) has forced a reassessment of the approach to treating cap ( heffelfinger et al 2000 ) . in this context , recent fluoroquinolones with antipneumococcal activity have been suggested as monotherapy in several international guidelines for the management of both outpatients and inpatients on a general medical ward and as a part of combination therapy for intensive care patients ( bts 2001 ; niederman et al 2001 ; mandell et al 2003 ) . among the newest generation of fluoroquinolones , moxifloxacin has been shown to display excellent activity against the most important respiratory pathogens including multi - drug resistant pneumococcal strains , and to possess satisfactory pharmacokinetic and pharmacodynamic characteristics at respiratory level ( balfour et al 2000 ; zhanel et al 2002 ) . this review will focus on the efficacy of moxifloxacin in the treatment of elderly patients with cap and on the safety and tolerability aspects of the drug in this population . pneumonia represents the leading infection - related cause of death and the fifth cause of overall mortality in the geriatric population ( schmidt - ioanas and lode 2006 ) . several factors such as alcoholism , asthma , immunosuppression , lung and heart diseases , institutionalization , and increasing age have been found to be associated with an increased risk of pneumonia in the elderly . the clinical presentation of cap in elderly patients is frequently characterized by a reduced prevalence of nonrespiratory symptoms and by the absence of the typical acute symptoms observed in young adults ( torres et al 1999 ; loeb 2003 ; niederman and ahmed 2003 ; schmidt - ioanas and lode 2006 ) . elderly people sometimes show only atypical clinical manifestations ( eg , weakness , urinary incontinence , and changes in mental status ) . these atypical findings could be responsible for a delay in diagnosis and treatment contributing to increased morbidity and mortality . the term cap should be reserved , in the elderly population , for pneumonia acquired outside the nursing home setting , since nursing home - acquired pneumonia ( nhap ) differs from cap in terms of its etiology and clinical manifestations ( lieberman and lieberman 2000 ) . atypical pathogens , mainly chlamydia pneumoniae , have been emphasized recently in old people as the cause of cap and nhap outbreaks with a high mortality rate in nursing homes . etiology of aspiration pneumonia , which represents 5%15% of cap in elderly patients , in outpatients is associated with s. aureus , s. pneumoniae , and h. influenzae , while in hospitalized or nursing home residents more likely results in infection by gram - negative rods . in addition , anerobes may also contribute to the pathogenesis of aspiration pneumonia , although evidence for their major role has not been confirmed in recent studies ( schmidt - ioanas and lode 2006 ) . despite advances in diagnosis and therapy , the management of pneumonia still represents a challenge to the physicians mainly in old patients . when respiratory infections occur , rapid diagnosis and prompt administration of appropriate antibacterial therapy that ensures adequate coverage of the major pathogens causing cap in old people is likely to increase the probability of a successful outcome , reducing the risk of hospitalization and death and preventing the spread of antimicrobial resistance ( rajagopalan and yoshikawa 2001 ; neralla and meyer 2004 ) . the initial antibiotic therapy for elderly patients with cap should be empirical and the selection of antibacterials should be based upon local resistance patterns of chosen drugs . the algorithm for therapy suggested by international guidelines recommends the use of either a selected beta - lactam combined with a macrolide or monotherapy with a new antipneumococcal quinolone for both adult outpatients and inpatients ( not in an intensive care unit ) ( niederman et al 2001 ; bts guidelines 2001 ; mandell et al 2003 ) . no difference in antimicrobial selection for elderly with cap has been suggested by the recent infectious diseases society of america update containing a new chapter on pneumonia in the elderly ( mandell et al 2003 ) . moxifloxacin , like other recent fluoroquinolones , has a broad antibacterial spectrum that provides excellent coverage of the major respiratory tract pathogens . it displays excellent activity against gram - negative bacilli ( enterobacteriaceae , h. influenzae , m. catarrhalis ) and improved gram - positive activity against s. pneumoniae ( both penicillin - susceptible and penicillin - resistant strains ) , and s. aureus compared with ciprofloxacin . moreover , it retains good activity against atypical pathogens with a significantly better antibacterial effect against legionella pneumophila compared with erythromycin , and displays improved activity against anerobes compared with ciprofloxacin ( zhanel et al 2002 ; tano et al 2005 ) . moxifloxacin may inhibit both dna gyrase and topoisomeras iv , but its mechanism of action is slightly different from that of other fluoroquinolones . owing to this difference , moxifloxacin may retain high activity against increasingly common pneumococcal strains bearing substitutions in topoisomerase iv due to the wide use of old derivatives . in addition , due to modification of the substituent at the c-7 position of the fluoroquinolone structure , moxifloxacin is a poor substrate for active efflux in s. pneumoniae ( pestova et al 2000 ) . like other fluoroquinolones , moxifloxacin can cause qt interval prolongation ; therefore it should be avoided in patients with known prolongation of the qt interval , patients with uncorrected hypokalemia or hypomagnesemia , and patients receiving class ia ( eg , quinidine , procainamide ) or class iii ( eg , amiodarone , sotalol ) antiarrhytmic agents or other drugs such as certain antimicrobials ( eg , erythromycin , halofantrine , pentamidine ) , certain antihistaminics ( eg , astemizole , mizolastine , terfenadine ) , neuroleptics , and tricyclic antidepressive agents ( stahlmann and lode 2003 ; miravitlles 2005 ) . moxifloxacin has a low propensity for causing phototoxic reactions and a low potential for causing excitatory effects and , like other agents of the group , can potentially cause tendon disorders mainly in aged patients in presence of concomitant use of corticosteroids and chronic renal diseases ( balfour and lamb 2000 ; zhanel et al 2002 ; stahlmann and lode 2003 ) . clinical efficacy of moxifloxacin in cap has been compared in some trials with different comparator agents both in adult and old patients ( table 1 ) . all studies excluding those of patel et al ( 2000 ) , and fogarty et al ( 2005 ) are comparative trials of moxifloxacin versus standard therapies including beta - lactams ( amoxicillin , amoxicillin - clavulanate , ceftriaxone ) alone or in combination with a macrolide ( erythromycin , roxithromycin , clarithromycin ) , a macrolide alone ( clarithromycin ) , or another fluoroquinolone , levofloxacin ( fogarty et al 1999 ; hoeffken et al 2001 ; petitpretz 2001 ; finch et al 2002 ; torres et al 2003 ; jardim et al 2003 ; katz et al 2004 ; portier et al 2005 ; welte et al 2006 ; anzueto et al 2006 ) . all these trials have confirmed that moxifloxacin is at least as effective as comparator regimens , since the overall clinical and bacteriological success rates for moxifloxacin ( range , 83%97% and 77%97% , respectively ) are comparable to those obtained for comparators ( range , 80%95% and 62%93% , respectively ) . aged subjects as a proportion of total number of patients included in these studies are not always available ; clinical trials providing such demographic data ( petitpretz et al 2001 ; torres et al 2003 ; jardim et al 2003 ; portier et al 2005 ; welte et al 2005 ) , and the only study entirely carried out on a population of elderly patients ( anzueto et al 2006 ) will be briefly summarized . welte et al ( 2005 ) compared the efficacy , safety , and speed and quality of defervescence of sequential intravenous or oral moxifloxacin ( 400 mg od ) and high dose ceftriaxone ( 2 g intravenously od ) with or without erythromycin ( 1 g intravenously every 68 hours ) for 714 days in patient with cap requiring parenteral therapy . clinical response rates at the test of cure ( toc ) visit in the validated pp population ( 317 patients ) were 85.7% in the moxifloxacin group ( 138 of 161 patients ) and 86.5% in the ceftriaxone erythromycin group ( 135 of 156 patients ) ; at the end of treatment , resolution was reported for 87.6% and 88.5% of patients for moxifloxacin and for the other group respectively . in subgroups of patients matched for age , sex , and pneumonia severity index score , no significant differences were observed at the toc visit in both treatment groups . compared with the ceftriaxone erythromycin , the moxifloxacin regimen was observed to shorten the duration of hospitalization by a mean of 1.3 days in the validated pp population . in addition , defervescence and relief of signs and symptoms associated with cap such as chest pain and weakness , occurred significantly earlier in the moxifloxacin - treated group than in the comparator - treated group . respective per - protocol clinical success rates at the toc visit for moxifloxacin and comparator were 131 of 151 ( 86.8% ) and 120 of 138 ( 87.0% ) , with a 95% confidence interval ( ci ) of 8.0 to 7.6 for the difference . the bacteriological success rates at the toc visit were also comparable for moxifloxacin- and comparator - treated patients with respective success rates of 23 of 30 ( 76.7% ) and 23 of 31 ( 74.2% ) . the clinical success rates were also maintained when subgroups were analyzed according to risk factors such as age ( > 65 years ) , comorbidities , prior hospitalization , and alcohol consumption . petitpretz et al ( 2001 ) compared the efficacy and safety of moxifloxacin with amoxicillin for the treatment of mild - to - moderate , suspected pneumococcal cap in adult patients ; 25% and 22% respectively of the pp population ( 362 patients ) were aged 70 years . finally , the study of anzueto et al ( 2006 ) was the first comparative trial entirely carried out in hospitalized elderly patients ( mean age 77.4 7.7 years ) . cure rates at the toc visit for the clinically valid ( pp ) population ( 141 and 140 patients in the moxifloxacin and levofloxacin group , respectively ) were 92.9% in the moxifloxacin arm and 87.9% in the levofloxacin arm ( 95% ci , 1.9 to 11.9 ; p = 0.2 ) . in the moxifloxacin group , cure rates were 92.6% for patients with mild or moderate cap and 94.7% for patients with severe cap , compared with cure rates of 88.6% and 84.6% , respectively , in the levofloxacin group . cure rates in the moxifloxacin arm were 90% for patients aged 6574 years and 94.5% for patients aged 75 years , compared with 85.0% and 90.0% , respectively , in the levofloxacin arm . no statistically significant differences were observed between the treatment groups in drug - related adverse events . aging is well known to be associated with physiological changes and a higher risk of drug interactions ; for these reasons special attention needs to be directed towards the safety of medications in elderly people . cumulative safety data from the most recent clinical trials and post - marketing surveillance studies including a large number of patients have shown that gastrointestinal complaints such as nausea , diarrhea , and dizziness were the most commonly reported drug - related adverse events ( 7.1 , 5.2 , and 2.6% , respectively ) following administration of an oral dosage of 400 mg od ( ball et al 2004 ) . gastrointestinal and central nervous system ( cns ) disturbances are in line with those of other fluoroquinolones ; however , as adverse cns reactions are of particular concern for the elderly , old patients with impairments of the cns ( eg , epilepsy , pronounced arteriosclerosis ) should be treated with a quinolone only under close supervision ( stahlmann and lode 2003 ) . the safety of oral moxifloxacin in adult and elderly patients pooled by age group ( 4939 patients aged < 65 years , 842 patients aged 6574 years , 489 patients aged 75 years ) has been evaluated in a retrospective analysis versus comparator ( cefuroxime and clarithromycin , the most frequently used comparators ) ( andriole et al 2005 ) . no arrhythmias related to corrected qt interval prolongation were reported in this large group of young and elderly patients and a similar number of deaths was observed between the treatment groups ( 17 moxifloxacin , 19 comparator ) . the cardiac rhythm safety of moxifloxacin versus levofloxacin in high - risk elderly patients with cap ( eg , comorbid conditions and multiple medications ) was recently evaluated in a randomized , double - blind trial ( morganroth et al 2005 ) . in the studied population ( 394 patients ; two - third of the patients were > 75 years old , and 74.1% had a history of cardiac disease ) , iv / oral moxifloxacin demonstrated a comparable cardiac rhythm safety profile to iv / oral levofloxacin ; moreover , no deaths clearly related to study drugs were reported to occur during the observation period . to date , sporadic moxifloxacin - related adverse events observed in the elderly population may be limited to very few cases : these include tendinitis , in a 65-year - old female ( burkhardt et al 2004 ) , nephrotoxicity in a 68-year - old woman who experienced acute tubulointerstitial nephritis developed approximately 10 days after the end of moxifloxacin therapy for a non - specific bronchial infection ( argirov et al 2005 ) , and cholestasis in a 69-year - old man treated with moxifloxacin because of a respiratory infection ( soto et al 2002 ) . of major concern , several case reports have recently documented , mainly in old patients , a clinically relevant interaction between moxifloxacin and warfarin with significantly elevated international normalized ratio ( inr ) in patients receiving concomitant therapy ( arnold et al 2005 ; elbe and chang 2005 ) . routine , frequent inr monitoring and a suitable warfarin dosage adjustment should be recommended mainly in old patients receiving this combination of drugs . the treatment of cap is often based on an empirical approach ; therefore , antibiotic choice must cover key pathogens and take into account , at the same time , the steady increase in resistance observed worldwide during recent years in important respiratory pathogens . basically , the antibiotic therapy of cap in the elderly should mainly cover s. pneumoniae , including the penicillin - resistant strains in countries with a high prevalence ; when previous antimicrobial treatment , prior hospitalization , or structural lung disease are present , pseudomonas aeruginosa should be suspected and should guide the antibiotic choice ; and enteric gram - negative rods and anerobes should be considered in residents of nursing homes and in suspicion of aspiration pneumonia . isolates of penicillin- or macrolide - resistant pneumococci are now a consideration in elderly patients , as an age of > 65 years represents a recognized risk factor for infection with these organisms . given the increasing resistance of s. pneumoniae to the traditional first - line agents employed in cap treatment , recent fluoroquinolones with improved antipneumococcal activity are emerging as important therapeutic options . moxifloxacin , thanks to its excellent microbiological , pharmacokinetic , and pharmacodynamic profile , fulfils all the requirements of an optimal antimicrobial agent useful for lower respiratory tract infections . results of clinical trials available so far have shown that generally moxifloxacin may achieve more than 90% cure in all patients irrespective of severity of pneumonia , patient s age , and underlying comorbidities , and may retain bacteriological and clinical efficacy also in presence of penicillin- , macrolide- and multidrug - resistant pneumococcal isolates ( petitpretz et al 2001 ; finch et al 2002 ; jardim et al 2003 ; fogarty et al 2005 ) . in addition , moxifloxacin therapy has been observed to be often associated with faster clinical recovery than comparator therapies . finally , based on pharmacoeconomic considerations , the option of moxifloxacin sequential therapy , leading to early discharge from the hospital , may save costs compared with standard therapy ( drummond et al 2003 ) . proven clinical and bacteriological efficacy and safety profile in line with established fluoroquinolones and comparator agents suggest that moxifloxacin should prove a successful agent for the treatment of elderly patients with cap . the growing use of the new - generation fluoroquinolones could , however , lead to emergence of resistant pneumococcal isolates ; although resistance is currently low , levofloxacin clinical failures in the management of pneumococcal pneumonia have been reported recently , requiring a re - evaluation of the newer agents ( ferrara 2005 ) . no reports have shown the same phenomenon in patients treated with the more active moxifloxacin , suggesting that the selection of the most potent fluoroquinolone , which may assure the best coverage against s. pneumoniae , will reduce the opportunity for resistance to develop . however , to preserve the activity of moxifloxacin for the future , it seems prudent to avoid the massive use of this new agent as long as effective standard antibiotics for the treatment of cap are still available .

epigenetic aberrations have been well established in cancer1 , 2 and occur in several other diseases including diabetes3 , lupus4 , asthma5 and a variety of neurological disorders2 , 6 , 7,8 ( table 1 and references within ) . in cancer , there is a global loss of dna methylation ( hypomethylation ) , particularly in gene bodies and intergenic regions , including repetitive elements , leading to genomic instability . this global hypomethylation is accompanied by increased de novo methylation ( hypermethylation ) of many promoters of tumor suppressor and other genes that are contained within cpg islands , resulting in permanent gene silencing ( figure 1 ) . in addition to changes in dna methylation , there is a global loss of h4k16 acetylation and h4k20 tri - methylation , as well as increased expression of bmi1 , a component of the polycomb repressive complex 1 ( prc1 ) , and ezh2 , a component of prc2 , which act to inhibit gene expression1 , 9 . interestingly , recent evidence has demonstrated that genes that are targets of the prc in embryonic stem cells are more likely than others to become methylated in cancer , potentially linking different epigenetic silencing mechanisms10 - 12 . h3 acetylation and h3k9 di - methylation can discriminate between prostate cancer ( pca ) and non - malignant prostate tissue and h3k4 tri - methylation can be a significant predictor of psa recurrence16 . ezh2 expression is an independent prognostic marker that correlates with the aggressiveness of prostate , breast and endometrial cancers17 . expression of the dna repair gene o(6)-methylguanine - dna methyltransferase ( mgmt ) antagonizes chemotherapy and radiation treatment18 thus , silencing of mgmt by hypermethylation correlates with positive treatment response . furthermore , epigenetic alterations can also precede tumor formation and thus are potential diagnostic indicators of disease risk19 . for example , h. pylori bacterial infection is associated with dna hypermethylation of specific genes , which are often methylated in cancer20 . thus , reversal of epigenetic alterations that occur as a result of an acute illness may prevent the progression to a more chronic disease state . with the growing development of technologies to analyze the epigenome , a new field : pharmaco - epigenomics - is emerging , where epigenetic profiles can be used to identify molecular pathways for cancer drug sensitivity21 and be used in determining the best therapeutic approach . in non - small - cell lung cancer , an unmethylated igfbp3 promoter is indicative of responsiveness to cisplatin based chemotherapy22 . a polymorphism in the cyp2c19 * 2 variant of cytochrome p450 requires higher valproic acid ( vpa ) doses to achieve target plasma concentration levels23 . furthermore , monitoring epigenetic changes can be used to measure treatment efficacy and disease progression . methylation of pitx2 can be used to predict outcome of early breast cancer patients following adjuvant tamoxifen therapy24 . patients with p16 hypermethylation had lower bladder cancer recurrence rates after il-2 treatment compared to patients with no p16 hypermethylation after il-2 treatment25 . since epigenetic mechanisms determine which genes and hence signaling pathways can be activated , the presence of distinct modifications on specific genes and subsets of genes can aid at several steps in determining and monitoring optimal therapeutic approaches . epigenetic modifications are somatically heritable yet also reversible , making them good candidates for potential drug treatments . multiple epigenetic abnormalities are typically present in diseased tissue leading to an altered epigenetic landscape . addicted to the aberrantly developed epigenetic landscape and , consequently , be more sensitive than normal cells to epigenetic therapy , in a process similar to an inverted oncogene addiction . one example of oncogene addiction is met , a tyrosine kinase that acts as a receptor for hepatocyte growth factor ( hgf ) and functions in normal cells to control tissue homeostasis26 . met can be aberrantly activated in cancer , by ligand dependent mechanisms or by overexpression26 . interestingly , while met plays roles in both normal and cancer cells , the latter are more sensitive to met inhibition due to increase reliance on met signaling26 . thus , cancer cells become dependent ( and consequently addicted ) to increased activity of a few highly important oncogenes . it is possible that cancer cells undergo a parallel process by which they become dependent ( and consequently addicted ) to aberrant silencing / inactivation of a few highly important tumor suppressor genes . since it is well known that several tumor suppressor genes are silenced in cancer by epigenetic mechanisms1 it is possible that cancer cells become addicted to their aberrant epigenetic landscape and , consequently , more sensitive to the epigenetic therapy than normal cells . in cancer there is global hypomethylation accompanied by hypermethylation of a subset of gene promoters contained within cpg islands leading to gene silencing ( figure 1)1 . this hypermethylation has recently been described to also extend past the boundaries of cpg islands into dna shores 27 . dna methyltransferases 3a / b are responsible for de novo dna methylation patterns , which are then copied to daughter cells during s - phase by dnmt1 . 5- azacytidine ( 5-aza - cr ; vidaza ) is a nucleoside analog that incorporates into rna and dna and is fda approved to treat high risk myelodysplastic syndromes ( mds ) patients and successful clinical results have recently been reported ( table 2)29 . 5-aza-2-deoxycytidine ( 5-aza - cdr ; decitabine ) is the deoxy derivative of 5-aza - cr and is incorporated only into dna . at low doses both azanucleosides act by sequestering dnmt enzymes after incorporation into dna thus leading to global demethylation as cells divide , while at higher doses they induce cytotoxicity . potential new drugs that can be used in the clinic include zebularine , a cytidine analog that acts similarly to 5-aza - cr , but has lower toxicity , increased stability and specificity30 and s110 , a decitabine derivative that has increased stability and activity , which has shown promise in pre - clinical studies ( figure 2)31 . in addition to inhibiting dna methyltransferase activity , azanucleosides also act through unspecific mechanisms , which likely contribute to their clinical effectiveness . promoter dna methylation can be used to molecularly classify21 , 32,33 predict progression of cancer34 , 35 and direct therapeutic approach36,37 . for example , dna methylation of specific promoters may identify a sub - population of colorectal cancer that is responsive to 5-fluorouracil36 . furthermore , reversing mlh1 silencing by dna methylation inhibitors restores sensitivity to cisplatin38 suggesting that inclusion of dna methylation inhibitors may increase the effectiveness of conventional chemotherapy . successful conventional chemotherapy depends on activation of pro - apoptotic genes that respond to cytotoxic agents leading to cell death . dna methylation of these pro - apoptotic genes can block cell death from occurring leading to chemotherapeutic resistance , thus reactivation of epigenetically silenced apoptotic genes can increase efficacy of chemotherapy . for example , apaf1 is silenced in metastatic melanoma cells and treatment with 5-aza - cdr restores expression and chemosensivity37 . conversely , methylation induced silencing of dna repair genes can be detrimental by leading to microsatellite instability39 or beneficial by preventing the repair of genes targeted by chemotherapy causing cells to undergo apoptosis rather than repair40 . methylation induced silencing of cancer - testis antigens , such as ny - eso-1 , can protect cancer cells from being recognized by t cells . treating cancer cells with demethylating agents can induce the expression of these antigens , allowing a reaction by engineered lymphocytes41 suggesting that epigenetic therapy can be combined with immunotherapy for better results ( figure 1 ) . despite the clinical successes achieved with dna methylation inhibitors currently available dna methylation inhibitors block dna methylation at the enzymatic level leading to global dna methylation inhibition . this is therapeutically beneficial as tumor suppressor genes are hypermethylated in cancer , however global hypomethylation may lead to activation of oncogenes and/or increased genomic instability . dna hypomethylation can activate promoters within repetitive elements , for example line-1 hypomethylation can activate an alternative transcript of the met oncogene in bladder cancer42 , suggesting a risk of global dna demethylation agents . developing dna methylation inhibitors that targeted specific genes or groups of genes would overcome this limitation . furthermore , dna methylation inhibitors act during s - phase of the cell cycle , thus they preferentially affect rapidly growing cells . this is advantageous when treating rapidly dividing cancer cells but may be less clinically useful in treating diseases that are not characterized by rapid cell cycling . furthermore , upon azanucleoside withdrawal the dna methylation levels return to pre - treatment levels6 demonstrating a continual need for dna methyltransferase inhibition . thus , while dna methylation inhibitors are clinically successful , their lack of specificity , cell cycle dependency and need for continual administration leave room for the development of better therapies . while dna methylation is considered to be a very stable epigenetic modification , histone modifications are comparably more labile with their levels being maintained by a balance between histone modifying enzymes , which add or remove specific modifications . aberrant histone modification levels result from an imbalance in these modifying enzymes in diseased tissue , thus correcting the increased or decreased level of a particular enzyme will restore the natural balance in the affected cells . in cancer , both histone methyltransferase and histone demethylase enzymes are misregulated and there is a high expression level of histone deacetylases ( hdac ) and a global reduction in histone acetylation levels1 , 9 , 43 , 44 , 45 . hdac inhibitors have long been studied in the clinical setting as potential therapies ( figure 2 ) and recent clinical trials have been extensively reviewed elsewhere ( table 2)46 . histone deacetylase inhibitors ( hdaci ) can affect acetylation of non - histone proteins , as well as histones , potentially leading to more global effects46 . furthermore , non - isoform selective hdaci only target approximately 10% of all acetylation sites 47 , thus more work is necessary to understand the underlying basis for target specification of global and isoform specific hdaci . currently , significant effort is underway to find new molecules that are able to selectively inhibit specific hdacs48 , 49 and thus avoid the side effects that occur with a global hdaci50 . to date , specific inhibitors of hdac6 ( class ii ) and hdac8 ( class i ) have been developed48 , 49 . the development of specific hdaci combined with a better understanding of the pathophysiology of diseases associated with alterations in hdacs will allow more rational therapy and , potentially , reduce side effects . for example , the hdac inhibitor pci-34051 , which is derived from a low molecular weight hydroxamic acid scaffold , was recently shown to selectively inhibit hdac8 and induce apoptosis specifically in t - cell lymphomas and not other tumor or normal cells , showing hdc8 plays an important role in the pathophysiology of this disease and suggesting the therapy with hdac8 specific inhibitor may lead to less side effects49 . while the identification of additional specific hdac inhibitors will increase specificity and the possibility of personalized treatments , it may also potentially limit the likelihood of success in combinatorial therapy . histone methyltransferase and demethylase enzymes are relatively more specific than hdacs in that they target a limited number of residues51 however , like hdacs , lysine and arginine methyltransferase enzymes methylate non - histone proteins as well as histone proteins52 , 53 . a great deal of effort is underway to find drugs able to revert specific histone methylation marks or to target histone methyltransferase or histone demethylases . in this regard , a new class of oligoamine analogs was recently found that act as potent inhibitors of lysine specific demethylase 1 ( lsd1 ) ( figure 2 ) . lsd1 targets the activating h3k4 mono and di - methylation mark but can also target the repressive h3k9me2 mark when complexed with the androgen receptor43 , 54 . treatment of colon cancer cells with lsd1 inhibitors ( e.g. sl11144 ) resulted in increased h3k4 methylation , decreased h3k9me2 , and re - expression of the sfrp2 gene55 , demonstrating context specificity of lsd1 and its inhibitors . lsd1 inhibition in neuroblastoma , resulted in decreased proliferation in vitro and reduced xenograft growth56 . interestingly , lsd1 can also demethylate dnmt1 resulting in destabilization and loss of global dna methylation maintenance57 . the ability of lsd1 to affect both histone and dna methylation , make it a promising target for epigenetic therapy . the repression mediated by the h3k27 tri - methylation mark , occurs through the actions of two multi - subunit complexes , prc1 and prc2 , h3k27me3 is deposited by ezh2 and then recognized and bound by prc1 , which can further recruit additional proteins to establish a repressed chromatin configuration1 . gene promoters which are marked by prc2 ( i.e. polycomb target genes ) in embryonic stem cells have recently been shown to be far more likely than other genes to become methylated in cancer10 - 12 . thus alterations in chromatin structure do not always coincide with changes in gene expression , rather dna methylation replacement of polycomb repressive marks acts to lock in an inactive chromatin state through a process called the mechanism underlying the predisposition of polycomb targets for dna methylation is not fully understood , however some links have recently been uncovered . cbx7 , a component of the prc1 complex , can directly interact with dnmt1 and 3b at polycomb target genes59 . while there is significant promise for drugs that target histone methylation enzymes , more work is necessary to determine their specificities and stability and there are currently no such drugs undergoing clinical trials . to date , the s - adenosylhomocysteine hydrolase inhibitor , 3-deazaneplanocin a ( dznep ) has the most promising pre - clinical results of drugs , which target histone methylating enzymes ( figure 2 ) . dznep depletes cellular levels of prc2 components ( ezh2 , eed and suz12 ) and consequently reduces h3k27me3 levels and induces apoptosis in breast cancer but not normal cells60 . the effect of dznep is similar to those observed when ezh2 is depleted by rnai , suggesting that this drug may be more effective in cancers like prostate and breast , which rely on abnormally high ezh2 expression levels61 . on the other hand , a subsequent study showed that dznep also decreased h4k20me3 demonstrating that dznep lacks specificity and acts more as a global histone methylation inhibitor , suggesting the need for further development of histone methylation inhibitors62 . for example , akt phosphorylates ezh2 at serine 21 , suppressing its methyltransferase activity and , consequently , reducing h3k27me363 . h3k27me3 levels can be restored using the pi3k / akt inhibitor ly294002 , opening a new therapeutic opportunity to repair epigenetic alterations by targeting upstream signaling pathways . furthermore , in prostate cancer , the oncogenic ets transcription factor erg can bind to the ezh2 promoter and induce over - expression . thus , the pharmacological disruption of erg activity could reduce ezh2 over - expression observed in cancer64 . ezh2 is a particularly important example because it is frequently over - expressed and aberrantly targeted to genes in cancer58 , a process termed prc reprogramming ( figure 1 ) . g9a and glp ( g9a like protein ) are histone methyl - transferases that catalyze h3k9 di - methylation and are often over - expressed in tumors65 . knockdown of g9a in prostate cancer cells demonstrates a critical role for this protein in regulating centrosome duplication and chromatin structure , suggesting that g9a is important to perpetuate the malignant phenotype and , thus , a putative target in cancer therapy66 . as a result there is significant interest in developing g9a / glp inhibitors and thus far the most efficient inhibitor is bix-01294 ( adiazepin - quinazolin - amine derivative ) , which is able to transiently reduce bulk h3k9me2 levels in several cell lines67 . bix-01294 binds to the set domain of glp in the same groove where the target lysine ( h3k9 ) binds , preventing the binding of the peptide substrate and , consequently , the deposition of methylation marks at h3k968 . several other histone methyltransferases and demethylases have also been associated with diseases and , thus , could be potential targets for epigenetic therapy . for instance , mmset , a h4k20 methyltransferase , is overexpressed in myeloma cell lines and required for cell viability69 . smyd3 , an h3k4 methyltransferase , is also highly expressed in cancer and seems to play a role in carcinogenesis as a coactivator of eralpha70 . gasc1 , an h3k9 and h3k36 demethylase , is often amplified in cancer and its inhibition results in decreases cell proliferation71 . despite a lack of specificity , hopefully the development of therapeutics that target specific histone modifying enzymes will retain or increase their therapeutic success while decreasing side effects due to the lack of specificity . on the other hand targeting individual histone modifying enzymes may decrease clinical efficacy due to compensation by other histone modifying enzymes potentially leading to resistance . designing personalized cocktails of inhibitors based on an individual 's need may help overcome the potential problems of compensation and resistance . mirnas are able to induce heritable changes in gene expression without altering dna sequence , and thus contribute to the epigenetic landscape . in addition mirnas for example , mir-101 targets ezh2 for degradation and is down - regulated in several types of cancer , resulting in increased ezh2 expression and consequently h3k27me3 levels and decreased tumor suppressor gene expression61 , 74 . re - expression of mir-101 leads to reduced h3k27me3 and inhibits colony formation and cancer cell proliferation61 , 74 . expression of mir-143 in colorectal cancer cells75 and the mir-29 family in lung cancer cells76 reduced dnmt3a and dnmt3a and b levels , respectively , and resulted in decreased cell growth and colony formation . treatment of cells with 5-aza - cdr and 4-phenylbutyric acid resulted in mir-127 activation , which in turn downregulated the bcl6 oncogene in bladder cancer cells77 . in fact , treatment with 5-aza - cdr alone is sufficient to reactivate mir148a , mir34b / c and mir-9 , a group of mirnas with metastasis suppression activity78 . in addition to inducing aberrantly repressed mirnas using epigenetic drugs , replacement gene therapy may also be useful in re - establishing mirna expression . viral vectors generated by cloning individual or groups of human mirnas has been successful in pre - clinical assays using a mouse model of hepatocellular carcinoma in which mir-26a expression by an adeno - associated virus ( aav ) resulted in apoptosis and inhibition of cancer cell proliferation in the absence of toxicity79 . microrna gene therapy has an advantage , over the conventional rnai , in that it is unlikely to generate a strong type i interferon response because double - strand rna is not introduced to the cell and furthermore , the aav vector is less immunogenic than previous generations of viral vectors used for gene transfer80 . abnormally high expression of mirnas can be targeted using recently developed locked - nucleic - acid ( lna)-modified phosphorothioate oligonucleotide technology . locked - nucleic - acid - modified oligonucleotides contain an extra bridge in their chemical composition leading to enhanced stability compared to their unmodified counterparts . mirnas can be generated using these lna modified phosphorothioate oligonucleotides to create lna - antimirs , which can be delivered systemically . in pre - clinical assays with primates , intravenous injections of lna - antimir complementary to the 5 end of mir-122 antagonized liver specific expression of this mirna without toxicity81 . based on these promising results , phase 1 trials lna - antimirs may be used to target aberrantly expressed mirnas in other diseases , such as cancer . for example , mir-155 is up - regulated in lung adenocarcinoma compared to non - cancerous lung tissue and patients with higher mir-155 expression had lower survival rates than patients with lower mir-155 expression , suggesting that mir-155 is a promising target for lna - antimir therapy ( table1)82 . several other mirnas are up - regulated in cancer and could , theoretically , be used as lna - antimir targets . for example , mir-21 , is up - regulated in several types of cancer ( lung , breast , colon , gastric and prostate carcinomas , endocrine pancreatic tumours , glioblastomas and cholangiocarcinomas ) and targets the tumor suppressor pten ( table 1)83 . thus , mirnas can alter the epigenetic machinery and also be regulated by epigenetic alterations , creating a highly controlled feedback mechanism , making it a suitable target for epigenetic therapy and possibly an epigenetic drug itself . one unique advantage of targeting mirnas is the ability of one mirna to regulate several target genes and multiple cellular process . in that way , if the level of one or a few mirnas has changed in a pathological state , several different pathways could be consequently altered . rather than trying to identify and directly target the proteins in multiple pathways it would be more effective to restore the physiological level and functions of the misregulated mirna(s ) . this clinical potential highlights the importance of better understanding mirna profiles in healthy and diseased tissues in order to develop better therapeutic strategies . furthermore , multiple mirnas that target different steps of an over - active pathway can be combined to increase efficacy and allow for customization of therapies to individual patients . while the unique composition of mirna based therapy provides many benefits , additional research is necessary to determine the best method of delivery and increase mirna stability to ensure efficacy . the presence of multiple epigenetic aberrations in a single tissue , the ability to develop resistance and the discovery that common sets of genes are regulated by distinct epigenetic mechanisms at different biological stages demonstrates the need and highlights the potential success of combinatorial therapy . combining epigenetic therapies to increase efficacy has been done for over 25 years84 , 85 and has demonstrated both additive and synergistic effects depending on the targets6 , 44 . extensive work has evaluated the clinical benefits of combined dnmt and hdac inhibition and comprehensive reviews have been published elsewhere1 , 39 , 46 , 86 , 87 . a recent phase ii multicenter study examining the combination of 5-aza - cr and the hdac inhibitor vpa in patients with higher risk mds found that therapeutic levels of vpa may increase 5-aza - cr efficacy23 . sequential administration of dnmt and hdac inhibitors demonstrated clinical efficacy in patients with hematologic malignancies46 , 86 . however , other studies found no correlation between baseline methylation levels or methylation reversal and positive clinical outcome in mds and aml patients following 5-aza - cr and entinostat treatment88 . the mechanism behind the clinical efficacy of combined dnmt and hdac remains controversial and additional studies investigating potential genetic or epigenetic determinants of responsiveness will be helpful . in addition to inducing apoptosis in cancer cells , another therapeutic approach is to induce differentiation of cancer cells . to this end , 5-aza - cr and vpa treatment followed by all trans - retinoic acid in patients with acute myeloid leukemia or high - risk myelodysplastic syndrome resulted in global hypomethylation and histone acetylation and clinical response in nearly half of treated patients89 . while targeting histone de - methylation enzymes is still in its infancy , early pre - clinical studies alone ( described above ) and combined with other epigenetic therapies are promising . sfrp2 ( a negative regulator of wnt signaling ) expression was re - established in vitro and in vivo in a human colon cancer model following lsd1 and dnmt inhibition resulting significant growth inhibition of established tumors55 . interestingly , in addition to histone residues , lsd1 can also demethylate dnmt1 providing the ability to target both histone methylation and dna methylation using a single compound . co - treatment with the hdac inhibitor , panobinostat , further enhanced dznep 's reduction of ezh2 levels leading to increased p16 , p21 , p27 and fbx032 expression and apoptosis in cultured aml cells and mouse models90 . thus , while clinical trials targeting histone methylation and demethylation enzymes are not currently underway it is an area of active investigation with significant promise . however , resistance to standard chemotherapy can be established through epigenetic and dna repair mechanisms22 . as a result , epigenetic therapeutics can be combined with more conventional therapies to induce responsiveness or overcome resistance to cytotoxic treatments . preconditioning with epigenetic drugs could reverse the epigenetic alteration(s ) that conferred resistance restoring chemotherapeutic sensitivity . for example , 5-aza - cr treatment can reverse dna methylation , thereby overcoming the gene silencing , which led to chemotherapeutic resistance91 . on the other hand , methylation induced silencing of dna repair genes ( i.e. mgmt ) correlates with a positive clinical response to chemotherapy . thus , whether the combination of dna methylation inhibitors and chemotherapy will be successful may depend on the epigenetic profile of an individual tumor . clinical findings in patients with previously untreated non - small cell lung cancer treated with the hdac inhibitor , vorinostat , in combination with carboplatin and paclitaxil were sufficiently promising to warrant a phase 2 study which also showed promising results ( table 2)92 , 93 . epigenetic therapy has been established as a successful treatment approach for a variety of malignancies . inhibition of dnmts and hdacs are two fda approved cancer treatments . while the mechanism(s ) behind the therapeutic benefit of dnmt and hdac inhibition are not fully understood , ongoing and future studies that combine genomic sequencing and expression data may provide the keys to understanding the mechanism(s ) underlying responsiveness . furthermore , histones can be phosphorylated , ubiquitylated and sumoylated in addition to methylated and acetylated , however these modifications have been less well studied in the context of disease and may offer additional therapeutic targets . one important challenge in epigenetic therapy is defining which genes are the drivers ( i.e. group of genes necessary to be epigenetically silenced for disease to occur ) and the passengers ( i.e. group of genes that are epigenetically silenced due to aberrant activity of the epigenetic machinery , but are not necessary for disease to occur ) . recent advances in high throughout technologies like genome - wide sequencing , combined with rna profiling , chromatin immunoprecipitation ( chip ) or bisulfite conversion , have led to the generation of large amounts of data that can be integrated to form a comprehensive understanding of the epigenetic alterations that are common and specific to various disease states . assimilating these large datasets will likely assist in identifying epigenetic alterations that are causational and those that are merely correlative . thus , in the future it may be possible for patients to be screened , using high throughout technologies , and classified by epigenetic alterations of the driver genes allowing the use of personalized targeted therapies . currently , epigenetic therapies are successfully used in the clinic to treat hematological malignancies , however little success has been achieved in treating solid cancers ( table 2)94 - 97 . initial clinical trials used treatment regimens later found to be less than optimal and resulted in few cases of positive clinical response . administering more recently developed dosing and treatment schedules in addition to subtyping tumor types based on molecular signatures may increase the efficacy of epigenetic therapy for solid tumors . furthermore , solid tumors are heterogeneous cell populations , often consisting of cells at a variety of stages of differentiation , thus determining which of these cells harbor epigenetic alterations and ensuring that therapeutic agents maintain stability and are able to penetrate the cellular mass and reach affected cells and will increase the likelihood of clinical success . the recognition of epigenetics as a significant contributor to normal development and disease opens an expanding new avenue for drug discovery and therapeutics . epigenetic therapies can be combined with conventional therapies to develop personalized treatments , used to convert unresponsive tumors and may allow for lower dosing which can limit side effects of treatment improving quality of life and treatment compliance .

epigenetic modifications work with genetic mechanisms to determine transcriptional activity and , while somatically heritable they are also reversible , making them good therapeutic candidates . epigenetic changes can precede disease pathology and thus are diagnostic indicators for risk , and can act as prognostic indicators for disease progression . histone deacetylase inhibitors and dna methylation inhibitors have been fda approved for several years and are clinically successful . more recently , histone methylation and micrornas have also gained attention as potential therapeutic targets . the presence of multiple epigenetic aberrations within a diseased tissue and the abilities of cells to develop resistance suggest that combination therapies may be most beneficial . this review will focus on recent examples of using epigenetic modifications to evaluate disease risk , progression and clinical response and will describe the latest clinical advances in epigenetic therapies concentrating on treatments which combine epigenetic therapeutics with each other and with cytotoxic agents to increase clinical response .

Epigenetic Disease Mechanisms DNA Methylation Histone Modifications microRNAs Combined Epigenetic Therapies Epigenetic and Cytotoxic Therapies Conclusions/Perspective

epigenetic aberrations have been well established in cancer1 , 2 and occur in several other diseases including diabetes3 , lupus4 , asthma5 and a variety of neurological disorders2 , 6 , 7,8 ( table 1 and references within ) . in cancer , there is a global loss of dna methylation ( hypomethylation ) , particularly in gene bodies and intergenic regions , including repetitive elements , leading to genomic instability . in addition to changes in dna methylation , there is a global loss of h4k16 acetylation and h4k20 tri - methylation , as well as increased expression of bmi1 , a component of the polycomb repressive complex 1 ( prc1 ) , and ezh2 , a component of prc2 , which act to inhibit gene expression1 , 9 . h3 acetylation and h3k9 di - methylation can discriminate between prostate cancer ( pca ) and non - malignant prostate tissue and h3k4 tri - methylation can be a significant predictor of psa recurrence16 . expression of the dna repair gene o(6)-methylguanine - dna methyltransferase ( mgmt ) antagonizes chemotherapy and radiation treatment18 thus , silencing of mgmt by hypermethylation correlates with positive treatment response . furthermore , epigenetic alterations can also precede tumor formation and thus are potential diagnostic indicators of disease risk19 . in non - small - cell lung cancer , an unmethylated igfbp3 promoter is indicative of responsiveness to cisplatin based chemotherapy22 . furthermore , monitoring epigenetic changes can be used to measure treatment efficacy and disease progression . since epigenetic mechanisms determine which genes and hence signaling pathways can be activated , the presence of distinct modifications on specific genes and subsets of genes can aid at several steps in determining and monitoring optimal therapeutic approaches . epigenetic modifications are somatically heritable yet also reversible , making them good candidates for potential drug treatments . multiple epigenetic abnormalities are typically present in diseased tissue leading to an altered epigenetic landscape . addicted to the aberrantly developed epigenetic landscape and , consequently , be more sensitive than normal cells to epigenetic therapy , in a process similar to an inverted oncogene addiction . one example of oncogene addiction is met , a tyrosine kinase that acts as a receptor for hepatocyte growth factor ( hgf ) and functions in normal cells to control tissue homeostasis26 . interestingly , while met plays roles in both normal and cancer cells , the latter are more sensitive to met inhibition due to increase reliance on met signaling26 . since it is well known that several tumor suppressor genes are silenced in cancer by epigenetic mechanisms1 it is possible that cancer cells become addicted to their aberrant epigenetic landscape and , consequently , more sensitive to the epigenetic therapy than normal cells . in cancer there is global hypomethylation accompanied by hypermethylation of a subset of gene promoters contained within cpg islands leading to gene silencing ( figure 1)1 . this hypermethylation has recently been described to also extend past the boundaries of cpg islands into dna shores 27 . dna methyltransferases 3a / b are responsible for de novo dna methylation patterns , which are then copied to daughter cells during s - phase by dnmt1 . 5- azacytidine ( 5-aza - cr ; vidaza ) is a nucleoside analog that incorporates into rna and dna and is fda approved to treat high risk myelodysplastic syndromes ( mds ) patients and successful clinical results have recently been reported ( table 2)29 . 5-aza-2-deoxycytidine ( 5-aza - cdr ; decitabine ) is the deoxy derivative of 5-aza - cr and is incorporated only into dna . at low doses both azanucleosides act by sequestering dnmt enzymes after incorporation into dna thus leading to global demethylation as cells divide , while at higher doses they induce cytotoxicity . potential new drugs that can be used in the clinic include zebularine , a cytidine analog that acts similarly to 5-aza - cr , but has lower toxicity , increased stability and specificity30 and s110 , a decitabine derivative that has increased stability and activity , which has shown promise in pre - clinical studies ( figure 2)31 . promoter dna methylation can be used to molecularly classify21 , 32,33 predict progression of cancer34 , 35 and direct therapeutic approach36,37 . for example , dna methylation of specific promoters may identify a sub - population of colorectal cancer that is responsive to 5-fluorouracil36 . furthermore , reversing mlh1 silencing by dna methylation inhibitors restores sensitivity to cisplatin38 suggesting that inclusion of dna methylation inhibitors may increase the effectiveness of conventional chemotherapy . successful conventional chemotherapy depends on activation of pro - apoptotic genes that respond to cytotoxic agents leading to cell death . dna methylation of these pro - apoptotic genes can block cell death from occurring leading to chemotherapeutic resistance , thus reactivation of epigenetically silenced apoptotic genes can increase efficacy of chemotherapy . for example , apaf1 is silenced in metastatic melanoma cells and treatment with 5-aza - cdr restores expression and chemosensivity37 . conversely , methylation induced silencing of dna repair genes can be detrimental by leading to microsatellite instability39 or beneficial by preventing the repair of genes targeted by chemotherapy causing cells to undergo apoptosis rather than repair40 . despite the clinical successes achieved with dna methylation inhibitors currently available dna methylation inhibitors block dna methylation at the enzymatic level leading to global dna methylation inhibition . developing dna methylation inhibitors that targeted specific genes or groups of genes would overcome this limitation . furthermore , dna methylation inhibitors act during s - phase of the cell cycle , thus they preferentially affect rapidly growing cells . this is advantageous when treating rapidly dividing cancer cells but may be less clinically useful in treating diseases that are not characterized by rapid cell cycling . furthermore , upon azanucleoside withdrawal the dna methylation levels return to pre - treatment levels6 demonstrating a continual need for dna methyltransferase inhibition . thus , while dna methylation inhibitors are clinically successful , their lack of specificity , cell cycle dependency and need for continual administration leave room for the development of better therapies . while dna methylation is considered to be a very stable epigenetic modification , histone modifications are comparably more labile with their levels being maintained by a balance between histone modifying enzymes , which add or remove specific modifications . aberrant histone modification levels result from an imbalance in these modifying enzymes in diseased tissue , thus correcting the increased or decreased level of a particular enzyme will restore the natural balance in the affected cells . hdac inhibitors have long been studied in the clinical setting as potential therapies ( figure 2 ) and recent clinical trials have been extensively reviewed elsewhere ( table 2)46 . histone deacetylase inhibitors ( hdaci ) can affect acetylation of non - histone proteins , as well as histones , potentially leading to more global effects46 . currently , significant effort is underway to find new molecules that are able to selectively inhibit specific hdacs48 , 49 and thus avoid the side effects that occur with a global hdaci50 . to date , specific inhibitors of hdac6 ( class ii ) and hdac8 ( class i ) have been developed48 , 49 . the development of specific hdaci combined with a better understanding of the pathophysiology of diseases associated with alterations in hdacs will allow more rational therapy and , potentially , reduce side effects . while the identification of additional specific hdac inhibitors will increase specificity and the possibility of personalized treatments , it may also potentially limit the likelihood of success in combinatorial therapy . a great deal of effort is underway to find drugs able to revert specific histone methylation marks or to target histone methyltransferase or histone demethylases . in this regard , a new class of oligoamine analogs was recently found that act as potent inhibitors of lysine specific demethylase 1 ( lsd1 ) ( figure 2 ) . lsd1 targets the activating h3k4 mono and di - methylation mark but can also target the repressive h3k9me2 mark when complexed with the androgen receptor43 , 54 . sl11144 ) resulted in increased h3k4 methylation , decreased h3k9me2 , and re - expression of the sfrp2 gene55 , demonstrating context specificity of lsd1 and its inhibitors . lsd1 inhibition in neuroblastoma , resulted in decreased proliferation in vitro and reduced xenograft growth56 . interestingly , lsd1 can also demethylate dnmt1 resulting in destabilization and loss of global dna methylation maintenance57 . the ability of lsd1 to affect both histone and dna methylation , make it a promising target for epigenetic therapy . thus alterations in chromatin structure do not always coincide with changes in gene expression , rather dna methylation replacement of polycomb repressive marks acts to lock in an inactive chromatin state through a process called the mechanism underlying the predisposition of polycomb targets for dna methylation is not fully understood , however some links have recently been uncovered . cbx7 , a component of the prc1 complex , can directly interact with dnmt1 and 3b at polycomb target genes59 . while there is significant promise for drugs that target histone methylation enzymes , more work is necessary to determine their specificities and stability and there are currently no such drugs undergoing clinical trials . to date , the s - adenosylhomocysteine hydrolase inhibitor , 3-deazaneplanocin a ( dznep ) has the most promising pre - clinical results of drugs , which target histone methylating enzymes ( figure 2 ) . the effect of dznep is similar to those observed when ezh2 is depleted by rnai , suggesting that this drug may be more effective in cancers like prostate and breast , which rely on abnormally high ezh2 expression levels61 . on the other hand , a subsequent study showed that dznep also decreased h4k20me3 demonstrating that dznep lacks specificity and acts more as a global histone methylation inhibitor , suggesting the need for further development of histone methylation inhibitors62 . for example , akt phosphorylates ezh2 at serine 21 , suppressing its methyltransferase activity and , consequently , reducing h3k27me363 . thus , the pharmacological disruption of erg activity could reduce ezh2 over - expression observed in cancer64 . g9a and glp ( g9a like protein ) are histone methyl - transferases that catalyze h3k9 di - methylation and are often over - expressed in tumors65 . knockdown of g9a in prostate cancer cells demonstrates a critical role for this protein in regulating centrosome duplication and chromatin structure , suggesting that g9a is important to perpetuate the malignant phenotype and , thus , a putative target in cancer therapy66 . as a result there is significant interest in developing g9a / glp inhibitors and thus far the most efficient inhibitor is bix-01294 ( adiazepin - quinazolin - amine derivative ) , which is able to transiently reduce bulk h3k9me2 levels in several cell lines67 . bix-01294 binds to the set domain of glp in the same groove where the target lysine ( h3k9 ) binds , preventing the binding of the peptide substrate and , consequently , the deposition of methylation marks at h3k968 . several other histone methyltransferases and demethylases have also been associated with diseases and , thus , could be potential targets for epigenetic therapy . smyd3 , an h3k4 methyltransferase , is also highly expressed in cancer and seems to play a role in carcinogenesis as a coactivator of eralpha70 . mirnas are able to induce heritable changes in gene expression without altering dna sequence , and thus contribute to the epigenetic landscape . in addition mirnas for example , mir-101 targets ezh2 for degradation and is down - regulated in several types of cancer , resulting in increased ezh2 expression and consequently h3k27me3 levels and decreased tumor suppressor gene expression61 , 74 . re - expression of mir-101 leads to reduced h3k27me3 and inhibits colony formation and cancer cell proliferation61 , 74 . expression of mir-143 in colorectal cancer cells75 and the mir-29 family in lung cancer cells76 reduced dnmt3a and dnmt3a and b levels , respectively , and resulted in decreased cell growth and colony formation . treatment of cells with 5-aza - cdr and 4-phenylbutyric acid resulted in mir-127 activation , which in turn downregulated the bcl6 oncogene in bladder cancer cells77 . in addition to inducing aberrantly repressed mirnas using epigenetic drugs , replacement gene therapy may also be useful in re - establishing mirna expression . based on these promising results , phase 1 trials lna - antimirs may be used to target aberrantly expressed mirnas in other diseases , such as cancer . for example , mir-155 is up - regulated in lung adenocarcinoma compared to non - cancerous lung tissue and patients with higher mir-155 expression had lower survival rates than patients with lower mir-155 expression , suggesting that mir-155 is a promising target for lna - antimir therapy ( table1)82 . for example , mir-21 , is up - regulated in several types of cancer ( lung , breast , colon , gastric and prostate carcinomas , endocrine pancreatic tumours , glioblastomas and cholangiocarcinomas ) and targets the tumor suppressor pten ( table 1)83 . thus , mirnas can alter the epigenetic machinery and also be regulated by epigenetic alterations , creating a highly controlled feedback mechanism , making it a suitable target for epigenetic therapy and possibly an epigenetic drug itself . in that way , if the level of one or a few mirnas has changed in a pathological state , several different pathways could be consequently altered . this clinical potential highlights the importance of better understanding mirna profiles in healthy and diseased tissues in order to develop better therapeutic strategies . furthermore , multiple mirnas that target different steps of an over - active pathway can be combined to increase efficacy and allow for customization of therapies to individual patients . while the unique composition of mirna based therapy provides many benefits , additional research is necessary to determine the best method of delivery and increase mirna stability to ensure efficacy . the presence of multiple epigenetic aberrations in a single tissue , the ability to develop resistance and the discovery that common sets of genes are regulated by distinct epigenetic mechanisms at different biological stages demonstrates the need and highlights the potential success of combinatorial therapy . combining epigenetic therapies to increase efficacy has been done for over 25 years84 , 85 and has demonstrated both additive and synergistic effects depending on the targets6 , 44 . extensive work has evaluated the clinical benefits of combined dnmt and hdac inhibition and comprehensive reviews have been published elsewhere1 , 39 , 46 , 86 , 87 . a recent phase ii multicenter study examining the combination of 5-aza - cr and the hdac inhibitor vpa in patients with higher risk mds found that therapeutic levels of vpa may increase 5-aza - cr efficacy23 . to this end , 5-aza - cr and vpa treatment followed by all trans - retinoic acid in patients with acute myeloid leukemia or high - risk myelodysplastic syndrome resulted in global hypomethylation and histone acetylation and clinical response in nearly half of treated patients89 . while targeting histone de - methylation enzymes is still in its infancy , early pre - clinical studies alone ( described above ) and combined with other epigenetic therapies are promising . interestingly , in addition to histone residues , lsd1 can also demethylate dnmt1 providing the ability to target both histone methylation and dna methylation using a single compound . co - treatment with the hdac inhibitor , panobinostat , further enhanced dznep 's reduction of ezh2 levels leading to increased p16 , p21 , p27 and fbx032 expression and apoptosis in cultured aml cells and mouse models90 . thus , while clinical trials targeting histone methylation and demethylation enzymes are not currently underway it is an area of active investigation with significant promise . however , resistance to standard chemotherapy can be established through epigenetic and dna repair mechanisms22 . as a result , epigenetic therapeutics can be combined with more conventional therapies to induce responsiveness or overcome resistance to cytotoxic treatments . for example , 5-aza - cr treatment can reverse dna methylation , thereby overcoming the gene silencing , which led to chemotherapeutic resistance91 . mgmt ) correlates with a positive clinical response to chemotherapy . thus , whether the combination of dna methylation inhibitors and chemotherapy will be successful may depend on the epigenetic profile of an individual tumor . inhibition of dnmts and hdacs are two fda approved cancer treatments . furthermore , histones can be phosphorylated , ubiquitylated and sumoylated in addition to methylated and acetylated , however these modifications have been less well studied in the context of disease and may offer additional therapeutic targets . one important challenge in epigenetic therapy is defining which genes are the drivers ( i.e. group of genes necessary to be epigenetically silenced for disease to occur ) and the passengers ( i.e. group of genes that are epigenetically silenced due to aberrant activity of the epigenetic machinery , but are not necessary for disease to occur ) . recent advances in high throughout technologies like genome - wide sequencing , combined with rna profiling , chromatin immunoprecipitation ( chip ) or bisulfite conversion , have led to the generation of large amounts of data that can be integrated to form a comprehensive understanding of the epigenetic alterations that are common and specific to various disease states . assimilating these large datasets will likely assist in identifying epigenetic alterations that are causational and those that are merely correlative . thus , in the future it may be possible for patients to be screened , using high throughout technologies , and classified by epigenetic alterations of the driver genes allowing the use of personalized targeted therapies . currently , epigenetic therapies are successfully used in the clinic to treat hematological malignancies , however little success has been achieved in treating solid cancers ( table 2)94 - 97 . initial clinical trials used treatment regimens later found to be less than optimal and resulted in few cases of positive clinical response . administering more recently developed dosing and treatment schedules in addition to subtyping tumor types based on molecular signatures may increase the efficacy of epigenetic therapy for solid tumors . furthermore , solid tumors are heterogeneous cell populations , often consisting of cells at a variety of stages of differentiation , thus determining which of these cells harbor epigenetic alterations and ensuring that therapeutic agents maintain stability and are able to penetrate the cellular mass and reach affected cells and will increase the likelihood of clinical success . epigenetic therapies can be combined with conventional therapies to develop personalized treatments , used to convert unresponsive tumors and may allow for lower dosing which can limit side effects of treatment improving quality of life and treatment compliance .

age - related macular degeneration ( amd ) is the leading cause of blindness in patients over the age of 65 years in industrialized nations . most affected patients ( approximately 90% ) have dry or nonexudative amd , characterized by drusen and retinal pigment epithelium ( rpe ) mottling , hyperplasia , and atrophy , but most cases of blindness ( approximately 90% ) are due to wet or exudative amd , which is characterized by the growth of choroidal neovascular membranes ( cnvm).1 wet amd results from repeated cycles of shedding , degradation , and resynthesis of photoreceptor outer segments , which induces metabolic stress within the outer retina and rpe . the resultant chronic ischemia and inflammation upregulates several inflammatory cytokines and growth factors , particularly vascular endothelial growth factor ( vegf ) , which promote the growth of cnvm from the choriocapillaris into the sub - rpe space ( type 1 cnvm ) , the subretinal space ( type 2 cnvm ) , or the inner retina as retinal angiomatous proliferation ( type 3 cnvm ) . the treatment of exudative amd has evolved considerably over the past 40 years , but the visual outcomes throughout most of this period have disappointed both patients and physicians . thermal laser photocoagulation , as performed in the macular photocoagulation study , slowed the rate of vision loss , but was complicated by recurrent neovascularization , and frequently left patients with dense scotomas.2 photodynamic therapy with intravenous verteporfin decreased the extent of photoreceptor and rpe scarring , but still resulted in vision loss for most patients.3 only with the introduction of drugs that directly inhibit the actions of vegf have surgeons been able to offer patients reasonable hope for improvement of vision . pivotal trials with ranibizumab , bevacizumab , and aflibercept show that patients receiving protocol - driven treatment have a 30%40% chance of achieving a 15-letter ( halving of the visual angle ) improvement in visual acuity.47 the rapid adoption of these drugs by physicians has yielded dramatic improvements in public health . between 2000 and 2010 , anti - vegf therapy resulted in a 50% reduction in the incident rate of blindness due to exudative amd in denmark , compared with a 33% reduction in the incidence of blindness from other causes.8 three anti - vegf drugs have been used for over 5 years , i.e. pegaptanib ( macugen ; eyetech inc , palm beach gardens , fl , introduced in 2004 ) , bevacizumab ( avastin ; genentech , south san francisco , ca / roche , basel , switzerland , first used off - label in 2005 ) and ranibizumab ( lucentis ; genentech , south san francisco , ca / roche , basel , switzerland , introduced in 2006 ) , whereas aflibercept ( vegf trap - eye , eylea ; regeneron , tarrytown , ny ) has been available only since being granted regulatory approval for the treatment of exudative amd by the united states food and drug administration on november 18 , 2011 . this paper discusses the pathophysiology of exudative amd with emphasis on the stimulatory role of vegf , the biochemical and pharmacologic characteristics of aflibercept , and the results of important clinical trials focusing on the treatment of patients with exudative amd . untreated cnvm due to amd carries a poor prognosis with 62% of subfoveal membranes and 65% of juxtafoveal membranes causing profound ( more than 6 lines ) loss of vision.9,10 treatment according to mps guidelines limits vision loss in some patients , but 87% of eyes with cnvm do not qualify for laser,11 and furthermore , only 18% of patients qualify for ocular photodynamic therapy.12 these sobering results underscore the need for both an improved understanding of the pathophysiology of amd and a non - destructive therapy that can be given to the majority of affected patients . the earliest observable signs of amd include mild atrophy and hypertrophy of the rpe , along with the formation of drusen . in addition to the development of confluent soft drusen , decreased choroidal blood flow , lipid deposition in bruch s membrane , oxidative stress in the rpe , and alteration in bruch s membrane all contribute to ischemia of the overlying rpe , secondary death of cells in the neural retina , and , finally to induction of cnvm.1318 whereas drusen and rpe atrophy generally cause a slow decrease in vision , a rapid decline usually accompanies the development of cnvm.19 the pathophysiology of amd actually begins with lipoprotein transport of carotenoids , vitamin e , and cholesterol to the photoreceptors through the rpe ( figure 1).20,21 nutrients are separated within the rpe and presented to the photoreceptors while , at the same time , the rpe performs near continuous , homeostatic phagocytosis of photoreceptor outer segments,2224 with assembly of apolipoprotein b from phagocytosed photoreceptor outer segments , plasma lipoprotein components , and endogenously synthesized lipids . with advancing age comes metabolic insufficiency which causes a decrease in protein and lipid degradation within the rpe . this results in accumulation of lipofuscin within lysozymes and a compromised ability to process photoreceptor outer segments and cytoplasmic proteins.2528 with the use of fundus autofluorescence imaging , lipofuscin accumulation within rpe cells has actually been identified prior to observable rpe damage.29,30 during the fourth decade of life , unprocessed lipid slowly exits the rpe cells . most molecules reach the systemic circulation via the choriocapillaris but , for unknown reasons , several layers of apolipoprotein slowly accumulate within bruch s membrane . both esterified and nonesterified cholesterol forms small vesicles within membrane fibrils.31,32 a wall of lipid material , high in several apolipoproteins including apolipoprotein - b , e , a - i , c - i , and c - ii , accumulates within bruch s membrane in a pattern consistent with secretion by the rpe rather than by the choriocapillaris . over time , proinflammatory molecules , such as linoleate hydroperoxide and 7-ketocholesterol appear , probably as a result of overlying oxidative stress of the rpe . as the highly demanding metabolic process within the photoreceptors and rpe continues , but oxygen diffusion from the choriocapillaris through the altered bruch s membrane decreases , the resultant oxidative stress produces reactive oxygen species , which leads to protein misfolding.33 additionally , lipofuscin contains vitamin a - derived photophores that inhibit mitochondrial respiration and cause misfolding of proteins.34 the rpe is highly dependent upon proper folding of proteins for optimal function , so improperly folded proteins react with heat shock proteins to facilitate repair.35,36 if this fails , the folded proteins are tagged with ubiquitin and directed to the proteosomes for degradation.37,38 however , because proteosomal function also decreases with age and under conditions of increased oxidative stress , the buildup of improperly folded proteins results in the release of proinflammatory cytokines , which leads to chronic inflammation and the formation of drusen.39 in addition to inducing the formation of reactive oxygen species , lipofuscin decreases lysozomal integrity , induces peroxidation , decreases phagocytosis , and promotes rpe death.40,41 apoptotic cell death is further enhanced by the presence of advanced glycation end products which activate their receptors ( rage)42,43 and upregulate nuclear factorkb . 44 additionally , rage upregulates vegf which leads to the development of cnvm.45 inflammation plays an important role in promoting angiogenesis through the innate immune system . membrane - bound toll - like receptors and cytosolic nod - like receptors are responsible for detecting invading pathogens and directing a compensatory inflammatory response . toll - like receptors recognize breakdown products of the intercellular matrix ( elastin , hyaluronic acid , and fibronectin ) and induce the expression of proinflammatory cytokines ( interleukins 6 and 8) , angiogenic chemokines ( cxcl8 and ccl2 ) , and adhesion molecules ( icam-1 and vcam-1).4650 these cytokines further increase production of reactive oxygen species and oxidative stress on the rpe.51,52 activation of nod - like receptors , through oxidative stress and lysozomal damage , leads to release of interleukin-1b and interleukin-18.53 choroidal dendritic cells become activated by damaged rpe and oxidized protein and lipid in bruch s membrane.54 activated dendritic cells promote secretion of immune response modulators such as apolipoprotein e and vibronectin from the rpe and initiate an autoimmune response with production of antiretinal and anti - rpe antibodies.55,56 additionally , inflammatory cells , including multinucleated giant cells , which are involved in the late stages of amd , secrete enzymes which degrade bruch s membrane , and cytokines which promote the growth of cnvm.57,58 eventually , chronic ischemia of the outer retina and inflammation directed against abnormalities in bruch s membrane result in the development of pathologic new blood vessels or angiogenesis . the growth of neovascular complexes requires sequential activation of several biochemical pathways , with upregulation of multiple growth factors , balanced with downregulation of key angiostatic molecules . angiogenesis plays an important role in several disparate human diseases , including tumor growth and metastases , rheumatoid arthritis , psoriasis , and intraocular neovascularization of both the choroid and retina . basic fibroblastic growth factor has been detected in excised cnvm59,60 and has been found to be sufficient61,62 but not necessary for the development of experimental cnvm.63 in addition to increased concentrations of growth factors , angiogenesis requires decreased levels of angiostatic molecules . 64 pigment epithelium - derived growth factor ( pedf ) leads to regression of neovascularization by promoting apoptosis of vascular endothelial cells.65,66 pedf synthesis is upregulated under hyperemic conditions and downregulated in hypoxia.67 pedf levels have been found to be low within choroidal neovascular tissues,68,69 and expression of pedf is inversely correlated with the formation of cnvm in animal models.70 angiopoietins regulate vascular integrity and also participate in pathologic neovascularization.71 angiopoietin-1 prevents leakage from the adult vasculature72 whereas angiopoietin- 2 , which is upregulated in vascular endothelial cells by both hypoxia and vegf,73 enhances the vasoproliferative effects of vegf . the opposite effects of these two closely related molecules are due to their agonist and antagonist effects on the tie2 receptor , which recruits pericytes and stabilizes new blood vessels . although other growth factors can induce development of blood vessels ( ie , transforming growth factor- , interleukins , insulin - like growth factor-1 , and epidermal growth factor ) , only vegf appears to be both sufficient and essential for both physiologic and pathologic angiogenesis . not surprisingly , the biochemical pathways involving vegf have been the most intensively studied and are the best understood . oxidative stress and inflammation due to accumulation of intracellular and extracellular waste material , including lipid in bruch s membrane and drusen , stimulate vegf synthesis . excessive local production of vegf causes multifocal sprouting of new vascular complexes from the choriocapillaris , with growth occurring beneath the rpe , or through the rpe into the subretinal space.74,75 the resultant serous detachment of the retina or rpe , often with associated hemorrhage , leads to neuroretinal dysfunction and late gliosis , fibrosis , and cell death.76 autopsies show that vegf is expressed in the rpe of eyes with amd , and vegf has been detected in surgically excised cnvm.7780 animal models that overexpress vegf are characterized by the growth of cnvm.8183 vegf levels are elevated in the vitreous of eyes with exudative amd.84,85 vegf is a member of the platelet - derived growth factor family,86 and is actually a collection of related molecules that segregate into distinct families , i.e. vegf - a vegf - b , vegf - c , vegf - d , vegf - e , and placental growth factor . native vegf ( of which vegf appears to be the dominant isomer ) exists as a homodimer with a molecular weight between 35 kda and 45 kda.87 isoforms of vegf - a are responsible for most of the processes essential in angiogenesis , i.e. endothelial cell proliferation and migration , recruitment of endothelial cell progenitors , endothelial cell survival , activation of matrix metalloproteinases , and phosphorylation of tight junction proteins . at least six major isoforms ( vegf121 , vegf145 , vegf165 , vegf183 , vegf189 , vegf206 ) and eight minor isoforms of vegf - a , all created by alternate splicing of mrna from the same vegf gene , have been discovered.88 shorter isoforms are water - soluble and biologically active whereas longer isoforms are bound to the intercellular matrix ( vegf165 is 50%70% matrix bound ) and only become biologically active when cleaved by matrix metalloproteinase-9 and plasmin into soluble vegf110 . vegf - a isoforms contain their receptor binding sequences in amino acids 81 through 92 and their heparin binding sequences within amino acids 110165 . vegf initiates its biological effects by binding to three transmembrane receptors , vegfr1 ( flt-1 ) , vegfr2 ( flk-1 ) , and vegfr3 ( flk-4 ) , which are expressed on vascular endothelial cells and pericytes , as well as on monocytes and macrophages.89,90 normal choriocapillaris expresses all of its receptors on the endothelial cells next to the rpe , suggesting a codependent paracrine relationship between the two tissues . 91 each receptor has extracellular binding domains for vegf , a transmembrane sequence and intracellular tyrosine kinase moieties . vegf binding to the extracellular receptor domain dimerizes the receptors and results in phosphorylation of the intracellular tyrosine kinase moieties . vegfr1 activation by vegf - a , vegf - b , and placental growth factor attracts mononuclear leukocytes and promotes development of myocardial arterioles . the role of vegfr1 in promoting noncoronary angiogenesis is not clear , but it may be a weak angiogenic mediator or may serve as a decoy receptor by binding placental growth factor , thereby displacing vegf - a and allowing it to bind to and activate vegfr2 . vegfr1 appears to assist endothelial cell assembly into vascular tubules . in the retina , vegf upregulation in rpe , mueller cells , pericytes , endothelial cells , glial cells , and ganglion cells92 is caused by inflammation ( interleukins , transforming growth factor alpha and beta ) and several growth factors ( fibroblastic growth factor , epidermal growth factor , keratinocyte growth factor , insulin - like growth factor-1 , and platelet - derived growth factor ) . however , the usual regulator of vegf synthesis in chorioretinal vascular conditions is tissue hypoxia . low tissue oxygen tension enables hypoxia inducible factor 1-alpha to dimerize with hypoxia inducible factor 1-beta , thereby preventing complex formation with the von hippel lindau factor and subsequent ubiquination within proteosomes . the stable hypoxia inducible factor dimer binds to the promoter zone of vegf mrna and upregulates vegf synthesis . reactive oxygen species and advanced glycation end products can stimulate the rpe to upregulate vegf.93,94 hypoxic rpe cells have also been shown to produce placental growth factor which has been detected within cnvm . activation of vegfr2 by vegf - a is necessary for many critical processes of physiologic and pathologic angiogenesis . activated vegfr2 upregulates nitric oxide synthase which promotes vascular endothelial cell proliferation.95 by upregulating matrix metalloproteinases and tissue - type plasminogen activators , and downregulating metalloproteinase inhibitors , vegf degrades the intercellular matrix , thereby enabling endothelial cell migration.76,96 matrix degradation causes the release of previously sequestered vegf , thereby amplifying the proliferative drive.97 vegf breaks down the blood retinal barrier by phosphorylating tight junction proteins , forming transcellular vesicles , and creating fenestrations.98 compared with the hyperpermeability effect of histamine , that of vegf is 50,000 times as potent.61 by activating vegfr1 , vegf - a attracts monocytes and macrophages . when creating a high - affinity vegf - binding protein , scientists at regeneron decided not to use the antibody technology used to create bevacizumab ( kd 58 pm ) or the affinityenhanced ranibizumab ( kd 46 pm).100 instead they spliced native receptor sequences from high - affinity vegfr1 ( kd 1020 pm ) onto the fc ( fragment crystallizable ) portion of a human igg1 molecule . vegf trapr1r1r1 with three binding domains from vegfr1 which tightly bound vegf165 , but exhibited poor serum pharmacokinetics because it was immediately sequestered within the intercellular matrix . after other intermediate molecules were tested , vegf trapr1r2 ( aflibercept ) was created . with the second binding domain from vegfr1 and the third binding domain from vegfr2 , this molecule exhibited excellent in vivo pharmacokinetics , with a strong binding affinity for vegf165 ( kd 0.5 pm ) , exceeding even that of the native receptors.101 whereas bevacizumab can simultaneously bind two vegf dimers , and two ranibizumab molecules can bind one vegf dimer , aflibercept tightly binds vegf - a and placental growth factor dimers in a 1 to 1 ratio with a powerful two - fisted grasp . the time - dependent intraocular concentration of aflibercept , like that of bevacizumab and ranibizumab , decreases according to first - order decay kinetics . the intravitreal half - life of aflibercept in rabbits is 4.7 days ( regeneron investigator manual ) , and although monkey and human data are not available , an adapted mathematical model calculates the half - life in human eyes to be 7.1 days.102 because the intraocular half - lives of macromolecules are primarily determined by their molecular weight , that of aflibercept ( molecular weight 115 kda ) should fall between bevacizumab ( molecular weight 149 kda ; t1/2 8.25 days ) and ranibizumab ( molecular weight 48 kda ; t1/2 4.75 days , estimated).102 based on its high binding affinity and estimated intraocular half - life , mathematical modeling suggests that intravitreally administered aflibercept should have a longer duration of clinical action ( possibly as long as 2.5 months ) than either ranibizumab or bevacizumab.103 aflibercept is believed to diffuse through the eye without being metabolized and enters the systemic circulation through either the choriocapillaris or trabecular meshwork . the half - life of unbound aflibercept is 13 days in the blood , whereas vegf - bound aflibercept has a half - life of 18 days . aflibercept is removed from the body via pinocytotic proteolysis and glomerular filtration after forming complexes with vegf.104 studies with animal models of corneal , iris , retina , and choroidal neovascularization have shown that vegf plays a central role in pathologic ocular neovascularization . in a matrigel model , aflibercept successfully prevented the growth of cnvm when administered 2 and 6 days after induction , and prevented leukocyte infiltration and halted collagen synthesis within cnvm when administered 10 days after induction.105 in mice , aflibercept prevented cnvm following laser photocoagulation to the rpe , prevented cnvm development after administration of exogenous vegf , and prevented development of cnvm in transgenic animals that secreted vegf from photoreceptors.106 aflibercept extends the survival of penetrating keratoplasties in mice107 and prevents induction of corneal neovascularization after treatment with fibroblast growth factor pellets.108 the successes achieved in these preclinical studies paved the way for aflibercept trials in human chorioretinal conditions ( table 1 ) . in the first exudative amd trial , intravenous aflibercept doses between 0.3 mg / kg and 3 mg / kg ( the usual oncologic dose is 4 mg / kg ) were administered every 2 weeks to 25 patients.109 macular thickness improved by an average of 66% and improvements in vision were modest . because two of five patients receiving the highest drug dose suffered dose - limiting hypertension and proteinuria , all subsequent intravenous administration of aflibercept was limited to oncology , because ophthalmology trials transitioned to intravitreal injections . the phase i clear - it 1 ( clinical evaluation of antiangiogenesis in the retina intravitreal trial ) investigation was designed to determine the safety , tolerability , maximum tolerated dose , and bioactivity of intravitreally administered aflibercept in patients with exudative amd.110 single doses of aflibercept between 0.05 mg and 4 mg were tolerated well by 21 patients . at 6 weeks after the injections , patients experienced mean visual acuity gains of + 4.4 letters and macular thickness changes of 104 m . based on the results of clear - it 1 , the developers hoped to show that aflibercept could be dosed less often than monthly . the phase ii clear - it 2 trial randomized 159 patients to receive three injections of 0.5 mg or 2 mg , or single doses of 0.5 mg , 2 mg , or 4 mg , with final evaluations at 12 weeks . at 12 weeks , the average macular thickness improved by 119 m from baseline ( p < 0.0001 ) although the reduction experienced by the patients receiving injections every 4 weeks exceeded that of patients treated only once . at 12 weeks , the average vision in all groups improved by + 5.7 letters ( p < 0.0001 ) , with the greatest improvement ( more than eight letters ) in patients treated monthly . visual improvement at week 8 was similar in patients receiving single doses or three doses.111 during the second phase of the clear - it 2 study , patients were followed from week 16 through 52 and given injections as needed.112 an average of two injections was required , with a mean time to first injection of 129 days . at week 52 , the average improvement in vision compared with baseline ( week 0 ) was + 5.3 letters ( p < 0.0001 ) , but patients initially treated with 2 mg every 4 weeks achieved an average improvement of + 9 letters . the area covered by the cnvm decreased by an average of 2.21 mm at 48 weeks . the clear - it 2 study demonstrated that patients treated as needed required few injections , yet maintained excellent gains in vision . additionally , patients receiving three monthly loading doses achieved superior visual results compared with those receiving single injections . two concurrent phase iii trials , i.e. view ( vascular endothelial growth factor [ vegf ] trap - eye : investigation of efficacy and safety in wet age - related macular degeneration studies ) , enrolled 1217 patients from north america ( view 1 ) and 1240 patients from south america , europe , asia and australia ( view 2 ) . patients were randomized 1:1:1:1 to receive ranibizumab 0.5 mg , aflibercept 0.5 mg , aflibercept 2 mg every 4 weeks , or aflibercept 2 mg every 8 weeks after three monthly loading doses . all of the aflibercept arms in both trials achieved the primary endpoint , i.e. noninferiority compared with ranibizumab for maintenance of vision ( decrease in vision less than 15 letters ) . between 95% and 96% of patients in all study groups maintained vision , compared with 94% of patients in both ranibizumab groups.7 gains in vision were comparable between patients in the aflibercept groups ( + 6.9 letters to + 10.9 letters ) and those receiving ranibizumab ( + 8.1 letters , + 9.4 letters ) but patients receiving aflibercept 2 mg every 4 weeks in view 1 gained more vision than those receiving ranibizumab ( + 10.9 letters versus + 8.1 letters ; p = 0.0054 ) . however , when similarly treated arms from view 1 and view 2 were pooled , there was no appreciable difference in letters gained between patients receiving aflibercept 2 mg every 4 weeks and those treated with ranibizumab . similar proportions of patients in all treatment arms gained > 0 letters and > 15 letters . improvements in macular thickness , which ranged from 218 m to 230 m in view 1 and from 130 m to 157 m in view 2 , were not statistically different among any of the treatment groups . patients receiving aflibercept 2 mg every 8 weeks in view 2 showed bimonthly fluctuations in macular thickness , from 17 m early in the trial to 8 m by week 52 . however , no corresponding fluctuations in visual acuity were noted . both aflibercept and ranibizumab were well tolerated by patients in both trials . there were no significant differences between aflibercept and ranibizumab in the incidences of serious ocular adverse events ( 1.6% and 2.3% versus 3.0% and 3.1% , respectively ) or systemic side effects ( 1.6% and 1.9% versus 1.7% and 1.7% ) . based on the one - year efficacy and safety results of the view trials , the united states food and drug administration approved the 2 mg dose of aflibercept for the treatment of exudative amd . the recommended treatment regimen includes three loading injections at 4-week intervals , followed by injections every 8 weeks . during year 2 of the view trials , patients continued receiving the same drug and dose as in year 1 , were evaluated every 4 weeks , and were given injections as needed , but at intervals not exceeding 12 weeks . between weeks 52 and 96 , patients initially receiving aflibercept 2 mg every 8 weeks and those initially receiving ranibizumab every 4 weeks maintained previous gains in vision ( + 8.4 letters + 7.6 letters versus + 8.7 letters + 7.9 letters).113 fewer patients receiving aflibercept required the most intensive treatments ; 15.9% of patients initially receiving aflibercept and 26.5% of patients initially receiving ranibizumab required at least six injections , and 1.9% and 3.1% required at least 11 injections . patients receiving aflibercept 2 mg required an average of 4.2 injections , whereas patients receiving ranibizumab required an average of 4.7 injections . forty - eight percent of patients receiving aflibercept 2 mg and 40% of patients receiving ranibizumab received the minimum number ( three ) of injections . because aflibercept has been used for only a short period of time since approval , surgeons can not accurately determine its durability , nor can they determine if the treatment interval can be extended beyond 8 weeks . however , the experience of the author and others ( philip rosenfeld , personal communication , march 10 , 2012 ) suggests that aflibercept works remarkably well as administration of aflibercept to eyes with persistent fluid despite prolonged bevacizumab or ranibizumab therapy results in rapid resolution of subretinal fluid and flattening of pigment epithelial detachments ( figure 2 ) . following its approval for exudative amd , aflibercept has entered a treatment landscape dominated by two drugs , ranibizumab and bevacizumab . two pivotal trials , marina ( the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular age- related macular degeneration)4 which evaluated patients with occult cnvm , and anchor ( the anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in age - related macular degeneration)5 which evaluated patients with classic cnvm , showed that monthly injections of ranibizumab resulted in better visual gains compared with both sham injections ( + 7.2 letters versus 10.4 letters ) and photodynamic therapy ( + 11.3 letters versus 9.5 letters ) . these trials established monthly ranibizumab injections as the standard against which all subsequent randomized trials have been compared . however , despite the results of the marina and anchor trials , nearly 60% of physicians prefer bevacizumab over ranibizumab . catt ( the complications of age - related macular degeneration treatment trials ) showed that monthly bevacizumab produced vision gains comparable with monthly ranibizumab ( + 8.0 letters versus + 8.5 letters).6 whereas improvements following prn dosing of ranibizumab were not statistically different from monthly injections ( + 6.8 letters versus + 8.5 letters ) , prn dosing of bevacizumab was termed indeterminate compared with monthly dosing ( + 5.9 letters versus + 8.0 letters ) . although the influence of the catt results on physicians choice of anti - vegf drugs is not fully known , catt investigators do not believe that many physicians have altered their drug preferences ( dan martin , retina 2012 , wailea , hi ) . in addition to demonstrating the efficacy of aflibercept given every 4 or 8 weeks , the view trials provided valuable information regarding peak efficacy and durability of anti- vegf therapy . increasing the monthly dose of aflibercept from 0.5 mg to 2 mg did not appear to improve peak efficacy ( maximum letters gained ) ; similarly , the harbor trial showed that increasing the monthly dose of ranibizumab from 0.5 mg to 2 mg did not lead to further gains in vision ( + 10.1 letters versus + 9.2 letters , data on file , genentech ) . these studies suggest that anti - vegf monotherapy for exudative amd has hit a therapeutic ceiling and that further gains in efficacy will require combination therapy with drugs that target other biological pathways . both trials suggest , but do not prove , that increasing the dose of a drug prolongs its duration of action ( durability ) . the view trials showed that aflibercept 2 mg every 8 weeks produced vision gains comparable with aflibercept 0.5 mg every 4 weeks ( + 7.9 and + 8.9 letters versus + 6.9 and + 9.7 letters ) . additionally , harbor showed that patients receiving prn ranibizumab 0.5 mg required 7.7 injections during the year whereas those receiving prn ranibizumab 2 mg required an average of 6.9 injections . these results are consistent with previously published models which predict that increasing the dose of an effective anti - vegf drug has a greater effect on its durability than its efficacy.114 the price of bevacizumab ( $ 30$75 us per dose ) is much lower than that of ranibizumab ( $ 1950 us per dose ) . physicians preferring bevacizumab because of its lower cost did so while believing that its efficacy was comparable with that of ranibizumab , and the results of catt provided level 1 evidence to support this position . other physicians prefer ranibizumab because of its excellent results in rigorous phase iii trials , on - label indications , and financial incentives.115 the price of aflibercept ( $ 1850 us per dose ) has been set just below that of ranibizumab , so for physicians following the treatment protocol used in the view trials , i.e. three loading doses given at 4-week intervals followed by injections every 8 weeks , patient visits and injections will be half as frequent and will incur half the cost . fewer injections should also decrease the cumulative incidence of ocular complications , including endophthalmitis . however , most physicians employ a treat - and - observe ( prn ) or treat - and - extend regimen , making it more difficult to estimate savings . because year 2 of the view trials used a 3-month capped prn protocol , we do not know the expected duration of action of aflibercept after one year of regular injections . the data suggest that aflibercept would have lasted an average of approximately 3 months , probably 23 weeks longer than ranibizumab . compared with ranibizumab , treat - and - observe with aflibercept would probably save 13 injections per year . however , compared with protocol - driven therapy , patients would probably receive eight fewer injections . now that aflibercept is available for amd , physicians considering a change in drugs will need to consider several factors . for many physicians using ranibizumab because of the strength of its phase iii testing and on - label indication , switching to aflibercept with its expected longer duration of action is a reasonable strategy . physicians will probably begin slowly , a few patients at a time , until they have accumulated sufficient experience . if the results obtained with post - approval use are similar to those seen in the view trials , then this group of physicians will likely drift to aflibercept . financial incentives ( high - volume discounts , difference between wholesale price and medicare reimbursement , and accrual of airline miles with credit card use ) have influenced others to use ranibizumab instead of bevacizumab . unless these incentives are reduced by either the government or the manufacturer , or matched by aflibercept , some physicians will continue to use ranibizumab for these reasons . whereas aflibercept has been approved only for the treatment of amd , phase iii trials for diabetic macular edema and vein occlusions are ongoing . ranibizumab has already received approval for vein occlusions and will likely receive approval for diabetic macular edema soon . therefore , physicians will be using ranibizumab for new indications , and this familiarity will probably keep many using ranibizumab for amd . despite the overall savings generated by aflibercept use ( compared with ranibizumab ) physicians preferring bevacizumab will now have the option of less frequent dosing with aflibercept , but at a higher overall cost . , significantly improve when the frequency of injections is increased from monthly to every 2 weeks.116 early postapproval experience shows that switching from bevacizumab or ranibizumab to monthly injections of aflibercept frequently improves these difficult - to - treat eyes . a slow shift by physicians from ranibizumab will decrease the number of patient visits to physicians offices , decrease the overall cost of therapy , and improve patient satisfaction with amd therapy . in the short term , physicians will notice a decrease in the burden of amd patients in their offices . this reprieve will likely be short - lived because the continuing epidemic of exudative amd patients will bring new patients to the offices for initiation of long - term therapy .

age - related macular degeneration ( amd ) has become a major public health problem and a leading cause of blindness in industrialized nations . amd results from the ageing eye s inability to metabolize and dispose completely of photoreceptor outer segments and other waste products . as a result , lipids , particularly apolipoproteins , accumulate within bruch s membrane , leading to chronic ischemia and inflammation . the subsequent upregulation of inflammatory cytokines and growth factors , including vascular endothelial growth factor ( vegf ) , induces the growth of neovascular membranes from the choriocapillaris into the subretinal or subretinal pigment epithelium spaces . to counter this , intravitreally administered drugs ( pegaptanib , bevacizumab , ranibizumab ) that specifically target vegf have become the standard treatment for exudative amd . aflibercept , a recently approved fusion protein , binds to all isoforms of both vegf - a and placental growth factor with high affinity . phase iii trials showed that monthly or every other month injections of aflibercept prevent vision loss ( fewer than 15 letters ) in 95% of patients . additionally , aflibercept injections every 4 or 8 weeks produce average vision gains of 6.9 letters to 10.9 letters , comparable with those achieved with monthly ranibizumab . after one year of regularly administered aflibercept injections , patients required an average of only 4.2 injections during the second year . aflibercept promises to decrease the injection frequency required for many patients and appears to serve as an effective salvage therapy for patients who respond poorly to other anti - vegf drugs .

Introduction Pathophysiology of AMD Vascular endothelial growth factor Aflibercept biochemistry and pharmacokinetics Preclinical and clinical studies Place in therapy Conclusion

age - related macular degeneration ( amd ) is the leading cause of blindness in patients over the age of 65 years in industrialized nations . most affected patients ( approximately 90% ) have dry or nonexudative amd , characterized by drusen and retinal pigment epithelium ( rpe ) mottling , hyperplasia , and atrophy , but most cases of blindness ( approximately 90% ) are due to wet or exudative amd , which is characterized by the growth of choroidal neovascular membranes ( cnvm).1 wet amd results from repeated cycles of shedding , degradation , and resynthesis of photoreceptor outer segments , which induces metabolic stress within the outer retina and rpe . the resultant chronic ischemia and inflammation upregulates several inflammatory cytokines and growth factors , particularly vascular endothelial growth factor ( vegf ) , which promote the growth of cnvm from the choriocapillaris into the sub - rpe space ( type 1 cnvm ) , the subretinal space ( type 2 cnvm ) , or the inner retina as retinal angiomatous proliferation ( type 3 cnvm ) . between 2000 and 2010 , anti - vegf therapy resulted in a 50% reduction in the incident rate of blindness due to exudative amd in denmark , compared with a 33% reduction in the incidence of blindness from other causes.8 three anti - vegf drugs have been used for over 5 years , i.e. pegaptanib ( macugen ; eyetech inc , palm beach gardens , fl , introduced in 2004 ) , bevacizumab ( avastin ; genentech , south san francisco , ca / roche , basel , switzerland , first used off - label in 2005 ) and ranibizumab ( lucentis ; genentech , south san francisco , ca / roche , basel , switzerland , introduced in 2006 ) , whereas aflibercept ( vegf trap - eye , eylea ; regeneron , tarrytown , ny ) has been available only since being granted regulatory approval for the treatment of exudative amd by the united states food and drug administration on november 18 , 2011 . this paper discusses the pathophysiology of exudative amd with emphasis on the stimulatory role of vegf , the biochemical and pharmacologic characteristics of aflibercept , and the results of important clinical trials focusing on the treatment of patients with exudative amd . in addition to the development of confluent soft drusen , decreased choroidal blood flow , lipid deposition in bruch s membrane , oxidative stress in the rpe , and alteration in bruch s membrane all contribute to ischemia of the overlying rpe , secondary death of cells in the neural retina , and , finally to induction of cnvm.1318 whereas drusen and rpe atrophy generally cause a slow decrease in vision , a rapid decline usually accompanies the development of cnvm.19 the pathophysiology of amd actually begins with lipoprotein transport of carotenoids , vitamin e , and cholesterol to the photoreceptors through the rpe ( figure 1).20,21 nutrients are separated within the rpe and presented to the photoreceptors while , at the same time , the rpe performs near continuous , homeostatic phagocytosis of photoreceptor outer segments,2224 with assembly of apolipoprotein b from phagocytosed photoreceptor outer segments , plasma lipoprotein components , and endogenously synthesized lipids . this results in accumulation of lipofuscin within lysozymes and a compromised ability to process photoreceptor outer segments and cytoplasmic proteins.2528 with the use of fundus autofluorescence imaging , lipofuscin accumulation within rpe cells has actually been identified prior to observable rpe damage.29,30 during the fourth decade of life , unprocessed lipid slowly exits the rpe cells . most molecules reach the systemic circulation via the choriocapillaris but , for unknown reasons , several layers of apolipoprotein slowly accumulate within bruch s membrane . both esterified and nonesterified cholesterol forms small vesicles within membrane fibrils.31,32 a wall of lipid material , high in several apolipoproteins including apolipoprotein - b , e , a - i , c - i , and c - ii , accumulates within bruch s membrane in a pattern consistent with secretion by the rpe rather than by the choriocapillaris . as the highly demanding metabolic process within the photoreceptors and rpe continues , but oxygen diffusion from the choriocapillaris through the altered bruch s membrane decreases , the resultant oxidative stress produces reactive oxygen species , which leads to protein misfolding.33 additionally , lipofuscin contains vitamin a - derived photophores that inhibit mitochondrial respiration and cause misfolding of proteins.34 the rpe is highly dependent upon proper folding of proteins for optimal function , so improperly folded proteins react with heat shock proteins to facilitate repair.35,36 if this fails , the folded proteins are tagged with ubiquitin and directed to the proteosomes for degradation.37,38 however , because proteosomal function also decreases with age and under conditions of increased oxidative stress , the buildup of improperly folded proteins results in the release of proinflammatory cytokines , which leads to chronic inflammation and the formation of drusen.39 in addition to inducing the formation of reactive oxygen species , lipofuscin decreases lysozomal integrity , induces peroxidation , decreases phagocytosis , and promotes rpe death.40,41 apoptotic cell death is further enhanced by the presence of advanced glycation end products which activate their receptors ( rage)42,43 and upregulate nuclear factorkb . toll - like receptors recognize breakdown products of the intercellular matrix ( elastin , hyaluronic acid , and fibronectin ) and induce the expression of proinflammatory cytokines ( interleukins 6 and 8) , angiogenic chemokines ( cxcl8 and ccl2 ) , and adhesion molecules ( icam-1 and vcam-1).4650 these cytokines further increase production of reactive oxygen species and oxidative stress on the rpe.51,52 activation of nod - like receptors , through oxidative stress and lysozomal damage , leads to release of interleukin-1b and interleukin-18.53 choroidal dendritic cells become activated by damaged rpe and oxidized protein and lipid in bruch s membrane.54 activated dendritic cells promote secretion of immune response modulators such as apolipoprotein e and vibronectin from the rpe and initiate an autoimmune response with production of antiretinal and anti - rpe antibodies.55,56 additionally , inflammatory cells , including multinucleated giant cells , which are involved in the late stages of amd , secrete enzymes which degrade bruch s membrane , and cytokines which promote the growth of cnvm.57,58 eventually , chronic ischemia of the outer retina and inflammation directed against abnormalities in bruch s membrane result in the development of pathologic new blood vessels or angiogenesis . the growth of neovascular complexes requires sequential activation of several biochemical pathways , with upregulation of multiple growth factors , balanced with downregulation of key angiostatic molecules . 64 pigment epithelium - derived growth factor ( pedf ) leads to regression of neovascularization by promoting apoptosis of vascular endothelial cells.65,66 pedf synthesis is upregulated under hyperemic conditions and downregulated in hypoxia.67 pedf levels have been found to be low within choroidal neovascular tissues,68,69 and expression of pedf is inversely correlated with the formation of cnvm in animal models.70 angiopoietins regulate vascular integrity and also participate in pathologic neovascularization.71 angiopoietin-1 prevents leakage from the adult vasculature72 whereas angiopoietin- 2 , which is upregulated in vascular endothelial cells by both hypoxia and vegf,73 enhances the vasoproliferative effects of vegf . although other growth factors can induce development of blood vessels ( ie , transforming growth factor- , interleukins , insulin - like growth factor-1 , and epidermal growth factor ) , only vegf appears to be both sufficient and essential for both physiologic and pathologic angiogenesis . oxidative stress and inflammation due to accumulation of intracellular and extracellular waste material , including lipid in bruch s membrane and drusen , stimulate vegf synthesis . excessive local production of vegf causes multifocal sprouting of new vascular complexes from the choriocapillaris , with growth occurring beneath the rpe , or through the rpe into the subretinal space.74,75 the resultant serous detachment of the retina or rpe , often with associated hemorrhage , leads to neuroretinal dysfunction and late gliosis , fibrosis , and cell death.76 autopsies show that vegf is expressed in the rpe of eyes with amd , and vegf has been detected in surgically excised cnvm.7780 animal models that overexpress vegf are characterized by the growth of cnvm.8183 vegf levels are elevated in the vitreous of eyes with exudative amd.84,85 vegf is a member of the platelet - derived growth factor family,86 and is actually a collection of related molecules that segregate into distinct families , i.e. vegf - a vegf - b , vegf - c , vegf - d , vegf - e , and placental growth factor . native vegf ( of which vegf appears to be the dominant isomer ) exists as a homodimer with a molecular weight between 35 kda and 45 kda.87 isoforms of vegf - a are responsible for most of the processes essential in angiogenesis , i.e. at least six major isoforms ( vegf121 , vegf145 , vegf165 , vegf183 , vegf189 , vegf206 ) and eight minor isoforms of vegf - a , all created by alternate splicing of mrna from the same vegf gene , have been discovered.88 shorter isoforms are water - soluble and biologically active whereas longer isoforms are bound to the intercellular matrix ( vegf165 is 50%70% matrix bound ) and only become biologically active when cleaved by matrix metalloproteinase-9 and plasmin into soluble vegf110 . vegfr1 activation by vegf - a , vegf - b , and placental growth factor attracts mononuclear leukocytes and promotes development of myocardial arterioles . the role of vegfr1 in promoting noncoronary angiogenesis is not clear , but it may be a weak angiogenic mediator or may serve as a decoy receptor by binding placental growth factor , thereby displacing vegf - a and allowing it to bind to and activate vegfr2 . with the second binding domain from vegfr1 and the third binding domain from vegfr2 , this molecule exhibited excellent in vivo pharmacokinetics , with a strong binding affinity for vegf165 ( kd 0.5 pm ) , exceeding even that of the native receptors.101 whereas bevacizumab can simultaneously bind two vegf dimers , and two ranibizumab molecules can bind one vegf dimer , aflibercept tightly binds vegf - a and placental growth factor dimers in a 1 to 1 ratio with a powerful two - fisted grasp . the intravitreal half - life of aflibercept in rabbits is 4.7 days ( regeneron investigator manual ) , and although monkey and human data are not available , an adapted mathematical model calculates the half - life in human eyes to be 7.1 days.102 because the intraocular half - lives of macromolecules are primarily determined by their molecular weight , that of aflibercept ( molecular weight 115 kda ) should fall between bevacizumab ( molecular weight 149 kda ; t1/2 8.25 days ) and ranibizumab ( molecular weight 48 kda ; t1/2 4.75 days , estimated).102 based on its high binding affinity and estimated intraocular half - life , mathematical modeling suggests that intravitreally administered aflibercept should have a longer duration of clinical action ( possibly as long as 2.5 months ) than either ranibizumab or bevacizumab.103 aflibercept is believed to diffuse through the eye without being metabolized and enters the systemic circulation through either the choriocapillaris or trabecular meshwork . in a matrigel model , aflibercept successfully prevented the growth of cnvm when administered 2 and 6 days after induction , and prevented leukocyte infiltration and halted collagen synthesis within cnvm when administered 10 days after induction.105 in mice , aflibercept prevented cnvm following laser photocoagulation to the rpe , prevented cnvm development after administration of exogenous vegf , and prevented development of cnvm in transgenic animals that secreted vegf from photoreceptors.106 aflibercept extends the survival of penetrating keratoplasties in mice107 and prevents induction of corneal neovascularization after treatment with fibroblast growth factor pellets.108 the successes achieved in these preclinical studies paved the way for aflibercept trials in human chorioretinal conditions ( table 1 ) . the phase i clear - it 1 ( clinical evaluation of antiangiogenesis in the retina intravitreal trial ) investigation was designed to determine the safety , tolerability , maximum tolerated dose , and bioactivity of intravitreally administered aflibercept in patients with exudative amd.110 single doses of aflibercept between 0.05 mg and 4 mg were tolerated well by 21 patients . at 12 weeks , the average vision in all groups improved by + 5.7 letters ( p < 0.0001 ) , with the greatest improvement ( more than eight letters ) in patients treated monthly . visual improvement at week 8 was similar in patients receiving single doses or three doses.111 during the second phase of the clear - it 2 study , patients were followed from week 16 through 52 and given injections as needed.112 an average of two injections was required , with a mean time to first injection of 129 days . at week 52 , the average improvement in vision compared with baseline ( week 0 ) was + 5.3 letters ( p < 0.0001 ) , but patients initially treated with 2 mg every 4 weeks achieved an average improvement of + 9 letters . view ( vascular endothelial growth factor [ vegf ] trap - eye : investigation of efficacy and safety in wet age - related macular degeneration studies ) , enrolled 1217 patients from north america ( view 1 ) and 1240 patients from south america , europe , asia and australia ( view 2 ) . between 95% and 96% of patients in all study groups maintained vision , compared with 94% of patients in both ranibizumab groups.7 gains in vision were comparable between patients in the aflibercept groups ( + 6.9 letters to + 10.9 letters ) and those receiving ranibizumab ( + 8.1 letters , + 9.4 letters ) but patients receiving aflibercept 2 mg every 4 weeks in view 1 gained more vision than those receiving ranibizumab ( + 10.9 letters versus + 8.1 letters ; p = 0.0054 ) . between weeks 52 and 96 , patients initially receiving aflibercept 2 mg every 8 weeks and those initially receiving ranibizumab every 4 weeks maintained previous gains in vision ( + 8.4 letters + 7.6 letters versus + 8.7 letters + 7.9 letters).113 fewer patients receiving aflibercept required the most intensive treatments ; 15.9% of patients initially receiving aflibercept and 26.5% of patients initially receiving ranibizumab required at least six injections , and 1.9% and 3.1% required at least 11 injections . patients receiving aflibercept 2 mg required an average of 4.2 injections , whereas patients receiving ranibizumab required an average of 4.7 injections . following its approval for exudative amd , aflibercept has entered a treatment landscape dominated by two drugs , ranibizumab and bevacizumab . two pivotal trials , marina ( the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular age- related macular degeneration)4 which evaluated patients with occult cnvm , and anchor ( the anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in age - related macular degeneration)5 which evaluated patients with classic cnvm , showed that monthly injections of ranibizumab resulted in better visual gains compared with both sham injections ( + 7.2 letters versus 10.4 letters ) and photodynamic therapy ( + 11.3 letters versus 9.5 letters ) . catt ( the complications of age - related macular degeneration treatment trials ) showed that monthly bevacizumab produced vision gains comparable with monthly ranibizumab ( + 8.0 letters versus + 8.5 letters).6 whereas improvements following prn dosing of ranibizumab were not statistically different from monthly injections ( + 6.8 letters versus + 8.5 letters ) , prn dosing of bevacizumab was termed indeterminate compared with monthly dosing ( + 5.9 letters versus + 8.0 letters ) . in addition to demonstrating the efficacy of aflibercept given every 4 or 8 weeks , the view trials provided valuable information regarding peak efficacy and durability of anti- vegf therapy . these studies suggest that anti - vegf monotherapy for exudative amd has hit a therapeutic ceiling and that further gains in efficacy will require combination therapy with drugs that target other biological pathways . the view trials showed that aflibercept 2 mg every 8 weeks produced vision gains comparable with aflibercept 0.5 mg every 4 weeks ( + 7.9 and + 8.9 letters versus + 6.9 and + 9.7 letters ) . additionally , harbor showed that patients receiving prn ranibizumab 0.5 mg required 7.7 injections during the year whereas those receiving prn ranibizumab 2 mg required an average of 6.9 injections . other physicians prefer ranibizumab because of its excellent results in rigorous phase iii trials , on - label indications , and financial incentives.115 the price of aflibercept ( $ 1850 us per dose ) has been set just below that of ranibizumab , so for physicians following the treatment protocol used in the view trials , i.e. because year 2 of the view trials used a 3-month capped prn protocol , we do not know the expected duration of action of aflibercept after one year of regular injections .

this paper explores the adaptive comfort model and its relationship to phenotypic ( within a life time ) adaptation . it goes hand in hand with notions of how to determine and facilitate the attainment of desirable environmental conditions , some of which are yet to be defined ; others that need re - evaluation . the architecturally oriented reader is invited to regard the present paper as an extension of downloadable material by auliciems and szokolay ( 2007).1 the psychophysiological framework of this thermal sensation model is based on herbert hensel ( 1959 ) and the auliciems ( 1981 , 1983 ) biometeorological techocultural construct the model provides an alternative of a mobile neutrality to the static solutions of fanger s ( 1970 ) predicted mean vote ( pmv ) . the basis for the model is both theoretical and empirical , not the least being the observation of neutral temperature variability in many field surveys using verbal scales . in these , seasonal differences in comfort sensation had been noted by yaglou and miller ( 1925 ) , partridge and maclean ( 1935 ) , hikish ( 1955 ) and auliciems ( 1969 , 1972 ) and humphreys ( 1975 , 1976 ) . the so - named adaptive model as presented to ashrae by de dear et al . ( 1997 ) was the auliciems ( 1981 , 1983 ) framework and equation . the american society of heating , refrigerating and airconditioning engineers have now finally approved the 55 - 2004 standards of thermal environmental conditions for human occupancy , with new amendments that describe adaptive comfort as a valid model that relates indoor design temperatures or acceptable temperature ranges to outdoor meteorological or climatological parameters . the parameter averaging period , now specified as 1 week or more , is a rational redefinition ( unless used as an argument for excluding the use of shorter term meteorotropic attractors ) . in brief , the original model justified the use of regression of comfort sensations , as hypothesized in auliciems ( 1989 , 1972 ) , namely , peoples neutrality shifts in response to prevailing warmth , or in biometeorological parlance , thermal sensation is meteorotropic . the most commonly observed attractor has been the convenient monthly outdoor mean as in the original equation:1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \mathrm{t}}_{\psi } = 17.6 + 0.31\kern0.5em { \mathrm{t}}_{mr}\kern7em \left(\mathrm{r}=0.88\right)\dots $ $ \end{document}where t is group neutrality and tmr outdoor monthly or running mean temperature as recommended in ansi / ashrae 2010 . the algorithm that allows estimation of the thermal indoor - outdoor gradient ( or ttmr ) that determines the external energy ex needed to achieve a mobile or maintain a static pmv neutrality is thus:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \%{\mathrm{e}}_{\mathrm{x}}=\left({\mathrm{t}}_{\psi } -{\mathrm{t}}_{\mathrm{pmv}}\right)/\left({\mathrm{t}}_{\mathrm{pmv}}-{\mathrm{t}}_{\mathrm{mr}}\right)\times 100 $ $ \end{document } obviously it will be in the most strenuous conditions that adaptation by mobility of neutrality will be of most benefit , but overall most locations ( see some annual estimates in auliciems 1989 ) benefit from a flexible response as related to the acclimatization possibilities by energy savings between 10 % and 30 % .2 richard de dear ( 2011 ) believes that due to the enabling clauses in ashrae standards ( 2004 ) and cen ( 2007 ) , more flexible tenets of the benefits of adaptation will prevail in sound economic management approaches and that the final frontier for adaptive thermal comfort researchers will be the engineering of building occupants attitudes and expectations . while not altogether in agreement with the accuracy of the historical attributions without historical referencing , the author agrees that the new enabling clauses in ashrae standards may offer a unique opportunity to advance sensation research and its application . unfortunately , there has been little effort in development of theoretical context and modeling beyond narrow disciplinary interest . as stated by andamon et al . ( 2006 ) , the lack of analysis of cultural factors goes ignored in adaptive models . even after the graphical multilingual scale developments as in woolard ( 1981 ) , . there has been little critical evaluation of linguistic difference in metaphor , cultural preference or experience . building design , microclimate management , ergonomics , exercise physiology and the yet unresolved fundamental question of a definition of optima , seem set to enter a new set of basic postulates . new interpretations of cell energy process and stress seem to provide missing logic and essential solutions , and represent a massive paradigm shift towards the molecular rather than the middle - sized processes of traditional thermophysiology . during the decades surrounding the 21st century , biological sciences , dealing with molecular scales capitalized on fast developing applications of computer enhanced imagery . using new technologies of silicon and integrated circuitry , fluorescence and electron microscopy in analysis of biological specimens ( see lewis et al . 1999 for some of the methodology in assessment of cell expression ) , breakthroughs in understanding of basic life processes appeared in many papers . at the turn of the century , the singer and nicholson ( 1972 ) cell membrane model had prevailed for three decades , and a vast amount of literature had already appeared on cell structures , shock proteins and energy interaction ( luis et al . 1990 ) . many specialist reviews ( in cell biology , neuroendocrinology , and genetics ) had appeared , but not surprisingly few were specifically interested in speculating about the implications to the whole person . noteworthy amongst these appeared to be moseley ( 1997 ) on shock proteins ( hsp ) and acclimatization , and romanovsky ( 2007 ) on neural science and thermoregulatory adaptation . the fields covered are huge and there seems to be little alternative but selective overview . genetic control of thermal response has been the focus of uncountable articles over the past decade . particularly useful general descriptions and classifications have come from kregel ( 2002 ) and sonna et al . there are now also many specialist monographs available on the thermal response as part of systems biology at the fundamental level ( as illustrated for cardiac arrhythmia by klber and rudy 2004 , and renal ischaemia and reperfusion injury by yazihan and kavas 2010 ) . the genome defense mechanism to thermal threat is based on more than 120 genes now known to have capacities for adaptation to temperature changes . they have been named , identified by their functional class , chromosome location , species / tissue , in all exposure modes ( in vitro , cell culture , whole animal , etc . ) , timing relative to stage of impact , and particular mechanisms involved . continuing mapping of genome controllers appears to provide a strong basis for future scientific integration ( omalley and soyer 2011 ) . contrary to some persisting misconceptions , the genome is not a fixed repository of information ( i.e. an unchangeable intergenerational database ) . it does provide a blueprint for adaptations within a lifetime that involves longer - term biochemical restructuring , but its response can also be proportional to temporary adjustments to rates of heat loss . focus on thermal response cycles can individually last only a fraction of a second in cells , and with real - time response capability , the genome is now increasingly recognized not as a passive keeper of evolutionary thresholds but as a dynamic controller of the organism s most sophisticated and at the same time fundamental responses to specific internal and external stimuli of any duration . this capacity for instantaneous response , especially once coupled to energy and electrical potentials in cell transduction , seems to explain some issues in biological control systems , and indeed questions the very notion of a fixed focus homeostatic mechanism as the centre of the whole spectrum of thermal response . such a dynamic gene control model also seems to be more commensurate with a sensitive proactive human thermoregulatory system that continuously readjusts energy flows , and adapts through neuroendocrinal sequences , instead of an automated exchange gateway with a passive high entropy store as in cannon s ( 1932 ) homeostasis , and mechanistic energy budget calculations that are unable to switch from the continuous algorithm of regulatory physics to the variable modes of hormonal adaptations . increasingly there has been growth of interest in the warning signals themselves that fit into the vast category of neuroendocrinal transmissions that provide the means of communications of information and energy between cells and all genome structures , including individual genes . this would seem to provide a missing link to gene control of the thermal and electrochemical dimensions of human response in general . the sources for cell energy are found in atp ad ( adinose triphosphate - diphosphate ) synthase , active mitochondria ( as discussed in goodsell 1996 ; polla et al . 1996 ; ban et al . 2001 ) and the protein maintained gradients across the critical ( singer and nicholson 1972 ) fluid membrane pore permeability ( isenberg and klaunig 2000 ; klber and rudy 2004 ; phillips et al . 2009 ; kimball 2011 ; berry and turberfield 2011 ) . within the cell energy domain , thermal signals are transmitted from highly receptive and specialized trp ( transient receptor potential ) temperature sensors as a series of electrical potential discharges , through calcium , potassium and other molecular ion channels as pathways to and presumably between individual genes . ( 1997 , 1999 ; caterina 2007 ; clapham 2003 ; voets et al . 2004 ; montell 2005 ; patapoutian 2005 ; dhaka et al . 2006 the trp channels are about an order of magnitude larger than hsp90 , weighing approximately 1000kd . ( 2006 ) and most others point to difficulties of explaining the relationship between temperature thresholds and those of electrical discharges , and the functional differences that seem to exist between hot and cold trp types . each is separately triggered by their specific threshold signals , as for example , the original trp1 is sensitive to a 10 c range about 43 c ( see some critical temperature thresholds in romanovsky 2007 , and voltage discharge derivations by clapham and miller 2011 ) . the largest concentrations of trps are seemingly selectively staged within nerve axons ( montell 2005 ; patapoutian 2005 ; dhaka et al . , the lack of homogeneity does not seem to prevent what would appear to be a rapid hebbean exchange of information ( hebbs 1976 ) , with adjacent thermally sensitive structures ( also see montague et al . thus , if hsp72 is the front line defense against thermal threats , the clapham and miller ( 2011 ) listed extreme temperature specialists ( high ) trpv1trpv4 and ( low ) trpa1trpm8trpc5 , may constitute early analysis and communication centres , or advanced signals outposts for upregulating hsp induction and maintenance of storage upkeep . clapham s ( 2003 ) caption to his review in nature clearly identified a critical role for the trp mechanism to thermal sensation as being both the derivative of electrical as well as thermal processes : trp channels are the vanguard of our sensory systems , responding to temperature , touch , pain , osmolarity , pheromones , taste and other stimuli . they are an ancient sensory apparatus for the cell , not just the multicellular organism , and they have been adapted to respond to all manner of stimuli , from both within and outside the cell . not surprisingly , such statements have created exceptional interest in the role of electrical discharges and especially the critical function of calcium ions as a prime trigger mechanism for thermal sensations ( clapham and miller 2011 ; liu et al . as observed through computer - enhanced microscopy and a variety of laboratory analyses of biological samples , gene expression has included shock proteins in structures and functions underlying cell performance and adaptation . genome expressions of shock proteins in most early reviews , including burdon ( 1986 ) , are a response to noxious temperatures . found in all living cells ( morimoto 1993 ) , heat shock or hsp macromolecules are referred to as being highly conservative , i.e. resistant to evolutionary adaptation . in the present selective overview , however , in as far as possible , items refer directly to humans and phenotypic adaptations , i.e. those within an individual s lifetime . the cell seemingly supports a routine production of maintenance , constitutional or basal hsps ( lindquist and craig 1988 ; maloyan et al . mosser et al . ( 1993 ) describe hsp as the living cell s first and immediate defense , but one that occurs in responses to many stressors , and with patterns and interactions varied according the hsp family , and species demands . feder and hofmann ( 1999 ) had tabulated observed correlations between cellular , cellular / organ , organism functions and protein induction . they noted that the magnitude of hsp expression did not correlate well with temperature as such , but did with the induction thresholds for hsp indicating the amount of already achieved adaptation . hsp are usually divided into six families according to their physical size , ranging from the small < 30 kda to large > 100 kda . heat was the first recognized of these stressors , hence the bewildering generic singular and plural usage of the acronym hsp and/or hsps to cover all , including the families of larger sized hsp70 and hsp90 , as well as cold shock proteins including hsp38 , some of which are also seemingly chaotically labeled by alphanumeric designation ( see sonna et al . unstressed cells appear to also support a resident store of hsp involved in continuous routine repair and essential chaperoning of errant polypeptides away from sensitive fluid membrane pores ( see especially phillips et al . 2009 , and berry and turberfield 2011 ) . under mild conditions , amongst other cell responses to increasing temperature stress , hsp70 ( especially hsp72 ) and hsp90 families are this is in concert with other different rna factors which create new bonding between the cell nucleus and dna ( shamovsky et al . hsps adjustment and adaptation mechanisms appear to vary in diversification of tasks that include folding , assembling and intracellular localization ; secretion , regulation , and selective degradation of other proteins seems to be the general universal response to most if not all stressors . as a result of the emergency measures , taken in the resolution of the traumatic protein fragmentation and new induction processes of transcription , splicing and translation , the hsps achieve an overall stabilization in cell activity and functions . they also carry out repair of structural damage to cell centrosomes and filaments , and the vitally essential maintenance of cell fluidity and seem to achieve a comprehensive set of programs variously described as structural integrity , cytoprotection , and cell fitness . in all levels there is a certain amount of functional overlap between the families , but by far the most active is family hsp70 . broken down by the categories , hsps can be examined to indicate potential symbiosis in function . within the human organism , threats to homeostasis and hsp induction changes can come from many sources : oxygen radicals , heavy metals , ethanol , amino acid and insults ( such as hemorrhage and organ malfunction ) , infection , fever , and inflammation . but the most frequent hsp expression does not require overtaxed organs , excessive thermal stress in clinically recognizable hypothermia or hyperthermia , it results from responses to temperature variability in cellular tissue . in broad reviews by javadpour et al . ( 2007 ) , hsp accumulation is typically reported to lead to reversible heat - induced changes in epithelial permeability and to increased general tolerance to endotoxin exposure , and to inhibition of cytokine production by inflammatory cells . the induction of hsps has been related to reduction of strain and damage to specific cell structures in seemingly most organs including the brain , heart , lungs and kidneys . there has been special interest in the interactions during acclimatization ( e.g. leukocytes yamada et al . ( 2006 ) and horowitz ( 2007 ) emphasize that the changes stimulated by enhanced induction of particular proteins during acclimatization leads to cross adaptation and may strengthen resistance in general . indeed , there is agreement that hsps fulfill essential roles within cells , and may become important as either indicators of health , or agents in cross adaptation . within any of these functions , hsps become critical items to indoor climate management , and this in itself should promote the realization that some understanding of immunity and adaptations and broad health issues is necessary , and also at least appreciation that human responses can not be assessed simply in terms of verbalized comfort or preference . traditionally , acclimatization has been thought of as increased whole person thermophysical response to thermal stress , but largely as an adjustment to ongoing thermostatic regulatory control and alteration of energy flow rates . on the warm side the transition from adjustment to adaptation is particularly notable in the peaking of sweating , its decreasing effectiveness and alteration of its electrolytic properties . under cold , there is a rapid increase in thermogenesis , and in both states there is alteration of core temperatures . there appears to be core temperature inflections during stha short - term heat acclimatization ( and lesser at the cold stca ) . these points probably occur at the maximum stress level , after which the main temperature control by thermophysical exchanges is replaced by neuroendocrinal action that operates with different methods and changed strategies . to what extent and under what circumstances such thresholds are crossed seems to become an important issue for further research . as suggested by various authors using different analogies and semantics , it seems certain that processes at cellular levels are also those at the larger scale . it is likely that the impacts of signals are mirrored and in general , thermal design has to cater simultaneously for both and interpretations of optima may require different approaches and concepts as at present . in the healthy , neutrality can be extended by some 13 degrees depending upon physical fitness , point of measurement , and other critical factors ( benzinger 1979 ; havenith 1997 ) . during this short term ( 35 day ) acclimatization there are a variety of physiologically assessable changes ( see armstrong 1998 ; sawka et al . 2011 ; taylor and cotter 2006 ; moran and pandolf 1999 ) that lead to decreased overall stress and elevated performance . depending upon the episode of noxious thermal signals , the acclimatization symptoms appear to prevail for periods of time variously listed as weeks , months and seasons if not longer , and the process of re - acclimatization is easier after initial exposure ( e.g. nielsen et al . 1993 ; garrett et al . given that differences in ion response times , thresholds and action potentials are measurable in fractions of millivolts and milliseconds , it is unlikely that the relatively huge overlaps and differences of natural magnitudes in cycles will ever enable a precise definition of when specific processes can be said to begin , or how much benefit is transferred , but it seems certain that the cell phases are those akin if not directly algorithmically calculable in units of whole body acclimatization . the same model in terms of attenuation rates and relative stress seems to be applicable at the molecular scale , and transduction appears to be the molecular equivalent of whole person acclimatization . a general similarity in patterns of response of different length acclimatization at both 2006 ) claim that during the shorter - term heat acclimatization , there is a vigorous upregulation of genes responsible for induction of hsp72 . the initial transition from a default thermoregulatory phenotypic condition ( such as seasonal neutrality ) can trigger transient anti - homeostatic integration and decoding of physical and chemical stimuli blueprints for perception and warning of other potentially harmful events ( maloyan and horowitz 2002 ; collier and zimbelman 2007 ) . the earliest linkages of shock proteins as part of acclimatization at the whole person level included those of nielsen et al . ( 1993 ) who noted that during fatigue testing there had been an influx of plasma proteins during first exposure to heat , but this molecular scale process was then interpreted only as a side effect of cardiac pressure changes , while endurance was identified as being directly related to seemingly more meaningful core temperature changes occurring during acclimatization . the recognizable dualism in the continuum of ( a ) thermoregulatory and ( b ) neuro - endocrine cascades appeared following publication of the horowitz et al . a follow up to the main hsp overexpression peak is in repair and maintenance of the cell s ability to cope with subsequent stressor signals . ( 2008 ) confirmed that in humans there was an elevation in basal hsp induction at least over a 10-day period . but constitutional hsp expression also appears to culminate within hours , following which , according to maloyan and horowitz ( 2002 ) and horowitz et al . ( 2004 ) , volatile thyroid hormones t3 and t4 concentrations decline to 3040 % . at that stage the body already seems to have at least started switching to a slower and presumably less demanding metabolic hormone regime . this switch seemingly would have been within the latter part of phase ii in table 1.table 1a neo - gas three phase concept for human thermal adaptability the avocado shape aims to portray the progress of the acclimatization process extended for a time period of minutes to weeks through cell transduction to full acclimatization . from the initial upregulation of hsps within phase i , there is mounting sress and progression from adjustment to adaptation mechanisms cresting at the maximized homeostatic evaporative flux at a ( stha ) in 35 days . the breaking crest of the log - normal aggregated thermal stress is represented by the drift downwards fom the core towards as the ultimate behavior stage in phase iiioperating within some 1416 c range of sensation responses of the normal 7-point ashrae scale , there seem to be are at least three main phases of thermal response that could be described on an adaptive continuum . i. adjustment ii transduction iii adaptation . such phases may be seen to be determined by the severity of the thermal stressor and the types of defensive mechanisms availabletable 1 also shows the original zones devised by hans selye ( e.g. 1936 , 1950 , 1974 ) , that constituted the general adaptation syndrome of gas , here simplified and labelled as arousal , adaptation and recovery being the main functions of his phases . adaptability as being the seemingly non - renewable genotypical adaptation energy availablethe transduction label for phase ii is used here to give emphasis to the purpose for the categorization being temperature- building design continuum , as opposed to thermo - physiological balance models , selye s ( 1936 ) model used to describe clinical stress management being also a forerunner of many biological works , cox ( 1978 ) sports medicine whole person transactional exercise focus and the allostasis load reduction system of sterling and eyer ( 1981 , 1988 ) and mcewen ( 1998a , b ) a neo - gas three phase concept for human thermal adaptability the avocado shape aims to portray the progress of the acclimatization process extended for a time period of minutes to weeks through cell transduction to full acclimatization . from the initial upregulation of hsps within phase i , there is mounting sress and progression from adjustment to adaptation mechanisms cresting at the maximized homeostatic evaporative flux at a ( stha ) in 35 days . the breaking crest of the log - normal aggregated thermal stress is represented by the drift downwards fom the core towards as the ultimate behavior stage in phase iii operating within some 1416 c range of sensation responses of the normal 7-point ashrae scale , there seem to be are at least three main phases of thermal response that could be described on an adaptive continuum . i. adjustment ii transduction iii adaptation . such phases may be seen to be determined by the severity of the thermal stressor and the types of defensive mechanisms available table 1 also shows the original zones devised by hans selye ( e.g. 1936 , 1950 , 1974 ) , that constituted the general adaptation syndrome of gas , here simplified and labelled as arousal , adaptation and recovery being the main functions of his phases . selye ( 1936 ) also used the term adaptation energy , or adaptability as being the seemingly non - renewable genotypical adaptation energy available the transduction label for phase ii is used here to give emphasis to the purpose for the categorization being temperature- building design continuum , as opposed to thermo - physiological balance models , selye s ( 1936 ) model used to describe clinical stress management being also a forerunner of many biological works , cox ( 1978 ) sports medicine whole person transactional exercise focus and the allostasis load reduction system of sterling and eyer ( 1981 , 1988 ) and mcewen ( 1998a , b ) while the time taken for hsp expression above basal levels depends on the family to which they belong , on average , accumulation in the intact body seems to be able to appear within minutes following stress exposure , and retain strong activity for a few days . heat shock proteins seem to appear soon after rises in core temperatures usually assumed to be 37 c , and cold stress asymmetrically some 5 degrees lower ( at approx 32 c ) . ( 2002 ) suggest a half life of 69 h , when at the conclusion of this front line cell defense , the hsf1 protein rebinds to newly synthesized hsp . in simple terms of amounts and rates of hsp induction in response to general environmental temperature cycles , some physiological periodicities have been reviewed for whole marine organisms and fish ( tomanek and somero 2002 ; basu et al . the significance of such observations however is difficult to assess in terms of human design temperature requirements . acclimatization is a broad process that does not necessarily require stress but possibly planned forays into marginal zones by sentient individuals . indeed the virtually instantaneous expression of the hsps , the control coordination by the genome and the remarkable logistical placements of trps and sequences are not chaotic with respect to time or location , which indicates that sentience exists at the fundamental level . 2008 ) regard trp channel signals as being the most sensitive of communications , at the forefront of our sensory stem . to assist visualization of such a multi - scaled and continuous process , fig . 1hypothetical cell energy transduction cycle ( based on moseley 1997 ; clapham 2003 ; romanovsky 2007 ; digel et al . reconstruction of trpv1ion channel atomic structure and a kv1.2 potassium channel as revealed by electron cryomicroscopy are used for notional illustration hypothetical cell energy transduction cycle ( based on moseley 1997 ; clapham 2003 ; romanovsky 2007 ; digel et al . reconstruction of trpv1ion channel atomic structure and a kv1.2 potassium channel as revealed by electron cryomicroscopy are used for notional illustration the cost of crossing the boundaries between adjustment and adaptation is complicated , and will depend upon the degree of exposure to stress , and the tolerance , that is , the physical fitness of the individual . the implications to indoor climate management are no longer a question of defining adjustment , neutrality , sensation or preference , and then designating arbitrarily simple comfort temperatures . it would seem that the new systems biology findings both support and reject the adaptive comfort model . in general the induction of hsp identifies a basic adaptive process at all scales and measurement may become possible of direct translation to both cell stress and integrated sensation . hsp may enable improved classification of atmospheric data in terms of direct indices of strain , and enable better prediction of neutralities and necessary thermal adjustment than semantic scales . there needs to be analysis that involves determination of what is the context , and what is meant by any optimum . obviously comfort statements should not be ones of simple ambient measure for a particular activity , or one amount of necessary adjustment defined by overgeneralized linear equations . perhaps most significantly there may be a need to radically review the designer s responsibility and basic understanding of the relative merits of achieving comfort , while also ensuring that there is adequate opportunity to encounter stimulating temperatures beyond the comfort zone . in the case of enhancing acclimatization , the general solution would be in maximizing an overall drift towards adaptation , i.e. from adjustment phase i categories towards phase iii in table 1 , without violating pre - established individual health requirements . until a more comprehensive design is produced , fig . 2 based on the above overview is offered as an alternative to the now defunct 1981 adaptive framework . the substantial difference is in differentiating between adjustment and adaptation as understood within gas , the former being a transient change in response intensity , the latter being persisting bio - chemical alteration . it is useful to also consider two independent controllers , being genetic for biological short - term cellular and whole body acclimatization , and decision making for the main contextual expectation . further , division can be made between cell , thermoregulatory physiological and psychological and technological levels . thermal stress and the traditional ans and cns are modified by both long- and short - term feedbacks from adjustments and adaptations.fig . 2a framework model of thermal adaptability suggested replacement for adaptive comfort a framework model of thermal adaptability suggested replacement for adaptive comfort gene control at the level of cellular molecular processes operates in expressions of trp and hsp , as part of cytological protection , increased thermotolerance and energy transduction at the beginning of the acclimatization and biological adaptation at the whole person scale . promotion of exercise and fitness through design and microclimate modification provides massive negative feedback from psychological adaptations that provide the necessary external control through the cognitive modules of decision - making and expectation . to provide for inclusion of these complexities , in fig . 2 the normal environmentally driven variable neutrality ( t ) , being the measured naturally occurring seasonal acclimatization neutrality value ( at present the context specific adaptive model prediction using monthly mean temperature for a location ) , is supplemented in topt by ttmax the increment by short - term acclimatization . should there be good reason to reject seasonality as a factor ( i.e. as may be the case of the medium - term itinerant soldier or athlete , or even the otherwise fit but habitually air - conditioned traveler ) , there may be reason to use fanger s tpmv in lieu of t . in looking at a future integration with other priorities , whatever the context , full acclimatization during warm and cold episodes will incur increased and seemingly upgraded neuroendocrinal adaptations , and less thermal adjustments . this adaptation will allow increased thermal tolerance and tempering in one sector , but also produce strain in other dimensions , at a cost to selye s adaptation energy . thus in any recommendation or design some safety margin needs to be identified the critical edge between net benefits of adaptation over its costs . it is suggested that this boundary is represented less as the onset of active sweating , but as maximum time in particular environments that can be enjoyed by acclimatized individuals . while not entirely symmetrical , active thermogenesis in cold should be encouraged , and until the easy availability of molecular thermometry or validation of some hsp expression can be used as an index , adaptation ( acclimatization ) plateaux should be used as basic criteria for recommendations . genome expressed heat shock proteins in particular , seem to overcome what may have been to some an impossible conceptual obstacle of size difference . if combined , trp and hsp expression , plus genome capacity for instantaneous response seems to explain some bewildering issues in biological control systems , and indeed questions the notion of a fixed focus homeostatic mechanism as the centre of the whole spectrum of thermal response . building design , microclimate management , ergonomics , exercise physiology and the yet unresolved fundamental questions of a definition of optima seem to enter a new set of basic postulates , which may require new interpretations of adaptability and time as episodic acclimatization processes . at this stage of development in the new systems biology , and until the full potential of expressions of hsps as indicators of adjustment and/or adaptation , and most suitable biological thermometers are assessed , any conclusions are likely to be interim solutions at best . however , with reference to the adaptive model , for the time being there are good economic and health reasons to continue promotion of variability in neutrality , but avoid emphasis upon vague principles of comfort , comfort zones or specification of precise thresholds . the merits of fitness and encouragement of exposure to stimulating environments should underlie all programs of planned activity and design . the opportunities for improved application and education as suggested by de dear ( 2011 ) are probably valid , but to do so , there is argument for improving scholarship and adhering to scientifically testable models that have appropriate capacities for assessment of human requirements . ( 2002 ) argument is that a fundamental issue for research in genomics and physiology is to resolve the relationship of cellular stress to organismal stress and optimum responses ( including adaptation limits ) at the higher level processes . ( 2004 ) it would also seem that the new paradigm must be a sentient one that recognizes that molecular systems are close to explanation of many of the fundamental mechanisms , including thermoregulation and adaptation . to them personalized medicine. such a move away from statistical generalization towards optimization of an individual s private climate , as based on a paradigm of deeper analysis and wider scholarship , would not be unwelcome to systems biology , architectural or biometeorological integration .

whole person adaptive comfort is discussed with reference to recent findings in molecular scale systems biology . the observations are upscaled to hypotheses relating to less traditional interpretations of thermal processes , which have new implications for indoor climate management and design . arguments are presented for a revision of current focus , model and paradigm . the issue is seen as a problem of integrating theoretical development , conceptual modeling and as an investigation of the extent to which environments and acclimatization can be used to achieve individual fitness and health , not only at the subjective comfort level , as hitherto promoted . it is argued that there are many questions yet to be asked about adaptability before celebrating a particular adaptive state .

Introduction: adaptive comfort model Developments in cognate fields The genome regulator TRP neural signals Shock proteins Acclimatization and transduction An adaptability model Conclusion

this paper explores the adaptive comfort model and its relationship to phenotypic ( within a life time ) adaptation . it goes hand in hand with notions of how to determine and facilitate the attainment of desirable environmental conditions , some of which are yet to be defined ; others that need re - evaluation . the architecturally oriented reader is invited to regard the present paper as an extension of downloadable material by auliciems and szokolay ( 2007).1 the psychophysiological framework of this thermal sensation model is based on herbert hensel ( 1959 ) and the auliciems ( 1981 , 1983 ) biometeorological techocultural construct the model provides an alternative of a mobile neutrality to the static solutions of fanger s ( 1970 ) predicted mean vote ( pmv ) . the basis for the model is both theoretical and empirical , not the least being the observation of neutral temperature variability in many field surveys using verbal scales . the american society of heating , refrigerating and airconditioning engineers have now finally approved the 55 - 2004 standards of thermal environmental conditions for human occupancy , with new amendments that describe adaptive comfort as a valid model that relates indoor design temperatures or acceptable temperature ranges to outdoor meteorological or climatological parameters . the parameter averaging period , now specified as 1 week or more , is a rational redefinition ( unless used as an argument for excluding the use of shorter term meteorotropic attractors ) . in brief , the original model justified the use of regression of comfort sensations , as hypothesized in auliciems ( 1989 , 1972 ) , namely , peoples neutrality shifts in response to prevailing warmth , or in biometeorological parlance , thermal sensation is meteorotropic . the algorithm that allows estimation of the thermal indoor - outdoor gradient ( or ttmr ) that determines the external energy ex needed to achieve a mobile or maintain a static pmv neutrality is thus:2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \%{\mathrm{e}}_{\mathrm{x}}=\left({\mathrm{t}}_{\psi } -{\mathrm{t}}_{\mathrm{pmv}}\right)/\left({\mathrm{t}}_{\mathrm{pmv}}-{\mathrm{t}}_{\mathrm{mr}}\right)\times 100 $ $ \end{document } obviously it will be in the most strenuous conditions that adaptation by mobility of neutrality will be of most benefit , but overall most locations ( see some annual estimates in auliciems 1989 ) benefit from a flexible response as related to the acclimatization possibilities by energy savings between 10 % and 30 % .2 richard de dear ( 2011 ) believes that due to the enabling clauses in ashrae standards ( 2004 ) and cen ( 2007 ) , more flexible tenets of the benefits of adaptation will prevail in sound economic management approaches and that the final frontier for adaptive thermal comfort researchers will be the engineering of building occupants attitudes and expectations . new interpretations of cell energy process and stress seem to provide missing logic and essential solutions , and represent a massive paradigm shift towards the molecular rather than the middle - sized processes of traditional thermophysiology . 1999 for some of the methodology in assessment of cell expression ) , breakthroughs in understanding of basic life processes appeared in many papers . at the turn of the century , the singer and nicholson ( 1972 ) cell membrane model had prevailed for three decades , and a vast amount of literature had already appeared on cell structures , shock proteins and energy interaction ( luis et al . many specialist reviews ( in cell biology , neuroendocrinology , and genetics ) had appeared , but not surprisingly few were specifically interested in speculating about the implications to the whole person . noteworthy amongst these appeared to be moseley ( 1997 ) on shock proteins ( hsp ) and acclimatization , and romanovsky ( 2007 ) on neural science and thermoregulatory adaptation . the fields covered are huge and there seems to be little alternative but selective overview . genetic control of thermal response has been the focus of uncountable articles over the past decade . there are now also many specialist monographs available on the thermal response as part of systems biology at the fundamental level ( as illustrated for cardiac arrhythmia by klber and rudy 2004 , and renal ischaemia and reperfusion injury by yazihan and kavas 2010 ) . focus on thermal response cycles can individually last only a fraction of a second in cells , and with real - time response capability , the genome is now increasingly recognized not as a passive keeper of evolutionary thresholds but as a dynamic controller of the organism s most sophisticated and at the same time fundamental responses to specific internal and external stimuli of any duration . this capacity for instantaneous response , especially once coupled to energy and electrical potentials in cell transduction , seems to explain some issues in biological control systems , and indeed questions the very notion of a fixed focus homeostatic mechanism as the centre of the whole spectrum of thermal response . such a dynamic gene control model also seems to be more commensurate with a sensitive proactive human thermoregulatory system that continuously readjusts energy flows , and adapts through neuroendocrinal sequences , instead of an automated exchange gateway with a passive high entropy store as in cannon s ( 1932 ) homeostasis , and mechanistic energy budget calculations that are unable to switch from the continuous algorithm of regulatory physics to the variable modes of hormonal adaptations . this would seem to provide a missing link to gene control of the thermal and electrochemical dimensions of human response in general . within the cell energy domain , thermal signals are transmitted from highly receptive and specialized trp ( transient receptor potential ) temperature sensors as a series of electrical potential discharges , through calcium , potassium and other molecular ion channels as pathways to and presumably between individual genes . each is separately triggered by their specific threshold signals , as for example , the original trp1 is sensitive to a 10 c range about 43 c ( see some critical temperature thresholds in romanovsky 2007 , and voltage discharge derivations by clapham and miller 2011 ) . , the lack of homogeneity does not seem to prevent what would appear to be a rapid hebbean exchange of information ( hebbs 1976 ) , with adjacent thermally sensitive structures ( also see montague et al . clapham s ( 2003 ) caption to his review in nature clearly identified a critical role for the trp mechanism to thermal sensation as being both the derivative of electrical as well as thermal processes : trp channels are the vanguard of our sensory systems , responding to temperature , touch , pain , osmolarity , pheromones , taste and other stimuli . they are an ancient sensory apparatus for the cell , not just the multicellular organism , and they have been adapted to respond to all manner of stimuli , from both within and outside the cell . heat was the first recognized of these stressors , hence the bewildering generic singular and plural usage of the acronym hsp and/or hsps to cover all , including the families of larger sized hsp70 and hsp90 , as well as cold shock proteins including hsp38 , some of which are also seemingly chaotically labeled by alphanumeric designation ( see sonna et al . as a result of the emergency measures , taken in the resolution of the traumatic protein fragmentation and new induction processes of transcription , splicing and translation , the hsps achieve an overall stabilization in cell activity and functions . they also carry out repair of structural damage to cell centrosomes and filaments , and the vitally essential maintenance of cell fluidity and seem to achieve a comprehensive set of programs variously described as structural integrity , cytoprotection , and cell fitness . broken down by the categories , hsps can be examined to indicate potential symbiosis in function . indeed , there is agreement that hsps fulfill essential roles within cells , and may become important as either indicators of health , or agents in cross adaptation . within any of these functions , hsps become critical items to indoor climate management , and this in itself should promote the realization that some understanding of immunity and adaptations and broad health issues is necessary , and also at least appreciation that human responses can not be assessed simply in terms of verbalized comfort or preference . traditionally , acclimatization has been thought of as increased whole person thermophysical response to thermal stress , but largely as an adjustment to ongoing thermostatic regulatory control and alteration of energy flow rates . there appears to be core temperature inflections during stha short - term heat acclimatization ( and lesser at the cold stca ) . these points probably occur at the maximum stress level , after which the main temperature control by thermophysical exchanges is replaced by neuroendocrinal action that operates with different methods and changed strategies . as suggested by various authors using different analogies and semantics , it seems certain that processes at cellular levels are also those at the larger scale . it is likely that the impacts of signals are mirrored and in general , thermal design has to cater simultaneously for both and interpretations of optima may require different approaches and concepts as at present . in the healthy , neutrality can be extended by some 13 degrees depending upon physical fitness , point of measurement , and other critical factors ( benzinger 1979 ; havenith 1997 ) . during this short term ( 35 day ) acclimatization there are a variety of physiologically assessable changes ( see armstrong 1998 ; sawka et al . given that differences in ion response times , thresholds and action potentials are measurable in fractions of millivolts and milliseconds , it is unlikely that the relatively huge overlaps and differences of natural magnitudes in cycles will ever enable a precise definition of when specific processes can be said to begin , or how much benefit is transferred , but it seems certain that the cell phases are those akin if not directly algorithmically calculable in units of whole body acclimatization . the same model in terms of attenuation rates and relative stress seems to be applicable at the molecular scale , and transduction appears to be the molecular equivalent of whole person acclimatization . the earliest linkages of shock proteins as part of acclimatization at the whole person level included those of nielsen et al . ( 1993 ) who noted that during fatigue testing there had been an influx of plasma proteins during first exposure to heat , but this molecular scale process was then interpreted only as a side effect of cardiac pressure changes , while endurance was identified as being directly related to seemingly more meaningful core temperature changes occurring during acclimatization . the recognizable dualism in the continuum of ( a ) thermoregulatory and ( b ) neuro - endocrine cascades appeared following publication of the horowitz et al . a follow up to the main hsp overexpression peak is in repair and maintenance of the cell s ability to cope with subsequent stressor signals . this switch seemingly would have been within the latter part of phase ii in table 1.table 1a neo - gas three phase concept for human thermal adaptability the avocado shape aims to portray the progress of the acclimatization process extended for a time period of minutes to weeks through cell transduction to full acclimatization . the breaking crest of the log - normal aggregated thermal stress is represented by the drift downwards fom the core towards as the ultimate behavior stage in phase iiioperating within some 1416 c range of sensation responses of the normal 7-point ashrae scale , there seem to be are at least three main phases of thermal response that could be described on an adaptive continuum . such phases may be seen to be determined by the severity of the thermal stressor and the types of defensive mechanisms availabletable 1 also shows the original zones devised by hans selye ( e.g. adaptability as being the seemingly non - renewable genotypical adaptation energy availablethe transduction label for phase ii is used here to give emphasis to the purpose for the categorization being temperature- building design continuum , as opposed to thermo - physiological balance models , selye s ( 1936 ) model used to describe clinical stress management being also a forerunner of many biological works , cox ( 1978 ) sports medicine whole person transactional exercise focus and the allostasis load reduction system of sterling and eyer ( 1981 , 1988 ) and mcewen ( 1998a , b ) a neo - gas three phase concept for human thermal adaptability the avocado shape aims to portray the progress of the acclimatization process extended for a time period of minutes to weeks through cell transduction to full acclimatization . from the initial upregulation of hsps within phase i , there is mounting sress and progression from adjustment to adaptation mechanisms cresting at the maximized homeostatic evaporative flux at a ( stha ) in 35 days . the breaking crest of the log - normal aggregated thermal stress is represented by the drift downwards fom the core towards as the ultimate behavior stage in phase iii operating within some 1416 c range of sensation responses of the normal 7-point ashrae scale , there seem to be are at least three main phases of thermal response that could be described on an adaptive continuum . such phases may be seen to be determined by the severity of the thermal stressor and the types of defensive mechanisms available table 1 also shows the original zones devised by hans selye ( e.g. selye ( 1936 ) also used the term adaptation energy , or adaptability as being the seemingly non - renewable genotypical adaptation energy available the transduction label for phase ii is used here to give emphasis to the purpose for the categorization being temperature- building design continuum , as opposed to thermo - physiological balance models , selye s ( 1936 ) model used to describe clinical stress management being also a forerunner of many biological works , cox ( 1978 ) sports medicine whole person transactional exercise focus and the allostasis load reduction system of sterling and eyer ( 1981 , 1988 ) and mcewen ( 1998a , b ) while the time taken for hsp expression above basal levels depends on the family to which they belong , on average , accumulation in the intact body seems to be able to appear within minutes following stress exposure , and retain strong activity for a few days . indeed the virtually instantaneous expression of the hsps , the control coordination by the genome and the remarkable logistical placements of trps and sequences are not chaotic with respect to time or location , which indicates that sentience exists at the fundamental level . reconstruction of trpv1ion channel atomic structure and a kv1.2 potassium channel as revealed by electron cryomicroscopy are used for notional illustration the cost of crossing the boundaries between adjustment and adaptation is complicated , and will depend upon the degree of exposure to stress , and the tolerance , that is , the physical fitness of the individual . the implications to indoor climate management are no longer a question of defining adjustment , neutrality , sensation or preference , and then designating arbitrarily simple comfort temperatures . it would seem that the new systems biology findings both support and reject the adaptive comfort model . there needs to be analysis that involves determination of what is the context , and what is meant by any optimum . obviously comfort statements should not be ones of simple ambient measure for a particular activity , or one amount of necessary adjustment defined by overgeneralized linear equations . perhaps most significantly there may be a need to radically review the designer s responsibility and basic understanding of the relative merits of achieving comfort , while also ensuring that there is adequate opportunity to encounter stimulating temperatures beyond the comfort zone . 2 based on the above overview is offered as an alternative to the now defunct 1981 adaptive framework . it is useful to also consider two independent controllers , being genetic for biological short - term cellular and whole body acclimatization , and decision making for the main contextual expectation . further , division can be made between cell , thermoregulatory physiological and psychological and technological levels . 2a framework model of thermal adaptability suggested replacement for adaptive comfort a framework model of thermal adaptability suggested replacement for adaptive comfort gene control at the level of cellular molecular processes operates in expressions of trp and hsp , as part of cytological protection , increased thermotolerance and energy transduction at the beginning of the acclimatization and biological adaptation at the whole person scale . 2 the normal environmentally driven variable neutrality ( t ) , being the measured naturally occurring seasonal acclimatization neutrality value ( at present the context specific adaptive model prediction using monthly mean temperature for a location ) , is supplemented in topt by ttmax the increment by short - term acclimatization . should there be good reason to reject seasonality as a factor ( i.e. as may be the case of the medium - term itinerant soldier or athlete , or even the otherwise fit but habitually air - conditioned traveler ) , there may be reason to use fanger s tpmv in lieu of t . it is suggested that this boundary is represented less as the onset of active sweating , but as maximum time in particular environments that can be enjoyed by acclimatized individuals . while not entirely symmetrical , active thermogenesis in cold should be encouraged , and until the easy availability of molecular thermometry or validation of some hsp expression can be used as an index , adaptation ( acclimatization ) plateaux should be used as basic criteria for recommendations . if combined , trp and hsp expression , plus genome capacity for instantaneous response seems to explain some bewildering issues in biological control systems , and indeed questions the notion of a fixed focus homeostatic mechanism as the centre of the whole spectrum of thermal response . building design , microclimate management , ergonomics , exercise physiology and the yet unresolved fundamental questions of a definition of optima seem to enter a new set of basic postulates , which may require new interpretations of adaptability and time as episodic acclimatization processes . at this stage of development in the new systems biology , and until the full potential of expressions of hsps as indicators of adjustment and/or adaptation , and most suitable biological thermometers are assessed , any conclusions are likely to be interim solutions at best . however , with reference to the adaptive model , for the time being there are good economic and health reasons to continue promotion of variability in neutrality , but avoid emphasis upon vague principles of comfort , comfort zones or specification of precise thresholds . the merits of fitness and encouragement of exposure to stimulating environments should underlie all programs of planned activity and design . ( 2002 ) argument is that a fundamental issue for research in genomics and physiology is to resolve the relationship of cellular stress to organismal stress and optimum responses ( including adaptation limits ) at the higher level processes . ( 2004 ) it would also seem that the new paradigm must be a sentient one that recognizes that molecular systems are close to explanation of many of the fundamental mechanisms , including thermoregulation and adaptation . such a move away from statistical generalization towards optimization of an individual s private climate , as based on a paradigm of deeper analysis and wider scholarship , would not be unwelcome to systems biology , architectural or biometeorological integration .

a woman 's preconception and pregnancy experience with the two most prevalent diseases of the mouth periodontal disease and dental caries not only influences her own oral health status but also may increase her risk of other diseases such as atherosclerosis [ 14 ] , rheumatoid arthritis , and diabetes , impact pregnancy outcome [ 79 ] , and her offspring 's risk of developing early and severe dental caries [ 1013 ] . although largely preventable through evidence - based interventions , both periodontal disease and caries in women of childbearing age are highly prevalent , particularly among low - income women and members of racial and ethnic minority groups . in addition , both periodontal disease and caries are typically asymptomatic for long periods of time with only intermittent painful exacerbations . the combination of high prevalence , insufficient treatment rates , missed preventive opportunities , and intermittent symptoms led the us surgeon general to publish a report in 2001 on oral health in america characterizing dental and oral disease as a silent epidemic . socioeconomic factors , lack of resources to pay for care , barriers to access to care , and lack of public understanding of the importance of oral health and effective self - care practices all represent underlying reasons cited for observed inadequacies in oral health . periodontal disease is a destructive inflammatory condition of the gingiva and bone that supports teeth . it is most commonly associated with a gram - negative anaerobic infection of these structures . fluid that bathes the tooth at the gingival margin , known as gingival crevicular fluid , often contains inflammatory mediators and oral pathogens associated with periodontal disease . the mechanisms underlying this destructive process involve both direct tissue damage resulting from plaque bacterial products , and indirect damage through bacterial induction of the host inflammatory and immune responses . destructive periodontal disease affects up to 15% of the population of childbearing age , with a relatively high proportion of pregnant women demonstrating some degree of periodontal disease [ 7 , 8 , 16 ] . advancing age , smoking , and diabetes are risk factors for the development of periodontal disease . whereas periodontal disease is a chronic , local oral infection , systemic inflammation may also occur . the second oral disease important to women of childbearing age because of its maternal - child health associations is dental caries . dental caries is the pathologic process by which teeth decay and develop cavities . it occurs when acid is produced at the tooth surface by cariogenic bacteria in the dental plaque that metabolize dietary carbohydrates . acquisition of these cariogenic bacteria , dietary practices that govern the caries process , use of fluorides that dampen the caries process , and utilization of dental care all link mothers and children 's experience with tooth decay through biological , behavioral , and social pathways . preterm birth , delivery at less than 37 weeks gestation , occurs in approximately 12% of all births [ 18 , 19 ] . prematurity is the leading cause of neonatal morbidity and mortality in non - anomalous infants . there are numerous and heterogeneous factors associated with preterm birth , such as low maternal body mass index , maternal smoking , and maternal infections . in 1996 , offenbacher and colleagues first reported a potential association between maternal periodontal disease and delivery of a preterm / low birthweight infant . in a case - control study of 124 pregnant women , they observed that women who delivered at less than 37 weeks gestation or an infant the adjusted odds ratio for delivery of a preterm , low birth weight infant was 7 ; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant . extrapolation from these data suggested that 18% of the preterm , low birth weight infants born annually might be attributable to periodontal disease , and thus account for a significant proportion of the $ 5.5 billion annual hospital costs associated with the care of preterm / low birthweight infants . in a subsequent case - control study , dasanayake et al . women in both of these case - control studies were examined at the end of pregnancy or after delivery , which does not convincingly prove an antecedent exposure and thus causality . despite this limitation , these early studies led to the hypothesis that periodontopathic bacteria , primarily gram - negative anaerobes , may serve as a source for endotoxin and lipopolysaccharides , which then increases local inflammatory mediators including pge2 , and cytokines , and that this increases systemic inflammatory mediators that can then lead to preterm birth . examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women , and found that moderate / severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth , independent of other traditional risk factors . despite these compelling data , it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth . in a case control study conducted in london , examined 236 infants born at < 37 weeks gestation or < 2500 g and compared them to a random sample of 507 control infants born at 38 weeks gestation and weighing 2500 g. the authors found no evidence for an association between delivery of a preterm , low birth weight infant and periodontal disease and somewhat surprisingly , found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm , low birth weight infant . the authors surmised that these discrepant findings might be due at least in part to racial differences in study populations . in a follow - up longitudinal study of 3738 women , moore et al however , there was an increase in second trimester fetal loss rates among women with periodontal disease . in an effort to better understand the possible mechanism behind the association between periodontal disease and preterm delivery , offenbacher and colleagues measured gingival crevicular levels of pge2 and il-1 in 48 mothers who delivered preterm , low birth weight infants compared to control women and discovered that gingival crevicular fluid levels of pge2 were significantly higher in case compared to control women . furthermore , among the primiparous women delivering preterm , low birth weight infants , a significant inverse association was demonstrated between birthweight and gestational age and gingival crevicular pge2 levels . it is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor . as further evidence to support the concept that maternal oral health is important for normal pregnancy outcome , other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk . three published studies of antepartum versus delayed ( postpartum ) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention . the effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young , minority , pregnant and postpartum women . of 164 women for whom birth outcome data were available , 74 were subjected to oral prophylaxis during pregnancy , and 90 received no periodontal treatment . the preterm / low birthweight rate was lower among women who received periodontal treatment compared to those who did not ( 13.5% vs. 18.9% ) . conducted a randomized clinical trial to assess the impact of periodontal treatment initiated during pregnancy versus delayed until postpartum on preterm low birthweight infant rates . the incidence of preterm / low birthweight infants in the antepartum treatment group was 1.8% ( 3/163 ) and in the delayed / postpartum group was 10.1% ( 19/188 ) , ( odds ratio [ or ] 5.5 , 95% confidence interval [ ci ] 1.718.2 , p=0.001 ) . multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm / low birthweight infant ( or 4.7 , 95% ci 1.317.1 ) . the data from these two studies suggest that treatment of periodontal disease during pregnancy could reduce preterm / low birthweight infant rates [ 26 , 27 ] . in a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth , jeffcoat et al . found that among women at high risk for preterm birth and presence of periodontal disease , scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth preeclampsia is a hypertensive disorder of pregnancy responsible for significant , maternal and fetal morbidity and mortality . some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia . in a retrospective analysis of data collected as part of the oral conditions and pregnancy study , boggess et al . reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery ( adjusted odds ratio 2.4 , 95% confidence interval 1.1 , 5.3 ) , or if they had periodontal disease progression during pregnancy ( adjusted odds ratio 2.1 , 95% confidence interval 1.0 , 4.4 ) . in a case - control study , canakci et al . found that pre - eclamptic patients were 3.5 ( 95% ci=1.111.9 ) times more likely to have periodontal disease than normotensive patients [ p < 0.01 ) . in a study of 30 pregnant women , significantly higher periodontal probing depth and clinical attachment level scores gingival crevicular fluid levels of pge2 , tnf- , and il-1 levels were all significantly higher in the preeclamptic group . further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative . if the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature , then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia . preterm birth , delivery at less than 37 weeks gestation , occurs in approximately 12% of all births [ 18 , 19 ] . prematurity is the leading cause of neonatal morbidity and mortality in non - anomalous infants . there are numerous and heterogeneous factors associated with preterm birth , such as low maternal body mass index , maternal smoking , and maternal infections . in 1996 , offenbacher and colleagues first reported a potential association between maternal periodontal disease and delivery of a preterm / low birthweight infant . in a case - control study of 124 pregnant women , they observed that women who delivered at less than 37 weeks gestation or an infant the adjusted odds ratio for delivery of a preterm , low birth weight infant was 7 ; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant . extrapolation from these data suggested that 18% of the preterm , low birth weight infants born annually might be attributable to periodontal disease , and thus account for a significant proportion of the $ 5.5 billion annual hospital costs associated with the care of preterm / low birthweight infants . in a subsequent case - control study , dasanayake et al . women in both of these case - control studies were examined at the end of pregnancy or after delivery , which does not convincingly prove an antecedent exposure and thus causality . despite this limitation , these early studies led to the hypothesis that periodontopathic bacteria , primarily gram - negative anaerobes , may serve as a source for endotoxin and lipopolysaccharides , which then increases local inflammatory mediators including pge2 , and cytokines , and that this increases systemic inflammatory mediators that can then lead to preterm birth . examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women , and found that moderate / severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth , independent of other traditional risk factors . despite these compelling data , it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth . in a case control study conducted in london , examined 236 infants born at < 37 weeks gestation or < 2500 g and compared them to a random sample of 507 control infants born at 38 weeks gestation and weighing 2500 g. the authors found no evidence for an association between delivery of a preterm , low birth weight infant and periodontal disease and somewhat surprisingly , found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm , low birth weight infant . the authors surmised that these discrepant findings might be due at least in part to racial differences in study populations . in a follow - up longitudinal study of 3738 women , moore et al however , there was an increase in second trimester fetal loss rates among women with periodontal disease . in an effort to better understand the possible mechanism behind the association between periodontal disease and preterm delivery , offenbacher and colleagues measured gingival crevicular levels of pge2 and il-1 in 48 mothers who delivered preterm , low birth weight infants compared to control women and discovered that gingival crevicular fluid levels of pge2 were significantly higher in case compared to control women . furthermore , among the primiparous women delivering preterm , low birth weight infants , a significant inverse association was demonstrated between birthweight and gestational age and gingival crevicular pge2 levels . it is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor . as further evidence to support the concept that maternal oral health is important for normal pregnancy outcome , other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk . three published studies of antepartum versus delayed ( postpartum ) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention . the effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young , minority , pregnant and postpartum women . of 164 women for whom birth outcome data were available , 74 were subjected to oral prophylaxis during pregnancy , and 90 received no periodontal treatment . the preterm / low birthweight rate was lower among women who received periodontal treatment compared to those who did not ( 13.5% vs. 18.9% ) . conducted a randomized clinical trial to assess the impact of periodontal treatment initiated during pregnancy versus delayed until postpartum on preterm low birthweight infant rates . the incidence of preterm / low birthweight infants in the antepartum treatment group was 1.8% ( 3/163 ) and in the delayed / postpartum group was 10.1% ( 19/188 ) , ( odds ratio [ or ] 5.5 , 95% confidence interval [ ci ] 1.718.2 , p=0.001 ) . multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm / low birthweight infant ( or 4.7 , 95% ci 1.317.1 ) . the data from these two studies suggest that treatment of periodontal disease during pregnancy could reduce preterm / low birthweight infant rates [ 26 , 27 ] . in a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth , jeffcoat et al . found that among women at high risk for preterm birth and presence of periodontal disease , scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth preeclampsia is a hypertensive disorder of pregnancy responsible for significant , maternal and fetal morbidity and mortality . some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia . in a retrospective analysis of data collected as part of the oral conditions and pregnancy study , boggess et al . reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery ( adjusted odds ratio 2.4 , 95% confidence interval 1.1 , 5.3 ) , or if they had periodontal disease progression during pregnancy ( adjusted odds ratio 2.1 , 95% confidence interval 1.0 , 4.4 ) . in a case - control study , found that pre - eclamptic patients were 3.5 ( 95% ci=1.111.9 ) times more likely to have periodontal disease than normotensive patients [ p < 0.01 ) . in a study of 30 pregnant women , significantly higher periodontal probing depth and clinical attachment level scores were found among preeclamptic women compared with non - preeclamptic women . were all significantly higher in the preeclamptic group . further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative . if the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature , then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia . cariogenic bacteria are typically acquired by young children through direct salivary transmission from their mothers . factors influencing transmission are the levels of these bacteria in maternal salivary reservoirs , frequency and efficiency of transmission , and the child 's receptivity to implantation , which is largely diet dependent . additional factors include timing of transmission , which is affected by the window of infectivity and the age of the child , and the composition and flow of the child 's saliva . the earlier the transmission and the more caries - supportive the diet , the earlier and more substantial the transfer will be . for this reason , mothers who have themselves experienced extensive tooth decay and therefore most likely harbor high titers of mutans streptococci in their saliva will more effectively transmit this infection vertically , thereby putting their young children at elevated risk for early childhood caries . although maternal cariogenic bacteria can be isolated in the pre - dentate infant 's mouth , these organisms become established in the dental plaque on the tooth surface only after teeth first appear at around six months of age . because oral flora tends to remain stable over time , a woman 's cariogenic flora before and during pregnancy anticipates her flora during the child 's first years of life as well as the likelihood of transmitting infection early to her offspring . the lag time between infection and expression of a discernable cavity in a tooth depends upon additional factors , including the frequency of simple carbohydrate exposure in a child 's diet , oral hygiene , and exposure to fluorides . the evidence that caries is frequently established as a pathologic process in the mouths of very young children is strong , as 28% of us children , over 4 million toddlers and preschoolers , experience one or more frank cavities by ages 25 years . given the biological and behavioral pathways that govern intergenerational transmission of caries activity , disease management , and use of dental care , it is not surprising that disparities in dental caries among adults are mimicked among their children . as with adults , children of color and children of low - income families experience substantially more extensive and severe disease and less treatment than their peers without these risk factors . fortunately , despite the high prevalence of caries in women and children , this disease is readily preventable or manageable though early and regular dental care , exposure to fluoridated water , use of appropriate topical fluorides including those in toothpastes , application of sealants to primary teeth , and adoption of a health - promoting diet like that suggested in the dietary guidelines for americans . it is intriguing to consider preconception , pregnancy , or intrapartum treatment of oral health conditions as a mechanism to improve women 's oral and general health , pregnancy outcomes , and their children 's dental health . evidence is currently weakest for interventions that seek to reduce the incidence of preterm low birth weight through oral care . the mechanism of periodontal disease - associated adverse pregnancy outcomes is as yet unclear , and althoughit is hypothesized that if the insult occurs early ( either at conception or implantation ) the risk is greater , no direct evidence to confirms that this is the case . however , given the strong relationship between oral health conditions and periodontal disease and general health and well - being , oral health care should be a goal in its own right for all individuals . if treatment of periodontal disease is going to impact pregnancy outcomes , then it is likely that the therapy will be of greatest benefit before or in very early pregnancy . the science supporting interventions before , during , and after pregnancy to reduce caries transmission is much stronger . educational and behavioral interventions that reduce caries activity through appropriate use of fluorides , dietary guidelines , chlorhexidine gels and varnishes , and xylitol , can reduce a woman 's caries activity and salivary cariogenic flora , thereby improving her own oral health and , at the same time , also reducing the risk of transmission to her offspring . in two landmark swedish studies [ 12 , 13 ] , children of mothers who had their cariogenic oral flora suppressed were less likely to experience cavities , more likely to develop cavities later if they were affected , and had fewer cavities than children of control mothers . pregnancy is itself often regarded as an opportune time for anticipatory guidance and oral health education , and is a suitable time , particularly during the second trimester , for dental repair . the cdc 's pregnancy risk assessment monitoring system ( prams ) reported that only 2343% of pregnant women received dental care during their pregnancies [ 36 , 37]a rate only half to two - thirds of us women 's overall use of dental services ( 67% ) ( 14 ) . the prams data revealed that overall , pregnant women covered by medicaid were 24%53% less likely to obtain a dental visit during pregnancy than women who are privately insured . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . in contrast , the behavioral risk factor surveillance system ( brfss ) revealed that 70% of pregnant women in the years 1999 and 2002 had received a dental visit in the prior 12 months . one possible explanation for the higher level reported by brfss is that it includes three or more months of pre - pregnancy time , during which dental care utilization would be expected to reflect the national norm for women . however , in contrast to recognized disparities in dental care utilization , race and ethnicity were not significantly associated with dental care during pregnancy in the brfss study . the authors suggest that , the prevalence of dental visits probably reflects factors such as prevailing attitudes toward dental care , provider availability and practice norms , and salient features of medical and dental care delivery within the state . overall , women covered by medicaid were 24%53% less likely to obtain a dental visit during pregnancy than women who are privately insured . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . an important additional consideration is that dentists are reportedly reluctant to provide care to pregnant women because of concern about possible risks . current practice typically limits non - urgent dental treatment of pregnant women to the second trimester , as there is concern about possible teratogenic consequences during the first trimester and about the woman 's comfort in the dental chair during the third trimester . a single study relating antepartum dental radiography with full term low birth weight raised concern about the safety of dental care during pregnancy , but was criticized for its methodology . neither professional associations nor government agencies have promulgated any authoritative guidance regarding dental care of pregnant women , although multi - center nih clinical trials are underway that will determine the impact of dental care for periodontal disease during pregnancy on preterm low birth weight outcomes . guidelines for prenatal care , oral health , and child health professionals that promotes routine use of dental care during pregnancy . independent of pregnancy , the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care . medicaid is particularly significant to dental care of pregnant women as this program covers approximately 1/3rd of births in the us . however , states vary widely in adult medicaid dental coverage , and at present only 7 jurisdictions providing comprehensive care to eligible adults . in contrast , low income pregnant women seeking dental services find themselves with no coverage in 8 states , coverage for only relief of pain or infection in 18 states or eligible for a limited range of services in 18 states . three states ( ut , la , ca ) have recently expanded dental benefits specifically to pregnant women in anticipation of reduced rates of unfavorable pregnancy outcomes . pregnancy may be the only time that some low - income woman can readily obtain dental care as some state medicaid programs provide adult dental coverage only to pregnant women or enhanced coverage during pregnancy . the cdc 's pregnancy risk assessment monitoring system ( prams ) reported that only 2343% of pregnant women received dental care during their pregnancies [ 36 , 37]a rate only half to two - thirds of us women 's overall use of dental services ( 67% ) ( 14 ) . the prams data revealed that overall , pregnant women covered by medicaid were 24%53% less likely to obtain a dental visit during pregnancy than women who are privately insured . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . in contrast , the behavioral risk factor surveillance system ( brfss ) revealed that 70% of pregnant women in the years 1999 and 2002 had received a dental visit in the prior 12 months . one possible explanation for the higher level reported by brfss is that it includes three or more months of pre - pregnancy time , during which dental care utilization would be expected to reflect the national norm for women . however , in contrast to recognized disparities in dental care utilization , race and ethnicity were not significantly associated with dental care during pregnancy in the brfss study . the authors suggest that , the prevalence of dental visits probably reflects factors such as prevailing attitudes toward dental care , provider availability and practice norms , and salient features of medical and dental care delivery within the state . overall , women covered by medicaid were 24%53% less likely to obtain a dental visit during pregnancy than women who are privately insured . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . an important additional consideration is that dentists are reportedly reluctant to provide care to pregnant women because of concern about possible risks . current practice typically limits non - urgent dental treatment of pregnant women to the second trimester , as there is concern about possible teratogenic consequences during the first trimester and about the woman 's comfort in the dental chair during the third trimester . a single study relating antepartum dental radiography with full term low birth weight raised concern about the safety of dental care during pregnancy , but was criticized for its methodology . neither professional associations nor government agencies have promulgated any authoritative guidance regarding dental care of pregnant women , although multi - center nih clinical trials are underway that will determine the impact of dental care for periodontal disease during pregnancy on preterm low birth weight outcomes . guidelines for prenatal care , oral health , and child health professionals that promotes routine use of dental care during pregnancy . independent of pregnancy , the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care . medicaid is particularly significant to dental care of pregnant women as this program covers approximately 1/3rd of births in the us . however , states vary widely in adult medicaid dental coverage , and at present only 7 jurisdictions providing comprehensive care to eligible adults . in contrast , low income pregnant women seeking dental services find themselves with no coverage in 8 states , coverage for only relief of pain or infection in 18 states or eligible for a limited range of services in 18 states . three states ( ut , la , ca ) have recently expanded dental benefits specifically to pregnant women in anticipation of reduced rates of unfavorable pregnancy outcomes . pregnancy may be the only time that some low - income woman can readily obtain dental care as some state medicaid programs provide adult dental coverage only to pregnant women or enhanced coverage during pregnancy . data are emerging to support a role for maternal periodontal disease as an infectious risk factor for preterm birth and other adverse outcomes of pregnancy . the prevalence of periodontal disease and the possibility of preterm birth prevention by treatment of oral infection make this a novel approach to improve the health and well being of our mothers and their soon - to - be born children . further studies to better understand the mechanism of periodontal disease - associated preterm birth will enable us to tailor treatment to those women who might benefit the most . data on the relationship between maternal and child experience with dental caries is well established . therefore , regardless of the potential for improved oral health to improve pregnancy outcomes , public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded , so not only their own oral and general health is safeguarded but also so that their children 's risk of caries is reduced . particularly if nih trials confirm that treating pregnant women for periodontal disease reduces the incidence of unfavorable birth outcomes , the centers for medicare and medicaid services should build on its september 2004 coverage expansions for pregnant women by stimulating the states to similarly expand oral health services for pregnant women . the power of prevention needs to be brought to bear , as both periodontal disease and caries are overwhelmingly preventable through well recognized strategies including regular and effective home care for periodontal disease and use of fluorides and sealants for caries . to the degree teachable moment in self - care and future child - care , prenatal education should universally adopt an oral health component . this educational intervention should prioritize those mothers who have suffered significantly from dental caries so that they can learn to effectively prevent transfer of this disease to their children . to be effective , oral health promotion must first seek to educate women and their health care providers about the importance of oral health and must promote an understanding of their ability to prevent and manage both periodontal disease and caries and to thereby limit the personal and intergenerational consequences of both conditions .

the mouth is an obvious portal of entry to the body , and oral health reflects and influences general health and well being . maternal oral health has significant implications for birth outcomes and infant oral health . maternal periodontal disease , that is , a chronic infection of the gingiva and supporting tooth structures , has been associated with preterm birth , development of preeclampsia , and delivery of a small - for - gestational age infant . maternal oral flora is transmitted to the newborn infant , and increased cariogenic flora in the mother predisposes the infant to the development of caries . it is intriguing to consider preconception , pregnancy , or intrapartum treatment of oral health conditions as a mechanism to improve women 's oral and general health , pregnancy outcomes , and their children 's dental health . however , given the relationship between oral health and general health , oral health care should be a goal in its own right for all individuals . regardless of the potential for improved oral health to improve pregnancy outcomes , public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded so that their own oral and general health is safeguarded and their children 's risk of caries is reduced . oral health promotion should include education of women and their health care providers ways to prevent oral disease from occurring , and referral for dental services when disease is present .

Introduction Is maternal oral health linked to pregnancy outcome? Preterm birth Other adverse pregnancy outcomes Is maternal oral health linked to children's experience with tooth decay? Is preconception preventive oral health care the answer? Access to oral health care during pregnancy Conclusions and future directions

a woman 's preconception and pregnancy experience with the two most prevalent diseases of the mouth periodontal disease and dental caries not only influences her own oral health status but also may increase her risk of other diseases such as atherosclerosis [ 14 ] , rheumatoid arthritis , and diabetes , impact pregnancy outcome [ 79 ] , and her offspring 's risk of developing early and severe dental caries [ 1013 ] . although largely preventable through evidence - based interventions , both periodontal disease and caries in women of childbearing age are highly prevalent , particularly among low - income women and members of racial and ethnic minority groups . the combination of high prevalence , insufficient treatment rates , missed preventive opportunities , and intermittent symptoms led the us surgeon general to publish a report in 2001 on oral health in america characterizing dental and oral disease as a silent epidemic . socioeconomic factors , lack of resources to pay for care , barriers to access to care , and lack of public understanding of the importance of oral health and effective self - care practices all represent underlying reasons cited for observed inadequacies in oral health . periodontal disease is a destructive inflammatory condition of the gingiva and bone that supports teeth . it is most commonly associated with a gram - negative anaerobic infection of these structures . fluid that bathes the tooth at the gingival margin , known as gingival crevicular fluid , often contains inflammatory mediators and oral pathogens associated with periodontal disease . the mechanisms underlying this destructive process involve both direct tissue damage resulting from plaque bacterial products , and indirect damage through bacterial induction of the host inflammatory and immune responses . destructive periodontal disease affects up to 15% of the population of childbearing age , with a relatively high proportion of pregnant women demonstrating some degree of periodontal disease [ 7 , 8 , 16 ] . advancing age , smoking , and diabetes are risk factors for the development of periodontal disease . whereas periodontal disease is a chronic , local oral infection , systemic inflammation may also occur . the second oral disease important to women of childbearing age because of its maternal - child health associations is dental caries . there are numerous and heterogeneous factors associated with preterm birth , such as low maternal body mass index , maternal smoking , and maternal infections . in 1996 , offenbacher and colleagues first reported a potential association between maternal periodontal disease and delivery of a preterm / low birthweight infant . in a case - control study of 124 pregnant women , they observed that women who delivered at less than 37 weeks gestation or an infant the adjusted odds ratio for delivery of a preterm , low birth weight infant was 7 ; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant . extrapolation from these data suggested that 18% of the preterm , low birth weight infants born annually might be attributable to periodontal disease , and thus account for a significant proportion of the $ 5.5 billion annual hospital costs associated with the care of preterm / low birthweight infants . despite this limitation , these early studies led to the hypothesis that periodontopathic bacteria , primarily gram - negative anaerobes , may serve as a source for endotoxin and lipopolysaccharides , which then increases local inflammatory mediators including pge2 , and cytokines , and that this increases systemic inflammatory mediators that can then lead to preterm birth . examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women , and found that moderate / severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth , independent of other traditional risk factors . despite these compelling data , it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth . in a case control study conducted in london , examined 236 infants born at < 37 weeks gestation or < 2500 g and compared them to a random sample of 507 control infants born at 38 weeks gestation and weighing 2500 g. the authors found no evidence for an association between delivery of a preterm , low birth weight infant and periodontal disease and somewhat surprisingly , found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm , low birth weight infant . in a follow - up longitudinal study of 3738 women , moore et al however , there was an increase in second trimester fetal loss rates among women with periodontal disease . it is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor . as further evidence to support the concept that maternal oral health is important for normal pregnancy outcome , other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk . three published studies of antepartum versus delayed ( postpartum ) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention . the effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young , minority , pregnant and postpartum women . of 164 women for whom birth outcome data were available , 74 were subjected to oral prophylaxis during pregnancy , and 90 received no periodontal treatment . multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm / low birthweight infant ( or 4.7 , 95% ci 1.317.1 ) . the data from these two studies suggest that treatment of periodontal disease during pregnancy could reduce preterm / low birthweight infant rates [ 26 , 27 ] . in a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth , jeffcoat et al . found that among women at high risk for preterm birth and presence of periodontal disease , scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth preeclampsia is a hypertensive disorder of pregnancy responsible for significant , maternal and fetal morbidity and mortality . some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia . reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery ( adjusted odds ratio 2.4 , 95% confidence interval 1.1 , 5.3 ) , or if they had periodontal disease progression during pregnancy ( adjusted odds ratio 2.1 , 95% confidence interval 1.0 , 4.4 ) . in a study of 30 pregnant women , significantly higher periodontal probing depth and clinical attachment level scores gingival crevicular fluid levels of pge2 , tnf- , and il-1 levels were all significantly higher in the preeclamptic group . further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative . if the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature , then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia . preterm birth , delivery at less than 37 weeks gestation , occurs in approximately 12% of all births [ 18 , 19 ] . there are numerous and heterogeneous factors associated with preterm birth , such as low maternal body mass index , maternal smoking , and maternal infections . in 1996 , offenbacher and colleagues first reported a potential association between maternal periodontal disease and delivery of a preterm / low birthweight infant . in a case - control study of 124 pregnant women , they observed that women who delivered at less than 37 weeks gestation or an infant the adjusted odds ratio for delivery of a preterm , low birth weight infant was 7 ; these data led the authors to conclude that periodontal disease may represent a previously unrecognized and clinically significant risk factor for delivery of a preterm low birth weight infant . extrapolation from these data suggested that 18% of the preterm , low birth weight infants born annually might be attributable to periodontal disease , and thus account for a significant proportion of the $ 5.5 billion annual hospital costs associated with the care of preterm / low birthweight infants . despite this limitation , these early studies led to the hypothesis that periodontopathic bacteria , primarily gram - negative anaerobes , may serve as a source for endotoxin and lipopolysaccharides , which then increases local inflammatory mediators including pge2 , and cytokines , and that this increases systemic inflammatory mediators that can then lead to preterm birth . examined the relationship between maternal periodontal disease and spontaneous preterm birth among 1313 pregnant women , and found that moderate / severe maternal periodontal disease identified early in pregnancy was associated with an increased risk for spontaneous preterm birth , independent of other traditional risk factors . despite these compelling data , it is important to recognize that other studies have failed to demonstrate any association between maternal periodontal disease and preterm birth . in a case control study conducted in london , examined 236 infants born at < 37 weeks gestation or < 2500 g and compared them to a random sample of 507 control infants born at 38 weeks gestation and weighing 2500 g. the authors found no evidence for an association between delivery of a preterm , low birth weight infant and periodontal disease and somewhat surprisingly , found that deeper mean tooth pocket depths at delivery was associated with a reduction in the risk of delivery of a preterm , low birth weight infant . in an effort to better understand the possible mechanism behind the association between periodontal disease and preterm delivery , offenbacher and colleagues measured gingival crevicular levels of pge2 and il-1 in 48 mothers who delivered preterm , low birth weight infants compared to control women and discovered that gingival crevicular fluid levels of pge2 were significantly higher in case compared to control women . furthermore , among the primiparous women delivering preterm , low birth weight infants , a significant inverse association was demonstrated between birthweight and gestational age and gingival crevicular pge2 levels . it is not yet clear whether the relationship between periodontal disease and adverse pregnancy outcomes is causal or is a surrogate for another maternal factor . as further evidence to support the concept that maternal oral health is important for normal pregnancy outcome , other investigators have examined the effect of antepartum treatment of periodontal disease on preterm birth risk . three published studies of antepartum versus delayed ( postpartum ) treatment of maternal periodontal disease demonstrate promise for this intervention for preterm birth prevention . the effect of periodontal interventions on pregnancy outcome was assessed in a prospective study designed to examine the relationship between periodontal disease and preterm low birthweight infants in a cohort of young , minority , pregnant and postpartum women . of 164 women for whom birth outcome data were available , 74 were subjected to oral prophylaxis during pregnancy , and 90 received no periodontal treatment . multivariable logistic regression analysis showed that periodontal disease was the strongest factor related to delivery of a preterm / low birthweight infant ( or 4.7 , 95% ci 1.317.1 ) . in a pilot intervention trial designed to assess the feasibility of conducting a trial to determine whether treatment of periodontal disease reduces the risk of spontaneous preterm birth , jeffcoat et al . found that among women at high risk for preterm birth and presence of periodontal disease , scaling and root planning therapy initiated during pregnancy is tolerated by pregnant women and may reduce spontaneous preterm birth preeclampsia is a hypertensive disorder of pregnancy responsible for significant , maternal and fetal morbidity and mortality . some investigators have hypothesized a potential role for maternal periodontal disease as a risk factor for preeclampsia . reported that women were at higher risk for preeclampsia if they had severe periodontal disease at delivery ( adjusted odds ratio 2.4 , 95% confidence interval 1.1 , 5.3 ) , or if they had periodontal disease progression during pregnancy ( adjusted odds ratio 2.1 , 95% confidence interval 1.0 , 4.4 ) . further study on the maternal and fetal inflammatory responses to chronic oral infection and on placental pathology in women with periodontal disease is needed to determine whether the relationship between periodontal disease and preeclampsia is causal or simply associative . if the relationship between maternal periodontal disease and preeclampsia risk proves causal in nature , then prevention of periodontal disease before pregnancy or treatment of periodontal disease during pregnancy may represent a novel approachs to the prevention of preeclampsia . additional factors include timing of transmission , which is affected by the window of infectivity and the age of the child , and the composition and flow of the child 's saliva . because oral flora tends to remain stable over time , a woman 's cariogenic flora before and during pregnancy anticipates her flora during the child 's first years of life as well as the likelihood of transmitting infection early to her offspring . the lag time between infection and expression of a discernable cavity in a tooth depends upon additional factors , including the frequency of simple carbohydrate exposure in a child 's diet , oral hygiene , and exposure to fluorides . the evidence that caries is frequently established as a pathologic process in the mouths of very young children is strong , as 28% of us children , over 4 million toddlers and preschoolers , experience one or more frank cavities by ages 25 years . given the biological and behavioral pathways that govern intergenerational transmission of caries activity , disease management , and use of dental care , it is not surprising that disparities in dental caries among adults are mimicked among their children . fortunately , despite the high prevalence of caries in women and children , this disease is readily preventable or manageable though early and regular dental care , exposure to fluoridated water , use of appropriate topical fluorides including those in toothpastes , application of sealants to primary teeth , and adoption of a health - promoting diet like that suggested in the dietary guidelines for americans . it is intriguing to consider preconception , pregnancy , or intrapartum treatment of oral health conditions as a mechanism to improve women 's oral and general health , pregnancy outcomes , and their children 's dental health . the mechanism of periodontal disease - associated adverse pregnancy outcomes is as yet unclear , and althoughit is hypothesized that if the insult occurs early ( either at conception or implantation ) the risk is greater , no direct evidence to confirms that this is the case . however , given the strong relationship between oral health conditions and periodontal disease and general health and well - being , oral health care should be a goal in its own right for all individuals . if treatment of periodontal disease is going to impact pregnancy outcomes , then it is likely that the therapy will be of greatest benefit before or in very early pregnancy . educational and behavioral interventions that reduce caries activity through appropriate use of fluorides , dietary guidelines , chlorhexidine gels and varnishes , and xylitol , can reduce a woman 's caries activity and salivary cariogenic flora , thereby improving her own oral health and , at the same time , also reducing the risk of transmission to her offspring . in two landmark swedish studies [ 12 , 13 ] , children of mothers who had their cariogenic oral flora suppressed were less likely to experience cavities , more likely to develop cavities later if they were affected , and had fewer cavities than children of control mothers . pregnancy is itself often regarded as an opportune time for anticipatory guidance and oral health education , and is a suitable time , particularly during the second trimester , for dental repair . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . however , in contrast to recognized disparities in dental care utilization , race and ethnicity were not significantly associated with dental care during pregnancy in the brfss study . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . current practice typically limits non - urgent dental treatment of pregnant women to the second trimester , as there is concern about possible teratogenic consequences during the first trimester and about the woman 's comfort in the dental chair during the third trimester . guidelines for prenatal care , oral health , and child health professionals that promotes routine use of dental care during pregnancy . independent of pregnancy , the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care . similarly , women who initiated prenatal care later than the first trimester , who did not intend the pregnancy , and who were poor were also less likely to obtain care . however , in contrast to recognized disparities in dental care utilization , race and ethnicity were not significantly associated with dental care during pregnancy in the brfss study . current practice typically limits non - urgent dental treatment of pregnant women to the second trimester , as there is concern about possible teratogenic consequences during the first trimester and about the woman 's comfort in the dental chair during the third trimester . guidelines for prenatal care , oral health , and child health professionals that promotes routine use of dental care during pregnancy . independent of pregnancy , the presence and source of dental insurance coverage is an important predictor of dental care utilization with publicly insured adults experiencing higher levels of oral diseases but less access to dental care . however , states vary widely in adult medicaid dental coverage , and at present only 7 jurisdictions providing comprehensive care to eligible adults . data are emerging to support a role for maternal periodontal disease as an infectious risk factor for preterm birth and other adverse outcomes of pregnancy . the prevalence of periodontal disease and the possibility of preterm birth prevention by treatment of oral infection make this a novel approach to improve the health and well being of our mothers and their soon - to - be born children . further studies to better understand the mechanism of periodontal disease - associated preterm birth will enable us to tailor treatment to those women who might benefit the most . data on the relationship between maternal and child experience with dental caries is well established . therefore , regardless of the potential for improved oral health to improve pregnancy outcomes , public policies that support comprehensive dental services for vulnerable women of childbearing age should be expanded , so not only their own oral and general health is safeguarded but also so that their children 's risk of caries is reduced . particularly if nih trials confirm that treating pregnant women for periodontal disease reduces the incidence of unfavorable birth outcomes , the centers for medicare and medicaid services should build on its september 2004 coverage expansions for pregnant women by stimulating the states to similarly expand oral health services for pregnant women . to the degree teachable moment in self - care and future child - care , prenatal education should universally adopt an oral health component . to be effective , oral health promotion must first seek to educate women and their health care providers about the importance of oral health and must promote an understanding of their ability to prevent and manage both periodontal disease and caries and to thereby limit the personal and intergenerational consequences of both conditions .

a hypertrophic scar is one of the fibrotic diseases , arising from fibroproliferation disorder which occurs after the damage of deep dermis by burns or trauma . patients with hypertrophic scar often suffer from difficulties in daily life and psycho - social dysfunction . the main pathological characteristics of the hypertrophic scar are an overproduction of extracellular matrix ( ecm ) and collagens . due to the severe functional disabilities caused by a hypertrophic scar , burn rehabilitation is very important for patients after burns , especially occupational therapy which bridges the transfer from hospitals into the community . it facilitates the scar maturation , improves the appearance of a hypertrophic scar , reduces the erythema , as well as alleviates pain and pruritus . however , the outcome of hypertrophic scar treatment was poor , and the mechanisms of how pressure therapy influences the scar maturation are not fully reviewed . lai et al . demonstrated that a static pressure of more than 20 mmhg could accelerate the hypertrophic scar remodeling . further work of this research team also illustrated the histopathological response of pressure therapy in patients with burn scar . suppression of dermal myofibroblasts by induction of apoptosis would result in reduced collagen overproduction . to investigate the mechanisms of pressure therapy on hypertrophic scar maturation , it is important for both surgeons and occupational therapists to identify the key molecule which promotes or inhibits the hypertrophic scar formation . our previous in vitro study demonstrated that the eukaryotic initiation factor 6 ( eif6 ) expression was significantly decreased in fibroblasts derived from hypertrophic scar when compared with that from normal skin ( unpublished data ) . eif6 ( also known as integrin 4 binding protein ) , is a ribosome anti - association factor that regulates translational initiation and ribosome synthesis due to its capacity to modulate ribosome 60s availability and 80s subunit , and plays a very important role in ribosome formation . additionally , eif6 was involved in connection with cytoskeleton , cell apoptosis , and cell cycle progression . moreover , eif6 has been identified as an interacting protein of the hypertrophic scar - related protein p311 , which indicates that it may be involved in the regulation of hypertrophic scar formation as well as myofibroblast differentiation . however , the spatial and temporal expression of eif6 in the process of human hypertrophic scar formation remains unknown . the current study was designed to study the eif6 protein expression pattern during the process of hypertrophic scar formation . because of the possible variation among the individuals , the cases enrolled in our study were only those who received hypertrophic scar excision followed by autoskin grafting , and there was remaining donor skin tissue after grafting , that is , the hypertrophic scar tissue and normal skin tissue were the homobody . immediately after the surgical procedure , the samples of hypertrophic scar tissues and pair - matched normal skin tissues were both kept either in liquid nitrogen or in 4% paraformaldehyde . therefore , total 18 cases ( whose hypertrophic scar tissues and remaining donor skin tissues were kept in the institute of burn research , southwest hospital , chongqing , china ) were enrolled in this study from october 2013 to august 2014 . the study was conducted according to the helsinki declaration and approved by the ethics committee of southwest hospital . samples used in this study were anonymous , and it is impossible for anyone to link the samples to the sources . therefore , the ethics committee of southwest hospital waivered the informed consent . due to the individual variation , we used vancouver scar scale ( vss ) to evaluate for height and color , and visual analog scale ( vas ) for pain and itch of the hypertrophic scar ( data not shown ) . all the patients were divided into three groups according to both clinical manifestation ( decided by vss and vas ) and scar age [ table 1 ] . the demographic information of the hs patients data are presented as the mean sd . pp : proliferative phase ; mp : mature phase ; rp : regressive phase ; sd : standard deviation ; hs : hypertrophic scar . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . we defined this period of the hypertrophic scar as proliferative phase.group ii : the scar age was about 12 years . we defined this period of the hypertrophic scar as mature phase.group iii : the scar age was more than 2 years . scars became flat and soft , and the color was closer to the normal skin . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . scars became flat and soft , and the color was closer to the normal skin . tissue samples were fixed in 4% paraformaldehyde in 0.1 mol / l phosphate buffered saline at ph 7.4 and embedded in paraffin . serial 4 m sections of the paraffin - embedded tissues were then collected . for immunohistochemical staining , the sections were incubated with proteinase k ( millipore , usa ) for 20 min ( 37c ) , and endogenous peroxidase was quenched with 3% h2 o2 in methanol for 20 min at room temperature . the sections were blocked with normal goat serum ( mouse sp kit , zsgb - bio , china ) for 30 min and then incubated in mouse polyclonal antibody against eif6 at a final dilution of 1:400 at 4c overnight . lastly , sections were incubated with a peroxidase - coupled secondary antibody plus streptavidin peroxidase complex and followed by diaminobenzidine staining . , total proteins were extracted in lysis buffer ( 50 mmol / l tris - hcl ph 7.5 ; 2% sodium deoxycholate stock ) from the frozen tissue samples , and the extracted proteins were quantified by the bicinchoninic acid method ( pierce , usa ) . twenty micrograms of proteins prepared in laemmli buffer , were loaded on 12% acrylamide reducing denaturing gel , transferred to polyvinylidene fluoride membranes ( millipore ) , and blotted with the mouse polyclonal antibody eif6 ( at a 1:2000 dilution with 3% bovine serum albumin ) . the revelation was performed with the ecl method ( pierce ) . additionally , we used glyceraldehyde-3-phosphate dehydrogenase for the normalization of data . pictures of each section were taken with a leica confocal microscope ( leica microsystems , wetzlar , germany ) . for the immunohistochemical staining analysis , the expression of eif6 was observed , and digital images were randomly obtained from five fields under 400 magnification each section . the expression intensity of the positive - labeled cells was measured and analyzed using image - pro plus 6.0 software ( media cybernetics , usa ) . data are expressed as the average optical density ( aod ) , aod = integral optical density ( iod)/area of the total field ( iod = optical intensity of positive cells area of positive cells ) . we performed statistical analyses using one - way analysis of variance followed by turkey multiple comparison . because of the possible variation among the individuals , the cases enrolled in our study were only those who received hypertrophic scar excision followed by autoskin grafting , and there was remaining donor skin tissue after grafting , that is , the hypertrophic scar tissue and normal skin tissue were the homobody . immediately after the surgical procedure , the samples of hypertrophic scar tissues and pair - matched normal skin tissues were both kept either in liquid nitrogen or in 4% paraformaldehyde . therefore , total 18 cases ( whose hypertrophic scar tissues and remaining donor skin tissues were kept in the institute of burn research , southwest hospital , chongqing , china ) were enrolled in this study from october 2013 to august 2014 . the study was conducted according to the helsinki declaration and approved by the ethics committee of southwest hospital . samples used in this study were anonymous , and it is impossible for anyone to link the samples to the sources due to the individual variation , there are no clear staging criteria for hypertrophic scar for the present . we used vancouver scar scale ( vss ) to evaluate for height and color , and visual analog scale ( vas ) for pain and itch of the hypertrophic scar ( data not shown ) . all the patients were divided into three groups according to both clinical manifestation ( decided by vss and vas ) and scar age [ table 1 ] . the demographic information of the hs patients data are presented as the mean sd . pp : proliferative phase ; mp : mature phase ; rp : regressive phase ; sd : standard deviation ; hs : hypertrophic scar . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . we defined this period of the hypertrophic scar as proliferative phase.group ii : the scar age was about 12 years . we defined this period of the hypertrophic scar as mature phase.group iii : the scar age was more than 2 years . scars became flat and soft , and the color was closer to the normal skin . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . scars became flat and soft , and the color was closer to the normal skin . tissue samples were fixed in 4% paraformaldehyde in 0.1 mol / l phosphate buffered saline at ph 7.4 and embedded in paraffin . serial 4 m sections of the paraffin - embedded tissues were then collected . for immunohistochemical staining , the sections were incubated with proteinase k ( millipore , usa ) for 20 min ( 37c ) , and endogenous peroxidase was quenched with 3% h2 o2 in methanol for 20 min at room temperature . the sections were blocked with normal goat serum ( mouse sp kit , zsgb - bio , china ) for 30 min and then incubated in mouse polyclonal antibody against eif6 at a final dilution of 1:400 at 4c overnight . lastly , sections were incubated with a peroxidase - coupled secondary antibody plus streptavidin peroxidase complex and followed by diaminobenzidine staining . western blot analysis was performed exactly according to the previously reported protocol . briefly , total proteins were extracted in lysis buffer ( 50 mmol / l tris - hcl ph 7.5 ; 2% sodium deoxycholate stock ) from the frozen tissue samples , and the extracted proteins were quantified by the bicinchoninic acid method ( pierce , usa ) . twenty micrograms of proteins prepared in laemmli buffer , were loaded on 12% acrylamide reducing denaturing gel , transferred to polyvinylidene fluoride membranes ( millipore ) , and blotted with the mouse polyclonal antibody eif6 ( at a 1:2000 dilution with 3% bovine serum albumin ) . pictures of each section were taken with a leica confocal microscope ( leica microsystems , wetzlar , germany ) . for the immunohistochemical staining analysis , the expression of eif6 was observed , and digital images were randomly obtained from five fields under 400 magnification each section . the expression intensity of the positive - labeled cells was measured and analyzed using image - pro plus 6.0 software ( media cybernetics , usa ) . data are expressed as the average optical density ( aod ) , aod = integral optical density ( iod)/area of the total field ( iod = optical intensity of positive cells area of positive cells ) . we performed statistical analyses using one - way analysis of variance followed by turkey multiple comparison . table 1 illustrates the demographic data of three patients groups . there were total 18 patients enrolled in this study with scar age ranging from 3 months to 32 years : group i , proliferative phase : n = 6 ; group ii , mature phase : n = 4 ; and group iii , regressive phase : n = 8 . hypertrophic scar sites included face , neck , preclavicle , hand , upper arm , lower limb , foot , perineum , and presternal . in normal skin , eif6 was mainly distributed in the cytoplasm of the basal layer of keratinocytes [ figure 1a c ] . in contrast , eif6 was mainly expressed in granular layer and stratum spinosum , as well as stratum corneum , but no obvious positive expression in basal layer and in the dermis of the hypertrophic scar [ figure 1d f ] . however , there is a small amount of keratinocytes in basal layer express eif6 in the hypertrophic scar of regressive phase [ figure 1f ] . the expression level of eif6 was significantly decreased in the proliferative phase when standardized to normal skin [ figure 2 and table 2 ] , eif6 expression level was significantly increased in regressive phase compared with proliferative phase ( p = 0.001 ) and mature phase ( p = 0.012 ) . there was no significant difference of eif6 expression between proliferative phase and mature phase of hypertrophic scar tissue ( p = 0.066 ) . immunohistochemistry staining of eukaryotic initiation factor 6 in a different phase of hypertrophic scar and normal skin . ( a ) normal skin of hypertrophic scar in proliferative phase ; ( b ) normal skin of hypertrophic scar in mature phase ; ( c ) normal skin of hypertrophic scar in regressive phase ; ( d ) hypertrophic scar in proliferative phase ; ( e ) hypertrophic scar in mature phase ; ( f ) hypertrophic scar in regressive phase ; the arrows point to the eukaryotic initiation factor 6 positive cells , and the yellow dotted lines presented the basement membrane ( basal layer was a single cell layer above the basement membrane ) . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . relative average optical density ratio of eukaryotic initiation factor 6 in hypertrophic scar / normal skin . the data are presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . aod : average optical density ; ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . the p values between different stages of hs hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . western blot analysis showed eif6 expression level was decreased in hypertrophic scar tissue compared with normal skin [ figure 3 ] . furthermore , when standardized to normal skin , the protein level of eif6 was significantly increased in mature phase and regressive phase of hypertrophic scar compared with proliferative phase of hypertrophic scar ( p = 0.01 and p = 0.001 , respectively ) [ figure 3b and table 2 ] . the degree of eif6 expression was markedly increased in the mature phase of hypertrophic scar compared with that in regressive phase ( p = 0.05 ) [ figure 3b and table 2 ] . the western blot analysis of eukaryotic initiation factor 6 expression in hypertrophic scar and normal skin . ( a ) western blot analysis of eukaryotic initiation factor 6 expression in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . ( b ) relative eukaryotic initiation factor 6 levels on western blot analysis in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . the data were presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . table 1 illustrates the demographic data of three patients groups . there were total 18 patients enrolled in this study with scar age ranging from 3 months to 32 years : group i , proliferative phase : n = 6 ; group ii , mature phase : n = 4 ; and group iii , regressive phase : n = 8 . hypertrophic scar sites included face , neck , preclavicle , hand , upper arm , lower limb , foot , perineum , and presternal . in normal skin , eif6 was mainly distributed in the cytoplasm of the basal layer of keratinocytes [ figure 1a c ] . in contrast , eif6 was mainly expressed in granular layer and stratum spinosum , as well as stratum corneum , but no obvious positive expression in basal layer and in the dermis of the hypertrophic scar [ figure 1d f ] . however , there is a small amount of keratinocytes in basal layer express eif6 in the hypertrophic scar of regressive phase [ figure 1f ] . the expression level of eif6 was significantly decreased in the proliferative phase when standardized to normal skin [ figure 2 and table 2 ] , eif6 expression level was significantly increased in regressive phase compared with proliferative phase ( p = 0.001 ) and mature phase ( p = 0.012 ) . there was no significant difference of eif6 expression between proliferative phase and mature phase of hypertrophic scar tissue ( p = 0.066 ) . immunohistochemistry staining of eukaryotic initiation factor 6 in a different phase of hypertrophic scar and normal skin . ( a ) normal skin of hypertrophic scar in proliferative phase ; ( b ) normal skin of hypertrophic scar in mature phase ; ( c ) normal skin of hypertrophic scar in regressive phase ; ( d ) hypertrophic scar in proliferative phase ; ( e ) hypertrophic scar in mature phase ; ( f ) hypertrophic scar in regressive phase ; the arrows point to the eukaryotic initiation factor 6 positive cells , and the yellow dotted lines presented the basement membrane ( basal layer was a single cell layer above the basement membrane ) . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . relative average optical density ratio of eukaryotic initiation factor 6 in hypertrophic scar / normal skin . the data are presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . aod : average optical density ; ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . the p values between different stages of hs hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . western blot analysis showed eif6 expression level was decreased in hypertrophic scar tissue compared with normal skin [ figure 3 ] . furthermore , when standardized to normal skin , the protein level of eif6 was significantly increased in mature phase and regressive phase of hypertrophic scar compared with proliferative phase of hypertrophic scar ( p = 0.01 and p = 0.001 , respectively ) [ figure 3b and table 2 ] . the degree of eif6 expression was markedly increased in the mature phase of hypertrophic scar compared with that in regressive phase ( p = 0.05 ) [ figure 3b and table 2 ] . the western blot analysis of eukaryotic initiation factor 6 expression in hypertrophic scar and normal skin . ( a ) western blot analysis of eukaryotic initiation factor 6 expression in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . ( b ) relative eukaryotic initiation factor 6 levels on western blot analysis in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . the data were presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . hypertrophic scar has been a problem since human being came to the earth , and it has been still a big challenge for human being , as we do not know much about the mystery of hypertrophic scar formation as well as we do not have great progress in the control of hypertrophic scar development . our previous pilot study found that the eif6 expression was significantly decreased in fibroblasts derived from hypertrophic scar compared with that from the normal skin in vitro . moreover , blocking transforming growth factor- ( tgf- ) signaling inhibits scar formation in rat cutaneous wounds . eif6 deficiency also promoted -smooth muscle actin and tgf-1 expression in fibroblasts , and cutaneous fibrosis in an animal model ( unpublished data ) . these unpublished data implied the involvement of eif6 in hypertrophic scar formation in a human being . therefore , the possible relationship between eif6 and hypertrophic scar formation was studied by the analysis of eif6 expression distribution and quantity in the development of hypertrophic scar in the human being through immunohistochemistry and western blot . in the current study , eif6 was down - regulated in human hypertrophic scar tissues compared to normal skin tissues as demonstrated by western blot and immunohistochemistry , which is consistent with our previous findings in scar - derived fibroblasts in vitro . importantly , eif6 expression was related to the moments of hypertrophic scar development , that is , eif6 was down - regulated in the proliferative phase of hypertrophic scar formation , and gradually increased during the regressive phase . it is known that eif6 regulates the biosynthesis of ribosome 60s as well as protein translation . eif6 is also highly expressed in the cycling cells and has an impact on cell cycle . it means that increased eif6 expression may promote cell proliferation . on the other hand , it is reasonable to speculate that decreased eif6 expression may impede the cell cycling , but promote the cell function , such as producing more ecm and cell differentiation , like epithelium - to - mesenchymal transition . therefore , down - regulated eif6 in the proliferative phase of hypertrophic scar formation may be related to the active function of both epidermal cells and fibroblasts . while , the gradually increased expression of eif6 in the regressive phase of hypertrophic scar formation indicates the process of reconstruction of the newly - formed regenerative tissues is close to the end . taken together , eif6 might be involved in the hypertrophic scar formation , and could be a marker for identifying the moment of hypertrophic scar formation . restoration of eif6 level in the cells might be helpful for hypertrophic scar control in the future . another finding in our present study was that eif6 was deficient in the basal layer of epidermis of proliferative phase and recurrence in regressive phase . eif6 is originally identified in mammals as a cytoplasmic interactor of 4 integrin , which is crucial in hemidesmosome formation , cell adhesion , and responsible for the blister formation of hypertrophic scar surface . in the epidermis of the skin , the reduction of eif6 in the early stage of hypertrophic scar implies that connection between newly - formed epidermis and dermis is fragile , and the hypertrophic scar remodeling is yet to be finished . based on the above analysis , we propose that eif6 expression level and distribution might be an indicator for the progress of hypertrophic scar development and be helpful for the evaluation of therapy effects , such as pressure therapy and massage . a future longitudinal study will be designed to investigate the effectiveness of pressure therapy on scars and the relationship between the clinical expressions and the change of eif6 in the cells of hypertrophic scar . in conclusion , eif6 could be one of the target molecules for hypertrophic scar control as well as a potential indicator for evaluation of hypertrophic scar management effects , such as pressure therapy and massage . 2012aa020504 ) , the national natural science foundation of china ( nos . 81372082 , 81401603 ) . 2012aa020504 ) , the national natural science foundation of china ( nos . 81372082 , 81401603 ) .

background : hypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients . therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation . to throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment . this study aimed to illustrate the negative role of eukaryotic initiation factor 6 ( eif6 ) in the process of human hypertrophic scar formation , and provide a possible indicator of hypertrophic scar treatment and a potential target molecule for hypertrophic scar.methods:in the present study , we investigated the protein expression of eif6 in the human hypertrophic scar of different periods by immunohistochemistry and western blot analysis.results:in the hypertrophic scar tissue , eif6 expression was significantly decreased and absent in the basal layer of epidermis in the early period , and increased slowly and began to appear in the basal layer of epidermis by the scar formation time.conclusions:this study confirmed that eif6 expression was significantly related to the development of hypertrophic scar , and the eif6 may be a target molecule for hypertrophic scar control or could be an indicator of the outcomes for other treatment modalities .

I M Clinical specimens Patient groups Immunohistochemistry study Western blot analysis Morphometric analysis of eukaryotic initiation factor 6 in hypertrophic scar and normal skin tissues Statistical analysis R Demography of the hypertrophic scar patients Eukaryotic initiation factor 6 expression was morphologically absent in the basal layer of epidermis of hypertrophic scar Eukaryotic initiation factor 6 was down-regulated during the hypertrophic scar development D Financial support and sponsorship Conflicts of interest

a hypertrophic scar is one of the fibrotic diseases , arising from fibroproliferation disorder which occurs after the damage of deep dermis by burns or trauma . patients with hypertrophic scar often suffer from difficulties in daily life and psycho - social dysfunction . the main pathological characteristics of the hypertrophic scar are an overproduction of extracellular matrix ( ecm ) and collagens . due to the severe functional disabilities caused by a hypertrophic scar , burn rehabilitation is very important for patients after burns , especially occupational therapy which bridges the transfer from hospitals into the community . it facilitates the scar maturation , improves the appearance of a hypertrophic scar , reduces the erythema , as well as alleviates pain and pruritus . however , the outcome of hypertrophic scar treatment was poor , and the mechanisms of how pressure therapy influences the scar maturation are not fully reviewed . demonstrated that a static pressure of more than 20 mmhg could accelerate the hypertrophic scar remodeling . to investigate the mechanisms of pressure therapy on hypertrophic scar maturation , it is important for both surgeons and occupational therapists to identify the key molecule which promotes or inhibits the hypertrophic scar formation . our previous in vitro study demonstrated that the eukaryotic initiation factor 6 ( eif6 ) expression was significantly decreased in fibroblasts derived from hypertrophic scar when compared with that from normal skin ( unpublished data ) . eif6 ( also known as integrin 4 binding protein ) , is a ribosome anti - association factor that regulates translational initiation and ribosome synthesis due to its capacity to modulate ribosome 60s availability and 80s subunit , and plays a very important role in ribosome formation . additionally , eif6 was involved in connection with cytoskeleton , cell apoptosis , and cell cycle progression . moreover , eif6 has been identified as an interacting protein of the hypertrophic scar - related protein p311 , which indicates that it may be involved in the regulation of hypertrophic scar formation as well as myofibroblast differentiation . however , the spatial and temporal expression of eif6 in the process of human hypertrophic scar formation remains unknown . the current study was designed to study the eif6 protein expression pattern during the process of hypertrophic scar formation . because of the possible variation among the individuals , the cases enrolled in our study were only those who received hypertrophic scar excision followed by autoskin grafting , and there was remaining donor skin tissue after grafting , that is , the hypertrophic scar tissue and normal skin tissue were the homobody . immediately after the surgical procedure , the samples of hypertrophic scar tissues and pair - matched normal skin tissues were both kept either in liquid nitrogen or in 4% paraformaldehyde . therefore , total 18 cases ( whose hypertrophic scar tissues and remaining donor skin tissues were kept in the institute of burn research , southwest hospital , chongqing , china ) were enrolled in this study from october 2013 to august 2014 . the study was conducted according to the helsinki declaration and approved by the ethics committee of southwest hospital . samples used in this study were anonymous , and it is impossible for anyone to link the samples to the sources . due to the individual variation , we used vancouver scar scale ( vss ) to evaluate for height and color , and visual analog scale ( vas ) for pain and itch of the hypertrophic scar ( data not shown ) . pp : proliferative phase ; mp : mature phase ; rp : regressive phase ; sd : standard deviation ; hs : hypertrophic scar . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . we defined this period of the hypertrophic scar as proliferative phase.group ii : the scar age was about 12 years . we defined this period of the hypertrophic scar as mature phase.group iii : the scar age was more than 2 years . scars became flat and soft , and the color was closer to the normal skin . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . scars became flat and soft , and the color was closer to the normal skin . serial 4 m sections of the paraffin - embedded tissues were then collected . , total proteins were extracted in lysis buffer ( 50 mmol / l tris - hcl ph 7.5 ; 2% sodium deoxycholate stock ) from the frozen tissue samples , and the extracted proteins were quantified by the bicinchoninic acid method ( pierce , usa ) . additionally , we used glyceraldehyde-3-phosphate dehydrogenase for the normalization of data . for the immunohistochemical staining analysis , the expression of eif6 was observed , and digital images were randomly obtained from five fields under 400 magnification each section . because of the possible variation among the individuals , the cases enrolled in our study were only those who received hypertrophic scar excision followed by autoskin grafting , and there was remaining donor skin tissue after grafting , that is , the hypertrophic scar tissue and normal skin tissue were the homobody . immediately after the surgical procedure , the samples of hypertrophic scar tissues and pair - matched normal skin tissues were both kept either in liquid nitrogen or in 4% paraformaldehyde . therefore , total 18 cases ( whose hypertrophic scar tissues and remaining donor skin tissues were kept in the institute of burn research , southwest hospital , chongqing , china ) were enrolled in this study from october 2013 to august 2014 . the study was conducted according to the helsinki declaration and approved by the ethics committee of southwest hospital . samples used in this study were anonymous , and it is impossible for anyone to link the samples to the sources due to the individual variation , there are no clear staging criteria for hypertrophic scar for the present . we used vancouver scar scale ( vss ) to evaluate for height and color , and visual analog scale ( vas ) for pain and itch of the hypertrophic scar ( data not shown ) . pp : proliferative phase ; mp : mature phase ; rp : regressive phase ; sd : standard deviation ; hs : hypertrophic scar . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . we defined this period of the hypertrophic scar as proliferative phase.group ii : the scar age was about 12 years . we defined this period of the hypertrophic scar as mature phase.group iii : the scar age was more than 2 years . scars became flat and soft , and the color was closer to the normal skin . group i : the scar age was within 1-year , and the clinical manifestation of scars was raised , erythematous and hard with or without itch and pain . scars became flat and soft , and the color was closer to the normal skin . western blot analysis was performed exactly according to the previously reported protocol . briefly , total proteins were extracted in lysis buffer ( 50 mmol / l tris - hcl ph 7.5 ; 2% sodium deoxycholate stock ) from the frozen tissue samples , and the extracted proteins were quantified by the bicinchoninic acid method ( pierce , usa ) . twenty micrograms of proteins prepared in laemmli buffer , were loaded on 12% acrylamide reducing denaturing gel , transferred to polyvinylidene fluoride membranes ( millipore ) , and blotted with the mouse polyclonal antibody eif6 ( at a 1:2000 dilution with 3% bovine serum albumin ) . for the immunohistochemical staining analysis , the expression of eif6 was observed , and digital images were randomly obtained from five fields under 400 magnification each section . the expression intensity of the positive - labeled cells was measured and analyzed using image - pro plus 6.0 software ( media cybernetics , usa ) . hypertrophic scar sites included face , neck , preclavicle , hand , upper arm , lower limb , foot , perineum , and presternal . in normal skin , eif6 was mainly distributed in the cytoplasm of the basal layer of keratinocytes [ figure 1a c ] . in contrast , eif6 was mainly expressed in granular layer and stratum spinosum , as well as stratum corneum , but no obvious positive expression in basal layer and in the dermis of the hypertrophic scar [ figure 1d f ] . however , there is a small amount of keratinocytes in basal layer express eif6 in the hypertrophic scar of regressive phase [ figure 1f ] . the expression level of eif6 was significantly decreased in the proliferative phase when standardized to normal skin [ figure 2 and table 2 ] , eif6 expression level was significantly increased in regressive phase compared with proliferative phase ( p = 0.001 ) and mature phase ( p = 0.012 ) . there was no significant difference of eif6 expression between proliferative phase and mature phase of hypertrophic scar tissue ( p = 0.066 ) . immunohistochemistry staining of eukaryotic initiation factor 6 in a different phase of hypertrophic scar and normal skin . ( a ) normal skin of hypertrophic scar in proliferative phase ; ( b ) normal skin of hypertrophic scar in mature phase ; ( c ) normal skin of hypertrophic scar in regressive phase ; ( d ) hypertrophic scar in proliferative phase ; ( e ) hypertrophic scar in mature phase ; ( f ) hypertrophic scar in regressive phase ; the arrows point to the eukaryotic initiation factor 6 positive cells , and the yellow dotted lines presented the basement membrane ( basal layer was a single cell layer above the basement membrane ) . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . relative average optical density ratio of eukaryotic initiation factor 6 in hypertrophic scar / normal skin . the data are presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . aod : average optical density ; ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . the p values between different stages of hs hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . western blot analysis showed eif6 expression level was decreased in hypertrophic scar tissue compared with normal skin [ figure 3 ] . furthermore , when standardized to normal skin , the protein level of eif6 was significantly increased in mature phase and regressive phase of hypertrophic scar compared with proliferative phase of hypertrophic scar ( p = 0.01 and p = 0.001 , respectively ) [ figure 3b and table 2 ] . the degree of eif6 expression was markedly increased in the mature phase of hypertrophic scar compared with that in regressive phase ( p = 0.05 ) [ figure 3b and table 2 ] . the western blot analysis of eukaryotic initiation factor 6 expression in hypertrophic scar and normal skin . ( a ) western blot analysis of eukaryotic initiation factor 6 expression in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . ( b ) relative eukaryotic initiation factor 6 levels on western blot analysis in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . the data were presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . there were total 18 patients enrolled in this study with scar age ranging from 3 months to 32 years : group i , proliferative phase : n = 6 ; group ii , mature phase : n = 4 ; and group iii , regressive phase : n = 8 . hypertrophic scar sites included face , neck , preclavicle , hand , upper arm , lower limb , foot , perineum , and presternal . in normal skin , eif6 was mainly distributed in the cytoplasm of the basal layer of keratinocytes [ figure 1a c ] . in contrast , eif6 was mainly expressed in granular layer and stratum spinosum , as well as stratum corneum , but no obvious positive expression in basal layer and in the dermis of the hypertrophic scar [ figure 1d f ] . however , there is a small amount of keratinocytes in basal layer express eif6 in the hypertrophic scar of regressive phase [ figure 1f ] . the expression level of eif6 was significantly decreased in the proliferative phase when standardized to normal skin [ figure 2 and table 2 ] , eif6 expression level was significantly increased in regressive phase compared with proliferative phase ( p = 0.001 ) and mature phase ( p = 0.012 ) . there was no significant difference of eif6 expression between proliferative phase and mature phase of hypertrophic scar tissue ( p = 0.066 ) . immunohistochemistry staining of eukaryotic initiation factor 6 in a different phase of hypertrophic scar and normal skin . ( a ) normal skin of hypertrophic scar in proliferative phase ; ( b ) normal skin of hypertrophic scar in mature phase ; ( c ) normal skin of hypertrophic scar in regressive phase ; ( d ) hypertrophic scar in proliferative phase ; ( e ) hypertrophic scar in mature phase ; ( f ) hypertrophic scar in regressive phase ; the arrows point to the eukaryotic initiation factor 6 positive cells , and the yellow dotted lines presented the basement membrane ( basal layer was a single cell layer above the basement membrane ) . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . relative average optical density ratio of eukaryotic initiation factor 6 in hypertrophic scar / normal skin . the data are presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . aod : average optical density ; ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . the p values between different stages of hs hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . western blot analysis showed eif6 expression level was decreased in hypertrophic scar tissue compared with normal skin [ figure 3 ] . furthermore , when standardized to normal skin , the protein level of eif6 was significantly increased in mature phase and regressive phase of hypertrophic scar compared with proliferative phase of hypertrophic scar ( p = 0.01 and p = 0.001 , respectively ) [ figure 3b and table 2 ] . the degree of eif6 expression was markedly increased in the mature phase of hypertrophic scar compared with that in regressive phase ( p = 0.05 ) [ figure 3b and table 2 ] . the western blot analysis of eukaryotic initiation factor 6 expression in hypertrophic scar and normal skin . ( a ) western blot analysis of eukaryotic initiation factor 6 expression in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . ( b ) relative eukaryotic initiation factor 6 levels on western blot analysis in normal skin and hypertrophic scar in proliferative phase , mature phase , and regressive phase . the data were presented as the mean standard deviation , and the statistic result was determined by one - way analysis of variance followed by turkey multiple comparison . ns : normal skin ; hs : hypertrophic scar ; pp : proliferative phase ; mp : mature phase ; rp : regressive phase . hypertrophic scar has been a problem since human being came to the earth , and it has been still a big challenge for human being , as we do not know much about the mystery of hypertrophic scar formation as well as we do not have great progress in the control of hypertrophic scar development . our previous pilot study found that the eif6 expression was significantly decreased in fibroblasts derived from hypertrophic scar compared with that from the normal skin in vitro . moreover , blocking transforming growth factor- ( tgf- ) signaling inhibits scar formation in rat cutaneous wounds . these unpublished data implied the involvement of eif6 in hypertrophic scar formation in a human being . therefore , the possible relationship between eif6 and hypertrophic scar formation was studied by the analysis of eif6 expression distribution and quantity in the development of hypertrophic scar in the human being through immunohistochemistry and western blot . in the current study , eif6 was down - regulated in human hypertrophic scar tissues compared to normal skin tissues as demonstrated by western blot and immunohistochemistry , which is consistent with our previous findings in scar - derived fibroblasts in vitro . importantly , eif6 expression was related to the moments of hypertrophic scar development , that is , eif6 was down - regulated in the proliferative phase of hypertrophic scar formation , and gradually increased during the regressive phase . it is known that eif6 regulates the biosynthesis of ribosome 60s as well as protein translation . on the other hand , it is reasonable to speculate that decreased eif6 expression may impede the cell cycling , but promote the cell function , such as producing more ecm and cell differentiation , like epithelium - to - mesenchymal transition . therefore , down - regulated eif6 in the proliferative phase of hypertrophic scar formation may be related to the active function of both epidermal cells and fibroblasts . while , the gradually increased expression of eif6 in the regressive phase of hypertrophic scar formation indicates the process of reconstruction of the newly - formed regenerative tissues is close to the end . taken together , eif6 might be involved in the hypertrophic scar formation , and could be a marker for identifying the moment of hypertrophic scar formation . restoration of eif6 level in the cells might be helpful for hypertrophic scar control in the future . another finding in our present study was that eif6 was deficient in the basal layer of epidermis of proliferative phase and recurrence in regressive phase . eif6 is originally identified in mammals as a cytoplasmic interactor of 4 integrin , which is crucial in hemidesmosome formation , cell adhesion , and responsible for the blister formation of hypertrophic scar surface . in the epidermis of the skin , the reduction of eif6 in the early stage of hypertrophic scar implies that connection between newly - formed epidermis and dermis is fragile , and the hypertrophic scar remodeling is yet to be finished . based on the above analysis , we propose that eif6 expression level and distribution might be an indicator for the progress of hypertrophic scar development and be helpful for the evaluation of therapy effects , such as pressure therapy and massage . a future longitudinal study will be designed to investigate the effectiveness of pressure therapy on scars and the relationship between the clinical expressions and the change of eif6 in the cells of hypertrophic scar . in conclusion , eif6 could be one of the target molecules for hypertrophic scar control as well as a potential indicator for evaluation of hypertrophic scar management effects , such as pressure therapy and massage .

soccer is the most popular sport consisting of exercise periods of high intensity interspersed between exercise periods of low intensity and involves activities that require numerous eccentric actions1,2,3 . participation in the unaccustomed or increased intensity and duration of eccentrically biased exercise often results in ultra - structural damage to skeletal muscle in both novel and elite athletes4,5,6 . this temporary cellular damage occurring in the skeletal muscle after unaccustomed exercises is called exercise induced muscle damage ( eimd)7 which is characterized by the disruption of contractile and non - contractile proteins with the decomposition of sarcolemma8 , increased concentration of muscle protein in bloodstream9 , delayed - onset muscle soreness ( doms)10 , decreased range of motion11 , swelling12 , impaired proprioceptive function and neuromuscular control13 , and prolonged loss of muscle function14 . moreover , this skeletal muscle damage can affect performance through a reduction in the joint range of motion , pain , and peak torque15 . researchers have utilized many modes of damaging exercise for inducing eimd , more specifically downhill running and plyometric exercises , however , these protocols still lack sports specificity and direct application to field sports16,17,18,19,20,21 and are mostly used for isolated muscle damage . thompson et al.22 found that an intermittent sprinting protocol resulted in muscle soreness by imposing a large mechanical stress on quadriceps and hamstrings as they work to decelerate the body s mass . other researchers have also provided a useful model utilizing sports - specific repeated sprint sessions with a rapid deceleration to induce eimd in the hamstrings , quadriceps , and the calf muscles23 , 24 . the current study , therefore , utilizes this sports specific task to elicit eimd in soccer players . soccer is characterized by a large number of sprints , accelerations and decelerations25 and direction changes . therefore , the players require good speed , power , agility , and balance for the optimal performance . balance in soccer is necessary to maintain single leg stance while shooting accurately , dribbling and passing the ball26 , 27 . balance or postural control is a dynamic process by which the body is maintained in equilibrium28 . dynamic and functional balances involve the maintenance of the center of gravity ( cog ) over a moving base of support which is more critical for athletic movement than static balance . several factors such as proprioceptive deficits , muscle weakness , muscle injury ( strain ) , and sports participation can affect balance29 , 31 . doms is often categorized as a type i muscle strain32 and clinically , acute strains and doms are very similar33 . previous studies reported that muscle damage following eccentric exercises can change the kinesthetic sense34 , 35 . poor balance has been associated with an increased risk of injuries in a number of sports37 . however , there is a dearth of information as to how eimd influences balance , as well as speed , agility and power , which are essential components of not only training and competition but also prevention and rehabilitation of sports injuries . therefore , the purpose of the present study was to examine the changes in physical performance ( speed , power and agility ) and balance performance ( static and dynamic balance ) resulting from eimd following sport - specific repeated sprints in male collegiate soccer players . fifteen male college soccer players ( mean age , 21.3 2 years , height 172.2 6.6 cm , weight 66.1 11 kg and bmi 22.3 3.2 ) from jamia millia islamia , new delhi , india , participated in the study . ethical approval was granted by the institutional ethics committee and the subjects were given a written informed consent . all the subjects regularly participated in the collegiate soccer and did sport - specific training at least 4 times / week . those involved in resistance training for at least 12 months prior to this study , martial arts or dancing , which may influence balance abilities or prior balance training were excluded . in addition , individuals having any history of lower limb injury , cerebral concussion and vestibular disorders for 3 months before testing were also excluded . participants were asked to refrain from competitive events , resistance exercise , and avoid use of alcohol , nutritional supplements , and non - steroidal anti - inflammatory drugs during the study duration . the sample size was calculated using the pass 11 software which showed that a sample of 15 subjects would achieve a 95% power to detect a difference of 1 in a design with 4 repeated measurements having a compound symmetrical co - variance structure when the standard deviation is 1 , the correlation between observation on the same subject is 0.5 and the level is 0.05 . prior to each test , the subjects were given a demonstration of the tests . on the first day of testing , subject s height , weight , knee range of motion ( rom ) , thigh circumference and muscle strength for quadriceps and hamstrings ( mvic ) were measured in the human performance lab , centre of physiotherapy and rehabilitation sciences , jamia millia islamia . two milliliter blood samples were drawn from the antecubital vein to determine baseline creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) levels . immediately thereafter , subjects were tested for physical performance parameters using the vertical jump , 20 meter ( m ) sprint and the illinois agility test , and static and dynamic balance . on the second day of testing , the subjects performed the repeated sprints protocol24 wherein , following a warm - up , the participants performed 15 30 meter sprints with a period of 1 minute rest between repetitions . subjects were instructed to maximally sprint between the cones and stop within the 10 meter deceleration zone , laid out immediately after the 30 meter sprint zone . after the completion of a repetition ( i.e. , the subject had come to a halt ) , the rest period was initiated . the criterion measures were recorded at 24 , 48 and 72 hours after the sprint protocol . muscle soreness , range of motion , thigh circumference , maximum voluntary contraction , ck , and ldh were assessed as markers of the muscle damage . the soreness in lower limb was assessed while squatting at a knee angle of approximately 90 degrees , using a 200 mm visual analogue scale with no soreness at one end and unbearably painful at the other18 . range of motion was measured for knee flexion using a plastic goniometer and universal landmarks ( lateral epicondyle of the femur , lateral malleolus and greater trochanter ) to ensure alignment38 . thigh circumference was measured at 5 , 10 and 15 cm above the patella by a gulick tape measure while the participant was in the long - sitting position with a relaxed quadriceps muscle . the mean of 2 measurements was taken from each marked site , and the average value of the 3 measurement sites was used for statistical analysis39 . maximum voluntary contraction of the knee flexors and extensors was measured using the lafayette mmt system ( model no . make contractions in which the patient used each tested muscle group to push maximally against the plate and the piston of the hand - held dynamometer for 45 seconds40 . venous blood was used to measure ck and ldh . the blood was then centrifuged at 3,000 rpm for 10 min41 . the serum was aspirated and stored at 20c until being analysed for ck and ldh activity39 , 42 . the serum ck and ldh activity was assessed by a spectrophotometer at 340 nm set wavelength using a ck kit ( nac act . crest biosystems , coral ) and ldh kit ( ldh , p - l , crest biosystems coral ) respectively . vertical jump performance , 20 m single sprint , and the agility ( illinois agility test ) were used to assess the physical performance . vertical jump performance is commonly used as an index for the power of the lower limb or explosive leg power43,44,45,46 . for squat jumps , the subjects were instructed to initiate from 90 knee flexion , and execute a maximum vertical jump while swinging their arms actively . to assess the counter movement jump , participants stood fully erect , and following a verbal command , initiated a countermovement followed by a maximal vertical jump in one continuous motion , keeping their legs straight whilst airborne , but using their arms during the jump47 . 20 m single sprint is a standard test for assessing the running speed in soccer players48 . the participants performed two 20 m trials separated by a 3 min recovery period . agility ( illinois agility test ) is commonly used to measure agility in soccer . the best score ( time ) of three trials performed by each subject the test measures the ability of the participant to balance on the ball of the foot with hands placed on the hips while positioning the non - supporting foot against the inside knee of the supporting leg . the time ( in seconds ) for which the participant is able to maintain this position is indicative of his balance performance . dynamic balance was assessed using the modified star excursion balance test ( sebt ) , where 3 reach directions ( anterior , posteromedial , posterolateral ) were measured53 . the farthest reach distance was recorded ( cm ) with a mark on the tape in each direction . if the evaluator felt the participant used the reaching leg for support , removed his foot from the centre of the grid , or was unable to maintain balance on the support leg , the test was discarded and repeated54 . data were assessed by a shapiro - wilk test for the normality of the distribution scores . sebt scores that demonstrated non - normal distribution were log- transformed for further analysis . fifteen participants were assessed during the experiment . to test for the difference across 4 assessments , a repeated measure anova time ( pre , 24 , 48 and 72 hours ) was employed . when the main effect of time was significant , a bonferroni test was employed as post hoc analysis to locate the time points having significant difference . for sebt , the average of three distances ( cm ) scores was taken for all the three directions over the three trials and was normalized to leg length . the normalized composite reach distances between the right and left leg were analyzed and compared using a paired t - test to examine the differences . the markers of muscle damage showed a significant effect for time ( table 1table 1.change in markers of muscle damage following exercise induced muscle damagevariablespre24 h48 h72 hmean semean semean semean sevasq0.0 0.086 5.61 * 115 6.94 * 73 4.80*vash0.0 0.058.3 6.80 * 81.3 7.80 * 47.6 4.70*mvcq258.2 15.56209.6 11.83 * 215.4 12.65 * 238.7 12.73mvch176.2 6.83147.8 5.84 * 149.2 8.83 * 161.5 8.10rom131.9 0.50124.1 0.68 * 127.5 0.83 * 131.9 0.58lg50.8 1.0752.2 1.15 * 52.1 1.17 * 50.9 1.07ck190.6 16.78620.7 61.0 * 502.4 51.7 * 341.5 38.7*ldh286.6 21.05563.8 52.7 * 467.7 42.5 * 392.6 18.3h : hours ; vasq : visual analogue scale quadriceps ; vash : visual analogue scale hamstrings ; mvcq : maximal voluntary contraction quadriceps ; mvch : maximal voluntary contraction hamstrings ; rom : range of motion knee ; lg : leg girth ; ck : creatine kinase ; ldh : lactate dehydrogenase ; significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) ) , as did the physical performance and balance variables ( table 2table 2.changes in balance and physical performance variables following exercise induced muscle damagevariablespre24 h48 h72 hmean semean semean semean sebalance performance variablessbtl47.7 1.6525.2 1.67 * 34 1.7747.3 1.77sbtr34.2 1.5521.7 1.58 * 26.7 1.6633 1.67antl79.8 2.7074.2 2.80 * 74.5 2.88 * 79 2.96antr80.4 2.9073.2 2.71 * 75.8 2.95 * 78.8 3.01platl102 1.4495.2 1.09 * 96.6 1.13 * 101.8 1.46platr99.6 1.4992.1 1.15 * 94.8 1.53 * 99.1 1.82pmedl99.2 1.3289.1 0.85 * 91.2 1.07 * 98.4 1.18pmedr98.9 1.2789.6 0.63 * 91.6 0.61 * 97.8 0.82physical performance variablessprint3.4 0.073.7 0.07 * 3.6 0.07 * 3.5 0.07iat17.4 0.2518.1 0.27 * 17.8 0.24 * 17.4 0.25sj42.8 1.3939.3 1.89 * 39.7 1.91 * 42.3 1.57cmj47.3 1.4741.8 2.18 * 42 1.94 * 45.5 1.53h : hours ; sbtl : static balance test left leg ; sbtr : static balance test right leg ; antl : star excursion balance test in anterior direction left leg ; antr : star excursion balance test in anterior direction right leg ; platl : star excursion balance test in postero - lateral direction left leg ; platr : star excursion balance test in postero - lateral direction right leg ; pmedl : star excursion balance test in postero - medial direction left leg ; pmedr : star excursion balance test in postero - medial direction right leg ; iat : ilionois agility test ; sj : squat jump ; cmj : counter - movement jump . significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) ) . moreover , the post hoc analyses revealed maximum differences between baseline and at 24 hours following eimd and non - significant differences between baseline values and those recorded at 72 hours ( table 2 ) . the paired t - test used to compare the scores for composite reach distances , indicated no significant difference between the right ( m=85.7 , sd=6.99 ) and left leg ( m=86.3 , sd=6.32 ) , p=0.336 . h : hours ; vasq : visual analogue scale quadriceps ; vash : visual analogue scale hamstrings ; mvcq : maximal voluntary contraction quadriceps ; mvch : maximal voluntary contraction hamstrings ; rom : range of motion knee ; lg : leg girth ; ck : creatine kinase ; ldh : lactate dehydrogenase ; significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) h : hours ; sbtl : static balance test left leg ; sbtr : static balance test right leg ; antl : star excursion balance test in anterior direction left leg ; antr : star excursion balance test in anterior direction right leg ; platl : star excursion balance test in postero - lateral direction left leg ; platr : star excursion balance test in postero - lateral direction right leg ; pmedl : star excursion balance test in postero - medial direction left leg ; pmedr : star excursion balance test in postero - medial direction right leg ; iat : ilionois agility test ; sj : squat jump ; cmj : counter - movement jump . significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) objective of the present study was to examine whether eimd following sport - specific repeated sprints would affect the physical and balance performance in the male collegiate soccer players . more pronounced increase in the muscle soreness , limb circumference , plasmatic ck and ldh activity and a decrease in rom and maximal voluntary contraction provide indirect evidence to suggest that eimd was present following the sport - specific repeated sprints . our findings suggest that the eimd was induced in both dominant and the non - dominant limbs . indeed , all the variables chosen to examine the changes in physical and balance performance showed significant change after the protocol . although , a previous study has investigated the eimd using the markers of muscle damage and physical performance in athletes55 . however , to the best of our knowledge , till date , no study has investigated the effects of eimd on dynamic balance performance . this is the first study that comprehensively examined the pain sensation , biochemical , physical and balance performance in both dominant and non - dominant legs in athletes using a sport - specific protocol . muscle soreness , increased 24 hours after exercise , peaked at 48 hours and finally decreased at 72 hours in both quadriceps and hamstring , showing a similar trend as that observed by howatson et al.24 using the same protocol . the trend to rise in the soreness that peaked at 48 hours further , the rom remained decreased even at 48 and 72 hours following eimd . this result is similar to previous studies which suggest rom decreased , after the exercise protocol and did not recover for the next 3 days58 , 59 . however , earlier studies have suggested that shortened non - contractile components , change in calcium homeostasis due to muscle damage , decreased strength , and/or swelling may be responsible for decreased rom12 , 60 . thigh circumference demonstrated a peak increase at 24 hours ( 3.2% ) , consistent with the findings of howatson et al.24 who used the same protocol and reported an increase in limb girth of 3% at 24 hours . the present study demonstrated reduction of swelling at 48 hours ( 2.5% ) and at 72 hours ( 0.2% ) . maximal voluntary contraction showed a significant drop at 24 hours , gradually recovering at 48 hours and the recovery continuing up to 72 hours in both quadriceps and hamstrings . measurement of the maximal force during isometric contractions is the most common method of assessing muscle function following eccentric exercise and is considered a reliable indicator of muscle damage9 . ck showed peak activity at 24 hours following exercise , gradually recovering at 48 and 72 hours . this trend was similar to the results of howatson et al.24 utilizing the same protocol for eimd . the ck response peaking at 24 h post - exercise was in accordance with previous study using a similar protocol to induce damage47 , 61 . it would appear that other lower limb exercise such as downhill running62 also follows a similar trend to this investigation . ldh showed a rise of 68% the baseline values at 24 hours , gradually decreasing to 40.2% and 1% from the baseline at 48 and 72 hours , respectively . armstrong et al.63 reported that ldh showed a significant secondary late phase elevations at 2448 hours post downhill running . however , sudhakar and naiya64 in their study found that that serum ldh level peaked at 48 hours after the eimd which is different from the findings of this study . in a 20 m sprint after 24 hours , the sprint time increased by 6.6% . at 48 hours , the sprint times improved showing an increase of 4.6% from the baseline values . at 72 hours , the sprint times these findings are consistent with highton et al.7 and twist and eston57 , who reported that sprint times was significantly increased at 24 and 48 hours following eimd before returning to baseline levels at 72 hours . but they have measured 5 m and 10 m sprint performance . on the other hand , contrary to these studies semark et al.41 reported no significant impairment in sprint running performance over 5 m , 10 m , 20 m , and 30 m following 70 drop jumps and reported that there was insufficient muscle damage to impair sprint performance . this is similar to the of study done by highton et al.7 who has reported a significant reduction in the agility performance following plyometric exercise and suggested that the reduction in agility performance may be due to a reduction in both strength and running speed . this study also showed that increase in sprint ( 6.6% ) is not exclusively responsible for decrements in agility performance ( 4.2% ) . vertical jump demonstrated a significant drop in both squat and counter - movement jump performance following repeated sprints . the squat jump performance showed a decrease of 7.1% in both 24 and 48 hours and 1.1% at 72 hours and the counter - movement jump performance showed a decline of 11.6% at 24 hours , 11.1% at 48 hours and the result of our study is in accordance with the study done by sarabon et al.65 , which showed a more pronounced drop in counter - movement jump height in relation to squat jump height 24 hours following eimd . however , in their study the recovery was slower for squat jump as compared to counter - movement jump , which is contrary to our study which suggests a comparatively slower return for the counter - movement jump . this may be because of different damaging protocol used as their protocol emphasized damage of the hamstring muscles and ours on the quadriceps . another study also observed a prolonged reduction in vertical jump height , by 11% in squat jump performance after 50 drop - jumps47 . static balance revealed similar results to the study of twist et al.66 who reported a significant impairment in unilateral balance from baseline at 24 hours following plyometric exercise and no significant impairment at 48 and 72 hours following plyometric exercise . however , sarabon et al.65 found no significant impairment on unilateral quite stance following drop jumps and leg curls emphasizing hamstring damage . the authors state that the reason for the non - significant changes might have been due to very less involvement of hamstring muscle during single leg stance . they further suggested that decreased hamstring muscle function may be due to minimal muscle activation and more degree of freedom of hip and thigh muscles . twist et al.66 proposed that balance performance was exacerbated by both central and peripheral mechanisms . olmsted et al.67 suggests that static postural impairment is caused by impairment of proprioception and neuromuscular control . the results suggest that the dynamic balance recorded by sebt declined significantly at 24 hours in all the directions . the balance recovered significantly in posteromedial and posterolateral directions and non - significantly in anterior directions at 48 hours . at 72 hours balance late recovery in anterior directions may be because the muscle damage was more in the quadriceps which is found to be more active during the anterior directions68 . to perform the anterior direction , gravity action on the upper body creates a larger knee - flexion moment , which must be controlled by the extension moment produced by the quadriceps . paterno et al.69,70,71 found that training experiences that improve joint strength , rom and neuromuscular coordination , are also likely to lead to improved balance . this suggests that if there is impairment in neuromuscular coordination , reduction in the strength , rom , and the balance is likely to be affected . further olmsted et al.67 believed that dynamic postural impairment may be influenced by impaired neuromuscular control , proprioception , rom and joint strength . palmieri et al.72 suggested that balance is influenced by sensory information obtained from the somatosensory , visual , and vestibular systems and motor responses that affect coordination , joint range of motion ( rom ) , and strength . previous researches suggest that eccentric exercise causing fatigue lead to desensitization of intramuscular receptors ( muscle spindles and golgi tendon organs ) which consequently result in balance impairment35 , 73 , 74 . moreover , the resting tension of the muscle is altered by repeated muscle contractions during sprints thereby , further modifying the proprioceptive control74 , 75 . studies have alluded that muscle damage results in change in kinaesthesia34 , muscle spindle activity76 and reflex sensitivity77 which increases muscle stiffness affecting performance of skilled movements . these findings on proprioceptive and kinaesthetic control concur with the results of the present study that also recorded deterioration in balance from 2448 hours . investigations studying the central responses to eccentric exercise state that soreness significantly influences proprioception74 , 78 . muscle strength showed a significant drop at 24 hours , gradually recovering at 48 hours and continuing up to 72 hours , in both quadriceps and hamstring . in this study force loss showed the same temporal trend of recovery as that of balance performance . hence , it may be that impairment in balance is because of a reduction in maximum voluntary contraction of quadriceps and hamstrings . hesari et al.68 demonstrated that there was co - contraction of the quadriceps and hamstring during all directions of excursion . the quadriceps muscle showed maximum activation during the anterior excursion ; vastuslateralis during the posteromedial excursion and biceps femoris during the posterolateral excursion . in the current study , the decline in activity of both quadriceps and hamstrings might have led to decrements in balance performance as shown by a decrease in the normalized reaching distance . the results also demonstrated decreased in rom at 24 hours , the rom remains decreased even at 48 and 72 hours following eimd . gribble et al.79 in their review article suggests that knee movements in the sagittal plane strongly influence sebt performance . differences in sagittal plane knee rom have been demonstrated among the different reach directions80 . the anterior , and posteromedial directions produced more knee flexion and it was less in the posterolateral direction . therefore , we suggest that alteration in rom as found in the present study might have affected the normalized reach distances . the findings of this study would benefit sports physiotherapist , athletic and fitness trainers quantify balance after eimd in order to reduce musculoskeletal injuries and improve performance . the study highlights the fact that even trained and reasonably well - conditioned players still experience eimd , despite being familiar to the exercise stress . athletes should make effort to prevent the onset of eimd that might compromise the efficiency of training . in eimd , an adequate recovery ( 72 hours ) with regeneration further application of this study is that athletic trainers may use these tests to check for athletes readiness for return to sports . the stork balance test and sebt measurements may be considered as limitations of this study because they could not quantify postural sway . force platform is considered as gold standard for measuring static balance81 and although no gold standard has been defined for dynamic balance , more sophisticated techniques such as dynamic postural control index and the time - to - stabilization test for dynamic balance are available . additionally , specific components of balance ( e.g. , proprioception , vision ) were also not examined in this study . future researches must be conducted to address these issues and include ultrasonography , mri images , and emg techniques to establish an advanced understanding of the physiological manifestations of eimd . other blood markers such as plasma interleukines , tnf- and crp may be used for a comprehensive evaluation of the inflammatory responses after eimd . in the conclusion , both the physical and balance performance studied were affected following repeated sprint protocol in soccer players . similar changes were found in ck activity , mvc , rom and balance performance with time suggesting a correlation between the biochemical markers and neuromuscular changes . moreover , the study found eimd can be induced even in an athletic population ; therefore , the trainer should be careful while adding intense eccentric contractions during the training . it is recommended that the balance performance of an athlete be continually assessed following eimd so as to determine the appropriate return to sport decision thereby , minimizing the risk of further injury .

[ purpose ] the present study aimed to determine the changes in physical and balance performance following exercise - induced muscle damage using a sport - specific protocol . [ subjects and methods ] fifteen collegiate soccer players were asked to perform a sport - specific sprint protocol to induce muscle damage . the markers of muscle damage ( soreness , range of motion , limb girth , muscle strength , creatine kinase and lactate dehydrogenase ) , physical performance ( speed , agility and power ) and balance ( static and dynamic balance ) were assessed at baseline and 24 , 48 and 72 hours following the sprint protocol . [ results ] all variables , including the markers of muscle damage , physical performance and balance showed a significant difference when assessed at the 4 time points . [ conclusion ] the study demonstrated that both the physical and balance performance were affected following repeated sprint protocol in soccer players . it is recommended the balance performance of an athlete be continually assessed following exercise - induced muscle damage so as to determine the appropriate return to sport decision thereby , minimizing the risk of further injury .

INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION

this temporary cellular damage occurring in the skeletal muscle after unaccustomed exercises is called exercise induced muscle damage ( eimd)7 which is characterized by the disruption of contractile and non - contractile proteins with the decomposition of sarcolemma8 , increased concentration of muscle protein in bloodstream9 , delayed - onset muscle soreness ( doms)10 , decreased range of motion11 , swelling12 , impaired proprioceptive function and neuromuscular control13 , and prolonged loss of muscle function14 . moreover , this skeletal muscle damage can affect performance through a reduction in the joint range of motion , pain , and peak torque15 . other researchers have also provided a useful model utilizing sports - specific repeated sprint sessions with a rapid deceleration to induce eimd in the hamstrings , quadriceps , and the calf muscles23 , 24 . the current study , therefore , utilizes this sports specific task to elicit eimd in soccer players . therefore , the players require good speed , power , agility , and balance for the optimal performance . several factors such as proprioceptive deficits , muscle weakness , muscle injury ( strain ) , and sports participation can affect balance29 , 31 . previous studies reported that muscle damage following eccentric exercises can change the kinesthetic sense34 , 35 . poor balance has been associated with an increased risk of injuries in a number of sports37 . however , there is a dearth of information as to how eimd influences balance , as well as speed , agility and power , which are essential components of not only training and competition but also prevention and rehabilitation of sports injuries . therefore , the purpose of the present study was to examine the changes in physical performance ( speed , power and agility ) and balance performance ( static and dynamic balance ) resulting from eimd following sport - specific repeated sprints in male collegiate soccer players . fifteen male college soccer players ( mean age , 21.3 2 years , height 172.2 6.6 cm , weight 66.1 11 kg and bmi 22.3 3.2 ) from jamia millia islamia , new delhi , india , participated in the study . all the subjects regularly participated in the collegiate soccer and did sport - specific training at least 4 times / week . participants were asked to refrain from competitive events , resistance exercise , and avoid use of alcohol , nutritional supplements , and non - steroidal anti - inflammatory drugs during the study duration . on the first day of testing , subject s height , weight , knee range of motion ( rom ) , thigh circumference and muscle strength for quadriceps and hamstrings ( mvic ) were measured in the human performance lab , centre of physiotherapy and rehabilitation sciences , jamia millia islamia . two milliliter blood samples were drawn from the antecubital vein to determine baseline creatine kinase ( ck ) and lactate dehydrogenase ( ldh ) levels . immediately thereafter , subjects were tested for physical performance parameters using the vertical jump , 20 meter ( m ) sprint and the illinois agility test , and static and dynamic balance . the criterion measures were recorded at 24 , 48 and 72 hours after the sprint protocol . muscle soreness , range of motion , thigh circumference , maximum voluntary contraction , ck , and ldh were assessed as markers of the muscle damage . the soreness in lower limb was assessed while squatting at a knee angle of approximately 90 degrees , using a 200 mm visual analogue scale with no soreness at one end and unbearably painful at the other18 . range of motion was measured for knee flexion using a plastic goniometer and universal landmarks ( lateral epicondyle of the femur , lateral malleolus and greater trochanter ) to ensure alignment38 . the serum ck and ldh activity was assessed by a spectrophotometer at 340 nm set wavelength using a ck kit ( nac act . crest biosystems , coral ) and ldh kit ( ldh , p - l , crest biosystems coral ) respectively . vertical jump performance , 20 m single sprint , and the agility ( illinois agility test ) were used to assess the physical performance . to assess the counter movement jump , participants stood fully erect , and following a verbal command , initiated a countermovement followed by a maximal vertical jump in one continuous motion , keeping their legs straight whilst airborne , but using their arms during the jump47 . the time ( in seconds ) for which the participant is able to maintain this position is indicative of his balance performance . dynamic balance was assessed using the modified star excursion balance test ( sebt ) , where 3 reach directions ( anterior , posteromedial , posterolateral ) were measured53 . data were assessed by a shapiro - wilk test for the normality of the distribution scores . to test for the difference across 4 assessments , a repeated measure anova time ( pre , 24 , 48 and 72 hours ) was employed . when the main effect of time was significant , a bonferroni test was employed as post hoc analysis to locate the time points having significant difference . the markers of muscle damage showed a significant effect for time ( table 1table 1.change in markers of muscle damage following exercise induced muscle damagevariablespre24 h48 h72 hmean semean semean semean sevasq0.0 0.086 5.61 * 115 6.94 * 73 4.80*vash0.0 0.058.3 6.80 * 81.3 7.80 * 47.6 4.70*mvcq258.2 15.56209.6 11.83 * 215.4 12.65 * 238.7 12.73mvch176.2 6.83147.8 5.84 * 149.2 8.83 * 161.5 8.10rom131.9 0.50124.1 0.68 * 127.5 0.83 * 131.9 0.58lg50.8 1.0752.2 1.15 * 52.1 1.17 * 50.9 1.07ck190.6 16.78620.7 61.0 * 502.4 51.7 * 341.5 38.7*ldh286.6 21.05563.8 52.7 * 467.7 42.5 * 392.6 18.3h : hours ; vasq : visual analogue scale quadriceps ; vash : visual analogue scale hamstrings ; mvcq : maximal voluntary contraction quadriceps ; mvch : maximal voluntary contraction hamstrings ; rom : range of motion knee ; lg : leg girth ; ck : creatine kinase ; ldh : lactate dehydrogenase ; significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) ) , as did the physical performance and balance variables ( table 2table 2.changes in balance and physical performance variables following exercise induced muscle damagevariablespre24 h48 h72 hmean semean semean semean sebalance performance variablessbtl47.7 1.6525.2 1.67 * 34 1.7747.3 1.77sbtr34.2 1.5521.7 1.58 * 26.7 1.6633 1.67antl79.8 2.7074.2 2.80 * 74.5 2.88 * 79 2.96antr80.4 2.9073.2 2.71 * 75.8 2.95 * 78.8 3.01platl102 1.4495.2 1.09 * 96.6 1.13 * 101.8 1.46platr99.6 1.4992.1 1.15 * 94.8 1.53 * 99.1 1.82pmedl99.2 1.3289.1 0.85 * 91.2 1.07 * 98.4 1.18pmedr98.9 1.2789.6 0.63 * 91.6 0.61 * 97.8 0.82physical performance variablessprint3.4 0.073.7 0.07 * 3.6 0.07 * 3.5 0.07iat17.4 0.2518.1 0.27 * 17.8 0.24 * 17.4 0.25sj42.8 1.3939.3 1.89 * 39.7 1.91 * 42.3 1.57cmj47.3 1.4741.8 2.18 * 42 1.94 * 45.5 1.53h : hours ; sbtl : static balance test left leg ; sbtr : static balance test right leg ; antl : star excursion balance test in anterior direction left leg ; antr : star excursion balance test in anterior direction right leg ; platl : star excursion balance test in postero - lateral direction left leg ; platr : star excursion balance test in postero - lateral direction right leg ; pmedl : star excursion balance test in postero - medial direction left leg ; pmedr : star excursion balance test in postero - medial direction right leg ; iat : ilionois agility test ; sj : squat jump ; cmj : counter - movement jump . moreover , the post hoc analyses revealed maximum differences between baseline and at 24 hours following eimd and non - significant differences between baseline values and those recorded at 72 hours ( table 2 ) . the paired t - test used to compare the scores for composite reach distances , indicated no significant difference between the right ( m=85.7 , sd=6.99 ) and left leg ( m=86.3 , sd=6.32 ) , p=0.336 . h : hours ; vasq : visual analogue scale quadriceps ; vash : visual analogue scale hamstrings ; mvcq : maximal voluntary contraction quadriceps ; mvch : maximal voluntary contraction hamstrings ; rom : range of motion knee ; lg : leg girth ; ck : creatine kinase ; ldh : lactate dehydrogenase ; significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) h : hours ; sbtl : static balance test left leg ; sbtr : static balance test right leg ; antl : star excursion balance test in anterior direction left leg ; antr : star excursion balance test in anterior direction right leg ; platl : star excursion balance test in postero - lateral direction left leg ; platr : star excursion balance test in postero - lateral direction right leg ; pmedl : star excursion balance test in postero - medial direction left leg ; pmedr : star excursion balance test in postero - medial direction right leg ; iat : ilionois agility test ; sj : squat jump ; cmj : counter - movement jump . significant effect of time ( pre , 24h , 48h , 72h ) using repeated measure anova ( p<0.05 ) ; * significant difference in comparison to baseline values , assessed using post hoc analysis ( p<0.05 ) objective of the present study was to examine whether eimd following sport - specific repeated sprints would affect the physical and balance performance in the male collegiate soccer players . more pronounced increase in the muscle soreness , limb circumference , plasmatic ck and ldh activity and a decrease in rom and maximal voluntary contraction provide indirect evidence to suggest that eimd was present following the sport - specific repeated sprints . indeed , all the variables chosen to examine the changes in physical and balance performance showed significant change after the protocol . although , a previous study has investigated the eimd using the markers of muscle damage and physical performance in athletes55 . however , to the best of our knowledge , till date , no study has investigated the effects of eimd on dynamic balance performance . this is the first study that comprehensively examined the pain sensation , biochemical , physical and balance performance in both dominant and non - dominant legs in athletes using a sport - specific protocol . muscle soreness , increased 24 hours after exercise , peaked at 48 hours and finally decreased at 72 hours in both quadriceps and hamstring , showing a similar trend as that observed by howatson et al.24 using the same protocol . the trend to rise in the soreness that peaked at 48 hours further , the rom remained decreased even at 48 and 72 hours following eimd . however , earlier studies have suggested that shortened non - contractile components , change in calcium homeostasis due to muscle damage , decreased strength , and/or swelling may be responsible for decreased rom12 , 60 . thigh circumference demonstrated a peak increase at 24 hours ( 3.2% ) , consistent with the findings of howatson et al.24 who used the same protocol and reported an increase in limb girth of 3% at 24 hours . the present study demonstrated reduction of swelling at 48 hours ( 2.5% ) and at 72 hours ( 0.2% ) . maximal voluntary contraction showed a significant drop at 24 hours , gradually recovering at 48 hours and the recovery continuing up to 72 hours in both quadriceps and hamstrings . ck showed peak activity at 24 hours following exercise , gradually recovering at 48 and 72 hours . the ck response peaking at 24 h post - exercise was in accordance with previous study using a similar protocol to induce damage47 , 61 . ldh showed a rise of 68% the baseline values at 24 hours , gradually decreasing to 40.2% and 1% from the baseline at 48 and 72 hours , respectively . armstrong et al.63 reported that ldh showed a significant secondary late phase elevations at 2448 hours post downhill running . at 72 hours , the sprint times these findings are consistent with highton et al.7 and twist and eston57 , who reported that sprint times was significantly increased at 24 and 48 hours following eimd before returning to baseline levels at 72 hours . this is similar to the of study done by highton et al.7 who has reported a significant reduction in the agility performance following plyometric exercise and suggested that the reduction in agility performance may be due to a reduction in both strength and running speed . vertical jump demonstrated a significant drop in both squat and counter - movement jump performance following repeated sprints . the squat jump performance showed a decrease of 7.1% in both 24 and 48 hours and 1.1% at 72 hours and the counter - movement jump performance showed a decline of 11.6% at 24 hours , 11.1% at 48 hours and the result of our study is in accordance with the study done by sarabon et al.65 , which showed a more pronounced drop in counter - movement jump height in relation to squat jump height 24 hours following eimd . static balance revealed similar results to the study of twist et al.66 who reported a significant impairment in unilateral balance from baseline at 24 hours following plyometric exercise and no significant impairment at 48 and 72 hours following plyometric exercise . the results suggest that the dynamic balance recorded by sebt declined significantly at 24 hours in all the directions . the balance recovered significantly in posteromedial and posterolateral directions and non - significantly in anterior directions at 48 hours . at 72 hours balance late recovery in anterior directions may be because the muscle damage was more in the quadriceps which is found to be more active during the anterior directions68 . to perform the anterior direction , gravity action on the upper body creates a larger knee - flexion moment , which must be controlled by the extension moment produced by the quadriceps . this suggests that if there is impairment in neuromuscular coordination , reduction in the strength , rom , and the balance is likely to be affected . palmieri et al.72 suggested that balance is influenced by sensory information obtained from the somatosensory , visual , and vestibular systems and motor responses that affect coordination , joint range of motion ( rom ) , and strength . studies have alluded that muscle damage results in change in kinaesthesia34 , muscle spindle activity76 and reflex sensitivity77 which increases muscle stiffness affecting performance of skilled movements . these findings on proprioceptive and kinaesthetic control concur with the results of the present study that also recorded deterioration in balance from 2448 hours . muscle strength showed a significant drop at 24 hours , gradually recovering at 48 hours and continuing up to 72 hours , in both quadriceps and hamstring . in this study force loss showed the same temporal trend of recovery as that of balance performance . in the current study , the decline in activity of both quadriceps and hamstrings might have led to decrements in balance performance as shown by a decrease in the normalized reaching distance . the results also demonstrated decreased in rom at 24 hours , the rom remains decreased even at 48 and 72 hours following eimd . therefore , we suggest that alteration in rom as found in the present study might have affected the normalized reach distances . in eimd , an adequate recovery ( 72 hours ) with regeneration further application of this study is that athletic trainers may use these tests to check for athletes readiness for return to sports . in the conclusion , both the physical and balance performance studied were affected following repeated sprint protocol in soccer players . similar changes were found in ck activity , mvc , rom and balance performance with time suggesting a correlation between the biochemical markers and neuromuscular changes . it is recommended that the balance performance of an athlete be continually assessed following eimd so as to determine the appropriate return to sport decision thereby , minimizing the risk of further injury .

the online version of this article ( doi:10.1007/s13353 - 012 - 0127 - 8 ) contains supplementary material , which is available to authorized users . preharvest sprouting ( phs ) is a major factor negatively affecting the quality of crops in years with high precipitation during a period of grain maturation . consequently , selection for genotypes showing no precocious germination in conditions of water stress is one of the priorities in many breeding programmes . this goal is rather difficult to achieve because phs is a complex trait largely dependent on weather conditions ( intensity and length of rain periods , profiles of temperatures ) and key genes for strong phs resistance have not yet been cloned . genomic studies carried out in major cereal species lead to the conclusion that phs and dormancy have a complex genetic basis , with numerous quantitative trait loci ( qtl ) distributed across the majority of chromosomes ( gale et al . 2012 ) dissected phs and dormancy in wheat into 18 chromosome bins , each containing a cluster of several genes . phs - related genes , even if they are clustered with other loci , may be identified by their different expression at the mrna and/or protein level in phs - resistant and phs - susceptible grain , since these contrasting phenotypes should be determined by specific profiles of genes expression and protein activities during development . this hypothesis was partially proved in wheat ( bykova et al . 2011 ) and rye ( masoj and kosmala 2012 ) by using two - dimensional gel electrophoresis ( 2-de ) and mass spectrometry ( ms ) to compare protein composition in mature grain of phs - resistant and phs - susceptible lines derived from a single cross . both investigations revealed associations between phs resistance and the abundance of specific proteins involved in responses to biotic and abiotic stresses , including oxidative stress . oxidative stress caused by the accumulation of reactive oxygen species ( ros ) in embryo cells was shown to be correlated with dormancy alleviation and germination ( oracz et al . this may explain the different accumulation of ros - scavenging enzymes in phs - resistant and phs - susceptible wheat ( bykova et al . rye proteome ( masoj and kosmala 2012 ) and genome ( masoj and milczarski 2009 ; masoj et al . 2009 ) studies show that phs resistance has a complex genetic basis involving at least 24 proteins of various functions and numerous qtl distributed on each of the seven chromosomes . this knowledge should be enriched with proteomic studies carried out on different stages of grain development . it is expected that proteome analysis of developing grain will generate more information on molecular mechanisms affecting predisposition to phs . this investigation is aimed at finding differences in protein composition between phs - resistant and phs - susceptible lines of rye at the early stages of grain development . two sets of rye recombinant inbred lines ( rils ) , 20 phs - resistant lines ( 0 % sprouting in spikes after 7 days of water spray at maturity ) and 20 phs - susceptible lines ( 90100 % sprouting in spikes after 7 days of water spray at maturity ) , were grown on the experimental field of the west pomeranian university of technology , szczecin in 2011 . these lines represented f10 generation of rils developed from the 541 ot1 - 3 intercross described in earlier studies ( masoj et al . two spikes per line were immersed in liquid nitrogen at the 25th day after anthesis ( 25 dpa ) and kept at 30 c for 1 month . frozen kernels with green pericarp and milky endosperm ( 10 per line ) were threshed out from spikes and freeze - dried in an alpha 1 - 2 ld plus freeze drier ( christ ) . dry kernels of the phs - resistant group of lines were bulked and ground in a retsch mm 200 mill . kernels from the phs - susceptible group of lines were bulked and ground in the same mill . two bulked samples of the rye flour representing 20 phs - resistant and 20 phs - susceptible lines were kept in a freezer prior to protein extraction . the proteomic work involved : ( 1 ) the analyses of rye grain protein abundance in the bulked samples of the resistant and the susceptible rye inbred lines using 2-de , ( 2 ) ms identification of proteins which were differentially accumulated between the analysed bulks . the protocol for proteomic research was the same as that described in detail by masoj and kosmala ( 2012 ) . protein extraction was performed according to the method described by hurkman and tanaka ( 1986 ) . this method was shown earlier to be useful to extract the proteins from different species and its use resulted in high - quality 2-d maps with proteins derived from different cell components , including membranes , chloroplasts , mitochondria and cytoplasm ( kosmala et al . briefly , the powdered tissue was homogenised with 500 l of extraction buffer ( 0.7 m sucrose , 0.5 m tris , 30 mm hcl , 50 mm edta , 2 % dtt , 0.1 m kcl ) . an equal volume of water - saturated phenol was then added and the sample was incubated for 5 min at 4 c , followed by 10 min of vortexing and centrifuging at 8,700 g for 30 min . the phenol phase ( upper ) was recovered and re - extracted with an equal volume of extraction buffer . after centrifuging , the proteins from the phenol phase were precipitated by the addition of five volumes of cold 0.1 m ammonium acetate in methanol and then incubated at 20 c at least overnight . the sample was then centrifuged at 20,500 g at 0 c for 30 min , the precipitate washed twice with the cold ammonium acetate in methanol and once in cold acetone , and dried . the pellet was finally dissolved in 100 l of the sample solution ( 7 m urea , 2 m thiourea , 2 % np-40 , 2 % ipg buffer ph range 47 or 310 , 40 mm dtt ) . the protein concentration was determined by the use of a 2-d quant kit ( ge healthcare ) . the aliquots of proteins extracted from 25 mg of rye flour were mixed with rehydration solution ( 7 m urea , 2 m thiourea , 2 % np-40 , 0.5 % ipg buffer ph range 47 , 0.002 % bromophenol blue , 18 mm dtt ) to the final volume of 450 l and used for 2-de , performed according to hochstrasser et al . this range was selected as the standard condition for resolving the proteins after the pre - selection performed with a relatively broad ph range ( 310 ) ( data not shown ) and followed our earlier work ( masoj and kosmala 2012 ) . rehydration and focusing was carried out in ettan ipgphor ii ( ge healthcare ) at 50 a per strip at 20 c , applying the following programme : 12 h of rehydration at 0 v and 9 h of focusing at 1 h/500 v , 2 h/1,000 v and 6 h/8,000 v. after ief , the strips were equilibrated for 15 min in sds equilibration buffer solution ( 6 m urea , 75 mm tris hcl ph = 8.8 , 29.3 % glycerol , 2 % sds , 0.002 % bromophenol blue , 65 mm dtt ) , followed for 15 min with the same buffer but containing 135 mm iodoacetamide instead of dtt . after equilibration , the proteins were separated in the second dimension , sodium dodecyl sulphate polyacrylamide gel electrophoresis ( sds - page ) , using 13 % polyacrylamide gels ( 1.5 255 196 mm ) at 4 w / gel for 30 min and then at 17 w / gel for 4 h. rainbow molecular weight marker ( ge healthcare ) was used as a standard to determine the molecular weights ( mws ) of proteins for particular spots . following electrophoresis , the gels were stained with colloidal coomassie brilliant blue g-250 , using the modified method of neuhoff et al . total separated protein spots on the gels were scanned by an imagescanner iii ( ge healthcare ) and subjected to processing by the labscan 6.0 program ( ge healthcare ) . spot detection and image analyses ( normalisation , spot matching , accumulation comparison ) were performed with the imagemaster 2d platinum software package ( ge healthcare ) . to compensate for subtle differences in sample loading , gel staining and destaining , the abundance of each protein spot was normalised as a relative volume ( % vol ) . the % vol of each spot was automatically calculated by the imagemaster software as a ratio of the volume of a particular spot to the total volume of all the spots present on the gel . the extraction procedure and electrophoretic separation were performed twice ( technical replicates ) , and the % vol for the spots from the two replicated gels were then used to calculate the means , which were used to make comparisons between the analysed rye lines . the protein spots which showed at least 2-fold ( p < 0.05 ) differences in protein abundance between two analysed lines ( quantitative analysis ) together with protein spots present only in one of the analysed lines ( qualitative analysis ) were subjected to ms analysis and identification . protein spots were excised from the gel and analysed by liquid chromatography coupled to the mass spectrometer in the laboratory of mass spectrometry , institute of biochemistry and biophysics , polish academy of sciences ( warsaw , poland ) . samples were concentrated and desalted on an rp - c18 pre - column ( waters ) and further peptide separation was achieved on a nano - ultra performance liquid chromatography ( uplc ) rp - c18 column ( waters , beh130 c18 column , 75 m i.d . , 250 mm long ) of a nanoacquity uplc system , using a 45-min linear acetonitrile gradient . the column outlet was directly coupled to the electrospray ionisation ( esi ) ion source of an orbitrap - type mass spectrometer ( thermo ) , working in the regime of data - dependent ms to ms / ms switch . raw data files were pre - processed with mascot distiller software ( version 2.3 , matrixscience ) . the obtained peptide masses and fragmentation spectra were matched to the national center biotechnology information ( ncbi ) non - redundant database with a viridiplantae filter ( 1,032,142 sequences ) using the mascot search engine ( mascot daemon v. 2.3 , mascot server v. 2.2.03 , matrixscience ) . the following search parameters were applied : enzyme specificity was set to trypsin , peptide mass tolerance to 40 ppm and fragment mass tolerance to 0.8 da . the protein mass was left as unrestricted and mass values as monoisotopic , with one missed cleavage being allowed . alkylation of cysteine by carbamidomethylation as fixed and oxidation of methionine was set as a variable modification . protein identification was performed using the mascot search probability - based mowse score . the ions score was 10 * log(p ) , where p was the probability that the observed match was a random event . the mascot - defined threshold , which indicated identity or extensive homology ( p < 0.05 ) , was 40 or less , therefore , an ion score of 40 was taken as a threshold for analysis . the proteins with the highest multidimensional protein identification technology ( mudpit ) scores and/or the highest number of peptide sequences were selected . if more than one reliable identification appeared in the single spot , they were all indicated . however , in these cases , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was shown . further research to estimate detailed protein contents of such multi - protein spots would be required according to , for example , the method described by ishihama et al . when the protein was identified as predicted protein , its amino acid sequence was blasted using the blastp algorithm . the protein with the highest score was then selected as the functional homologue of the predicted protein . following our earlier work ( masoj and kosmala 2012 ) , the ph = 47 range was selected as the standard condition for resolving the proteins to achieve the best compromise between the number and resolution of the separated spots . only the spots which were detected within two replicate gels out of all protein spots separated on 2-de gels , 27 showed significant quantitative differences between phs - resistant and phs - susceptible lines ( figs . 1 and 2 ) . thirteen of them ( spot nos . 2 , 3 , 4 , 5 , 6 , 9 , 10 , 11 , 14 , 16 , 18 , 21 , 22 ) were 24.1-fold more abundant in phs - susceptible lines than in phs - resistant lines . fourteen others ( spot nos . 1 , 7 , 8 , 12 , 13 , 15 , 17 , 19 , 20 , 23 , 24 , 25 , 26 , 27 ) were 24.3-fold more abundant in phs - resistant lines . two protein spots ( nos . 29 and 30 ) were found only in phs - susceptible lines ( fig . 1two - dimensional gel electrophoresis ( 2-de ) map of proteins from developing rye grain [ 25th day after anthesis ( 25 dpa ) ] of 20 recombinant inbred lines ( f10 ) highly resistant to pre - harvest sprouting ( phs ) , derived from the 541 ot1 - 3 cross . proteins were separated by isoelectric focusing ( ief ) at ph range 47 , followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate ( sds - page ) and staining with colloidal coomassie brilliant blue g-250 . spot detection and image analyses ( normalisation , spot matching , protein accumulation analyses ) were performed using the imagemaster 2d platinum software package ( ge healthcare ) . the normalised volumes of matched spots were used for comparisons made between the phs - resistant and phs - susceptible secale cereale lines ( in the case of each spot , the means derived from two replicated gels were applied ) . 28 ) present only in phs - resistant lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel . the molecular weight ( mw ) and ph scales are shownfig . 22-de map of proteins from developing rye grain ( 25 dpa ) of 20 recombinant inbred lines ( f10 ) highly susceptible to phs , derived from the 541 ot1 - 3 cross . 29 and 30 ) present only in phs - susceptible lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel two - dimensional gel electrophoresis ( 2-de ) map of proteins from developing rye grain [ 25th day after anthesis ( 25 dpa ) ] of 20 recombinant inbred lines ( f10 ) highly resistant to pre - harvest sprouting ( phs ) , derived from the 541 ot1 - 3 cross . proteins were separated by isoelectric focusing ( ief ) at ph range 47 , followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate ( sds - page ) and staining with colloidal coomassie brilliant blue g-250 . spot detection and image analyses ( normalisation , spot matching , protein accumulation analyses ) were performed using the imagemaster 2d platinum software package ( ge healthcare ) . the normalised volumes of matched spots were used for comparisons made between the phs - resistant and phs - susceptible secale cereale lines ( in the case of each spot , the means derived from two replicated gels were applied ) . 28 ) present only in phs - resistant lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel . the molecular weight ( mw ) and ph scales are shown 2-de map of proteins from developing rye grain ( 25 dpa ) of 20 recombinant inbred lines ( f10 ) highly susceptible to phs , derived from the 541 ot1 - 3 cross . two protein spots ( spot nos . 29 and 30 ) present only in phs - susceptible lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel ms of individual spots revealed that the majority of them were homogenous , containing one specific protein ( table 1 ) . however , spot nos . 3 , 4 , 5 , 6 , 7 , 9 , 10 , 11 , 22 , 25 , 26 , 27 and 28 were found to be heterogeneous , with 2 or 3 ( in the case of spot no . 10 ) different proteins identified in each spot . in these spots , the relative abundance of particular proteins was not established . in total , 44 proteins were found in 30 spots , showing an association with phs.table 1the results of mass spectrometry ( ms ) performed on the selected protein spots representing developing grain [ 25th day after anthesis ( 25 dpa ) ] of preharvest sprouting ( phs)-resistant ( rl ) and phs - susceptible ( sl ) recombinant inbred lines of ryespot no.accessionidentified proteincell pathway / molecular functionscore / coverageprotein abundance1baj93636predicted protein ( hordeum vulgare subsp . % 2.1 higher in rlrubisco large subunit - binding protein subunit alpha , chloroplastic - like ( brachypodium distachyon ) ; xp_003558045carbon fixation2q43772utp vulgare)l - ascorbate biosynthesis1,942/63 % 3.7 higher in slsucrose biosynthesis3p12783phosphoglycerate kinase , cytosolic ( triticum aestivum)glycolysis , sucrose biosynthesis , xylose degradation220/12 % 2.1 higher in slp17614atp synthase subunit beta , mitochondrial ( nicotiana plumbaginifolia)energy metabolism , transport of ions212/10 % 4baj90922predicted protein ( hordeum vulgare subsp . vulgare)lysine biosynthesis557/22 % 2.6 higher in slaspartate - semialdehyde dehydrogenase - like ( brachypodium distachyon ) ; xp_003559131baj88969predicted protein ( hordeum vulgare subsp . vulgare ) succinyl - coa ligase ( adp - forming ) subunit beta , mitochondrial - like ( brachypodium distachyon ) ; xp_003575382the citric acid cycle ( energy metabolism)548/23 % 5baj88969predicted protein ( hordeum vulgare subsp . vulgare ) succinyl - coa ligase ( adp - forming ) subunit beta , mitochondrial - like ( brachypodium distachyon ) ; xp_003575382the citric acid cycle ( energy metabolism)457/23 % 2.5 higher in slaco44683fructose - bisphosphate aldolase ( secale cereale)glycolysis , sucrose biosynthesis359/25 % 6p93693serpin - z1b ( triticum aestivum)storage protein and defence against insect pathogens1,425/20 % 2.2 higher in slbak02194predicted protein ( hordeum vulgare subsp . vulgare ) heat shock cognate 70-kda protein - like ( brachypodium distachyon ) ; xp_003558220protection against abiotic stress618/17 % 7bak04960predicted protein ( hordeum vulgare subsp . vulgare ) lea ( late embryogenesis abundant ) protein d-34-like ( brachypodium distachyon ) ; xp_003562052protection against dessication527/16 % 2.4 higher in rlcaa7459214 - 3 - 3 protein ( hordeum vulgare)other protein activity and targeting396/27 % 8aab99745hsp70 ( triticum aestivum)protection against abiotic stress436/14 % 2.0 higher in rl9bak04746predicted protein ( hordeum vulgare subsp . vulgare ) vicilin - like antimicrobial peptides 2 - 2-like ( brachypodium distachyon ) ; xp_003561946defence2,135/11 % 3.6 higher in slxp_003563776ras - related protein ric2-like ( brachypodium distachyon)gtp - binding protein628/45 % 10bak04746predicted protein ( hordeum vulgare subsp . vulgare ) vicilin - like antimicrobial peptides 2 - 2-like(brachypodium distachyon ) ; xp_003561946defence734/4 % 4.1 higher in slaay43813beta1 proteasome-7d ( aegilops tauschii)protein degradation384/31 % baj85410predicted protein ( hordeum vulgare subsp . vulgare ) lea ( late embryogenesis abundant ) protein , group 3-like ( brachypodium distachyon ) ; xp_003569641protection against cold stress445/22 % 11ace82290peroxiredoxin ( triticum aestivum)antioxidant and ros - scavenger pathway445/41 % 3.1 higher in slq9frv0basic endochitinase c ( secale cereale)defence346/21 % 12ace82290peroxiredoxin ( triticum aestivum)antioxidant and ros - scavenger pathway855/48 % 3.9 higher in rl13bak03707predicted protein ( hordeum vulgare subsp . vulgare ) hypothetical protein sorbidraft_10g010410 ( sorghum bicolor ) ; xp_002436874protection against cold stress345/34 % 2.9 higher in rl14p1281016.9-kda class i heat shock protein 1 ( triticum aestivum)molecular chaperone / defence2006/43 % 2.2 higher in sl15p1281016.9-kda class i heat shock protein 1 ( triticum aestivum)molecular chaperone / defence404/43 % 3.2 higher in rl16cad42633immunophilin ( hordeum vulgare subsp . vulgare]defence416/36 % 2.9 higher in sl17acp40912dimeric alpha - amylase inhibitor ( secale cereale)defence369/59 % 2.5 higher in rl18aaz67071cereal - type amylase inhibitor , partial ( secale cereale)defence591/40 % 2.2 higher in sl19baf30986glycine - rich rna - binding protein ( triticum aestivum)gene expression on post - transcriptional level787/36 % 2.7 higher in rl20acp40912dimeric alpha - amylase inhibitor ( secale cereale)defence485/59 % 4.3 higher in rl21abo45934monomeric alpha - amylase inhibitor , partial ( triticum monococcum)defence1,287/51 % 2.5 higher in sl22acq83667monomeric alpha - amylase inhibitor ( triticum dicoccoides)defence214/26 % 3.4 higher in slacp40912dimeric alpha - amylase inhibitor ( secale cereale)defence160/17 % 23cab88093early - methionine - labelled polypeptide ( secale cereale)unknown608/81 % 2.3 higher in rl24cae46332xylanase inhibitor , partial ( secale cereale)defence601/26 % 2.8 higher in rl25yp_001561420elongation factor tu ( delftia acidovorans sph-1)transport of t - rna to ribosomes / molecular chaperone278/12 % 2.6 higher in rleef09621predicted protein , partial ( populus trichocarpa ) chaperonin groel ( delftia acidovorans sph-1 ) ; yp_001566674molecular chaperone210/14 % 26eef09621predicted protein , partial ( populus trichocarpa)molecular chaperone290/14 % 3.8 higher in rlchaperonin groel ( delftia acidovorans sph-1 ) ; yp_001566674acg37173histone h4 ( zea mays)chromatin structure and interaction with proteins231/31 % 27p16062subtilisin - chymotrypsin inhibitor ci-1a ( hordeum vulgare subsp . tu ( delftia acidovorans sph-1)transport of t - rna to ribosomes / molecular chaperone181/8 % 28p46226triosephosphate isomerase , cytosolic ; ( secale cereale)glycolysis111/9 % present only in rlp16062subtilisin - chymotrypsin inhibitor ci-1a ( hordeum vulgare subsp . vulgare)protection against dessication403/15 % present only in slembryonic protein dc-8-like ( brachypodium distachyon ) ; xp_00358051930acg37173histone h4 ( zea mays)chromatin structure and interaction with proteins297/35 % present only in slspot numbering was the same as in figs . 1 and 2database accession ( according to ncbinr ) of a homologous proteinhomologous protein and organism from which it originates , identified also with the help of the blastp algorithm ( xp _ , yp _ s1protein abundance was calculated using the mean of relative volumes ( % vol ) of two replicates of particular protein spotsif more than one reliable identification appeared in the single spot , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was compared . the names of identified proteins which were also found to be associated with phs in wheat by bykova et al . ( 2011 ) are underlined and the names of those revealed also by rehman arif et al . proteins showing an association with phs both in mature ( masoj and kosmala 2012 ) and in developing rye grain have accession numbers in bold the results of mass spectrometry ( ms ) performed on the selected protein spots representing developing grain [ 25th day after anthesis ( 25 dpa ) ] of preharvest sprouting ( phs)-resistant ( rl ) and phs - susceptible ( sl ) recombinant inbred lines of rye spot numbering was the same as in figs . 1 and 2 database accession ( according to ncbinr ) of a homologous protein homologous protein and organism from which it originates , identified also with the help of the blastp algorithm ( xp _ , yp _ annotations ) score / coverage for the primary identifications indicated also in supplementary fig . s1 protein abundance was calculated using the mean of relative volumes ( % vol ) of two replicates of particular protein spots if more than one reliable identification appeared in the single spot , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was compared . the names of identified proteins which were also found to be associated with phs in wheat by bykova et al . ( 2011 ) are underlined and the names of those revealed also by rehman arif et al . proteins showing an association with phs both in mature ( masoj and kosmala 2012 ) and in developing rye grain have accession numbers in bold a predominant function of proteins more abundant in phs - susceptible lines was defence against pathogens , as shown by serpin - z1b ( spot no . 6 ) , cereal - type alpha - amylase inhibitor ( spot no . 18 ) , monomeric alpha - amylase inhibitor ( spot nos . 11 ) and vicilin - like antimicrobial peptides ( spot nos . 9 and 10 ) . sensitivity to phs was also associated with a higher amount of several enzymes that sustain energy metabolism ( spot nos . 3 , 4 and 5 ) , 6 , 14 and 29 ) and act as ros scavengers ( spot nos . 2 , 11 and 29 ) . sprouting - resistant lines contained higher amounts of proteins protecting against : desiccation ( spot no . 7 ) , heat shock and other abiotic stresses ( spot nos . 8 , 13 , 25 and 26 ) and pests ( spot nos . 17 and 20 ) , subtilisin - chymotrypsin inhibitor and triosephosphate isomerase ( spot nos . 27 and 28 ) , glycine - rich rna - binding protein ( spot no . there were examples of the same proteins present in different spots , of which one molecular form was more abundant in phs - resistant lines and another was associated with phs - susceptible lines ( 16.9-kda class i heat shock protein 1 , spot nos . 11 and 12 ; histone h4 , spot nos . 26 and 30 ) . on the other hand , multiple spots for one protein more abundant in phs - resistant lines ( dimeric alpha - amylase inhibitor , spot nos . 25 and 27 ) or in phs - susceptible lines ( monomeric alpha - amylase inhibitor , spot nos . 21 and 22 ; vicilin - like antimicrobial peptides , spot nos . 9 and 10 ) were also found . the majority of the detected proteins were involved in response to abiotic and biotic stresses and in energy metabolism , which suggest that predisposition to phs is , above all , a genetically determined molecular mechanism of response to various kinds of stresses acting during grain desiccation followed by prolonged contact with water . since the inability of mature grain to germinate in water - sprayed spikes showed by phs - resistant lines should be imposed by the genetic control of down- and/or up - regulation of genes expression , it is not surprising that some regulatory proteins were revealed in this study . they were found in spot no . 7 ( 14 - 3 - 3 regulatory protein ) , 9 ( ras - related protein ric2-like ) , spot no . 19 ( glycine - rich rna - binding protein ) and in spot nos . 25 and 27 ( elongation factor tu ) . 10 is another example of a protein whose proteolytic activity may influence other proteins composition and activity within different parts of developing grain . the consequences for gene expression on the genomic scale might result from apparently substantial differences in the abundance of histone h4 isoforms , which control chromatin structure and dna protein interaction . 30 ) was found only in phs - susceptible lines , whereas the second ( spot no . 26 ) was shown to be more abundant in phs - resistant lines . at the present stage of knowledge on molecular mechanisms leading to phs resistance , it is difficult to explain the different abundances of three other proteins in the grain of phs - susceptible and phs - resistant lines . a single molecular form of rubisco ( spot no . 1 ) , a basic enzyme for carbon fixation during photosynthesis taking place in the green pericarp of developing grain , showed a relatively high level of accumulation in the phs - resistant group of lines . 23 ) was found to be associated with high resistance to phs . on the other hand , the developing grain of phs - susceptible lines contained more aspartate - semialdehyde dehydrogenase ( spot no . differences in protein composition of the grain at 25 dpa , found between the two groups of rye lines with extremely different predispositions to phs , clearly show that the process of preparing grain to resist germination in mature spike during the rainy season starts in early development ( this paper ) and continues up to the end of grain maturation ( masoj and kosmala 2012 ) . proteomes of phs - resistant and phs - susceptible grain revealed more differences in protein abundance 25 days post - anthesis than at the maturity stage . there were , however , several common features of the phs - related protein patterns at both stages of grain development . in both studies , phs - resistant grain contained higher levels of dimeric alpha - amylase inhibitor and phs - susceptible grain had increased levels of atp synthase , serpin - z1b and monomeric alpha - amylase inhibitor . differences in the abundance of 16.9-kda class i heat - shock protein , triosephosphate isomerase and 14 - 3 - 3 regulatory protein were depicted in both studied stages of seed formation . generally , both young and mature seeds of phs - resistant and phs - susceptible lines showed differentiation in respect to proteins responding to biotic and abiotic stresses , including oxidative stress . the set of differentially accumulated proteins related to biotic stress showed higher complexity in developing seeds . similarly , a more complex set of proteins affecting genes expression and protein synthesis and degradation rates was associated with phs at the early stage of grain development . this observation raises the question about the significance of transiently expressed differences in protein abundance during grain development for attaining phs resistance or susceptibility at maturity . there are two possible answers : ( 1 ) differences observed at 25 dpa persist until seed maturity but they decrease below the 2-fold threshold value for 2-de analysis and ( 2 ) differences in concentrations of particular proteins decline with the time but they have a long - lasting impact on the grain properties . similarly to in a previous study ( masoj and kosmala 2012 ) , individual proteins associated with phs were detected in different spots separated by means of 2-de . various reasons might underlie the multiple molecular forms of proteins , including multi - gene families ( isoforms ) , heterozygosity ( allozymes ) , post - translational modifications or even protein degradation during sample preparation . change in protein abundance due to different frequencies of allelic variants within each of the two extreme groups of lines would be an indirect proof for the involvement of respective structural genes in phs resistance / susceptibility . verification of allelic forms among 2-de protein spots should be possible by comparing patterns of individual rils , including parental lines . it is known that resistance to phs can be reduced when seed coat is ruptured by pests at any stage of development . the persistence of dimeric alpha - amylase inhibitor in higher amounts in developing and mature grain of phs - resistant lines and higher amounts of xylanase inhibitor and subtilisin - chymotrypsin inhibitor at 25 dpa gives strong evidence that the accumulation of these particular defence proteins is important for the phs resistance of rye . different sets of defence proteins seem to be important for phs susceptibility , since it is associated with a higher abundance of serpins and monomeric alpha - amylase inhibitor at both 25 dpa and maturity , and with increased levels of immunophilin and endochitinase c at 25 dpa and tritin at maturity . another specific feature of phs - susceptible lines detected in both proteomic studies of rye grain is the relatively higher accumulation level of some proteins related to energy metabolism , including those involved in the citric acid cycle and in glycolysis . this is , above all , atp synthase overproduced at 25 dpa and at maturity , glucose and ribitol dehydrogenase , triosephosphate isomerase and phosphoglucomutase ( maturity ) , phosphoglycerate kinase , succinyl - coa ligase , fructose bisphosphate aldolase ( at 25 dpa ) . elevated synthesis of these proteins might be necessary to achieve unusually high rates of germination observed in phs - susceptible rils . a developmental programme of predisposing grain to phs can also be based on different rates of protein degradation due to the activity of proteasomes . this hypothesis is supported by the detection of beta1 proteasome 7d protein among those which are up - regulated in phs - susceptible lines at 25 dpa . a higher level of anti - oxidant protection is seemingly attributed to the grain of phs - susceptible lines , which accumulated ros - scavenging enzymes , such as : glutathione transferase , dehydroascorbate reductase and superoxide dismutase ( mature grain ) , but also produced possibly more l - ascorbic acid due to the up - regulation of utp - glucose-1-phosphate uridylyltransferase synthesis at 25 dpa . seed imbibition in water followed by the germination process is connected with the production of high levels of ros ( bykova et al . it is possible that increased levels of ros - scavenging enzymes in phs - susceptible grain play a protective role during premature germination . many proteins protecting seeds against desiccation , low temperature and heat shock , including chaperones for other proteins , were found to be differentially distributed in phs - resistant and phs - susceptible grain at the 25 dpa . among them , one isoform of 16.9-kda heat shock protein ( spot no . 14 ) was overproduced in phs - susceptible grain and another isoform ( spot no . this protein also showed higher abundance in phs - susceptible mature grain ( masoj and kosmala 2012 ) . evidently , rye lines extremely different in respect to phs exhibited individual profiles of anti - stress protein synthesis during grain development . it is especially interesting in respect to proteins acting against desiccation stress , since phs - resistant lines must be prepared to pass more rounds of seed imbibitions and desiccation before their dormancy is alleviated . 7 ) , hsp70 ( spot . 8) , groel chaperone ( spot nos . 25 and 26 ) and sorbidraft protein ( spot no . 13 ) should be further evaluated in respect to their role in sprouting resistance . more examples of regulatory proteins being differentially accumulated in the grain of phs - resistant and phs - susceptible lines were revealed through proteomic analysis performed at 25 dpa in respect to that carried out on mature grain ( masoj and kosmala 2012 ) . identified proteins can affect other genes expression at different levels , such as transcription ( 14 - 3 - 3 , ras - related protein ric2-like ) , post - transcription ( glycine - rich rna - binding protein ) , translation ( elongation factor tu ) , post - translation ( beta1 proteasome ) and epigenetically ( histone h4 ) . the different molecular compositions of these potent proteins at early stages of grain development could have a strong impact on later molecular processes , leading to extreme phenotypes in respect to phs . although wheat and rye could have evolved quite different strategies of phs resistance , it is interesting to compare proteomic ( bykova et al . 2011 ) and genomic ( rehman arif et al . 2012 ) analyses of this trait performed separately for each of these related species . as it was shown in table 1 , there are several genes and proteins which are common for wheat and rye as candidate genes for phs resistance / susceptibility . this list contains serpins , heat shock protein hsp70 , endochitinase , alpha - amylase inhibitors , subtilisin - chymotrypsin inhibitors , vicilin - like antimicrobial peptides , i.e. a complex set of proteins involved in plant response to biotic and abiotic stresses . proteins found to be related to phs in both wheat and rye are also peroxiredoxin , xylanase inhibitor and rna - binding protein ( kamal et al . 2009 , and this paper ) . in addition , a study on transcriptomes from rice grain representing cultivars of high and low dormancy ( qin et al . 2010 ) showed differences in the expression level of genes encoding heat shock proteins , which were also associated with phs in wheat ( bykova et al . . further work on genes controlling phs should combine a map - based approach with the analysis of seed proteome and/or transcriptome . these potent research tools will lead to the identification of genes , their expression profiles and interactions , which are necessary in order to understand the molecular biology of phs .

significant differences in the two - dimensional electrophoresis patterns of proteins from developing rye grain were found to be associated with resistance and susceptibility to preharvest sprouting ( phs ) . mass spectrometry of individual spots showing different abundance in phs - resistant and phs - susceptible lines identified proteins involved in : reaction to biotic and abiotic stresses , including oxidative stress , energy metabolism and regulation of gene expression . highly differentiated abundance of proteins found in developing grain suggest that the diversification of processes leading to developing phs resistance or phs susceptibility starts from an early stage of grain development . a part of the identified proteins in rye grain were also reported to be associated with phs in wheat and rice , which suggests that some mechanisms affecting precocious germination might be common for different cereal species.electronic supplementary materialthe online version of this article ( doi:10.1007/s13353 - 012 - 0127 - 8 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction Materials and methods Results Discussion Electronic supplementary material

the online version of this article ( doi:10.1007/s13353 - 012 - 0127 - 8 ) contains supplementary material , which is available to authorized users . preharvest sprouting ( phs ) is a major factor negatively affecting the quality of crops in years with high precipitation during a period of grain maturation . consequently , selection for genotypes showing no precocious germination in conditions of water stress is one of the priorities in many breeding programmes . 2012 ) dissected phs and dormancy in wheat into 18 chromosome bins , each containing a cluster of several genes . phs - related genes , even if they are clustered with other loci , may be identified by their different expression at the mrna and/or protein level in phs - resistant and phs - susceptible grain , since these contrasting phenotypes should be determined by specific profiles of genes expression and protein activities during development . 2011 ) and rye ( masoj and kosmala 2012 ) by using two - dimensional gel electrophoresis ( 2-de ) and mass spectrometry ( ms ) to compare protein composition in mature grain of phs - resistant and phs - susceptible lines derived from a single cross . both investigations revealed associations between phs resistance and the abundance of specific proteins involved in responses to biotic and abiotic stresses , including oxidative stress . oxidative stress caused by the accumulation of reactive oxygen species ( ros ) in embryo cells was shown to be correlated with dormancy alleviation and germination ( oracz et al . this may explain the different accumulation of ros - scavenging enzymes in phs - resistant and phs - susceptible wheat ( bykova et al . 2009 ) studies show that phs resistance has a complex genetic basis involving at least 24 proteins of various functions and numerous qtl distributed on each of the seven chromosomes . this knowledge should be enriched with proteomic studies carried out on different stages of grain development . it is expected that proteome analysis of developing grain will generate more information on molecular mechanisms affecting predisposition to phs . this investigation is aimed at finding differences in protein composition between phs - resistant and phs - susceptible lines of rye at the early stages of grain development . two sets of rye recombinant inbred lines ( rils ) , 20 phs - resistant lines ( 0 % sprouting in spikes after 7 days of water spray at maturity ) and 20 phs - susceptible lines ( 90100 % sprouting in spikes after 7 days of water spray at maturity ) , were grown on the experimental field of the west pomeranian university of technology , szczecin in 2011 . dry kernels of the phs - resistant group of lines were bulked and ground in a retsch mm 200 mill . kernels from the phs - susceptible group of lines were bulked and ground in the same mill . two bulked samples of the rye flour representing 20 phs - resistant and 20 phs - susceptible lines were kept in a freezer prior to protein extraction . the proteomic work involved : ( 1 ) the analyses of rye grain protein abundance in the bulked samples of the resistant and the susceptible rye inbred lines using 2-de , ( 2 ) ms identification of proteins which were differentially accumulated between the analysed bulks . this method was shown earlier to be useful to extract the proteins from different species and its use resulted in high - quality 2-d maps with proteins derived from different cell components , including membranes , chloroplasts , mitochondria and cytoplasm ( kosmala et al . the aliquots of proteins extracted from 25 mg of rye flour were mixed with rehydration solution ( 7 m urea , 2 m thiourea , 2 % np-40 , 0.5 % ipg buffer ph range 47 , 0.002 % bromophenol blue , 18 mm dtt ) to the final volume of 450 l and used for 2-de , performed according to hochstrasser et al . after equilibration , the proteins were separated in the second dimension , sodium dodecyl sulphate polyacrylamide gel electrophoresis ( sds - page ) , using 13 % polyacrylamide gels ( 1.5 255 196 mm ) at 4 w / gel for 30 min and then at 17 w / gel for 4 h. rainbow molecular weight marker ( ge healthcare ) was used as a standard to determine the molecular weights ( mws ) of proteins for particular spots . to compensate for subtle differences in sample loading , gel staining and destaining , the abundance of each protein spot was normalised as a relative volume ( % vol ) . the extraction procedure and electrophoretic separation were performed twice ( technical replicates ) , and the % vol for the spots from the two replicated gels were then used to calculate the means , which were used to make comparisons between the analysed rye lines . the protein spots which showed at least 2-fold ( p < 0.05 ) differences in protein abundance between two analysed lines ( quantitative analysis ) together with protein spots present only in one of the analysed lines ( qualitative analysis ) were subjected to ms analysis and identification . protein spots were excised from the gel and analysed by liquid chromatography coupled to the mass spectrometer in the laboratory of mass spectrometry , institute of biochemistry and biophysics , polish academy of sciences ( warsaw , poland ) . however , in these cases , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was shown . only the spots which were detected within two replicate gels out of all protein spots separated on 2-de gels , 27 showed significant quantitative differences between phs - resistant and phs - susceptible lines ( figs . 2 , 3 , 4 , 5 , 6 , 9 , 10 , 11 , 14 , 16 , 18 , 21 , 22 ) were 24.1-fold more abundant in phs - susceptible lines than in phs - resistant lines . 1 , 7 , 8 , 12 , 13 , 15 , 17 , 19 , 20 , 23 , 24 , 25 , 26 , 27 ) were 24.3-fold more abundant in phs - resistant lines . 29 and 30 ) were found only in phs - susceptible lines ( fig . 1two - dimensional gel electrophoresis ( 2-de ) map of proteins from developing rye grain [ 25th day after anthesis ( 25 dpa ) ] of 20 recombinant inbred lines ( f10 ) highly resistant to pre - harvest sprouting ( phs ) , derived from the 541 ot1 - 3 cross . the normalised volumes of matched spots were used for comparisons made between the phs - resistant and phs - susceptible secale cereale lines ( in the case of each spot , the means derived from two replicated gels were applied ) . 28 ) present only in phs - resistant lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel . 22-de map of proteins from developing rye grain ( 25 dpa ) of 20 recombinant inbred lines ( f10 ) highly susceptible to phs , derived from the 541 ot1 - 3 cross . 29 and 30 ) present only in phs - susceptible lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel two - dimensional gel electrophoresis ( 2-de ) map of proteins from developing rye grain [ 25th day after anthesis ( 25 dpa ) ] of 20 recombinant inbred lines ( f10 ) highly resistant to pre - harvest sprouting ( phs ) , derived from the 541 ot1 - 3 cross . the normalised volumes of matched spots were used for comparisons made between the phs - resistant and phs - susceptible secale cereale lines ( in the case of each spot , the means derived from two replicated gels were applied ) . 28 ) present only in phs - resistant lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel . the molecular weight ( mw ) and ph scales are shown 2-de map of proteins from developing rye grain ( 25 dpa ) of 20 recombinant inbred lines ( f10 ) highly susceptible to phs , derived from the 541 ot1 - 3 cross . 29 and 30 ) present only in phs - susceptible lines and 27 protein spots ( spot nos . 127 ) with quantitative differences in protein abundance between phs - resistant and phs - susceptible lines are indicated and numbered on the gel ms of individual spots revealed that the majority of them were homogenous , containing one specific protein ( table 1 ) . 3 , 4 , 5 , 6 , 7 , 9 , 10 , 11 , 22 , 25 , 26 , 27 and 28 were found to be heterogeneous , with 2 or 3 ( in the case of spot no . in total , 44 proteins were found in 30 spots , showing an association with phs.table 1the results of mass spectrometry ( ms ) performed on the selected protein spots representing developing grain [ 25th day after anthesis ( 25 dpa ) ] of preharvest sprouting ( phs)-resistant ( rl ) and phs - susceptible ( sl ) recombinant inbred lines of ryespot no.accessionidentified proteincell pathway / molecular functionscore / coverageprotein abundance1baj93636predicted protein ( hordeum vulgare subsp . 1 and 2database accession ( according to ncbinr ) of a homologous proteinhomologous protein and organism from which it originates , identified also with the help of the blastp algorithm ( xp _ , yp _ s1protein abundance was calculated using the mean of relative volumes ( % vol ) of two replicates of particular protein spotsif more than one reliable identification appeared in the single spot , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was compared . the names of identified proteins which were also found to be associated with phs in wheat by bykova et al . proteins showing an association with phs both in mature ( masoj and kosmala 2012 ) and in developing rye grain have accession numbers in bold the results of mass spectrometry ( ms ) performed on the selected protein spots representing developing grain [ 25th day after anthesis ( 25 dpa ) ] of preharvest sprouting ( phs)-resistant ( rl ) and phs - susceptible ( sl ) recombinant inbred lines of rye spot numbering was the same as in figs . s1 protein abundance was calculated using the mean of relative volumes ( % vol ) of two replicates of particular protein spots if more than one reliable identification appeared in the single spot , it was impossible to evaluate the abundance of particular proteins present in the spot and the total protein abundance was compared . the names of identified proteins which were also found to be associated with phs in wheat by bykova et al . proteins showing an association with phs both in mature ( masoj and kosmala 2012 ) and in developing rye grain have accession numbers in bold a predominant function of proteins more abundant in phs - susceptible lines was defence against pathogens , as shown by serpin - z1b ( spot no . sensitivity to phs was also associated with a higher amount of several enzymes that sustain energy metabolism ( spot nos . sprouting - resistant lines contained higher amounts of proteins protecting against : desiccation ( spot no . there were examples of the same proteins present in different spots , of which one molecular form was more abundant in phs - resistant lines and another was associated with phs - susceptible lines ( 16.9-kda class i heat shock protein 1 , spot nos . on the other hand , multiple spots for one protein more abundant in phs - resistant lines ( dimeric alpha - amylase inhibitor , spot nos . 25 and 27 ) or in phs - susceptible lines ( monomeric alpha - amylase inhibitor , spot nos . the majority of the detected proteins were involved in response to abiotic and biotic stresses and in energy metabolism , which suggest that predisposition to phs is , above all , a genetically determined molecular mechanism of response to various kinds of stresses acting during grain desiccation followed by prolonged contact with water . since the inability of mature grain to germinate in water - sprayed spikes showed by phs - resistant lines should be imposed by the genetic control of down- and/or up - regulation of genes expression , it is not surprising that some regulatory proteins were revealed in this study . they were found in spot no . the consequences for gene expression on the genomic scale might result from apparently substantial differences in the abundance of histone h4 isoforms , which control chromatin structure and dna protein interaction . 30 ) was found only in phs - susceptible lines , whereas the second ( spot no . 26 ) was shown to be more abundant in phs - resistant lines . at the present stage of knowledge on molecular mechanisms leading to phs resistance , it is difficult to explain the different abundances of three other proteins in the grain of phs - susceptible and phs - resistant lines . 1 ) , a basic enzyme for carbon fixation during photosynthesis taking place in the green pericarp of developing grain , showed a relatively high level of accumulation in the phs - resistant group of lines . 23 ) was found to be associated with high resistance to phs . on the other hand , the developing grain of phs - susceptible lines contained more aspartate - semialdehyde dehydrogenase ( spot no . differences in protein composition of the grain at 25 dpa , found between the two groups of rye lines with extremely different predispositions to phs , clearly show that the process of preparing grain to resist germination in mature spike during the rainy season starts in early development ( this paper ) and continues up to the end of grain maturation ( masoj and kosmala 2012 ) . proteomes of phs - resistant and phs - susceptible grain revealed more differences in protein abundance 25 days post - anthesis than at the maturity stage . there were , however , several common features of the phs - related protein patterns at both stages of grain development . in both studies , phs - resistant grain contained higher levels of dimeric alpha - amylase inhibitor and phs - susceptible grain had increased levels of atp synthase , serpin - z1b and monomeric alpha - amylase inhibitor . differences in the abundance of 16.9-kda class i heat - shock protein , triosephosphate isomerase and 14 - 3 - 3 regulatory protein were depicted in both studied stages of seed formation . generally , both young and mature seeds of phs - resistant and phs - susceptible lines showed differentiation in respect to proteins responding to biotic and abiotic stresses , including oxidative stress . the set of differentially accumulated proteins related to biotic stress showed higher complexity in developing seeds . similarly , a more complex set of proteins affecting genes expression and protein synthesis and degradation rates was associated with phs at the early stage of grain development . this observation raises the question about the significance of transiently expressed differences in protein abundance during grain development for attaining phs resistance or susceptibility at maturity . similarly to in a previous study ( masoj and kosmala 2012 ) , individual proteins associated with phs were detected in different spots separated by means of 2-de . various reasons might underlie the multiple molecular forms of proteins , including multi - gene families ( isoforms ) , heterozygosity ( allozymes ) , post - translational modifications or even protein degradation during sample preparation . change in protein abundance due to different frequencies of allelic variants within each of the two extreme groups of lines would be an indirect proof for the involvement of respective structural genes in phs resistance / susceptibility . verification of allelic forms among 2-de protein spots should be possible by comparing patterns of individual rils , including parental lines . the persistence of dimeric alpha - amylase inhibitor in higher amounts in developing and mature grain of phs - resistant lines and higher amounts of xylanase inhibitor and subtilisin - chymotrypsin inhibitor at 25 dpa gives strong evidence that the accumulation of these particular defence proteins is important for the phs resistance of rye . different sets of defence proteins seem to be important for phs susceptibility , since it is associated with a higher abundance of serpins and monomeric alpha - amylase inhibitor at both 25 dpa and maturity , and with increased levels of immunophilin and endochitinase c at 25 dpa and tritin at maturity . another specific feature of phs - susceptible lines detected in both proteomic studies of rye grain is the relatively higher accumulation level of some proteins related to energy metabolism , including those involved in the citric acid cycle and in glycolysis . elevated synthesis of these proteins might be necessary to achieve unusually high rates of germination observed in phs - susceptible rils . this hypothesis is supported by the detection of beta1 proteasome 7d protein among those which are up - regulated in phs - susceptible lines at 25 dpa . a higher level of anti - oxidant protection is seemingly attributed to the grain of phs - susceptible lines , which accumulated ros - scavenging enzymes , such as : glutathione transferase , dehydroascorbate reductase and superoxide dismutase ( mature grain ) , but also produced possibly more l - ascorbic acid due to the up - regulation of utp - glucose-1-phosphate uridylyltransferase synthesis at 25 dpa . it is possible that increased levels of ros - scavenging enzymes in phs - susceptible grain play a protective role during premature germination . many proteins protecting seeds against desiccation , low temperature and heat shock , including chaperones for other proteins , were found to be differentially distributed in phs - resistant and phs - susceptible grain at the 25 dpa . 14 ) was overproduced in phs - susceptible grain and another isoform ( spot no . this protein also showed higher abundance in phs - susceptible mature grain ( masoj and kosmala 2012 ) . it is especially interesting in respect to proteins acting against desiccation stress , since phs - resistant lines must be prepared to pass more rounds of seed imbibitions and desiccation before their dormancy is alleviated . more examples of regulatory proteins being differentially accumulated in the grain of phs - resistant and phs - susceptible lines were revealed through proteomic analysis performed at 25 dpa in respect to that carried out on mature grain ( masoj and kosmala 2012 ) . the different molecular compositions of these potent proteins at early stages of grain development could have a strong impact on later molecular processes , leading to extreme phenotypes in respect to phs . although wheat and rye could have evolved quite different strategies of phs resistance , it is interesting to compare proteomic ( bykova et al . as it was shown in table 1 , there are several genes and proteins which are common for wheat and rye as candidate genes for phs resistance / susceptibility . a complex set of proteins involved in plant response to biotic and abiotic stresses . proteins found to be related to phs in both wheat and rye are also peroxiredoxin , xylanase inhibitor and rna - binding protein ( kamal et al . 2010 ) showed differences in the expression level of genes encoding heat shock proteins , which were also associated with phs in wheat ( bykova et al .

surgeon general 's report on oral health published in may 2000 , dental caries is the most common chronic childhood disease . in addition , most ( 75%80% ) of the caries in children in this country occur in a small segment of the population ( 20%25% ) , and this problem is particularly prevalent in minorities and immigrants and lower - income children . unfortunately , the funds to provide preventive care to all children who are either from a minority population or the lower ses levels are simply not available . therefore , it is reasonable to attempt to identify those at highest risk in these populations and concentrate what limited financial and manpower resources are available in these highest of the high . additionally , the cultural , and behavioral determinants of disease and the barriers to access dental health services in these populations may be dissimilar to those in other social groups . understanding these differences may eventually influence different preventive strategies and alternative ways to help health providers to communicate with these groups in order to enhance health related behaviors and conditions . therefore , identifying a child 's level of risk for development of dental caries and the reasons behind it are necessary first steps in managing dental caries . risk - based prevention and disease management have been recognized as the cornerstones of modern caries management [ 68 ] , especially in young children [ 810 ] . the fact that the existence of past restorations is one of the greatest indicators of risk for the development of new caries lesions [ 9 , 11 ] only proves that the act of surgically treating the caries lesion does little to reduce the risk of developing the next lesion , generally makes no significant difference to bacterial loading , nor on the enactment of self - promoting health behaviors such as brushing one 's teeth [ 1214 ] . the etiology of the dental caries process is multifactorial in nature and involves a combination of factors including diet , a susceptible host , and microflora , which interplay with a variety of social , cultural and behavioral factors . additionally , most young children appear to acquire some cariogenic microorganisms ( i.e. , mutans streptococci - ms ) from their mothers or primary caregivers . transmission happens through saliva and can be affected among other variables by the frequency of the contact ( e.g. , sharing of food and utensils , kissing , etc . ) , which could have cultural and behavioral determinants and , therefore , may vary among different ethnic and cultural population groups . because of the multifactorial nature of the dental caries disease process , and the fact that the disease is very dynamic , but not continuous ( e.g. , lesions can progress and/or regress ) , studies on risk assessment tend to be complex , with a multitude of variables challenging the prediction at different times during the life of an individual . for a clinician , the concepts of assessment of risk and prognosis are an important part of clinical decision making , individualized counseling , and anticipatory guidance . in addition , risk factors may vary based on race , culture , and ethnicity [ 1621 ] . unfortunately , there are very few high - quality , longitudinal caries risk studies focusing on infants and toddlers [ 10 , 22 ] . furthermore , existing studies have been conducted primarily in selective populations in northern europe [ 2329 ] , diminishing the generalizability of these results to the us population . one recent study has been conducted in a low ses , african - american u.s . community . in addition , gao et al . have recently suggested that practical biopsychosocial caries risk models without biological markers , such as the one tested here , are effective ( sensitivity / specificity was 82%/73% ) and promise to be cost - effective to reach children in a variety of settings . others have studied dental habits , attitudes , and beliefs in a range of settings . however , these studies have drawbacks in application to caregivers of toddlers due to the populations studied ( age , race / ethnicity , and/or geographic location ) and due to the range of topics covered . dental habits , beliefs , and attitudes have been studied in adults [ 3240 ] , finding variability in beliefs and attitudes which may affect their own dental outcomes , but was not necessarily examined in the context of adults who were caring for toddlers . in parents of young children in great britain , knowledge and attitudes were found to vary due to education , ethnicity , and area of residence . dental knowledge , attitudes , and practices may also be impacted by the overall health of the child . research has shown beliefs were found to differ between parents of children with and without congenital heart disease . relationships of caries in 3-year olds in japan with child - rearing behaviors and mother 's health behaviors were examined , finding a stronger association with the child - related behaviors than the mother 's behaviors . the purpose of this study was to evaluate how known caries risk factors evaluated longitudinally in young u.s . these factors had been identified through previous research as possible risk factors and were included as part of a one - year longitudinal risk study . if differences were found in the risk factors , as expected , this could indicate the need to target caregiver / patient education and preventive care intervention strategies based on the characteristics of the population or individual . the study population included caregiver - toddler pairs in indianapolis and connersville , indiana , usa . subjects were recruited through four sites : ( 1 ) a primary - care - based study - recruitment system affiliated with a large metropolitan hospital serving a generally underserved and lower - income population , ( 2 ) the oral health research institute of the indiana university school of dentistry , ( 3 ) the hispanic center of indianapolis , and ( 4 ) the rural town of connersville . at sites 24 above , recruitment was done by radio and newspaper advertisements , as well as contacting an irb - approved database of people who had participated in previous studies with us at those locations . the adult accompanying the child was required to self - identify as being the primary caregiver for the child . ( pcg ) as the individual consistently responsible for the housing , health , and safety of the child . toddlers ranged in age from 16 to 36 months at the time of recruitment , and were generally healthy based on the caregivers ' responses to a medical history questionnaire . the study protocol , letter of informational consent , and other supporting documents were approved by the indiana university medical center institutional review board prior to their use . written informed consent was obtained from all pcgs ( and parent / legal guardian if different from the child 's pcg ) prior to their enrollment . a caries risk questionnaire was developed to include questions related both to the pcg and the child regarding social , cultural , functional , psychological , sociodemographic , dietary , and biological factors that may affect transmission , development of caries , and access to care in these populations . many of the questions were taken or modified from other risk assessment questionnaires and tools . an external review panel , which ranged from practitioners ( pediatric dentists and pediatric physicians ) to experts in the area of cariology , predictive modeling , and behavioral science , were provided a copy of the questionnaire and asked to review / edit the questionnaire to ensure that the initial draft of the questionnaire was reasonable in scope and that no established risk indicator had been omitted . after receiving separate irb approval , the draft questionnaire was tested in a panel of 25 caregivers ( nearly equal numbers of english and spanish speaking ) , similar to the target population ( had to consent to participate and have a child between 18 and 36 months of age ) , to ensure that the questions that were asked were worded appropriately for nonprofessionals , to eliminate jargon , to define or eliminate confusing terminology ( e.g. , words such as frequent , often , etc . ) , to ensure use of culturally - sensitive language , to finalize the organization of the items , and to verify the consistency of the structure of similar items . in most cases , it was believed that the majority of persons to be interviewed as pcg would be the mothers , but others ( e.g. , grandmothers , fathers ) were to be included if it was found that they were responsible for providing the largest percentage of the child 's care . based on the results of the focus group data , some changes were made in the wording of questions , some questions were eliminated and some were reordered , and the questionnaire was finalized . the final version of the questionnaire , which included 105 items ( see appendix ) , was administered by study personnel to the pcg ( n = 396 ) using a multiple choice format , with responses recorded directly into a web - based database system . topics included in the questionnaire were categorized into : demographics , access to care , possible routes for oral bacteria transmission , usual dental and medical health practices of the caregiver and the toddler , dental beliefs of the caregiver , and snacking and drinking habits of the caregiver and the toddler . in addition , a subset of the caregivers ( n = 250 ) was invited to participate in an additional investigation of health literacy . after additional informed consent , caregivers were administered the short test of functional health literacy in adults ( s - tofhla ) , with the caregiver given the option of using either the english or spanish version . the associations of pcg education and household income with race / ethnicity were tested using anova , and spearman correlation coefficients were calculated to measure the association between pcg education and household income . for the analyses , education levels were collapsed into 8th grade or lower , some high school , completed high school , some college , 4-year college , and postgraduate . we analyzed each survey item individually to assess the need to modify caregiver / patient education and preventive care intervention strategies based on demographic factors . to examine the associations of individual survey items ( dependent variables in separate models ) with the caregiver 's race / ethnicity , the caregiver 's education , and the household income simultaneously ( three independent variables ) , multivariable logistic and linear regression analyses were used for survey items with qualitative responses and quantitative responses , respectively ; thus race / ethnicity comparisons are adjusted for income and education , income comparisons are adjusted for race / ethnicity and education , and education comparisons are adjusted for race / ethnicity and income . p - values presented for the race / ethnicity comparisons are for the overall tests for any effect among the three groups ; individual pairwise results are presented when significant but the p - values are not provided . odds ratios presented for education and income are for a one - level change in the response categories . a 5% significance level was used for all analyses ; although a large number of tests were performed , we did not adjust for multiple testing . a less restrictive cutoff without a multiple - testing adjustment provides a larger pool of possible differences that can be targeted when revising caregiver / patient education and preventive care intervention strategies . the study enrolled 396 caregiver - toddler pairs at baseline ( two additional pairs were screened but did not qualify due to the child 's medical condition ) , which is estimated to be approximately 70% of those invited to participate . nearly all of the primary caregivers ( 378 ) were the child 's mother , with the remaining caregivers consisting of 14 fathers , 2 grandmothers , 1 aunt , and 1 other . the caregivers ' ages ranged from 18 to 64 years , with an average age of 28 ( sd = 6 ) years . the children ranged in age from 16 to 36 months , with a mean of 26 ( sd = 6 ) months , and ages did not differ significantly by race / ethnicity , income , or education of the caregiver . one hundred seventy - five ( 44% ) of the caregivers self - identified themselves as non - hispanic african - american , 141 ( 36% ) were non - hispanic white , 75 ( 19% ) were hispanic ( all races ) , and 5 ( 1% ) did not fall into one of the previous three categories . nearly one - third of hispanic caregivers reported difficulty understanding information they receive from physicians and dentists , while the rate was less than ten percent in non - hispanic african - americans and non - hispanic whites ( table 1 ) . furthermore , health literacy , collected on a subset of 250 caregivers , was not different among race / ethnicity groups but was weakly associated with education ( r = 0.18 , p = .02 ) . non - hispanic whites were more likely to use city water as their primary drinking water source as opposed to bottled or well water . interestingly , drinking water source was not related to income or education in this cohort . there was a moderately high correlation between education and income ( r = 0.56 , p = .0001 ) and moderate correlations for caregiver age with education ( r = 0.42 , p = .0001 ) and income ( r = 0.38 , p = .0001 ) . habits of the caregivers that might lead to transmission of bacteria to the toddler differed by race / ethnicity ( figure 1 ) , education , and income . hispanic caregivers were less likely than non - hispanic african - american and non - hispanic white caregivers to put the toddler 's pacifier in their own mouth ( 12% versus 37% and 31% , p = .0156 ) , which was also associated with higher education ( odds ratio 1.3 , 95% ci 1.01.7 , p = .0212 ) but not with income ( odds ratio 1.0 , 95% ci 0.81.1 , p = .44 ) . tasting the child 's food or drink using the same fork / spoon or glass was common in all race / ethnicity groups ( approximately 70% , p = .87 ) , but was more common with those reporting a higher income ( odds ratio 1.3 , 95% ci 1.11.4 , p = .0013 ) . sharing food with the child using the same bowl / plate / glass and kissing the child on the lips occurred with nearly all non - hispanic african - american and non - hispanic white caregivers but was less frequent among hispanics ( p = .0001 ) and was more common with higher income ( odds ratio 1.3 , 95% ci 1.11.6 , p = .0028 ) . however , 87% of hispanics ever breast - fed compared to 50% of non - hispanic african - americans and 62% of non - hispanic whites ( p = .0004 ) ; breast - feeding was also more common with higher education ( odds ratio 1.6 , 95% ci 1.22.1 , p = .0004 ) and higher income ( odds ratio 1.1 , 95% ci 1.01.3 , p = .0458 ) . although caregivers with more education more often reported that their child had a dentist ( table 2 ) , there were no differences in whether the child had ever been to the dentist . because the toddlers may have similar access to care as their caregivers , the questionnaire also asked about dentist and physician visits made by the caregiver . seventy - one percent of non - hispanic white caregivers , 53% of non - hispanic african - american caregivers , and 29% of hispanic caregivers had a dentist ( table 1 ) , and having a dentist was also associated with higher education attainment and higher income . approximately half of non - hispanic african - americans caregivers reported going to the dentist for regular checkups , while nearly 40% of hispanic caregivers reported never going to the dentist . interestingly , higher income was associated with caregivers going to the dentist for checkups , while lower education but not income was associated with never going to the dentist . in addition , patterns of caregiver visits to the physician differed by race / ethnicity ( table 1 ) but were not as affected by income or education , where only regular visits to the physician were associated with higher income . hispanic caregivers reported their children 's teeth were brushed less frequently than teeth of non - hispanic african - americans and non - hispanic whites ( table 2 ) . caregivers with lower income were more likely to have problems with dry mouth when eating . hispanic caregivers were more likely to be bothered by the appearance of their own teeth , which was not associated with education or income . while there were differences among the race / ethnicity groups in how the caregivers felt about their child 's and their own dental and medical health , education and income were generally not related to these ratings . beliefs and knowledge ( figure 2 ) differed by race / ethnicity adults eventually losing all their teeth ( p = .0001 , higher response of false for non - hispanic whites ) , most children getting cavities ( p = .0304 , lower response of false for hispanics ) , bad teeth being mostly inherited from parents ( p = .0119 , lower response of false for hispanics ) , and when tooth cleaning should start ( p = .0001 , earlier for non - hispanic - whites ) , with also a trend for when the child 's first dental visit should be ( p = .06 , earliest for non - hispanic african - americans and latest for non - hispanic whites ) . belief that adults will eventually lose all their teeth was associated with less education ( odds ratio 1.4 , 95% ci 1.11.8 , p = .0072 ) and lower income ( odds ratio 1.1 , 95% ci 1.01.3 , p = .0376 ) , and belief that most children will eventually get cavities was associated with less education ( odds ratio 1.3 , 95% ci 1.01.6 , p = .0258 ) , while none of the other beliefs / knowledge assessed were significantly associated with education or income . hispanic toddlers were more likely drink from a bottle ( 29% ) compared to non - hispanic whites toddlers ( 11% ) and non - hispanic african - american toddlers ( 4% ) , while non - hispanic white toddlers and non - hispanic african - american toddlers were not significantly different . non - hispanic african - american toddlers were also less likely to drink from a sippy cup ( 67% ) compared to non - hispanic whites ( 84% ) and hispanic ( 87% ) toddlers , who were not significantly different from each other ( table 3 ) . hispanic children were most likely to receive a bottle or sippy cup at bedtime or naptime . although hispanic caregivers cleaned their child 's teeth after removing the drink more frequently than non - hispanic african - americans or non - hispanic whites , cleaning the child 's teeth after removing the drink was rare for all races . less than half of hispanic children regularly sipped on drinks between meals , while nearly all non - hispanic african - american and non - hispanic white children did . types of snacks and drinks usually eaten / drank between meals varied considerably among race / ethnicity groups for toddlers ( table 3 ) and for pcgs ( table 4 ) , while snacking and between - meals drinks were typically not associated with education or income , with a specific exception of nondiet soda being associated with less education . despite a decrease in dental caries prevalence in permanent teeth for most americans since the early 1970s , oral health disparities remain across some population groups , and dental caries is still the most prevalent chronic disease of childhood . furthermore , between 19881994 and 19992004 , caries experience in primary teeth of children aged 25 years has significantly increased from 24% to 28% , primarily due to an increase in the percent with fillings . unfortunately , as mentioned earlier , our current understanding of caries risk and etiological factors derived from longitudinal studies in young children in the united states is limited . available caries risk questionnaire tools are , for the most part , expert - based tools . examples include the caries risk tool of the american academy of pediatric dentistry , the ada 's caries risk tool for children younger than 6 , and the caries management by risk assessment ( cambra ) tool for children younger than 6 [ 48 , 49 ] . while other studies have identified caries risk factors in low - ses rural and low - ses african - american communities , the prevalence of the risk factors may affect both the disease prevalence and the types of interventions that may be effective in preventing and/or treating caries . age , socioeconomic status , and race / ethnicity differences as well as in non - us populations studied previously provided individual risk factor prevalence estimates , but only indirect evaluations of the effects of the sociodemographic factors on the risk factors could be made . in the present study , multiple factors from the caries risk questionnaire within the access to care , oral bacterial transmission , dental and medical health practices of the caregiver and the toddler , and snacking and drinking habits of the caregiver and the toddler areas were directly compared and differed by race / ethnicity , income , and/or education . having general and pediatric dentists understand that these differences exist is only a first step . the information must be incorporated in improved strategies to treat and/or prevent caries in toddlers . with the limited sample size and single location sampled in this study , it is difficult to differentiate the effects of cultural influences , health knowledge gained through educational background , and income - based health utilization disparities on the risk factors ; in other words , we were unable to look at the influence of interactions among the three factors or stratify the analyses . and while the study included three race / ethnicity groups , the single location of the study ( indiana ) may not fully represent responses nationwide . a larger multisite study would be needed for increased generalizability as well as provide the sample size needed to differentiate among the cultural , income , and education influences on the risk factors . a large number of risk factors were examined , based on the extensive list of factors proposed or identified previously . some of the risk factors differing by sociodemographic factors are likely to be false positives . nevertheless the information from our study can provide useful risk factor prevalence data when revising caregiver / patient education and preventive care intervention strategies . as mentioned above , our sample size was not large enough to justify a detailed examination of the 3-way interaction among race / ethnicity , income , and education to differentiate the effects of cultural influences , health knowledge gained through educational background , and income - based health utilization disparities on the risk factors . regardless of the underlying cause , as others have suggested based on observations in various populations [ 32 , 36 ] , education and intervention strategies can be targeted generally to the population seen in the practice and specifically to individual patients . it is noteworthy to mention efforts in this country by medical ( e.g. , american academy of pediatrics and american medical association [ 51 , 52 ] ) and dental ( e.g. , american dental association , american academy of pediatric dentistry [ 46 , 53 ] ) associations , among others , to stress not only the importance of a dental home early in life , but also the importance of risk - based preventive interventions and anticipatory guidance provided in a variety of settings to reach young children . in fact , a variety of programs have evolved in different places around the country . ( ibm ) program in north carolina is one of the best examples of the effort resulting from the partnership between dentists and pediatricians to improve the oral health of children . the imb program was initiated in 2000 and has led to a substantial increase in access to preventive dental services by enabling medicaid children younger than 3 years of age to receive dental screening , counseling , and fluoride varnish in physicians ' offices . more work will certainly be needed to evaluate the acceptability and effectiveness of education and intervention strategies in targeted populations . one problem hindering treatment and prevention of caries in high - risk children is that they may not seek care from dentists regularly , if at all . despite the importance of establishing a dental home in the first year of life , most children do not receive a dental examination , nor do the parents receive needed education on oral health . while 89% of infants and one - year - olds have been examined by a physician , only 1.5% has had a dental appointment . some of the factors identified above could be included in discussions of healthy behaviors with the caregivers at well - child checkups . patient education materials could also be developed to be made available through pediatrician and family practice offices . the results from our study may be useful to future investigators to focus the materials on factors prevalent in specific offices , such transmission of bacteria through sharing drinks or foods in higher income practices and providing drinks at bedtime or naptime in offices that have a high proportion of hispanics . in conclusion , significant differences were found in all areas of the questionnaire related to race / ethnicity , income , and/or education . a larger followup study may be able to explore more detailed differentiation of the effects of cultural influences , health knowledge gained through educational background , and income - based health utilization disparities on the risk factors . patient education and preventive care intervention studies may need to be targeted based on the characteristics of the population to achieve increase effectiveness . first , i 'd like to ask about the child 's ( i.e. , refers to the child in this study ) eating and health habits . does the child usually drink from a bottle ? does the child usually drink from a sippy cup ? is the child currently being breast - fed ? if not currently breast - fed , was the child ever breast - fed ? ( if yes , how long was the child breastfeed for : _ _ _ _ _ _ months ) does the child share a toothbrush with anyone ? ( if yes , indicate who : _ _ _ _ _ _ ) yes no does not use one do you help the child brush his or her teeth ? when you or the child brushes his / her teeth , do you use toothpaste ? does the child 's toothpaste have fluoride in it ? yes no do not know do you ever check the child 's teeth for cavities ? does the child have cavities now ? yes no do not know has the child had cavities or fillings in the past ? has the child had teeth pulled because of cavities ? have other children ( brothers , sisters , or others ) in the child 's household had cavities or fillings ? does the child have problems chewing ? does the child have a tooth that hurts ? does the child have a dentist ? has the child ever been to the dentist ? does the child use other products for his / her teeth ( mouth rinse , prescription toothpaste , tablets , drops , or other ) with fluoride ? ( do not count water ) yes no do not know do or did you ever put the child 's pacifier in your mouth before giving it to him / her ? yes no does not use it do you ever kiss the child on the lips ? when the child was born , were you told by his / her doctor that his / her birth - weight was low ? yes no do not know did the child 's mother get prenatal care ? yes no do not know do you ever taste the child 's food and/or drinks using the same spoon , fork , glass , or other ? do you ever share food or drinks with the child from the same plate , bowl , or glass ? does the child regularly sip on drinks between meals on most days ? does the child use antibiotics more than every three months ? 30 ) do you regularly brush the child 's teeth after use of the medication ? 32 ) what kinds of foods does he / she usually snack on ? ( a snack is food eaten in between regular meals . ) please read the list , and check all foods that apply . candy cakes / cupcakes cereal with milk chips cookies crackers dried fruit ( e.g. , raisins ) dry cereal fresh fruit ice cream popcorn yogurt other ( list or specify : _ _ _ _ ) dried fruit ( e.g. , raisins ) other ( list or specify : _ _ _ _ _ _ ) how many regular meals ( e.g. , breakfast , lunch , dinner , or other ) does the child eat per day ? _ _ _ _ _ _ what does he / she usually drink with a snack or in between meals water juice ( 100% juice ) milk soda ( with sugar ) fruit drink ( with sugar ) tea soda ( diet or sugar free ) fruit drink ( sugar - free ) other ( list or specify : _ _ _ _ _ _ ) fruit drink ( with sugar ) soda ( diet or sugar free ) fruit drink ( sugar - free ) other ( list or specify : _ _ _ _ _ _ ) the child 's main source of drinking water is : city bottled well other ( list or specify : _ _ _ _ ) other ( list or specify : _ _ _ _ _ _ ) the child got his / her first tooth at _ _ _ _ _ _ months of age . when did brushing / cleaning of the child 's teeth start ? ( check all that apply ) when the first tooth came into the mouth younger than 12 months 1324 months 2536 months older than 36 months not brushing / cleaning teeth yet when the first tooth came into the mouth younger than 12 months older than 36 months not brushing / cleaning teeth yet how often do you or the child clean or brush the child 's teeth more than once a day once a day several times a week several times a month a few times a year never several times a month how often do you brush your own teeth ? more than once a day once a day several times a week several times a month a few times a year never several times a month how often do you floss your own teeth ? more than once a day once a day several times a week several times a month a few times a year never several times a month how often does the child get a bottle / sippy cup in bed , at either bedtime or naptime with something other than water in it ? more than once a day once a day several times a week several times a month a few times a year never several times a month how often does the child get a bottle / sippy cup filled with something other than water in it during the day ( do not count mealtimes ) ? more than once a day once a day several times a week several times a month a few times a year never several times a month how often do you clean your child 's teeth after you remove the bottle / sippy cup at night ( after going to bed ) ? more than once a day once a day several times a week several times a month a few times a year never does not drink at night several times a month does not drink at night how often does your child breast - feed at night ? more than once a day once a day several times a week several times a month a few times a year never does not breast - feed several times a month how frequently does your child drink tap water or drinks prepared with tap water ? more than once a day once a day several times a week several times a month a few times a year never several times a month which sentence or sentences below describe how you decide ( or intend to decide ) when to take the child to the dentist ? ( check all that apply ) i only take the child to the dentist if he / she has pain or a problem with his / her teeth . i take the child to the dentist regularly because he / she has problems with the teeth or gums . i do not take the child to the dentist as often as the dentist wants me to . i only take the child to the dentist if he / she has pain or a problem with his / her teeth . i take the child to the dentist regularly because he / she has problems with the teeth or gums . i do not take the child to the dentist as often as the dentist wants me to . which sentence or sentences below describe how you decide when to take the child to the doctor ? ( check all that apply ) i only take the child to the doctor if he / she has pain or is sick . i take the child to the doctor regularly because he / she has a health problem . i do n't take the child to the doctor as often as the doctor wants me to . i only take the child to the doctor if he / she has pain or is sick . i take the child to the doctor regularly because he / she has a health problem . i do n't take the child to the doctor as often as the doctor wants me to . i only go to the dentist if i have pain or if i have a problem with my teeth or gums . i see my dentist regularly because i have problems with my teeth or gums . i do n't see my dentist as often as the dentist wants me to . i only go to the dentist if i have pain or if i have a problem with my teeth or gums . ( check all that apply ) i only go to the doctor if i have pain or if i 'm sick . i do n't see my doctor as often as the doctor wants me to . i only go to the doctor if i have pain or if i 'm sick . excellent very good good fair poor how would you describe how you take care of the child 's dental ( teeth and gums ) health ? excellent very good good fair poor how would you describe the child 's medical health ? excellent very good good fair poor how would you describe how you take care of the child 's medical health ? excellent very good good fair poor how would you describe your dental ( teeth and gums ) health ? excellent very good good fair poor how would you describe how you take care of your dental ( teeth and gums ) health ? excellent very good good fair poor how would you describe your medical health ? excellent very good good fair poor how would you describe how you take care of your medical health ? excellent very good good fair poor how satisfied are you with the child 's dentist / dental care ? very satisfied somewhat satisfied somewhat dissatisfied very dissatisfied not applicable somewhat dissatisfied how satisfied are you with the child 's doctor / medical care ? very satisfied somewhat satisfied somewhat dissatisfied very dissatisfied not applicable somewhat dissatisfied how satisfied are you with your dentist / dental care ? very satisfied somewhat satisfied somewhat dissatisfied very dissatisfied not applicable somewhat dissatisfied how satisfied are you with your doctor / medical care ? very satisfied somewhat satisfied somewhat dissatisfied very dissatisfied not applicable somewhat dissatisfied the next questions focus on your dental beliefs most adults will lose all their teeth as they get older true false do not know most young children will get cavities true false do not know only children need fluoride true false do not know the type of food and drink a child eats or drinks may cause cavities true false do not know baby teeth are important to take care of true false do not know bad teeth are mostly inherited from the parents true false do not know cleaning of the mouth of a child should begin : ( check all that apply ) before the first tooth comes in as soon as the first tooth comes in 1224 months - of - age after 24 months - of - age when the adult teeth come in do not know before the first tooth comes in as soon as the first tooth comes in after 24 months - of - age when the adult teeth come in a child 's first routine dental visit should be : ( check all that apply ) before the first tooth comes in as soon as the first tooth comes in 1224 months - of - age after 24 months - of - age when the adult teeth come in do not know before the first tooth comes in as soon as the first tooth comes in after 24 months - of - age when the adult teeth come in the next questions focus on your own eating and health habits . have you had any cavities in the past , which are now restored / fixed ? yes no do not know are you bothered by how your teeth look ? do you think you need dental treatment ( other than a cleaning ) now ? yes no do not know do you use other products for your teeth ( mouth rinse , prescription toothpaste , other ) with fluoride ? do not know have you had more than half of your adult teeth pulled ? candy cakes / cupcakes cereal with milk chips cookies crackers dried fruit ( e.g. , raisins ) dry cereal fresh fruit ice cream popcorn yogurt other ( list or specify : _ _ _ _ _ _ ) dried fruit ( e.g. , raisins ) other ( list or specify : _ _ _ _ _ _ ) what do you usually drink with a snack or in between meals ? please read the list , and check all drinks that apply . water juice ( 100% juice ) milk coffee without sugar soda ( with sugar ) fruit drink ( with sugar ) tea coffee with sugar soda ( diet or sugar - free ) fruit drink ( sugar - free ) other ( list or specify _ _ _ _ _ _ ) fruit drink ( with sugar ) soda ( diet or sugar - free ) fruit drink ( sugar - free ) other ( list or specify _ _ _ _ _ _ ) now we are going to ask some questions about you , the child 's family , and the child . grade school ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8) high school ( 9 , 10 , 11 , 12 ) college ( 13 , 14 , 15 , 16 ) post graduate ( 17 + ) grade school ( 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8) high school ( 9 , 10 , 11 , 12 ) college ( 13 , 14 , 15 , 16 ) does the child have medicaid or hoosier healthwise ? does the child have health insurance ? ( private health insurance ) does the child receive free care ( medical or dental ) through any other program ? another country list : _ _ _ _ _ _ ( skip to 92 ) another country list : _ _ _ _ _ _ english another language list : _ _ _ _ _ _ _ because of language , do you have difficulty talking to the child 's dentist or doctor ? yes no regularly use an interpreter regularly use an interpreter is it sometimes difficult for you to understand the information given by the child 's doctor or dentist ? what is the child 's racial or ethnic background ? ( mark one or more races to indicate the race or races you consider the child to be ) white or caucasian african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ _ ) african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ ( mark one or more races to indicate the race or races you consider yourself to be ) white or caucasian african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ _ ) african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ number of adults / children _ _ _ _ _ _ number of adults / children _ _ _ _ _ _ how many adults in the child 's household have paid jobs ? number of adults / children _ _ _ _ _ _ number of adults / children _ _ _ _ _ _ how many adults other than yourself take care of the child regularly ? number of adults / children _ _ _ _ _ _ number of adults / children _ _ _ _ _ _ how many children live in the child 's household , including the study child ? number of adults / children _ _ _ _ _ _ number of adults / children _ _ _ _ _ _ do you have transportation to go to the doctor or dentist ? which of the following categories best represents the combined income for all family members in your household added together for the past 12 months ? please read the list of income categories and check the one that applies to you . less than $ 5000 $ 5,000$9,999 $ 10,000$19,999 $ 20,000$29,999 $ 30,000$39,999 $ 40,000$49,999

objectives . dental caries is the most common chronic childhood disease , with numerous identified risk factors . risk factor differences could indicate the need to target caregiver / patient education / preventive care intervention strategies based on population and/or individual characteristics . the purpose of this study was to evaluate caries risk factors differences by race / ethnicity , income , and education . methods . we enrolled 396 caregiver - toddler pairs and administered a 105-item questionnaire addressing demographics , access to care , oral bacteria transmission , caregiver's / toddler 's dental and medical health practices , caregiver 's dental beliefs , and caregiver's / toddler 's snacking / drinking habits . logistic regressions and anovas were used to evaluate the associations of questionnaire responses with caregiver 's race / ethnicity , income , and education . results . caregivers self - identified as non - hispanic african - american ( 44% ) , non - hispanic white ( 36% ) , hispanic ( 19% ) , and other ( 1% ) . differences related to race / ethnicity , income , and education were found in all risk factor categories . conclusions . planning of caregiver / patient education / preventive care intervention strategies should be undertaken with these caries risk factor differences kept in mind .

1. Introduction 2. Methods 3. Results 4. Discussion Questionnaire

surgeon general 's report on oral health published in may 2000 , dental caries is the most common chronic childhood disease . in addition , most ( 75%80% ) of the caries in children in this country occur in a small segment of the population ( 20%25% ) , and this problem is particularly prevalent in minorities and immigrants and lower - income children . the etiology of the dental caries process is multifactorial in nature and involves a combination of factors including diet , a susceptible host , and microflora , which interplay with a variety of social , cultural and behavioral factors . , lesions can progress and/or regress ) , studies on risk assessment tend to be complex , with a multitude of variables challenging the prediction at different times during the life of an individual . in addition , risk factors may vary based on race , culture , and ethnicity [ 1621 ] . one recent study has been conducted in a low ses , african - american u.s . however , these studies have drawbacks in application to caregivers of toddlers due to the populations studied ( age , race / ethnicity , and/or geographic location ) and due to the range of topics covered . dental habits , beliefs , and attitudes have been studied in adults [ 3240 ] , finding variability in beliefs and attitudes which may affect their own dental outcomes , but was not necessarily examined in the context of adults who were caring for toddlers . in parents of young children in great britain , knowledge and attitudes were found to vary due to education , ethnicity , and area of residence . the purpose of this study was to evaluate how known caries risk factors evaluated longitudinally in young u.s . if differences were found in the risk factors , as expected , this could indicate the need to target caregiver / patient education and preventive care intervention strategies based on the characteristics of the population or individual . the study population included caregiver - toddler pairs in indianapolis and connersville , indiana , usa . toddlers ranged in age from 16 to 36 months at the time of recruitment , and were generally healthy based on the caregivers ' responses to a medical history questionnaire . the study protocol , letter of informational consent , and other supporting documents were approved by the indiana university medical center institutional review board prior to their use . a caries risk questionnaire was developed to include questions related both to the pcg and the child regarding social , cultural , functional , psychological , sociodemographic , dietary , and biological factors that may affect transmission , development of caries , and access to care in these populations . the final version of the questionnaire , which included 105 items ( see appendix ) , was administered by study personnel to the pcg ( n = 396 ) using a multiple choice format , with responses recorded directly into a web - based database system . topics included in the questionnaire were categorized into : demographics , access to care , possible routes for oral bacteria transmission , usual dental and medical health practices of the caregiver and the toddler , dental beliefs of the caregiver , and snacking and drinking habits of the caregiver and the toddler . after additional informed consent , caregivers were administered the short test of functional health literacy in adults ( s - tofhla ) , with the caregiver given the option of using either the english or spanish version . the associations of pcg education and household income with race / ethnicity were tested using anova , and spearman correlation coefficients were calculated to measure the association between pcg education and household income . we analyzed each survey item individually to assess the need to modify caregiver / patient education and preventive care intervention strategies based on demographic factors . to examine the associations of individual survey items ( dependent variables in separate models ) with the caregiver 's race / ethnicity , the caregiver 's education , and the household income simultaneously ( three independent variables ) , multivariable logistic and linear regression analyses were used for survey items with qualitative responses and quantitative responses , respectively ; thus race / ethnicity comparisons are adjusted for income and education , income comparisons are adjusted for race / ethnicity and education , and education comparisons are adjusted for race / ethnicity and income . p - values presented for the race / ethnicity comparisons are for the overall tests for any effect among the three groups ; individual pairwise results are presented when significant but the p - values are not provided . a less restrictive cutoff without a multiple - testing adjustment provides a larger pool of possible differences that can be targeted when revising caregiver / patient education and preventive care intervention strategies . the study enrolled 396 caregiver - toddler pairs at baseline ( two additional pairs were screened but did not qualify due to the child 's medical condition ) , which is estimated to be approximately 70% of those invited to participate . nearly all of the primary caregivers ( 378 ) were the child 's mother , with the remaining caregivers consisting of 14 fathers , 2 grandmothers , 1 aunt , and 1 other . the children ranged in age from 16 to 36 months , with a mean of 26 ( sd = 6 ) months , and ages did not differ significantly by race / ethnicity , income , or education of the caregiver . one hundred seventy - five ( 44% ) of the caregivers self - identified themselves as non - hispanic african - american , 141 ( 36% ) were non - hispanic white , 75 ( 19% ) were hispanic ( all races ) , and 5 ( 1% ) did not fall into one of the previous three categories . nearly one - third of hispanic caregivers reported difficulty understanding information they receive from physicians and dentists , while the rate was less than ten percent in non - hispanic african - americans and non - hispanic whites ( table 1 ) . furthermore , health literacy , collected on a subset of 250 caregivers , was not different among race / ethnicity groups but was weakly associated with education ( r = 0.18 , p = .02 ) . non - hispanic whites were more likely to use city water as their primary drinking water source as opposed to bottled or well water . habits of the caregivers that might lead to transmission of bacteria to the toddler differed by race / ethnicity ( figure 1 ) , education , and income . hispanic caregivers were less likely than non - hispanic african - american and non - hispanic white caregivers to put the toddler 's pacifier in their own mouth ( 12% versus 37% and 31% , p = .0156 ) , which was also associated with higher education ( odds ratio 1.3 , 95% ci 1.01.7 , p = .0212 ) but not with income ( odds ratio 1.0 , 95% ci 0.81.1 , p = .44 ) . tasting the child 's food or drink using the same fork / spoon or glass was common in all race / ethnicity groups ( approximately 70% , p = .87 ) , but was more common with those reporting a higher income ( odds ratio 1.3 , 95% ci 1.11.4 , p = .0013 ) . sharing food with the child using the same bowl / plate / glass and kissing the child on the lips occurred with nearly all non - hispanic african - american and non - hispanic white caregivers but was less frequent among hispanics ( p = .0001 ) and was more common with higher income ( odds ratio 1.3 , 95% ci 1.11.6 , p = .0028 ) . however , 87% of hispanics ever breast - fed compared to 50% of non - hispanic african - americans and 62% of non - hispanic whites ( p = .0004 ) ; breast - feeding was also more common with higher education ( odds ratio 1.6 , 95% ci 1.22.1 , p = .0004 ) and higher income ( odds ratio 1.1 , 95% ci 1.01.3 , p = .0458 ) . because the toddlers may have similar access to care as their caregivers , the questionnaire also asked about dentist and physician visits made by the caregiver . seventy - one percent of non - hispanic white caregivers , 53% of non - hispanic african - american caregivers , and 29% of hispanic caregivers had a dentist ( table 1 ) , and having a dentist was also associated with higher education attainment and higher income . approximately half of non - hispanic african - americans caregivers reported going to the dentist for regular checkups , while nearly 40% of hispanic caregivers reported never going to the dentist . in addition , patterns of caregiver visits to the physician differed by race / ethnicity ( table 1 ) but were not as affected by income or education , where only regular visits to the physician were associated with higher income . hispanic caregivers reported their children 's teeth were brushed less frequently than teeth of non - hispanic african - americans and non - hispanic whites ( table 2 ) . while there were differences among the race / ethnicity groups in how the caregivers felt about their child 's and their own dental and medical health , education and income were generally not related to these ratings . beliefs and knowledge ( figure 2 ) differed by race / ethnicity adults eventually losing all their teeth ( p = .0001 , higher response of false for non - hispanic whites ) , most children getting cavities ( p = .0304 , lower response of false for hispanics ) , bad teeth being mostly inherited from parents ( p = .0119 , lower response of false for hispanics ) , and when tooth cleaning should start ( p = .0001 , earlier for non - hispanic - whites ) , with also a trend for when the child 's first dental visit should be ( p = .06 , earliest for non - hispanic african - americans and latest for non - hispanic whites ) . belief that adults will eventually lose all their teeth was associated with less education ( odds ratio 1.4 , 95% ci 1.11.8 , p = .0072 ) and lower income ( odds ratio 1.1 , 95% ci 1.01.3 , p = .0376 ) , and belief that most children will eventually get cavities was associated with less education ( odds ratio 1.3 , 95% ci 1.01.6 , p = .0258 ) , while none of the other beliefs / knowledge assessed were significantly associated with education or income . hispanic toddlers were more likely drink from a bottle ( 29% ) compared to non - hispanic whites toddlers ( 11% ) and non - hispanic african - american toddlers ( 4% ) , while non - hispanic white toddlers and non - hispanic african - american toddlers were not significantly different . non - hispanic african - american toddlers were also less likely to drink from a sippy cup ( 67% ) compared to non - hispanic whites ( 84% ) and hispanic ( 87% ) toddlers , who were not significantly different from each other ( table 3 ) . although hispanic caregivers cleaned their child 's teeth after removing the drink more frequently than non - hispanic african - americans or non - hispanic whites , cleaning the child 's teeth after removing the drink was rare for all races . less than half of hispanic children regularly sipped on drinks between meals , while nearly all non - hispanic african - american and non - hispanic white children did . types of snacks and drinks usually eaten / drank between meals varied considerably among race / ethnicity groups for toddlers ( table 3 ) and for pcgs ( table 4 ) , while snacking and between - meals drinks were typically not associated with education or income , with a specific exception of nondiet soda being associated with less education . despite a decrease in dental caries prevalence in permanent teeth for most americans since the early 1970s , oral health disparities remain across some population groups , and dental caries is still the most prevalent chronic disease of childhood . examples include the caries risk tool of the american academy of pediatric dentistry , the ada 's caries risk tool for children younger than 6 , and the caries management by risk assessment ( cambra ) tool for children younger than 6 [ 48 , 49 ] . while other studies have identified caries risk factors in low - ses rural and low - ses african - american communities , the prevalence of the risk factors may affect both the disease prevalence and the types of interventions that may be effective in preventing and/or treating caries . age , socioeconomic status , and race / ethnicity differences as well as in non - us populations studied previously provided individual risk factor prevalence estimates , but only indirect evaluations of the effects of the sociodemographic factors on the risk factors could be made . in the present study , multiple factors from the caries risk questionnaire within the access to care , oral bacterial transmission , dental and medical health practices of the caregiver and the toddler , and snacking and drinking habits of the caregiver and the toddler areas were directly compared and differed by race / ethnicity , income , and/or education . with the limited sample size and single location sampled in this study , it is difficult to differentiate the effects of cultural influences , health knowledge gained through educational background , and income - based health utilization disparities on the risk factors ; in other words , we were unable to look at the influence of interactions among the three factors or stratify the analyses . and while the study included three race / ethnicity groups , the single location of the study ( indiana ) may not fully represent responses nationwide . a larger multisite study would be needed for increased generalizability as well as provide the sample size needed to differentiate among the cultural , income , and education influences on the risk factors . a large number of risk factors were examined , based on the extensive list of factors proposed or identified previously . nevertheless the information from our study can provide useful risk factor prevalence data when revising caregiver / patient education and preventive care intervention strategies . as mentioned above , our sample size was not large enough to justify a detailed examination of the 3-way interaction among race / ethnicity , income , and education to differentiate the effects of cultural influences , health knowledge gained through educational background , and income - based health utilization disparities on the risk factors . regardless of the underlying cause , as others have suggested based on observations in various populations [ 32 , 36 ] , education and intervention strategies can be targeted generally to the population seen in the practice and specifically to individual patients . the imb program was initiated in 2000 and has led to a substantial increase in access to preventive dental services by enabling medicaid children younger than 3 years of age to receive dental screening , counseling , and fluoride varnish in physicians ' offices . more work will certainly be needed to evaluate the acceptability and effectiveness of education and intervention strategies in targeted populations . in conclusion , significant differences were found in all areas of the questionnaire related to race / ethnicity , income , and/or education . patient education and preventive care intervention studies may need to be targeted based on the characteristics of the population to achieve increase effectiveness . very satisfied somewhat satisfied somewhat dissatisfied very dissatisfied not applicable somewhat dissatisfied the next questions focus on your dental beliefs most adults will lose all their teeth as they get older true false do not know most young children will get cavities true false do not know only children need fluoride true false do not know the type of food and drink a child eats or drinks may cause cavities true false do not know baby teeth are important to take care of true false do not know bad teeth are mostly inherited from the parents true false do not know cleaning of the mouth of a child should begin : ( check all that apply ) before the first tooth comes in as soon as the first tooth comes in 1224 months - of - age after 24 months - of - age when the adult teeth come in do not know before the first tooth comes in as soon as the first tooth comes in after 24 months - of - age when the adult teeth come in a child 's first routine dental visit should be : ( check all that apply ) before the first tooth comes in as soon as the first tooth comes in 1224 months - of - age after 24 months - of - age when the adult teeth come in do not know before the first tooth comes in as soon as the first tooth comes in after 24 months - of - age when the adult teeth come in the next questions focus on your own eating and health habits . water juice ( 100% juice ) milk coffee without sugar soda ( with sugar ) fruit drink ( with sugar ) tea coffee with sugar soda ( diet or sugar - free ) fruit drink ( sugar - free ) other ( list or specify _ _ _ _ _ _ ) fruit drink ( with sugar ) soda ( diet or sugar - free ) fruit drink ( sugar - free ) other ( list or specify _ _ _ _ _ _ ) now we are going to ask some questions about you , the child 's family , and the child . ( mark one or more races to indicate the race or races you consider the child to be ) white or caucasian african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ _ ) african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ ( mark one or more races to indicate the race or races you consider yourself to be ) white or caucasian african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ _ ) african american or black ( black refers to people with ancestors from sub - saharan africa , the west indies , the caribbean ( including haiti , jamaica , barbados , and cape verde ) asian ( specify subgroup _ _ _ _ _ _ ) native hawaiian or other pacific islander american indian or alaskan native other ( specify : _ _ _ _ _ number of adults / children _ _ _ _ _ _ number of adults / children _ _ _ _ _ _ how many adults in the child 's household have paid jobs ?

nigeria has one of the highest maternal mortality ratios ( mmr ) in the world . between 2010 - 2012 , the world health organization ( who ) estimated that the country has an mmr of 630 per 100,000 live births . this figure indicates that the country only surpassed war torned and or politically unstable countries like sierra leone ( 890 per 100,000 ) , liberia ( 777 per100 , 000 ) and chad ( 1,100 per 100,000 ) live births respectively . the poor maternal outcome might not be unrelated to the low utilization rates of maternal health care services . for example , about half of the estimated eight million annual numbers of pregnancies have not had antenatal care ( anc ) in 2010 . furthermore , among those that had anc , 45% have made less than the minimum four anc visits as recommended by the who . this national average does not reveal the disparities that exist within and between the six geopolitical zones of the country which are fairly heterogeneous in terms of religious and cultural affiliations . women from the north east zone of nigeria are more likely to die due to pregnancy related complications . for instance , while the north east zonal average for the proportion of women who had anc was 43% , yobe state had the lowest with only 36% anc utilization rate . these figures might even be lower since the state is largely rural , lowest number of skilled health workers , and poor records of vital events . further , the sensitivity of the surveillance system is low based on recent reports that indicated the state has low anc utilization and having mmr that is two fold higher than the reported national average of 630 per 100,000 live births . although yobe state has 528 public and private health facilities , however , these facilities lack both technical and institutional capacity , which invariability might have contributed to the low utilization of anc services . with an estimated annual number of pregnancies of 600,000 and only 45 public employed midwives , a midwife is expected to attend to more than 50 pregnancies per day which could compromise the quality of services rendered . considering the yobe state strategic health development plan 2010 2015 is in its final year , there is the need to assess the the predictors of anc utilization in the state in order to sustain the achievements recorded towards millenium development goal number five ( mdg 5 ) which is to reduce maternal deaths by two thirds compared to what was recorded in 1990 . unfortunately , there seems to be a dearth of local research studies on maternal mortality in nigeria . for instance , despite nigeria being among the areas with high mmr in the world,[8 - 10 ] reports of systematic reviews of 2,500 and 5,575 showed that only 3 ( 1.2% ) and 5 ( 0.5% ) articles respectively were from nigeria and non from yobe state . one of the plausible reasons for such findings is that most published studies in nigeria were hospital - based and focused more on medical causes of death such as hemorrhage , eclampsia , obstructed labor , ruptured uterus , sepsis , malaria and anemia in pregnancy , which does not provide information on antecedents before arrival to a health facility . this paper is an effort to bridge this gap aimed to find out whether women s individual and community level factors influence the utilization of anc services in yobe state as measured by number of antenatal care visits . this is a cross - sectional study using data from the 2008 nigeria demographic and health survey ( 2008 ndhs ) after receiving approval from orc macro and icf international based in calverton maryland , usa . a total of 967 women between 15 - 49 years who had given birth between january 2003 and december 2008 participated in the study . bivariate pearson s chi square test statistic and two stages of multivariate regression analysis were conducted in order to identify factors that predict the utilization of anc in yobe state . first , logistic regression of one variable at a time was conducted to get the unadjusted odds ratio and thereafter , a multivariate logistic regression was conducted to obtain adjusted odds ratios aimed at accounting for the effect of each of the study covariates . we utilized the anderson health behavior model as a framework for analyzing the determinants of utilization of health services . the model is composed of three sets of individual and community level factors that provide constructs to assess individuals capacity to access and use health services . the three main set of factors are a ) predisposing characteristics at individual level ; b ) enabling characteristics which focus on community level factors such as health care financing mechanism and health resources and c ) need characteristics which is the perceived state of health by individual and health workere . this unique characteristic of the anderson s model was underscored by its application in a range of medical research such as the utilization of health care services[15 - 17 ] and other social issues.[18 - 24 ] out of the 967 respondents , a total of 648 ( 67% ) had at least one anc visit during the last pregnancy before the 2008 ndhs survey . among those that had anc , only 328 had four or more recommended anc visits constituting only 33.9% of the total study sample . moreover , 319 ( 33% ) and 320 ( 33.1% ) of repondents had zero or less than four anc visits respectively . about 26.4% of the respondents commenced their anc visits in their third trimester ( table 1 ) . bio - socio - demographic characteristics of study respondents the age group 20 34 years old constituted the majority ( 44.9% ) of the respondents with a mean age of 27.2 + 3.2 years . the extreme of ages ( < 20 years and 35 years ) had the lowest proportion of women who had four or more anc visits . majority of the women ( 87% ) had at least two children ( table 1 ) . majority of the women were married ( 89.7% ) , belonging to kanuri / baribari ethnic group ( 42.2% ) , muslims ( 98.7% ) , lack any form of autonomy ( 79.8% ) and lived in rural areas ( 72.8% ) . living in urban areas ( 27.2% ) , yoruba ethnicity ( 60% ) , christians ( 30% ) and having full autonomy ( 30% ) had higher proportion of women that had four or more anc visits ( table 1 ) . majority of the respondents were illiterate ( 82% ) , unemployed ( 50% ) and belonged to the poorest / poor wealth quintile ( 78.3% ) , and only 11.7% , 14% and 24,7% , respectively of these categories of women had met the recommended minimum of four anc by the who ( table 1 ) . distance to the nearest health facility and lacking health insurance policy ( 99.6% ) showed lower proportion of women that had appropriate number of anc visits . majority of the respondents ( 91% ) were not attended by skilled health care workers and fewer still ( 19.8% ) had achieved the who recommendation of at least four anc visits ( table 1 ) . age is significantly associated with the number of anc visits ( = 43.11 ; df = 2 ; p < 0.00 ) . the age group 15 19 years were less likely to make four or more anc visits compared to women who were aged 20 34 years even after controlling for the covariates ( aor = 1.72 ; ci 0.75 3.91 ) ( table 2 ) . factors associated with the number of antenatal care visits attended . notes : or = odds ratio ; ci = confidence intervals ; attended at least one anc visit=648 parity of 2 ( aor = 0.47 ; ci 0.20 1.11 ) , religion ( aor = 0.99 ; ci 0.17 2.31 ) , distance to the nearest health facility ( aor = 0.99 ; ci 0.22 4.50 ) , availability of health insurance policy ( aor = 1.12 ; ci 0.99 1.71 ) and access to the media as a source of information on mhs ( aor = 0.99 ; ci 0.37 2.71 ) have no impact on the use of anc services after controlling for education , family wealth index , and availably of skilled health workers ( table 2 ) . place of domicile ( = 90.36 ; df = 1 ; p < 0.00 ) , ethnicity ( = 46.94 ; df = 5 ; p < 0.00 ) , level of education ( = 73.03 ; df = 3 ; p < 0.00 ) , occupation ( = 39.21 ; df = 3 ; p < 0.00 ) , family wealth index ( = 139.35 ; df = 4 ; p < 0.00 ) and availability of skilled health workers ( = 78.86 ; df = 1 ; p < 0.00 ) are associated with the number of anc visits ( table 2 ) . after adjusting for covariates , women of yoruba and igbo ethnic groups were 44 and 94 times respectively more likely to achieve the recommended four or more anc visits compared to women belonging to hausa ethnic group ( table 2 ) . women living in urban areas ( aor = 2.14 ; ci 1.23 3.73 ) , having some form of autonomy ( aor = 8.74 ; ci 0.62 12.45 ) and those in the highest wealth quintile ( aor = 4.44 ; ci 0.64 30.64 ) had four or more number of anc visits compared to their rural colleagues , those without any form of autonomy and belonging the poorest wealth quintile respectively ( table 2 ) . similarly , women with at least primary level education ( aor = 2.40 ; ci 1.24 4.67 ) , belonging to professional employment category such as lawyers , health workers , teachers etc ( aor = 12.07 ; ci 0.19 75.74 ) and those who have access to skilled health workers ( aor = 5.13 ; ci 2.50 10.52 ) are more likely to make the required number of anc visits compared to those who are illiterates , unemployed and had no access to skilled health workers respectively ( table 2 ) . the model contained six predictive variables ( age , family wealth quintile , religious affiliation , highest educational attainment , parity , and distance to health facilityr ) . these variables were selected based on the result of the bivariate pearson chi square test , binary logistic regression and or known theoretical facts . these variables were found to be statistically significant ( 28 , n=967 ) 53.10 ; p < 0.00 , indicating that , the model was able to distinguish between participants who have had less than four anc visits and those who had four or more anc visits . the variables accounted for 39.1% ( cox & snell r square ) and 54.4% ( nagelkerke r square ) of variability among participants , correctly classified 80.4% of cases and together with hosmer and lemeshow goodness of fit test indicated the model being useful ( p = 0.77 ) since the p - value is larger than the alpha level ( table not shown ) . out of the 967 respondents , a total of 648 ( 67% ) had at least one anc visit during the last pregnancy before the 2008 ndhs survey . among those that had anc , only 328 had four or more recommended anc visits constituting only 33.9% of the total study sample . moreover , 319 ( 33% ) and 320 ( 33.1% ) of repondents had zero or less than four anc visits respectively . about 26.4% of the respondents commenced their anc visits in their third trimester ( table 1 ) . the age group 20 34 years old constituted the majority ( 44.9% ) of the respondents with a mean age of 27.2 + 3.2 years . the extreme of ages ( < 20 years and 35 years ) had the lowest proportion of women who had four or more anc visits . majority of the women ( 87% ) had at least two children ( table 1 ) . majority of the women were married ( 89.7% ) , belonging to kanuri / baribari ethnic group ( 42.2% ) , muslims ( 98.7% ) , lack any form of autonomy ( 79.8% ) and lived in rural areas ( 72.8% ) . living in urban areas ( 27.2% ) , yoruba ethnicity ( 60% ) , christians ( 30% ) and having full autonomy ( 30% ) had higher proportion of women that had four or more anc visits ( table 1 ) . majority of the respondents were illiterate ( 82% ) , unemployed ( 50% ) and belonged to the poorest / poor wealth quintile ( 78.3% ) , and only 11.7% , 14% and 24,7% , respectively of these categories of women had met the recommended minimum of four anc by the who ( table 1 ) . distance to the nearest health facility and lacking health insurance policy ( 99.6% ) showed lower proportion of women that had appropriate number of anc visits . majority of the respondents ( 91% ) were not attended by skilled health care workers and fewer still ( 19.8% ) had achieved the who recommendation of at least four anc visits ( table 1 ) . age is significantly associated with the number of anc visits ( = 43.11 ; df = 2 ; p < 0.00 ) . the age group 15 19 years were less likely to make four or more anc visits compared to women who were aged 20 34 years even after controlling for the covariates ( aor = 1.72 ; ci 0.75 3.91 ) ( table 2 ) . notes : or = odds ratio ; ci = confidence intervals ; attended at least one anc visit=648 parity of 2 ( aor = 0.47 ; ci 0.20 1.11 ) , religion ( aor = 0.99 ; ci 0.17 2.31 ) , distance to the nearest health facility ( aor = 0.99 ; ci 0.22 4.50 ) , availability of health insurance policy ( aor = 1.12 ; ci 0.99 1.71 ) and access to the media as a source of information on mhs ( aor = 0.99 ; ci 0.37 2.71 ) have no impact on the use of anc services after controlling for education , family wealth index , and availably of skilled health workers ( table 2 ) . place of domicile ( = 90.36 ; df = 1 ; p < 0.00 ) , ethnicity ( = 46.94 ; df = 5 ; p < 0.00 ) , level of education ( = 73.03 ; df = 3 ; p < 0.00 ) , occupation ( = 39.21 ; df = 3 ; p < 0.00 ) , family wealth index ( = 139.35 ; df = 4 ; p < 0.00 ) and availability of skilled health workers ( = 78.86 ; df = 1 ; p < 0.00 ) are associated with the number of anc visits ( table 2 ) . after adjusting for covariates , women of yoruba and igbo ethnic groups were 44 and 94 times respectively more likely to achieve the recommended four or more anc visits compared to women belonging to hausa ethnic group ( table 2 ) . women living in urban areas ( aor = 2.14 ; ci 1.23 3.73 ) , having some form of autonomy ( aor = 8.74 ; ci 0.62 12.45 ) and those in the highest wealth quintile ( aor = 4.44 ; ci 0.64 30.64 ) had four or more number of anc visits compared to their rural colleagues , those without any form of autonomy and belonging the poorest wealth quintile respectively ( table 2 ) . similarly , women with at least primary level education ( aor = 2.40 ; ci 1.24 4.67 ) , belonging to professional employment category such as lawyers , health workers , teachers etc ( aor = 12.07 ; ci 0.19 75.74 ) and those who have access to skilled health workers ( aor = 5.13 ; ci 2.50 10.52 ) are more likely to make the required number of anc visits compared to those who are illiterates , unemployed and had no access to skilled health workers respectively ( table 2 ) . the model contained six predictive variables ( age , family wealth quintile , religious affiliation , highest educational attainment , parity , and distance to health facilityr ) . these variables were selected based on the result of the bivariate pearson chi square test , binary logistic regression and or known theoretical facts . these variables were found to be statistically significant ( 28 , n=967 ) 53.10 ; p < 0.00 , indicating that , the model was able to distinguish between participants who have had less than four anc visits and those who had four or more anc visits . the variables accounted for 39.1% ( cox & snell r square ) and 54.4% ( nagelkerke r square ) of variability among participants , correctly classified 80.4% of cases and together with hosmer and lemeshow goodness of fit test indicated the model being useful ( p = 0.77 ) since the p - value is larger than the alpha level ( table not shown ) . maternal and child health services ( mchs ) is among the top priority of yobe state government as enshrined in the state health strategic plan for 2010 - 2015 . despite significant progress in mchs the mmr in the state was reported to be 1,549 per 100 , 000 live births which is about three times higher than the national average of 545 per 100,000 live births . furthermore , the anc utilization rate was 36% and of these only 9.8% had access to appropriate skilled health workers . in general , wide disparities exist between the different parts of the country with the states in the north east zone which includes yobe state having the worst maternal health indicators . in order to identify the root issues for the abysmal performance of the state , this study disaggregated data in line with the social determinants of health as advanced by the who . the low anc utilization rates in the north eastern states of nigeria which include yobe state is consistent with areas with the high levels of poverty , low female literacy and empowerment this is a reflection of the socioeconomic development , access and utilization mchs rendered in the state . while it might have cultural connotations , however , mchs will only be used when geographical and economic access is guaranteed . for instance , the yobe state health strategic plan for 2010 2015 clearly indicated the lack of technical and institutional capacity which could have contributed to the low utilization of anc services . the worsening of the current boko haram islamist extremist insurgency particularly from 2011 to date , could have further decimated the availability of skilled health workers based on recent report that the doctor population ratio is 1 for every 54,000 people . furthermore , a study on the state of health system following the boko haram insurgency has indicated that health workers have been abducted and/or killed and many health facilities were closed . the findings of this study showed that few respondent have access to skilled health workers ( 9% ) , very high proportion of illiterates ( 82% ) , families living in poverty ( 78.3% ) , high risk pregnancies among teenagers ( 22.1% ) and women more than 34 years ( 33% ) and lack of female autonomy ( 79.8% ) in terms of decision making , economic independence and freedom of going out of their matrimonial home on health grounds . these findings suggest that these women are likely to have high proportion of complicated pregnancies , poor access and utilization of mchs , which might have contributed to the high mmr as was similarly observed in other studies.[29 - 33 ] furthermore , these high risk pregnancies coupled with short interval between births ( 25.3% ) and grand multipara ( 50.6% ) as observed in this study , could be the underlying root causes for most of the high proportion of preventable maternal deaths . however , the low proportion of women who had four or more number of anc visits means that , many high risk pregnant women may not be detected and may result in life threatening condition and in extreme cases could lead to the death of a woman and or her baby . the lack of autonomy further compound the scenario , since it may lead to delays to decide to seek modern medical care when early signs of danger are noticed . although , the low anc utilization rate could be influenced by place of domicile , ethnicity , religion and female autonomy , however , these factors were not found to be consistently statistically significant after controlling for covariates such as education and income levels . the low utilization of anc services as observed in this study is not in keeping with the findings of another study in nigeria that reported muslim women are less likely to have four or more number of anc visits compared to their christian counterparts . this assertion is not consistent when confounders such as tribe , income and level of education were controlled as was the case in this study . moreover , after controlling for age , wealth index , education and distance , parity and religion have no significant impact on the number of anc visits and its main effect associations shifted from been statistically significant to not significant ( table 2 ) . hence , the role of ethnicity and religion needs to be studied as both shape the attitude and behaviors of the populace . the reason been that , each of the major ethnic group particularly hausa , igbo , fulani and kanuris are predorminantly ( > 90% ) adherents of either christianity or islam and each has different ethnically driven norms on pregnancy and childbirth . it is important to underscore that the study being cross sectional design has only demonstrated the strength of associations rather than causal factors . moreover , dhs data is individual based and therefore might not fully represent community level factors and hence the need for studies with robust designs . this study demonstrated that , educational level , family wealth income , and availability of skilled health worker are consistently associated with the number of anc even after controlling for covariates . these three predicor variables are in tandem with mdg 1 ( eradication of extreme poverty and hunger ) , mdg 2 ( universal basic education ) , mdg 3 ( gender equality ) and mdg 4 ( maternal mortality ) . these three variables have significant impact on population health outcome and need for intersectoral collaboration with ministries of education , agricultural and other social services using primary health care as the springboard . a sustainable approach is to provide compulsory free universal basic education for girls ( mdg 2 ) to emhance uptake and retention of girl s up to secondary level of education . this will have a multiplier effect towards reduction in poverty ( mdg 1 ) , improve female autonomy and equality ( mdg 3 ) and improve health cultural capital that will ultimately reduce maternal deaths ( mdg 5 ) . hence , the provision of health care services should be driven by results based management approach in order to enhance realistic multipronged planning , ownership , commitment , accountability and transparency among various stakeholders . there is therefore , the need for an independent periodic program reviews to guide timely and appropriate interventions . more than 67% of pregnant women in yobe state , nigeria did not receive the recommended four antenatal care visits visits.lack of education is the most consistent variable associated with antenatal care utilization in yobe state , nigeria.educating all girls beyond primary school level could enhance improvement in female empowerment and ability to make the most appropriate healthcare related choices.girl-child education is among the most promising interventions to address global issues like extreme poverty and hunger ( mdg 1 ) , low female literacy rate ( mdg 2 ) , gender inequality ( mdg 3 ) , high maternal and infant morbidity and mortality ( mdg 4 & 5 ) in yobe state , nigeria . more than 67% of pregnant women in yobe state , nigeria did not receive the recommended four antenatal care visits visits . lack of education is the most consistent variable associated with antenatal care utilization in yobe state , nigeria . educating all girls beyond primary school level could enhance improvement in female empowerment and ability to make the most appropriate healthcare related choices . girl - child education is among the most promising interventions to address global issues like extreme poverty and hunger ( mdg 1 ) , low female literacy rate ( mdg 2 ) , gender inequality ( mdg 3 ) , high maternal and infant morbidity and mortality ( mdg 4 & 5 ) in yobe state , nigeria .

objective : in nigeria , wide disparities exist between the different parts of the country , with the states in the north east zone having poor health resources . the objective of this study is to assess whether women s biological , sociocultural , and economic characteristics are associated with utilization of ante natal care services as measured by number of antenatal care ( anc ) visits in yobe state.methods:this is a secondary data analysis of the 2008 nigeria demographic and health survey with records from 33,385 women between 15 - 49 years who had given birth between january 2003 and december 2008 in yobe state . bivariate pearson s chi square test and two stages of multivariate regression analysis were conducted.results:women with at least primary level education ( adjusted or ( aor ) = 2.40 ; ci 1.24 4.67 ) , belonging to professional employment category ( aor = 12.07 ; ci 0.19 75.74 ) and those who had access to skilled health workers ( aor = 5.13 ; ci 2.50 10.52 ) are more likely to make the required number of anc visits compared to those who are illiterates , unemployed and had no access to skilled health workers.conclusion and global health implications : this study demonstrated that educational level , family wealth income , and availability of skilled health worker were consistently associated with the number of anc visits even after controlling for covariates . these three covariates are in tandem with the millenium development goals ( mdg ) 1 - eradication of extreme poverty and hunger ; mdg 2 - universal basic education ; mdg 3 - gender equality ; and mdg 4 - maternal mortality . there is the need for inter - sectoral holistic intervention approach .

Background and Objective Methodology Results Descriptive Biological characteristics of respondents Cultural characteristics of respondents Economic characteristics of respondents Respondents health system characteristics Results of bivariate and logistic regression analysis Predictive model on the utilization of ANC services Discussions Conclusions and Global Health Implications

nigeria has one of the highest maternal mortality ratios ( mmr ) in the world . this figure indicates that the country only surpassed war torned and or politically unstable countries like sierra leone ( 890 per 100,000 ) , liberia ( 777 per100 , 000 ) and chad ( 1,100 per 100,000 ) live births respectively . for example , about half of the estimated eight million annual numbers of pregnancies have not had antenatal care ( anc ) in 2010 . this national average does not reveal the disparities that exist within and between the six geopolitical zones of the country which are fairly heterogeneous in terms of religious and cultural affiliations . women from the north east zone of nigeria are more likely to die due to pregnancy related complications . for instance , while the north east zonal average for the proportion of women who had anc was 43% , yobe state had the lowest with only 36% anc utilization rate . these figures might even be lower since the state is largely rural , lowest number of skilled health workers , and poor records of vital events . although yobe state has 528 public and private health facilities , however , these facilities lack both technical and institutional capacity , which invariability might have contributed to the low utilization of anc services . considering the yobe state strategic health development plan 2010 2015 is in its final year , there is the need to assess the the predictors of anc utilization in the state in order to sustain the achievements recorded towards millenium development goal number five ( mdg 5 ) which is to reduce maternal deaths by two thirds compared to what was recorded in 1990 . for instance , despite nigeria being among the areas with high mmr in the world,[8 - 10 ] reports of systematic reviews of 2,500 and 5,575 showed that only 3 ( 1.2% ) and 5 ( 0.5% ) articles respectively were from nigeria and non from yobe state . this paper is an effort to bridge this gap aimed to find out whether women s individual and community level factors influence the utilization of anc services in yobe state as measured by number of antenatal care visits . this is a cross - sectional study using data from the 2008 nigeria demographic and health survey ( 2008 ndhs ) after receiving approval from orc macro and icf international based in calverton maryland , usa . a total of 967 women between 15 - 49 years who had given birth between january 2003 and december 2008 participated in the study . bivariate pearson s chi square test statistic and two stages of multivariate regression analysis were conducted in order to identify factors that predict the utilization of anc in yobe state . the three main set of factors are a ) predisposing characteristics at individual level ; b ) enabling characteristics which focus on community level factors such as health care financing mechanism and health resources and c ) need characteristics which is the perceived state of health by individual and health workere . this unique characteristic of the anderson s model was underscored by its application in a range of medical research such as the utilization of health care services[15 - 17 ] and other social issues. [18 - 24 ] out of the 967 respondents , a total of 648 ( 67% ) had at least one anc visit during the last pregnancy before the 2008 ndhs survey . moreover , 319 ( 33% ) and 320 ( 33.1% ) of repondents had zero or less than four anc visits respectively . about 26.4% of the respondents commenced their anc visits in their third trimester ( table 1 ) . majority of the women ( 87% ) had at least two children ( table 1 ) . majority of the women were married ( 89.7% ) , belonging to kanuri / baribari ethnic group ( 42.2% ) , muslims ( 98.7% ) , lack any form of autonomy ( 79.8% ) and lived in rural areas ( 72.8% ) . living in urban areas ( 27.2% ) , yoruba ethnicity ( 60% ) , christians ( 30% ) and having full autonomy ( 30% ) had higher proportion of women that had four or more anc visits ( table 1 ) . majority of the respondents were illiterate ( 82% ) , unemployed ( 50% ) and belonged to the poorest / poor wealth quintile ( 78.3% ) , and only 11.7% , 14% and 24,7% , respectively of these categories of women had met the recommended minimum of four anc by the who ( table 1 ) . distance to the nearest health facility and lacking health insurance policy ( 99.6% ) showed lower proportion of women that had appropriate number of anc visits . majority of the respondents ( 91% ) were not attended by skilled health care workers and fewer still ( 19.8% ) had achieved the who recommendation of at least four anc visits ( table 1 ) . age is significantly associated with the number of anc visits ( = 43.11 ; df = 2 ; p < 0.00 ) . the age group 15 19 years were less likely to make four or more anc visits compared to women who were aged 20 34 years even after controlling for the covariates ( aor = 1.72 ; ci 0.75 3.91 ) ( table 2 ) . factors associated with the number of antenatal care visits attended . notes : or = odds ratio ; ci = confidence intervals ; attended at least one anc visit=648 parity of 2 ( aor = 0.47 ; ci 0.20 1.11 ) , religion ( aor = 0.99 ; ci 0.17 2.31 ) , distance to the nearest health facility ( aor = 0.99 ; ci 0.22 4.50 ) , availability of health insurance policy ( aor = 1.12 ; ci 0.99 1.71 ) and access to the media as a source of information on mhs ( aor = 0.99 ; ci 0.37 2.71 ) have no impact on the use of anc services after controlling for education , family wealth index , and availably of skilled health workers ( table 2 ) . place of domicile ( = 90.36 ; df = 1 ; p < 0.00 ) , ethnicity ( = 46.94 ; df = 5 ; p < 0.00 ) , level of education ( = 73.03 ; df = 3 ; p < 0.00 ) , occupation ( = 39.21 ; df = 3 ; p < 0.00 ) , family wealth index ( = 139.35 ; df = 4 ; p < 0.00 ) and availability of skilled health workers ( = 78.86 ; df = 1 ; p < 0.00 ) are associated with the number of anc visits ( table 2 ) . after adjusting for covariates , women of yoruba and igbo ethnic groups were 44 and 94 times respectively more likely to achieve the recommended four or more anc visits compared to women belonging to hausa ethnic group ( table 2 ) . women living in urban areas ( aor = 2.14 ; ci 1.23 3.73 ) , having some form of autonomy ( aor = 8.74 ; ci 0.62 12.45 ) and those in the highest wealth quintile ( aor = 4.44 ; ci 0.64 30.64 ) had four or more number of anc visits compared to their rural colleagues , those without any form of autonomy and belonging the poorest wealth quintile respectively ( table 2 ) . similarly , women with at least primary level education ( aor = 2.40 ; ci 1.24 4.67 ) , belonging to professional employment category such as lawyers , health workers , teachers etc ( aor = 12.07 ; ci 0.19 75.74 ) and those who have access to skilled health workers ( aor = 5.13 ; ci 2.50 10.52 ) are more likely to make the required number of anc visits compared to those who are illiterates , unemployed and had no access to skilled health workers respectively ( table 2 ) . the model contained six predictive variables ( age , family wealth quintile , religious affiliation , highest educational attainment , parity , and distance to health facilityr ) . these variables were selected based on the result of the bivariate pearson chi square test , binary logistic regression and or known theoretical facts . these variables were found to be statistically significant ( 28 , n=967 ) 53.10 ; p < 0.00 , indicating that , the model was able to distinguish between participants who have had less than four anc visits and those who had four or more anc visits . out of the 967 respondents , a total of 648 ( 67% ) had at least one anc visit during the last pregnancy before the 2008 ndhs survey . among those that had anc , only 328 had four or more recommended anc visits constituting only 33.9% of the total study sample . moreover , 319 ( 33% ) and 320 ( 33.1% ) of repondents had zero or less than four anc visits respectively . about 26.4% of the respondents commenced their anc visits in their third trimester ( table 1 ) . majority of the women were married ( 89.7% ) , belonging to kanuri / baribari ethnic group ( 42.2% ) , muslims ( 98.7% ) , lack any form of autonomy ( 79.8% ) and lived in rural areas ( 72.8% ) . living in urban areas ( 27.2% ) , yoruba ethnicity ( 60% ) , christians ( 30% ) and having full autonomy ( 30% ) had higher proportion of women that had four or more anc visits ( table 1 ) . majority of the respondents were illiterate ( 82% ) , unemployed ( 50% ) and belonged to the poorest / poor wealth quintile ( 78.3% ) , and only 11.7% , 14% and 24,7% , respectively of these categories of women had met the recommended minimum of four anc by the who ( table 1 ) . distance to the nearest health facility and lacking health insurance policy ( 99.6% ) showed lower proportion of women that had appropriate number of anc visits . majority of the respondents ( 91% ) were not attended by skilled health care workers and fewer still ( 19.8% ) had achieved the who recommendation of at least four anc visits ( table 1 ) . age is significantly associated with the number of anc visits ( = 43.11 ; df = 2 ; p < 0.00 ) . the age group 15 19 years were less likely to make four or more anc visits compared to women who were aged 20 34 years even after controlling for the covariates ( aor = 1.72 ; ci 0.75 3.91 ) ( table 2 ) . notes : or = odds ratio ; ci = confidence intervals ; attended at least one anc visit=648 parity of 2 ( aor = 0.47 ; ci 0.20 1.11 ) , religion ( aor = 0.99 ; ci 0.17 2.31 ) , distance to the nearest health facility ( aor = 0.99 ; ci 0.22 4.50 ) , availability of health insurance policy ( aor = 1.12 ; ci 0.99 1.71 ) and access to the media as a source of information on mhs ( aor = 0.99 ; ci 0.37 2.71 ) have no impact on the use of anc services after controlling for education , family wealth index , and availably of skilled health workers ( table 2 ) . place of domicile ( = 90.36 ; df = 1 ; p < 0.00 ) , ethnicity ( = 46.94 ; df = 5 ; p < 0.00 ) , level of education ( = 73.03 ; df = 3 ; p < 0.00 ) , occupation ( = 39.21 ; df = 3 ; p < 0.00 ) , family wealth index ( = 139.35 ; df = 4 ; p < 0.00 ) and availability of skilled health workers ( = 78.86 ; df = 1 ; p < 0.00 ) are associated with the number of anc visits ( table 2 ) . after adjusting for covariates , women of yoruba and igbo ethnic groups were 44 and 94 times respectively more likely to achieve the recommended four or more anc visits compared to women belonging to hausa ethnic group ( table 2 ) . women living in urban areas ( aor = 2.14 ; ci 1.23 3.73 ) , having some form of autonomy ( aor = 8.74 ; ci 0.62 12.45 ) and those in the highest wealth quintile ( aor = 4.44 ; ci 0.64 30.64 ) had four or more number of anc visits compared to their rural colleagues , those without any form of autonomy and belonging the poorest wealth quintile respectively ( table 2 ) . similarly , women with at least primary level education ( aor = 2.40 ; ci 1.24 4.67 ) , belonging to professional employment category such as lawyers , health workers , teachers etc ( aor = 12.07 ; ci 0.19 75.74 ) and those who have access to skilled health workers ( aor = 5.13 ; ci 2.50 10.52 ) are more likely to make the required number of anc visits compared to those who are illiterates , unemployed and had no access to skilled health workers respectively ( table 2 ) . the model contained six predictive variables ( age , family wealth quintile , religious affiliation , highest educational attainment , parity , and distance to health facilityr ) . these variables were selected based on the result of the bivariate pearson chi square test , binary logistic regression and or known theoretical facts . these variables were found to be statistically significant ( 28 , n=967 ) 53.10 ; p < 0.00 , indicating that , the model was able to distinguish between participants who have had less than four anc visits and those who had four or more anc visits . maternal and child health services ( mchs ) is among the top priority of yobe state government as enshrined in the state health strategic plan for 2010 - 2015 . furthermore , the anc utilization rate was 36% and of these only 9.8% had access to appropriate skilled health workers . in general , wide disparities exist between the different parts of the country with the states in the north east zone which includes yobe state having the worst maternal health indicators . in order to identify the root issues for the abysmal performance of the state , this study disaggregated data in line with the social determinants of health as advanced by the who . the low anc utilization rates in the north eastern states of nigeria which include yobe state is consistent with areas with the high levels of poverty , low female literacy and empowerment this is a reflection of the socioeconomic development , access and utilization mchs rendered in the state . for instance , the yobe state health strategic plan for 2010 2015 clearly indicated the lack of technical and institutional capacity which could have contributed to the low utilization of anc services . the worsening of the current boko haram islamist extremist insurgency particularly from 2011 to date , could have further decimated the availability of skilled health workers based on recent report that the doctor population ratio is 1 for every 54,000 people . the findings of this study showed that few respondent have access to skilled health workers ( 9% ) , very high proportion of illiterates ( 82% ) , families living in poverty ( 78.3% ) , high risk pregnancies among teenagers ( 22.1% ) and women more than 34 years ( 33% ) and lack of female autonomy ( 79.8% ) in terms of decision making , economic independence and freedom of going out of their matrimonial home on health grounds . [29 - 33 ] furthermore , these high risk pregnancies coupled with short interval between births ( 25.3% ) and grand multipara ( 50.6% ) as observed in this study , could be the underlying root causes for most of the high proportion of preventable maternal deaths . however , the low proportion of women who had four or more number of anc visits means that , many high risk pregnant women may not be detected and may result in life threatening condition and in extreme cases could lead to the death of a woman and or her baby . although , the low anc utilization rate could be influenced by place of domicile , ethnicity , religion and female autonomy , however , these factors were not found to be consistently statistically significant after controlling for covariates such as education and income levels . the low utilization of anc services as observed in this study is not in keeping with the findings of another study in nigeria that reported muslim women are less likely to have four or more number of anc visits compared to their christian counterparts . moreover , after controlling for age , wealth index , education and distance , parity and religion have no significant impact on the number of anc visits and its main effect associations shifted from been statistically significant to not significant ( table 2 ) . moreover , dhs data is individual based and therefore might not fully represent community level factors and hence the need for studies with robust designs . this study demonstrated that , educational level , family wealth income , and availability of skilled health worker are consistently associated with the number of anc even after controlling for covariates . these three predicor variables are in tandem with mdg 1 ( eradication of extreme poverty and hunger ) , mdg 2 ( universal basic education ) , mdg 3 ( gender equality ) and mdg 4 ( maternal mortality ) . these three variables have significant impact on population health outcome and need for intersectoral collaboration with ministries of education , agricultural and other social services using primary health care as the springboard . a sustainable approach is to provide compulsory free universal basic education for girls ( mdg 2 ) to emhance uptake and retention of girl s up to secondary level of education . this will have a multiplier effect towards reduction in poverty ( mdg 1 ) , improve female autonomy and equality ( mdg 3 ) and improve health cultural capital that will ultimately reduce maternal deaths ( mdg 5 ) . there is therefore , the need for an independent periodic program reviews to guide timely and appropriate interventions . more than 67% of pregnant women in yobe state , nigeria did not receive the recommended four antenatal care visits visits.lack of education is the most consistent variable associated with antenatal care utilization in yobe state , nigeria.educating all girls beyond primary school level could enhance improvement in female empowerment and ability to make the most appropriate healthcare related choices.girl-child education is among the most promising interventions to address global issues like extreme poverty and hunger ( mdg 1 ) , low female literacy rate ( mdg 2 ) , gender inequality ( mdg 3 ) , high maternal and infant morbidity and mortality ( mdg 4 & 5 ) in yobe state , nigeria . more than 67% of pregnant women in yobe state , nigeria did not receive the recommended four antenatal care visits visits . lack of education is the most consistent variable associated with antenatal care utilization in yobe state , nigeria . girl - child education is among the most promising interventions to address global issues like extreme poverty and hunger ( mdg 1 ) , low female literacy rate ( mdg 2 ) , gender inequality ( mdg 3 ) , high maternal and infant morbidity and mortality ( mdg 4 & 5 ) in yobe state , nigeria .

nanotechnology has the potential to create new materials and devices with wide - ranging applications in medicine,13 agriculture,4 and energy or electronic production.5,6 the size - dependent optical , electrical , and magnetic properties of nanoparticles make nanotechnology a promising candidate for bioapplications such as in vivo imaging , sensing , catalysis , therapeutics , and cell targeting.79 based on different approaches , physicians , physicists , chemists , biologists as well as bioengineers share a common interest to treat severe diseases through nanotechnology . theoretically , nanoparticles can be tailored to reach the right target at the right time . pathogenic agents such as viruses or bacteria , and cancer cells could be precisely targeted and affected without disturbing healthy tissues . this crucial task has been one of the highest priorities for the past 10 years . among several medical applications , nanoparticles could be largely employed as carriers of therapeutical biomolecules.10 the combination of nanoparticles with biomolecules such as proteins or specific polypeptides offers opportunities for the design of very precise and versatile hybrid systems mostly useful in helping to fight cancer and immunological diseases.1113 there are more than 50,000 different proteins in the human body.14 proteins are present in complex biological processes such as muscle contraction , immune protection and transmission of nerve impulses . all enzymes and most hormones are proteins ; hence , proteins are vital sources for the body s metabolism and their lack can result in several diseases ( eg , lack of insulin in type 1 diabetes ) . the latest development of protein engineering allows the production of proteins having desired properties and great potential market;15 however , protein fragility is one of the major drawbacks for their utilization . consequently , the discovery and development of new therapeutic proteins have also created new opportunities for drug - delivery systems involving the design of appropriate nanocarriers such as liposomes , micro- , and nanoparticles.1619 the oral route is a comfortable way for drug administration especially when repeated or routine dosing is necessary.20 nevertheless , the development of oral carriers for many proteins remains a challenge due to the fact that bioavailability of these molecules is limited.21 indeed , most polypeptides and proteins are quickly degraded in the gastrointestinal ( gi ) tract by proteolytic enzymes.22,23 moreover , the intestinal epithelium is a major barrier to the absorption of hydrophilic drugs that can not easily diffuse across the cells through the lipid - bilayer cell membranes . numerous investigations have shown that nanocarriers can improve the stability of therapeutic agents against enzymatic degradation and achieve desired therapeutic levels in target tissues for the required duration . nanoparticle drug - delivery systems ( nano - dds ) could permit an optimal pharmacokinetic profile and meet specific needs . for example , nanoparticles as oral protein carriers could protect the active ingredient in the gi tract and/or prolong the residence time of its contents on the mucous membrane . after administration , nano - dds can be taken up and transported across the intestinal mucosa by enterocytes or m cells in the peyer s patches because of their small size.24 several articles and reviews on the use of nanoparticles or microparticles for oral drug delivery are dedicated to insulin.2528 in 1980 , couvreur and colleagues performed the first study on hypoglycemic effects after oral and parenteral administration of insulin - loaded nanoparticles to diabetic rats.26 proteins are also difficult to be delivered via topical or transdermal routes and therefore their parenteral administration is still largely applied . besides the general complications of the parenteral route ( such as local infections , thrombophlebitis , rarely tissue necrosis ) , small proteins ( < 30 kd ) are quickly filtered out by the kidneys . without an appropriate drug carrier , proteins can also cause unwanted allergic reactions , can be targeted by the immune system and be rapidly degraded . for example , rapid clearance from the circulation can be an explanation of the modest in vivo antitumor effects of the antiangiogenic rgd ( arg gly asp ) peptides.29 bone morphogenetic proteins ( bmps ) induce bone formation after implantation ; their orthopedic application in repair of bone fractures and defects is focused in local device and spinal fusion procedures . the problem of bmp is its rapid diffusion from the administration site when applied without a carrier . currently , one of the most effective and biocompatible carriers for bmp delivery is the type i bovine absorbable collagen sponge ( acs ) . however the bmp release rate is difficult to control and to maintain constant for long term because of a high initial burst release of this device . the use of new nanotechnologies could maintain bmps at the treatment site preventing extraneous bond formation and optimizing the drug release.30 synthetic antigenic peptides are specific sections or a variant sequence of viral / bacterial proteins able to induce an immune response in the host . these small peptides are very useful in the vaccine development compared to the use of the whole protein / antigen . to date , several antigenic peptides have been identified but delivery problems still limit their application . even in this case , the design of an effective delivery system is an important challenge in nanotechnology field . an efficient protein carrier should solve different problems allowing the access to the target sites , at the right time and for the proper duration . in order to choose the best nanosystem , five factors must be considered : nature of the protein , route of administration , pattern of drug release , method of delivery and formulation.31,32 proteins such as albumin , antibody , growth factors , transferrin , cytokines and low - density lipoprotein can be also used as active ligands to help nanoparticles loaded with chemotherapeutic or other drugs to reach particular sites in the body.3335 abraxane ( abraxis bioscience , los angeles , ca ; astrazeneca , wilmington , de ) , albumin bound nanoparticle of paclitaxel , is an example of us food and drug administration ( fda)-approved protein - based active ligand for the treatment of metastatic breast cancer.35 monoclonal antibodies ( mab ) has been widely used as bioprobes in diagnostics as well as delivery drug to specific tumors.36,37 ox26 mab can help nanoparticles to cross the blood brain barrier and diffuse in the brain tissue in order to transport drugs ( eg , the anticaptase peptide , z - devd - fmk ) for the treatment of neurological and psychiatric disorders.38 nanoparticles can be also coated with mab for cell surface antigen and used as a bait for detection or isolation of various kind of cells including lymphocyte and tumor cells.39,40 despite many potential applications , the interaction of nanoparticles with biomolecules and living systems is still not fully understood.4150 continuous study on this subject contributes to the current knowledge and stimulates the development of novel therapies such as nonviral vectors for gene therapies or as precise anticancer molecules.37,5153 furthermore , by clarifying these aspects , specific protein - based nanovectors with optimized functions could be developed . this review aims to provide an overall picture on current progress and general aspect of the most successful approaches used to combine proteins with nanosystems . these methods and the correlated problems of protein denaturation are discussed in turn in this review . the interaction between biological and synthetic materials impacts on a vast range of medical issues from implants to pharmacokinetic aspects . the study of the materials bio - compatibility starts , therefore , with the analysis of protein absorption on surfaces.54 synthetic materials for biomedical applications are immediately covered by proteins when put in contact with a biological environment.55,56 after protein binding , nanoparticles are quickly cleared by the mononuclear phagocytic system ( mps ) , also known as the reticuloendothelial system ( res).57,58 these macrophages , which are typically kupffer cells of the liver , can not directly identify the nanoparticles themselves , but rather recognize specific opsonin proteins bound to the surface of the particles.59 the interaction between proteins and nanoparticles surface leads to the formation of proteins corona around nanoparticles that largely defines their biological identity as well their potential toxicity.49,50,58,6064 recently , lynch and dawson postulated the importance of the protein corona as the vehicle and the biological identity of a nanoparticle for its transport through cell membranes.60 the nanoparticle surface is immediately occupied by proteins with high concentrations and high association rate constants and successively by proteins having lower concentrations but a higher affinity.47 competitive absorption of proteins is influenced by several factors such as electrostatic interactions , protein stability , and kinetic parameters.65 as the protein corona could affect the nanoparticle behavior , including its biological effect , the nanoparticle could also have an effect on the protein behavior . some nanoparticles seem able to promote the protein assembly into amyloid fibrils in vitro by assisting the nucleation process.66 bellezza and colleagues found that nanoparticles affect the morphology of the myoglobin absorbed onto phosphate - grafted zirconia nanoparticles , inducing prefibrillar - like aggregates.67 this phenomenon could have important implications for medical application of nanoparticles because the self - assembly of a variety of proteins and peptides is known to be the cause of human amyloid diseases where fibrous protein aggregates are formed , resulting in amyloid plaque deposition in the extra - cellular tissues.6874 moreover , fibrillar structure seems to be related to heavy human disorders such as alzheimer s disease , parkinson s disease , and spongiform encephalopathies . for instance , there are nanosystems such as c60 hydrated fullerenes that can relax fibrillar structures.60,68 certainly , the control of the protein absorption on nanoparticle surfaces is an important issue to control their fate in biological systems.75 in order to prevent or control the opsonization , several methods of disguising nanoparticles have been developed . in these methods , generally , nanoparticles are coated with biocompatible polymers that have the double function of preventing their aggregation and retarding the protein absorption.57,76,77 a common strategy to improve blood compatibility and to increase the blood circulation half - life of the nanoparticles is the construction of a protein - coated surface resistant to the absorption of the other opsonines.78 a thin layer of protein appears to minimize adhesion and aggregation of nanoparticles , avoiding subsequent macrophage recognition or , in the worst case , a thrombus formation . moreover , it is possible to properly tune the cells uptake of the nanoparticles using specific proteins.35 proteins are mainly amphiphatic molecules that typically adhere to the surface of a biomaterial in a nonspecific way . in various cases , this nonspecific adhesion is sufficient to artificially immobilize proteins on the nanoparticles surface , and no surface modification is necessary . despite of the large number of studies , the absorption of a protein on whatever the solid surface is still a complex and not well understood process.60,7985 in the case of nanoparticles , size and radius of curvature become significant when compared to the protein size resulting in new interactions not shown with the bulk materials.47 the high hydrophobicity of many proteins seems to play an important role in their absorption on the nanoparticles surface.86 several models of protein absorption on surfaces identify two main steps in the process . the first step could involve the arrival of the protein at the interface , through a diffusion process following the brownian law of motion , and its further collision with the solid surface . depending on the balance of the energetic interaction if the protein has been absorbed , the second step could lead to conformational changes ( because of van der waals interactions ) , surface charge , protein dipole moment , and protein size or solution ionic strength.84,85,8790 this second step often involves irreversible changes in the protein structure up to denaturation.9193 proteins can be divided in two groups : hard and soft proteins . the first group includes proteins with high internal stability , while proteins in the second group have a low internal stability . soft proteins seem to be able to change their conformation better than the hard ones . this characteristic results in a gain in conformational entropy when absorbed on solid surfaces , improving the efficacy of the absorption process when compared to the hard proteins . on the other hand , it seems that some degree of denaturation upon absorption is more probable for soft proteins than for the hard ones , especially on hydrophobic surfaces.84,9497 during the artificial absorption of protein to nanoparticles surface , the use of a large excess of the target material could allow the retention of sufficient biological activity and native epitopes , even if some proteins are denatured . however , problems associated with denaturation of the protein over time , or its exchange with other proteins in solution , could make this strategy satisfactory only for short - term uses . the success of an absorption strategy to deliver drug or therapeutical proteins using protein - based nanoparticles as a carrier can be influenced by several factors such as the type of nanoparticles , delivery route and the nature of proteins to be absorbed . for this reason , nanoparticle protein affinity needs to be intensely examined case - by - case . the knowledge of how the protein - based nanoparticles interact with other proteins present in the blood is fundamental for the understanding of their biological and toxicological properties.77 many methods based on established techniques could be applied such as size - exclusion chromatography , isothermal titration calorimetry , surface plasmon resonance , atomic force microscopy , differential scanning calorimeter , and circular dichroisim ( cd ) spectroscopy.47,67 even if several existing characterization methods for measuring the nature and the amount of absorbed protein on solid surfaces could be applied to nanoparticle systems,96 the development of new physical and biophysical methods may be necessary to fully understand the relationship between proteins and nanomaterials . conjugation of biomolecules on nanoparticle surfaces has attracted widespread interest in biotechnology and medicine.7,98100 the conjugation of specific proteins with nanoparticles has introduced a new advancement in molecular and cellular biology which has further led to a vast improvement of in vivo gene delivery , clinical diagnosis , medical / cancer imaging , receptor - targeted delivery.40,101105 a preferred method used in many areas of biochemistry to couple specific protein to solid surface is the bioconjugation by covalent binding . while protein absorption on solid surfaces such as nanoparticles can be reversible depending on ph , salt concentration , temperature or other environment physicochemical characteristics , protein covalent bounds are highly stable . to fulfill the purpose of stable covalent binding , a large number of reactions have been proposed and many protein modifications using new techniques have been developed.7,106111 the choice of the bioconjugation procedure depends strictly on physicochemical and biochemical properties of nanomaterials and proteins . protein made by various side chains and residues can interact by multiple coating ligands with the same nanoparticles or even with more nanoparticles . moreover , nanoparticles can be more or less polydispersed and have different physicochemical surface properties such as area , porosity , and charge . these aspects are very important since the hydrophobicity , charge and site affinity could affect the interaction and thus jeopardize the stability of final covalent - coupled products . the most popular approach for coupling covalently nanoparticle to protein is based on the existence on proteins of specific and reactive functional groups such as amino nh2 ( lysine ) , carboxylic acid cooh ( aspartic , glutamic ) , hydroxyl oh ( serine , tyrosine ) and sh ( cysteine).112 proteins can be chemically coupled to different kinds of nanoparticles using established reagents such bifunctional cross - linker molecules . in this case , nanoparticles need to be functionalized with functional groups such as carboxylic acid , hydroxyl , sulfhydryl and amino groups . proteins , including antibodies , generally have several primary amines in the side chain of lysine residues and the n - terminus of each polypeptide that are available as targets for n - hydroxysuccinimide - ester and carbodiimide reagents . cysteine residues on proteins can react with maleimides and iodoacetamides reagents to give thioether - coupled products.113 these reagents react rapidly at physiological ph and can be usually coupled with thiol groups selectively in the presence of amine groups . maleimides and iodoacetamides have the same application but the first reagent seems to have better selectivity than the second one , not apparently reacting with histidine or methionine . cross - linking reagents contain reactive ends to specific functional groups ( such as primary amines , sulfhydryls ) on proteins or other molecules . they can be divided into homobifunctional ( same reactive groups ) and heterobifunctional ( different reactive groups ) which chemical cross - links may or may not be reversed.114 homobifunctional cross - linkers have a disadvantage of potentially connecting two neighboring groups , either on the nanoparticle surface or on the protein inducing undesired cross - linking . sulfosuccinimidyl-4-(n - maleimidomethyl)-cyclohexane-1-carboxylate ( sulfo - smcc ) can be used to couple thiol - containing biomolecules with amine coated nanoparticles , or vice versa . whereas the hetero - bifunctional cross - linker 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide ( edc ) is commonly used to link nh2 and cooh groups ( table 1).114118 many cross - linkers are available in the market and they can be chosen for specific needs ( such as chemical specificity , spacer arm length , cleavability ) . among several cross - linkers , the zero - length ones such as carbodiimides are widely used allowing covalent bonds between nanoparticles and proteins without insertion of an exogenous spacer . nevertheless , the direct attachment of a protein to a surface without a spacer can cause steric constraint modifying the protein reactivity compared to the protein in solution . in addition , without a spacer , multiple contacts between protein and nanoparticle surface are more probable favoring total or partial protein denaturation and thus decreasing protein activity.119 when protein does not have the suitable residue necessary for the specific conjugation , the most common way to get it is the chemical introduction of sulfhydryl groups . this process ( figures 1a and 1b ) can be mainly made by the following four methods : 1 ) reduction of protein disulfide bonds using reductive agents such as dithiotreitol ( dtt = clelands reagent ) . 3 ) quenching of reactive protein aldehyde residues with cystaminiumdichloride reagents or 4 ) coupling of cystaminiumdichloride to carboxyl groups via 1-ethyl-3-(3-dimethyl - aminopropyl)carbodiimide ( edc ) ; both cases followed by the disulfide bonds reduction with dtt as outlined above.112,120123 the avidin / streptavidin biotin bound is the strongest noncovalent biological interaction known ; for this reason this technology is commonly used in biological labs.124,125 biotinylated proteins / antibodies / enzymes can be efficiently coupled on amino nanoparticle surfaces by streptavidin - biotin technology accomplished by streptavidin activation through carbodiimide ( edc ) chemistry . biotin binds strongly to this biochemically modified surface in the most specific and sensitive way . furthermore , streptavidin through carbodiimide ( edc ) chemistry can be covalently coupled with different ligands such as mab and enzymes which make the biotin streptavidin system widely used in a variety of biotinylated nanoparticles.38,126128 proteins having cysteine residues can be directly attached to some metal nanoparticle surfaces such as gold and silver by stable metal sulfur bonds.129,130 in the other cases , the covalent coupling of proteins on nanoparticle surfaces is always a long experimental procedure . covalent bioconjugation procedure can be summarized in : 1 ) coating of nanoparticles with the selected active functional groups . 2 ) chemical activation of thiol groups on the protein side with specific reductive agents , if necessary . 3 ) total removal of the reduction agent in excess ; this step can create unplanned reactions and spoil the whole coupling process . in addition to the disadvantage of the long experimental procedure , covalent bioconjugation can affect the protein structure and function resulting in its partial denaturation ( figure 2 ) . moreover , modification of enzymes under strong denaturing conditions can result in their complete loss of activity . proteins can be denaturated during manipulations or formulations mainly by two mechanisms : conformational denaturation ( eg , reversible unfolding and irreversible aggregation via noncovalent interactions ) and chemical denaturation ( covalent bonds such as deamidation , hydrolysis , oxidation , -elimination , incorrect disulfide formation , maillard reaction , and transamidation ) . even if the first denaturation mechanism can happen during the nanoparticles bioconjugation process , the second one is often necessary to obtain high efficacy of the coupling . for example , the sh or s - groups in cysteine sh or disulfide s s bridges are important in maintaining the conformation of the proteins . as a result , the engineering introduction of sulfhydryl groups in the protein changes its natural disulfide bonds resulting in partial conformational and chemical denaturation . the dtt reagent , widely used to reduce disulfide bonds in biochemical systems , can alter protein function not only by thiol - disulfide exchanging but also by interacting with protein domains in the absence of cysteine residues.131 while carboxyl groups seem to play an important role in enzymes catalytic activity,132 their modification likely results in a change of protein secondary and tertiary structure . the - amino groups of lysine are often specifically targeted because of their high reactivity and their modification seems to have fewer effects on protein properties . unfortunately , the high abundance of these groups in many proteins can lead to increased heterogeneity and restricted conformational flexibility owing to multipoint attachment on a nanoparticles surface . it is also possible that other reagents used during the coupling chemical process can contribute to the protein denaturation and to its activity loss . therefore biological function checking as well as close monitoring of the quality and quantity of conjugated protein are extremely important to be assessed before being used.111 gold or silver nanoparticles too , due to the similar strength bond between au / ag s and s s , can potentially break up protein disulfide s s bridges leading to denaturation . specific elisa kits can be used to explore the activity of the proteins coupled to nanosystems . however there are nanoparticles such as quantum dots ( qd ) that can have an overlap in the absorption spectra and the elisa essay end product . in this case the proper specific activity of the protein needs to be assessed directly by in vitro testing.133 therapeutic biomolecules based on peptides , proteins or enzymes can be extremely fragile and easily aggressed by external agent such as proteases . encapsulation of these fragile drugs in nanocarriers is a possible strategy for preventing their aggression and denaturation . this process can also improve the drug pharmacokinetic pathway and reduce immunological reactions.19 an optimal drug delivery system should be biocompatible , biodegradable and should not cause any immunological adverse reaction in the human body . among several candidates , liposomes are considered as the most promising vectors for proteins delivery due to their biocompatibility and their capacity to improve the drug pharmacokinetic . liposomes , also known as lipid - based vesicles , are generally composed of concentric amphiphilic lipids , such as phospholipids , containing a water compartment . these carriers are versatile and their physico chemical characteristics can be properly tuned.19 liposomes synthesized from dehydrated rehydrated vesicles are widely used due to the ease of this preparation process and the low amount of stress applied to the proteins.134 liposome formulations are most frequently considered for parental administration of the drug , but may also be a potential formulation principle for alternative routes such as topical and nasal administration . several liposomes have immunoadjuvant properties and their application in vaccines based on recombinant protein subunits and synthetic peptide antigens is attractive . the first liposome based vaccine ( against hepatitis a ) that has been licensed for human use is commercially known as epaxal berna vaccine.135 the main drawback of liposomes is their instability in biological media as well as their sensitivity to many external parameters such as temperature or osmotic pressure . theoretically , it could be possible to increase their stability following several strategies such as the polymerization of a two dimensional network in the hydrophobic core of the membrane , coating the liposome with a polyelectrolyte shell or adding surface active polymers to form mixed vesicular structures.136138 however , poor loading and partial protein / enzyme denaturation during the entrapment process can occur . another well established technique to encapsulate biological species such as enzymes , antibodies and other proteins in a functional state is based on the sol gel chemistry method.139 silica is indeed considered a very appealing material for drug delivery systems because it is relatively inexpensive , chemically inert , thermally stable , and biocompatible . amorphous silica , used for decades as a food additive and for specific applications , is generally regarded as safe . up until now , the fda has not established if existing silica safety data can be applied to nanoscale forms of the material . in this approach , polypeptides , especially enzymes , could be entrapped inside silica matrix allowing the retention of enzymatic activity.139141 on the other hand , process difficulties such as uncontrolled release , denaturation and the hardness control of the protein orientation can be found.142,143 the control of the drug release of such silica nanoparticles is the most important and difficult parameter that needs to be properly tuned . the encapsulation efficacy of insoluble protein is greatly different compared to the soluble one and the existence of soluble and insoluble part of polypeptides in the same therapeutic protein subunit complicates the synthesis process . additionally , in the crowded environment of a silica matrix , the physical and chemical properties of the silica can directly influence protein structure and activity . furthermore , functional activity of proteins entrapped into the sol - gel matrix needs to be accurately analyzed case - by - case using several techniques such as cd spectropolarimetry.144,145 the drug release and the capability of the carrier to be metabolized can be important factors to be considered when chronic or repeated treatments are necessary . the disadvantage associated with inorganic and synthetic carriers are the poor or slow biodegradability and possible inflammatory responses.146 biodegradable polymers nanosystems are an attractive alternative to liposomes since they have the advantages of longer circulation in the blood stream and generally higher drug carrying capacity.147 polymers such as poly(lactic acid ) ( pla ) , poly(lactic - co - glycolic acid ) ( plga ) have been extensively investigated for their biocompatibility and potential capability of releasing therapeutically proteins in a controlled way even over a prolonged period of time.148154 these polymers are degradable by bulk erosion through hydrolysis of the ester bonds . the hydrolysis rate depends on several nanoparticles physicochemical parameters and can be tailored according to the desired release pattern of the protein to be incorporated . pla and plga are fda - approved as excipients to achieve sustained release of the active ingredient . however , their application in protein delivery systems is often characterized by low entrapment efficiency , burst release , instability of encapsulated hydrophilic protein and partial protein release.155158 to improve the performance of these polymer nanoparticles , polysaccharides such as alginate ( alg ) and chitosan ( cs ) could be applied.151,159 cs and its derivatives have been intensively studied as carriers for proteins and drugs . more specifically these nanoparticles can be totally made by cs or used in several copolymer combinations.25,160 copolymers made by the combination of cs / alg are able to generate a more friendly environment which protects peptides and proteins from stressing conditions and allows their stabilization during encapsulation , storage and release.161166 glycol chitosan nanoparticles modified with hydrophobic bile acid analogs self - assemble into polymeric nanoparticles with hydrophilic shells of glycol chitosan and hydrophobic cores of bile acid derivatives have been reported as possible vehicle for rgd ( arg gly asp ) peptide.29,30 regardless of the nanomaterial chosen for protein encapsulation , an important issue that needs to be considered is the understanding of protein protein interactions . protein interactions that occur in the cell , which in turn control a large number of cellular processes . these transient interactions of protein complexes can cause several effects such as activation / inactivation of certain proteins , resulting in the formation of a new binding site.167,168 kinetics properties of enzymes can be also altered by denaturation during the entrapment process allowing potential change of the protein specificity to its substrate.169171 gaining a clear picture of these basics knowledge will definitely lead to a change of object design to increase the protein load , to control the protein release and to retain the protein integrity and efficacy . the interaction between biological and synthetic materials impacts on a vast range of medical issues from implants to pharmacokinetic aspects . the study of the materials bio - compatibility starts , therefore , with the analysis of protein absorption on surfaces.54 synthetic materials for biomedical applications are immediately covered by proteins when put in contact with a biological environment.55,56 after protein binding , nanoparticles are quickly cleared by the mononuclear phagocytic system ( mps ) , also known as the reticuloendothelial system ( res).57,58 these macrophages , which are typically kupffer cells of the liver , can not directly identify the nanoparticles themselves , but rather recognize specific opsonin proteins bound to the surface of the particles.59 the interaction between proteins and nanoparticles surface leads to the formation of proteins corona around nanoparticles that largely defines their biological identity as well their potential toxicity.49,50,58,6064 recently , lynch and dawson postulated the importance of the protein corona as the vehicle and the biological identity of a nanoparticle for its transport through cell membranes.60 the nanoparticle surface is immediately occupied by proteins with high concentrations and high association rate constants and successively by proteins having lower concentrations but a higher affinity.47 competitive absorption of proteins is influenced by several factors such as electrostatic interactions , protein stability , and kinetic parameters.65 as the protein corona could affect the nanoparticle behavior , including its biological effect , the nanoparticle could also have an effect on the protein behavior . some nanoparticles seem able to promote the protein assembly into amyloid fibrils in vitro by assisting the nucleation process.66 bellezza and colleagues found that nanoparticles affect the morphology of the myoglobin absorbed onto phosphate - grafted zirconia nanoparticles , inducing prefibrillar - like aggregates.67 this phenomenon could have important implications for medical application of nanoparticles because the self - assembly of a variety of proteins and peptides is known to be the cause of human amyloid diseases where fibrous protein aggregates are formed , resulting in amyloid plaque deposition in the extra - cellular tissues.6874 moreover , fibrillar structure seems to be related to heavy human disorders such as alzheimer s disease , parkinson s disease , and spongiform encephalopathies . for instance , there are nanosystems such as c60 hydrated fullerenes that can relax fibrillar structures.60,68 certainly , the control of the protein absorption on nanoparticle surfaces is an important issue to control their fate in biological systems.75 in order to prevent or control the opsonization , several methods of disguising nanoparticles have been developed . in these methods , generally , nanoparticles are coated with biocompatible polymers that have the double function of preventing their aggregation and retarding the protein absorption.57,76,77 a common strategy to improve blood compatibility and to increase the blood circulation half - life of the nanoparticles is the construction of a protein - coated surface resistant to the absorption of the other opsonines.78 a thin layer of protein appears to minimize adhesion and aggregation of nanoparticles , avoiding subsequent macrophage recognition or , in the worst case , a thrombus formation . moreover , it is possible to properly tune the cells uptake of the nanoparticles using specific proteins.35 proteins are mainly amphiphatic molecules that typically adhere to the surface of a biomaterial in a nonspecific way . in various cases , this nonspecific adhesion is sufficient to artificially immobilize proteins on the nanoparticles surface , and no surface modification is necessary . despite of the large number of studies , the absorption of a protein on whatever the solid surface is still a complex and not well understood process.60,7985 in the case of nanoparticles , size and radius of curvature become significant when compared to the protein size resulting in new interactions not shown with the bulk materials.47 the high hydrophobicity of many proteins seems to play an important role in their absorption on the nanoparticles surface.86 several models of protein absorption on surfaces identify two main steps in the process . the first step could involve the arrival of the protein at the interface , through a diffusion process following the brownian law of motion , and its further collision with the solid surface . depending on the balance of the energetic interaction if the protein has been absorbed , the second step could lead to conformational changes ( because of van der waals interactions ) , surface charge , protein dipole moment , and protein size or solution ionic strength.84,85,8790 this second step often involves irreversible changes in the protein structure up to denaturation.9193 proteins can be divided in two groups : hard and soft proteins . the first group includes proteins with high internal stability , while proteins in the second group have a low internal stability . soft proteins seem to be able to change their conformation better than the hard ones . this characteristic results in a gain in conformational entropy when absorbed on solid surfaces , improving the efficacy of the absorption process when compared to the hard proteins . on the other hand , it seems that some degree of denaturation upon absorption is more probable for soft proteins than for the hard ones , especially on hydrophobic surfaces.84,9497 during the artificial absorption of protein to nanoparticles surface , the use of a large excess of the target material could allow the retention of sufficient biological activity and native epitopes , even if some proteins are denatured . however , problems associated with denaturation of the protein over time , or its exchange with other proteins in solution , could make this strategy satisfactory only for short - term uses . the success of an absorption strategy to deliver drug or therapeutical proteins using protein - based nanoparticles as a carrier can be influenced by several factors such as the type of nanoparticles , delivery route and the nature of proteins to be absorbed . for this reason , nanoparticle protein affinity needs to be intensely examined case - by - case . the knowledge of how the protein - based nanoparticles interact with other proteins present in the blood is fundamental for the understanding of their biological and toxicological properties.77 many methods based on established techniques could be applied such as size - exclusion chromatography , isothermal titration calorimetry , surface plasmon resonance , atomic force microscopy , differential scanning calorimeter , and circular dichroisim ( cd ) spectroscopy.47,67 even if several existing characterization methods for measuring the nature and the amount of absorbed protein on solid surfaces could be applied to nanoparticle systems,96 the development of new physical and biophysical methods may be necessary to fully understand the relationship between proteins and nanomaterials . conjugation of biomolecules on nanoparticle surfaces has attracted widespread interest in biotechnology and medicine.7,98100 the conjugation of specific proteins with nanoparticles has introduced a new advancement in molecular and cellular biology which has further led to a vast improvement of in vivo gene delivery , clinical diagnosis , medical / cancer imaging , receptor - targeted delivery.40,101105 a preferred method used in many areas of biochemistry to couple specific protein to solid surface is the bioconjugation by covalent binding . while protein absorption on solid surfaces such as nanoparticles can be reversible depending on ph , salt concentration , temperature or other environment physicochemical characteristics , protein covalent bounds are highly stable . to fulfill the purpose of stable covalent binding , a large number of reactions have been proposed and many protein modifications using new techniques have been developed.7,106111 the choice of the bioconjugation procedure depends strictly on physicochemical and biochemical properties of nanomaterials and proteins . protein made by various side chains and residues can interact by multiple coating ligands with the same nanoparticles or even with more nanoparticles . moreover , nanoparticles can be more or less polydispersed and have different physicochemical surface properties such as area , porosity , and charge . these aspects are very important since the hydrophobicity , charge and site affinity could affect the interaction and thus jeopardize the stability of final covalent - coupled products . the most popular approach for coupling covalently nanoparticle to protein is based on the existence on proteins of specific and reactive functional groups such as amino nh2 ( lysine ) , carboxylic acid cooh ( aspartic , glutamic ) , hydroxyl oh ( serine , tyrosine ) and sh ( cysteine).112 proteins can be chemically coupled to different kinds of nanoparticles using established reagents such bifunctional cross - linker molecules . in this case , nanoparticles need to be functionalized with functional groups such as carboxylic acid , hydroxyl , sulfhydryl and amino groups . proteins , including antibodies , generally have several primary amines in the side chain of lysine residues and the n - terminus of each polypeptide that are available as targets for n - hydroxysuccinimide - ester and carbodiimide reagents . cysteine residues on proteins can react with maleimides and iodoacetamides reagents to give thioether - coupled products.113 these reagents react rapidly at physiological ph and can be usually coupled with thiol groups selectively in the presence of amine groups . maleimides and iodoacetamides have the same application but the first reagent seems to have better selectivity than the second one , not apparently reacting with histidine or methionine . cross - linking reagents contain reactive ends to specific functional groups ( such as primary amines , sulfhydryls ) on proteins or other molecules . they can be divided into homobifunctional ( same reactive groups ) and heterobifunctional ( different reactive groups ) which chemical cross - links may or may not be reversed.114 homobifunctional cross - linkers have a disadvantage of potentially connecting two neighboring groups , either on the nanoparticle surface or on the protein inducing undesired cross - linking . sulfosuccinimidyl-4-(n - maleimidomethyl)-cyclohexane-1-carboxylate ( sulfo - smcc ) can be used to couple thiol - containing biomolecules with amine coated nanoparticles , or vice versa . whereas the hetero - bifunctional cross - linker 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide ( edc ) is commonly used to link nh2 and cooh groups ( table 1).114118 many cross - linkers are available in the market and they can be chosen for specific needs ( such as chemical specificity , spacer arm length , cleavability ) . among several cross - linkers , the zero - length ones such as carbodiimides are widely used allowing covalent bonds between nanoparticles and proteins without insertion of an exogenous spacer . nevertheless , the direct attachment of a protein to a surface without a spacer can cause steric constraint modifying the protein reactivity compared to the protein in solution . in addition , without a spacer , multiple contacts between protein and nanoparticle surface are more probable favoring total or partial protein denaturation and thus decreasing protein activity.119 when protein does not have the suitable residue necessary for the specific conjugation , the most common way to get it is the chemical introduction of sulfhydryl groups . this process ( figures 1a and 1b ) can be mainly made by the following four methods : 1 ) reduction of protein disulfide bonds using reductive agents such as dithiotreitol ( dtt = clelands reagent ) . 3 ) quenching of reactive protein aldehyde residues with cystaminiumdichloride reagents or 4 ) coupling of cystaminiumdichloride to carboxyl groups via 1-ethyl-3-(3-dimethyl - aminopropyl)carbodiimide ( edc ) ; both cases followed by the disulfide bonds reduction with dtt as outlined above.112,120123 the avidin / streptavidin biotin bound is the strongest noncovalent biological interaction known ; for this reason this technology is commonly used in biological labs.124,125 biotinylated proteins / antibodies / enzymes can be efficiently coupled on amino nanoparticle surfaces by streptavidin - biotin technology accomplished by streptavidin activation through carbodiimide ( edc ) chemistry . biotin binds strongly to this biochemically modified surface in the most specific and sensitive way . furthermore , streptavidin through carbodiimide ( edc ) chemistry can be covalently coupled with different ligands such as mab and enzymes which make the biotin streptavidin system widely used in a variety of biotinylated nanoparticles.38,126128 proteins having cysteine residues can be directly attached to some metal nanoparticle surfaces such as gold and silver by stable metal sulfur bonds.129,130 in the other cases , the covalent coupling of proteins on nanoparticle surfaces is always a long experimental procedure . covalent bioconjugation procedure can be summarized in : 1 ) coating of nanoparticles with the selected active functional groups . 2 ) chemical activation of thiol groups on the protein side with specific reductive agents , if necessary . 3 ) total removal of the reduction agent in excess ; this step can create unplanned reactions and spoil the whole coupling process . in addition to the disadvantage of the long experimental procedure , covalent bioconjugation can affect the protein structure and function resulting in its partial denaturation ( figure 2 ) . moreover , modification of enzymes under strong denaturing conditions can result in their complete loss of activity . proteins can be denaturated during manipulations or formulations mainly by two mechanisms : conformational denaturation ( eg , reversible unfolding and irreversible aggregation via noncovalent interactions ) and chemical denaturation ( covalent bonds such as deamidation , hydrolysis , oxidation , -elimination , incorrect disulfide formation , maillard reaction , and transamidation ) . even if the first denaturation mechanism can happen during the nanoparticles bioconjugation process for example , the sh or s - groups in cysteine sh or disulfide s s bridges are important in maintaining the conformation of the proteins . as a result , the engineering introduction of sulfhydryl groups in the protein changes its natural disulfide bonds resulting in partial conformational and chemical denaturation . the dtt reagent , widely used to reduce disulfide bonds in biochemical systems , can alter protein function not only by thiol - disulfide exchanging but also by interacting with protein domains in the absence of cysteine residues.131 while carboxyl groups seem to play an important role in enzymes catalytic activity,132 their modification likely results in a change of protein secondary and tertiary structure . the - amino groups of lysine are often specifically targeted because of their high reactivity and their modification seems to have fewer effects on protein properties . unfortunately , the high abundance of these groups in many proteins can lead to increased heterogeneity and restricted conformational flexibility owing to multipoint attachment on a nanoparticles surface . it is also possible that other reagents used during the coupling chemical process can contribute to the protein denaturation and to its activity loss . therefore biological function checking as well as close monitoring of the quality and quantity of conjugated protein are extremely important to be assessed before being used.111 gold or silver nanoparticles too , due to the similar strength bond between au / ag s and s s , can potentially break up protein disulfide s s bridges leading to denaturation . specific elisa kits can be used to explore the activity of the proteins coupled to nanosystems . however there are nanoparticles such as quantum dots ( qd ) that can have an overlap in the absorption spectra and the elisa essay end product . in this case the proper specific activity of the protein needs to be assessed directly by in vitro testing.133 therapeutic biomolecules based on peptides , proteins or enzymes can be extremely fragile and easily aggressed by external agent such as proteases . encapsulation of these fragile drugs in nanocarriers is a possible strategy for preventing their aggression and denaturation . this process can also improve the drug pharmacokinetic pathway and reduce immunological reactions.19 an optimal drug delivery system should be biocompatible , biodegradable and should not cause any immunological adverse reaction in the human body . among several candidates , liposomes are considered as the most promising vectors for proteins delivery due to their biocompatibility and their capacity to improve the drug pharmacokinetic . liposomes , also known as lipid - based vesicles , are generally composed of concentric amphiphilic lipids , such as phospholipids , containing a water compartment . these carriers are versatile and their physico chemical characteristics can be properly tuned.19 liposomes synthesized from dehydrated rehydrated vesicles are widely used due to the ease of this preparation process and the low amount of stress applied to the proteins.134 liposome formulations are most frequently considered for parental administration of the drug , but may also be a potential formulation principle for alternative routes such as topical and nasal administration . several liposomes have immunoadjuvant properties and their application in vaccines based on recombinant protein subunits and synthetic peptide antigens is attractive . the first liposome based vaccine ( against hepatitis a ) that has been licensed for human use is commercially known as epaxal berna vaccine.135 the main drawback of liposomes is their instability in biological media as well as their sensitivity to many external parameters such as temperature or osmotic pressure . theoretically , it could be possible to increase their stability following several strategies such as the polymerization of a two dimensional network in the hydrophobic core of the membrane , coating the liposome with a polyelectrolyte shell or adding surface active polymers to form mixed vesicular structures.136138 however , poor loading and partial protein / enzyme denaturation during the entrapment process can occur . another well established technique to encapsulate biological species such as enzymes , antibodies and other proteins in a functional state is based on the sol gel chemistry method.139 silica is indeed considered a very appealing material for drug delivery systems because it is relatively inexpensive , chemically inert , thermally stable , and biocompatible . amorphous silica , used for decades as a food additive and for specific applications , is generally regarded as safe . up until now , the fda has not established if existing silica safety data can be applied to nanoscale forms of the material . in this approach , polypeptides , especially enzymes , could be entrapped inside silica matrix allowing the retention of enzymatic activity.139141 on the other hand , process difficulties such as uncontrolled release , denaturation and the hardness control of the protein orientation can be found.142,143 the control of the drug release of such silica nanoparticles is the most important and difficult parameter that needs to be properly tuned . the encapsulation efficacy of insoluble protein is greatly different compared to the soluble one and the existence of soluble and insoluble part of polypeptides in the same therapeutic protein subunit complicates the synthesis process . additionally , in the crowded environment of a silica matrix , the physical and chemical properties of the silica can directly influence protein structure and activity . furthermore , functional activity of proteins entrapped into the sol - gel matrix needs to be accurately analyzed case - by - case using several techniques such as cd spectropolarimetry.144,145 the drug release and the capability of the carrier to be metabolized can be important factors to be considered when chronic or repeated treatments are necessary . the disadvantage associated with inorganic and synthetic carriers are the poor or slow biodegradability and possible inflammatory responses.146 biodegradable polymers nanosystems are an attractive alternative to liposomes since they have the advantages of longer circulation in the blood stream and generally higher drug carrying capacity.147 polymers such as poly(lactic acid ) ( pla ) , poly(lactic - co - glycolic acid ) ( plga ) have been extensively investigated for their biocompatibility and potential capability of releasing therapeutically proteins in a controlled way even over a prolonged period of time.148154 these polymers are degradable by bulk erosion through hydrolysis of the ester bonds . the hydrolysis rate depends on several nanoparticles physicochemical parameters and can be tailored according to the desired release pattern of the protein to be incorporated . pla and plga are fda - approved as excipients to achieve sustained release of the active ingredient . however , their application in protein delivery systems is often characterized by low entrapment efficiency , burst release , instability of encapsulated hydrophilic protein and partial protein release.155158 to improve the performance of these polymer nanoparticles , polysaccharides such as alginate ( alg ) and chitosan ( cs ) could be applied.151,159 cs and its derivatives have been intensively studied as carriers for proteins and drugs . more specifically these nanoparticles can be totally made by cs or used in several copolymer combinations.25,160 copolymers made by the combination of cs / alg are able to generate a more friendly environment which protects peptides and proteins from stressing conditions and allows their stabilization during encapsulation , storage and release.161166 glycol chitosan nanoparticles modified with hydrophobic bile acid analogs self - assemble into polymeric nanoparticles with hydrophilic shells of glycol chitosan and hydrophobic cores of bile acid derivatives have been reported as possible vehicle for rgd ( arg gly asp ) peptide.29,30 regardless of the nanomaterial chosen for protein encapsulation , an important issue that needs to be considered is the understanding of protein protein interactions . protein interactions that occur in the cell , which in turn control a large number of cellular processes . these transient interactions of protein complexes can cause several effects such as activation / inactivation of certain proteins , resulting in the formation of a new binding site.167,168 kinetics properties of enzymes can be also altered by denaturation during the entrapment process allowing potential change of the protein specificity to its substrate.169171 gaining a clear picture of these basics knowledge will definitely lead to a change of object design to increase the protein load , to control the protein release and to retain the protein integrity and efficacy . although new proteins are available for medical purposes , their administration as therapeutics still remains difficult . nanosystems seem to be the optimal solution to improve protein bioavailability , biodistribution and safety . moreover , the combination of nanoparticles with proteins could also be a valid system to achieve the design of efficient nanovectors for drug delivery . indeed , nanoparticles can be properly tuned for specific applications and could be precisely designed to meet biological needs . however , to completely fulfill this purpose , it is necessary to better clarify the nature of interaction between nanoparticles and biomolecules . the control of the protein denaturation is another important parameter that needs a deeper understanding . further investigations should help to manage these hybrid nanosystems , opening new therapeutic and diagnostic perspectives as well as new challenges in the near future .

the latest development of protein engineering allows the production of proteins having desired properties and large potential markets , but the clinical advances of therapeutical proteins are still limited by their fragility . nanotechnology could provide optimal vectors able to protect from degradation therapeutical biomolecules such as proteins , enzymes or specific polypeptides . on the other hand , some proteins can be also used as active ligands to help nanoparticles loaded with chemotherapeutic or other drugs to reach particular sites in the body . the aim of this review is to provide an overall picture of the general aspects of the most successful approaches used to combine proteins with nanosystems . this combination is mainly achieved by absorption , bioconjugation and encapsulation . interactions of nanoparticles with biomolecules and caveats related to protein denaturation are also pointed out . a clear understanding of nanoparticle - protein interactions could make possible the design of precise and versatile hybrid nanosystems . this could further allow control of their pharmacokinetics as well as activity , and safety .

Introduction and background Nanoparticleprotein absorption, bioconjugation, and encapsulation Absorption of proteins on nanoparticles surface Bioconjugation of proteins on nanoparticle surfaces Protein encapsulation Conclusion

among several medical applications , nanoparticles could be largely employed as carriers of therapeutical biomolecules.10 the combination of nanoparticles with biomolecules such as proteins or specific polypeptides offers opportunities for the design of very precise and versatile hybrid systems mostly useful in helping to fight cancer and immunological diseases.1113 there are more than 50,000 different proteins in the human body.14 proteins are present in complex biological processes such as muscle contraction , immune protection and transmission of nerve impulses . the latest development of protein engineering allows the production of proteins having desired properties and great potential market;15 however , protein fragility is one of the major drawbacks for their utilization . consequently , the discovery and development of new therapeutic proteins have also created new opportunities for drug - delivery systems involving the design of appropriate nanocarriers such as liposomes , micro- , and nanoparticles.1619 the oral route is a comfortable way for drug administration especially when repeated or routine dosing is necessary.20 nevertheless , the development of oral carriers for many proteins remains a challenge due to the fact that bioavailability of these molecules is limited.21 indeed , most polypeptides and proteins are quickly degraded in the gastrointestinal ( gi ) tract by proteolytic enzymes.22,23 moreover , the intestinal epithelium is a major barrier to the absorption of hydrophilic drugs that can not easily diffuse across the cells through the lipid - bilayer cell membranes . after administration , nano - dds can be taken up and transported across the intestinal mucosa by enterocytes or m cells in the peyer s patches because of their small size.24 several articles and reviews on the use of nanoparticles or microparticles for oral drug delivery are dedicated to insulin.2528 in 1980 , couvreur and colleagues performed the first study on hypoglycemic effects after oral and parenteral administration of insulin - loaded nanoparticles to diabetic rats.26 proteins are also difficult to be delivered via topical or transdermal routes and therefore their parenteral administration is still largely applied . in order to choose the best nanosystem , five factors must be considered : nature of the protein , route of administration , pattern of drug release , method of delivery and formulation.31,32 proteins such as albumin , antibody , growth factors , transferrin , cytokines and low - density lipoprotein can be also used as active ligands to help nanoparticles loaded with chemotherapeutic or other drugs to reach particular sites in the body.3335 abraxane ( abraxis bioscience , los angeles , ca ; astrazeneca , wilmington , de ) , albumin bound nanoparticle of paclitaxel , is an example of us food and drug administration ( fda)-approved protein - based active ligand for the treatment of metastatic breast cancer.35 monoclonal antibodies ( mab ) has been widely used as bioprobes in diagnostics as well as delivery drug to specific tumors.36,37 ox26 mab can help nanoparticles to cross the blood brain barrier and diffuse in the brain tissue in order to transport drugs ( eg , the anticaptase peptide , z - devd - fmk ) for the treatment of neurological and psychiatric disorders.38 nanoparticles can be also coated with mab for cell surface antigen and used as a bait for detection or isolation of various kind of cells including lymphocyte and tumor cells.39,40 despite many potential applications , the interaction of nanoparticles with biomolecules and living systems is still not fully understood.4150 continuous study on this subject contributes to the current knowledge and stimulates the development of novel therapies such as nonviral vectors for gene therapies or as precise anticancer molecules.37,5153 furthermore , by clarifying these aspects , specific protein - based nanovectors with optimized functions could be developed . this review aims to provide an overall picture on current progress and general aspect of the most successful approaches used to combine proteins with nanosystems . the study of the materials bio - compatibility starts , therefore , with the analysis of protein absorption on surfaces.54 synthetic materials for biomedical applications are immediately covered by proteins when put in contact with a biological environment.55,56 after protein binding , nanoparticles are quickly cleared by the mononuclear phagocytic system ( mps ) , also known as the reticuloendothelial system ( res).57,58 these macrophages , which are typically kupffer cells of the liver , can not directly identify the nanoparticles themselves , but rather recognize specific opsonin proteins bound to the surface of the particles.59 the interaction between proteins and nanoparticles surface leads to the formation of proteins corona around nanoparticles that largely defines their biological identity as well their potential toxicity.49,50,58,6064 recently , lynch and dawson postulated the importance of the protein corona as the vehicle and the biological identity of a nanoparticle for its transport through cell membranes.60 the nanoparticle surface is immediately occupied by proteins with high concentrations and high association rate constants and successively by proteins having lower concentrations but a higher affinity.47 competitive absorption of proteins is influenced by several factors such as electrostatic interactions , protein stability , and kinetic parameters.65 as the protein corona could affect the nanoparticle behavior , including its biological effect , the nanoparticle could also have an effect on the protein behavior . some nanoparticles seem able to promote the protein assembly into amyloid fibrils in vitro by assisting the nucleation process.66 bellezza and colleagues found that nanoparticles affect the morphology of the myoglobin absorbed onto phosphate - grafted zirconia nanoparticles , inducing prefibrillar - like aggregates.67 this phenomenon could have important implications for medical application of nanoparticles because the self - assembly of a variety of proteins and peptides is known to be the cause of human amyloid diseases where fibrous protein aggregates are formed , resulting in amyloid plaque deposition in the extra - cellular tissues.6874 moreover , fibrillar structure seems to be related to heavy human disorders such as alzheimer s disease , parkinson s disease , and spongiform encephalopathies . in these methods , generally , nanoparticles are coated with biocompatible polymers that have the double function of preventing their aggregation and retarding the protein absorption.57,76,77 a common strategy to improve blood compatibility and to increase the blood circulation half - life of the nanoparticles is the construction of a protein - coated surface resistant to the absorption of the other opsonines.78 a thin layer of protein appears to minimize adhesion and aggregation of nanoparticles , avoiding subsequent macrophage recognition or , in the worst case , a thrombus formation . despite of the large number of studies , the absorption of a protein on whatever the solid surface is still a complex and not well understood process.60,7985 in the case of nanoparticles , size and radius of curvature become significant when compared to the protein size resulting in new interactions not shown with the bulk materials.47 the high hydrophobicity of many proteins seems to play an important role in their absorption on the nanoparticles surface.86 several models of protein absorption on surfaces identify two main steps in the process . depending on the balance of the energetic interaction if the protein has been absorbed , the second step could lead to conformational changes ( because of van der waals interactions ) , surface charge , protein dipole moment , and protein size or solution ionic strength.84,85,8790 this second step often involves irreversible changes in the protein structure up to denaturation.9193 proteins can be divided in two groups : hard and soft proteins . on the other hand , it seems that some degree of denaturation upon absorption is more probable for soft proteins than for the hard ones , especially on hydrophobic surfaces.84,9497 during the artificial absorption of protein to nanoparticles surface , the use of a large excess of the target material could allow the retention of sufficient biological activity and native epitopes , even if some proteins are denatured . the success of an absorption strategy to deliver drug or therapeutical proteins using protein - based nanoparticles as a carrier can be influenced by several factors such as the type of nanoparticles , delivery route and the nature of proteins to be absorbed . the knowledge of how the protein - based nanoparticles interact with other proteins present in the blood is fundamental for the understanding of their biological and toxicological properties.77 many methods based on established techniques could be applied such as size - exclusion chromatography , isothermal titration calorimetry , surface plasmon resonance , atomic force microscopy , differential scanning calorimeter , and circular dichroisim ( cd ) spectroscopy.47,67 even if several existing characterization methods for measuring the nature and the amount of absorbed protein on solid surfaces could be applied to nanoparticle systems,96 the development of new physical and biophysical methods may be necessary to fully understand the relationship between proteins and nanomaterials . the most popular approach for coupling covalently nanoparticle to protein is based on the existence on proteins of specific and reactive functional groups such as amino nh2 ( lysine ) , carboxylic acid cooh ( aspartic , glutamic ) , hydroxyl oh ( serine , tyrosine ) and sh ( cysteine).112 proteins can be chemically coupled to different kinds of nanoparticles using established reagents such bifunctional cross - linker molecules . furthermore , streptavidin through carbodiimide ( edc ) chemistry can be covalently coupled with different ligands such as mab and enzymes which make the biotin streptavidin system widely used in a variety of biotinylated nanoparticles.38,126128 proteins having cysteine residues can be directly attached to some metal nanoparticle surfaces such as gold and silver by stable metal sulfur bonds.129,130 in the other cases , the covalent coupling of proteins on nanoparticle surfaces is always a long experimental procedure . these carriers are versatile and their physico chemical characteristics can be properly tuned.19 liposomes synthesized from dehydrated rehydrated vesicles are widely used due to the ease of this preparation process and the low amount of stress applied to the proteins.134 liposome formulations are most frequently considered for parental administration of the drug , but may also be a potential formulation principle for alternative routes such as topical and nasal administration . in this approach , polypeptides , especially enzymes , could be entrapped inside silica matrix allowing the retention of enzymatic activity.139141 on the other hand , process difficulties such as uncontrolled release , denaturation and the hardness control of the protein orientation can be found.142,143 the control of the drug release of such silica nanoparticles is the most important and difficult parameter that needs to be properly tuned . more specifically these nanoparticles can be totally made by cs or used in several copolymer combinations.25,160 copolymers made by the combination of cs / alg are able to generate a more friendly environment which protects peptides and proteins from stressing conditions and allows their stabilization during encapsulation , storage and release.161166 glycol chitosan nanoparticles modified with hydrophobic bile acid analogs self - assemble into polymeric nanoparticles with hydrophilic shells of glycol chitosan and hydrophobic cores of bile acid derivatives have been reported as possible vehicle for rgd ( arg gly asp ) peptide.29,30 regardless of the nanomaterial chosen for protein encapsulation , an important issue that needs to be considered is the understanding of protein protein interactions . these transient interactions of protein complexes can cause several effects such as activation / inactivation of certain proteins , resulting in the formation of a new binding site.167,168 kinetics properties of enzymes can be also altered by denaturation during the entrapment process allowing potential change of the protein specificity to its substrate.169171 gaining a clear picture of these basics knowledge will definitely lead to a change of object design to increase the protein load , to control the protein release and to retain the protein integrity and efficacy . the study of the materials bio - compatibility starts , therefore , with the analysis of protein absorption on surfaces.54 synthetic materials for biomedical applications are immediately covered by proteins when put in contact with a biological environment.55,56 after protein binding , nanoparticles are quickly cleared by the mononuclear phagocytic system ( mps ) , also known as the reticuloendothelial system ( res).57,58 these macrophages , which are typically kupffer cells of the liver , can not directly identify the nanoparticles themselves , but rather recognize specific opsonin proteins bound to the surface of the particles.59 the interaction between proteins and nanoparticles surface leads to the formation of proteins corona around nanoparticles that largely defines their biological identity as well their potential toxicity.49,50,58,6064 recently , lynch and dawson postulated the importance of the protein corona as the vehicle and the biological identity of a nanoparticle for its transport through cell membranes.60 the nanoparticle surface is immediately occupied by proteins with high concentrations and high association rate constants and successively by proteins having lower concentrations but a higher affinity.47 competitive absorption of proteins is influenced by several factors such as electrostatic interactions , protein stability , and kinetic parameters.65 as the protein corona could affect the nanoparticle behavior , including its biological effect , the nanoparticle could also have an effect on the protein behavior . some nanoparticles seem able to promote the protein assembly into amyloid fibrils in vitro by assisting the nucleation process.66 bellezza and colleagues found that nanoparticles affect the morphology of the myoglobin absorbed onto phosphate - grafted zirconia nanoparticles , inducing prefibrillar - like aggregates.67 this phenomenon could have important implications for medical application of nanoparticles because the self - assembly of a variety of proteins and peptides is known to be the cause of human amyloid diseases where fibrous protein aggregates are formed , resulting in amyloid plaque deposition in the extra - cellular tissues.6874 moreover , fibrillar structure seems to be related to heavy human disorders such as alzheimer s disease , parkinson s disease , and spongiform encephalopathies . in these methods , generally , nanoparticles are coated with biocompatible polymers that have the double function of preventing their aggregation and retarding the protein absorption.57,76,77 a common strategy to improve blood compatibility and to increase the blood circulation half - life of the nanoparticles is the construction of a protein - coated surface resistant to the absorption of the other opsonines.78 a thin layer of protein appears to minimize adhesion and aggregation of nanoparticles , avoiding subsequent macrophage recognition or , in the worst case , a thrombus formation . depending on the balance of the energetic interaction if the protein has been absorbed , the second step could lead to conformational changes ( because of van der waals interactions ) , surface charge , protein dipole moment , and protein size or solution ionic strength.84,85,8790 this second step often involves irreversible changes in the protein structure up to denaturation.9193 proteins can be divided in two groups : hard and soft proteins . on the other hand , it seems that some degree of denaturation upon absorption is more probable for soft proteins than for the hard ones , especially on hydrophobic surfaces.84,9497 during the artificial absorption of protein to nanoparticles surface , the use of a large excess of the target material could allow the retention of sufficient biological activity and native epitopes , even if some proteins are denatured . the success of an absorption strategy to deliver drug or therapeutical proteins using protein - based nanoparticles as a carrier can be influenced by several factors such as the type of nanoparticles , delivery route and the nature of proteins to be absorbed . the knowledge of how the protein - based nanoparticles interact with other proteins present in the blood is fundamental for the understanding of their biological and toxicological properties.77 many methods based on established techniques could be applied such as size - exclusion chromatography , isothermal titration calorimetry , surface plasmon resonance , atomic force microscopy , differential scanning calorimeter , and circular dichroisim ( cd ) spectroscopy.47,67 even if several existing characterization methods for measuring the nature and the amount of absorbed protein on solid surfaces could be applied to nanoparticle systems,96 the development of new physical and biophysical methods may be necessary to fully understand the relationship between proteins and nanomaterials . the most popular approach for coupling covalently nanoparticle to protein is based on the existence on proteins of specific and reactive functional groups such as amino nh2 ( lysine ) , carboxylic acid cooh ( aspartic , glutamic ) , hydroxyl oh ( serine , tyrosine ) and sh ( cysteine).112 proteins can be chemically coupled to different kinds of nanoparticles using established reagents such bifunctional cross - linker molecules . furthermore , streptavidin through carbodiimide ( edc ) chemistry can be covalently coupled with different ligands such as mab and enzymes which make the biotin streptavidin system widely used in a variety of biotinylated nanoparticles.38,126128 proteins having cysteine residues can be directly attached to some metal nanoparticle surfaces such as gold and silver by stable metal sulfur bonds.129,130 in the other cases , the covalent coupling of proteins on nanoparticle surfaces is always a long experimental procedure . these carriers are versatile and their physico chemical characteristics can be properly tuned.19 liposomes synthesized from dehydrated rehydrated vesicles are widely used due to the ease of this preparation process and the low amount of stress applied to the proteins.134 liposome formulations are most frequently considered for parental administration of the drug , but may also be a potential formulation principle for alternative routes such as topical and nasal administration . in this approach , polypeptides , especially enzymes , could be entrapped inside silica matrix allowing the retention of enzymatic activity.139141 on the other hand , process difficulties such as uncontrolled release , denaturation and the hardness control of the protein orientation can be found.142,143 the control of the drug release of such silica nanoparticles is the most important and difficult parameter that needs to be properly tuned . more specifically these nanoparticles can be totally made by cs or used in several copolymer combinations.25,160 copolymers made by the combination of cs / alg are able to generate a more friendly environment which protects peptides and proteins from stressing conditions and allows their stabilization during encapsulation , storage and release.161166 glycol chitosan nanoparticles modified with hydrophobic bile acid analogs self - assemble into polymeric nanoparticles with hydrophilic shells of glycol chitosan and hydrophobic cores of bile acid derivatives have been reported as possible vehicle for rgd ( arg gly asp ) peptide.29,30 regardless of the nanomaterial chosen for protein encapsulation , an important issue that needs to be considered is the understanding of protein protein interactions . these transient interactions of protein complexes can cause several effects such as activation / inactivation of certain proteins , resulting in the formation of a new binding site.167,168 kinetics properties of enzymes can be also altered by denaturation during the entrapment process allowing potential change of the protein specificity to its substrate.169171 gaining a clear picture of these basics knowledge will definitely lead to a change of object design to increase the protein load , to control the protein release and to retain the protein integrity and efficacy .

the study group consisted of 50 children born very preterm ( i.e. , gestational age 30 weeks , established by weeks and days after the mother s last menstrual period ) , and 50 controls . for the purposes of the current study , our very preterm sample was consecutively and randomly acquired from the total population of very preterm survivors ( n = 276 ) born and admitted between 1998 - 1999 to the neonatal intensive care unit ( nicu ) of the sophia children s hospital rotterdam . our sample did not differ from the total population of very preterm survivors in terms of gender , (1 , 115 ) = 1.15 , p = 0.30 ; gestational age , f(1 , 113 ) = 1.16 , p = 0.24 ; birthweight , f(1 , 113 ) = .96 , p = 0.33 ; days of ventilation , f(1 , 113 ) = 0.04 , p = 0.84 ; days of added oxygen , f(1 , 113 ) = 0.34 , p = 0.54 ; or days of intensive care , f(1 , 113 ) = 0.28 , p = 0.66 . the control group ( mean gestational age = 39.7 , sd = 1.3 ; mean birthweight = 3579 , sd = 510 ) was recruited from local elementary schools as a part of a normative study of the vu university amsterdam . included in the control group were normally developing children without histories of prematurity ( i.e. , gestational age > 37 weeks ) , perinatal complications , psychiatric and neurological disorders . exclusion criteria for both groups were mental and/or motor handicaps too profound to allow task execution . table 1 presents the sample characteristics of the very preterm and the control group . no significant group differences were found for age , level of maternal education , or for the distribution of both genders . very preterm children obtained lower iq scores ( f(1 , 98 ) = 20.2 , p < 0.001 ) , and comprised of more twins and triplets ( (1 , 100 ) = 29.9 , p < 0.001 ) , than the controls . cerebral palsy was classified according to standards of the surveillance of cerebral palsy in europe ( scpe ) . the scpe standards ( 2000 ) differentiate between spastic ( unilateral or bilateral ) , ataxic and dyskinetic ( dystonic or choreo - athetotic ) cp . thirteen ( 26% ) very preterm children had neurosensory impairments ( eight with visual impairment , two with hearing impairment , one with cerebral palsy , and one with both cerebral palsy as well as with visual impairment ) . visual and hearing impairments , and cp , are hereafter referred to as neurosensory impairments . three ( 6% ) very preterm children were formally diagnosed with pervasive developmental disorder not otherwise specified ( pdd - nos ) , of whom two participated in special education . table 1sample characteristics of the very preterm and the control groupvery pretermcontrolsample characteristicsage ( in years ) , mean ( sd)5.9 ( .4)6.0 ( .6)level of maternal education , mean ( sd)3.9 ( .9)4.2 ( .8)iq , mean ( sd , range)92.5 ( 17.5 , 70 - 140)109.0 ( 19.2 , 71 - 150)***boys , n ( % ) 27 ( 54.0)23 ( 46.0)twins or triplets , n ( % ) 11 ( 22.0)0 ( 0.0)***visual impairmentimpaired , use of glasses , n ( % ) 9 ( 18.0)0 ( 0.0)***blind or perceives light only , n ( % ) 0 ( 0.0)0 ( 0.0)hearing impairmentimpaired , use of hearing aid , n ( % ) 2 ( 4.0)0 ( 0.0)deafness , n ( % ) 0 ( 0.0)0 ( 0.0)cerebral palsyspastic ( unilateral ) , n ( % ) 3 ( 6.0)0 ( 0.0)ataxic , n ( % ) 0 ( 0.0)0 ( 0.0)dyskinetic , n ( % ) 0 ( 0.0)0 ( 0.0)level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004).*p < 0.05.**p < 0.01.***p < 0.001 . sample characteristics of the very preterm and the control group level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004 ) . the severity of neonatal illness is expressed in the neurobiological risk score ( nbrs ) total score ( brazy et al . 1991 ) . the nbrs total score is a composite measure of neonatal risk that summarizes neonatal medical events , with higher scores indicating higher degree of neurobiological risk . table 2neonatal characteristics of the very preterm groupneonatal characteristicsbirthweight in grams , mean ( sd , range)1042.6 ( 31.8 , 605.01640.0)gestational age in weeks , mean ( sd , range)28.0 ( 1.4 , 25.030.0)duration of nicu stay in days , mean ( sd)78.7 ( 22.9)<750 g birthweight , n ( % ) 3.0 ( 6.0)<28 weeks gestational age , n ( % ) 23.0 ( 46.0)outborn , n ( % ) 4.0 ( 8.0)assisted ventilation , n ( % ) 5.0 ( 84.0)grade i / ii intra ventricular hemorrhage , n ( % ) 11.0 ( 22.0)grade iii / iv intra ventricular hemorrhage , n ( % ) 0.0 ( 0.0)periventricular leukomalacia , n ( % ) 2.0 ( 4.0)hypoglycemia , n ( % ) 0.0 ( 0.0)meningitis , n ( % ) 2.0 ( 4.0)necrotizing enterocolitis , n ( % ) 0.0 ( 0.0)chronic lung disease , n ( % ) 27.0 ( 54.0)rop ( grade i / ii / iii ) , n ( % ) 7.0/8.0/1.0 ( 14.0/16.0/2.0)small for gestational age , n ( % ) 3.0 ( 6.0)neurobiological risk score , mean ( sd)3.5 ( .9)outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . small for gestational age is defined as birthweight less than the 3rd percentile for gestational age ( usher and mclean 1969).0 - 4 = low . neonatal characteristics of the very preterm group outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . small for gestational age is defined as birthweight less than the 3rd percentile for gestational age ( usher and mclean 1969 ) . go / nogo the go / nogo task is a well - established measure of inhibition with adequate psychometric properties ( casey et al . 1997 ; drewe 1975 ; picton et al . in this study an adaptation of the original go / nogo paradigm was used ( smidts 2003 ) , which has previously been employed by raaijmakers et al . ( children completed a go / nogo task in which images of an elephant or a dog appeared on a computer screen . children were instructed to respond to the elephant ( go - stimulus ) and to withhold their response when the dog appeared ( nogo - stimulus ) . after a 300 ms delay , the go- or nogo - stimulus was presented for 1000 ms , with a fixed interstimulus interval of 1500 ms . a fixed interstimulus interval was used as variable intervals ( specifically shorter ones ) would have made the task too difficult for the youngest children . fifty percent of trials were go - trials , and the trials were shown in a random order . after an initial practice block of 12 stimuli , where the child was required to respond correctly to at least 5 consecutive stimuli in order to proceed to the experimental trials , an experimental block consisting of 24 stimuli was completed . the total number of correct responses and efficiency of responding ( total number of correct responses divided by the mean reaction time of correct responses ) was used as an index of inhibition . measures of efficiency have been used in previous studies on ef performance in preschoolers ( espy 1997 ; isquith et al . efficiency measures comprise both accuracy and response time and take into account speed accuracy trade off ( sato ) . as response time improves significantly during early childhood , the use of efficiency measures is valuable specifically in studies with young children . the shape school the original shape school task is a storybook for preschoolers , designed to measure inhibition and switching processes ( espy 1997 ) . adequate psychometric properties have been established for the shape school task ( espy et al . , we used a computerized , modified version of the shape school ( smidts and groot 2005 ) . children were asked to respond using response buttons ( see procedure for details regarding the response buttons ) . children responded by pressing either the red or yellow button , depending on the color of the figure and the rule accompanying the condition . three conditions were administered : the control , inhibition , and switching condition . in the control condition , the child was asked to respond to the color of the figures by pressing the corresponding button as quickly as possible . in the inhibition condition , children had to respond whenever they saw a figure with a happy face ( fifty percent of the trials were inhibitory trials ) , but were instructed to suppress a response whenever they saw a figure with a sad face . in the switching condition , children had to give an opposite response ( switch ) by pressing the button that was originally linked with the other color whenever the figure was wearing a hat ( fifty percent of the trials were switch trials ) . all conditions started with an initial practice block of 12 stimuli , where the child was required to respond correctly to at least 5 consecutive stimuli in order to proceed to the experimental trials , after which an experimental block consisting of 24 stimuli was completed . stimuli were preceded by a 200 ms fixation cross and a 300 ms delay , and were presented for 2000 ms in condition a and b , and for 3000 ms in condition c , with a fixed interstimulus interval of 1500 ms . dependent variables used in this study were : mean reaction time ( rt ) in ms on all trials from the control condition ( measure for speed of processing ) ; and the total number of correct responses and efficiency of responding ( i.e. , total number of correct responses divided by mean rt of correct responses ) from the inhibition and switching conditions . day - night task the day - night task is a well - validated measure of prepotent response inhibition in young children ( diamond et al . 1994 ; simpson and riggs 2005 ) . in the day - night task ( gerstadt et al . 1994 ) , children were shown a set of 16 cards with pictures of either a sun or a moon with stars . there were two conditions : ( 1 ) a control condition , in which the child had to say " day " in response to a sun card and " night " in response to a moon card , and ( 2 ) an experimental condition , where the child was asked to respond to the sun card by saying " night " and vice versa . in both conditions , the same set of cards was used , shown in a pseudorandom order . response time for each condition for the total of 16 cards was recorded manually using a stopwatch . the dependent variables used in this study were the total number of correct responses and the efficiency of responding in the control condition and experimental condition ( i.e. , total number of correct responses divided by the total naming time ) . 1991 ) , children were asked to name as many examples from two specific categories : " animals " and " things you can eat or drink " within a 40-second time frame . two examples of each category were provided before the beginning of the task . an item named for the second time was scored as incorrect , as well as examples that fell outside above - mentioned categories . the total number of correct words across both categories was used as an index for verbal fluency . word span this task , based on the digit span subtest of the wechsler iq scale for children ( wisc : wechsler 1997 ) , was used to assess verbal working memory ( smidts 2003 ) . a string of words was read aloud , and the child was asked to repeat the words . similar to the wisc subtest , the number of words increased across trials , to a maximum of six words . the child had to repeat at least one string correctly in order to proceed to the next trial . in the forward condition , words had to be repeated in the same order as read by the examiner , and in the backward condition , words were to be repeated in the reverse order . the dependent variables used in this study were the total number of correctly recalled strings in the forward and backward condition , of which the latter served as an index for working memory . object classification task for children ( octc ) the original object classification task for children ( smidts et al . 2004 ) is a concept - shifting task that requires the child to group six toys according to three predetermined groupings : color ( red or yellow ) , size ( big or small ) , and function ( car or plane ) . in this study , as opposed to toys , we used cards . these cards depicted yellow or red cars or planes , and could be sorted according to the same predetermined groupings as the toys in the original task . there were three conditions characterized by three increasing levels of structure in terms of help supplied by the examiner : ( 1 ) free generation , where the child is required to sort the cards without any help of the examiner , ( 2 ) identification , where the examiner constructs a category and the child is asked to identify the sort , and ( 3 ) explicit cueing , where the child is explicitly told how to sort the cards . first , there were two practice trials , where the child was asked to sort four cards depicting two different disney figures ( two cards showed identical pictures of mickey mouse , the other pair contained images of donald duck ) . the child was asked to " put the ones that are the same on this side of the table and the other ones that are the same on the other side of the table " . these practice trials were designed to assess whether a child was able to sort according to overall appearance.after these practice trials , the experimental trials started with presenting six cards to the child . in contrast to the practice trials , these cards did not show identical images that needed to be matched , but instead the child was required to sort the cards according to color ( three cards showed red images , the other three cards displayed images in yellow ) , size ( three cards depicted small images , the other three images were large ) , or function ( three cards displayed cars , the other three had planes on them ) . the child was told , " there is something the same about these images " , and was then asked to put the ones that are the same on this side of the table and the other ones that are the same on the other side of the table " . after a correct sort of one of the three groupings ( i.e. , color , size or function ) , the child was encouraged to verbally name the identified grouping " so why did you place these cards on this side of the table and the other ones over there ? what s the same about these pictures ? " . the child s answer was recorded and the examiner then mixed up the cards and asked the child to " make two groups again , but this time , something else has to be the same " . this procedure was repeated until the child had correctly sorted the cards according to the three different groupings . if the child had arranged the cards correctly according to color , size or function , but was unable to sort the cards again for a second ( or third ) time , the examiner sorted the cards according to one of the remaining categories . the child was then asked to identify the sort ( " so can you tell me what s the same about these cards ? " ) . if the child was unable to identify the sort , the examiner specifically asked the child to sort the cards according to a particular grouping ( " can you put all the red ones over there , and all the yellow ones over there ? " ) . this was called the explicit cueing condition , where the child received one point for each correct sort . however , if the child did not understand task instructions when first presented with the six cards , one dimension was removed , and the child was shown four cards , which could be sorted according to either color or size . testing procedures and point scoring system were similar to those described for the six cards . the total raw score was calculated by summing all the points earned and was used as an indication of childrens ability to shift between concepts . intelligence four subtests of the wechsler primary and preschool scale intelligence - revised ( wechsler 1997 ; dutch version by vander steene and bos 1997 ) were used to estimate full scale iq : picture completion , vocabulary , block design and similarities . the vocabulary and similarities ( verbal scale ) subtest scores were added up , and then multiplied by three . the same procedure was followed for the picture completion and block design subtests ( performance scale ) . both the verbal and performance scale scores were then added up into a composite score , of which the corresponding full scale iq could be derived from the manual ( sattler 1992 ) . scores on these subtests correlate highly ( 0.90 range ) with full scale iq ( groth - marnat 1997 ) . half of the children in each group performed the tasks according to order a ( intelligence subtests day night task go / nogo octc shape school control condition and inhibition condition verbal fluency shape school switching condition word span ) , while the other half of the children of in each group performed the tests according to order b ( intelligence subtests go / nogo word span shape school control condition and inhibition condition verbal fluency shape school switching condition octc day - night task ) . computerized tasks were administered using the e - prime software package ( psychology software tools , pittsburgh , pa ) and a dell latitude d800 laptop with a 15.4-inch color screen . children responded by making a button press with one hand , but were required to keep both hands placed on top of the buttons so that they could react as quickly as possible . the buttons were converted emergency stop switches , with an external diameter of 94 mm ( moeller safety products ; model number : fak - r / v / kc11/1y ) . the stimuli were 700 pixels high and 500 pixels in width and presented with a 45 visual angle . total duration of testing was ninety minutes , and frequent breaks were introduced to avoid fatigue . the children were examined individually in a quiet room while one of their parents was present . the observations in this study were not strictly independent , given the large number of multiple births . therefore , we applied the method of mixed modeling , i.e. , random regression modeling ( rrm ) , to take the relatedness of the multiple births into account . the error structure was assumed to be related ( compound symmetry ) which implies that both correlations and variances within the multiple births did not differ significantly . group differences for the ef task dependent variables were analyzed with group ( very preterm versus control ) as the between subjects factor . we also examined group differences both with and without controlling for maternal education , and both with and without inclusion of the subset of very preterm children with neurosensory impairments . chi - square statistics were carried out to determine if there were group differences in rates of ef impairments . an impairment in ef was defined by a mean score on the ef dependent variable greater than one sd below the control group mean ( e.g. , taylor et al . analyses were run while controlling for mean rt on the control condition of each specific task . thus , group differences in performance on the go / nogo task and the shape school inhibition and switching conditions ( both tasks parallel in main task characteristics ) were reanalyzed while entering the mean rt on the shape school control condition as a covariate . similar analyses were performed for the day - night task experimental condition , with mean rt on the day - night task control condition serving as a covariate . pearson s correlation coefficients were calculated for the relationship between iq and the ef dependent variables . cohen s guidelines were followed to indicate the strength of the correlation coefficients , with 0.10 , 0.30 , and 0.50 referring to small , medium , and large coefficients , respectively ( cohen 1992 ) . next , group differences in ef were reanalyzed with iq as a covariate , and vice versa . cohen s guidelines were followed to indicate the strength of effect sizes , with 0.20 , 0.50 , and 0.80 referring to small , medium , and large effect sizes , respectively ( cohen 1992 ) . hierarchical , multiple regression analyses were conducted to test the impact of demographic and neonatal variables on the ef dependent variables of the very preterm group . the demographic predictor variables gender and maternal education were entered in the first block , gestational age in the next block to examine the impact of gestational age over and above background demographics , and finally the nbrs total score as an index of neonatal illness was entered in the last block . for all analyses , the threshold for significance was set at p < 0.05 ( two - sided ) . missing data and extreme values missing data resulted from either examiner error or child noncompliance and was less than 4% for each of the dependent variables . due to not pressing the response button hard enough , the percentage of missing data for the dependent variables of the go / nogo task was 9% . missing data was replaced by means of expectation maximization ( tabachnick and fidell 2001 ) . extreme values were defined as having an absolute z - score exceeding 3 sds from the group mean and identified in both groups separately . if an extreme value occurred due to examiner error ( n = 1 ) , the case was removed from the analyses . if due to child non - compliance ( n = 1 ) , the extreme value was truncated to either 0.5 sd beyond the next most extreme score if that score was z < 3.0 ( nigg et al . 2002 ) . the study group consisted of 50 children born very preterm ( i.e. , gestational age 30 weeks , established by weeks and days after the mother s last menstrual period ) , and 50 controls . for the purposes of the current study , our very preterm sample was consecutively and randomly acquired from the total population of very preterm survivors ( n = 276 ) born and admitted between 1998 - 1999 to the neonatal intensive care unit ( nicu ) of the sophia children s hospital rotterdam . our sample did not differ from the total population of very preterm survivors in terms of gender , (1 , 115 ) = 1.15 , p = 0.30 ; gestational age , f(1 , 113 ) = 1.16 , p = 0.24 ; birthweight , f(1 , 113 ) = .96 , p = 0.33 ; days of ventilation , f(1 , 113 ) = 0.04 , p = 0.84 ; days of added oxygen , f(1 , 113 ) = 0.34 , p = 0.54 ; or days of intensive care , f(1 , 113 ) = 0.28 , p = 0.66 . the control group ( mean gestational age = 39.7 , sd = 1.3 ; mean birthweight = 3579 , sd = 510 ) was recruited from local elementary schools as a part of a normative study of the vu university amsterdam . included in the control group were normally developing children without histories of prematurity ( i.e. , gestational age > 37 weeks ) , perinatal complications , psychiatric and neurological disorders . exclusion criteria for both groups were mental and/or motor handicaps too profound to allow task execution . table 1 presents the sample characteristics of the very preterm and the control group . no significant group differences were found for age , level of maternal education , or for the distribution of both genders . very preterm children obtained lower iq scores ( f(1 , 98 ) = 20.2 , p < 0.001 ) , and comprised of more twins and triplets ( (1 , 100 ) = 29.9 , p < 0.001 ) , than the controls . cerebral palsy was classified according to standards of the surveillance of cerebral palsy in europe ( scpe ) . the scpe standards ( 2000 ) differentiate between spastic ( unilateral or bilateral ) , ataxic and dyskinetic ( dystonic or choreo - athetotic ) cp . thirteen ( 26% ) very preterm children had neurosensory impairments ( eight with visual impairment , two with hearing impairment , one with cerebral palsy , and one with both cerebral palsy as well as with visual impairment ) . visual and hearing impairments , and cp , are hereafter referred to as neurosensory impairments . three ( 6% ) very preterm children were formally diagnosed with pervasive developmental disorder not otherwise specified ( pdd - nos ) , of whom two participated in special education . table 1sample characteristics of the very preterm and the control groupvery pretermcontrolsample characteristicsage ( in years ) , mean ( sd)5.9 ( .4)6.0 ( .6)level of maternal education , mean ( sd)3.9 ( .9)4.2 ( .8)iq , mean ( sd , range)92.5 ( 17.5 , 70 - 140)109.0 ( 19.2 , 71 - 150)***boys , n ( % ) 27 ( 54.0)23 ( 46.0)twins or triplets , n ( % ) 11 ( 22.0)0 ( 0.0)***visual impairmentimpaired , use of glasses , n ( % ) 9 ( 18.0)0 ( 0.0)***blind or perceives light only , n ( % ) 0 ( 0.0)0 ( 0.0)hearing impairmentimpaired , use of hearing aid , n ( % ) 2 ( 4.0)0 ( 0.0)deafness , n ( % ) 0 ( 0.0)0 ( 0.0)cerebral palsyspastic ( unilateral ) , n ( % ) 3 ( 6.0)0 ( 0.0)ataxic , n ( % ) 0 ( 0.0)0 ( 0.0)dyskinetic , n ( % ) 0 ( 0.0)0 ( 0.0)level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004).*p < 0.05.**p < 0.01.***p < 0.001 . sample characteristics of the very preterm and the control group level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004 ) . the severity of neonatal illness is expressed in the neurobiological risk score ( nbrs ) total score ( brazy et al . 1991 ) . the nbrs total score is a composite measure of neonatal risk that summarizes neonatal medical events , with higher scores indicating higher degree of neurobiological risk . table 2neonatal characteristics of the very preterm groupneonatal characteristicsbirthweight in grams , mean ( sd , range)1042.6 ( 31.8 , 605.01640.0)gestational age in weeks , mean ( sd , range)28.0 ( 1.4 , 25.030.0)duration of nicu stay in days , mean ( sd)78.7 ( 22.9)<750 g birthweight , n ( % ) 3.0 ( 6.0)<28 weeks gestational age , n ( % ) 23.0 ( 46.0)outborn , n ( % ) 4.0 ( 8.0)assisted ventilation , n ( % ) 5.0 ( 84.0)grade i / ii intra ventricular hemorrhage , n ( % ) 11.0 ( 22.0)grade iii / iv intra ventricular hemorrhage , n ( % ) 0.0 ( 0.0)periventricular leukomalacia , n ( % ) 2.0 ( 4.0)hypoglycemia , n ( % ) 0.0 ( 0.0)meningitis , n ( % ) 2.0 ( 4.0)necrotizing enterocolitis , n ( % ) 0.0 ( 0.0)chronic lung disease , n ( % ) 27.0 ( 54.0)rop ( grade i / ii / iii ) , n ( % ) 7.0/8.0/1.0 ( 14.0/16.0/2.0)small for gestational age , n ( % ) 3.0 ( 6.0)neurobiological risk score , mean ( sd)3.5 ( .9)outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . small for gestational age is defined as birthweight less than the 3rd percentile for gestational age ( usher and mclean 1969).0 - 4 = low . neonatal characteristics of the very preterm group outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . small for gestational age is defined as birthweight less than the 3rd percentile for gestational age ( usher and mclean 1969 ) . go / nogo the go / nogo task is a well - established measure of inhibition with adequate psychometric properties ( casey et al . an adaptation of the original go / nogo paradigm was used ( smidts 2003 ) , which has previously been employed by raaijmakers et al . children completed a go / nogo task in which images of an elephant or a dog appeared on a computer screen . children were instructed to respond to the elephant ( go - stimulus ) and to withhold their response when the dog appeared ( nogo - stimulus ) . after a 300 ms delay , the go- or nogo - stimulus was presented for 1000 ms , with a fixed interstimulus interval of 1500 ms . a fixed interstimulus interval was used as variable intervals ( specifically shorter ones ) would have made the task too difficult for the youngest children . fifty percent of trials were go - trials , and the trials were shown in a random order . after an initial practice block of 12 stimuli , where the child was required to respond correctly to at least 5 consecutive stimuli in order to proceed to the experimental trials , an experimental block consisting of 24 stimuli was completed . the total number of correct responses and efficiency of responding ( total number of correct responses divided by the mean reaction time of correct responses ) was used as an index of inhibition . measures of efficiency have been used in previous studies on ef performance in preschoolers ( espy 1997 ; isquith et al . efficiency measures comprise both accuracy and response time and take into account speed accuracy trade off ( sato ) . as response time improves significantly during early childhood , the use of efficiency measures is valuable specifically in studies with young children . the shape school the original shape school task is a storybook for preschoolers , designed to measure inhibition and switching processes ( espy 1997 ) . adequate psychometric properties have been established for the shape school task ( espy et al . , we used a computerized , modified version of the shape school ( smidts and groot 2005 ) . children were asked to respond using response buttons ( see procedure for details regarding the response buttons ) . children responded by pressing either the red or yellow button , depending on the color of the figure and the rule accompanying the condition . three conditions were administered : the control , inhibition , and switching condition . in the control condition , the child was asked to respond to the color of the figures by pressing the corresponding button as quickly as possible . in the inhibition condition , children had to respond whenever they saw a figure with a happy face ( fifty percent of the trials were inhibitory trials ) , but were instructed to suppress a response whenever they saw a figure with a sad face . in the switching condition , children had to give an opposite response ( switch ) by pressing the button that was originally linked with the other color whenever the figure was wearing a hat ( fifty percent of the trials were switch trials ) . all conditions started with an initial practice block of 12 stimuli , where the child was required to respond correctly to at least 5 consecutive stimuli in order to proceed to the experimental trials , after which an experimental block consisting of 24 stimuli was completed . stimuli were preceded by a 200 ms fixation cross and a 300 ms delay , and were presented for 2000 ms in condition a and b , and for 3000 ms in condition c , with a fixed interstimulus interval of 1500 ms . dependent variables used in this study were : mean reaction time ( rt ) in ms on all trials from the control condition ( measure for speed of processing ) ; and the total number of correct responses and efficiency of responding ( i.e. , total number of correct responses divided by mean rt of correct responses ) from the inhibition and switching conditions . day - night task the day - night task is a well - validated measure of prepotent response inhibition in young children ( diamond et al . 1994 ; simpson and riggs 2005 ) . in the day - night task ( gerstadt et al . 1994 ) , children were shown a set of 16 cards with pictures of either a sun or a moon with stars . there were two conditions : ( 1 ) a control condition , in which the child had to say " day " in response to a sun card and " night " in response to a moon card , and ( 2 ) an experimental condition , where the child was asked to respond to the sun card by saying " night " and vice versa . in both conditions , the same set of cards was used , shown in a pseudorandom order . response time for each condition for the total of 16 cards was recorded manually using a stopwatch . the dependent variables used in this study were the total number of correct responses and the efficiency of responding in the control condition and experimental condition ( i.e. , total number of correct responses divided by the total naming time ) . 1991 ) , children were asked to name as many examples from two specific categories : " animals " and " things you can eat or drink " within a 40-second time frame . two examples of each category were provided before the beginning of the task . an item named for the second time was scored as incorrect , as well as examples that fell outside above - mentioned categories . the total number of correct words across both categories was used as an index for verbal fluency . word span this task , based on the digit span subtest of the wechsler iq scale for children ( wisc : wechsler 1997 ) , was used to assess verbal working memory ( smidts 2003 ) . a string of words was read aloud , and the child was asked to repeat the words . similar to the wisc subtest , the number of words increased across trials , to a maximum of six words . the child had to repeat at least one string correctly in order to proceed to the next trial . in the forward condition , words had to be repeated in the same order as read by the examiner , and in the backward condition , words were to be repeated in the reverse order . the dependent variables used in this study were the total number of correctly recalled strings in the forward and backward condition , of which the latter served as an index for working memory . object classification task for children ( octc ) the original object classification task for children ( smidts et al . 2004 ) is a concept - shifting task that requires the child to group six toys according to three predetermined groupings : color ( red or yellow ) , size ( big or small ) , and function ( car or plane ) . in this study , as opposed to toys , we used cards . these cards depicted yellow or red cars or planes , and could be sorted according to the same predetermined groupings as the toys in the original task . there were three conditions characterized by three increasing levels of structure in terms of help supplied by the examiner : ( 1 ) free generation , where the child is required to sort the cards without any help of the examiner , ( 2 ) identification , where the examiner constructs a category and the child is asked to identify the sort , and ( 3 ) explicit cueing , where the child is explicitly told how to sort the cards . first , there were two practice trials , where the child was asked to sort four cards depicting two different disney figures ( two cards showed identical pictures of mickey mouse , the other pair contained images of donald duck ) . the child was asked to " put the ones that are the same on this side of the table and the other ones that are the same on the other side of the table " . these practice trials were designed to assess whether a child was able to sort according to overall appearance.after these practice trials , the experimental trials started with presenting six cards to the child . in contrast to the practice trials , these cards did not show identical images that needed to be matched , but instead the child was required to sort the cards according to color ( three cards showed red images , the other three cards displayed images in yellow ) , size ( three cards depicted small images , the other three images were large ) , or function ( three cards displayed cars , the other three had planes on them ) . the child was told , " there is something the same about these images " , and was then asked to put the ones that are the same on this side of the table and the other ones that are the same on the other side of the table " . after a correct sort of one of the three groupings ( i.e. , color , size or function ) , the child was encouraged to verbally name the identified grouping " so why did you place these cards on this side of the table and the other ones over there ? what s the same about these pictures ? " . the child s answer was recorded and the examiner then mixed up the cards and asked the child to " make two groups again , but this time , something else has to be the same " . this procedure was repeated until the child had correctly sorted the cards according to the three different groupings . if the child had arranged the cards correctly according to color , size or function , but was unable to sort the cards again for a second ( or third ) time , the examiner sorted the cards according to one of the remaining categories . the child was then asked to identify the sort ( " so can you tell me what s the same about these cards ? " ) . if the child was unable to identify the sort , the examiner specifically asked the child to sort the cards according to a particular grouping ( " can you put all the red ones over there , and all the yellow ones over there ? " ) . this was called the explicit cueing condition , where the child received one point for each correct sort . however , if the child did not understand task instructions when first presented with the six cards , one dimension was removed , and the child was shown four cards , which could be sorted according to either color or size . testing procedures and point scoring system were similar to those described for the six cards . the total raw score was calculated by summing all the points earned and was used as an indication of childrens ability to shift between concepts . intelligence four subtests of the wechsler primary and preschool scale intelligence - revised ( wechsler 1997 ; dutch version by vander steene and bos 1997 ) were used to estimate full scale iq : picture completion , vocabulary , block design and similarities . the vocabulary and similarities ( verbal scale ) subtest scores were added up , and then multiplied by three . the same procedure was followed for the picture completion and block design subtests ( performance scale ) . both the verbal and performance scale scores were then added up into a composite score , of which the corresponding full scale iq could be derived from the manual ( sattler 1992 ) . scores on these subtests correlate highly ( 0.90 range ) with full scale iq ( groth - marnat 1997 ) . specifically trained experimenters administered all measures using standardized instructions . to control for order effects , half of the children in each group performed the tasks according to order a ( intelligence subtests day night task go / nogo octc shape school control condition and inhibition condition verbal fluency shape school switching condition word span ) , while the other half of the children of in each group performed the tests according to order b ( intelligence subtests go / nogo word span shape school control condition and inhibition condition verbal fluency shape school switching condition octc day - night task ) . computerized tasks were administered using the e - prime software package ( psychology software tools , pittsburgh , pa ) and a dell latitude d800 laptop with a 15.4-inch color screen . children responded by making a button press with one hand , but were required to keep both hands placed on top of the buttons so that they could react as quickly as possible . the buttons were converted emergency stop switches , with an external diameter of 94 mm ( moeller safety products ; model number : fak - r / v / kc11/1y ) . the stimuli were 700 pixels high and 500 pixels in width and presented with a 45 visual angle . total duration of testing was ninety minutes , and frequent breaks were introduced to avoid fatigue . the children were examined individually in a quiet room while one of their parents was present . the observations in this study were not strictly independent , given the large number of multiple births . therefore , we applied the method of mixed modeling , i.e. , random regression modeling ( rrm ) , to take the relatedness of the multiple births into account . the error structure was assumed to be related ( compound symmetry ) which implies that both correlations and variances within the multiple births did not differ significantly . group differences for the ef task dependent variables were analyzed with group ( very preterm versus control ) as the between subjects factor . we also examined group differences both with and without controlling for maternal education , and both with and without inclusion of the subset of very preterm children with neurosensory impairments . chi - square statistics were carried out to determine if there were group differences in rates of ef impairments . an impairment in ef was defined by a mean score on the ef dependent variable greater than one sd below the control group mean ( e.g. , taylor et al . , analyses were run while controlling for mean rt on the control condition of each specific task . thus , group differences in performance on the go / nogo task and the shape school inhibition and switching conditions ( both tasks parallel in main task characteristics ) were reanalyzed while entering the mean rt on the shape school control condition as a covariate . similar analyses were performed for the day - night task experimental condition , with mean rt on the day - night task control condition serving as a covariate . pearson s correlation coefficients were calculated for the relationship between iq and the ef dependent variables . cohen s guidelines were followed to indicate the strength of the correlation coefficients , with 0.10 , 0.30 , and 0.50 referring to small , medium , and large coefficients , respectively ( cohen 1992 ) . next , group differences in ef were reanalyzed with iq as a covariate , and vice versa . cohen s guidelines were followed to indicate the strength of effect sizes , with 0.20 , 0.50 , and 0.80 referring to small , medium , and large effect sizes , respectively ( cohen 1992 ) . hierarchical , multiple regression analyses were conducted to test the impact of demographic and neonatal variables on the ef dependent variables of the very preterm group . the demographic predictor variables gender and maternal education were entered in the first block , gestational age in the next block to examine the impact of gestational age over and above background demographics , and finally the nbrs total score as an index of neonatal illness was entered in the last block . for all analyses , the threshold for significance was set at p < 0.05 ( two - sided ) . missing data and extreme values missing data resulted from either examiner error or child noncompliance and was less than 4% for each of the dependent variables . due to not pressing the response button hard enough , the percentage of missing data for the dependent variables of the go / nogo task was 9% . missing data was replaced by means of expectation maximization ( tabachnick and fidell 2001 ) . extreme values were defined as having an absolute z - score exceeding 3 sds from the group mean and identified in both groups separately . if an extreme value occurred due to examiner error ( n = 1 ) , the case was removed from the analyses . if due to child non - compliance ( n = 1 ) , the extreme value was truncated to either 0.5 sd beyond the next most extreme score if that score was z < 3.0 ( nigg et al . 2002 ) . convergent and divergent validity coefficients the convergent validity coefficient for the two measures of processing speed in the current study ( mean rt on the shape school control condition and mean rt on the day - night task control condition ) was .45 , p < 0.01 . convergent validity coefficients between the inhibitory control tasks ranged from 0.22 to 0.58 , all ps < 0.001 . for each of the other measured ef domains , i.e. , working memory , switching , verbal fluency and concept generation , we have employed one task per domain . therefore , convergent validity coefficients could not be calculated for these measures . divergent validity coefficients between the ef measures employed ranged from 0.15 to 0.39 , all ps < 0.001 ( details are available from first author ) . ef task performance all participating children met the performance criteria for continuing on to the experimental trials during the practice phases of the go / gono task and the shape school task . table 3 shows the means and standard deviations , and the statistical values indicating whether group differences were significant for the ef dependent variables . the very preterm group performed significantly poorer than the controls on all ef measures , except for the total number of correct responses and efficiency on the shape school inhibition condition , or for total correct for the word span forward , for which group differences were nonsignificant . analyses with and without inclusion of the subset of very preterm children with neurosensory impairments , or with and without inclusion of the three very preterm children with pdd - nos revealed similar results.1table 3means and standard deviations of the dependent variables , and statistical values indicating group differences between the very preterm and the control groupafter controlling forafter controlling forvery pretermcontrolgroupprocessing speediqdependent variablesmsdmsdfpdfpdfpdss control time in ms90825475316713.54<0.010.74 - --9.06<0.010.61ss inhibition total correct21.300.6322.540.222.360.150.310.940.350.200.570.460.15ss inhibition efficiency0.020.010.020.010.730.410.172.870.110.342.380.150.31ss switching total correct18.840.7322.220.287.660.020.563.150.100.131.830.200.27ss switching efficiency0.01<0.010.02<0.014.290.040.420.040.840.040.000.970.01go / nogo total correct20.800.5722.600.397.980.020.574.62<0.050.193.240.090.36go / nogo efficiency0.030.010.040.0116.92<0.010.835.320.040.476.280.030.51dn exp total correct13.150.3714.400.237.400.020.551.040.330.210.860.370.19dn exp efficiency0.410.100.620.2044.88<0.0011.3518.85<0.010.8815.26<0.010.79vf total correct11.873.7114.905.2110.86<0.010.67 - --5.400.02.47ws total correct forwards5.300.235.840.173.770.070.39 - --0.970.340.20ws total correct backwards1.900.692.760.8715.61<0.010.80 - --6.900.020.53octc total points7.322.108.802.0114.69<0.010.77 - --4.780.040.44dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span.processing speed is measured by the mean rt on the shape school control condition.df = 1 , 98.df = 1 , 97 . means and standard deviations of the dependent variables , and statistical values indicating group differences between the very preterm and the control group dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span . table 4 depicts the rates of ef impairments in the very preterm group and control group . in comparison to the control group , very preterm children exhibited significant impairments in all measured efs , except for the shape school inhibition condition , or verbal fluency for which group differences in impairment rates were not significant , all (1 , n = 100 ) < 2.10 , p > 0.05 . table 4rates of executive function impairments in the very preterm and control groupvery pretermcontroldependent variablesn ( % ) n ( % ) ss control time in ms23 ( 46)7 ( 14)12.90***ss inhibition total correct14 ( 28)12 ( 24).21ss inhibition efficiency0 ( 0)2 ( 4)2.04ss switching total correct19 ( 38)8 ( 16)6.14**ss switching efficiency12 ( 24)3 ( 6)6.35*go / nogo total correct11 ( 22)4 ( 8)3.84*go / nogo efficiency18 ( 26)6 ( 12)7.90***dn exp total correct31 ( 62)21 ( 42)4.01*dn exp efficiency33 ( 66)10 ( 20)21.58***vf total correct12 ( 24)8 ( 16)1.00ws total correct forwards23 ( 46)19 ( 38).66ws total correct backwards18 ( 36)1 ( 2)18.78***octc total points18 ( 36)5 ( 10)9.54**definition of an impairment is given in the text.dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span.*p < 0.05.**p < 0.01.***p < 0.001 . rates of executive function impairments in the very preterm and control group definition of an impairment is given in the text . dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span . speed of processing and iq to determine the impact of baseline processing speed on the results , we reanalyzed group differences for efficiency on the go / nogo task and the shape school inhibition and switching conditions while covarying for mean rt on the shape school control condition ( as a baseline measure of processing speed ) . group differences for the go / nogo task remained significant after taking into account processing speed . group differences for the shape school switching condition , however , became nonsignificant after covarying for processing speed . group differences for efficiency on the day - night task experimental condition were adjusted for mean rt on the day - night task control condition . strong , nearly large ( cohen 1992 ) correlation coefficients were found for word span backwards ( r = 0.43 , p < 0.01 ) , octc total points ( r = 0.44 , p < 0.001 ) , and efficiency on the day - night task experimental condition ( r = 0.46 , p < 0.001 ) . the majority of the ef group differences remained significant after controlling for iq , except for the shape school inhibition and switching conditions , for which group differences became nonsignificant . additional , exploratory analyses were conducted to examine whether group differences in iq between the very preterm children and the controls persisted while controlling for ef . for the purpose of this analysis , we extracted a composite ef factor from eight ef dependent variables ( i.e. , total number of correct responses for each task ) using principal components analysis . one variable of each task was chosen to prevent an artificial clustering of variables from the same task . an unrotated covariance matrix revealed one factor with an eigenvalue greater than 1 , which explained 49% of the variance . group differences for iq remained significant after entering the ef factor as covariate , f(1 , 97 ) = 12.04 , p < 0.001 . the impact of demographic and neonatal risk factors on ef of the demographic factors gender and maternal education , which were entered in the first block , gender was not associated with any of the ef dependent variables . maternal education explained 12% of the variance ( r = 0.12 ; f(2 , 47 ) = 3.26 , p < 0.05 ) in efficiency on the shape school inhibition condition ( = 0.31 , p < 0.05 ) , and did not predict performance on any of the other ef dependent variables ( variance explained 4% , all ps > 0.25 ) . gestational age , entered in the second block , explained 12% of the variance ( r = 0.08 ; f(1 , 46 ) = 4.12 , p < 0.05 ) in performance on the octc ( = 0.29 , p < 0.05 ) , however was not predictive for the other ef dependent variables , ( variance explained < 6% , all ps > 0.09 ) . the nbrs total score , which was entered in the third or final block , did not predict performance on any of the ef measures ( variance explained 7% , all ps > 0.08 ) . this study compared test performance of 50 very preterm children at early school age to that of 50 age - matched controls on a comprehensive ef battery . the findings demonstrated that very preterm children with average iq performed significantly poorer than the healthy term born children on ef tests of inhibition , switching , working memory , verbal fluency , and concept generation . group differences were not attributable to maternal education , and remained significant when very preterm children with neurosensory impairments were excluded from the analyses . in addition , very preterm children displayed significant higher rates of impairments in processing speed , inhibition , switching , working memory , and concept generation , than the controls . very preterm children demonstrated poorer inhibitory control than the controls on the go / nogo task and the day - night task . group differences remained significant after controlling for processing speed , which suggests that very preterm children exhibit a deficit in inhibitory control in addition to slower processing speed . group differences for switching , however , became nonsignificant after covarying for processing speed , which suggests that switching difficulties in very preterm children might be explained by slow processing speed . different cognitive processes are involved in switching , i.e. , holding the switching rule in mind ( working memory ) , inhibiting the incorrect response ( inhibition ) , and switching response set ( diamond 2002 ) . the developmental pathways of these processes differ , and inhibition is one of the first efs to emerge ( barkley 1997 ; brocki and bohlin 2004 ) . at early school age switching is still immature ( anderson et al . heavily appeals to speed ( isquith et al . 2005 ) , and as response time improves significantly during childhood ( isquith et al . 2005 ) , it seems that our results point to the fact that switching processes in very preterm children are so immature that these childrens performance in switching tasks is dominated by processing speed . the very preterm group obtained a mean iq within the average range , which however was significantly lower than the mean iq of the control group . it should be noted that the high average mean iq of the control group might be associated with the high level of maternal education , though the groups did not differ significantly in level of maternal education . group differences between the very preterm children and the controls could not be explained by differences in iq . our results are in line with research stating that ef is related to , yet distinct from iq ( friedman et al . 2006 ) . among studies into ef in very preterm children , there is substantial variation in whether poor ef in these children is independent of iq ( e.g. , bayless and stevenson 2007 ; bohm et al . for example , abbreviated iq measures may not be as reliable as more comprehensive iq measures , as extreme scores have far greater influence . in addition , some iq measures have a greater focus on fluid intelligence in contrast to crystallized intelligence , than others , which is likely to result in higher correlations with ef ( blair 2006 ) . in our study three of the four subtests employed to estimate iq had a fluid component ( similarities , picture arrangement and block design ; blair 2006 ) . iq is suggested to mostly influence more complex functions that require a greater degree of conceptual problem - solving ability and higher levels of cognitive efficiency ( blair 2006 ; seidenberg et al . 1983 ) , which was supported by our findings showing a substantial overlap between iq and measures of concept generation ( octc ) , working memory , and ( verbal ) inhibition ( word span backwards , and day - night task ) . in conclusion , to obtain a thorough understanding of very preterm childrens neurocognitive difficulties , both ef and iq should be measured , since ef and iq are related yet distinct concepts . in the present study , we investigated the relationship between demographic and neonatal risk factors and ef . we found that gender was not associated with ef . although some studies with normally developing children found gender differences in performance on ef tasks ( anderson et al . 2000a , b ; krikorian and bartok 1998 ) , most research agrees on that boys and girls show similar development of ef ( e.g. , welsh et al . 1991 ) . in line with previous research ( ardila et al . 2005 ) , maternal education was , though marginally , associated with ef . this finding suggests a modest role for stimulating environmental aspects to improve ef , though more specific environmental factors , such as family functioning , parenting style , and the presence of resources and opportunities , might even have a greater contribution ( aylward 1992 ) . however , these factors were not targeted in the present study , and our sample size limited the inclusion of more than 5 predictors in the analyses . creating a stimulating environment yet early in development should focus on parent instruction to enhance parent - child interaction ( als et al . other environmental focused intervention techniques that have been shown to be successful in children with executive dysfunction include computer guided behavioral training ( dowsett and livesey 2000 ; klingberg et al . , the degree of neonatal illness was not associated with poor performance on the ef tasks , although luciana et al . ( 1999 ) previously demonstrated that a high level of neonatal illness was associated with poor working memory . our findings might be related to the fact that in our study the incidence of neonatal medical events such as infections or ivh was fairly low . paralleling previous findings ( saavalainen et al . b ) , we did find that gestational age was related to ef , in particular to concept generation . it might not be neonatal illness associated with preterm birth in particular that results in deficits in ef , but rather the preterm birth itself that constitutes the risk for ef deficits strengths of the study concern the sample , which comprises consecutive admissions , comparison to an age - matched control group , assessment at early school age , and statistical control for both iq and speed of processing in the analyses . a limitation is that reliability and validity of our battery of neurocognitive measures have not been fully assessed for all measures . however , the use of experimental measures tapping into a comprehensive range of ef abilities with differing levels of complexity helps to chart the nature of the neurocognitive difficulties in very preterm children under various levels of executive demand . some of our tasks have been specifically developed to capture neurocognitive processes underlying task performance ( e.g. , espy et al . in addition , verbal fluency and go / nogo tasks , as employed in the present study , have been found fruitful in elucidating functioning of the corpus callosum , cerebellum , cingulate gyrus , and prefrontal cortex in very preterm children and adolescents ( lawrence et al . 2009 ; narberhaus et al . future studies , using techniques such as functional imaging ( fmri ) or diffusion tensor imaging ( dti ) , should be conducted to cast more light on how ef deficits in these children are related to white and grey matter pathology . in conclusion , our findings add to the relatively small but rapidly growing literature on early school - aged very preterm children , and demonstrate poor performance on ef measures related to very preterm birth , which could not be explained by iq . furthermore , it shows that speed of processing is marginally related to ef in very preterm children . the results show that very preterm children are at high risk for ef impairments , beside the risk for adverse outcome at later ages already constituted by lower iq scores and slow speed of processing ( mcdermott et al . an unresolved issue is whether ef deficits in very preterm children reflect a maturational lag or a permanent impairment . this question calls for a longitudinal approach . nevertheless , the ef deficits observed may have important implications for their later academic and behavioral functioning ( bull and scerif 2001 ; martinussen and tannock 2006 ; pennington and ozonoff 1996 ) . many follow - up studies document the outcomes of very preterm children in terms of neurosensory handicaps and iq scores . however , of significant concern is the trend of worsening outcome in the non - disabled very preterm survivors ( aylward 2005 ) . an important role in this issue may be played by subtle deficits in cognitive processes such as ef which hamper the ability to function in an increasingly complex and demanding environment ( salt and redshaw 2006 ) . our findings underline the need in neonatal follow - up care to extend the regular use of iq assessments with the assessments of efs and processing speed .

we examined whether very preterm ( 30 weeks gestation ) children at early school age have impairments in executive function ( ef ) independent of iq and processing speed , and whether demographic and neonatal risk factors were associated with ef impairments . a consecutive sample of 50 children ( 27 boys and 23 girls ) born very preterm ( mean age = 5.9 years , sd = 0.4 , mean gestational age = 28.0 weeks , sd = 1.4 ) was compared to a sample of 50 age - matched full - term controls ( 23 girls and 27 boys , mean age = 6.0 years , sd = 0.6 ) with respect to performance on a comprehensive ef battery , assessing the domains of inhibition , working memory , switching , verbal fluency , and concept generation . the very preterm group demonstrated poor performance compared to the controls on all ef domains , even after partialing out the effects of iq . processing speed was marginally related to ef . analyses with demographic and neonatal risk factors showed maternal education and gestational age to be related to ef . this study adds to the emerging body of literature showing that very preterm birth is associated with ef impairments .

Method Participants Measures Procedure Statistical Analyses Results Discussion

the study group consisted of 50 children born very preterm ( i.e. , gestational age 30 weeks , established by weeks and days after the mother s last menstrual period ) , and 50 controls . our sample did not differ from the total population of very preterm survivors in terms of gender , (1 , 115 ) = 1.15 , p = 0.30 ; gestational age , f(1 , 113 ) = 1.16 , p = 0.24 ; birthweight , f(1 , 113 ) = .96 , p = 0.33 ; days of ventilation , f(1 , 113 ) = 0.04 , p = 0.84 ; days of added oxygen , f(1 , 113 ) = 0.34 , p = 0.54 ; or days of intensive care , f(1 , 113 ) = 0.28 , p = 0.66 . the control group ( mean gestational age = 39.7 , sd = 1.3 ; mean birthweight = 3579 , sd = 510 ) was recruited from local elementary schools as a part of a normative study of the vu university amsterdam . table 1sample characteristics of the very preterm and the control groupvery pretermcontrolsample characteristicsage ( in years ) , mean ( sd)5.9 ( .4)6.0 ( .6)level of maternal education , mean ( sd)3.9 ( .9)4.2 ( .8)iq , mean ( sd , range)92.5 ( 17.5 , 70 - 140)109.0 ( 19.2 , 71 - 150)***boys , n ( % ) 27 ( 54.0)23 ( 46.0)twins or triplets , n ( % ) 11 ( 22.0)0 ( 0.0)***visual impairmentimpaired , use of glasses , n ( % ) 9 ( 18.0)0 ( 0.0)***blind or perceives light only , n ( % ) 0 ( 0.0)0 ( 0.0)hearing impairmentimpaired , use of hearing aid , n ( % ) 2 ( 4.0)0 ( 0.0)deafness , n ( % ) 0 ( 0.0)0 ( 0.0)cerebral palsyspastic ( unilateral ) , n ( % ) 3 ( 6.0)0 ( 0.0)ataxic , n ( % ) 0 ( 0.0)0 ( 0.0)dyskinetic , n ( % ) 0 ( 0.0)0 ( 0.0)level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004). table 2neonatal characteristics of the very preterm groupneonatal characteristicsbirthweight in grams , mean ( sd , range)1042.6 ( 31.8 , 605.01640.0)gestational age in weeks , mean ( sd , range)28.0 ( 1.4 , 25.030.0)duration of nicu stay in days , mean ( sd)78.7 ( 22.9)<750 g birthweight , n ( % ) 3.0 ( 6.0)<28 weeks gestational age , n ( % ) 23.0 ( 46.0)outborn , n ( % ) 4.0 ( 8.0)assisted ventilation , n ( % ) 5.0 ( 84.0)grade i / ii intra ventricular hemorrhage , n ( % ) 11.0 ( 22.0)grade iii / iv intra ventricular hemorrhage , n ( % ) 0.0 ( 0.0)periventricular leukomalacia , n ( % ) 2.0 ( 4.0)hypoglycemia , n ( % ) 0.0 ( 0.0)meningitis , n ( % ) 2.0 ( 4.0)necrotizing enterocolitis , n ( % ) 0.0 ( 0.0)chronic lung disease , n ( % ) 27.0 ( 54.0)rop ( grade i / ii / iii ) , n ( % ) 7.0/8.0/1.0 ( 14.0/16.0/2.0)small for gestational age , n ( % ) 3.0 ( 6.0)neurobiological risk score , mean ( sd)3.5 ( .9)outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . neonatal characteristics of the very preterm group outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . hierarchical , multiple regression analyses were conducted to test the impact of demographic and neonatal variables on the ef dependent variables of the very preterm group . the study group consisted of 50 children born very preterm ( i.e. , gestational age 30 weeks , established by weeks and days after the mother s last menstrual period ) , and 50 controls . our sample did not differ from the total population of very preterm survivors in terms of gender , (1 , 115 ) = 1.15 , p = 0.30 ; gestational age , f(1 , 113 ) = 1.16 , p = 0.24 ; birthweight , f(1 , 113 ) = .96 , p = 0.33 ; days of ventilation , f(1 , 113 ) = 0.04 , p = 0.84 ; days of added oxygen , f(1 , 113 ) = 0.34 , p = 0.54 ; or days of intensive care , f(1 , 113 ) = 0.28 , p = 0.66 . the control group ( mean gestational age = 39.7 , sd = 1.3 ; mean birthweight = 3579 , sd = 510 ) was recruited from local elementary schools as a part of a normative study of the vu university amsterdam . table 1sample characteristics of the very preterm and the control groupvery pretermcontrolsample characteristicsage ( in years ) , mean ( sd)5.9 ( .4)6.0 ( .6)level of maternal education , mean ( sd)3.9 ( .9)4.2 ( .8)iq , mean ( sd , range)92.5 ( 17.5 , 70 - 140)109.0 ( 19.2 , 71 - 150)***boys , n ( % ) 27 ( 54.0)23 ( 46.0)twins or triplets , n ( % ) 11 ( 22.0)0 ( 0.0)***visual impairmentimpaired , use of glasses , n ( % ) 9 ( 18.0)0 ( 0.0)***blind or perceives light only , n ( % ) 0 ( 0.0)0 ( 0.0)hearing impairmentimpaired , use of hearing aid , n ( % ) 2 ( 4.0)0 ( 0.0)deafness , n ( % ) 0 ( 0.0)0 ( 0.0)cerebral palsyspastic ( unilateral ) , n ( % ) 3 ( 6.0)0 ( 0.0)ataxic , n ( % ) 0 ( 0.0)0 ( 0.0)dyskinetic , n ( % ) 0 ( 0.0)0 ( 0.0)level of maternal education : 1 and 2 = primary education / secondary education not finished ; 3 = secondary education ; 4 = intermediate vocational education ; 5 = higher vocational education ; 6 and 7 = university ( central office of statistics 2004). table 2neonatal characteristics of the very preterm groupneonatal characteristicsbirthweight in grams , mean ( sd , range)1042.6 ( 31.8 , 605.01640.0)gestational age in weeks , mean ( sd , range)28.0 ( 1.4 , 25.030.0)duration of nicu stay in days , mean ( sd)78.7 ( 22.9)<750 g birthweight , n ( % ) 3.0 ( 6.0)<28 weeks gestational age , n ( % ) 23.0 ( 46.0)outborn , n ( % ) 4.0 ( 8.0)assisted ventilation , n ( % ) 5.0 ( 84.0)grade i / ii intra ventricular hemorrhage , n ( % ) 11.0 ( 22.0)grade iii / iv intra ventricular hemorrhage , n ( % ) 0.0 ( 0.0)periventricular leukomalacia , n ( % ) 2.0 ( 4.0)hypoglycemia , n ( % ) 0.0 ( 0.0)meningitis , n ( % ) 2.0 ( 4.0)necrotizing enterocolitis , n ( % ) 0.0 ( 0.0)chronic lung disease , n ( % ) 27.0 ( 54.0)rop ( grade i / ii / iii ) , n ( % ) 7.0/8.0/1.0 ( 14.0/16.0/2.0)small for gestational age , n ( % ) 3.0 ( 6.0)neurobiological risk score , mean ( sd)3.5 ( .9)outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . neonatal characteristics of the very preterm group outborn refers to infants born in community hospitals and referred to the perinatal center for neonatal intensive care . there were two conditions : ( 1 ) a control condition , in which the child had to say " day " in response to a sun card and " night " in response to a moon card , and ( 2 ) an experimental condition , where the child was asked to respond to the sun card by saying " night " and vice versa . we also examined group differences both with and without controlling for maternal education , and both with and without inclusion of the subset of very preterm children with neurosensory impairments . hierarchical , multiple regression analyses were conducted to test the impact of demographic and neonatal variables on the ef dependent variables of the very preterm group . , working memory , switching , verbal fluency and concept generation , we have employed one task per domain . the very preterm group performed significantly poorer than the controls on all ef measures , except for the total number of correct responses and efficiency on the shape school inhibition condition , or for total correct for the word span forward , for which group differences were nonsignificant . analyses with and without inclusion of the subset of very preterm children with neurosensory impairments , or with and without inclusion of the three very preterm children with pdd - nos revealed similar results.1table 3means and standard deviations of the dependent variables , and statistical values indicating group differences between the very preterm and the control groupafter controlling forafter controlling forvery pretermcontrolgroupprocessing speediqdependent variablesmsdmsdfpdfpdfpdss control time in ms90825475316713.54<0.010.74 - --9.06<0.010.61ss inhibition total correct21.300.6322.540.222.360.150.310.940.350.200.570.460.15ss inhibition efficiency0.020.010.020.010.730.410.172.870.110.342.380.150.31ss switching total correct18.840.7322.220.287.660.020.563.150.100.131.830.200.27ss switching efficiency0.01<0.010.02<0.014.290.040.420.040.840.040.000.970.01go / nogo total correct20.800.5722.600.397.980.020.574.62<0.050.193.240.090.36go / nogo efficiency0.030.010.040.0116.92<0.010.835.320.040.476.280.030.51dn exp total correct13.150.3714.400.237.400.020.551.040.330.210.860.370.19dn exp efficiency0.410.100.620.2044.88<0.0011.3518.85<0.010.8815.26<0.010.79vf total correct11.873.7114.905.2110.86<0.010.67 - --5.400.02.47ws total correct forwards5.300.235.840.173.770.070.39 - --0.970.340.20ws total correct backwards1.900.692.760.8715.61<0.010.80 - --6.900.020.53octc total points7.322.108.802.0114.69<0.010.77 - --4.780.040.44dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span.processing speed is measured by the mean rt on the shape school control condition.df = 1 , 98.df = 1 , 97 . means and standard deviations of the dependent variables , and statistical values indicating group differences between the very preterm and the control group dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span . table 4 depicts the rates of ef impairments in the very preterm group and control group . in comparison to the control group , very preterm children exhibited significant impairments in all measured efs , except for the shape school inhibition condition , or verbal fluency for which group differences in impairment rates were not significant , all (1 , n = 100 ) < 2.10 , p > 0.05 . table 4rates of executive function impairments in the very preterm and control groupvery pretermcontroldependent variablesn ( % ) n ( % ) ss control time in ms23 ( 46)7 ( 14)12.90***ss inhibition total correct14 ( 28)12 ( 24).21ss inhibition efficiency0 ( 0)2 ( 4)2.04ss switching total correct19 ( 38)8 ( 16)6.14**ss switching efficiency12 ( 24)3 ( 6)6.35*go / nogo total correct11 ( 22)4 ( 8)3.84*go / nogo efficiency18 ( 26)6 ( 12)7.90***dn exp total correct31 ( 62)21 ( 42)4.01*dn exp efficiency33 ( 66)10 ( 20)21.58***vf total correct12 ( 24)8 ( 16)1.00ws total correct forwards23 ( 46)19 ( 38).66ws total correct backwards18 ( 36)1 ( 2)18.78***octc total points18 ( 36)5 ( 10)9.54**definition of an impairment is given in the text.dn exp = day - night task experimental condition , octc = object classification task for children , ss control = shape school control condition , ss inhibition = shape school inhibition condition , ss switching = shape school switching condition , vf = verbal fluency , ws = word span. rates of executive function impairments in the very preterm and control group definition of an impairment is given in the text . the impact of demographic and neonatal risk factors on ef of the demographic factors gender and maternal education , which were entered in the first block , gender was not associated with any of the ef dependent variables . maternal education explained 12% of the variance ( r = 0.12 ; f(2 , 47 ) = 3.26 , p < 0.05 ) in efficiency on the shape school inhibition condition ( = 0.31 , p < 0.05 ) , and did not predict performance on any of the other ef dependent variables ( variance explained 4% , all ps > 0.25 ) . this study compared test performance of 50 very preterm children at early school age to that of 50 age - matched controls on a comprehensive ef battery . the findings demonstrated that very preterm children with average iq performed significantly poorer than the healthy term born children on ef tests of inhibition , switching , working memory , verbal fluency , and concept generation . in addition , very preterm children displayed significant higher rates of impairments in processing speed , inhibition , switching , working memory , and concept generation , than the controls . group differences remained significant after controlling for processing speed , which suggests that very preterm children exhibit a deficit in inhibitory control in addition to slower processing speed . group differences for switching , however , became nonsignificant after covarying for processing speed , which suggests that switching difficulties in very preterm children might be explained by slow processing speed . at early school age switching is still immature ( anderson et al . 1983 ) , which was supported by our findings showing a substantial overlap between iq and measures of concept generation ( octc ) , working memory , and ( verbal ) inhibition ( word span backwards , and day - night task ) . in the present study , we investigated the relationship between demographic and neonatal risk factors and ef . b ) , we did find that gestational age was related to ef , in particular to concept generation . it might not be neonatal illness associated with preterm birth in particular that results in deficits in ef , but rather the preterm birth itself that constitutes the risk for ef deficits strengths of the study concern the sample , which comprises consecutive admissions , comparison to an age - matched control group , assessment at early school age , and statistical control for both iq and speed of processing in the analyses . in addition , verbal fluency and go / nogo tasks , as employed in the present study , have been found fruitful in elucidating functioning of the corpus callosum , cerebellum , cingulate gyrus , and prefrontal cortex in very preterm children and adolescents ( lawrence et al . in conclusion , our findings add to the relatively small but rapidly growing literature on early school - aged very preterm children , and demonstrate poor performance on ef measures related to very preterm birth , which could not be explained by iq . furthermore , it shows that speed of processing is marginally related to ef in very preterm children . our findings underline the need in neonatal follow - up care to extend the regular use of iq assessments with the assessments of efs and processing speed .

injury is a serious public health issue in canada . in addition to being the leading cause of death in the first four decades of life , injuries are estimated to cost the canadian healthcare system $ 10.7 billion in direct healthcare costs and the canadian economy $ 9.1 billion in indirect costs resulting from hospitalisation , disability and premature death.1 every year , injuries result in approximately 13 677 deaths , 211 000 hospitalisations and 3 million emergency room visits.1 what sets injuries apart from the other leading causes of death in canada is that they are almost always preventable . the aim of this study was to explore the relationship between neighbourhood socioeconomic status ( nses ) and the rate of adult severe injury in greater vancouver using a variety of descriptive statistics and exploratory spatial data analysis methods . more specifically , this study sought to determine whether there was a statistically significant relationship between nses and the rate of all - cause , unintentional and intentional adult severe injury in greater vancouver . if so , the second objective of this study was to determine whether this relationship was spatially consistent or varied from one region to another . understanding the factors that influence a person 's risk for severe injury can help public health organisations develop effective injury prevention programmes and policies . likewise , knowing what population groups are at greatest risk of severe injury can inform where these programmes and other healthcare services ( eg , trauma centres ) should be located . investigating the degree of spatial heterogeneity in the nses - injury relationship can help to further target these resources by identifying places where the relationship is strongest and thus , where modifying the socioeconomic environment would potentially result in the largest reduction of severe injury.2 for the purposes of this study , greater vancouver was defined as the vancouver and abbotsford census metropolitan areas . this is a largely urban region located within south - western british columbia , canada and is home to approximately 2.4 million people.3 greater vancouver was chosen for this analysis because it is comprised of a socioeconomically diverse collection of communities . in fact , this region contains some of the most socioeconomically privileged as well as some of the most socioeconomically deprived neighbourhoods in canada . vancouver 's downtown eastside ( dtes ) , for example , is a neighbourhood notorious for its extreme levels of poverty , homelessness , mental illness and drug addiction.4 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . a severe injury was defined as an injury that results in death prior to hospital admission or one that is treated at one of british columbia 's eight trauma centres and given an iss greater than 12 . an adult was considered to be someone 20 years of age or older so that the injury data aligned with the denominator census population groupings used to calculate the rates . once identified , the severe injury cases were aggregated by the 2006 census dissemination areas ( das ) using their home residence postal codes and statistics canada 's postal code conversion file . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . nses was measured at the da level using area - based indices of material and social deprivation based on the conceptualisation of deprivation proposed by peter townsend.5 the social deprivation index is comprised of the following 2006 census variables : the proportion of individuals living alone ; the proportion of individuals who are separated , divorced or widowed ; and the proportion of single - parent families . the material deprivation index , on the other hand , includes : the proportion of people with no high school diploma ; the employment / population ratio ; and the average income of adults . we used the composite scores of these indices in our regression models and the quintile ranks in our descriptive analyses . the deprivation quintiles were population weighted so that each quintile contains approximately 20% of the study region 's population ( figure 1 ) . therefore , the index enables a comparison and identification of highly deprived areas relative to their corresponding region . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . each census dissemination area is symbolised based on their quintile rank , with light green and light blue depicting neighbourhoods in the least deprived quintile ( q1 ) and dark green and dark blue depicting neighbourhoods in the most deprived quintile ( q5 ) . in the map at the top of the figure , only the neighbourhoods that are in the least deprived quintile of both indices ( q1q1 ) and in the most deprived quintile of both indices ( q5q5 ) are highlighted . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . we than used the anselin local moran 's i statistic , a local measure of spatial autocorrelation , to identify hot spots . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . then , we added the two control variables to each model to control for the age and gender structures of the neighbourhoods . once the final model for each dependent variable was identified , we tested the residuals for spatial autocorrelation using the global moran 's i tool . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . unlike traditional regression methods , gwr does not assume that the observations are spatially independent.9 instead , gwr is based on the assumption that spatial autocorrelation does exist and enables the researcher to objectively measure and visualise how a relationship varies over space.10 a more detailed description of gwr can be found elsewhere.11 12 for the purposes of this study , greater vancouver was defined as the vancouver and abbotsford census metropolitan areas . this is a largely urban region located within south - western british columbia , canada and is home to approximately 2.4 million people.3 greater vancouver was chosen for this analysis because it is comprised of a socioeconomically diverse collection of communities . in fact , this region contains some of the most socioeconomically privileged as well as some of the most socioeconomically deprived neighbourhoods in canada . vancouver 's downtown eastside ( dtes ) , for example , is a neighbourhood notorious for its extreme levels of poverty , homelessness , mental illness and drug addiction.4 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . a severe injury was defined as an injury that results in death prior to hospital admission or one that is treated at one of british columbia 's eight trauma centres and given an iss greater than 12 . an adult was considered to be someone 20 years of age or older so that the injury data aligned with the denominator census population groupings used to calculate the rates . once identified , the severe injury cases were aggregated by the 2006 census dissemination areas ( das ) using their home residence postal codes and statistics canada 's postal code conversion file . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . nses was measured at the da level using area - based indices of material and social deprivation based on the conceptualisation of deprivation proposed by peter townsend.5 the social deprivation index is comprised of the following 2006 census variables : the proportion of individuals living alone ; the proportion of individuals who are separated , divorced or widowed ; and the proportion of single - parent families . the material deprivation index , on the other hand , includes : the proportion of people with no high school diploma ; the employment / population ratio ; and the average income of adults . we used the composite scores of these indices in our regression models and the quintile ranks in our descriptive analyses . the deprivation quintiles were population weighted so that each quintile contains approximately 20% of the study region 's population ( figure 1 ) . therefore , the index enables a comparison and identification of highly deprived areas relative to their corresponding region . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . each census dissemination area is symbolised based on their quintile rank , with light green and light blue depicting neighbourhoods in the least deprived quintile ( q1 ) and dark green and dark blue depicting neighbourhoods in the most deprived quintile ( q5 ) . in the map at the top of the figure , only the neighbourhoods that are in the least deprived quintile of both indices ( q1q1 ) and in the most deprived quintile of both indices ( q5q5 ) are highlighted . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . we than used the anselin local moran 's i statistic , a local measure of spatial autocorrelation , to identify hot spots . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . then , we added the two control variables to each model to control for the age and gender structures of the neighbourhoods . once the final model for each dependent variable was identified , we tested the residuals for spatial autocorrelation using the global moran 's i tool . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . unlike traditional regression methods , gwr does not assume that the observations are spatially independent.9 instead , gwr is based on the assumption that spatial autocorrelation does exist and enables the researcher to objectively measure and visualise how a relationship varies over space.10 a more detailed description of gwr can be found elsewhere.11 12 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . a severe injury was defined as an injury that results in death prior to hospital admission or one that is treated at one of british columbia 's eight trauma centres and given an iss greater than 12 . an adult was considered to be someone 20 years of age or older so that the injury data aligned with the denominator census population groupings used to calculate the rates . once identified , the severe injury cases were aggregated by the 2006 census dissemination areas ( das ) using their home residence postal codes and statistics canada 's postal code conversion file . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . nses was measured at the da level using area - based indices of material and social deprivation based on the conceptualisation of deprivation proposed by peter townsend.5 the social deprivation index is comprised of the following 2006 census variables : the proportion of individuals living alone ; the proportion of individuals who are separated , divorced or widowed ; and the proportion of single - parent families . the material deprivation index , on the other hand , includes : the proportion of people with no high school diploma ; the employment / population ratio ; and the average income of adults . we used the composite scores of these indices in our regression models and the quintile ranks in our descriptive analyses . the deprivation quintiles were population weighted so that each quintile contains approximately 20% of the study region 's population ( figure 1 ) . therefore , the index enables a comparison and identification of highly deprived areas relative to their corresponding region . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . each census dissemination area is symbolised based on their quintile rank , with light green and light blue depicting neighbourhoods in the least deprived quintile ( q1 ) and dark green and dark blue depicting neighbourhoods in the most deprived quintile ( q5 ) . in the map at the top of the figure , only the neighbourhoods that are in the least deprived quintile of both indices ( q1q1 ) and in the most deprived quintile of both indices ( q5q5 ) are highlighted . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . we than used the anselin local moran 's i statistic , a local measure of spatial autocorrelation , to identify hot spots . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . then , we added the two control variables to each model to control for the age and gender structures of the neighbourhoods . once the final model for each dependent variable was identified , we tested the residuals for spatial autocorrelation using the global moran 's i tool . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . unlike traditional regression methods , gwr does not assume that the observations are spatially independent.9 instead , gwr is based on the assumption that spatial autocorrelation does exist and enables the researcher to objectively measure and visualise how a relationship varies over space.10 a more detailed description of gwr can be found elsewhere.11 12 of these , 4742 ( 72% ) were unintentional , 1704 ( 26% ) were intentional and 190 ( 3% ) had an unknown intentionality . over our 5 year study period , the annual adult incidence rate of all - cause severe injury was 76 per 100 000 person - years . when broken down by intentionality , the rate of unintentional injuries ( 54 per 100 000 person - years ) was more than double the rate of intentional severe injuries ( 19 per 100 000 person - years ) . the median age was 46 years and the highest age - specific incidence rate ( 228 per 100 000 person - years ) was observed in the very elderly ( ie , 85 years of age and older ) population , which is consistent with the results of other injury surveillance research in canada.7 13 there was also a significant gender gap in the rate of severe injury , with men having an all - cause severe injury incidence rate ( 115 per 100 000 person - years ) almost triple that of women ( 40 per 100 000 person - years ) . interestingly , a gender gap in the incidence rate of all - cause severe injury was observed in every age category , not just the young . the crude all - cause severe injury incidence rates for each social and material deprivation quintile are shown in figure 2 . as shown , the rate of severe injury in the most socially deprived neighbourhoods ( 101 per 100 000 person - years ) was much higher than in the least socially deprived neighbourhoods ( 60 per 100 000 person - years ) . similarly , the neighbourhoods with the greatest material deprivation had a much higher rate of severe injury ( 96 per 100 000 person - years ) than those with the least amount of material deprivation ( 65 per 100 000 person - years ) . when the nses quintiles were considered together ( ie , q1q1 vs q5q5 ) the gap was even more pronounced , with the rate of severe injury almost four times higher in the most deprived neighbourhoods ( 205 per 100 000 person - years ) compared with least deprived neighbourhoods ( 56 per 100 000 person - years ) . crude annual incident rates of all - cause severe injury by neighbourhood socio - economic status ( nses ) quintiles . the crude annual incidence rate of all - cause severe injury is given for each social and material deprivation quintile . neighbourhoods in the least deprived social and material deprivation quintiles ( q1-q1 ) are also compared with the neighbourhoods in the most deprived social and material deprivation quintiles ( q5-q5 ) . the dark bars correspond with the least deprived quintiles ( q1 ) and with the most deprived quintiles ( q5 ) . figure 2 also indicates that the rate of severe injury increases with each incremental step down the nses ladder suggesting that nses affects a person 's risk of severe injury regardless of where they are located along the socioeconomic continuum . however , the largest jump in the crude rate of severe injury was observed between the fourth and fifth quintiles ( ie , the second most deprived neighbourhoods and the most deprived neighbourhoods ) . this suggests that nses may play a larger role in influencing the rate of severe injury in the most deprived neighbourhoods than at other levels of nses . when comparing the slopes of the graphs shown in figure 2 , it is also evident that social deprivation may have a slightly stronger relationship with the rate of severe injury than material deprivation . the pearson correlations between the rates of severe injury and the explanatory variables used in our regression analyses are shown in table 1 . these were calculated using spss software v.19.0 ( spss , chicago , illinois , usa ) . the only insignificant association was between the rate of intentional severe injury and the proportion of the population aged 85 years or older ( % 85 + ) . thus , this variable was not included in any of the intentional severe injury regression models . pearson correlations between the dependent and explanatory variables used in the regression analyses the strongest association for each dependent variable is shown in bold text . * significant correlation at the 0.01 level . mat , material deprivation ; soc , social deprivation . all of the explanatory and outcome variables exhibited statistically significant positive spatial autocorrelation at the global and local levels . this was not surprising given the spatial patterns that were evident after initially mapping the data sets . these tests of spatial autocorrelation confirm that our observations are not independent and thus , the use of ols to measure the relationship between our dependent and independent variables may be inappropriate.14 as shown in figure 3 , there was a fair amount of overlap between the local clusters of unintentional and intentional severe injury rates in greater vancouver . clusters of high ( hh ) and low ( ll ) rates , unintentional and intentional severe injury . these maps show spatial clusters of high ( hh ) and low ( ll ) rates of unintentional and intentional severe injury in greater vancouver . although each of our initial ols models were statistically significant , they all had fairly low adjusted r values meaning they only explained a small proportion of the variation in the rates of severe injury . however , closer examination of the models shows that each of our initial models was greatly underpredicting the rates of severe injury in and around vancouver 's dtes . therefore , we created a dummy variable ( dtes ) to account for this regional variation and added it to the final model for each dependent variable . the addition of this dummy variable significantly improved the adjusted r and alkaline information criterion ( aicc ) values in each of the models , suggesting the relationships between nses and the rates of severe injury are indeed location dependent . the ols models with the highest adjusted r for each dependent variable included material and social deprivation as explanatory variables . the coefficients of the explanatory variables in all the models shown in table 2 were statistically significant and the variance inflation factor for each explanatory variable was less than 1.5 , meaning there were no issues of multicollinearity . however , as shown in table 2 , the residuals for every ols model exhibited statistically significant spatial autocorrelation . this indicates that our ols results may be unreliable because the assumption of residual independence has been violated . ols regression models dtes , downtown eastside ; mat , material deprivation ; ols , ordinary least squares ; soc , social deprivation . unlike with our ols models , the inclusion of the age and gender variables ( % male and % 85 + ) in our gwr models resulted in significant local multicollinearity . also , because gwr allows the model parameters to vary over space , the inclusion of the dtes dummy variable was unnecessary . the results of our gwr analysis are shown in table 3 . because the parameter estimates are allowed to vary over space , the adjusted r values produced from gwr are often much higher than stationary regression methods and thus , are best interpreted as relative rather than absolute measures of model performance . interestingly , the gwr models that performed the best for each dependent variable had social deprivation as the only explanatory variable . also , the gwr models with the rate of unintentional severe injury as the dependent variable performed slightly better than the models with the rate of intentional severe injury as the dependent variable . this was consistent with the results of our ols analysis , but conflicted with our pearson correlation results , both of which measured the strength of these associations at the global level ( figure 4 ) . this map shows where the relationship between the incidence rate of all - cause severe injury and social deprivation is the strongest and the weakest . the map also identifies the neighbourhoods where there were clusters of high all - cause severe injury rates and social deprivation coefficient values . in other words , it depicts specific regions within our study area where policies and programmes aimed at reducing the rates of all - cause severe injury by augmenting the social environment would have the greatest benefit . as shown by their higher adjusted r values and the lower aicc values , the gwr models provided a better fit with the observed data than the ols models ( tables 2 and 3 ) . the residuals of the gwr models also exhibited very little or no spatial autocorrelation , meaning their parameter estimates are more reliable than their ols counterparts ( table 3 ) . furthermore , the condition numbers for the gwr models including social and material deprivation were all far less than 30 , meaning there were no issues with local multicollinearity.15 unlike with our ols models , the inclusion of the age and gender variables ( % male and % 85 + ) in our gwr models resulted in significant local multicollinearity . also , because gwr allows the model parameters to vary over space , the inclusion of the dtes dummy variable was unnecessary . the results of our gwr analysis are shown in table 3 . because the parameter estimates are allowed to vary over space , the adjusted r values produced from gwr are often much higher than stationary regression methods and thus , are best interpreted as relative rather than absolute measures of model performance . interestingly , the gwr models that performed the best for each dependent variable had social deprivation as the only explanatory variable . also , the gwr models with the rate of unintentional severe injury as the dependent variable performed slightly better than the models with the rate of intentional severe injury as the dependent variable . this was consistent with the results of our ols analysis , but conflicted with our pearson correlation results , both of which measured the strength of these associations at the global level ( figure 4 ) . this map shows where the relationship between the incidence rate of all - cause severe injury and social deprivation is the strongest and the weakest . the map also identifies the neighbourhoods where there were clusters of high all - cause severe injury rates and social deprivation coefficient values . in other words , it depicts specific regions within our study area where policies and programmes aimed at reducing the rates of all - cause severe injury by augmenting the social environment would have the greatest benefit . as shown by their higher adjusted r values and the lower aicc values , the gwr models provided a better fit with the observed data than the ols models ( tables 2 and 3 ) . the residuals of the gwr models also exhibited very little or no spatial autocorrelation , meaning their parameter estimates are more reliable than their ols counterparts ( table 3 ) . furthermore , the condition numbers for the gwr models including social and material deprivation were all far less than 30 , meaning there were no issues with local multicollinearity.15 our study identified a statistically significant inverse relationship between two measures of nses and the rates of all - cause , unintentional and intentional severe injury in greater vancouver . in other words , neighbourhoods ( ie , das ) with high social and material deprivation were associated with higher rates of severe injury ( figure 4 ) . this supports the findings of a literature review on injury and socioeconomic status conducted by cubbin et al,16 which found that injuries resulting in a death or hospitalisation had an inverse relationship with individual and area level measures of socioeconomic status . interestingly , our results were mixed as to whether nses had a stronger relationship with the rates of intentional or unintentional severe injuries . however , our analysis did find that social deprivation explained slightly more of the variation in the rates of severe injury than material deprivation . this suggests that the sociocultural milieu of a neighbourhood plays at least an equally important role as material deprivation in determining the risk of severe injury in greater vancouver . although much of the literature investigating the relationship between injury and socioeconomic status has used material measures of socioeconomic status , the few studies that have used measures related to the social components of socioeconomic status , have also detected an inverse association.17 18 in a recent study investigating the relationship between material deprivation and unintentional injury deaths across canada , the authors found that area level socioeconomic status only played a significant role in determining the risk of injury death in the most deprived neighbourhoods.19 similarly , the results of our analysis suggest that the relationship between nses and severe injury is the strongest in the most socioeconomically deprived regions of greater vancouver . in fact , given the dramatic improvement in our ols models after the addition of the dummy variable for vancouver 's dtes , it is plausible that these strong local relationships are the driving force behind the identified relationships at the global level . this highlights the importance of explicitly testing whether or not an identified relationship is stationary , because if it 's not , important local variations and local drivers could be overlooked.12 this study has several limitations . first , we had to omit 800 cases of severe injury from our analysis because they lacked sufficient home address information . however , because we used provincial data sets , not all of these cases would have resided within greater vancouver . also , many of these cases were homeless individuals and thus , would likely also be missing from the denominator census population figures used to calculate the rates of severe injury . nonetheless , the exclusion of these cases from our analysis may have influenced our results . second , although we attempted to control for potential confounders such as the age and gender structure of the neighbourhoods , the results of our regression analyses may still suffer from omitted variable or residual confounding bias and thus , should be interpreted with caution . similarly , because we did not control for individual level socioeconomic status , we were unable to infer whether the observed relationship was due to mechanisms working at the individual or neighbourhood levels . because of this , and due to the cross - sectional nature of the data used and the lack of temporal order between the explanatory and dependent variables , we were unable to infer causality . also , it is important to note that our results may have been different if we had used another rate smoothing technique or unit of analysis . likewise , results may have varied if we had used other measures of socioeconomic status , if we had studied a different population or age group , if we had studied injuries with different outcome , or if we had studied a different type of severe injury.16 in addition , our analysis was unable to examine specific causes of injuries , such as motor vehicle collisions or falls , which alone may have had a different relationship with neighbourhood socioeconomic status than when combined . more detailed research will be needed to determine how neighbourhood social deprivation may influence the rate of severe injury and whether it varies by the cause of injury . for example , are persons living alone more likely to suffer from injuries caused by falls because they have nobody around to ask for help when partaking in risky household activities ? or are individuals who are separated , divorced or widowed more likely to incur injuries resulting from self - harm . this study found statistically significant global as well as local relationships between the rates of all - cause , unintentional and intentional severe injury and two measures of nses in greater vancouver . this suggests that a combination of region - wide and neighbourhood - specific injury prevention programmes and policies may be warranted . however , the strength of the local relationships varied from place to place , with the strongest associations located in the most socioeconomically deprived neighbourhoods of our study region , such as vancouver 's dtes . this variation in the nses - injury relationship may mean that injury prevention efforts would be more successful in some regions than in others . many studies show that severe injuries resulting in death and hospitalisation typically occur at a higher rate in economically deprived populations . our analysis did find that social deprivation explained slightly more of the variation in the rates of severe injury than material deprivation . this suggests that the social and cultural structure of a neighbourhood plays a role at least as important as material deprivation in determining the risk of severe injury in greater vancouver .

backgroundevery year , injuries cost the canadian healthcare system billions of dollars and result in thousands of emergency room visits , hospitalisations and deaths . the purpose of this study was to explore the relationship between neighbourhood socioeconomic status ( nses ) and the rates of all - cause , unintentional and intentional severe injury in greater vancouver adults . a second objective was to determine whether the identified associations were spatially consistent or non-stationary.methodssevere injury cases occurring between 2001 and 2006 were identified using the british columbia 's coroner 's service records and the british columbia trauma registry , and mapped by census dissemination areas using a geographical information system . descriptive statistics and exploratory spatial data analysis methods were used to gain a better understanding of the data sets and to explore the relationship between the rates of severe injury and two measures of nses ( social and material deprivation ) . ordinary least squares and geographically weighted regression were used to model these relationships at the global and local levels.resultsinverse relationships were identified between both measures of nses and the rates of severe injury with the strongest associations located in greater vancouver 's most socioeconomically deprived neighbourhoods . social deprivation was found to have a slightly stronger relationship with the rates of severe injury than material deprivation.conclusionsresults of this study suggest that policies and programmes aimed at reducing the burden of severe injury in greater vancouver should take into account social and material deprivation , and should target the most socioeconomically deprived neighbourhoods in greater vancouver .

Background Methods Study region Data Rate of severe injury Neighbourhood socioeconomic status Covariates Descriptive statistics and exploratory spatial data analysis Modelling the NSES-injury relationship Results GWR results Discussion Conclusion

in addition to being the leading cause of death in the first four decades of life , injuries are estimated to cost the canadian healthcare system $ 10.7 billion in direct healthcare costs and the canadian economy $ 9.1 billion in indirect costs resulting from hospitalisation , disability and premature death.1 every year , injuries result in approximately 13 677 deaths , 211 000 hospitalisations and 3 million emergency room visits.1 what sets injuries apart from the other leading causes of death in canada is that they are almost always preventable . the aim of this study was to explore the relationship between neighbourhood socioeconomic status ( nses ) and the rate of adult severe injury in greater vancouver using a variety of descriptive statistics and exploratory spatial data analysis methods . more specifically , this study sought to determine whether there was a statistically significant relationship between nses and the rate of all - cause , unintentional and intentional adult severe injury in greater vancouver . if so , the second objective of this study was to determine whether this relationship was spatially consistent or varied from one region to another . investigating the degree of spatial heterogeneity in the nses - injury relationship can help to further target these resources by identifying places where the relationship is strongest and thus , where modifying the socioeconomic environment would potentially result in the largest reduction of severe injury.2 for the purposes of this study , greater vancouver was defined as the vancouver and abbotsford census metropolitan areas . in fact , this region contains some of the most socioeconomically privileged as well as some of the most socioeconomically deprived neighbourhoods in canada . vancouver 's downtown eastside ( dtes ) , for example , is a neighbourhood notorious for its extreme levels of poverty , homelessness , mental illness and drug addiction.4 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . nses was measured at the da level using area - based indices of material and social deprivation based on the conceptualisation of deprivation proposed by peter townsend.5 the social deprivation index is comprised of the following 2006 census variables : the proportion of individuals living alone ; the proportion of individuals who are separated , divorced or widowed ; and the proportion of single - parent families . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . each census dissemination area is symbolised based on their quintile rank , with light green and light blue depicting neighbourhoods in the least deprived quintile ( q1 ) and dark green and dark blue depicting neighbourhoods in the most deprived quintile ( q5 ) . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . in fact , this region contains some of the most socioeconomically privileged as well as some of the most socioeconomically deprived neighbourhoods in canada . vancouver 's downtown eastside ( dtes ) , for example , is a neighbourhood notorious for its extreme levels of poverty , homelessness , mental illness and drug addiction.4 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . once identified , the severe injury cases were aggregated by the 2006 census dissemination areas ( das ) using their home residence postal codes and statistics canada 's postal code conversion file . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . in the map at the top of the figure , only the neighbourhoods that are in the least deprived quintile of both indices ( q1q1 ) and in the most deprived quintile of both indices ( q5q5 ) are highlighted . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . unlike traditional regression methods , gwr does not assume that the observations are spatially independent.9 instead , gwr is based on the assumption that spatial autocorrelation does exist and enables the researcher to objectively measure and visualise how a relationship varies over space.10 a more detailed description of gwr can be found elsewhere.11 12 british columbia 's coroner 's service records and the british columbia 's trauma registry were used to identify all adults that had sustained a severe injury between 1 april 2001 and 31 march 2006 . once identified , the severe injury cases were aggregated by the 2006 census dissemination areas ( das ) using their home residence postal codes and statistics canada 's postal code conversion file . next , the crude annual incidence rates of all - cause , unintentional and intentional severe injury per 100 000 person - years was calculated for all das within our study region . a detailed methodology for the creation of the index can be found here.6 maps of social deprivation , material deprivation , and social and material deprivation ( c ) in greater vancouver . these maps show the spatial distribution of social deprivation , material deprivation , and social and material deprivation across greater vancouver . in the map at the top of the figure , only the neighbourhoods that are in the least deprived quintile of both indices ( q1q1 ) and in the most deprived quintile of both indices ( q5q5 ) are highlighted . as shown , there is very little overlap between the spatial distribution of social and material deprivation in greater vancouver . to account for the influence of the age and gender structure of neighbourhoods on the rates of severe injury , we included two control variables in our regression analyses : the proportion of the population that was male ( % male ) and the population that was 85 years of age or older ( % 85 + ) . we chose to control for this age group because it had the highest age - specific severe injury incidence rate and because it had been identified previously as an age group with a significantly higher risk of severe injury in canada.7 we used the global moran 's i statistic , as implemented in arcgis v.10 , to determine the degree of global spatial autocorrelation ( ie , clustering ) present in each data set . a detailed explanation of how the moran 's i works can be found here.8 next , we calculated the pearson correlations between the rates of severe injury and our explanatory variables to determine the direction and magnitude of their associations . lastly , we calculated the crude annual incidence rates of severe injury per 100 000 person - years for each social and material deprivation quintile in greater vancouver . first , an ordinary least squares ( ols ) regression was used on each dependent variable ( eg , all - cause severe injury rate , intentional severe injury rate and unintentional severe injury rate ) to determine which combination of explanatory variables resulted in the final model . three models were built , one with social deprivation as the explanatory variable , one with material deprivation as the explanatory variable , and the last with social deprivation and material deprivation as the explanatory variables . next , we used geographically weighted regression ( gwr ) to explicitly test whether the nses - injury relationship was spatially consistent or heterogeneous . the median age was 46 years and the highest age - specific incidence rate ( 228 per 100 000 person - years ) was observed in the very elderly ( ie , 85 years of age and older ) population , which is consistent with the results of other injury surveillance research in canada.7 13 there was also a significant gender gap in the rate of severe injury , with men having an all - cause severe injury incidence rate ( 115 per 100 000 person - years ) almost triple that of women ( 40 per 100 000 person - years ) . interestingly , a gender gap in the incidence rate of all - cause severe injury was observed in every age category , not just the young . the crude all - cause severe injury incidence rates for each social and material deprivation quintile are shown in figure 2 . as shown , the rate of severe injury in the most socially deprived neighbourhoods ( 101 per 100 000 person - years ) was much higher than in the least socially deprived neighbourhoods ( 60 per 100 000 person - years ) . similarly , the neighbourhoods with the greatest material deprivation had a much higher rate of severe injury ( 96 per 100 000 person - years ) than those with the least amount of material deprivation ( 65 per 100 000 person - years ) . when the nses quintiles were considered together ( ie , q1q1 vs q5q5 ) the gap was even more pronounced , with the rate of severe injury almost four times higher in the most deprived neighbourhoods ( 205 per 100 000 person - years ) compared with least deprived neighbourhoods ( 56 per 100 000 person - years ) . crude annual incident rates of all - cause severe injury by neighbourhood socio - economic status ( nses ) quintiles . the crude annual incidence rate of all - cause severe injury is given for each social and material deprivation quintile . neighbourhoods in the least deprived social and material deprivation quintiles ( q1-q1 ) are also compared with the neighbourhoods in the most deprived social and material deprivation quintiles ( q5-q5 ) . however , the largest jump in the crude rate of severe injury was observed between the fourth and fifth quintiles ( ie , the second most deprived neighbourhoods and the most deprived neighbourhoods ) . this suggests that nses may play a larger role in influencing the rate of severe injury in the most deprived neighbourhoods than at other levels of nses . when comparing the slopes of the graphs shown in figure 2 , it is also evident that social deprivation may have a slightly stronger relationship with the rate of severe injury than material deprivation . the pearson correlations between the rates of severe injury and the explanatory variables used in our regression analyses are shown in table 1 . the only insignificant association was between the rate of intentional severe injury and the proportion of the population aged 85 years or older ( % 85 + ) . all of the explanatory and outcome variables exhibited statistically significant positive spatial autocorrelation at the global and local levels . these tests of spatial autocorrelation confirm that our observations are not independent and thus , the use of ols to measure the relationship between our dependent and independent variables may be inappropriate.14 as shown in figure 3 , there was a fair amount of overlap between the local clusters of unintentional and intentional severe injury rates in greater vancouver . clusters of high ( hh ) and low ( ll ) rates , unintentional and intentional severe injury . these maps show spatial clusters of high ( hh ) and low ( ll ) rates of unintentional and intentional severe injury in greater vancouver . although each of our initial ols models were statistically significant , they all had fairly low adjusted r values meaning they only explained a small proportion of the variation in the rates of severe injury . however , closer examination of the models shows that each of our initial models was greatly underpredicting the rates of severe injury in and around vancouver 's dtes . the addition of this dummy variable significantly improved the adjusted r and alkaline information criterion ( aicc ) values in each of the models , suggesting the relationships between nses and the rates of severe injury are indeed location dependent . ols regression models dtes , downtown eastside ; mat , material deprivation ; ols , ordinary least squares ; soc , social deprivation . this map shows where the relationship between the incidence rate of all - cause severe injury and social deprivation is the strongest and the weakest . in other words , it depicts specific regions within our study area where policies and programmes aimed at reducing the rates of all - cause severe injury by augmenting the social environment would have the greatest benefit . furthermore , the condition numbers for the gwr models including social and material deprivation were all far less than 30 , meaning there were no issues with local multicollinearity.15 unlike with our ols models , the inclusion of the age and gender variables ( % male and % 85 + ) in our gwr models resulted in significant local multicollinearity . this map shows where the relationship between the incidence rate of all - cause severe injury and social deprivation is the strongest and the weakest . the map also identifies the neighbourhoods where there were clusters of high all - cause severe injury rates and social deprivation coefficient values . in other words , it depicts specific regions within our study area where policies and programmes aimed at reducing the rates of all - cause severe injury by augmenting the social environment would have the greatest benefit . furthermore , the condition numbers for the gwr models including social and material deprivation were all far less than 30 , meaning there were no issues with local multicollinearity.15 our study identified a statistically significant inverse relationship between two measures of nses and the rates of all - cause , unintentional and intentional severe injury in greater vancouver . in other words , neighbourhoods ( ie , das ) with high social and material deprivation were associated with higher rates of severe injury ( figure 4 ) . this supports the findings of a literature review on injury and socioeconomic status conducted by cubbin et al,16 which found that injuries resulting in a death or hospitalisation had an inverse relationship with individual and area level measures of socioeconomic status . interestingly , our results were mixed as to whether nses had a stronger relationship with the rates of intentional or unintentional severe injuries . however , our analysis did find that social deprivation explained slightly more of the variation in the rates of severe injury than material deprivation . this suggests that the sociocultural milieu of a neighbourhood plays at least an equally important role as material deprivation in determining the risk of severe injury in greater vancouver . although much of the literature investigating the relationship between injury and socioeconomic status has used material measures of socioeconomic status , the few studies that have used measures related to the social components of socioeconomic status , have also detected an inverse association.17 18 in a recent study investigating the relationship between material deprivation and unintentional injury deaths across canada , the authors found that area level socioeconomic status only played a significant role in determining the risk of injury death in the most deprived neighbourhoods.19 similarly , the results of our analysis suggest that the relationship between nses and severe injury is the strongest in the most socioeconomically deprived regions of greater vancouver . in fact , given the dramatic improvement in our ols models after the addition of the dummy variable for vancouver 's dtes , it is plausible that these strong local relationships are the driving force behind the identified relationships at the global level . also , many of these cases were homeless individuals and thus , would likely also be missing from the denominator census population figures used to calculate the rates of severe injury . because of this , and due to the cross - sectional nature of the data used and the lack of temporal order between the explanatory and dependent variables , we were unable to infer causality . likewise , results may have varied if we had used other measures of socioeconomic status , if we had studied a different population or age group , if we had studied injuries with different outcome , or if we had studied a different type of severe injury.16 in addition , our analysis was unable to examine specific causes of injuries , such as motor vehicle collisions or falls , which alone may have had a different relationship with neighbourhood socioeconomic status than when combined . more detailed research will be needed to determine how neighbourhood social deprivation may influence the rate of severe injury and whether it varies by the cause of injury . this study found statistically significant global as well as local relationships between the rates of all - cause , unintentional and intentional severe injury and two measures of nses in greater vancouver . however , the strength of the local relationships varied from place to place , with the strongest associations located in the most socioeconomically deprived neighbourhoods of our study region , such as vancouver 's dtes . our analysis did find that social deprivation explained slightly more of the variation in the rates of severe injury than material deprivation . this suggests that the social and cultural structure of a neighbourhood plays a role at least as important as material deprivation in determining the risk of severe injury in greater vancouver .

polychlorinated biphenyls ( pcbs ) are a class of industrial chemicals and unintentional byproducts of industrial processes banned under the stockholm convention on persistent organic pollutants . pcbs remain an environmental and human health concern because of their ongoing , inadvertent production , their environmental persistence , and the presence of pcbs in the environment , diet , and in human serum and tissues . nineteen pcb congeners and various hydroxylated ( and other ) metabolites with three or four ortho chlorine substituents and an unsymmetrical substitution pattern in both phenyl rings are chiral . these pcb derivatives exist as nonsuperimposable rotational isomers , called atropisomers , which are mirror images of each other . chiral pcb congeners , in particular congeners with a 2,3,6 substitution pattern in one phenyl ring , have been linked to neurodevelopmental toxicity in humans and laboratory animals and shown to cause effects on neurotransmitter functions in the central nervous system and alter cellular processes related to calcium signaling . chiral pcbs are present in commercial pcb mixtures as racemates ( i.e. , a 1:1 mixture of atropisomers ) and , due to pcbs chemical and thermal stability , are released into the environment as racemates . studies of the atropisomeric enrichment of chiral pcbs reveal near racemic signatures in diet , house dust , and air , but highly variable atropisomeric enrichment in wildlife , especially at higher tropic levels , and humans ( reviewed in ref ( 5 ) ) . because physical and chemical transport ( e.g. , passive diffusion ) and transformation processes ( e.g. , photodegradation ) are not atropselective , the variable atropisomeric enrichment of pcbs in environmental samples is due to atropselective biotransformation and/or biological transport processes . pcbs can undergo atropselective bacterial biodegradation and are atropselectively metabolized in plant and animal models ( reviewed in ref ( 5 ) ) . laboratory studies have shown that cytochrome p450 enzymes , such as different cyp2b isoforms , can atropselectively metabolize pcbs to oh - pcbs and , thus , contribute to nonracemic signatures of pcbs at higher trophic levels . oh - pcbs are found in many species , including humans , and represent an environmental and human health concern . in mammals , oh - pcbs can adversely affect neurodevelopment by altering processes related to calcium signaling or thyroid function . activity relationship studies show that the oh - pcb metabolites of chiral pcbs are ryanodine receptor- ( ryr-)active and display different modes of action depending on the position of the hydroxyls group on the biphenyl moiety in vitro . oh - pcb profiles are highly species dependent and can display interindividual variability . these differences in oh - pcb profiles are due to differences in the isoform composition , expression , and activities of pcb and oh - pcb metabolizing enzymes ( e.g. , p450 enzymes , sulfotransferases , glucuronosyl transferases , and others ) . furthermore , differences in the composition and oh - pcb binding affinity of various transport proteins ( e.g. , transthyretin ) may contribute to species and interindividual differences in oh - pcb profiles in vivo . since pcbs are atropselectively metabolized by p450 enzymes to oh - pcbs , it is likely that , similar to the parent pcbs , p450 enzyme - mediated metabolism contributes to differences in the atropisomeric enrichment of chiral oh - pcbs in wildlife and humans . consistent with this hypothesis , we have recently reported differences in the atropselective formation of oh - pcbs in rats and mice in in vitro metabolism studies ; however , systematic laboratory and environmental studies of the atropselective formation of oh - pcbs by p450 enzymes from different species have not been reported to date . the present study investigates the atropselective formation of oh - pcb from 2,2,3,3,6,6-hexachlorobiphenyl ( pcb 136 ) by liver microsomes from humans and several other mammalian species . pooled liver microsomes from nave animals were used to assess representative oh - pcb profiles and chiral signatures in the species investigated . pcb 136 was selected for this study as a prototypical chiral pcb congener of environmental relevance . untreated beagle dog ( female ) , cynomolgus monkey ( male ) , new zealand rabbit ( male ) , golden syrian hamster ( male ) , hartley albino guinea pig ( male ) , and human liver microsomes pooled from 50 donors with mixed age , sex , and race ( hlms ) were purchased from xenotech ( lenexa , ks , usa ) . microsomes obtained from female instead of male beagle dogs were used because of the higher enzymatic activity of the respective microsomal preparations . mouse liver microsomes were prepared by pooling livers from saline and corn oil treated male c57bi/6 mice and characterized as described previously . the microsomal cytochrome p450 content is described in the supporting information ( tables s1 and s2 ) . dimethyl sulfoxide ( dmso ) , sodium phosphate dibasic ( na2hpo4 ) , sodium phosphate monobasic ( nah2po4 ) , magnesium chloride ( mgcl2 ) , tetrabutylammonium sulfite , sodium sulfite , and pesticide grade solvents were obtained from fisher scientific ( pittsburgh , pa , usa ) . nicotinamide adenine dinucleotide phosphate reduced ( nadph ) was purchased from sigma - aldrich co. ( st . louis , mo , usa ) . racemic pcb 136 was synthesized by the ullmann coupling of 2,3,6-trichloro-1-iodobenzene , and the atropisomers of pcb 136 were separated using two serially connected nucleodex -pm columns ( macherey - nagel , dren , germany ) . the enantiomeric fractions ( ef = area(+)-pcb 136 /(area(+)-pcb 136 + area()-pcb 136 ) ) of ( )-pcb 136 and ( + ) -pcb 136 were 0.01 and 1.00 , respectively . 2,2,3,3,6,6-hexachlorobiphenyl-4-ol ( 4136 ) , 2,2,3,3,6,6-hexachlorobiphenyl-5-ol ( 5136 ) , 4,5-dimethoxy-2,2,3,3,6,6-hexachlorobiphenyl , and 2,2,3,4,6,6-hexachloro-3-methoxybiphenyl were prepared as described elsewhere . recovery standards ( 2,3,4,4,5,6-hexachlorobiphenyl , pcb 166 ; 2,3,4,5,6-pentachlorobiphenyl , pcb 117 ; 2,3,3,4,5,5-hexachlorobiphenyl-4-ol , 4-159 ) and the internal standard ( 2,2,3,4,4,5,6,6-octachlorobiphenyl , pcb 204 ) were purchased from accustandard ( new haven , ct ) . incubation conditions were initially optimized for microsomal protein content and napdh concentration using human and dog liver microsomes as described previously . subsequently , time - course experiments were performed in triplicate with the optimized experimental condition . briefly , an incubation mixture ( 12 ml ) consisting of phosphate buffer ( 0.1 m , ph 7.4 ) , nadph ( 0.5 mm in hlms or 1.5 mm in all animal microsomes ) , magnesium chloride ( 3 mm ) , and hepatic microsomal protein ( 1.0 mg / ml for all animal microsomes or 0.5 mg / ml for human microsomes ) was preincubated for 5 min at 37 1 c in a shaking water bath . pcb 136 in dmso ( 0.5% ) was added with a final concentration of 50 m . these incubation conditions , including the high pcb 136 concentrations , were selected to ensure the formation of sufficient oh - pcb quantities for atropselective analyses . experiments with hlms used ( )- , ( )- , or ( + ) -pcb 136 . an aliquot ( 2 ml ) of the incubation mixture was removed after 5 , 10 , 15 , 20 , 25 , and 30 min . a total of 2 ml of ice cold sodium hydroxide ( 0.5 m ) was added to each aliquot to stop the reaction . for the 0 min time point , a separate sample ( 1990 l ) was preincubated for 5 min as described above , followed by sequential addition of the sodium hydroxide ( 2 ml ) and pcb 136 solution ( 10 l ) . control incubations without microsomes or nadph or containing heat - inactivated microsomes were performed in parallel . extraction of pcb 136 and its hydroxylated metabolites was performed using a published method . in short , surrogate standards ( 500 ng of pcb 117 in animal microsomes or pcb 166 in human microsomes ; 274 ng of 4-159 ) were added to each sample , followed by hydrochloric acid ( 6 m , 1 ml ) and 2-propanol ( 3 ml ) . the samples were extracted with hexane - mtbe ( 1:1 v / v , 5 ml ) and hexane ( 3 ml ) . the combined organic extracts were washed with an aqueous kcl solution ( 1% , 3 ml ) . after removal of the organic phase , the kcl phase was re - extracted with hexane ( 3 ml ) , and the combined extracts were reduced under a gentle stream of nitrogen to 1 ml . the hydroxylated metabolites were derivatized with diazomethane and subjected to a sulfur cleanup as described previously . pcb 204 ( 200 ng ) was added as an internal standard prior to analysis . levels of oh - pcb 136 metabolites were determined using an agilent 6890n gas chromatograph with a ni-ecd detector and a db1-ms capillary column ( 60 m 0.25 mm i d 0.25 m film thickness ; agilent , santa clara , ca , usa ) . relative rates of oh - pcb formation were determined in the linear range of metabolite formation ( i.e. , 5 min ) by adjusting the amount of oh - pcb by the total p450 content . the limits of detection and background pcbs levels are listed in table s3 . to further verify the formation of specific metabolites , samples from 30 min incubations were analyzed on an agilent 7890a gas chromatograph with a 5975 c mass selective detector in both total and selective ion monitoring modes with an hp-5 ms column ( 30 m 0.32 mm i.d . atropselective analysis of the derivatized hydroxylated pcb 136 atropisomers was performed using an agilent 7890a gas chromatograph with a ni ecd detector . the atropisomers of 5136 and 4136 were separated on chirasil - dex ( cd column , 25 m 0.25 mm i d 0.25 m film thickness ; varian , palo alto , ca , us ) and cyclosil - b columns ( cb column , 30 m 0.25 mm i d 0.25 m film thickness ; agilent , santa clara , ca , us ) , respectively , following a published method . enantiomeric fractions ( efs ) were determined as ef = a2/(a1 + a2 ) , where a1 and a2 are the peak areas of the first and second eluting atropisomers , respectively . the species dependent formation of metabolites was studied using one way anova and proc in the statistical analysis package sas ( version 9.3 , sas institute , cary , nc , usa ) . metabolites formation and ef values were compared by tukey s studentized range ( hsd ) test . a paired t test was used to compare the ef values of 5136 and 4136 to racemic standards and the formation rates of 4136 and 5136 between each species . a p value < 0.05 was used to indicate a significant difference between species . racemic pcb 136 or its atropisomers were incubated with hlms to investigate if potentially neurotoxic oh - pcbs are formed atropselectively in humans . only a small percentage ( < 1% ) of the total pcb 136 was converted to oh - pcbs under the incubation conditions ( table s4 ) . 4136 and 5136 were the major metabolites for racemic pcb 136 and pure pcb 136 atropisomers , with more 4136 being formed ( 5136/4136 ratio = 0.39 after 5 min , table s5 ) . 4,5136 and the 1,2-shift product of pcb 136 ( 3150 ) were minor metabolites . one unknown metabolite peak ( m / z = 420.0 ) was observed at a later retention time , indicating the formation of a second dihydroxylated metabolite ( figure s1 ) . the amounts of 3150 , 4136 , 5136 , and 4,5136 increased with time in all hlm incubations and depended on the pcb atropisomer composition ( figure 1 ) . for all metabolites , the rate of formation followed the order ( + ) -pcb 136 > racemic pcb 136 > ( )-pcb 136 ( figure 1 and table s4 ) . the time - dependent formation of ( a ) 3150 , ( b ) 5136 , ( c ) 4136 , ( d ) 4 , 5136 , and ( e ) oh-136 followed the rank order ( + ) -pcb 136 > ( )-pcb 136 > ( )-pcb 136 in incubations with human liver microsomes . ( f ) the relative rates of oh - pcb formation showed a significantly slower metabolite formation in the incubations with ( )-pcb 136 compared to incubations with ( + ) -pcb 136 and ( )-pcb 136 . different letters indicate statistically significant differences in the oh - pcb formation rates ( p < 0.05 ) as determined by a tukey student range test using sas . * p = 0.05 for comparison of the 4,5136 formation rates between incubations with ( + ) - and ( )-pcb 136 . racemic pcb 136 was incubated with liver microsomes obtained from different species ( i.e. , male monkey , guinea pig , mouse , hamster , and rabbit ; female dog ) to explore differences in typical metabolite profiles and chiral signatures between humans and toxicologically relevant mammalian species . only a small percentage of pcb 136 ( < 3% ) was converted to oh - pcbs ( table s4 ) . similar to experiments with hlms , 4136 was the major metabolite formed in incubations with microsomes obtained from male monkeys and rabbits ( figure 2 ) , with 5136/4136 ratios of 0.17 and 0.64 at 5 min , respectively ( table s5 ) . in contrast , 5136 was the major metabolite in incubations using microsomes from dogs , guinea pigs , mice , and hamsters . after a 5 min incubation time , the 5136/4136 ratios were 14 , 10 , 2.2 , and 6.9 for incubations with dog , guinea pig , mouse , and hamster microsomes , respectively ( table s5 ) . 4,5136 was a minor metabolite observed in the incubations with microsomes prepared from dogs , guinea pigs , mice , hamsters , and rabbits . the formation of 3150 , but not 4,5136 , was observed in microsomes obtained from monkeys . the formation of these oh - pcb metabolites was confirmed by gc - ms ( figure s1 ) . time- and species - dependent formation of oh - pcbs in incubations of pcb 136 with liver microsomes from ( a ) humans ( pooled ) , ( b ) dog , ( c ) monkey , ( d ) guinea pig , ( e ) mouse , ( f ) hamster , and ( g ) rabbit . 4136 was the major metabolite in incubations using human , monkey and rabbit liver microsomes . 5136 was the major metabolite in experiments with dog , guinea pig , mouse , and hamster liver microsomes . the 1,2-shift metabolite , 3150 , was only observed in incubations using human and monkey liver microsomes . the relative rates of formation of 5136 and 4136 were determined for the 5 min incubation time by expressing oh - pcb levels per nanomole of total p450 content . this adjustment accounts for the differences in total p450 content between different microsomal preparation and allows a comparison across species ( figure 3 ) . the rates of formation of 5136 followed the order rat ( estimated based on published data , see ref ( 30 ) ) > dog guinea pig hamster > human > monkey mouse rabbit . however , the rate of 5136 formation by hlms was only significantly different compared to incubations using microsomes obtained from dogs and guinea pigs . the rate of 5136 formation by hlms was similar compared to the rate observed in incubations with microsomes from hamster , monkey , mouse , and rabbit . the rate of 4136 formation by hlms was significantly faster compared to experiments using microsomes from other species and followed the order human > monkey > rabbit rat > dog guinea pig mouse hamster ( figure 3 ) . the formation rate of 5136 was significantly different from that of 4136 in all species after paired t test within species . comparison of the formation rates of ( a ) 5136 and ( b ) 4136 in incubations with liver microsomes obtained from different species . different letters indicate statistically significant differences in the oh - pcb formation rates ( p < 0.05 ) as determined by a tukey student range test using sas . the formation rate of 4136 was significantly different from that of 5136 within each species ( p < 0.05 ; paired t test ) . the atropisomeric enrichment of oh - pcb 136 metabolites was determined in microsomal incubations with racemic pcb 136 using atropselective gas chromatography . the objective was to determine if oh - pcbs are formed atropselectively in incubations with hlm and how this enrichment differs compared to toxicologically relevant species . the second eluting atropisomers of 5136 ( e2 - 5136 ) , which is formed from ( + ) -pcb 136 , was enriched in incubations using human , dog , monkey , guinea pig , and rabbit microsomes ( figure 4a ) . the atropselective formation of the 5136 resulted in near constant ef values with time ( data not shown ) . therefore , ef values at 5 min were statistically analyzed and presented in figure 4c . the atropisomeric enrichment of 5136 and 4136 formed from liver microsomes is species - dependent . ( a ) representive chromatograms showing an enrichment of the second eluting 5136 atropisomer in incubations with human ( pooled ) , dog , monkey , guinea pig , and rabbit liver microsomes and an enrichment of the first eluting 5136 atropisomer in experiments with mouse and hamster liver microsomes . ( b ) representive chromatograms showing an enrichment of the second eluting 4136 atropisomer in incubations with human ( pooled ) , dog , monkey , guinea pig , hasmster , and rabbit liver microsomes and an enrichment of the first eluting 4136 atropisomer in experiments with mouse liver microsomes . different letters indicate statistically significant differences in the ef values ( p < 0.05 ) as determined by a tukey student range test using sas . * ef values significantly different from control ( p < 0.05 , paired t test ) . ef values of 4136 in incubations with dog microsomes showed a trend to significance from control ( p = 0.054 ) . the extent of the atropisomeric enrichment of 5136 in microsomal incubations followed the order dog guinea pig > monkey human rabbit ( figure 4c ) . interestingly , the first eluting atropisomer of 5136 ( e1 - 5136 ) , the 5136 metabolite formed from ( -)-pcb 136 , displayed atropisomeric enrichment in experiments with mouse and , to a lesser extent , hamster microsomes . the ef values of 5136 were significantly different from the racemic standard , with the exception of incubations using hamster microsomes . similar to 5136 , the second eluting atropisomers of 4136 ( e2 - 4136 ) , a metabolite formed from ( + ) -pcb 136 , was enriched in incubations using human , dog , monkey , guinea pig , hamster , and rabbit microsomes ( figure 4b ) . the extent of the atropisomeric enrichment of 4136 formed in microsomal incubations followed the order human monkey > guinea pig hamster rabbit > dog . in contrast , the first eluting atropisomer of 4136 ( e1 - 4136 ) , which is formed from ( )-pcb 136 , was enriched in experiments with mouse microsomes . the ef values of 4136 were significantly different from racemic standards , with incubation using dog microsomes displaying only a trend of e2 - 4136 enrichment ( p = 0.054 ; figure 4d ) . the present study uses hepatic microsomes to gain insights into typical oh - pcb 136 metabolite profiles and chiral signatures formed by p450 enzymes in different mammalian species , including humans . 4136 and 5136 were the two major monohydroxylated pcb 136 metabolites formed atropselectively by hlms , which is consistent with an earlier study by schnellmann and co - workers . in addition , a few other mono- and dihydroxylated pcb metabolites were observed as minor metabolites . this includes 3150 , a 1,2-shift product of pcb 136 formed via an arene oxide intermediate . the formation of such a 1,2-shift metabolite by hlms has not been reported previously . the 5136/4136 ratios in our study ranged from 0.4 to 0.8:1 ( for incubation times from 5 to 30 min ) . on the basis of our re - evaluation of the published mass spectra , this difference in the metabolite profile is most likely due to differences in the p450 enzyme composition of the respective hlms . the relative rate of formation of all oh - pcbs was different for incubations using ( + ) -pcb 136 , ( )-pcb 136 , and racemic pcb 136 , with ( + ) -pcb 136 being more rapidly oxidized compared to ( )-pcb 136 . these atropisomer - specific differences in the oh - pcb formation rates explain the atropisomeric enrichment of pcbs observed in in vitro studies and are consistent with a role of p450 enzymes in their atropisomeric enrichment observed human samples . analogous to hlms , 5136 , 4136 , and 4,5136 were formed by most animal microsomal preparations studied . there is considerable evidence that cyp2b enzymes are involved in the oxidation of pcb 136 and structurally related pcb congeners in the meta position . studies with recombinant enzymes demonstrate that rat cyp2b1 and dog cyp2b11 selectively oxidize pcb 136 to 5136 . cyp2b1 also metabolizes 4-oh - pcbs and 5-oh - pcbs to the corresponding 4,5-dihydroxylated metabolites , such as 4,5136 . warner and co - workers demonstrated that pcb 136 is oxidized by human cyp2b6 to a single , unidentified oh - pcb . this oh - pcb metabolite is most likely 5136 because cyp2b6 oxidizes other pcbs in the meta position . the p450 isoforms responsible for the formation of 4136 remain elusive , as cyp3a enzymes are probably not involved in its formation in rats or mice . we also observed no change in 4136 levels in liver microsomes after induction of cyp1a enzymes in rats pretreated with pcb 126 , which suggests that 4136 is not formed by cyp1a enzymes ( wu and lehmler , unpublished data ) . while essentially the same metabolites were formed by liver microsomes from different species , the ratios , relative formation rates , and chiral signatures of the oh - pcbs differed considerably depending on the species . experiments with hlms displayed the fastest formation rate for 4136 and one of the lowest formation rates for 5136 of all microsomal preparations investigated . it is important to emphasize that our result represents an average oh - pcb profile formed by a pool of liver microsomes from 50 individual donors ; however , there can be considerable interindividual variability in humans due to genetic polymorphisms , diseases , and exposure to other xenobiotics . for example , a recent pcb 146 metabolism experiment with hlms from individual donors revealed considerable interindividual metabolism of pcb in humans associated with cyp2b6 activity . 4136 was also the major metabolite in incubations using microsomes from monkeys and rabbits . in contrast , 5136 was the major metabolite in experiments with microsomes from dogs , guinea pigs , mice , hamsters , and , as reported previously , rats . the faster formation of 5136 in rat compared to dog microsomes in the current study is consistent with the differences in the oxidation of pcb 136 reported by waller et al . for recombinant rat cyp2b1 and dog cyp2b11 . 5136 and 4136 are also the major pcb 136 metabolites formed in rats after intraperitoneal administration of pcb 136 , with a rank order of 5136 > 4136 . these species differences in the oh - pcb ratios and formation rates are not surprising because of the considerable interspecies differences in the constitutive expression and catalytic activity of p450 enzymes . it is important to emphasize that our results represent oh - pcb ratios and formation rates obtained with pooled microsomal preparations from nave animals . as with humans , genetic and environmental factors can result in interindividual variability in the oh - pcb profiles and formation rates in these species . however , these differences are likely small in toxicologically relevant animal models because the animals under investigation are inbred and maintained under rigorously controlled environmental and dietary conditions . more significant interindividual variability in the p450 enzyme - mediated oxidation of chiral pcbs is likely to occur in wildlife . the present study also revealed considerable differences in the atropisomeric enrichment of 5136 and 4136 formed by microsomal preparations obtained from different species . interestingly , the e2 - 5136 and e2 - 4136 were enriched in incubations with liver microsomes from most species , albeit to a different extent . microsomes from mice were a notable exception , because e1 - 5136 and e1 - 4136 were enriched . the same direction of the atropisomeric enrichment of 5136 and 4136 has been observed in mouse tissue slices . it is currently unclear to which extent our in vitro metabolism studies predict chiral oh - pcb signatures in vivo . in particular , subsequent metabolism ( e.g. , sulfation and glucuronidation ) and transport of oh - pcbs may modulate the atropisomeric enrichment of oh - pcbs in vivo ; however , the atropselectivity of these biological processes has not been investigated to date . both e1 - 5136 and e1 - 4136 consequently , most mammalian species , including humans , metabolize and eliminate the ( )-pcb 136 atropisomer less rapidly than the ( + ) -pcb 136 atropisomer . in contrast , mice metabolize ( )-pcb 136 more rapidly , at least compared to the other mammalian species investigated in our study . consistent with this interpretation , ( + ) -pcb 136 undergoes atropisomeric enrichment in mice , whereas only a slight enrichment of ( )-pcb 136 is observed in rats . this observation is important because ( )-pcb 136 , but not ( + ) -pcb 136 , is a potent sensitizer of ryrs and alters neuronal connectivity via a ryr - dependent mechanism . it is therefore possible that differences in the metabolism of pcb 136 atropisomers across species , sex , or individuals play a role in the developmental neurotoxicity of pcb 136 and structurally related congeners . the toxicological relevance of the atropisomeric enrichment of oh - pcb metabolites of pcb 136 and other chiral pcbs is currently unknown and warrants further investigation . like the parent pcbs , pure oh - pcb atropisomers may display atropselectivity toward cellular targets and , thus , cause atropselective toxicity in wildlife and humans . developmental neurotoxicity is a particular concern in humans because oh - pcbs cross the placenta and accumulate in fetal target tissues . oh - pcbs have several modes of action and , for example , disrupt cellular calcium homeostasis by mechanisms involving ryrs or cause thyroid dysfunction . oh - pcb 136 metabolites and structurally related , chiral oh - pcbs have not been detected in humans , partly because suitable analytical standards are not readily available ; however , their parent compounds can be present at high levels in indoor air , including in school buildings in the united states . it is therefore likely that potentially neurotoxic , chiral oh - pcbs are present in humans , especially in school children and other susceptible human populations . similar to the parent pcbs , our observation that the atropisomeric enrichment of oh - pcbs is highly species - dependent will be useful for source apportionment studies of oh - pcb . oh - pcbs have not only been detected in laboratory animals and humans , but also in species at different tropic levels , such as fish , sea birds , marine mammals , and plants . although studies with liver microsomes demonstrate that p450 enzymes are involved in the formation of oh - pcbs in many species , several studies demonstrate the presence of oh - pcbs in abiotic samples . oh - pcbs are also formed by the reaction of oh radicals with pcbs and have been detected in surface water and precipitation . as with pcbs , chiral oh - pcb formed by abiotic processes will be racemic , whereas oh - pcbs in biological samples will be nonracemic due to atropselective biological transport and biotransformation processes . consequently , chiral signatures can be used to distinguish abiotic from biotic oh - pcb sources . furthermore , species - dependent differences on chiral signatures may be useful to study how oh - pcbs move through aquatic and terrestrial food webs . similarly , chiral signatures are a powerful tool to study the movement of chiral pcbs through aquatic and terrestrial food webs .

chiral polychlorinated biphenyls ( pcbs ) display variable atropisomeric enrichment in wildlife and animal models , especially at higher trophic levels . these differences in pcbs chiral signatures are , at least in part , due to species - dependent oxidation of pcbs to hydroxylated pcb metabolites ( oh - pcbs ) . here , we investigate the hypothesis that the cytochrome p450 ( p450 ) enzyme - mediated oxidation of chiral pcbs results in species - dependent differences in the chiral signatures of oh - pcbs ( i.e. , the direction and extent of oh - pcbs atropisomeric enrichment ) . to investigate this hypothesis , we incubated pcb 136 , a representative chiral pcb , with pooled human liver microsomes ( hlms ) or liver microsomes from male guinea pig , hamster , monkey , mouse , and rabbit or female dog and determined average profiles and chiral signatures of the oh - pcbs . 2,2,3,3,6,6-hexachlorobiphenyl-4-ol ( 4136 ) was the major metabolite in incubations with hlms and monkey and rabbit microsomes . 2,2,3,3,6,6-hexachlorobiphenyl-5-ol ( 5136 ) was the major metabolite formed by microsomes from all other species . both 4136 and 5136 were formed atropselectively in all microsomal incubations ; however , the direction and extent of the atropisomeric enrichment of both oh - pcb metabolites showed considerable differences across microsomal preparations obtained from different species . these differences in oh - pcbs atropisomeric enrichment may not only be toxicologically relevant but may also be useful to study sources and transport of oh - pcbs in the environment .

Introduction Experimental Section Results Discussion

polychlorinated biphenyls ( pcbs ) are a class of industrial chemicals and unintentional byproducts of industrial processes banned under the stockholm convention on persistent organic pollutants . pcbs remain an environmental and human health concern because of their ongoing , inadvertent production , their environmental persistence , and the presence of pcbs in the environment , diet , and in human serum and tissues . , a 1:1 mixture of atropisomers ) and , due to pcbs chemical and thermal stability , are released into the environment as racemates . studies of the atropisomeric enrichment of chiral pcbs reveal near racemic signatures in diet , house dust , and air , but highly variable atropisomeric enrichment in wildlife , especially at higher tropic levels , and humans ( reviewed in ref ( 5 ) ) . , photodegradation ) are not atropselective , the variable atropisomeric enrichment of pcbs in environmental samples is due to atropselective biotransformation and/or biological transport processes . laboratory studies have shown that cytochrome p450 enzymes , such as different cyp2b isoforms , can atropselectively metabolize pcbs to oh - pcbs and , thus , contribute to nonracemic signatures of pcbs at higher trophic levels . activity relationship studies show that the oh - pcb metabolites of chiral pcbs are ryanodine receptor- ( ryr-)active and display different modes of action depending on the position of the hydroxyls group on the biphenyl moiety in vitro . these differences in oh - pcb profiles are due to differences in the isoform composition , expression , and activities of pcb and oh - pcb metabolizing enzymes ( e.g. furthermore , differences in the composition and oh - pcb binding affinity of various transport proteins ( e.g. , transthyretin ) may contribute to species and interindividual differences in oh - pcb profiles in vivo . since pcbs are atropselectively metabolized by p450 enzymes to oh - pcbs , it is likely that , similar to the parent pcbs , p450 enzyme - mediated metabolism contributes to differences in the atropisomeric enrichment of chiral oh - pcbs in wildlife and humans . consistent with this hypothesis , we have recently reported differences in the atropselective formation of oh - pcbs in rats and mice in in vitro metabolism studies ; however , systematic laboratory and environmental studies of the atropselective formation of oh - pcbs by p450 enzymes from different species have not been reported to date . the present study investigates the atropselective formation of oh - pcb from 2,2,3,3,6,6-hexachlorobiphenyl ( pcb 136 ) by liver microsomes from humans and several other mammalian species . pooled liver microsomes from nave animals were used to assess representative oh - pcb profiles and chiral signatures in the species investigated . untreated beagle dog ( female ) , cynomolgus monkey ( male ) , new zealand rabbit ( male ) , golden syrian hamster ( male ) , hartley albino guinea pig ( male ) , and human liver microsomes pooled from 50 donors with mixed age , sex , and race ( hlms ) were purchased from xenotech ( lenexa , ks , usa ) . 2,2,3,3,6,6-hexachlorobiphenyl-4-ol ( 4136 ) , 2,2,3,3,6,6-hexachlorobiphenyl-5-ol ( 5136 ) , 4,5-dimethoxy-2,2,3,3,6,6-hexachlorobiphenyl , and 2,2,3,4,6,6-hexachloro-3-methoxybiphenyl were prepared as described elsewhere . briefly , an incubation mixture ( 12 ml ) consisting of phosphate buffer ( 0.1 m , ph 7.4 ) , nadph ( 0.5 mm in hlms or 1.5 mm in all animal microsomes ) , magnesium chloride ( 3 mm ) , and hepatic microsomal protein ( 1.0 mg / ml for all animal microsomes or 0.5 mg / ml for human microsomes ) was preincubated for 5 min at 37 1 c in a shaking water bath . these incubation conditions , including the high pcb 136 concentrations , were selected to ensure the formation of sufficient oh - pcb quantities for atropselective analyses . for the 0 min time point , a separate sample ( 1990 l ) was preincubated for 5 min as described above , followed by sequential addition of the sodium hydroxide ( 2 ml ) and pcb 136 solution ( 10 l ) . levels of oh - pcb 136 metabolites were determined using an agilent 6890n gas chromatograph with a ni-ecd detector and a db1-ms capillary column ( 60 m 0.25 mm i d 0.25 m film thickness ; agilent , santa clara , ca , usa ) . relative rates of oh - pcb formation were determined in the linear range of metabolite formation ( i.e. , 5 min ) by adjusting the amount of oh - pcb by the total p450 content . racemic pcb 136 or its atropisomers were incubated with hlms to investigate if potentially neurotoxic oh - pcbs are formed atropselectively in humans . only a small percentage ( < 1% ) of the total pcb 136 was converted to oh - pcbs under the incubation conditions ( table s4 ) . 4136 and 5136 were the major metabolites for racemic pcb 136 and pure pcb 136 atropisomers , with more 4136 being formed ( 5136/4136 ratio = 0.39 after 5 min , table s5 ) . the time - dependent formation of ( a ) 3150 , ( b ) 5136 , ( c ) 4136 , ( d ) 4 , 5136 , and ( e ) oh-136 followed the rank order ( + ) -pcb 136 > ( )-pcb 136 > ( )-pcb 136 in incubations with human liver microsomes . ( f ) the relative rates of oh - pcb formation showed a significantly slower metabolite formation in the incubations with ( )-pcb 136 compared to incubations with ( + ) -pcb 136 and ( )-pcb 136 . different letters indicate statistically significant differences in the oh - pcb formation rates ( p < 0.05 ) as determined by a tukey student range test using sas . racemic pcb 136 was incubated with liver microsomes obtained from different species ( i.e. , male monkey , guinea pig , mouse , hamster , and rabbit ; female dog ) to explore differences in typical metabolite profiles and chiral signatures between humans and toxicologically relevant mammalian species . only a small percentage of pcb 136 ( < 3% ) was converted to oh - pcbs ( table s4 ) . similar to experiments with hlms , 4136 was the major metabolite formed in incubations with microsomes obtained from male monkeys and rabbits ( figure 2 ) , with 5136/4136 ratios of 0.17 and 0.64 at 5 min , respectively ( table s5 ) . in contrast , 5136 was the major metabolite in incubations using microsomes from dogs , guinea pigs , mice , and hamsters . after a 5 min incubation time , the 5136/4136 ratios were 14 , 10 , 2.2 , and 6.9 for incubations with dog , guinea pig , mouse , and hamster microsomes , respectively ( table s5 ) . time- and species - dependent formation of oh - pcbs in incubations of pcb 136 with liver microsomes from ( a ) humans ( pooled ) , ( b ) dog , ( c ) monkey , ( d ) guinea pig , ( e ) mouse , ( f ) hamster , and ( g ) rabbit . 4136 was the major metabolite in incubations using human , monkey and rabbit liver microsomes . 5136 was the major metabolite in experiments with dog , guinea pig , mouse , and hamster liver microsomes . the rate of 5136 formation by hlms was similar compared to the rate observed in incubations with microsomes from hamster , monkey , mouse , and rabbit . the rate of 4136 formation by hlms was significantly faster compared to experiments using microsomes from other species and followed the order human > monkey > rabbit rat > dog guinea pig mouse hamster ( figure 3 ) . comparison of the formation rates of ( a ) 5136 and ( b ) 4136 in incubations with liver microsomes obtained from different species . different letters indicate statistically significant differences in the oh - pcb formation rates ( p < 0.05 ) as determined by a tukey student range test using sas . the atropisomeric enrichment of oh - pcb 136 metabolites was determined in microsomal incubations with racemic pcb 136 using atropselective gas chromatography . the objective was to determine if oh - pcbs are formed atropselectively in incubations with hlm and how this enrichment differs compared to toxicologically relevant species . the second eluting atropisomers of 5136 ( e2 - 5136 ) , which is formed from ( + ) -pcb 136 , was enriched in incubations using human , dog , monkey , guinea pig , and rabbit microsomes ( figure 4a ) . the atropisomeric enrichment of 5136 and 4136 formed from liver microsomes is species - dependent . ( a ) representive chromatograms showing an enrichment of the second eluting 5136 atropisomer in incubations with human ( pooled ) , dog , monkey , guinea pig , and rabbit liver microsomes and an enrichment of the first eluting 5136 atropisomer in experiments with mouse and hamster liver microsomes . ( b ) representive chromatograms showing an enrichment of the second eluting 4136 atropisomer in incubations with human ( pooled ) , dog , monkey , guinea pig , hasmster , and rabbit liver microsomes and an enrichment of the first eluting 4136 atropisomer in experiments with mouse liver microsomes . the extent of the atropisomeric enrichment of 5136 in microsomal incubations followed the order dog guinea pig > monkey human rabbit ( figure 4c ) . interestingly , the first eluting atropisomer of 5136 ( e1 - 5136 ) , the 5136 metabolite formed from ( -)-pcb 136 , displayed atropisomeric enrichment in experiments with mouse and , to a lesser extent , hamster microsomes . similar to 5136 , the second eluting atropisomers of 4136 ( e2 - 4136 ) , a metabolite formed from ( + ) -pcb 136 , was enriched in incubations using human , dog , monkey , guinea pig , hamster , and rabbit microsomes ( figure 4b ) . the extent of the atropisomeric enrichment of 4136 formed in microsomal incubations followed the order human monkey > guinea pig hamster rabbit > dog . the present study uses hepatic microsomes to gain insights into typical oh - pcb 136 metabolite profiles and chiral signatures formed by p450 enzymes in different mammalian species , including humans . 4136 and 5136 were the two major monohydroxylated pcb 136 metabolites formed atropselectively by hlms , which is consistent with an earlier study by schnellmann and co - workers . on the basis of our re - evaluation of the published mass spectra , this difference in the metabolite profile is most likely due to differences in the p450 enzyme composition of the respective hlms . the relative rate of formation of all oh - pcbs was different for incubations using ( + ) -pcb 136 , ( )-pcb 136 , and racemic pcb 136 , with ( + ) -pcb 136 being more rapidly oxidized compared to ( )-pcb 136 . these atropisomer - specific differences in the oh - pcb formation rates explain the atropisomeric enrichment of pcbs observed in in vitro studies and are consistent with a role of p450 enzymes in their atropisomeric enrichment observed human samples . analogous to hlms , 5136 , 4136 , and 4,5136 were formed by most animal microsomal preparations studied . this oh - pcb metabolite is most likely 5136 because cyp2b6 oxidizes other pcbs in the meta position . while essentially the same metabolites were formed by liver microsomes from different species , the ratios , relative formation rates , and chiral signatures of the oh - pcbs differed considerably depending on the species . experiments with hlms displayed the fastest formation rate for 4136 and one of the lowest formation rates for 5136 of all microsomal preparations investigated . it is important to emphasize that our result represents an average oh - pcb profile formed by a pool of liver microsomes from 50 individual donors ; however , there can be considerable interindividual variability in humans due to genetic polymorphisms , diseases , and exposure to other xenobiotics . 4136 was also the major metabolite in incubations using microsomes from monkeys and rabbits . in contrast , 5136 was the major metabolite in experiments with microsomes from dogs , guinea pigs , mice , hamsters , and , as reported previously , rats . the faster formation of 5136 in rat compared to dog microsomes in the current study is consistent with the differences in the oxidation of pcb 136 reported by waller et al . 5136 and 4136 are also the major pcb 136 metabolites formed in rats after intraperitoneal administration of pcb 136 , with a rank order of 5136 > 4136 . these species differences in the oh - pcb ratios and formation rates are not surprising because of the considerable interspecies differences in the constitutive expression and catalytic activity of p450 enzymes . it is important to emphasize that our results represent oh - pcb ratios and formation rates obtained with pooled microsomal preparations from nave animals . as with humans , genetic and environmental factors can result in interindividual variability in the oh - pcb profiles and formation rates in these species . however , these differences are likely small in toxicologically relevant animal models because the animals under investigation are inbred and maintained under rigorously controlled environmental and dietary conditions . more significant interindividual variability in the p450 enzyme - mediated oxidation of chiral pcbs is likely to occur in wildlife . the present study also revealed considerable differences in the atropisomeric enrichment of 5136 and 4136 formed by microsomal preparations obtained from different species . interestingly , the e2 - 5136 and e2 - 4136 were enriched in incubations with liver microsomes from most species , albeit to a different extent . the same direction of the atropisomeric enrichment of 5136 and 4136 has been observed in mouse tissue slices . , sulfation and glucuronidation ) and transport of oh - pcbs may modulate the atropisomeric enrichment of oh - pcbs in vivo ; however , the atropselectivity of these biological processes has not been investigated to date . the toxicological relevance of the atropisomeric enrichment of oh - pcb metabolites of pcb 136 and other chiral pcbs is currently unknown and warrants further investigation . like the parent pcbs , pure oh - pcb atropisomers may display atropselectivity toward cellular targets and , thus , cause atropselective toxicity in wildlife and humans . oh - pcb 136 metabolites and structurally related , chiral oh - pcbs have not been detected in humans , partly because suitable analytical standards are not readily available ; however , their parent compounds can be present at high levels in indoor air , including in school buildings in the united states . it is therefore likely that potentially neurotoxic , chiral oh - pcbs are present in humans , especially in school children and other susceptible human populations . similar to the parent pcbs , our observation that the atropisomeric enrichment of oh - pcbs is highly species - dependent will be useful for source apportionment studies of oh - pcb . oh - pcbs have not only been detected in laboratory animals and humans , but also in species at different tropic levels , such as fish , sea birds , marine mammals , and plants . although studies with liver microsomes demonstrate that p450 enzymes are involved in the formation of oh - pcbs in many species , several studies demonstrate the presence of oh - pcbs in abiotic samples . oh - pcbs are also formed by the reaction of oh radicals with pcbs and have been detected in surface water and precipitation . as with pcbs , chiral oh - pcb formed by abiotic processes will be racemic , whereas oh - pcbs in biological samples will be nonracemic due to atropselective biological transport and biotransformation processes . consequently , chiral signatures can be used to distinguish abiotic from biotic oh - pcb sources . furthermore , species - dependent differences on chiral signatures may be useful to study how oh - pcbs move through aquatic and terrestrial food webs . similarly , chiral signatures are a powerful tool to study the movement of chiral pcbs through aquatic and terrestrial food webs .

noninfectious uveitis patients and healthy controls were recruited from the national eye institute ( nei ) clinic and the national institutes of health ( nih ) clinical center blood bank , respectively . patients with infectious uveitis or systemic disease lacking ocular involvement were excluded from this study . healthy controls were required to be nonpregnant ( if female ) with no history of heart , lung , kidney , or hematologic diseases and no history of intravenous injection drug use , high - risk activity , or experimental drug use in order to donate blood to the blood bank . all study subjects provided written informed consent before participation in the study commenced . disease activity was assessed by the treating physicians and reported according to the standardization of uveitis nomenclature ( sun criteria ) . for the purpose of this study we categorized activity as follows : quiescent ( 0 anterior chamber cell and 0 vitreous haze ) and active ( 0.5 + or greater anterior chamber cells and/or any vitreous haze ) . those patients that reactivated within 6 months after the initial evaluation were retrospectively categorized as clinical reactivation . peripheral blood ( 0.4 ml ) was obtained from each participant for flow cytometry analysis . cd1c mdcs and plasmacytoid dendritic cells ( pdcs ) were identified as the lineage cocktail 1 ( lin1)hladrcd1c and lin1hladrcd303 populations , respectively , as previously reported . antibodies were purchased from miltenyi biotec ( san diego , ca , usa ) or bd biosciences ( san diego , ca , usa ) . the same procedure for handling blood samples was used for hcs and patients . in the mlr assay , monocyte - derived dendritic cells ( modcs ) with or without p38-mapk inhibitor ( sb203580 ; cell signaling , danvers , ma , usa ) treatment were cocultured with allogeneic cd4 or cd8 t cells for 5 days with concurrent stimulation of 1 g / ml anti - cd3 ( clone ucht1 ; bd biosciences ) . t - cell proliferation was characterized by flow cytometry as the percentage of cells exhibiting carboxyfluorescein succinimidyl ester ( cfse ) fluorescence dilution . statistical comparisons were performed by using the student 's t - test when analyzing data for two groups or analysis of variance ( anova ) when analyzing data for three or more groups . receiver operating characteristic ( roc ) curves were plotted to evaluate the accuracy of cd1c mdcs proportion as a risk classifier for clinical activity and predicting future activity . the measurements with the largest area under the curve ( auc ) were chosen as a significant classifier and , therefore , eligible for calculation of sensitivity and specificity ( test ) . analyses were accomplished by using prism 6 software ( graphpad software , inc . , la jolla , ca , usa ) . noninfectious uveitis patients and healthy controls were recruited from the national eye institute ( nei ) clinic and the national institutes of health ( nih ) clinical center blood bank , respectively . patients with infectious uveitis or systemic disease lacking ocular involvement were excluded from this study . healthy controls were required to be nonpregnant ( if female ) with no history of heart , lung , kidney , or hematologic diseases and no history of intravenous injection drug use , high - risk activity , or experimental drug use in order to donate blood to the blood bank . all study subjects provided written informed consent before participation in the study commenced . disease activity was assessed by the treating physicians and reported according to the standardization of uveitis nomenclature ( sun criteria ) . for the purpose of this study we categorized activity as follows : quiescent ( 0 anterior chamber cell and 0 vitreous haze ) and active ( 0.5 + or greater anterior chamber cells and/or any vitreous haze ) . those patients that reactivated within 6 months after the initial evaluation were retrospectively categorized as clinical reactivation . peripheral blood ( 0.4 ml ) was obtained from each participant for flow cytometry analysis . cd1c mdcs and plasmacytoid dendritic cells ( pdcs ) were identified as the lineage cocktail 1 ( lin1)hladrcd1c and lin1hladrcd303 populations , respectively , as previously reported . antibodies were purchased from miltenyi biotec ( san diego , ca , usa ) or bd biosciences ( san diego , ca , usa ) . the same procedure for handling blood samples was used for hcs and patients . in the mlr assay , monocyte - derived dendritic cells ( modcs ) with or without p38-mapk inhibitor ( sb203580 ; cell signaling , danvers , ma , usa ) treatment were cocultured with allogeneic cd4 or cd8 t cells for 5 days with concurrent stimulation of 1 g / ml anti - cd3 ( clone ucht1 ; bd biosciences ) . t - cell proliferation was characterized by flow cytometry as the percentage of cells exhibiting carboxyfluorescein succinimidyl ester ( cfse ) fluorescence dilution . statistical comparisons were performed by using the student 's t - test when analyzing data for two groups or analysis of variance ( anova ) when analyzing data for three or more groups . receiver operating characteristic ( roc ) curves were plotted to evaluate the accuracy of cd1c mdcs proportion as a risk classifier for clinical activity and predicting future activity . the measurements with the largest area under the curve ( auc ) were chosen as a significant classifier and , therefore , eligible for calculation of sensitivity and specificity ( test ) . analyses were accomplished by using prism 6 software ( graphpad software , inc . , la jolla , ca , usa ) . noninfectious uveitis patients ( n = 89 ) seen at the nei and healthy controls ( n = 111 ) from the nih blood bank between january 2012 and july 2015 were included in the study . demographics ( age , sex , and race ) and clinical information ( underlying systemic diseases , anatomic locations of uveitis , medications , and disease activity ) of patients were recorded ( table ) . cd1c mdc levels were collected longitudinally on 44 of 89 patients who visited the eye clinic multiple times throughout the study period . the frequency of visits depended on their disease activity , disease development , and response to therapy . in general , patients with active disease were followed up every 2 weeks and quiescent patients were followed up every 2 months . therefore , some of patients were able to give blood samples at least twice . characteristics of uveitis patients and healthy controls consistent with our prior observation , the proportions of cd1c mdcs in the total dc populations were significantly elevated in uveitis patients when compared to hcs ( hcs = 111 , uveitis = 89 , p = 0.0025 ; fig . furthermore , cd1c mdcs were significantly higher in patients with active disease as compared to those who were clinically quiescent ( p = 0.0052 ; fig . in addition to mdcs , pdcs also play a role in uveitis although their function is not fully understood yet . to assess any relative changes in the pdc populations in uveitis patients compared to hcs , we also measured the proportions of pdcs in the total dc populations by flow cytometry . in contrast to cd1c mdc proportions , pdcs proportions were decreased in uveitis patients compared to those in hcs ( p = 0.01 ; fig . 1c ) , and no difference was detected between patients with active and quiescent uveitis ( fig . cd1c mdcs and cd303 pdcs were gated on total blood dc population ( lin1hladr ) . ( a ) the proportions of cd1c mdcs were compared between hcs and noninfectious uveitis patients ( uveitis ) . ( b ) the percentages of cd1c mdcs were compared between quiescent and active uveitis patients . ( c ) the proportions of cd303 pdcs were compared between hcs and uveitis patients . ( d ) the proportions of cd303 pdcs were compared between quiescent and active uveitis patients . indeed , the diversity in underlying systemic disease implies that there are key differences in pathogenesis among these diseases , but the commonality of noninfectious ocular inflammation also implies common mechanisms . subgroup univariate analyses revealed no difference in cd1c mdc proportions among uveitis patients with different underlying diseases including sarcoidosis , idiopathic , birdshot , vogt - koyanagi - harada ( vkh ) , behcet 's diseases , or others ( fig . 2a ) , and different anatomic locations ( anterior , intermediate , posterior , or panuveitis ; fig . uveitis patients were divided into subgroups on the basis of their underlying systemic diseases and locations of uveitis . ( a ) proportions of cd1c mdcs were compared in different systemic diseases including sarcoidosis , idiopathic , birdshot , vkh , behcet 's disease , and others ( anova test , p = 0.40 ) . ( b ) proportions of cd1c mdcs were compared in anatomic locations of uveitis including anterior , intermediate , posterior , and panuveitis ( anova test , p = 0.76 ) . ( c ) cd1c mdc proportions were compared among four subgroups : no therapy , prednisone only ( pred only ) , other systemic immunosuppressive treatment ( others ) , and prednisone plus other systemic immunosuppressive treatment ( pred + others ) ( anova test , p = 0.75 ) . ( d ) cd1c mdc proportions were compared in the subgroups on the basis of the dosage of prednisone used daily including 0 mg , 1 to 10 mg , 11 to 20 mg , and > 20 mg ( anova test , p = 0.16 ) . ( e , f ) monocyte - derived dendritic cells were treated with dexamethasone ( dex , n = 6 ) , cyclosporine ( csa , n = 6 ) for 24 hours . monocyte - derived dendritic cells without treatment were the control group ( con , n = 6 ) and those treated with dimethyl sulfoxide ( dmso ) were the vehicle group ( veh , n = 6 ) because dex and csa were dissolved in dmso . proportions of cd1c mdcs and mean fluorescence intensity ( mfi ) of cd1c on mdcs were calculated by flow cytometry . the data are presented as mean sd ( anova test , p = 0.23 [ e ] ; p = 0.99 [ f ] ) . during the study period , 73% of patients were on immunosuppressive therapy , which is known to alter t - cell phenotype and function . therefore , we also investigated the effects of treatment with immunosuppressive medications on cd1c mdc proportions and found no significant difference ( fig . there was also no difference on cd1c mdc proportions between patients treated with different doses of prednisone ranging from no prednisone , 10 mg prednisone per day , 11 to 20 mg per day , and > 20 mg per day ( fig . moreover , in vitro treatment of modcs with dexamethasone or cyclosporine a did not alter cd1c mdc proportions or mean fluorescence intensity ( figs . cd1c mdc elevation was not significantly associated with the etiologic and anatomic classifications of uveitis , or types and dosages of immunosuppressive therapy . previous publications have shown that sex differences may alter susceptibility to autoimmune disease and that aging is associated with immunosenescence . therefore , subgroup univariate analyses of cd1c mdc proportions in hcs ( figs . 3a cd1c mdc proportions from hcs ( a c ) or uveitis patients ( d f ) were divided into subgroups according to their ages including 30 , 31 to 40 , 41 to 50 , 51 to 60 , and > 60 years ( a , d ) ; sex including female and male ( b , e ) ; and race including caucasian , african american , and others ( e , f ) . analysis of variance test was used for statistical analysis in ( a ) , ( c ) , ( d ) , and ( f ) . unpaired student 's t - test was used in ( b ) and ( e ) . among 44 patients who had longitudinal data of cd1c mdc levels with multiple time points , cd1c mdc proportions were not altered significantly in those patients who were initially clinically quiescent ( q ) and remained quiescent ( q - q , n = 25 , p = 0.35 ; fig . 4a ) , or in patients with active disease ( a ) who remained active between blood draws ( a - a , n = 10 , p = 0.67 ; fig . 4b ) . however , active patients who became quiescent after treatment showed decreased cd1c mdc proportions ( a - q , n = 14 , p = 0.03 ; fig . 4c ) and quiescent patients showed increased cd1c mdc levels when they developed active uveitis after tapering off medications ( q - a , n = 4 , p = 0.02 ; fig . 4d ) . plotting the average level of cd1c mdc proportions in continually quiescent patients ( q - q ) , continually active patients ( a - a ) , and patients with changes in disease activity from active to quiescent ( a - q ) or quiescent to active ( q - a ) , showed continually low , continually high , significantly decreasing , and significantly increasing cd1c mdc proportions , respectively ( fig . fold changes of cd1c mdc proportions between two time points from each patient were calculated in the groups of q - q ( q / q = 1.05 ) , a - a ( a / a = 1.07 ) , a - q ( q / a = 0.88 ) and q - a ( a / q = 1.40 ) . the fold changes in the q - a group were significantly increased as compared to groups of q - q and a - a ( q - a versus q - q , p = 0.017 ; q - a versus a - a , p = 0.022 ; fig . the fold changes in a - q group were decreased as compared to the groups of q - q and a - a although not statistically significantly ( fig . these results suggest that dynamic levels of cd1c mdc proportions are associated with disease activity . blood samples were collected at multiple time points from each patient longitudinally , followed by the measurement of cd1c mdc proportions by flow cytometry . the data were compared in the groups of patients who maintained the quiescent uveitis ( [ a ] , quiescent to quiescent , q - q ) , of patients whose diseases were active during sampling ( [ b ] , active to active , a - a ) , of patients whose disease activity changed from the active to quiescent ( [ c ] , a - q ) or from the quiescent to active ( [ d ] , q - a ) . ( e ) the average proportion of cd1c mdcs from each group ( q - q , a - a , a - q , q - a ) was compared . lines q - q , a - a , a - q , and q - a represent the four groups , respectively . ( f ) fold change of cd1c mdc proportions in each patient from groups of q - q , a - a , a - q , q - a was calculated and compared by anova analysis ( p = 0.005 ) . ( f ) receiver operating characteristic curve was plotted and the area under the curve was obtained ( a = 0.64 , p < 0.035 ) . paired student 's t - test was used in ( a c ) . to evaluate the potential role of cd1c mdc proportion as a biomarker for uveitic activity , an roc curve was plotted , resulting in an auc of 0.64 ( p = 0.035 ; fig . further investigation is needed to evaluate whether cd1c mdc proportion could be a biomarker for uveitic activity . although current uveitic activity can be determined by clinical examination and imaging findings , an oncoming activity is difficult to predict . there is currently no adequate laboratory or clinical biomarker to predict reactivation . in our cross - sectional data ( fig . 1b ) , we noticed that while most quiescent patients had low proportions of cd1c mdcs , 26% of quiescent patients still had cd1c mdc proportions that were higher than the average proportion in active uveitis patients ( 60% of cd1c mdcs ) . we hypothesized that these quiescent patients with elevated cd1c mdc proportions were at increased risks for disease reactivation . to test this hypothesis , we divided quiescent patients ( n = 34 ) into high ( n = 10 ) and low ( n = 24 ) cd1c mdc proportion subgroups on the basis of a cutoff of cd1c mdc proportion in total dc population , which was 60% . interestingly , 6 of 24 quiescent patients ( 25% ) had disease reactivation in the low cd1c subgroup compared to 7 of 10 patients ( 70% ) in high cd1c subgroup ( p = 0.01 ; fig . 5a ) . additionally , the proportions of cd1c mdcs among patients who eventually reactivated were higher than for those who remained clinically quiet ( p = 0.02 ; fig . 5b ) , indicating that cd1c mdc proportions were associated with subsequent uveitis reactivation . given the potential clinical use of cd1c mdc proportion to detect clinical reactivation , a roc curve was plotted , with an auc of 0.68 ( p = 0.03 ; fig . the level of cd1c mdcs at 60% was set as the cutoff , which was the average proportional level in active uveitis patients . ( a ) patients ( n = 34 ) were divided into low levels of cd1c proportion ( 60% of cd1c mdcs ) and high levels of cd1c proportion ( > 60% of cd1c mdcs ) groups ( low cd1c versus high cd1c ) . six of 24 patients had disease reactivation ( 25% ) in low cd1c proportion group , and 7 of 10 patients ( 70% ) had disease recurrence in high cd1c proportion group . ( b ) cd1c mdcs proportions were compared in the groups of quiescent patients with or without disease reactivation ( no reactivation versus reactivation ) . ( c ) receiver operating characteristic curve was plotted and the area under the curve was obtained ( a = 0.68 , p < 0.03 ) . unpaired student 's t - test was used in ( a , b ) . to investigate whether the elevated cd1c mdc proportions observed in noninfectious uveitis patients had effects on adaptive immune response , we performed an in vitro study to evaluate the effects of cd1c expression on mdc t - cell interactions . our published data have shown that blocking p38-mapk pathway during modc differentiation impairs cd1c expression on modcs in vitro . in this experiment , proportions of cd1c modcs were decreased after p38-mapk inhibitor treatment ( fig . 6a ) , and then these modcs ( p38i - modcs ) cocultured with allogeneic cd4 or cd8 t cells . t - cell proliferation was significantly reduced in cultures with modcs treated with p38-mapk inhibitor ( figs . monocyte - derived dendritic cells with low cd1c proportions were associated with impaired allogeneic t - cell proliferation . monocyte - derived dendritic cells were induced with or without treatment of p38 mapk inhibitor in vitro . ( a ) proportions of cd1c modcs were measured by flow cytometry ( n = 4 ) . monocyte - derived dendritic cells with or without p38 mapk inhibitor treatment ( p38i - modcs versus modcs ) cocultured with allogeneic cd4 ( b ; n = 5 ) or cd8 t cells ( c ; n = 5 ) for 5 days , respectively . noninfectious uveitis patients ( n = 89 ) seen at the nei and healthy controls ( n = 111 ) from the nih blood bank between january 2012 and july 2015 were included in the study . demographics ( age , sex , and race ) and clinical information ( underlying systemic diseases , anatomic locations of uveitis , medications , and disease activity ) of patients were recorded ( table ) . cd1c mdc levels were collected longitudinally on 44 of 89 patients who visited the eye clinic multiple times throughout the study period . the frequency of visits depended on their disease activity , disease development , and response to therapy . in general , patients with active disease were followed up every 2 weeks and quiescent patients were followed up every 2 months . therefore , some of patients were able to give blood samples at least twice . characteristics of uveitis patients and healthy controls consistent with our prior observation , the proportions of cd1c mdcs in the total dc populations were significantly elevated in uveitis patients when compared to hcs ( hcs = 111 , uveitis = 89 , p = 0.0025 ; fig . furthermore , cd1c mdcs were significantly higher in patients with active disease as compared to those who were clinically quiescent ( p = 0.0052 ; fig . in addition to mdcs , pdcs also play a role in uveitis although their function is not fully understood yet . to assess any relative changes in the pdc populations in uveitis patients compared to hcs , we also measured the proportions of pdcs in the total dc populations by flow cytometry . in contrast to cd1c mdc proportions , pdcs proportions were decreased in uveitis patients compared to those in hcs ( p = 0.01 ; fig . 1c ) , and no difference was detected between patients with active and quiescent uveitis ( fig . cd1c mdcs and cd303 pdcs were gated on total blood dc population ( lin1hladr ) . ( a ) the proportions of cd1c mdcs were compared between hcs and noninfectious uveitis patients ( uveitis ) . ( b ) the percentages of cd1c mdcs were compared between quiescent and active uveitis patients . ( c ) the proportions of cd303 pdcs were compared between hcs and uveitis patients . ( d ) the proportions of cd303 pdcs were compared between quiescent and active uveitis patients . indeed , the diversity in underlying systemic disease implies that there are key differences in pathogenesis among these diseases , but the commonality of noninfectious ocular inflammation also implies common mechanisms . subgroup univariate analyses revealed no difference in cd1c mdc proportions among uveitis patients with different underlying diseases including sarcoidosis , idiopathic , birdshot , vogt - koyanagi - harada ( vkh ) , behcet 's diseases , or others ( fig . 2a ) , and different anatomic locations ( anterior , intermediate , posterior , or panuveitis ; fig uveitis patients were divided into subgroups on the basis of their underlying systemic diseases and locations of uveitis . ( a ) proportions of cd1c mdcs were compared in different systemic diseases including sarcoidosis , idiopathic , birdshot , vkh , behcet 's disease , and others ( anova test , p = 0.40 ) . ( b ) proportions of cd1c mdcs were compared in anatomic locations of uveitis including anterior , intermediate , posterior , and panuveitis ( anova test , p = 0.76 ) . ( c ) cd1c mdc proportions were compared among four subgroups : no therapy , prednisone only ( pred only ) , other systemic immunosuppressive treatment ( others ) , and prednisone plus other systemic immunosuppressive treatment ( pred + others ) ( anova test , p = 0.75 ) . ( d ) cd1c mdc proportions were compared in the subgroups on the basis of the dosage of prednisone used daily including 0 mg , 1 to 10 mg , 11 to 20 mg , and > 20 mg ( anova test , p = 0.16 ) . ( e , f ) monocyte - derived dendritic cells were treated with dexamethasone ( dex , n = 6 ) , cyclosporine ( csa , n = 6 ) for 24 hours . monocyte - derived dendritic cells without treatment were the control group ( con , n = 6 ) and those treated with dimethyl sulfoxide ( dmso ) were the vehicle group ( veh , n = 6 ) because dex and csa were dissolved in dmso . proportions of cd1c mdcs and mean fluorescence intensity ( mfi ) of cd1c on mdcs were calculated by flow cytometry . the data are presented as mean sd ( anova test , p = 0.23 [ e ] ; p = 0.99 [ f ] ) . during the study period , 73% of patients were on immunosuppressive therapy , which is known to alter t - cell phenotype and function . therefore , we also investigated the effects of treatment with immunosuppressive medications on cd1c mdc proportions and found no significant difference ( fig . 2c ) . there was also no difference on cd1c mdc proportions between patients treated with different doses of prednisone ranging from no prednisone , 10 mg prednisone per day , 11 to 20 mg per day , and > 20 mg per day ( fig . moreover , in vitro treatment of modcs with dexamethasone or cyclosporine a did not alter cd1c mdc proportions or mean fluorescence intensity ( figs . cd1c mdc elevation was not significantly associated with the etiologic and anatomic classifications of uveitis , or types and dosages of immunosuppressive therapy . previous publications have shown that sex differences may alter susceptibility to autoimmune disease and that aging is associated with immunosenescence . therefore , subgroup univariate analyses of cd1c mdc proportions in hcs ( figs . 3a cd1c mdc proportions from hcs ( a c ) or uveitis patients ( d f ) were divided into subgroups according to their ages including 30 , 31 to 40 , 41 to 50 , 51 to 60 , and > 60 years ( a , d ) ; sex including female and male ( b , e ) ; and race including caucasian , african american , and others ( e , f ) . analysis of variance test was used for statistical analysis in ( a ) , ( c ) , ( d ) , and ( f ) . unpaired student 's t - test was used in ( b ) and ( e ) . among 44 patients who had longitudinal data of cd1c mdc levels with multiple time points , cd1c mdc proportions were not altered significantly in those patients who were initially clinically quiescent ( q ) and remained quiescent ( q - q , n = 25 , p = 0.35 ; fig . 4a ) , or in patients with active disease ( a ) who remained active between blood draws ( a - a , n = 10 , p = 0.67 ; fig . 4b ) . however , active patients who became quiescent after treatment showed decreased cd1c mdc proportions ( a - q , n = 14 , p = 0.03 ; fig . 4c ) and quiescent patients showed increased cd1c mdc levels when they developed active uveitis after tapering off medications ( q - a , n = 4 , p = 0.02 ; fig . 4d ) . plotting the average level of cd1c mdc proportions in continually quiescent patients ( q - q ) , continually active patients ( a - a ) , and patients with changes in disease activity from active to quiescent ( a - q ) or quiescent to active ( q - a ) , showed continually low , continually high , significantly decreasing , and significantly increasing cd1c mdc proportions , respectively ( fig . fold changes of cd1c mdc proportions between two time points from each patient were calculated in the groups of q - q ( q / q = 1.05 ) , a - a ( a / a = 1.07 ) , a - q ( q / a = 0.88 ) and q - a ( a / q = 1.40 ) . the fold changes in the q - a group were significantly increased as compared to groups of q - q and a - a ( q - a versus q - q , p = 0.017 ; q - a versus a - a , p = 0.022 ; fig . the fold changes in a - q group were decreased as compared to the groups of q - q and a - a although not statistically significantly ( fig . these results suggest that dynamic levels of cd1c mdc proportions are associated with disease activity . blood samples were collected at multiple time points from each patient longitudinally , followed by the measurement of cd1c mdc proportions by flow cytometry . the data were compared in the groups of patients who maintained the quiescent uveitis ( [ a ] , quiescent to quiescent , q - q ) , of patients whose diseases were active during sampling ( [ b ] , active to active , a - a ) , of patients whose disease activity changed from the active to quiescent ( [ c ] , a - q ) or from the quiescent to active ( [ d ] , q - a ) . ( e ) the average proportion of cd1c mdcs from each group ( q - q , a - a , a - q , q - a ) was compared . lines q - q , a - a , a - q , and q - a represent the four groups , respectively . ( f ) fold change of cd1c mdc proportions in each patient from groups of q - q , a - a , a - q , q - a was calculated and compared by anova analysis ( p = 0.005 ) . ( f ) receiver operating characteristic curve was plotted and the area under the curve was obtained ( a = 0.64 , p < 0.035 ) . paired student 's t - test was used in ( a c ) . to evaluate the potential role of cd1c mdc proportion as a biomarker for uveitic activity , an roc curve was plotted , resulting in an auc of 0.64 ( p = 0.035 ; fig . further investigation is needed to evaluate whether cd1c mdc proportion could be a biomarker for uveitic activity . although current uveitic activity can be determined by clinical examination and imaging findings , an oncoming activity is difficult to predict . there is currently no adequate laboratory or clinical biomarker to predict reactivation . in our cross - sectional data ( fig . 1b ) , we noticed that while most quiescent patients had low proportions of cd1c mdcs , 26% of quiescent patients still had cd1c mdc proportions that were higher than the average proportion in active uveitis patients ( 60% of cd1c mdcs ) . we hypothesized that these quiescent patients with elevated cd1c mdc proportions were at increased risks for disease reactivation . to test this hypothesis , we divided quiescent patients ( n = 34 ) into high ( n = 10 ) and low ( n = 24 ) cd1c mdc proportion subgroups on the basis of a cutoff of cd1c mdc proportion in total dc population , which was 60% . interestingly , 6 of 24 quiescent patients ( 25% ) had disease reactivation in the low cd1c subgroup compared to 7 of 10 patients ( 70% ) in high cd1c subgroup ( p = 0.01 ; fig . 5a ) . additionally , the proportions of cd1c mdcs among patients who eventually reactivated were higher than for those who remained clinically quiet ( p = 0.02 ; fig . 5b ) , indicating that cd1c mdc proportions were associated with subsequent uveitis reactivation . given the potential clinical use of cd1c mdc proportion to detect clinical reactivation , a roc curve was plotted , with an auc of 0.68 ( p = 0.03 ; fig . the level of cd1c mdcs at 60% was set as the cutoff , which was the average proportional level in active uveitis patients . ( a ) patients ( n = 34 ) were divided into low levels of cd1c proportion ( 60% of cd1c mdcs ) and high levels of cd1c proportion ( > 60% of cd1c mdcs ) groups ( low cd1c versus high cd1c ) . six of 24 patients had disease reactivation ( 25% ) in low cd1c proportion group , and 7 of 10 patients ( 70% ) had disease recurrence in high cd1c proportion group . ( b ) cd1c mdcs proportions were compared in the groups of quiescent patients with or without disease reactivation ( no reactivation versus reactivation ) . ( c ) receiver operating characteristic curve was plotted and the area under the curve was obtained ( a = 0.68 , p < 0.03 ) . to investigate whether the elevated cd1c mdc proportions observed in noninfectious uveitis patients had effects on adaptive immune response , we performed an in vitro study to evaluate the effects of cd1c expression on mdc t - cell interactions . our published data have shown that blocking p38-mapk pathway during modc differentiation impairs cd1c expression on modcs in vitro . in this experiment , proportions of cd1c modcs were decreased after p38-mapk inhibitor treatment ( fig . 6a ) , and then these modcs ( p38i - modcs ) cocultured with allogeneic cd4 or cd8 t cells . t - cell proliferation was significantly reduced in cultures with modcs treated with p38-mapk inhibitor ( figs . monocyte - derived dendritic cells with low cd1c proportions were associated with impaired allogeneic t - cell proliferation . monocyte - derived dendritic cells were induced with or without treatment of p38 mapk inhibitor in vitro . ( a ) proportions of cd1c modcs were measured by flow cytometry ( n = 4 ) . monocyte - derived dendritic cells with or without p38 mapk inhibitor treatment ( p38i - modcs versus modcs ) cocultured with allogeneic cd4 ( b ; n = 5 ) or cd8 t cells ( c ; n = 5 ) for 5 days , respectively . the data are presented as mean sd . paired student 's t - test was used for statistical analysis . uveitis is a significant cause of ocular morbidity and vision loss , and the lack of adequate biomarkers for predicting disease recurrence makes long - term clinical management challenging . our study showed that cd1c mdc proportions were increased in uveitis patients and that this elevation appeared to be correlated with clinical activity . in univariate analyses , high proportions of cd1c mdcs were significantly associated with disease activity , while other possible clinical parameters , such as underlying systemic diseases , anatomic locations of uveitis , current immunosuppressive medications , age , sex , and race , were not . these patients subsequently had a higher rate of disease reactivation as compared to quiescent patients with low cd1c mdc proportions . thus , cd1c mdc proportion may have the potential to be a biomarker for uveitic disease activity and reactivation . increased cd1c mdc number and activity have been demonstrated in a variety of autoimmune and other immune response related diseases such as inflammatory bowel disease , rheumatoid arthritis , and asthma . in our previous publications , we have reported elevated proportions of cd1c mdcs in a small cohort of noninfectious uveitis patients when compared to healthy controls and have demonstrated that cd1c proportion rises and decreases with changes in clinical uveitis activity . in this study , with a larger sample size , we supported our previous findings of ( 1 ) elevated cd1c mdc proportions in uveitis patients compared to healthy controls and ( 2 ) a correlation between cd1c mdc proportion and disease activity . owing to the existing evidence that aging causes t - cell immunosenescence and our recent publication showing increased cd16 expression on monocytes after dexamethasone treatment in vivo and in vitro , we expected that age or systemic prednisone treatment would have some associations with the levels of cd1c mdc proportions . moreover , there was no significant association between proportions of cd1c mdcs and the various individual etiologic or anatomic diagnoses associated with uveitis . this could be due to the small number of patients within each individual diagnostic subgroup or possibly a common autoimmune mechanism shared by these different causes and locations of noninfectious uveitis . in the longitudinal data analysis , proportions of cd1c mdc closely correlated with disease activity . of our 44 patients with longitudinal cd1c data , only 4 quiescent patients activated during the blood sampling period as compared to 14 active patients who became quiescent . this is good for our patients who are undergoing treatment for uveitis ; however , it gave us a small number of patients who activated after becoming quiescent for analysis . in addition , there were several apparent exceptions to this association in the data : one quiescent patient remained quiescent despite an increase in cd1c mdc proportion from 45% to 75% , and two active patients remained active despite a decrease in cd1c mdc proportion from 87% to 66% and 65% to 44% . further clinical follow - up ultimately validated the overall correlation between cd1c mdc proportion and disease activity : the quiescent patient suffered disease recurrence 7 weeks after the second blood draw and the two active patients became quiescent 1 and 3 weeks after the second blood draw . these findings suggest that cd1c mdcs may predict disease activity in the near future , which led us to further investigate the levels of cd1c mdcs in disease recurrence . although most quiescent patients had low proportions of cd1c mdcs , there was still a relatively small population of quiescent patients ( 26% ) that maintained high cd1c mdc proportions in our cohort as defined by the average proportion of cd1c mdcs in active uveitis patients ( > 60% of cd1c mdcs ) . these quiescent patients with high cd1c mdc proportions had significantly higher rate of subsequent disease reactivation as compared to quiescent patients with low cd1c mdc proportions ( reactivation rate in high versus low cd1c groups : 70% vs. 25% ) . however , we were unable to find a correlation between cd1c mdc proportions and time to reactivation in clinically quiescent patients owing to the lack of a consistent follow - up schedule for all patients . in addition , we have previously found that the proportions of cd1c mdc subpopulation in total cd1c mdcs are associated with uveitis disease activity . although the current data of cd1c mdcs are consistent with the published data , we were unable to correlate the proportions of cd1c mdc subpopulation with disease reactivation . additionally , we showed in vitro that modcs with low cd1c mdc proportions were associated with their impaired function in assisting allogeneic t - cell proliferation . this observation , which is consistent with our previously published data that modcs with high cd1c expression levels are correlated with high hladr expression and are partially regulated by p38-mapk pathway , suggests a possible explanation for the role of cd1c mdcs in the pathophysiology of noninfectious uveitis and a potential therapeutic target . receiver operating characteristic analysis showed that cd1c mdcs had an auc of 0.68 with low sensitivity ( 44% ) but high specificity ( 90% ) for disease reactivation at the cutoff level of 60% of cd1c mdcs . this cutoff corresponded to the average cd1c mdc proportion in active uveitis patients in our cohort . the high specificity of this test means that the likelihood of a false - positive result is low and a true - positive result is high . this is clinically valuable as it allows physicians to reserve prolonged immunosuppressive therapy for only those patients at highest risk for disease reactivation . on the other hand , the low sensitivity of this test means that some patients at risk for reactivation may have erroneously negative results . though interesting , it is apparent that our study is a preliminary investigation and that prospective studies with larger sample sizes and more rigorous controls are needed to validate cd1c mdcs as a biomarker for uveitis activity and reactivation . firstly , as we mentioned above , this was a single - center investigation with 89 patients and 111 healthy controls , which may limit the generalizability of our results . secondly , we were unable to follow up patients from the beginning of their treatment course , since all the patients were referred from different local clinics to nei . thirdly , while we were able to generate longitudinal data for patients , these patients were not seen at regular intervals and thus changes in disease course in the interim could be missed . fourthly , other factors may affect the cd1c mdc proportions , such as smoking , diet , and body mass index , which were not controlled in this study . fifthly , further mechanistic studies are required to understand the cause and effect of increased cd1c mdc proportion in uveitis , such as overexpression or knockout cd1c expression on modcs . in conclusion , the proportion of cd1c mdcs reflects disease activity and was associated with high risk of disease reactivation in some clinically quiescent patients with noninfectious uveitis .

purposeto test the association between elevated proportions of cd1c+ myeloid dendritic cells ( mdcs ) and disease activation / reactivation in noninfectious uveitis.methodsnoninfectious uveitis patients ( n = 89 ) and healthy controls ( n = 111 ) were recruited . the proportion of cd1c+ mdcs in the total dendritic cell ( dc ) population of peripheral blood was measured by flow cytometry ( cd1c+ mdcs gated on lineage 1+hladr+ dcs ) . disease activity was assessed per standardization of uveitis nomenclature criteria . uveitis reactivation was ascribed to clinically quiescent patients who developed reactivation of intraocular inflammation within 6 months.resultsthe proportions of cd1c+ mdcs were increased in noninfectious uveitis patients , especially in active disease , compared to healthy controls . this cd1c+ mdc elevation was not associated with underlying systemic diseases , anatomic locations of uveitis , medications , or demographic factors . longitudinal data showed that the dynamics of cd1c+ mdc levels were correlated with disease activity . the average proportion of cd1c+ mdcs in active uveitis patients was 60% so we set this as the cutoff between high and low cd1c+ mdc levels . although 74% of quiescent patients had low proportions of cd1c+ mdcs , 26% still had high proportions . quiescent patients with high cd1c+ mdc proportions showed increased risk of disease reactivation , compared to quiescent patients with low cd1c+ mdc proportions.conclusionsincreased proportions of cd1c+ mdcs were associated with clinical activity , and quiescent patients with elevated cd1c+ mdcs were more likely to undergo reactivation . this suggests that cd1c+ mdc proportion may be a potential biomarker for assessing clinical activation and reactivation in noninfectious uveitis .

Materials and Methods Study Design Clinical Evaluation CD1c Mixed Lymphocyte Reaction (MLR) Assay Statistical Analysis Results Study Participant Characteristics CD1c Elevated CD1c Elevated CD1c Dynamic Proportions of CD1c Elevated CD1c Monocyte-Derived Dendritic Cells With Low CD1c Discussion

noninfectious uveitis patients and healthy controls were recruited from the national eye institute ( nei ) clinic and the national institutes of health ( nih ) clinical center blood bank , respectively . disease activity was assessed by the treating physicians and reported according to the standardization of uveitis nomenclature ( sun criteria ) . cd1c mdcs and plasmacytoid dendritic cells ( pdcs ) were identified as the lineage cocktail 1 ( lin1)hladrcd1c and lin1hladrcd303 populations , respectively , as previously reported . noninfectious uveitis patients and healthy controls were recruited from the national eye institute ( nei ) clinic and the national institutes of health ( nih ) clinical center blood bank , respectively . disease activity was assessed by the treating physicians and reported according to the standardization of uveitis nomenclature ( sun criteria ) . cd1c mdcs and plasmacytoid dendritic cells ( pdcs ) were identified as the lineage cocktail 1 ( lin1)hladrcd1c and lin1hladrcd303 populations , respectively , as previously reported . noninfectious uveitis patients ( n = 89 ) seen at the nei and healthy controls ( n = 111 ) from the nih blood bank between january 2012 and july 2015 were included in the study . demographics ( age , sex , and race ) and clinical information ( underlying systemic diseases , anatomic locations of uveitis , medications , and disease activity ) of patients were recorded ( table ) . in general , patients with active disease were followed up every 2 weeks and quiescent patients were followed up every 2 months . characteristics of uveitis patients and healthy controls consistent with our prior observation , the proportions of cd1c mdcs in the total dc populations were significantly elevated in uveitis patients when compared to hcs ( hcs = 111 , uveitis = 89 , p = 0.0025 ; fig . furthermore , cd1c mdcs were significantly higher in patients with active disease as compared to those who were clinically quiescent ( p = 0.0052 ; fig . to assess any relative changes in the pdc populations in uveitis patients compared to hcs , we also measured the proportions of pdcs in the total dc populations by flow cytometry . ( a ) the proportions of cd1c mdcs were compared between hcs and noninfectious uveitis patients ( uveitis ) . subgroup univariate analyses revealed no difference in cd1c mdc proportions among uveitis patients with different underlying diseases including sarcoidosis , idiopathic , birdshot , vogt - koyanagi - harada ( vkh ) , behcet 's diseases , or others ( fig . uveitis patients were divided into subgroups on the basis of their underlying systemic diseases and locations of uveitis . ( a ) proportions of cd1c mdcs were compared in different systemic diseases including sarcoidosis , idiopathic , birdshot , vkh , behcet 's disease , and others ( anova test , p = 0.40 ) . ( b ) proportions of cd1c mdcs were compared in anatomic locations of uveitis including anterior , intermediate , posterior , and panuveitis ( anova test , p = 0.76 ) . monocyte - derived dendritic cells without treatment were the control group ( con , n = 6 ) and those treated with dimethyl sulfoxide ( dmso ) were the vehicle group ( veh , n = 6 ) because dex and csa were dissolved in dmso . proportions of cd1c mdcs and mean fluorescence intensity ( mfi ) of cd1c on mdcs were calculated by flow cytometry . cd1c mdc elevation was not significantly associated with the etiologic and anatomic classifications of uveitis , or types and dosages of immunosuppressive therapy . 3a cd1c mdc proportions from hcs ( a c ) or uveitis patients ( d f ) were divided into subgroups according to their ages including 30 , 31 to 40 , 41 to 50 , 51 to 60 , and > 60 years ( a , d ) ; sex including female and male ( b , e ) ; and race including caucasian , african american , and others ( e , f ) . among 44 patients who had longitudinal data of cd1c mdc levels with multiple time points , cd1c mdc proportions were not altered significantly in those patients who were initially clinically quiescent ( q ) and remained quiescent ( q - q , n = 25 , p = 0.35 ; fig . 4a ) , or in patients with active disease ( a ) who remained active between blood draws ( a - a , n = 10 , p = 0.67 ; fig . 4c ) and quiescent patients showed increased cd1c mdc levels when they developed active uveitis after tapering off medications ( q - a , n = 4 , p = 0.02 ; fig . plotting the average level of cd1c mdc proportions in continually quiescent patients ( q - q ) , continually active patients ( a - a ) , and patients with changes in disease activity from active to quiescent ( a - q ) or quiescent to active ( q - a ) , showed continually low , continually high , significantly decreasing , and significantly increasing cd1c mdc proportions , respectively ( fig . these results suggest that dynamic levels of cd1c mdc proportions are associated with disease activity . the data were compared in the groups of patients who maintained the quiescent uveitis ( [ a ] , quiescent to quiescent , q - q ) , of patients whose diseases were active during sampling ( [ b ] , active to active , a - a ) , of patients whose disease activity changed from the active to quiescent ( [ c ] , a - q ) or from the quiescent to active ( [ d ] , q - a ) . 1b ) , we noticed that while most quiescent patients had low proportions of cd1c mdcs , 26% of quiescent patients still had cd1c mdc proportions that were higher than the average proportion in active uveitis patients ( 60% of cd1c mdcs ) . we hypothesized that these quiescent patients with elevated cd1c mdc proportions were at increased risks for disease reactivation . to test this hypothesis , we divided quiescent patients ( n = 34 ) into high ( n = 10 ) and low ( n = 24 ) cd1c mdc proportion subgroups on the basis of a cutoff of cd1c mdc proportion in total dc population , which was 60% . interestingly , 6 of 24 quiescent patients ( 25% ) had disease reactivation in the low cd1c subgroup compared to 7 of 10 patients ( 70% ) in high cd1c subgroup ( p = 0.01 ; fig . 5b ) , indicating that cd1c mdc proportions were associated with subsequent uveitis reactivation . the level of cd1c mdcs at 60% was set as the cutoff , which was the average proportional level in active uveitis patients . ( a ) patients ( n = 34 ) were divided into low levels of cd1c proportion ( 60% of cd1c mdcs ) and high levels of cd1c proportion ( > 60% of cd1c mdcs ) groups ( low cd1c versus high cd1c ) . six of 24 patients had disease reactivation ( 25% ) in low cd1c proportion group , and 7 of 10 patients ( 70% ) had disease recurrence in high cd1c proportion group . ( b ) cd1c mdcs proportions were compared in the groups of quiescent patients with or without disease reactivation ( no reactivation versus reactivation ) . to investigate whether the elevated cd1c mdc proportions observed in noninfectious uveitis patients had effects on adaptive immune response , we performed an in vitro study to evaluate the effects of cd1c expression on mdc t - cell interactions . monocyte - derived dendritic cells with low cd1c proportions were associated with impaired allogeneic t - cell proliferation . ( a ) proportions of cd1c modcs were measured by flow cytometry ( n = 4 ) . noninfectious uveitis patients ( n = 89 ) seen at the nei and healthy controls ( n = 111 ) from the nih blood bank between january 2012 and july 2015 were included in the study . demographics ( age , sex , and race ) and clinical information ( underlying systemic diseases , anatomic locations of uveitis , medications , and disease activity ) of patients were recorded ( table ) . in general , patients with active disease were followed up every 2 weeks and quiescent patients were followed up every 2 months . characteristics of uveitis patients and healthy controls consistent with our prior observation , the proportions of cd1c mdcs in the total dc populations were significantly elevated in uveitis patients when compared to hcs ( hcs = 111 , uveitis = 89 , p = 0.0025 ; fig . furthermore , cd1c mdcs were significantly higher in patients with active disease as compared to those who were clinically quiescent ( p = 0.0052 ; fig . to assess any relative changes in the pdc populations in uveitis patients compared to hcs , we also measured the proportions of pdcs in the total dc populations by flow cytometry . ( a ) the proportions of cd1c mdcs were compared between hcs and noninfectious uveitis patients ( uveitis ) . subgroup univariate analyses revealed no difference in cd1c mdc proportions among uveitis patients with different underlying diseases including sarcoidosis , idiopathic , birdshot , vogt - koyanagi - harada ( vkh ) , behcet 's diseases , or others ( fig . 2a ) , and different anatomic locations ( anterior , intermediate , posterior , or panuveitis ; fig uveitis patients were divided into subgroups on the basis of their underlying systemic diseases and locations of uveitis . ( a ) proportions of cd1c mdcs were compared in different systemic diseases including sarcoidosis , idiopathic , birdshot , vkh , behcet 's disease , and others ( anova test , p = 0.40 ) . ( b ) proportions of cd1c mdcs were compared in anatomic locations of uveitis including anterior , intermediate , posterior , and panuveitis ( anova test , p = 0.76 ) . monocyte - derived dendritic cells without treatment were the control group ( con , n = 6 ) and those treated with dimethyl sulfoxide ( dmso ) were the vehicle group ( veh , n = 6 ) because dex and csa were dissolved in dmso . proportions of cd1c mdcs and mean fluorescence intensity ( mfi ) of cd1c on mdcs were calculated by flow cytometry . cd1c mdc elevation was not significantly associated with the etiologic and anatomic classifications of uveitis , or types and dosages of immunosuppressive therapy . 3a cd1c mdc proportions from hcs ( a c ) or uveitis patients ( d f ) were divided into subgroups according to their ages including 30 , 31 to 40 , 41 to 50 , 51 to 60 , and > 60 years ( a , d ) ; sex including female and male ( b , e ) ; and race including caucasian , african american , and others ( e , f ) . among 44 patients who had longitudinal data of cd1c mdc levels with multiple time points , cd1c mdc proportions were not altered significantly in those patients who were initially clinically quiescent ( q ) and remained quiescent ( q - q , n = 25 , p = 0.35 ; fig . 4a ) , or in patients with active disease ( a ) who remained active between blood draws ( a - a , n = 10 , p = 0.67 ; fig . 4c ) and quiescent patients showed increased cd1c mdc levels when they developed active uveitis after tapering off medications ( q - a , n = 4 , p = 0.02 ; fig . plotting the average level of cd1c mdc proportions in continually quiescent patients ( q - q ) , continually active patients ( a - a ) , and patients with changes in disease activity from active to quiescent ( a - q ) or quiescent to active ( q - a ) , showed continually low , continually high , significantly decreasing , and significantly increasing cd1c mdc proportions , respectively ( fig . these results suggest that dynamic levels of cd1c mdc proportions are associated with disease activity . the data were compared in the groups of patients who maintained the quiescent uveitis ( [ a ] , quiescent to quiescent , q - q ) , of patients whose diseases were active during sampling ( [ b ] , active to active , a - a ) , of patients whose disease activity changed from the active to quiescent ( [ c ] , a - q ) or from the quiescent to active ( [ d ] , q - a ) . 1b ) , we noticed that while most quiescent patients had low proportions of cd1c mdcs , 26% of quiescent patients still had cd1c mdc proportions that were higher than the average proportion in active uveitis patients ( 60% of cd1c mdcs ) . we hypothesized that these quiescent patients with elevated cd1c mdc proportions were at increased risks for disease reactivation . to test this hypothesis , we divided quiescent patients ( n = 34 ) into high ( n = 10 ) and low ( n = 24 ) cd1c mdc proportion subgroups on the basis of a cutoff of cd1c mdc proportion in total dc population , which was 60% . interestingly , 6 of 24 quiescent patients ( 25% ) had disease reactivation in the low cd1c subgroup compared to 7 of 10 patients ( 70% ) in high cd1c subgroup ( p = 0.01 ; fig . 5b ) , indicating that cd1c mdc proportions were associated with subsequent uveitis reactivation . the level of cd1c mdcs at 60% was set as the cutoff , which was the average proportional level in active uveitis patients . ( a ) patients ( n = 34 ) were divided into low levels of cd1c proportion ( 60% of cd1c mdcs ) and high levels of cd1c proportion ( > 60% of cd1c mdcs ) groups ( low cd1c versus high cd1c ) . six of 24 patients had disease reactivation ( 25% ) in low cd1c proportion group , and 7 of 10 patients ( 70% ) had disease recurrence in high cd1c proportion group . ( b ) cd1c mdcs proportions were compared in the groups of quiescent patients with or without disease reactivation ( no reactivation versus reactivation ) . to investigate whether the elevated cd1c mdc proportions observed in noninfectious uveitis patients had effects on adaptive immune response , we performed an in vitro study to evaluate the effects of cd1c expression on mdc t - cell interactions . monocyte - derived dendritic cells with low cd1c proportions were associated with impaired allogeneic t - cell proliferation . ( a ) proportions of cd1c modcs were measured by flow cytometry ( n = 4 ) . our study showed that cd1c mdc proportions were increased in uveitis patients and that this elevation appeared to be correlated with clinical activity . in univariate analyses , high proportions of cd1c mdcs were significantly associated with disease activity , while other possible clinical parameters , such as underlying systemic diseases , anatomic locations of uveitis , current immunosuppressive medications , age , sex , and race , were not . these patients subsequently had a higher rate of disease reactivation as compared to quiescent patients with low cd1c mdc proportions . thus , cd1c mdc proportion may have the potential to be a biomarker for uveitic disease activity and reactivation . in our previous publications , we have reported elevated proportions of cd1c mdcs in a small cohort of noninfectious uveitis patients when compared to healthy controls and have demonstrated that cd1c proportion rises and decreases with changes in clinical uveitis activity . in this study , with a larger sample size , we supported our previous findings of ( 1 ) elevated cd1c mdc proportions in uveitis patients compared to healthy controls and ( 2 ) a correlation between cd1c mdc proportion and disease activity . in the longitudinal data analysis , proportions of cd1c mdc closely correlated with disease activity . of our 44 patients with longitudinal cd1c data , only 4 quiescent patients activated during the blood sampling period as compared to 14 active patients who became quiescent . further clinical follow - up ultimately validated the overall correlation between cd1c mdc proportion and disease activity : the quiescent patient suffered disease recurrence 7 weeks after the second blood draw and the two active patients became quiescent 1 and 3 weeks after the second blood draw . although most quiescent patients had low proportions of cd1c mdcs , there was still a relatively small population of quiescent patients ( 26% ) that maintained high cd1c mdc proportions in our cohort as defined by the average proportion of cd1c mdcs in active uveitis patients ( > 60% of cd1c mdcs ) . these quiescent patients with high cd1c mdc proportions had significantly higher rate of subsequent disease reactivation as compared to quiescent patients with low cd1c mdc proportions ( reactivation rate in high versus low cd1c groups : 70% vs. 25% ) . however , we were unable to find a correlation between cd1c mdc proportions and time to reactivation in clinically quiescent patients owing to the lack of a consistent follow - up schedule for all patients . in addition , we have previously found that the proportions of cd1c mdc subpopulation in total cd1c mdcs are associated with uveitis disease activity . additionally , we showed in vitro that modcs with low cd1c mdc proportions were associated with their impaired function in assisting allogeneic t - cell proliferation . this observation , which is consistent with our previously published data that modcs with high cd1c expression levels are correlated with high hladr expression and are partially regulated by p38-mapk pathway , suggests a possible explanation for the role of cd1c mdcs in the pathophysiology of noninfectious uveitis and a potential therapeutic target . receiver operating characteristic analysis showed that cd1c mdcs had an auc of 0.68 with low sensitivity ( 44% ) but high specificity ( 90% ) for disease reactivation at the cutoff level of 60% of cd1c mdcs . this cutoff corresponded to the average cd1c mdc proportion in active uveitis patients in our cohort . in conclusion , the proportion of cd1c mdcs reflects disease activity and was associated with high risk of disease reactivation in some clinically quiescent patients with noninfectious uveitis .

the snf2 protein was originally identified as a result of genetic screens for genes involved in regulating mating type switching ( swi ) and sucrose fermentation ( sucrose non - fermenting ) . it was subsequently found to be the catalytic subunit of the multi - subunit swi / snf complex that acts to alter chromatin structure ( 1 ) . since this time , many proteins have been identified that are related to snf2p through sequence similarity . the common feature of all such snf2 family proteins is a region of sequence similarity that includes seven helicase - related sequence motifs that are also found in dexx box helicases ( 25 ) . helicase - related proteins are classified in superfamilies ( sf1 , sf2 , sf3 etc ) based from the sequence and spacing of these motifs . snf2 family proteins fall into sf2 whereas dexx box helicases are classified variously into sf1 , sf2 and sf3 ( 6 ) . however , snf2 family proteins are unusual compared to typical sf2 members , such as dead box helicases : the spacing between helicase - related motifs iii and iv is significantly elongated , their helicase - related motifs ia , iii , iv , v and vi have a specific and conserved character , and they contain a number of other conserved sequence blocks ( 2,7 ) . the rapid progress of genome sequencing has revealed a significant breadth and diversity within the snf2 family . for example , there are 17 genes for snf2 family proteins in saccharomyces cerevisiae ( table 1 ) and at least 32 in humans ( 8) . it has been appreciated for some time that snf2 family members fall into various groupings or subfamilies ( 2 ) , and a current survey shows they can be divided into some 24 different subfamilies on the basis of primary sequence of the common helicase - like region ( 8) . for example , the 17 yeast snf2 family proteins fall into at least 12 distinct subfamilies ( table 1 ) . these subfamilies correlate well with known biological functions , suggesting that the helicase - like region is not a generic motor but is instead highly tuned for its biochemical role . this is supported by the recent observation that the swapping of the helicase - like region between snf2h and brg1 has a dominant effect on the activity of the resulting chimeric atpases ( 9 ) . for instance , a snf2h brg1 chimera that contains the helicase - related domain of brg1 and the n- and c - terminal domains of snf2h exhibits brg1 like remodelling properties , and vice versa . many snf2 family proteins are part of larger complexes . for instance , the swi / snf , rsc , nurf , acf and ino80 complexes each contain many polypeptides with combined molecular weights in the megadalton range ( 1014 ) . however , in several cases the isolated snf2 family polypeptides alone have biochemical activity , but reduced efficiency relative to the intact complexes ( 1517 ) . some snf2 family polypeptides , such as chd1p ( 18 ) and rad54p ( 19 ) can be purified from yeast and show function without additional proteins . this suggests that in many cases the additional proteins of the large remodeller complexes may function to enhance specificity , processivity or targeting but are not central to the generation of mechanical force . snf2 family polypeptides also almost invariably contain one or more domains in addition to the helicase - like region . a number of these extra domains have been shown to interact with surfaces on the nucleosome , often involving post - translational modifications . bromodomains which bind acetyl lysine ( 20 ) , whereas drosophila iswi and its homologues contain myb - like sant domains ( 2124 ) . although sant domains are structurally related to the dna - binding domain of myb , they are functionally diverged to bind to unmodified histone tails ( 25 ) . mouse chd1 and homologues contain chromodomains which bind methyl lysine ( 26,27 ) and also nucleic acids ( 28,29 ) . it has been demonstrated that removing the myb - like domains of iswi ( 22 ) or the chromodomains of mi-2 ( 29 ) compromises remodelling in vitro . historically , the involvement of the archetypal yeast snf2 protein in chromatin remodelling has led to assumptions that all snf2 family proteins are chromatin remodellers . although snf2 family proteins are ubiquitous in eukaryotes , they are also found in a significant number of the sequenced prokaryotic and archaeal genomes , which lack eukaryotic - type chromatin . the more distant of these , on the border of the snf2 family , includes escherichia coli rapa ( also known as hepa ) , which has been shown to be involved in polymerase recycling under high salt conditions when dna is more highly supercoiled ( 30 ) . similarly , a number of eukaryotic snf2 family members have functions which do not seem to be directly linked to chromatin . for example , yeast mot1p acts to displace the transcription factor tbp from dna ( 31,32 ) , the transcription - coupled repair factor cockayne syndrome protein b rescues rna polymerase that is stalled at dna lesions ( 3336 ) , and the rad16 complex facilitates nucleotide excision repair ( 37 ) . despite the diversity of biochemical activity , the presence of the conserved snf2 family atpase domain engine suggests that shared mechanistic features may underlie the way by which these proteins act . in other dexx box enzymes the helicase - related motifs act to transduce the energy of atp - hydrolysis into a conformational stress required for the remodelling of nucleic acid or protein nucleic acid structure . like many helicases , atp - hydrolysis activity of snf2 family enzymes is stimulated by dna or dna protein substrates ( 15,31,3841 ) . however , a longstanding puzzle stems from the fact that snf2 family enzymes do not show the dna unwinding activity which defines helicases . recently , a variety of biochemical and structural results indicate that instead of duplex unwinding , snf2 family enzymes use the energy of atp - hydrolysis to translocate on duplex dna by a mechanism that does not require strand - separation ( 17,4245 ) . in this respect , they may act similarly to other members of sf2 , such as the translocating subunits of type i restriction enzymes ( 46 ) . significantly , the passage of a translocase along dna provides a direct means of altering dna protein contacts . in addition , because the path of dna is helical , translocation is also likely to be associated with rotation of dna , which could indirectly manipulate protein dna interactions . consistent with this , several snf2 family proteins have been shown to generate torsion in dna ( 37,43,45,4749 ) . are there common mechanistic principles linking snf2 family enzymes and dexx box helicases ? amongst the various groups of proteins within the sf1 and sf2 superfamilies of helicase - like proteins , a great deal of progress has recently been made in understanding the mechanism of dna translocation and unwinding by dexx box helicases . in particular , detailed structural knowledge of the interaction of helicases with dna has been obtained for the sf1 helicase bacillus stearothermophilus pcra in complex with a 3-tail partial duplex dna and in presence or absence of ampnp ( 50,51 ) , the sf2 helicase ns3 from hepatitis c virus in complex with deoxyuridine octamer ( du8 ) ( 52 ) , the sf2 thermatoga maritima recg in complex with a three way junction ( 53 ) , the sf2 dead box rna helicase vasa in complex with ssrna ( 54 ) and the sf1 related helicase complex recbcd in complex with a partially unwound dsdna substrate duplex ( 55 ) . these crystal structures revealed an underlying common structural fold and a modular structural organization ( 6 ) , and showed that these helicases typically consist of a dna translocation module linked to a strand - separation module ( 56 ) . a notable exception is vasa , where the dexx box atpase module appears to bend dna instead of translocating on it . the translocation module is highly conserved among helicases and consists of two reca - like domains , plus their associated structural elements ( domain 1 and 2 ) ( 50 ) . residues of the seven helicase - related motifs line the interdomain cleft separating the two reca - like domains and are involved in atp - binding / hydrolysis as well as dna - binding ( 57,58 ) . in addition , atp - binding into the interface cleft has been shown to induce a conformational change that is linked to dna translocation ( 51 ) . the available structures suggest that nucleic acids bind across the interface of the two reca - like domains ( 51,52,54 ) . depending on the nature of the particular helicase , the translocation module can bind duplex dna ( e.g. recg ) or single - stranded nucleic acids ( e.g. ns3 ) . biochemical analysis suggests that nucleic acid translocation and duplex dna unwinding are separable processes in the reaction cycle of helicases ( 53,59,60 ) . consistent with earlier observations , recent structures of the sf1 helicase uvrd highlight the fact that the structural movements of sf1 helicase domains , which underlie the translocation and unwinding processes , are closely correlated ( w. yang , personal communication ) . two dna - binding sites alternate in high affinity for dna and move the enzyme along the dna in a process that resembles the movement of an inchworm . during translocation , atp - driven conformational changes between the two reca - like domains result in a closure of the cleft between them , advancing dna by one base at a time , as judged from the pcra dna complex crystal structures ( 51 ) . the closed arrangement of the domains is largely stabilized by interaction of a conserved arginine residue in helicase - related motif vi with the -phosphate of the bound atp . atp - dependent closure and opening of the active site cleft could result in an alternating sliding of one domain , while the other domain tightly grips the dna and serves as an anchor to generate inchworm - like progress . recent biochemical observations indicate that within this cycle , single - strand binding provides a large part of the energy for unwinding , while atp - binding weakens the interaction with dna and allows the advance on the product strand ( 61 ) . however , the detailed mechanism of helicases can be more complex , involving cycles of rapid advancement by many bases followed by pausing ( 62 ) . the fold similarity suggests that sf2 helicases might in principle function in an analogous way . a mechanism similar to that of sf1 enzymes was postulated for the sf2 helicases ns3 and recg ( 53,63 ) . the crystal structure of ns3 revealed that both reca - like domains contact the single - stranded dna ( 52 ) . atp mediated weakening of the dna - binding strength of domain 1 , followed by a change of the relative orientation of domain 1 with respect to domain 2 that tightly grips dna via val432 , would result in concomitant translocation of the dna in ns3 . this is consistent with atp - dependent changes of the dna - binding properties of ns3 , which become weaker in the presence of atp ( 64 ) . . a closer inspection of the crystal structure of sf1 and sf2 helicases in complex with dna reveals a divergence in the mode of dna - binding . for instance , pcra binds ssdna mainly through hydrophobic contacts , which are formed by aromatic side chains that stack against the dna base moieties . in contrast , the sf2 helicases ns3 and vasa bind ssdna predominantly by recognition of the dna phosphate backbone ( 54,56,65 ) . for high affinity ssdna - binding , ns3 possesses a specialized domain that is attached to domain 1 and specifically interacts with the bases of single - strand nucleic acids . on the other hand , the sf2 helicase recg lacks the ssdna - binding domain of ns3 and translocates double - strand dna ( 63 ) . unfortunately , the dna in the recg crystal structure does not extend as far as the reca - like domains . biochemical studies of nucleic acid recognition by the sf2 helicase nph - ii suggest that sf2 helicases maintain continuous contact with the phosphodiester linkage of one substrate strand ( 65 ) . this suggests a molecular wire stripper like mechanism , where one domain maintains contact with the substrate strand during the cycles of tight binding and sliding , while the other domain grabs and releases the nucleic acid strand . overall , the generation of force to move the dna phosphate backbone across the surface of sf2 helicases is much less understood than the pulling of dna bases by pcra . snf2 family proteins were placed within sf2 in the original studies of gorbalenya and koonin ( 66 ) . in the last decade , biochemical characterization and recent crystal structures have suggested that the catalytic core of the snf2 family enzymes is a structure and sequence unspecific translocase for duplex dna ( 17,4244,67 ) . the two new crystal structures of zebrafish rad54 and the archaeal sulfolobus solfataricus sso1653 gene product in complex with a dsdna substrate have revealed the first structural insights into this motor ( 43,68 ) . the crystallized fragments are active atpases that can translocate on dna , introduce superhelical tension in dna , and/or remodel chromatin . additional high - resolution structural information is also available for the nucleosome binding domain of iswi ( 23 ) , although the nature of the interaction between this region and the catalytic motor is still unknown . the crystal structures of the catalytic domains from the two snf2 family members revealed that the enzymes possess two domains ( domain 1 and 2 ) , each containing a core reca - like fold ( 1a and 2a ) , fused to snf2 family specific helical domains ( 1b and 2b ) ( figure 1 ) . the two reca - like domains are related to the equivalent region of the dexx box helicases and contain the seven characteristic helicase - related motifs . this similarity suggests that snf2 family atpases and dexx box helicases possess a related atp - hydrolysis mechanism and probably exhibit related atp - driven conformational changes . comparison of the zebrafish and s.solfataricus structures revealed a highly similar fold , but with large conformational difference in the orientation of the two reca - like domains ( 43,68 ) : the orientation of the second reca - like domain with respect to the first differs by 180 between the crystal structures . biochemical analysis of mutant sso1653 suggests that the second reca - like domain can adopt a domain orientation that is similar to zebrafish rad54 during the atp - hydrolysis cycle . only in the conventional conformation are all conserved helicase - related sequence motifs located in the atp - binding cleft . such a conformational change is also consistent with data for yeast mot1p , where a mutation in the proposed pivot point of the two reca - like domains abolishes atp - hydrolysis activity ( 69 ) . whether this proposed large scale conformational change is part of the functional atp - binding and hydrolysis cycle of swi2/snf2 enzymes or represents a dna loading conformation , is not known . the recently determined structure of trcf also has a sf2 helicase - like region related to recg . in it , the second reca - like domain is rotated by some 90 relative to the conventional orientation ( 70 ) . this suggests a highly flexible domain connection between the two reca - like domains of sf2 atpases . in this respect , it might be interesting to probe conformational sub - strates of snf2 family enzymes in the presence of various nucleotides by proteolysis or fluorescence resonance energy transfer experiments . in any case , both crystallographic studies revealed the presence of the typical seven helicase - related motifs ( 43,68 ) . mutational analysis has revealed the significance of these motifs in remodeling processes or translocation ( 38,43,68 ) . interestingly , the structural and mutational analysis also suggested a variety of additional motifs that could be involved in dsdna stimulated atpase activity [ iia in ref . motif iva , also dubbed the qxxr motif , has recently been identified as a nucleic acid phosphate - binding motif in the vasa dead box rna helicase ( 54 ) . the structure of the sso1653 region was determined bound to duplex dna and provides a first detailed structural insight into how the snf2 family atpase domain interacts with dna ( 43 ) . duplex dna binds along the domain 1a : domain 2a interface in a position that is in principle well suited for dna translocation by atp - driven conformational changes between domains 1a and 2a . comparison with the sf2 helicase recg ( 53 ) suggests that the translocase module of recg could interact with dna in an analogous way ( figure 1 ) . the total footprint of both phosphate chains amounts to about 67 nt , comparable with footprint of ssdna bound to helicases . the snf2 family specific domains 1b and 2b might , by analogy with the accessory domains of the sf1 helicases , play a key role in dna translocation . do not directly contact dna in the available crystal structure so their precise role is unclear . duplex dna is bound to the s.solfataricus enzyme in b - form conformation without evidence for strand - separation . pin-like features that force the duplex apart , or to bind the nucleic acids in a sharp bend , which is incompatible with base pairing . the lack of either type of duplex - destabilizing region in snf2 family enzymes parallels the lack of helicase activity . instead , the minor groove binding of b - form dna suggests that snf2 family enzymes probably track on dna without strand - separation activity . this is similar to the non - helicase sf2 motor of a type i restriction enzyme , which can move on dna with crosslinked strands ( 46 ) . what can we infer about the mechanism of dna translocation by snf2 family enzymes from dexx box helicases ? the remarkable structural and topological similarities of the core architecture in the two reca - like domains suggest that snf2 family atpases and sf2 helicases share a fundamentally similar atp - hydrolysis mechanism ( figure 2 ) . in particular , the structure and arrangement of the atp and mg binding motifs i ( walker a ) , ii ( walker b ) and vi correspond closely to equivalent motifs of other dexx box enzymes . importantly , an invariant arginine residue of motif vi that conducts domain communication and senses atp - hydrolysis is conserved in snf2 family enzymes ( 39 ) . dna binds to the sso1653 snf2 family structure along a similar surface path to both sf1 and sf2 helicases ( 43,51,52 ) . most intriguingly , the 35 strand of the dna duplex bound to the sso1653 structure overlays very well with the 35 oligo(du ) strand bound to the dna helicase pcra and the rna helicases ns3 and vasa ( 43,52,54 ) . therefore , it is likely that atp - driven opening and closing of the cleft between the reca - like domains transports the 35 strand in helicases and snf2 enzymes in an analogous manner . in support of such a mechanistic similarity , biochemical studies indicated that the integrity of this 35 strand is more important for activity , while gaps in the 53 strand can be tolerated during translocation ( 17,71 ) . recent single molecule and bulk solution measurements on the dsdna motor protein ecor124i , an enzyme related to snf2 atpases , also indicate that an intact 35 strand is required for translocation , whereas the 53 strand only assists in processivity ( 46 ) . we do not currently know how the force is generated to propel dna along its binding groove in snf2 family enzymes . based on present insights , for example , in analogy to the inchworm model , alternating high and low affinity binding sites in domain 1 and 2 could transport dna along the dna backbone . however , biochemical data show that domain 2 of sso1653 has no significant dna - binding affinity by itself , whereas domain 1 readily binds dna ( 43 ) . alternatively , atp - driven closure of the active site cleft might push on upstream dna . such a push could for instance propel the enzyme forward in analogy to the punting technique of fishermen ( k. theis , personal communication ) . alternative mechanisms are also consistent with current information , such as the formation of small loops in the 35 strands ( 46 ) . for instance , an adp - driven conformational change has been identified in the isw2 complex ( 72 ) . whether similar adp dependent conformational changes exist in other snf2 enzymes , in addition , the crystallographic analysis is only a first snapshot , and structures of snf2 enzymes in complex with dna plus adp or atp are needed to provide further insights into the dna transport mechanism . although , all chromatin remodelling complexes have a snf2 family polypeptide at their core , they contain divergent subunit compositions and exhibit many different functional roles in vivo and in vitro . for example , the action of different complexes can result in nucleosome sliding in cis , histone transfer in trans and histone variant exchange . support for the idea that remodelling complexes use atp - dependent translocation on duplex dna to generate the force in remodelling processes comes from recent single molecule biophysical analysis of the rsc complex ( 45 ) . in principle , translocation along dna by a motor domain could disrupt dna protein complexes or slide nucleosomes simply by collision . such a sliding effect has been found to occur when polymerases collide with nucleosomes ( 73 ) . however , the action of specialized remodellers is probably much more complex and involves the specific positioning of the motor domain relative to the remodelled substrate . for example , some of the larger chromatin remodeling complexes are big enough to encapsulate nucleosomes or bind to substrates via multiple binding sites ( 7476 ) . in these cases , differences in the location and orientation with which the translocating motor engages dna may influence the outcome of remodeling reactions . for example , recent studies point to an important role for dna contacts within the nucleosome for action of the swi / snf and rsc complexes ( 71,77 ) whereas nucleosome spacing enzymes , such as isw2 make contact both with nucleosomes and the adjacent dna ( 77,78 ) . once engaged , translocation of the motor domain along the minor groove will necessarily lead to a pulling or pushing force due to the translocation component , and also to a twisting force due to the rotation component of minor groove tracking . this would be expected to result in the application of a force between the atpase motor and other contacts restraining the enzyme relative to the dna or remodelled substrate . for instance , the sant and slide domains could anchor iswi to nucleosomes ( 23 ) . likewise , the n - terminal region of mot1 binds the tata box binding ( tbp ) protein ( 31 ) . in the case of nucleosome sliding , the force created by the motor domains could for example result in the generation of dna loops that peel dna away from the surface of the histone octamer . alternatively , the rotation of dna could lead to an altered twist that disrupts the histone dna complex . diffusion of these types of distortion around the histone octamer provides an attractive means by which these enzymes might alter chromatin structure ( 1 ) . in this way , transient or non - processive alterations may be sufficient to cause persistent changes to chromatin structure . while it is possible to conceive how directed dna translocation may underlie the mechanism by which many atp - dependent chromatin remodelling enzymes act , this need not necessarily be the case for all snf2 family proteins . for example , mot1p has no detectable dna translocase activity , but disrupts tbp dna complexes ( 79 ) . the csb protein has been observed to wrap dna around itself in an atp - dependent reaction ( 80 ) . further investigation will be required to determine how the mechanisms by which these proteins act are related to their chromatin remodeling siblings ! structural homology of the two reca - like domains suggest that snf2 family dsdna translocases and sf2 helicases use related mechanisms for atp - driven transport of their nucleic acid substrates across the active site cleft . based on these structures , a model of dsdna translocation by snf2 family enzymes and possibly other dsdna translocases can be envisioned that is similar to the ssdna translocation by dexx box helicases . however , detailed insights how atp - driven conformational changes propel duplex dna along the active site of the snf2 atpase domain are still missing and need to be addressed in future studies . in this regard , it will be interesting to probe conformational changes of snf2 enzymes at the single molecule level , or to probe the precise structure of dna during the translocation process . recent breakthroughs in the application of this technique to remodelling complexes and helicases can reveal a wealth of mechanistic insights how these enzymes move on dna ( 45,81,82 ) . with structures of snf2 enzymes in complex with atp or adp , and analysis of conformational changes using tools like fluorescence energy resonance transfer or small angle solution scattering , we should be able to dissect the conformational substates of remodellers and the mechanistic coupling of atp - binding with dna transport . in addition , we are only at the beginning of our understanding of how the force generated by the snf2 translocase module is used by complex multidomain remodelling factors . for instance , more detailed studies on the reaction cycle of snf2 family enzymes , how they engage with their substrates and the nature and role of conformational changes within the reca - like domains are needed . in this regard , what is the role of the various domains that flank the translocase module of snf2 family enzymes ? do they target the enzyme to particular places on the genome , do they grip the substrate to provide a handle for the action of the atpase module , or do they do both ? ultimately , we need to understand how dna tracking by the translocase domains generates the diverse range of macromolecular changes in substrate dna protein complexes during the course of remodelling reactions . comparison of structures of snf2 family enzymes and the recg helicase . structural comparison of the s.solfataricus ( sso ) sso1653 catalytic domain [ cd , ( 43 ) , the zebrafish ( zf ) rad54 cd ( 68 ) and t.maritima ( tm ) recg ( 53 ) . the two reca - like domains ( 1a : orange , 2a : green ) are shared across dexx box atpases , and form the atp - binding site in their interface cleft . the location of the atp - binding site , as well as locations of the seven conserved helicase - related atpase / dna - binding motifs ( ia , i , ii , iii , iv , v , vi ) are indicated in tmrecg . the two reca - like domains interact with additional domains ( 1b and 2b : blue ) that are suggested to convert atp - driven rearrangements of 1a and 2a into the translocase or dna unwinding function . for instance , these domains bind to the replication fork substrate in recg , which is dragged like a plough through dna by the action of the translocase module . the role of the helical domains of snf2 family enzymes is not as well understood , but they could play a role in advancing the enzyme by atp - driven conformational changes . note that domain 2 of sso1653 ( 2a and 2b ) is flipped by 180 with respect to more typical conformations found in zfrad54cd and recg ( double arrow ) . schematic comparison of ( a ) dsdna translocases ( e.g. snf2 ) and ( b ) ssdna translocases ( e.g. pcra and ns3 helicases ) . both enzyme families contain a conserved reca - like domain core ( orange / green ) , but differ in other subunits ( data not shown ) . helicases transport product ssdna and often contain an upstream dna unwinding element ( grey triangle ) . in contrast , snf2 family enzymes also recognize the 53 strand ( blue ) and track along the minor groove . despite many functional differences , however , both enzymes families bind the 35 strands at an equivalent site across the two reca - like domains , indicating that atp - driven conformational changes transport dna substrates via the 35 strands in analogous ways ( arrows ) . snf2 family genes identified in s.cerevisiae official gene name and chromosomal locus as recorded in the s.cerevisiae genome database ( sgd ; ) .

proteins with sequence similarity to the yeast snf2 protein form a large family of atpases that act to alter the structure of a diverse range of dna protein structures including chromatin . snf2 family enzymes are related in sequence to dexx box helicases , yet they do not possess helicase activity . recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves atp - dependent translocation on dna . crystal structures suggest that these enzymes travel along the minor groove , a process that can generate the torque or energy in remodelling processes . we review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both snf2 family and dexx box helicases .

PRIMARY SEQUENCE PROPERTIES OF THE SNF2 FAMILY BIOCHEMICAL ACTIVITIES OF SNF2 PROTEINS REVIEW OF HELICASE STRUCTURE AND MECHANISM THE STRUCTURE OF THE SNF2 ATPASE DOMAIN UNIFIED MECHANISM FOR SNF2 ENZYMES AND DEXX BOX HELICASES HOW DO REMODELLING MACHINES WORK? CONCLUSIONS AND OUTLOOK Figures and Tables

the snf2 protein was originally identified as a result of genetic screens for genes involved in regulating mating type switching ( swi ) and sucrose fermentation ( sucrose non - fermenting ) . it was subsequently found to be the catalytic subunit of the multi - subunit swi / snf complex that acts to alter chromatin structure ( 1 ) . since this time , many proteins have been identified that are related to snf2p through sequence similarity . the common feature of all such snf2 family proteins is a region of sequence similarity that includes seven helicase - related sequence motifs that are also found in dexx box helicases ( 25 ) . snf2 family proteins fall into sf2 whereas dexx box helicases are classified variously into sf1 , sf2 and sf3 ( 6 ) . however , snf2 family proteins are unusual compared to typical sf2 members , such as dead box helicases : the spacing between helicase - related motifs iii and iv is significantly elongated , their helicase - related motifs ia , iii , iv , v and vi have a specific and conserved character , and they contain a number of other conserved sequence blocks ( 2,7 ) . the rapid progress of genome sequencing has revealed a significant breadth and diversity within the snf2 family . it has been appreciated for some time that snf2 family members fall into various groupings or subfamilies ( 2 ) , and a current survey shows they can be divided into some 24 different subfamilies on the basis of primary sequence of the common helicase - like region ( 8) . for example , the 17 yeast snf2 family proteins fall into at least 12 distinct subfamilies ( table 1 ) . these subfamilies correlate well with known biological functions , suggesting that the helicase - like region is not a generic motor but is instead highly tuned for its biochemical role . this is supported by the recent observation that the swapping of the helicase - like region between snf2h and brg1 has a dominant effect on the activity of the resulting chimeric atpases ( 9 ) . for instance , a snf2h brg1 chimera that contains the helicase - related domain of brg1 and the n- and c - terminal domains of snf2h exhibits brg1 like remodelling properties , and vice versa . many snf2 family proteins are part of larger complexes . however , in several cases the isolated snf2 family polypeptides alone have biochemical activity , but reduced efficiency relative to the intact complexes ( 1517 ) . some snf2 family polypeptides , such as chd1p ( 18 ) and rad54p ( 19 ) can be purified from yeast and show function without additional proteins . this suggests that in many cases the additional proteins of the large remodeller complexes may function to enhance specificity , processivity or targeting but are not central to the generation of mechanical force . snf2 family polypeptides also almost invariably contain one or more domains in addition to the helicase - like region . although sant domains are structurally related to the dna - binding domain of myb , they are functionally diverged to bind to unmodified histone tails ( 25 ) . historically , the involvement of the archetypal yeast snf2 protein in chromatin remodelling has led to assumptions that all snf2 family proteins are chromatin remodellers . similarly , a number of eukaryotic snf2 family members have functions which do not seem to be directly linked to chromatin . despite the diversity of biochemical activity , the presence of the conserved snf2 family atpase domain engine suggests that shared mechanistic features may underlie the way by which these proteins act . in other dexx box enzymes the helicase - related motifs act to transduce the energy of atp - hydrolysis into a conformational stress required for the remodelling of nucleic acid or protein nucleic acid structure . like many helicases , atp - hydrolysis activity of snf2 family enzymes is stimulated by dna or dna protein substrates ( 15,31,3841 ) . however , a longstanding puzzle stems from the fact that snf2 family enzymes do not show the dna unwinding activity which defines helicases . recently , a variety of biochemical and structural results indicate that instead of duplex unwinding , snf2 family enzymes use the energy of atp - hydrolysis to translocate on duplex dna by a mechanism that does not require strand - separation ( 17,4245 ) . significantly , the passage of a translocase along dna provides a direct means of altering dna protein contacts . in addition , because the path of dna is helical , translocation is also likely to be associated with rotation of dna , which could indirectly manipulate protein dna interactions . consistent with this , several snf2 family proteins have been shown to generate torsion in dna ( 37,43,45,4749 ) . are there common mechanistic principles linking snf2 family enzymes and dexx box helicases ? amongst the various groups of proteins within the sf1 and sf2 superfamilies of helicase - like proteins , a great deal of progress has recently been made in understanding the mechanism of dna translocation and unwinding by dexx box helicases . these crystal structures revealed an underlying common structural fold and a modular structural organization ( 6 ) , and showed that these helicases typically consist of a dna translocation module linked to a strand - separation module ( 56 ) . a notable exception is vasa , where the dexx box atpase module appears to bend dna instead of translocating on it . residues of the seven helicase - related motifs line the interdomain cleft separating the two reca - like domains and are involved in atp - binding / hydrolysis as well as dna - binding ( 57,58 ) . in addition , atp - binding into the interface cleft has been shown to induce a conformational change that is linked to dna translocation ( 51 ) . the available structures suggest that nucleic acids bind across the interface of the two reca - like domains ( 51,52,54 ) . consistent with earlier observations , recent structures of the sf1 helicase uvrd highlight the fact that the structural movements of sf1 helicase domains , which underlie the translocation and unwinding processes , are closely correlated ( w. yang , personal communication ) . two dna - binding sites alternate in high affinity for dna and move the enzyme along the dna in a process that resembles the movement of an inchworm . during translocation , atp - driven conformational changes between the two reca - like domains result in a closure of the cleft between them , advancing dna by one base at a time , as judged from the pcra dna complex crystal structures ( 51 ) . atp - dependent closure and opening of the active site cleft could result in an alternating sliding of one domain , while the other domain tightly grips the dna and serves as an anchor to generate inchworm - like progress . recent biochemical observations indicate that within this cycle , single - strand binding provides a large part of the energy for unwinding , while atp - binding weakens the interaction with dna and allows the advance on the product strand ( 61 ) . the crystal structure of ns3 revealed that both reca - like domains contact the single - stranded dna ( 52 ) . this is consistent with atp - dependent changes of the dna - binding properties of ns3 , which become weaker in the presence of atp ( 64 ) . a closer inspection of the crystal structure of sf1 and sf2 helicases in complex with dna reveals a divergence in the mode of dna - binding . snf2 family proteins were placed within sf2 in the original studies of gorbalenya and koonin ( 66 ) . in the last decade , biochemical characterization and recent crystal structures have suggested that the catalytic core of the snf2 family enzymes is a structure and sequence unspecific translocase for duplex dna ( 17,4244,67 ) . the two new crystal structures of zebrafish rad54 and the archaeal sulfolobus solfataricus sso1653 gene product in complex with a dsdna substrate have revealed the first structural insights into this motor ( 43,68 ) . the crystallized fragments are active atpases that can translocate on dna , introduce superhelical tension in dna , and/or remodel chromatin . the crystal structures of the catalytic domains from the two snf2 family members revealed that the enzymes possess two domains ( domain 1 and 2 ) , each containing a core reca - like fold ( 1a and 2a ) , fused to snf2 family specific helical domains ( 1b and 2b ) ( figure 1 ) . the two reca - like domains are related to the equivalent region of the dexx box helicases and contain the seven characteristic helicase - related motifs . this similarity suggests that snf2 family atpases and dexx box helicases possess a related atp - hydrolysis mechanism and probably exhibit related atp - driven conformational changes . comparison of the zebrafish and s.solfataricus structures revealed a highly similar fold , but with large conformational difference in the orientation of the two reca - like domains ( 43,68 ) : the orientation of the second reca - like domain with respect to the first differs by 180 between the crystal structures . biochemical analysis of mutant sso1653 suggests that the second reca - like domain can adopt a domain orientation that is similar to zebrafish rad54 during the atp - hydrolysis cycle . only in the conventional conformation are all conserved helicase - related sequence motifs located in the atp - binding cleft . whether this proposed large scale conformational change is part of the functional atp - binding and hydrolysis cycle of swi2/snf2 enzymes or represents a dna loading conformation , is not known . the recently determined structure of trcf also has a sf2 helicase - like region related to recg . in this respect , it might be interesting to probe conformational sub - strates of snf2 family enzymes in the presence of various nucleotides by proteolysis or fluorescence resonance energy transfer experiments . interestingly , the structural and mutational analysis also suggested a variety of additional motifs that could be involved in dsdna stimulated atpase activity [ iia in ref . the structure of the sso1653 region was determined bound to duplex dna and provides a first detailed structural insight into how the snf2 family atpase domain interacts with dna ( 43 ) . duplex dna binds along the domain 1a : domain 2a interface in a position that is in principle well suited for dna translocation by atp - driven conformational changes between domains 1a and 2a . comparison with the sf2 helicase recg ( 53 ) suggests that the translocase module of recg could interact with dna in an analogous way ( figure 1 ) . the total footprint of both phosphate chains amounts to about 67 nt , comparable with footprint of ssdna bound to helicases . the snf2 family specific domains 1b and 2b might , by analogy with the accessory domains of the sf1 helicases , play a key role in dna translocation . do not directly contact dna in the available crystal structure so their precise role is unclear . the lack of either type of duplex - destabilizing region in snf2 family enzymes parallels the lack of helicase activity . instead , the minor groove binding of b - form dna suggests that snf2 family enzymes probably track on dna without strand - separation activity . this is similar to the non - helicase sf2 motor of a type i restriction enzyme , which can move on dna with crosslinked strands ( 46 ) . what can we infer about the mechanism of dna translocation by snf2 family enzymes from dexx box helicases ? the remarkable structural and topological similarities of the core architecture in the two reca - like domains suggest that snf2 family atpases and sf2 helicases share a fundamentally similar atp - hydrolysis mechanism ( figure 2 ) . in particular , the structure and arrangement of the atp and mg binding motifs i ( walker a ) , ii ( walker b ) and vi correspond closely to equivalent motifs of other dexx box enzymes . importantly , an invariant arginine residue of motif vi that conducts domain communication and senses atp - hydrolysis is conserved in snf2 family enzymes ( 39 ) . dna binds to the sso1653 snf2 family structure along a similar surface path to both sf1 and sf2 helicases ( 43,51,52 ) . most intriguingly , the 35 strand of the dna duplex bound to the sso1653 structure overlays very well with the 35 oligo(du ) strand bound to the dna helicase pcra and the rna helicases ns3 and vasa ( 43,52,54 ) . in support of such a mechanistic similarity , biochemical studies indicated that the integrity of this 35 strand is more important for activity , while gaps in the 53 strand can be tolerated during translocation ( 17,71 ) . we do not currently know how the force is generated to propel dna along its binding groove in snf2 family enzymes . based on present insights , for example , in analogy to the inchworm model , alternating high and low affinity binding sites in domain 1 and 2 could transport dna along the dna backbone . alternatively , atp - driven closure of the active site cleft might push on upstream dna . such a push could for instance propel the enzyme forward in analogy to the punting technique of fishermen ( k. theis , personal communication ) . although , all chromatin remodelling complexes have a snf2 family polypeptide at their core , they contain divergent subunit compositions and exhibit many different functional roles in vivo and in vitro . for example , the action of different complexes can result in nucleosome sliding in cis , histone transfer in trans and histone variant exchange . support for the idea that remodelling complexes use atp - dependent translocation on duplex dna to generate the force in remodelling processes comes from recent single molecule biophysical analysis of the rsc complex ( 45 ) . however , the action of specialized remodellers is probably much more complex and involves the specific positioning of the motor domain relative to the remodelled substrate . once engaged , translocation of the motor domain along the minor groove will necessarily lead to a pulling or pushing force due to the translocation component , and also to a twisting force due to the rotation component of minor groove tracking . this would be expected to result in the application of a force between the atpase motor and other contacts restraining the enzyme relative to the dna or remodelled substrate . in the case of nucleosome sliding , the force created by the motor domains could for example result in the generation of dna loops that peel dna away from the surface of the histone octamer . diffusion of these types of distortion around the histone octamer provides an attractive means by which these enzymes might alter chromatin structure ( 1 ) . while it is possible to conceive how directed dna translocation may underlie the mechanism by which many atp - dependent chromatin remodelling enzymes act , this need not necessarily be the case for all snf2 family proteins . the csb protein has been observed to wrap dna around itself in an atp - dependent reaction ( 80 ) . further investigation will be required to determine how the mechanisms by which these proteins act are related to their chromatin remodeling siblings ! structural homology of the two reca - like domains suggest that snf2 family dsdna translocases and sf2 helicases use related mechanisms for atp - driven transport of their nucleic acid substrates across the active site cleft . based on these structures , a model of dsdna translocation by snf2 family enzymes and possibly other dsdna translocases can be envisioned that is similar to the ssdna translocation by dexx box helicases . however , detailed insights how atp - driven conformational changes propel duplex dna along the active site of the snf2 atpase domain are still missing and need to be addressed in future studies . in this regard , it will be interesting to probe conformational changes of snf2 enzymes at the single molecule level , or to probe the precise structure of dna during the translocation process . recent breakthroughs in the application of this technique to remodelling complexes and helicases can reveal a wealth of mechanistic insights how these enzymes move on dna ( 45,81,82 ) . with structures of snf2 enzymes in complex with atp or adp , and analysis of conformational changes using tools like fluorescence energy resonance transfer or small angle solution scattering , we should be able to dissect the conformational substates of remodellers and the mechanistic coupling of atp - binding with dna transport . for instance , more detailed studies on the reaction cycle of snf2 family enzymes , how they engage with their substrates and the nature and role of conformational changes within the reca - like domains are needed . in this regard , what is the role of the various domains that flank the translocase module of snf2 family enzymes ? do they target the enzyme to particular places on the genome , do they grip the substrate to provide a handle for the action of the atpase module , or do they do both ? ultimately , we need to understand how dna tracking by the translocase domains generates the diverse range of macromolecular changes in substrate dna protein complexes during the course of remodelling reactions . comparison of structures of snf2 family enzymes and the recg helicase . the two reca - like domains ( 1a : orange , 2a : green ) are shared across dexx box atpases , and form the atp - binding site in their interface cleft . the location of the atp - binding site , as well as locations of the seven conserved helicase - related atpase / dna - binding motifs ( ia , i , ii , iii , iv , v , vi ) are indicated in tmrecg . the two reca - like domains interact with additional domains ( 1b and 2b : blue ) that are suggested to convert atp - driven rearrangements of 1a and 2a into the translocase or dna unwinding function . for instance , these domains bind to the replication fork substrate in recg , which is dragged like a plough through dna by the action of the translocase module . the role of the helical domains of snf2 family enzymes is not as well understood , but they could play a role in advancing the enzyme by atp - driven conformational changes . in contrast , snf2 family enzymes also recognize the 53 strand ( blue ) and track along the minor groove . despite many functional differences , however , both enzymes families bind the 35 strands at an equivalent site across the two reca - like domains , indicating that atp - driven conformational changes transport dna substrates via the 35 strands in analogous ways ( arrows ) . snf2 family genes identified in s.cerevisiae official gene name and chromosomal locus as recorded in the s.cerevisiae genome database ( sgd ; ) .

the purity of new tested compounds was determined to be 95% by analytical hplc . hplc analyses were carried out on a waters 2695 alliance module equipped with a waters 2996 photodiode array detector ( 210350 nm ) . the chromatographic system consisted of a hichrom guard column for hplc and a phenomenex synergi 4 max - rp 80a column ( 150 mm 4.60 mm ) , eluted at 1 ml / min with the following ion - pair buffer : 0.17% ( m / v ) cetrimide and 45% ( v / v ) phosphate buffer ( ph 6.4 ) in meoh . nadase ( from neurospora crassa ; sigma ; 0.9 u ) in tris - hcl buffer ( 2 ml , 0.1 m , ph 7.27.4 ) was added to a solution of the nad analogue ( 30 mol ) in tris - hcl buffer ( 1 ml , 0.1 m , ph 7.27.4 ) . the reaction mixture was stirred at 37 c until complete , followed by purification of the product by ion - exchange ( q - sepharose ) chromatography eluting with a gradient ( 050% ) of teab ( 1.0 m ) in milli - q water . the residue was coevaporated several times with meoh to remove excess teab to yield the desired adpr analogue as a glassy solid in its triethylammonium ( tea ) form . cesium carbonate ( 0.24 mmol , 2.9 equiv ) was added in one portion to a stirred solution of the corresponding boronic acid ( 0.103 mmol , 1.2 equiv ) , palladium acetate ( 0.004 mmol , 0.05 equiv ) , tppts ( 0.02 mmol , 0.24 equiv ) , and 8-br - adpr ( tea salt , 0.0823 mmol ) in degassed mecn h2o ( 1:2 v / v ; 2.4 ml ) under an argon atmosphere . the reaction mixture was heated at 125 c for 5 min ; the reaction mixture turned black and hplc analysis confirmed the reaction was complete . the reaction mixture was cooled to room temperature , quadrapure tu ( 100 mg ) added , and the mixture stirred for 16 h. the mixture was filtered and evaporated under reduced pressure to leave a crude product that was purified by ion - exchange ( q - sepharose ) chromatography eluted with a gradient ( 040% ) of teab ( 1.0 m ) in milli - q water followed by reverse phase ( rp-18 ) column chromatography , eluted with 020% mecn in teab ( 0.05 m ) to isolate the desired 8-substituted adpr product . to a solution of amine ( 0.443 mmol ) and dipea ( 42 l , 0.239 mmol ) in etoh ( 5 ml ) was added diethylsquarate ( 72 l , 0.487 mmol ) . the reaction was stirred at rt until tlc indicated completion of the reaction ( ca . 1 h ) . the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm acetone , 8:2 v / v ) to yield the desired product . the protected compound ( 0.1 mmol ) was stirred in a 75% aq tfa solution ( 5 ml ) at rt for 1 h. the solvents were evaporated under reduced pressure , and the residue was coevaporated with meoh to remove any residual tfa . the remaining residue was purified on an isco purification system ( dcm meoh , 8:2 v / v ) to yield the desired compound as a white solid . phenylboronic acid ( 0.103 mmol , 21 mg ) and 8-br - adpr 4 ( tea salt , 0.0823 mmol ) were reacted under the general protocol b , yielding 8-ph - adpr ( tea salt , 6.0 by h nmr ) ( 18 mg , 14.3 mol , 19% ) as a colorless solid . h ( 400 mhz , d2o ) 8.14 ( br s , 1h , h-2 ) , 7.567.48 ( br m , 5h , ph ) , 5.78 ( d , 1h , j = 5.9 , h-1 ) , 5.16 ( br , 0.4h , h-1 ) , 5.08 ( br , 1h , h-2 ) , 5.05 ( br , 0.6h , h-1 ) , 4.30 ( br , 1h , h-2 ) , 3.824.18 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . c ( 100 mhz , d2o ) 154.5 , 153.4 , 152.3 , 150.3 ( c-2 ) , 131.3 ( ph , c h ) , 129.7 ( ph , c h ) , 129.2 ( ph , c h ) , 127.9 , 118.6 , 101.3 ( c/-1 ) , 96.5 ( c/-1 ) , 89.0 ( c-1 ) , 83.2 ( c-4 or c/-4 ) , 81.9 ( c-4 or c/-4 ) 81.3 ( c-4 or c/-4 ) , 75.3 , 70.8 , 70.6 , 70.5 ( c-2 ) , 70.2 , 69.7 ( c-2 ) , 66.5 ( c-5 or c/-5 ) 65.4 ( c-5 or c/-5 ) and 65.5 ( c-5 or c/-5 ) ; p ( 162 mhz , d2o ) 10.1 ( very br ) . hrms ( es ) calcd for c21h26n5o14p2 , 634.0957 m ; found , 634.0970 ; and rt = 26.7 min . 3-acetylphenylboronic acid ( 0.1 mmol , 17 mg ) and 8-br - adpr 4 ( 2 equiv tea salt , 67 mg , 0.079 mmol ) were reacted under the general protocol b to yield 8-(3-ac - ph)-adpr ( tea salt , 3.0 equiv by h nmr ) ( 11 mg , 9.3 mol , 12% ) as a colorless solid . h ( 400 mhz , d2o ) 8.138.19 ( m , 2h , ar 2-h and h-2 ) , 8.05 ( d , 1h , j = 6.9 , ar 6-h ) , 7.86 ( d , 1h , j = 6.9 , ar 4-h ) , 7.62 ( t , 1h , j = 6.9 , ar 5-h ) , 5.78 ( d , 1h , j = 5.9 , h-1 ) , 5.195.23 ( m , 1.4h , ( 1h ) h-2 and ( 0.4h ) h-1 ) , 5.09 ( d , 0.6h , j = 2.4 , h-1 ) , 4.374.41 ( m , 1h , h-2 ) , 3.874.21 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) and 2.62 ( s , 3h , arcoch3 ) . c ( 100 mhz , d2o ) 202.4 ( c = o ) , 155.1 , 152.8 ( c-2 ) , 151.9 , 150.1 , 136.7 , 134.4 ( ar 4-c ) , 130.6 ( ar 6-c ) , 129.6 , 129.4 ( ar 5-c ) , 128.3 ( ar 2-c ) , 118.5 , 101.1 ( c/-1 ) , 96.3 ( c/-1 ) , 88.8 ( c-1 ) , 83.0 ( c-4 or c/-4 , d , j 9.4 ) , 81.7 ( c-4 or c/-4 ) 81.0 ( c-4 or c/-4 , d , j = 9.4 ) , 75.1 , 70.6 , 70.3 , 70.3 ( c-2 ) , 69.9 , 69.5 ( c/-2 ) , 66.2 ( c-5 or c/-5 , d , j = 7.1 ) , 65.3 ( c-5 or c/-5 , d , j = 7.1 ) , 65.3 ( c-5 or c/-5 , d , j = 7.1 ) and 26.3 ( coch3 ) . p ( 162 mhz , d2o ) 11.2 ( br ) and 11.4 ( br ) . hrms ( es ) calcd for c23h28n5o15p2 , 676.1063 m ; found , 676.1076 ; and rt = 17.2 min . thiophene-3-boronic acid ( 0.12 mmol , 16 mg ) and 8-br - adpr 4 ( tea salt , 0.097 mmol ) were reacted under the general protocol b to give 8-(3-thiophenyl)-adpr ( tea salt , 2.3 equiv by h nmr ) ( 25 mg , 24.7 mol , 25% ) as a colorless solid . h ( 400 mhz , d2o ) 8.14 ( s , 1h , h-2 ) , 7.88 ( br s , 1h , thiophenyl 2-h ) , 7.54 ( dd , 1h , j = 4.7 , 3.2 , thiophenyl 4-h ) , 7.35 ( d , 1h , j = 4.7 , thiophenyl 5-h ) , 5.90 ( d , 1h , j = 5.9 , h-1 ) , 5.175.21 ( m , 1.4h , ( 1h ) h-2 and ( 0.4h ) h-1 ) , 5.09 ( d , 0.6h , j = 1.9 , h-1 ) , 4.374.40 ( m , 1h , h-2 ) and 3.884.18 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . c ( 100 mhz , d2o ) 154.5 , 152.1 ( c-2 ) , 149.9 , 148.8 , 129.3 , 127.9 , 127.8 , 127.7 , 118.2 , 101.1 ( c/-1 ) , 96.3 ( c/-1 ) , 88.6 ( c-1 ) , 82.8 ( c-4 or c/-4 , d , = j 8.5 ) , 81.7 ( c-4 or c/-4 ) , 81.1 ( c-4 or c/-4 , d , j = 8.5 ) , 75.1 , 70.6 , 70.3 , 70.1 , 69.9 , 69.4 , 66.2 ( c-5 or c/-5 ) , 65.3 ( c-5 or c/-5 ) , 65.3 ( c-5 or c/-5 ) . p ( 162 mhz , d2o ) 11.2 ( br ) and 11.3 ( br ) . hrms ( es ) calcd for c19h24n5o14p2s , 640.0521 m ; found , 640.0527 ; and rt = 24.9 min . dl-4-boronophenylalanine ( 0.1 mmol , 21 mg ) and 8-br - amp 8 ( 0.75 equiv tea salts , 40 mg , 0.08 mmol ) were reacted under the general protocol b to give 8-(4-(2-aminopropanoic acid)phenyl)-amp ( tea salt , 2.2 equiv by h nmr ) ( 19 mg , 14.4 mol , 18% ) as a colorless solid . h ( 400 mhz , d2o ) 8.17 ( s , 1h , h-2 ) , 7.61 ( d , 2h , j = 8.2 , ar h ) , 5.77 ( d , 1h , j = 5.8 , h-1 ) , 5.16 ( t , 1h , j = 6.3 , h-2 ) , 4.35 ( dd , 1h , j = 6.2 , 5.1 , h-3 ) , 3.884.09 ( m , 4h , h-4 , h-5 and nh2chch2 ) , 3.26 ( dd , 1h , j = 14.9 , 5.3 , nh2chcha / b ) and 3.08 ( 1h , obscured by et3n salt peak , nh2chcha / b ) . c ( 100 mhz , d2o ) 192.2 ( c = o ) , 155.1 , 152.8 , 152.6 ( c-2 ) , 150.1 , 138.6 , 129.9 , 129.8 , 126.9 , 118.4 , 88.6 ( c-1 ) , 83.6 ( c-4 ) , 70.0 ( c-2 ) , 69.5 ( c-3 ) , 63.4 ( c-5 ) , 55.8 ( nh2chch2 ) and 36.5 ( arch2 ) . hrms ( es ) calcd for c19h22n6o9p , 509.1191 m ; found , 509.1174 ; and rt = 6.96 min . triphenylphosphine ( 130 mg , 0.5 mmol ) , morpholine ( 92 ml , 1.06 mmol ) , and 2,2-dipyridyldisulfide ( 110 mg , 0.5 mmol ) were added to a solution of 8-nh2-amp 10 ( 55 mg , 0.15 mmol ) in dry dmso ( 600 l ) . the mixture was stirred at rt for 4 h , and then a solution of sodium iodide in acetone ( 0.1 m ) was added dropwise . the precipitate that formed was collected , washed with acetone , and redissolved in water and lyophilized to leave the crude morpholidate intermediate ( 39 mg ) as a pale - yellow solid . the morpholidate was dissolved in a solution of mncl2 in formamide ( 1 ml , 0.2 m ) , mgso4 ( 48 mg , 0.4 mmol ) and -nmn ( 67 mmol , 0.2 mmol ) were added , and the mixture was stirred for 2 days . the crude product was precipitated from the reaction mixture by the dropwise addition of mecn , and the precipitate was collected , washed with mecn , and dried . the crude product was purified by reverse phase column chromatography , eluting with 020% mecn in teab ( 0.05 m ) . the sample was then treated with chelex 100 ( sodium form ) to remove any paramagnetic material and lyophilized to yield the 8-amino - nad ( 13 mg , 0.02 mmol , 13% ) as a colorless solid . h ( 270 mhz , d2o ) broad , possibly small amount of remaining mn . hrms ( es ) calcd for c21h29n8o14p2 , 679.1273 m ; found , 679.1252 ; and rt = 3.03 min . nadase ( from neurospora crassa ; sigma ; 0.52 u ) in tris - hcl buffer ( 1 ml , 0.1 m , ph 7.27.4 ) was added to a solution of 8-nh2-nad11 ( 13 mg ) in tris - hcl buffer ( 4 ml , 0.1 m , ph 7.27.4 ) . after 4 h , all of the starting material had been consumed , the reaction mixture was diluted with water until the conductivity < 200 s / cm , and the product purified by ion - exchange ( q - sepaharose ) chromatography eluting with a gradient ( 050% ) of teab ( 1.0 m ) in milli - q . subsequent purification by reverse phase column chromatography , eluting with 030% mecn in teab ( 0.05 m ) , left the desired 8-nh2-adpr product ( 4.5 mg , 7.65 mol , 40% ) as a colorless solid . h ( 400 mhz , d2o ) 7.98 ( s , 1h , h-2 ) , 5.99 ( d , 1h , j = 7.6 , h-1 ) , 5.245.31 ( br , 0.4h , h-1 ) , 5.115.17 ( br , 0.6h , h-1 ) , 4.684.64 ( br m , 1h , h-2 ) , 4.384.44 ( br m , 1h , h-2 ) , 3.914.31 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . hrms ( es ) calcd for c15h23n6o14p2 , 573.0753 m ; found , 573.0775 ; and rt = 12.2 min . nhd ( 30 mol ) and nadase were reacted under the general protocol a to afford idpr as a glassy solid ( 24.6 mol , 82% ) . h ( 400 mhz , d2o ) 8.44 ( s , 1h , h-2 ) , 8.19 ( s , 1h , h-8 ) , 6.11 ( d , 1h , j1,2 = 6.1 , h-1 ) , 5.31 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.17 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.764.72 ( m , 1h , h-2 ) and 4.533.96 ( m , 9h , h-3 , h-4 , 2 h-5 , h-2 , h-3 , h-4 and 2 h-5 ) . p ( decoupled , 162 mhz , d2o ) 10.2 ( d , ab system , j = 18.8 ) , 10.6 ( d , ab system , j = 18.8 ) . hrms ( es ) calcd for c15h21n4o15p2 , 559.0484 ( m h ) ; found , 559.0480 . uv ( h2o , ph 7.2 ) max 248 nm ( 14500 ) . nicotinamide-7-deaza-8-bromoadenine-5-dinucletide ( 7-deaza-8-bromo - nad , 15 mol ) was treated with nadase under the general procedure a to afford 7-deaza-8-br - adpr as a glassy solid ( 12.7 mol , 85% ) . h ( 270 mhz , d2o ) 8.03 ( s , 1h , h-2 ) , 6.66 ( s , 1h , h-7 ) , 6.17 ( d , 1h , j1,2 = 6.1 , h-1 ) , 5.215.17 ( m , 2h , h-2 and h-1 ) , 4.544.50 ( m , 1h , h-3 ) and 4.183.94 ( m , 8h , h - ribose ) . p ( decoupled , 109 mhz , d2o ) 10.5 ( m ) and 10.7 ( m ) . hrms ( es ) calcd for c12h22n4o14p2br , 634.9797 ( m h ) ; found 634.9787 . hrms ( es ) calcd for c12h22n4o14p2br , 636.9776 ( m h ) ; found , 636.9778 . to a solution of 1,2,3,5-o - tetraacetate ribofuranose 18 ( 4.7 g , 14.7 mmol ) , 6-chloropurine 17 ( 2.5 g , 16.17 mmol ) , and dbu ( 6.5 ml , 44.1 mmol ) in dry mecn ( 100 ml ) was added dropwise tmsotf ( 10 ml , 58.8 mmol ) at 0 c . the resulting clear brown solution was stirred for 2 h at 60 c , after which it was cooled to room temperature and aq satd nahco3 ( 400 ml ) was added . the aqueous phase was extracted with dcm ( 3 300 ml ) , dried ( na2so4 ) , filtered , and evaporated under reduced pressure , giving a brown oil . the crude was purified by column chromatography on silica gel ( dcm acetone , 9:1 v / v ) to afford the desired product as a white foam ( 4.9 g , 91% ) . h ( 270 mhz , cdcl3 ) 8.75 ( s , 1h , h-8 ) , 8.28 ( s , 1h , h-2 ) , 6.21 ( d , 1h , j1,2 = 5.1 , h-1 ) , 5.92 ( app t , 1h , j2,1 = j2,3 = 5.1 , h-2 ) , 5.62 ( app t , 1h , j3,2 = j3,4 = 5.1 , h-3 ) , 4.484.33 ( m , 3h , h-4 , h-5a and h-5b ) , 2.13 ( s , 3h , ch3 ) , 2.10 ( s , 3h , ch3 ) and 2.06 ( s , 3h , ch3 ) . c ( 68 mhz , cdcl3 ) 170.4 , 169.7 , 169.5 ( all c = o ) , 152.4 ( c-2 ) , 151.7 , 151.3 ( 2 c ) , 143.7 ( c-8 ) , 86.9 ( c-1 ) , 80.6 ( c-4 ) , 73.2 ( c-2 ) , 70.5 ( c-3 ) , 62.9 ( c-5 ) , 20.8 , 20.6 , and 20.5 ( 3 ch3 ) . rf = 0.57 ( dcm acetone , 9:1 v / v ) . 2,3,5-tri - o - acetyl-6-chloro adenosine 19 ( 1.45 g , 3.52 mmol ) was added to a freshly prepared solution of naome in meoh ( 7.04 mmol in 10 ml ) . the solution was refluxed for one hour , after which it was cooled to rt and neutralized with acoh . the solvent was evaporated , and the residue was purified by column chromatography on silica gel ( dcm acetone , 6:4 v / v ) to yield the desired product as a white foam ( 943 mg , 95% ) . h ( 270 mhz , meoh - d4 ) 8.49 ( s , 1h , h-8 ) , 8.42 ( s , 1h , h-2 ) , 6.04 ( d , 1h , j1,2 = 5.9 , h-1 ) , 4.774.73 ( m , 1h , h-2 ) , 4.38 ( dd , 1h , j3,2 = 5.1 and j3,4 = 3.1 , h-3 ) , 4.184.15 ( m , 1h , h-4 ) , 4.13 ( s , 3h , ch3 ) , 3.91 ( dd , 1h , j5a,5b = 12.5 and j5a,4 = 2.6 , h-5a ) and 3.77 ( dd , 1h , j5b,5a = 12.5 and j5b,4 = 3.5 , h-5b ) . c ( 68 mhz , meoh - d4 ) 160.5 ( c-6 ) , 151.7 ( c-2 ) , 150.8 ( c ) , 142.6 ( c-8 ) , 121.3 ( c ) , 89.9 ( c-1 ) , 86.6 ( c-4 ) , 74.3 ( c-2 ) , 71.1 ( c-3 ) , 61.9 ( c-5 ) and 53.7 ( ch3 ) ; rf = 0.09 ( dcm acetone , 6:4 v / v ) . ms ( apci ) m / z 283.4 [ ( mh ) , 100% ] . hrms ( es ) calcd for c11h15n4o5 , 283.1037 ( mh ) ; found , 283.1038 . 6-o - methylinosine 20 ( 80 mg , 0.264 mmol ) was dissolved in triethylphosphate ( 1 ml ) by heating with a heatgun . the resulting colorless solution was cooled to 0 c , and water ( 2 l ) was added followed by pocl3 ( 0.1 ml , 1.056 mmol ) . it was stirred at 0 c until disappearance of starting material and formation of a single peak as shown by hplc . after 1 h , the reaction mixture was quenched by addition of ice / water ( 15 ml ) and stirred for 15 min at 0 c , after which it was warmed up to rt . triethylphosphate was extracted with etoac ( 6 6 ml ) , and the aqueous phase was neutralized with 2 m naoh . it was then applied to a reverse phase gradifrac column eluted with a gradient of 565% mecn in 0.05 m teab . the appropriate fractions were collected and lyophilized overnight to afford the desired monophosphate as its triethylammonium salt . h ( 270 mhz , d2o ) 9.01 ( s , 1h , h-8 ) , 8.51 ( s , 1h , h-2 ) , 6.14 ( d , 1h , j1,2 = 3.7 , h-1 ) , 4.63 ( app t , 1h , j2,1 = j2,3 = 4.2 , h-2 ) , 4.414.37 ( m , 1h , h-3 ) , 4.31 ( br s , 1h , h-3 ) , 4.224.15 ( m , 1h , h-5a ) and 4.11 ( m , 4h , och3 and h-5b ) . c ( 68 mhz , cdcl3 ) 159.5 ( c-6 ) , 153.6 ( c-2 ) , 149.4 ( c-4 ) , 129.6 ( c-8 ) , 115.7 ( c-5 ) , 89.6 ( c-1 ) , 83.7 ( c-4 , j = 8.7 ) , 74.7 ( c-2 ) , 69.5 ( c-3 ) , 64.2 ( c-5 ) and 55.9 ( och3 ) . ms : ( es ) m / z 361.5 [ ( m h ) , 100% ] . hrms ( es ) calcd for c11h14n4o8p , 361.0555 [ ( m h ) ] ; found , 361.0558 . 6-o - me - imp 21 ( 120 mg , 0.331 mmol ) was dissolved in dry dmso ( 2 ml ) and coevaporated with dry dmf ( 5 3 ml ) . the residue was dissolved in dmso ( 1 ml ) to which was added morpholine ( 150 l , 1.724 mmol ) , dipyridyldisulfide ( 182 mg , 0.827 mmol ) , and triphenylphosphine ( 217 mg , 0.827 mmol ) , at which point the solution became bright yellow . it was stirred for 1 h at rt , after which hplc analysis showed formation of a new peak . precipitation of the product occurred by dropwise addition of a solution of nai in acetone ( 0.1 m ) . the resulting precipitate was filtered and washed with acetone to yield the desired product as a pale - yellow solid , which was used in the next step without further purification . 6-o - me - imp morpholidate ( 100 mg , 0.232 mmol ) , -nmn ( 85 mg , 0.253 mmol ) , and mgso4 ( 54 mg , 0.464 mmol ) were dissolved in a 0.2 m solution of mncl2 in formamide ( 1.7 ml ) and stirred at rt for 16 h , after which hplc analysis showed completion of the reaction ( rt ( -nmn ) = 2.1 min and rt ( 6-o - me - nhd ) = 3.8 min ) . mecn was added to precipitate the product , which was filtered , dissolved in milli - q , and applied to a reverse phase gradifrac column eluted with a gradient of 565% mecn in 0.05 m teab . further treatment with chelex 100 to remove any paramagnetic particles afforded the desired product as the sodium salt ( 18 mg , 8% ) . h ( 400 mhz , d2o ) 9.21 ( s , 1h , hn2 ) , 9.07 ( d , 1h , j6,5 = 6.3 , hn6 ) , 8.67 ( d , 1h , j4,5 = 8.2 , hn4 ) , 8.39 ( s , 1h , h-8 ) , 8.27 ( s , 1h , h-2 ) , 8.098.06 ( m , 1h , hn5 ) , 5.96 ( d , 1h , j1,2 = 5.9 , h-1 ) , 5.94 ( d , 1h , j1,2 = 5.5 , h-1 ) , 4.62 ( app t , 1h , j2,1 = j2,3 = 5.5 , h-2 ) and 4.384.06 ( m , 9h , hsugar ) . c ( 100 mhz , d2o ) 165.1 ( c = o ) , 160.7 ( c-6 ) , 151.1 ( c-8 ) , 151.0 ( c-4 ) , 145.7 ( cn4 ) , 142.4 ( cn6 ) , 141.5 ( c-2 ) , 139.8 ( cn2 ) , 133.6 ( cn3 ) , 128.6 ( cn5 ) , 120.4 ( c-5 ) , 99.9 ( c-1 ) , 87.0 ( c-1 ) , 86.8 ( c-4 , d , j = 9.2 ) , 83.7 ( c-4 , d , j = 9.2 ) , 77.4 ( c-2 ) , 74.0 ( c-2 ) , 70.4 ( c-3 ) , 70.2 ( c-3 ) , 64.8 , 63.3 ( 2 ch2 ) and 54.9 ( ch3 ) . p ( 109 mhz , d2o ) 11.4 ( d , j = 20.7 ) and 11.7 ( d , j = 20.7 ) . ms ( es ) m / z 678.2 [ ( m h ) , 100% ] . hrms ( es ) calcd for c22h28n6o15p2 , 678.1093 [ ( m h ) ] ; found , 678.1088 . 6-o - me - nhd sodium salt 23 ( 17.3 mg , 25.5 mol ) was incubated with aplysia cyclase ( 40 l ) in a 25 mm hepes buffer ( 35 ml , ph 7.4 ) at rt . after 4 h at rt , hplc analysis showed completion of the reaction ( rt ( nicotinamide ) = 1.7 min and rt ( product ) = 15.9 min ) . the mixture was then applied to a q - sepharose ion exchange column eluted with 1 m teab buffer . the appropriate fractions were collected and evaporated under vacuum , and the residue was coevaporated with meoh to afford the hydrolyzed product 6-o - me - idpr as a triethylammonium salt . h ( 270 mhz , d2o ) 8.58 ( s , 1h , h-2 ) , 8.45 ( s , 1h , h-8 ) , 6.15 ( d , 1h , j1,2 = 5.6 , h-1 ) , 5.26 ( d , 0.5 h , j1,2 = 4.2 , h-1 ) , 5.16 ( d , 0.5 h , j1,2 = 2.2 , h-1 ) , 4.78 ( 1h , hidden under hdo peak ) , 4.484.47 ( m , 1h ) , 4.34 ( br s , 1h ) , 4.274.17 ( m , 3h ) , 4.12 ( s , 3h , ome ) , 4.083.92 ( m , 3h ) and 3.843.82 ( m , 1h ) . p ( 109 mhz , d2o ) 10.2 ( d , j = 21.1 ) and 10.6 ( d , j = 21.1 ) . ms : ( es ) m / z 573.4 [ ( m h ) , 80% ] . hrms ( es ) calcd for c16h23n4o15p2 , 573.0641 [ ( m h ) ] ; found , 573.0646 . 2-deoxy - nad32 ( 22 mol ) was reacted with nadase under general protocol b to yield the desired hydrolyzed product ( 18.7 mol , 85% ) . h ( 270 mhz , d2o ) 8.41 ( s , 1h , h-2 ) , 8.17 ( s , 1h , h-8 ) , 6.486.43 ( m , 1h , h-1 ) , 5.26 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.15 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.71 ( m , 1h , partially hidden under hdo peak , h-2 ) , 4.273.87 ( m , 8h , h-3 , h-4 , h-5 , h-3 , h-4 and h-5 ) , 2.832.78 ( m , 1h , h-2a ) and 2.55 ( ddd , 1h , j2b,2a = 14.0 , j2b,1 = 6.3 and j2b,3 = 3.3 , h-2b ) . p ( decoupled , 109 mhz , d2o ) 10.4 ( br s ) , 10.5 ( br s ) . hrms ( es ) calcd for c15h22n5o13p2 , 542.0695 ( m h ) ; found , 542.0681 . uv ( h2o , ph 7.4 ) max 259 nm ( 15400 ) . to a solution of 3-deoxy - nad42 ( 16 mol ) in tris buffer ( 100 mm , ph 7.3 , 5 ml ) was added nadase ( 200 l ) . the reaction was left for 2 h at 37 c , after which hplc analysis showed no remaining starting material . the volatiles were evaporated under reduced pressure , and the residue was applied to a semipreparative c18 column developed with a linear gradient of 0.1 m teab against mecn . the appropriate fractions were evaporated , and excess tea salt was removed by coevaporation with meoh to leave the desired adpr analogue ( 2.6 mol , 20% ) as a glassy solid tea salt . h ( 400 mhz , d2o ) 8.37 ( s , 1h , h-8 ) , 8.16 ( s , 1h , h-2 ) , 6.03 ( d , 1h , j1,2 = 5.0 , h-1 ) , 5.20 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.10 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.714.63 ( m , 2h , h - sugar ) , 4.233.85 ( m , 7h , h - sugar ) , 2.35 ( dd , 1h , j3a,3b = 10.0 and j3a,4 = 5.8 , h-3a ) and 2.172.12 ( m , 1h , h-3b ) . p ( decoupled , 162 mhz , d2o ) 11.1 ( br m ) . hrms ( es ) calcd for c15h22n5o13p2 , 542.3090 ( m h ) ; found , 542.3098 . 9-(4-hydroxybutyl)adenine 27 ( 80 mg , 0.386 mmol ) was dissolved in trimethylphosphate ( 1.3 ml ) by heating with a heatgun . phosphorus oxychloride ( 144 l , 1.545 mmol ) and water ( 2 l ) were added at 0 c , and the resulting solution was stirred at rt for 3 h. ice / water ( 15 ml ) was then added , and the mixture was stirred for further 15 min , after which it was extracted with etoac ( 6 ) . the aqueous layer was neutralized with 5 n naoh and applied to a reverse phase column and the product eluted with a gradient of 0.05 m teab against mecn . the residue obtained was coevaporated with meoh to remove excess tea salt , leaving the desired monophosphate as its triethylammonium salt ( 92 mg , 72% ) . h ( 270 mhz , dmso - d6 ) 8.13 ( s , 1h , h-2 or h-8 ) , 7.92 ( s , 1h , h-8 or h-2 ) , 7.88 ( br s , 2h , nh2 ) , 4.03 ( t , 2h , j = 7.2 , ch2-n ) , 3.85 ( q , 2h , j = 7.2 , ch2-o ) , 1.801.75 ( m , 2h , o - ch2-ch2 ) and 1.561.49 ( m , 2h , o - ch2-ch2 ) . 9-(4-acetoxybutyl)adenine-5-monophosphate1tea 28 ( 92 mg , 0.277 mmol ) was dissolved in dry dmso ( 1 ml ) and coevaporated with dry dmf ( 5 3 ml ) . the residue was dissolved in dmso ( 400 l ) to which was added morpholine ( 106 l , 1.233 mmol ) , dipyridyldisulfide ( 130 mg , 0.592 mmol ) , and triphenylphosphine ( 155 mg , 0.592 mmol ) , at which point the solution became bright yellow . it was stirred for 2 h at rt , after which hplc analysis showed completion of the reaction . precipitation of the product occurred by dropwise addition of a solution of nai in acetone ( 0.1 m , 20 ml ) . the resulting precipitate was filtered , washed with acetone , and dried ( p : = 6.7 ppm ) . it was then reacted with -nmn ( 84 mg , 0.250 mmol ) and mgso4 ( 53 mg , 0.454 mmol ) in a 0.2 m solution of mncl2 in formamide ( 1.5 ml ) at rt overnigh , t after which hplc analysis showed completion of the reaction ( rt = 2.9 min ) . the precipitate was filtered , dissolved in milli - q , and applied to a reverse phase column eluted with a 565% gradient of mecn in 0.05 m teab . further treatment with chelex 100 to remove any paramagnetic particles afforded the desired dinucleotide as its sodium salt . nicotinamide-9-(4-acetoxybutyl)adenine-5-dinucleotide 29 ( 10 mol ) was treated with nadase under general procedure b to leave the desired acyclic - adpr ( 8.1 mol , 81% ) . h ( 270 mhz , d2o ) 8.09 ( s , 2h , h-2 and h-8 ) , 5.25 ( d , 0.4h , j1,2 = 3.8 , h-1 ) , 5.17 ( d , 0.6h , j1,2 = 1.9 , h-1 ) , 4.204.02 ( m , 3h , h-2 and ch2-n ) , 3.973.90 ( m , 5h , h-3 , h-4 , h-5 and ch2-o ) , 1.901.83 ( m , 2h , o - ch2ch2 ) and 1.621.55 ( m , 2h , o - ch2ch2 ) . p ( decoupled , 109 mhz , do ) 10.2 ( m ) . hrms ( es ) calcd for c14h22n5o11p2 , 498.0795 ( m h ) ; found , 498.0786 . uv ( h2o , ph 7.2 ) max 261 nm ( 16000 ) . a solution of catpr 46 ( 5 mol ) in tris hcl ( 100 mm , ph 7 ) was heated to 100 c for 1 h , after which hplc analysis showed conversion to a new product . the solution was applied to a reverse phase column eluted with a 565% gradient of mecn in 0.05 m teab . the appropriate fractions were collected and evaporated to afford the desired nucleotide as its triethylammonium salt ( 2.7 mol , 54% ) . h ( 270 mhz , do ) 8.54 ( s , 1h , h-2 ) , 8.26 ( s , 1h , h-8 ) , 6.11 ( d , 1h , j1,2 = 5.8 , h-1 ) , 5.31 ( d , 0.4h , j1,2 = 4.1 , h-1 ) , 5.15 ( d , 0.6h , j1,2 = 2.3 , h-1 ) , 4.554.52 ( m , 1h , h-2 ) and 4.373.96 ( m , 9h , h-3 , h-4 , h-5 , h-2 , h-3 , h-4 and h-5 ) . p ( decoupled , 109 mhz , d2o ) 11.6 ( br s ) , 23.4 ( br s , o - p - o ) . hrms ( es ) calcd for c15h23n5o17p2 , 638.0307 ( m h ) ; found , 638.0331 . uv ( h2o , ph 7.2 ) max 259 nm ( 17180 ) . synthesis was carried out without protection of the 6-amino group to generate sal - ams . for details , see supporting information . 10% pd / c ( 110 mg ) was added to a solution of 2,3-o - isopropylidene-5-azido-5-deoxyadenosine ( 1.0 g , 3.01 mmol ) in etoh . the mixture was stirred for 16 h under a hydrogen atmosphere , after which the palladium was filtered and the solvent was removed under vacuum , yielding the desired compound as a white solid ( 0.9 g , 95% ) . h ( 400 mhz , dmso - d6 ) 8.34 ( s , 1h , h-8 ) , 8.14 ( s , 1h , h-2 ) , 7.29 ( br s , 2h , nh2 ) , 6.11 ( d , 1h , j1,2 = 3.0 , h-1 ) , 5.42 ( dd , 1h , j2,3 = 6.2 and j2,1 = 3.0 , h-2 ) , 5.01 ( dd , 1h , j3,2 = 6.2 and j3,4 = 2.9 , h-3 ) , 4.204.16 ( m , 1h , h-4 ) , 2.912.81 ( m , 2h , 2 h-5 ) , 1.52 ( s , 3h , ch3 ) and 1.30 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.7 ( c-8 ) , 148.8 ( c-4 ) , 140.0 ( c-2 ) , 119.2 ( c-5 ) , 113.3 ( c ) , 89.2 ( c-1 ) , 878.0 ( c-4 ) , 82.7 ( c-2 ) , 81.6 ( c-3 ) , 43.7 ( c-5 ) , 27.0 and 25.2 ( 2 ch3 ) . hrms ( es ) calcd for c13h19n6o3 , 307.1513 ( mh ) ; found , 307.1511 . to a solution of 1-o - methyl-2,3-o - isopropylidene--d - ribofuranose 49 ( 0.61 g , 2.989 mmol ) in dry pyridine ( 1 ml ) , externally cooled with ice , was added p - toluenesulfonyl chloride ( 0.7 g , 3.668 mmol ) and a catalytic amount of dmap . the reaction mixture was stirred at rt under nitrogen for 5 h. water ( 0.3 ml ) was added and stirring continued for 30 min . this mixture was extracted with chloroform ( 3 10 ml ) and the combined organic phases washed sequentially with cuso4 ( 10% w / v , aq satd ) , nahco3 ( aq satd ) and water and then dried over anhydrous sodium sulfate . the solvent was evaporated , and the residue was purified on an isco chromatographic system ( petrol etoac , 7:3 v / v ) to yield the desired compound as a white solid ( 0.92 g , 81% ) . h ( 400 mhz , cdcl3 ) 7.71 ( d , 2h , j = 8.7 2 ar h ) , 7.26 ( d , 2h , j = 8.0 , ar h ) , 4.83 ( s , 1h , h-1 ) , 4.51 ( dd , 1h , j3,2 = 6.0 and j3,4 = 0.6 , h-3 ) , 4.44 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.21 ( dt , 1h , j4,5 = 7.1 and j4,3 = 0.6 , h-4 ) , 3.933.91 ( m , 2h , h-5 ) , 3.14 ( s , 3h , ome ) , 2.36 ( s , 3h , ch3 ) , 1.35 ( s , 3h , ch3 ) and 1.19 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 144.9 ( c - so2 ) , 132.8 ( c - me ) , 129.8 ( 2c ) , 127.9 ( 2c ) ( all ch ) , 112.6 ( c ) , 109.4 ( c-1 ) , 84.8 ( c-4 ) , 83.5 ( c-2 ) , 81.3 ( c-3 ) , 69.1 ( c-5 ) , 54.9 ( ome ) , 26.2 , 24.8 ( 2 ch3 ) and 21.5 ( ch3-ph ) . hrms ( es ) calcd for c16h22nao7s , 381.0978 ( mh ) ; found , 381.0969 . to a solution of 1-o - methyl-2,3-o - isopropylidene-5-o - p - toluenesulfonyl--d - ribofuranose 50 ( 2.4 g , 6.7 mmol ) in dmf ( 20 ml ) was added nan3 ( 5.2 g , 80.4 mmol ) , and the reaction mixture was stirred at 120 c for 16 h. after cooling to rt , acetone ( 20 ml ) was added and the solid was removed by filtration . the solvents were evaporated under reduced pressure , and the residue was dissolved in dcm ( 50 ml ) and washed successively with water ( 50 ml ) , satd aq nahco3 ( 50 ml ) , and water ( 50 ml ) . the organic layer was dried ( na2so4 ) , filtered , and evaporated to leave an oil which was purified on an isco chromatographic system ( petrol etoac , 1:1 v / v ) , yielding the title compound as a colorless oil ( 1.4 g , 91% ) . h ( 400 mhz , cdcl3 ) 4.90 ( s , 1h , h-1 ) , 4.50 ( s , 2h , h-2 and h-3 ) , 4.19 ( ddd , 1h , j4,5a = 7.6 , j4,5b = 6.8 and j4,3 = 0.6 , h-4 ) , 3.35 ( dd , 1h , j5a,5b = 12.5 and j5a,4 = 7.6 , h-5a ) , 3.28 ( s , 3h , ome ) , 3.17 ( dd , 1h , j5b,5a = 12.5 and j5b,4 = 6.8 , h-5b ) , 1.39 ( s , 3h , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.6 ( c ) , 109.8 ( c-1 ) , 85.3 ( c-4 ) , 85.1 ( c-2 ) , 82.0 ( c-3 ) , 55.1 ( ome ) , 53.7 ( c-5 ) , 26.4 and 24.9 ( 2 ch3 ) . hrms ( es ) calcd for c9h15n3nao4 , 252.0955 ( mh ) ; found , 252.0949 . pph3 ( 1.95 g , 7.45 mmol ) was added to a solution of 1-o - methyl-2,3-o - isopropylidene-5-azido-5-deoxy--d - ribofuranose 51 ( 1.4 g , 6.11 mmol ) in thf ( 7 ml ) . the reaction mixture was stirred at rt for 16 h , after which water ( 7 ml ) was added and it was stirred for further 7 h. evaporation of the solvents followed by purification on an isco chromatographic system ( petrol etoac , 1:1 v / v ) gave the title compound as a colorless oil ( 1.04 g , 85% ) . h ( 400 mhz , cdcl3 ) 4.84 ( s , 1h , h-1 ) , 4.494.46 ( s , 2h , h-2 and h-3 ) , 4.054.01 ( m , 1h , h-4 ) , 3.24 ( s , 3h , ome ) , 2.712.62 ( m , 2h , 2 h-5 ) , 1.36 ( s , 3h , ch3 ) and 1.19 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.2 ( c ) , 109.5 ( c-1 ) , 88.8 ( c-4 ) , 85.4 ( c-2 ) , 82.1 ( c-3 ) , 54.9 ( ome ) , 45.4 ( c-5 ) , 26.4 and 24.8 ( 2 ch3 ) . hrms ( es ) calcd for c9h18no4 , 204.1230 ( mh ) ; found , 204.1226 . 1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose 52 ( 90 mg , 0.443 mmol ) , dipea ( 42 l , 0.239 mmol ) , and diethylsquarate ( 72 l , 0.487 mmol ) were reacted under the general protocol c to yield the desired product as a white foam ( 137 mg , 95% ) . h ( 400 mhz , cdcl3 ) 4.91 ( s , 1h , h-1 ) , 4.66 ( q , 4h , j = 6.9 , ch2 ) , 4.534.49 ( m , 2h , h-2 and h-3 ) , 4.29 ( app t , 1h , j = 5.6 , h-4 ) , 3.733.71 ( br m , 1h , h-5a ) , 3.513.49 ( br m , 1h , h-5b , 3.31 ( s , 3h , ome ) , 1.38 ( s , 3h , ch3 ) , 1.37 ( t , 3h , j = 6.9 , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 189.4 ( c = o ) , 184.3 ( c = o ) , 172.6 ( 2 c = c ) , 112.8 ( c ) , 109.9 ( c-1 ) , 85.8 ( c-4 ) , 85.2 ( c-2 ) , 81.5 ( c-3 ) , 69.7 ( ch2 ) , 55.5 ( ome ) , 46.4 ( c-5 ) , 26.3 , 24.8 ( 2 ch3 isopropyl ) and 15.7 ( ch3 ) . hrms ( es ) calcd for c15h22no7 , 328.1391 ( mh ) ; found , 328.1408 . to a solution of 3-(1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose)-4-ethoxycyclobut-3-ene-1,2-dione ( 91 mg , 0.305 mmol ) and dipea ( 26 l , 0.152 mmol ) in etoh ( 2 ml ) the reaction was stirred at rt for 1 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 106 mg , 60% ) . h ( 400 mhz , dmso - d6 ) 8.30 ( s , 1h , h-2 ) , 8.16 ( s , 1h , h-8 ) , 7.29 ( br s , 2h , nh2 ) , 6.18 ( d , 1h , j1,2 = 2.5 , h-1 ) , 5.42 ( dd , 1h , j2,3 = 6.3 and j2,1 = 2.5 , h-2 ) , 5.0 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.5 , h-3 ) , 4.91 ( s , 1h , h-1 ) , 4.63 ( d , 1h , j3,2 = 6.0 , h-3 ) , 4.55 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.264.22 ( m , 1h , h-4 ) , 4.12 ( app t , 1h , j4,5 = 7.0 , h-4 ) , 3.91 ( br s , 1h , h-5a ) , 3.753.68 ( m , 1h , h-5b ) , 3.64 ( br s , 1h , h-5a ) , 3.493.47 ( m , 1h , h-5b ) , 3.27 ( s , 3h , ome ) , 1.52 ( s , 3h , ch3 ) , 1.34 ( s , 3h , ch3 ) , 1.30 ( s , 3h , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 , 182.6 ( 2 c = o ) , 167.6 ( 2 c = c ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.2(c-5 ) , 113.7 , 111.6 ( 2 c ) , 108.8 ( c-1 ) , 88.7 ( c-1 ) , 85.5 ( c-4 ) , 85.1 ( c-4 ) , 84.5 ( c-2 ) , 83.2 ( c-2 ) , 81.2 ( c-3 ) , 81.1 ( c-3 ) , 54.4 ( och3 ) , 46.4 ( c-5 ) , 45.1 ( c-5 ) , 27.0 , 26.2 , 25.3 , and 24.7 ( 4 ch3 ) . hrms ( es ) calcd for c26h34n7o9 , 588.2413 ( mh ) ; found , 588.2429 . 3-(2,3-isopropylidene-5-amino-5-deoxyadenosine)-4-(1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose ) cyclobut-3-ene-1,2-dione 60 ( 40 mg , 0.07 mmol ) was deprotected by stirring in 0.1 m h2so4 for 16 h at 80 c to yield the desired compound as a white solid ( 15 mg , 45% ) . h ( 400 mhz , dmso - d6 ) 8.31 ( s , 1h , h-2 ) , 8.16 ( s , 1h , h-8 ) , 7.27 ( br s , 2h , nh2 ) , 6.17 ( d , 1h , j1,2 = 2.5 , h-1 ) , 4.91 ( s , 1h , h-1 ) , 4.354.24 ( m , 4h , h-2 , h-2 , h-3 , h-3 ) , 4.224.18 ( m , 1h , h-4 ) , 4.12 ( app t , 1h , j4,5 = 7.0 , h-4 ) , 3.92 ( br s , 1h , h-5a ) , 3.753.68 ( m , 1h , h-5b ) , 3.65 ( br s , 1h , h-5a ) , 3.493.47 ( m , 1h , h-5b ) . c ( 100 mhz , dmso - d6 ) 182.8 , 182.6 ( 2 c = o ) , 167.7 ( 2 c = c ) , 156.2 ( c-6 ) , 152.8 ( c-2 ) , 148.9 ( c-4 ) , 139.9 ( c-8 ) , 119.2(c-5 ) , 108.8 ( c-1 ) , 88.7 ( c-1 ) , 85.5 ( c-4 ) , 85.1 ( c-4 ) , 84.5 ( c-2 ) , 83.2 ( c-2 ) , 81.2 ( c-3 ) , 81.1 ( c-3 ) , 46.4 ( c-5 ) , 45.1 ( c-5 ) . hrms ( es ) calcd for c19h23n7o9 , 493.1557 ( mh ) ; found , 493.1564 . cyclopentylamine ( 104 l , 0.863 mmol ) , dipea ( 99 l , 0.570 mmol ) , and diethylsquarate ( 172 l , 1.162 mmol ) were reacted under the general protocol c to yield the desired product as a white foam ( 137 mg , 95% ) . h ( 400 mhz , cdcl3 ) 7.04 ( br s , 1h , nh ) , 4.754.73 ( m , 2h , och2 ) , 4.084.03 ( m , 1h , ch ) , 1.991.96 ( m , 2h ) , 1.741.72 ( m , 2h ) , 1.591.56 ( m , 4h ) ( 4 ch2 ) and 1.41 ( t , 3h , j = 6.6 , ch3 ) . c ( 100 mhz , cdcl3 ) 189.6 ( c-2 ) , 182.6 ( c-1 ) , 177.3 ( c-3 ) , 171.8 ( c-4 ) , 69.4 ( ch2 ) , 56.4 ( ch ) , 33.8 ( ch2 ) , 23.5 ( ch2 ) and 15.7 ( ch3 ) . hrms ( es ) calcd for c11h15no3 , 210.1130 ( mh ) ; found , 211.1127 . rf = 0.3 ( petrol etoac , 6:4 v / v ) . to a solution of 3-cyclopentylamino-4-ethoxycyclobut-3-ene-1,2-dione 55 ( 110 mg , 0.526 mmol ) and dipea ( 50 l , 0.284 mmol ) in etoh ( 3 ml ) the reaction mixture was stirred at rt for 18 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white solid ( 106 mg , 43% ) . h ( 400 mhz , meoh - d4 ) 8.32 ( s , 1h , h-8 ) , 8.29 ( s , 1h , h-2 ) , 6.25 ( d , 1h , j1,2 = 2.6 , h-1 ) , 5.55 ( dd , 1h , j2,3 = 6.4 and j2,1 = 2.6 , h-2 ) , 5.16 ( dd , 1h , j3,2 = 6.4 and j3,4 = 3.8 , h-3 ) , 4.444.40 ( m , 1h , h-4 ) , 4.07 ( dd , 1h , j5a,5b = 14.1 and j5a,4 = 4.4 , h-5a ) , 3.93 ( dd , 1h , j5ba,5a = 14.1 and j5b,4 = 6.7 , h-5b ) , 3.78 ( sept , 1h , j = 6.6 , ch ) , 1.722.07 ( m , 8h , 4 ch2 ) , 1.64 ( s , 3h , ch3 ) and 1.43 ( s , 3h , ch3 ) . c ( 100 mhz , meoh - d4 ) 183.9 ( c-2 ) , 183.5 ( c-1 ) , 169.4 ( c-3 ) , 169.0 ( c-4 ) , 157.4 ( c-6 ) , 154.2 ( c-2 ) , 150.3 ( c-4 ) , 141.9 ( c-8 ) , 120.7 ( c-5 ) , 115.9 ( c ) , 91.4 ( c-1 ) , 87.0 ( c-4 ) , 85.2 ( c-2 ) , 82.9 ( c-3 ) , 55.9 ( ch ) , 46.6 ( c-5 ) , 43.8 , 35.1 ( 4 ch2 ) , 27.5 and 25.6 ( 2 ch3 ) . hrms ( es ) calcd for c22h28n7o5 , 470.2146 ( mh ) ; found , 470.2158 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-(cyclopentylamino)-cyclobut-3-ene-1,2dione 61 ( 50 mg , 0.11 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid ( 40 mg , 85% ) . h ( 400 mhz , dmso - d6 ) 9.32 , 9.17 ( 2 nh ) , 8.39 ( s , 1h , h-8 ) , 8.30 ( s , 1h , h-2 ) , 7.74 ( br s , 2h , nh2 ) , 5.94 ( d , 1h , j = 5.9 , h-1 ) , 4.744.63 ( m , 4h , 2 ch2 ) , 4.254.14 ( m , 2h , h-2 , h-3 ) , 3.993.96 ( m , 1h , h-4 ) , 3.843.80 ( m , 1h , h-5a ) , 3.753.67 ( m , 2h , ch , h-5b ) , 1.42 ( t , 2h , j = 6.8 ) and 1.33 ( t , 2h , j = 6.8)(2 ch2 ) . c ( 100 , dmso - d6 ) 189.2 ( c = o ) , 182.3 ( c = o ) , 176.8 ( c = c ) , 172.8 ( c = c ) , 155.3 ( c-6 ) , 151.3 ( c-2 ) , 148.8 ( c-4 ) , 140.7 ( c-2 ) , 119.5 ( c-5 ) , 88.3 ( c-1 ) , 83.5 ( c-2 ) , 72.5 ( c-3 ) , 70.8 ( c-4 ) , 68.8 ( 2c , 2 ch2 ) , 45.8 ( c-5 ) , 15.5 ( 2c , 2 ch2 ) . hrms ( es ) calcd for c19h24n7o5 , 430.1833 ( mh ) ; found , 418.1838 . butylamine ( 135 l , 1.367 mmol ) , dipea ( 141 l , 0.811 mmol ) , and diethylsquarate 53 ( 222 l , 1.503 mmol ) were reacted under general protocol c to leave the desired product as a white foam ( 230 mg , 91% ) . h ( 400 mhz , cdcl3 ) 4.77 ( q , 2h , j = 7.2 , och2-me ) , 3.66 ( t , 1h , j = 7.0 , chh ) , 3.48 ( t , 1h , j = 7.0 , chh ) , 1.691.62 ( m , 2h , ch2 ) , 1.531.40 ( br m , 5h , ch2 and ch3 ) and 1.01 ( t , 3h , j = 7.2 , ch3 ) . c ( 100 mhz , cdcl3 ) 189.9 ( c-2 ) , 184.5 ( c-1 ) , 177.6 ( c-3 ) , 174.7 ( c-4 ) , 70.7 , 45.3 , 33.7 , 20.6 ( 4 ch2 ) , 16.2 ( et : ch3 ) and 14.0 ( bu : ch3 ) . hrms ( es ) calcd for c10h16no3 , 198.1125 ( mh ) ; found , 198.1124 . rf = 0.5 ( petrol etoac , 6:4 v / v ) . to a solution of 3-butylamino-4-ethoxycyclobut-3-ene-1,2-dione 56 ( 200 mg , 1.081 mmol ) and dipea ( 92 l , 0.531 mmol ) in etoh ( 5 ml ) the reaction mixture was stirred at rt for 24 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 364 mg , 81% ) . h ( 400 mhz , dmso - d6 ) 8.38 ( s , 1h , h-8 ) , 8.23 ( s , 1h , h-2 ) , 7.39 ( br s , 2h , nh2 ) , 6.25 ( d , 1h , j1,2 = 2.4 , h-1 ) , 5.50 ( dd , 1h , j2,3 = 6.3 and j2,1 = 2.4 , h-2 ) , 5.04 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.5 , h-3 ) , 4.334 .. 29 ( m , 1h , h-4 ) , 3.95 ( br , 1h , h-5a ) , 3.813.75 ( br , 1h , h-5b ) , 3.51 ( br , 2h , ch2 ) , 3.383.34 ( m , 1h , chh ) , 1.60 ( s , 3h , ch3 ) , 1.51 ( br , 1h , chh ) , 1.32 ( s , 3h , ch3 ) , 1.32 ( q , 2h , j = 7.3 , ch2-me ) and 0.91 ( t , 3h , j = 7.3 , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 ( c = o ) , 182.2 ( c = o ) , 167.7 ( c = c ) , 167.1 ( c = c ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.1 ( c-5 ) , 113.7 ( c ) , 88.7 ( c-1 ) , 85.1 ( c-4 ) , 83.6 ( c-2 ) , 81.2 ( c-3 ) , 45.0 ( ch2 ) , 42.9 ( c-5 ) , 32.7 ( ch2 ) , 27.0 , 25.2 ( 2 ch3 ) , 18.9 ( ch2 ) and 13.4 ( ch3 ) . hrms ( es ) calcd for c21h28n7o5 , 458.2146 ( mh ) ; found , 458.2142 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-butylamino - cyclobut-3-ene-1,2-dione 62 ( 50 mg , 0.109 mmol ) was deprotected under general protocol d to give the desired compound as a white solid ( 41 mg , 91% ) . h ( 400 mhz , dmso - d6 ) 8.40 ( s , 1h , h-8 ) , 8.26 ( s , 1h , h-2 ) , 7.567.45 ( br m , 4h , nh2 and 2 nh ) , 5.98 ( d , 1h , j1,2 = 5.8 , h-1 ) , 4.73 ( app t , 1h , j2,3 = j2,1 = 5.8 , h-2 ) , 4.23 ( app t , 1h , j3,2 = j3,4 = 5.8 , h-3 ) , 4.104.06 ( m , 1h , h-4 ) , 3.863.79 ( m , 2h , 2 h-5 ) , 3.535.52 ( m , 2h , ch2 ) , 1.511.49 ( m , 2h , ch2 ) , 1.34 ( q , 2h , j = 7.3 ) and 0.92 ( t , 3h , j = 7.3 ) . c ( 100 mhz , dmso - d6 ) 182.2 ( 2 c = o ) , 167.6 ( c-3 ) , 167.2 ( c-4 ) , 154.9 ( c-6 ) , 151.2 ( c-2 ) , 149.2 ( c-4 ) , 140.4 ( c-8 ) , 119.2 ( c-5 ) , 87.8 ( c-1 ) , 83.7 ( c-4 ) , 72.9 ( c-2 ) , 70.8 ( c-3 ) , 45.2 ( c-5 ) , 33.4 , 24.8 , 23.8 ( 3 ch2 ) and 15.8 ( ch3 ) . hrms ( es ) calcd for c18h24n7o5 , 418.1833 ( mh ) ; found , 418.1834 . hexylamine ( 130 l , 0.988 mmol ) , dipea ( 92 l , 0.533 mmol ) , and diethylsquarate 53 ( 161 l , 1.087 mmol ) were reacted under general protocol c to yield the desired product as a white foam ( 200 mg , 95% ) . h ( 400 mhz , cdcl3 ) 4.804.75 ( m , 2h , ch2-me ) , 3.65 ( t , 1h , j = 7.0 , chh - nh ) , 3.48 ( t , 1h , j = 7.0 , chh - nh ) , 1.691.64 ( m , 2h , ch2 ) , 1.531.44 ( m , 9h , 3 ch2 and ch3 ) and 0.990.96 ( m , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 189.9 ( c-2 ) , 184.5 ( c-1 ) , 177.5 ( c-3 ) , 174.8 ( c-4 ) , 70.7 ( et : ch2 ) , 45.6 , 32.5 , 31.6 , 27.1 , 23.6 ( 5 ch2 ) , 16.2 ( et : ch3 ) and 14.5 ( hex : ch3 ) . hrms ( es ) calcd for c12h20no3 , 226.1438 ( mh ) ; found , 226.1443 . rf = 0.62 ( petrol etoac , 6:4 v / v ) . to a solution of 3-hexylamino-4-ethoxycyclobut-3-ene-1,2-dione 57 ( 190 mg , 0.892 mmol ) and dipea ( 76 l , 0.438 mmol ) in etoh ( 5 ml ) the reaction was stirred at rt for 20 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 291 mg , 74% ) . h ( 400 mhz , dmso - d6 ) 8.37 ( s , 1h , h-8 ) , 8.23 ( s , 1h , h-2 ) , 7.38 ( br s , 4h , nh2 and 2 nh ) , 6.26 ( d , 1h , j1,2 = 2.4 , h-1 ) , 5.49 ( dd , 1h , j2,3 = 6.2 and j2,1 = 2.4 , h-2 ) , 5.07 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.334.29 ( m , 1h , h-4 ) , 3.96 ( br , 1h , h-5a ) , 3.813.74 ( br , 1h , h-5b ) , 3.50 ( br , 2h , ch2 ) , 1.60 ( s , 3h , ch3 ) , 1.51 ( br , 1h , chh ) , 1.38 ( s , 3h , ch3 ) , 1.30 ( br , 7h , 3 ch2 and chh ) and 0.910.88 ( m , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 ( c-2 ) , 182.2 ( c-1 ) , 167.9 ( c-3 ) , 168.5 ( c-4 ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.2 ( c-5 ) , 113.7 ( c ) , 88.7 ( c-1 ) , 85.2 ( c-4 ) , 83.1 ( c-2 ) , 81.2 ( c-3 ) , 45.1 ( ch2 ) , 43.2 ( c-5 ) , 30.7 , 30.6 ( 2 ch2 ) , 27.0 ( ch3 ) , 25.4 ( ch2 ) , 25.3 ( ch3 ) 21.9 ( ch2 ) and 13.8 ( ch3 ) . hrms ( es ) calcd for c23h32n7o5 , 486.2459 ( mh ) ; found , 486.2475 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-(hexylamino)cyclobut-3-ene-1,2-dione 63 ( 50 mg , 0.102 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid ( 41 mg , 91% ) . h ( 400 mhz , dmso - d6 ) 8.42 ( s , 1h , h-8 ) , 8.27 ( s , 1h , h-2 ) , 7.65 ( br s , 2h , nh2 ) , 7.44 ( br s , 2h , 2 nh ) , 5.98 ( d , 1h , j1,2 = 5.7 , h-1 ) , 4.72 ( app t , 1h , j = 5.0 , h-2 ) , 4.22 ( app t , 1h , j = 4.3 , h-3 ) , 4.104.06 ( m , 2h , h-4 and h-5a ) , 3.853.79 ( m , 1h , h-5b ) , 3.51 ( br s , 2h , ch2-nh ) , 1.511.29 ( m , 8h , 4 ch2 ) and 0.910.88 ( m , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.6 ( c-2 ) , 182.3 ( c-1 ) , 167.9 ( 2 c = c ) , 155.2 ( c-6 ) , 151.5 ( c-2 ) , 149.2 ( c-4 ) , 140.2 ( c-8 ) , 119.2 ( c-5 ) , 87.6 ( c-1 ) , 83.7 ( c-4 ) , 72.9 ( c-2 ) , 70.8 ( c-3 ) , 45.5 ( ch2 ) , 43.2 ( c-5 ) , 30.7 , 30.6 , 25.4 , 21.9 ( 4 ch2 ) and 13.8 ( ch3 ) . hrms ( es ) calcd for c20h28n7o5 , 446.2146 ( mh ) ; found , 446.2157 . a solution of 1-o - methyl-2,3-o - isopropylidene--d - ribofuranose 49 ( 600 mg , 2.94 mmol ) in dmf ( 40 ml ) was cooled to 0 c , and nah ( 156 mg , 3.91 mmol , 60% in mineral oil ) was added . the mixture was stirred at 0 c for 30 min , after which tbai ( 65 mg , 0.176 mmol ) and propargyl chloride ( 0.25 ml , 3.528 mmol ) were added . the reaction mixture was stirred at rt for 16 h , the solvent was removed under reduced pressure , and the residue was purified by column chromatography using an isco chromatographic system ( petrol etoac , 1:0 0:1 v / v ) . the product was obtained as a colorless liquid ( 512 mg , 72% ) . h ( 400 mhz , cdcl3 ) 4.95 ( s , 1h , h-1 ) , 4.65 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.55 ( d , 1h , j3,2 = 6.0 , h-3 ) , 4.334.30 ( m , 1h , h-4 ) , 4.17 ( d , 2h , j = 2.4 , ch2 ) , 3.58 ( dd , 1h , j5a,5b = 9.5 and j5a,4 = 6.5 , h-5a ) , 3.523.47 ( m , 1h , h-5b ) , 3.32 ( s , 3h , ome ) , 2.42 ( t , 1h , j = 6.3 hz , ch ) , 1.46 ( s , 3h , ch3 ) and 1.30 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.3 ( c ) , 109.2 ( c-1 ) , 85.0 ( c-4 ) , 84.8 ( c-2 ) , 81.9 ( c-3 ) , 79.3 ( c ) , 74.6 ( hc ) , 70.5 ( c-5 ) , 58.3 ( ch2 ) , 54.8 ( och3 ) , 26.4 and 24.9 ( 2 ch3 ) . hrms ( es ) calcd for c12h18nao5 , 265.1046 ( mh ) ; found , 265.1042 . 2,3-o - isopropylidene-5-azido-5-deoxyadenosine 68 ( 100 mg , 0.30 mmol ) was taken up in naoac buffer ( ph 4 , 1 m , 15 ml ) and br2 ( 12 l , 0.45 mmol ) added . the resulting solution was stirred in the dark for 24 h and then a solution of nahso3 ( 4 m , aq ) added until the solution was colorless . all solvents were evaporated and the residue purified by column chromatography using an isco chromatographic system ( dcm acetone , 6:4 v / v ) . the title compound was obtained as an off - white solid ( 123 mg , 99% ) . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 6.20 ( d , 1h , j1,2 = 1.8 hz , h-1 ) , 5.99 ( br s , 2h , nh2 ) , 5.68 ( dd , 1h , j2,3 = 6.3 and j2,1 = 1.8 , h-2 ) , 5.15 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.6 , h-4 ) , 4.364.31 ( m , 1h , h-4 ) , 3.543.43 ( m , 2h , 2 h-5 ) , 1.61 ( s , 3h , ch3 ) and 1.39 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 154.4 ( c-6 ) , 152.8 ( c-2 ) , 150.3 ( c-4 ) , 127.5 ( c-8 ) , 120.1 ( c-5 ) , 114.5 ( c ) , 91.2 ( c-1 ) , 86.4 ( c-4 ) , 83.4 ( c-2 ) , 82.4 ( c-3 ) , 52.1 ( c-5 ) , 27.0 and 25.3 ( 2 ch3 ) . hrms ( es ) calcd for c13h16n8o3br , 411.0523 ( mh ) ; found , 411.0532 ; and calcd for c13h16n8o3br , 413.0503 ( mh ) ; found , 413.0522 . rf = 0.58 ( dcm acetone , 3:2 v / v ) . to a solution of 2,3-o - isopropylidene-5-deoxy-5-azido-8-bromoadenosine 69 ( 123 mg , 0.30 mmol ) in a mixture of buoh - h2o ( 1:1 v / v , 6 ml ) was added cuso45h2o ( 2 mg , 0.015 mmol ) , sodium ascorbate ( 6 mg , 0.03 mmol ) , and 1-o - methyl-2,3-o - isopropylidene-5-o - propargyl--d - ribofuranose 70 ( 73 mg , 0.30 mmol ) . the reaction mixture was stirred at rt for 16 h , the solvents were removed under vacuum , and the residue was purified on an isco chromatographic system ( dcm acetone , 6:4 v / v ) to yield the product as a pale - yellow solid ( 140 mg , 71% ) . h ( 400 mhz , cdcl3 ) 8.22 ( s , 1h , h-2 ) , 7.32 ( s , 1h , ch - triazole ) , 6.29 ( br s , 2h , nh2 ) , 6.17 ( d , 1h , j1,2 = 1.7 hz , h-1 ) , 5.55 ( dd , 1h , j2,3 = 6.3 and j2,1 = 1.7 , h-2 ) , 5.22 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.9 , h-3 ) , 4.88 ( s , 1h , h-1 ) , 4.74 ( dd , 1h , j5a,5b = 13.9 and j5a,4 = 4.1 , h-5a ) , 4.624.48 ( m , 6h , h-4 , h-2 , h-3 , h-5b and ch2-triazole ) , 4.254.21 ( m , 1h , h-4 ) , 3.503.39 ( m , 2h , 2 h-5 ) , 3.20 ( s , 3h , ome ) , 1.55 ( s , 3h , ch3 ) , 1.40 ( s , 3h , ch3 ) , 1.33 ( s , 3h , ch3 ) and 1.21 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 154.6 ( c-6 ) , 153.1 ( c-2 ) , 150.1 ( c-4 ) , 144.9 ( c - triazole ) , 127.0 ( c-8 ) , 123.5 ( ch - triazole ) , 120.1 ( c-5 ) , 114.7 , 112.3 ( 2 c ) , 109.2 ( c-1 ) , 90.9 ( c-1 ) , 85.9 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.9 ( c-2 ) , 81.9 ( 2c , c-3 and c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.5 ( c-5 ) , 27.0 , 26.3 , 25.3 , and 24.9 ( 4 ch3 ) . hrms ( es ) calcd for c25h33n8nao8br , 675.1497 ( mh ) ; found , 675.1469 ; calcd for c25h33n8nao8br , 677.1476 ( mh ) ; found , 677.1451 . rf = 0.58 ( dcm acetone , 3:2 v / v ) . to 1-(2,3-o - isopropylidene-5-deoxy-8-bromoadenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 71 ( 140 mg , 0.21 mmol ) , na2cl4pd ( 3 mg , 5 mol % ) , phb(oh2 ) ( 32 mg , 0.27 mmol ) , tppts ( 30 mg , 25 mol % ) , and na2co3 ( 68 mg , 0.64 mmol ) was added degassed mecn h2o ( 3 ml , 1:2 v / v ) and the resulting solution stirred at 80 c for 1 h. all solvents were evaporated and the residue purified by column chromatography using an isco chromatography system ( dcm acetone , 6:4 v / v ) to yield the product ( 30 mg , 21% ) . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 7.707.48 ( m , 5h , ph ) , 7.35 ( s , 1h , ch - triazole ) , 6.90 ( br s , 2h , nh2 ) , 6.04 ( d , 1h , j1,2 = 1.6 , h-1 ) , 5.57 ( dd , 1h , j2,3 = 6.2 and j2,1 = 1.6 , h-2 ) , 5.28 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.87 ( s , 1h , h-1 ) , 4.82 ( dd , 1h , j5a,5b = 14.2 and j5a,4 = 4.7 , h-5a ) , 4.71 ( dd , 1h , j5b,5a = 14.2 and j5b,4 = 8.0 , h-5a ) , 4.594.46 ( m , 5h , h-4 , h-2 , h-3 and ch2-triazole ) , 4.234.20 ( m , 1h , h-4 ) , 3.47 ( dd , 1h , j5a,5b = 9.7 and j5a,4 = 6.5 , h-5a ) , 3.433.40 ( m , 1h , h-5b ) , 3.18 ( s , 3h , ome ) , 1.45 ( s , 3h , ch3 ) , 1.38 ( s , 3h , ch3 ) , 1.29 ( s , 3h , ch3 ) and 1.21 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 114.3 , 112.2 ( 2 c ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.8 ( c-5 ) , 26.9 , 26.3 , 25.2 , and 24.9 ( 4 ch3 ) . hrms ( es ) calcd for c31h38n8nao8 , 673.2705 ( mh ) ; found , 673.2678 . 1-(2,3-o - isopropylidene-5-deoxy-8-phenyladenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 72 ( 30 mg , 0.046 mmol ) was deprotected by stirring in 0.1 m h2so4 for 16 h at 80 c to yield the desired compound ( 6 mg , 24% ) as a white solid . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 7.707.48 ( m , 5h , ph ) , 7.35 ( s , 1h , ch - triazole ) , 6.90 ( br s , 2h , nh2 ) , 6.04 ( d , 1h , j1,2 = 1.6 , h-1 ) , 5.57 ( dd , 1h , j2,3 = 6.2 and j2,1 = 1.6 , h-2 ) , 5.28 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.87 ( s , 1h , h-1 ) , 4.82 ( dd , 1h , j5a,5b = 14.2 and j5a,4 = 4.7 , h-5a ) , 4.71 ( dd , 1h , j5b,5a = 14.2 and j5b,4 = 8.0 , h-5a ) , 4.594.46 ( m , 5h , h-4 , h-2 , h-3 and ch2-triazole ) , 4.234.20 ( m , 1h , h-4 ) , 3.47 ( dd , 1h , j5a,5b = 9.7 and j5a,4 = 6.5 , h-5a ) and 3.433.40 ( m , 1h , h-5b ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) and 51.8 ( c-5 ) . hrms ( es ) calcd for c24h29n8o8 , 557.2103 ( mh ) ; found , 557.2097 . to a solution of tetrazole ( 81 mg , 1.16 mmol ) and diisopropyl - dibenzylphosphoramidite ( 300 mg , 0.871 mmol ) in dcm ( 10 ml ) was added cyclopentanol 77 ( 50 mg , 0.58 mmol ) . the reaction mixture was stirred at rt for 20 min , after which tlc analysis showed total conversion of starting material to a single phosphite ( petrol etoac , 6:4 v / v , rf = 0.32 ) . the solution was cooled to 0 c , and mcpba ( 200 mg , 1.16 mmol ) was added in one portion . the mixture was warmed up to rt , diluted with etoac ( 20 ml ) , and washed with 10% na2so3 ( 20 ml ) , satd aq nahco3 ( 20 ml ) , and brine ( 20 ml ) . the organic phase was collected , dried ( na2so4 ) , filtered , and evaporated to dryness . the residue was purified with an isco chromatographic system ( petrol etoac , 7:3 v / v ) to yield the title compound as a colorless oil ( 173 mg , 86% ) . h ( 400 mhz , cdcl3 ) 7.367.20 ( m , 10h , h - benzyl ) , 5.064.97 ( m , 4h , 2 ch2 ) , 4.904.85 ( m , 1h , ch p ( 161 mhz , cdcl3 , decoupled ) 1.6 ( s ) . the above material ( 78 , 173 mg , 0.5 mmol ) cyclohexane ( 10:1:5 v / v / v , 16 ml ) to which was added pd(oh)2/c ( 20% ) . the solution was heated to 80 c for 2 h , after which the palladium was removed by filtration through celite and the filtrate was evaporated under reduced pressure , leaving a residue which was used directly in the next step . ampna salt ( 190 mg , 0.547 mmol ) was passed through a small dowex column ( tea form ) and eluted with milli - q water . the solvent was evaporated to leave a residue , which was dissolved in dmso and coevaporated with dmf ( 3 3 ml ) . the residue obtained was dissolved in dmso ( 3 ml ) and morpholine ( 0.25 ml , 2.845 mmol ) , dipyridyldisulfide ( 301 mg , 1.367 mmol ) , and triphenylphosphine ( 358 mg , 1.367 mmol ) were added in this order . the resulting yellow solution was stirred for 90 min , after which a 0.1 m solution of nai in acetone was added . the precipitate obtained was collected by filtration and used directly in the next step . to a solution of amp - morpholidate ( 154 mg , 0.350 mmol ) and cyclopentane monophosphate 79 ( 64 mg , 0.380 mmol ) in 0.2 n mncl2 in formamide ( 2 ml ) was added mgso4 ( 82 mg , 0.70 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . precipitation of the product occurred on addition of mecn and purification on rp-18 afforded ( after treatment with chelex 100 ) the desired dinucleotide as a glassy solid ( 55 mol , 14% over 2 steps ) . h ( 400 mhz , d2o ) 8.44 ( s , 1h , h-2 ) , 8.19 ( s , 1h , h-8 ) , 6.07 ( s , 1h , h-1 ) , 4.734.71 ( m , 1h , ch o ) , 4.66 ( br s , 1h , h-2 ) , 4.48 ( br s , 1h , h-3 ) , 4.32 ( br s , 1h , h-4 ) , 4.15 ( br s , 2h , 2 h-5 ) , 1.621.60 ( m , 4h ) , 1.521.50 ( m , 2h ) and 1.361.34 ( m , 2h ) ( 4 ch2 ) . c ( 100 mhz , d2o ) 158.2 ( c-6 ) , 153.0 ( c-8 ) , 149.3 ( c-4 ) , 140.0 ( c-2 ) , 113.3 ( c-5 ) , 87.0 ( c-1 ) , 84.0 ( c-4 ) , 79.9 ( ch ) , 74.3 ( c-2 ) , 70.5 ( c-3 ) , 65.2 ( c-5 ) , 33.4 and 22.7 ( 2 ch2 ) . a flask containing 8-bromo-2,3-o - isopropylidene - adenosine 81 ( 200 mg , 0.519 mmol ) , na2cl4pd ( 5 mol % ) , tppts ( 25 mol % ) , phb(oh)2 ( 190 mg , 1.562 mmol ) , and na2co3 ( 165 mg , 1.557 mmol ) was purged with argon , and a degassed mixture of mecn h2o ( 1:1 v / v , 6 ml ) was added . the resulting mixture was refluxed for 1 h , then water ( 6 ml ) was added and the solution neutralized with 1 m hcl . the white precipitate obtained was collected by filtration and dried under vacuum ( 161 mg , 81% ) . h ( 400 mhz , dmso - d6 ) 8.16 ( s , 1h , h-2 ) , 7.737.71 ( m , 2h , ar h ) , 7.44 ( s , 2h , nh2 ) , 5.84 ( d , 1h , j1,2 = 3.4 , h-1 ) , 5.56 ( dd , 1h , j2,3 = 6.1 and j2,1 = 3.4 , h-2 ) , 5.385.36 ( m , 1h , 5oh ) , 5.03 ( dd , 1h , j3,2 = 6.1 and j3,4 = 2.5 , h-3 ) , 4.17 ( dd , 1h , j4,5 = 5.1 and j4,3 = 2.5 , h-4 ) , 3.63 ( dd , 1h , j5a,5b = 11.5 and j5a,4 = 5.1 , h-5a ) , 3.563.51 ( m , 1h , h-5b ) , 1.41 ( s , 3h , ch3 ) and 1.28 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.4 ( c-2 ) , 150.1 ( c-4 ) , 149.6 ( c - ph ) , 130.3 , 129.6 ( 2 ch ) , 129.1 ( c-8 ) , 128.8 ( ch ) , 118.8 ( c-5 ) , 113.0 ( c ) , 90.4 ( c-1 ) , 86.5 ( c-4 ) , 81.9 ( c-2 ) , 81.8 ( c-3 ) , 61.9 ( c-5 ) , 27.0 and 25.2 ( 2 ch3 ) . hrms ( es ) calcd for c19h21n5o4 , 384.1672 ( mh ) ; found , 384.1686 . 8-phenyl-2,3-o - isopropylidene - adenosine ( 82 , 150 mg , 0.39 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid which was used directly in the next step . 8-phenyl - adenosine ( 0.39 mmol ) was dissolved in triethylphosphate ( 1 ml ) by heating with a heatgun . the resulting colorless solution was cooled to 0 c , and water ( 2 l ) was added followed by pocl3 ( 0.15 ml , 1.56 mmol ) , then stirred at 0 c until disappearance of the starting material and formation of a single peak was observed as shown by hplc . water ( 15 ml ) and the mixture was stirred for 15 min at 0 c , after which it was warmed to rt . triethylphosphate was extracted with etoac ( 6 6 ml ) , and the aqueous phase was neutralized with 2 n naoh . it was then applied to a reverse phase gradifrac column eluted with a 565% gradient of mecn in 0.05 m teab . the appropriate fractions were collected and lyophilized to afford the desired monophosphate as its triethylammonium salt . h ( 400 mhz , dmso - d6 ) 8.16 ( s , 1h , h-2 ) , 7.737.71 ( m , 2h , ar h ) , 7.44 ( s , 2h , nh2 ) , 5.84 ( d , 1h , j1,2 = 3.4 , h-1 ) , 5.56 ( dd , 1h , j2,3 = 6.1 and j2,1 = 3.4 , h-2 ) , 5.03 ( dd , 1h , j3,2 = 6.1 and j3,4 = 2.5 , h-3 ) , 4.17 ( dd , 1h , j4,5 = 5.1 and j4,3 = 2.5 , h-4 ) , 3.63 ( dd , 1h , j5a,5b = 11.5 and j5a,4 = 5.1 , h-5a ) and 3.563.51 ( m , 1h , h-5b ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.4 ( c-2 ) , 150.1 ( c-4 ) , 149.6 ( c - ph ) , 130.3 , 129.6 ( 2 ch ) , 129.1 ( c-8 ) , 128.8 ( ch ) , 118.8 ( c-5 ) , 113.0 ( c ) , 90.4 ( c-1 ) , 86.5 ( c-4 , d , j = 8.3 hz ) , 81.9 ( c-2 ) , 81.8 ( c-3 ) , 61.9 ( c-5 , d , j = 8.8 hz ) . hrms ( es ) calcd for c16h17n5o7 , 422.0871 ( m h ) ; found , 422.0868 . 8-ph - ampna salt ( 83 , 53 mg , 0.092 mmol ) was passed through a small dowex column ( tea form ) and eluted with milli - q water . the solvent was evaporated to leave a residue , which was dissolved in dmso and coevaporated with dmf ( 3 3 ml ) . the residue obtained was dissolved in dmso ( 90 l ) and morpholine ( 42 l , 0.478 mmol ) , dipyridyldisulfide ( 51 mg , 0.23 mmol ) , and triphenylphosphine ( 60 mg , 0.23 mmol ) were added in this order . the resulting yellow solution was stirred for 90 min , after which a 0.1 m solution of nai in acetone was added . the precipitate obtained was collected by filtration and used directly in the next step . to a solution of 8-ph - amp - morpholidate ( 31 mg , 0.060 mmol ) and cyclopentane monophosphate 79 ( 11 mg , 0.066 mmol ) in 0.2 n mncl2 in formamide ( 0.5 ml ) was added mgso4 ( 14 mg , 0.12 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . precipitation of the product occurred on addition of mecn and purification on rp-18 afforded ( after treatment with chelex 100 ) the desired dinucleotide as a glassy solid ( 11 mol , 12% over 2 steps ) . h ( 400 mhz , d2o ) 8.21 ( s , 1h , h-2 ) , 7.67 ( d , 1h , j = 6.2 ) , 7.607.56 ( m , 2h ) , 7.28 ( d , 2h , j = 8.1 ) ( 5 arh ) , 5.82 ( d , 1h , j = 6.4 , h-1 ) , 5.15 ( app t , 1h , h-2 ) , 4.31 ( dd , 1h , j = 6.4 , 4.5 , h-3 ) , 4.083.98 ( m , 4h , h-4 , 2 h-5 , ch o ) , 1.621.60 ( m , 4h ) , 1.521.50 ( m , 2h ) and 1.361.34 ( m , 2h ) ( 4 ch2 ) . c ( 100 mhz , d2o ) 158.3 ( c-6 ) , 152.1 ( c-8 ) , 149.3 ( c-4 ) , 140.1 ( c-2 ) , 132.4 ( 2c ) , 129.7 ( 2c ) , 128.6 ( 5 arch ) , 113.2 ( c-5 ) , 87.2 ( c-1 ) , 83.8 ( c-4 ) , 79.7 ( ch ) , 73.9 ( c-2 ) , 70.4 ( c-3 ) , 65.3 ( c-5 ) , 33.6 and 22.9 ( 2 ch2 ) . hrms ( es ) calcd for c21h27n5o10p2 , 570.1155 ( m h ) ; found , 570.1149 . uv ( h2o , ph 7.4 ) max 276 nm ( 17600 ) . 8-phenyl-2-deoxy - cadpr 85 ( 13 mol ) was incubated in kh2po4 buffer ( 100 mm , ph 7.4 ) at 70 c for 2.5 h , after which hplc analysis showed a new peak at rt = 28 min . the volatiles were removed under reduced pressure , and the residue was applied to a c18 semipreparative column eluted with a gradient of 0.1 m teab in mecn . the appropriate fractions were combined and evaporated to leave the desired product as a glassy solid in its triethylammonium form ( 5.06 mol , 39% ) . h ( 500 mhz , d2o ) 8.21 ( s , 1h , h-2 ) , 7.657.55 ( m , 5h , ar - h ) , 6.296.27 ( m , 1h , h-1 ) , 5.21 ( d , 1h , j1,2 = 4.5 , h-1 ) , 5.10 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.543.81 ( m , 9h , h - ribose ) , 3.223.16 ( m , 1h , h-2a ) and 2.202.13 ( m , 1h , h-2b ) . p ( decoupled , 109 mhz , d2o ) 11.1 ( br s ) . c ( 100 m , d2o ) 153.0 ( c-6 ) , 152.6 ( c-8 ) , 150.3 ( c-2 ) , 148.4 ( c-4 ) , 131.1 , 129.7 , 129.0 , 128.5 ( 5 ch phenyl ) , 101.2 ( c-1 ) , 91.1 ( c-1 ) , 86.6 ( c-4/c-4 ) , 75.2 ( c-2 ) , 74.8 ( c-2 ) , 70.9 ( c-3 ) , 70.4 ( c-3 ) , 65.4 ( c-5 ) and 62.8 ( c-5 ) . hrms ( es ) calcd for c21h26n5o13p2 , 618.1008 ( m h ) ; found , 618.1013 . bsa ( bovine serum albumin ) , fura-2/am , nacl , egta , nmdg , tris base , and histopaque-1119 were purchased from sigma aldrich ( mnchen , germany ) . kcl , mgso4 , mgcl2 , cacl2 , nah2po4 , d - glucose , l - ascorbic acid , tween , ionomycin , and edta were obtained from merck ( darmstadt , germany ) . fibronectin , dmem , and penicillin / streptomycin were supplied by invitrogen ( darmstadt , germany ) . fbs ( fetal bovine serum ) and g418 sulfate were purchased from biochrom ( berlin , germany ) . the anti - trpm2 antibody was procured from novus biologicals ( littleton , usa ) . the goat antirabbit antiserum was purchased from dianova ( hamburg , germany ) . percoll and ecl western blotting detection reagents were supplied by ge healthcare ( uppsala , sweden ) . hek293 cells were maintained in dmem medium containing glutamax i complemented with 10% fbs , 100 units / ml penicillin , and 100 g / ml streptomycin at 37 c in the presence of 5% co2 . hek293 clones expressing trpm2/egfp ( or egfp for control ) were cultured under the same conditions , while the medium was supplemented with 400 g / ml g418 sulfate . hek293 wild - type cells were transfected with two different expression vectors coding either for human trpm2 and egfp ( pires2-egfp - trpm2 ) or for egfp alone ( pires2-egfp ) as described previously . cells carrying the expression constructs were selected by addition of 400 g / ml g418 sulfate ( biochrom ) to the culture medium . cells were seeded at low density the day before use . during the experiments , cells were kept at room temperature in bath solution ( 1 mm cacl2 , 140 mm nmdg , 5 mm kcl , 3.3 mm mgcl2 , 1 mm cacl2 , 5 mm d - glucose , 10 mm hepes , ph 7.4 ) . patch pipets with resistances of 1.73.5 m were pulled from 1.5 mm diameter borosilicate glass capillaries and filled with pipet solution ( 120 mm kcl , 8 mm nacl , 1 mm mgcl2 , 10 mm hepes , 10 mm egta , 5.6 mm cacl2 ) , resulting in 200 nm free [ ca ] as calculated by cabuf software ( g. droogmans , formerly available from ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip ) . data were acquired with an epc10 amplifier and patchmaster software ( heka elektronik , germany ) and were compensated for fast and slow capacity transients . the cells were held at 50 mv and current was measured during 140 ms voltage ramps from 85 to 20 mv every 5 s over a period of 450 s. series resistance was compensated by 70% . for activation of trpm2 , adpr was added to the pipet solution at a concentration of 100 m . antagonist activity of the adpr analogues was tested by adding them at different concentrations to a pipet solution with 100 m adpr . during some experiments , the pipet solution contained 0.1% dmso because stock solutions of the more lipophilic adpr analogues were prepared in dmso . neutrophils were isolated as described elsewhere . in brief , the blood was fractionated with a histopaque-1119 density gradient and subsequently neutrophils were further purified by the use of percoll layers ranging from 65 to 85% in density . after a final washing step , the cells were resuspended in ca measurement buffer ( 140 mm nacl , 5 mm kcl , 1 mm mgso4 , 1 mm cacl2 , 1 mm nah2po4 , 4 mm glucose , 20 mm hepes , ph 7.4 ) and kept on ice until use . to avoid premature activation of the neutrophils , all buffers used during the isolation were supplemented with 2 mm edta , cell concentrations exceeding 5 10 cells / ml were avoided , and only endotoxin free materials and solutions were used . neutrophils were incubated with 4 m fura2/am for 30 min at 37 c in the dark , washed twice , and resuspended in ca measurement buffer ( see above ) at a concentration of 1 10 cells / ml . for each measurement , 5 10 cells were transferred to a small chamber consisting of a rubber o - ring fixed with silicon grease on a glass coverslip coated subsequently with 25 ng of bsa and 250 ng of fibronectin . the cells were incubated for 15 min at ambient temperature in the presence of 10 mm l - ascorbic acid ( ph 7.4 ) and , if applicable , varying concentrations of 8-phenyl - adpr . the loaded coverslip was mounted on the stage of a perkinelmer / imrovision imaging system built around a leica dm ire 2 fluorescence microscope . approximately 70 s after the beginning of the measurement , the cells were stimulated by addition of fmlp ( final concentration 1 m ) or a5 peptide ( final concentration 10 m ) . the migration of neutrophils was observed microscopically in microfluidic devices ( -slide chemotaxis , ibidi , martinsried , germany ) . first the -slides were coated with 50 g / ml fibronectin for 30 min at ambient temperature before washing three times and drying . isolated neutrophils were resuspended to a concentration of 3 10 cells / ml in ca measurement buffer supplemented with 10% ( v / v ) plasma obtained from the same donor . if applicable , 8-phenyl - adpr or egta was added and the whole slide was loaded according to the manufacturer s instructions and incubated at room temperature for 15 min . adding 18 l of fmlp ( 125 nm ) to the upper reservoir resulted in a chemotactical gradient from 0 50 nm fmlp across the observation chamber . the slide was mounted on the stage of the imaging system , and the main chamber observed at 10 times magnification in bright - field mode . after a 5 min resting period , greyscale images with a resolution of 672 510 pixels were recorded every 30 s for 1 h using openlab software 4.0.4 . cell migration was tracked manually with a 5 5 pixel maximum intensity centering correction using the manual - tracking plugin for imagej ( 1.45e ) . migrational parameters were calculated from the movement paths using the chemotaxis and migration tool software ( v2.0 , ibidi gmbh ) . phenylboronic acid ( 0.103 mmol , 21 mg ) and 8-br - adpr 4 ( tea salt , 0.0823 mmol ) were reacted under the general protocol b , yielding 8-ph - adpr ( tea salt , 6.0 by h nmr ) ( 18 mg , 14.3 mol , 19% ) as a colorless solid . h ( 400 mhz , d2o ) 8.14 ( br s , 1h , h-2 ) , 7.567.48 ( br m , 5h , ph ) , 5.78 ( d , 1h , j = 5.9 , h-1 ) , 5.16 ( br , 0.4h , h-1 ) , 5.08 ( br , 1h , h-2 ) , 5.05 ( br , 0.6h , h-1 ) , 4.30 ( br , 1h , h-2 ) , 3.824.18 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . c ( 100 mhz , d2o ) 154.5 , 153.4 , 152.3 , 150.3 ( c-2 ) , 131.3 ( ph , c h ) , 129.7 ( ph , c h ) , 129.2 ( ph , c h ) , 127.9 , 118.6 , 101.3 ( c/-1 ) , 96.5 ( c/-1 ) , 89.0 ( c-1 ) , 83.2 ( c-4 or c/-4 ) , 81.9 ( c-4 or c/-4 ) 81.3 ( c-4 or c/-4 ) , 75.3 , 70.8 , 70.6 , 70.5 ( c-2 ) , 70.2 , 69.7 ( c-2 ) , 66.5 ( c-5 or c/-5 ) 65.4 ( c-5 or c/-5 ) and 65.5 ( c-5 or c/-5 ) ; p ( 162 mhz , d2o ) 10.1 ( very br ) . hrms ( es ) calcd for c21h26n5o14p2 , 634.0957 m ; found , 634.0970 ; and rt = 26.7 min . 3-acetylphenylboronic acid ( 0.1 mmol , 17 mg ) and 8-br - adpr 4 ( 2 equiv tea salt , 67 mg , 0.079 mmol ) were reacted under the general protocol b to yield 8-(3-ac - ph)-adpr ( tea salt , 3.0 equiv by h nmr ) ( 11 mg , 9.3 mol , 12% ) as a colorless solid . h ( 400 mhz , d2o ) 8.138.19 ( m , 2h , ar 2-h and h-2 ) , 8.05 ( d , 1h , j = 6.9 , ar 6-h ) , 7.86 ( d , 1h , j = 6.9 , ar 4-h ) , 7.62 ( t , 1h , j = 6.9 , ar 5-h ) , 5.78 ( d , 1h , j = 5.9 , h-1 ) , 5.195.23 ( m , 1.4h , ( 1h ) h-2 and ( 0.4h ) h-1 ) , 5.09 ( d , 0.6h , j = 2.4 , h-1 ) , 4.374.41 ( m , 1h , h-2 ) , 3.874.21 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) and 2.62 ( s , 3h , arcoch3 ) . c ( 100 mhz , d2o ) 202.4 ( c = o ) , 155.1 , 152.8 ( c-2 ) , 151.9 , 150.1 , 136.7 , 134.4 ( ar 4-c ) , 130.6 ( ar 6-c ) , 129.6 , 129.4 ( ar 5-c ) , 128.3 ( ar 2-c ) , 118.5 , 101.1 ( c/-1 ) , 96.3 ( c/-1 ) , 88.8 ( c-1 ) , 83.0 ( c-4 or c/-4 , d , j 9.4 ) , 81.7 ( c-4 or c/-4 ) 81.0 ( c-4 or c/-4 , d , j = 9.4 ) , 75.1 , 70.6 , 70.3 , 70.3 ( c-2 ) , 69.9 , 69.5 ( c/-2 ) , 66.2 ( c-5 or c/-5 , d , j = 7.1 ) , 65.3 ( c-5 or c/-5 , d , j = 7.1 ) , 65.3 ( c-5 or c/-5 , d , j = 7.1 ) and 26.3 ( coch3 ) . p ( 162 mhz , d2o ) 11.2 ( br ) and 11.4 ( br ) . hrms ( es ) calcd for c23h28n5o15p2 , 676.1063 m ; found , 676.1076 ; and rt = 17.2 min . thiophene-3-boronic acid ( 0.12 mmol , 16 mg ) and 8-br - adpr 4 ( tea salt , 0.097 mmol ) were reacted under the general protocol b to give 8-(3-thiophenyl)-adpr ( tea salt , 2.3 equiv by h nmr ) ( 25 mg , 24.7 mol , 25% ) as a colorless solid . h ( 400 mhz , d2o ) 8.14 ( s , 1h , h-2 ) , 7.88 ( br s , 1h , thiophenyl 2-h ) , 7.54 ( dd , 1h , j = 4.7 , 3.2 , thiophenyl 4-h ) , 7.35 ( d , 1h , j = 4.7 , thiophenyl 5-h ) , 5.90 ( d , 1h , j = 5.9 , h-1 ) , 5.175.21 ( m , 1.4h , ( 1h ) h-2 and ( 0.4h ) h-1 ) , 5.09 ( d , 0.6h , j = 1.9 , h-1 ) , 4.374.40 ( m , 1h , h-2 ) and 3.884.18 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . c ( 100 mhz , d2o ) 154.5 , 152.1 ( c-2 ) , 149.9 , 148.8 , 129.3 , 127.9 , 127.8 , 127.7 , 118.2 , 101.1 ( c/-1 ) , 96.3 ( c/-1 ) , 88.6 ( c-1 ) , 82.8 ( c-4 or c/-4 , d , = j 8.5 ) , 81.7 ( c-4 or c/-4 ) , 81.1 ( c-4 or c/-4 , d , j = 8.5 ) , 75.1 , 70.6 , 70.3 , 70.1 , 69.9 , 69.4 , 66.2 ( c-5 or c/-5 ) , 65.3 ( c-5 or c/-5 ) , 65.3 ( c-5 or c/-5 ) . p ( 162 mhz , d2o ) 11.2 ( br ) and 11.3 ( br ) . hrms ( es ) calcd for c19h24n5o14p2s , 640.0521 m ; found , 640.0527 ; and rt = 24.9 min . dl-4-boronophenylalanine ( 0.1 mmol , 21 mg ) and 8-br - amp 8 ( 0.75 equiv tea salts , 40 mg , 0.08 mmol ) were reacted under the general protocol b to give 8-(4-(2-aminopropanoic acid)phenyl)-amp ( tea salt , 2.2 equiv by h nmr ) ( 19 mg , 14.4 mol , 18% ) as a colorless solid . h ( 400 mhz , d2o ) 8.17 ( s , 1h , h-2 ) , 7.61 ( d , 2h , j = 8.2 , ar h ) , 5.77 ( d , 1h , j = 5.8 , h-1 ) , 5.16 ( t , 1h , j = 6.3 , h-2 ) , 4.35 ( dd , 1h , j = 6.2 , 5.1 , h-3 ) , 3.884.09 ( m , 4h , h-4 , h-5 and nh2chch2 ) , 3.26 ( dd , 1h , j = 14.9 , 5.3 , nh2chcha / b ) and 3.08 ( 1h , obscured by et3n salt peak , nh2chcha / b ) . c ( 100 mhz , d2o ) 192.2 ( c = o ) , 155.1 , 152.8 , 152.6 ( c-2 ) , 150.1 , 138.6 , 129.9 , 129.8 , 126.9 , 118.4 , 88.6 ( c-1 ) , 83.6 ( c-4 ) , 70.0 ( c-2 ) , 69.5 ( c-3 ) , 63.4 ( c-5 ) , 55.8 ( nh2chch2 ) and 36.5 ( arch2 ) . p ( 162 mhz , d2o ) 5.6 ( s ) . hrms ( es ) calcd for c19h22n6o9p , 509.1191 m ; found , 509.1174 ; and rt = 6.96 min . triphenylphosphine ( 130 mg , 0.5 mmol ) , morpholine ( 92 ml , 1.06 mmol ) , and 2,2-dipyridyldisulfide ( 110 mg , 0.5 mmol ) were added to a solution of 8-nh2-amp 10 ( 55 mg , 0.15 mmol ) in dry dmso ( 600 l ) . the mixture was stirred at rt for 4 h , and then a solution of sodium iodide in acetone ( 0.1 m ) was added dropwise . the precipitate that formed was collected , washed with acetone , and redissolved in water and lyophilized to leave the crude morpholidate intermediate ( 39 mg ) as a pale - yellow solid . the morpholidate was dissolved in a solution of mncl2 in formamide ( 1 ml , 0.2 m ) , mgso4 ( 48 mg , 0.4 mmol ) and -nmn ( 67 mmol , 0.2 mmol ) were added , and the mixture was stirred for 2 days . the crude product was precipitated from the reaction mixture by the dropwise addition of mecn , and the precipitate was collected , washed with mecn , and dried . the crude product was purified by reverse phase column chromatography , eluting with 020% mecn in teab ( 0.05 m ) . the sample was then treated with chelex 100 ( sodium form ) to remove any paramagnetic material and lyophilized to yield the 8-amino - nad ( 13 mg , 0.02 mmol , 13% ) as a colorless solid . h ( 270 mhz , d2o ) broad , possibly small amount of remaining mn . hrms ( es ) calcd for c21h29n8o14p2 , 679.1273 m ; found , 679.1252 ; and rt = 3.03 min . nadase ( from neurospora crassa ; sigma ; 0.52 u ) in tris - hcl buffer ( 1 ml , 0.1 m , ph 7.27.4 ) was added to a solution of 8-nh2-nad11 ( 13 mg ) in tris - hcl buffer ( 4 ml , 0.1 m , ph 7.27.4 ) . after 4 h , all of the starting material had been consumed , the reaction mixture was diluted with water until the conductivity < 200 s / cm , and the product purified by ion - exchange ( q - sepaharose ) chromatography eluting with a gradient ( 050% ) of teab ( 1.0 m ) in milli - q . subsequent purification by reverse phase column chromatography , eluting with 030% mecn in teab ( 0.05 m ) , left the desired 8-nh2-adpr product ( 4.5 mg , 7.65 mol , 40% ) as a colorless solid . h ( 400 mhz , d2o ) 7.98 ( s , 1h , h-2 ) , 5.99 ( d , 1h , j = 7.6 , h-1 ) , 5.245.31 ( br , 0.4h , h-1 ) , 5.115.17 ( br , 0.6h , h-1 ) , 4.684.64 ( br m , 1h , h-2 ) , 4.384.44 ( br m , 1h , h-2 ) , 3.914.31 ( m , 8h , h-3 , h-4 , 2 h-5 , h-3 , h-4 and 2 h-5 ) . hrms ( es ) calcd for c15h23n6o14p2 , 573.0753 m ; found , 573.0775 ; and rt = 12.2 min . nhd ( 30 mol ) and nadase were reacted under the general protocol a to afford idpr as a glassy solid ( 24.6 mol , 82% ) . h ( 400 mhz , d2o ) 8.44 ( s , 1h , h-2 ) , 8.19 ( s , 1h , h-8 ) , 6.11 ( d , 1h , j1,2 = 6.1 , h-1 ) , 5.31 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.17 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.764.72 ( m , 1h , h-2 ) and 4.533.96 ( m , 9h , h-3 , h-4 , 2 h-5 , h-2 , h-3 , h-4 and 2 h-5 ) . p ( decoupled , 162 mhz , d2o ) 10.2 ( d , ab system , j = 18.8 ) , 10.6 ( d , ab system , j = 18.8 ) . hrms ( es ) calcd for c15h21n4o15p2 , 559.0484 ( m h ) ; found , 559.0480 . uv ( h2o , ph 7.2 ) max 248 nm ( 14500 ) . nicotinamide-7-deaza-8-bromoadenine-5-dinucletide ( 7-deaza-8-bromo - nad , 15 mol ) was treated with nadase under the general procedure a to afford 7-deaza-8-br - adpr as a glassy solid ( 12.7 mol , 85% ) . h ( 270 mhz , d2o ) 8.03 ( s , 1h , h-2 ) , 6.66 ( s , 1h , h-7 ) , 6.17 ( d , 1h , j1,2 = 6.1 , h-1 ) , 5.215.17 ( m , 2h , h-2 and h-1 ) , 4.544.50 ( m , 1h , h-3 ) and 4.183.94 ( m , 8h , h - ribose ) . p ( decoupled , 109 mhz , d2o ) 10.5 ( m ) and 10.7 ( m ) . hrms ( es ) calcd for c12h22n4o14p2br , 634.9797 ( m h ) ; found 634.9787 . to a solution of 1,2,3,5-o - tetraacetate ribofuranose 18 ( 4.7 g , 14.7 mmol ) , 6-chloropurine 17 ( 2.5 g , 16.17 mmol ) , and dbu ( 6.5 ml , 44.1 mmol ) in dry mecn ( 100 ml ) was added dropwise tmsotf ( 10 ml , 58.8 mmol ) at 0 c . the resulting clear brown solution was stirred for 2 h at 60 c , after which it was cooled to room temperature and aq satd nahco3 ( 400 ml ) was added . the aqueous phase was extracted with dcm ( 3 300 ml ) , dried ( na2so4 ) , filtered , and evaporated under reduced pressure , giving a brown oil . the crude was purified by column chromatography on silica gel ( dcm acetone , 9:1 v / v ) to afford the desired product as a white foam ( 4.9 g , 91% ) . h ( 270 mhz , cdcl3 ) 8.75 ( s , 1h , h-8 ) , 8.28 ( s , 1h , h-2 ) , 6.21 ( d , 1h , j1,2 = 5.1 , h-1 ) , 5.92 ( app t , 1h , j2,1 = j2,3 = 5.1 , h-2 ) , 5.62 ( app t , 1h , j3,2 = j3,4 = 5.1 , h-3 ) , 4.484.33 ( m , 3h , h-4 , h-5a and h-5b ) , 2.13 ( s , 3h , ch3 ) , 2.10 ( s , 3h , ch3 ) and 2.06 ( s , 3h , ch3 ) . c ( 68 mhz , cdcl3 ) 170.4 , 169.7 , 169.5 ( all c = o ) , 152.4 ( c-2 ) , 151.7 , 151.3 ( 2 c ) , 143.7 ( c-8 ) , 86.9 ( c-1 ) , 80.6 ( c-4 ) , 73.2 ( c-2 ) , 70.5 ( c-3 ) , 62.9 ( c-5 ) , 20.8 , 20.6 , and 20.5 ( 3 ch3 ) . rf = 0.57 ( dcm acetone , 9:1 v / v ) . 2,3,5-tri - o - acetyl-6-chloro adenosine 19 ( 1.45 g , 3.52 mmol ) was added to a freshly prepared solution of naome in meoh ( 7.04 mmol in 10 ml ) . the solution was refluxed for one hour , after which it was cooled to rt and neutralized with acoh . the solvent was evaporated , and the residue was purified by column chromatography on silica gel ( dcm acetone , 6:4 v / v ) to yield the desired product as a white foam ( 943 mg , 95% ) . h ( 270 mhz , meoh - d4 ) 8.49 ( s , 1h , h-8 ) , 8.42 ( s , 1h , h-2 ) , 6.04 ( d , 1h , j1,2 = 5.9 , h-1 ) , 4.774.73 ( m , 1h , h-2 ) , 4.38 ( dd , 1h , j3,2 = 5.1 and j3,4 = 3.1 , h-3 ) , 4.184.15 ( m , 1h , h-4 ) , 4.13 ( s , 3h , ch3 ) , 3.91 ( dd , 1h , j5a,5b = 12.5 and j5a,4 = 2.6 , h-5a ) and 3.77 ( dd , 1h , j5b,5a = 12.5 and j5b,4 = 3.5 , h-5b ) . c ( 68 mhz , meoh - d4 ) 160.5 ( c-6 ) , 151.7 ( c-2 ) , 150.8 ( c ) , 142.6 ( c-8 ) , 121.3 ( c ) , 89.9 ( c-1 ) , 86.6 ( c-4 ) , 74.3 ( c-2 ) , 71.1 ( c-3 ) , 61.9 ( c-5 ) and 53.7 ( ch3 ) ; rf = 0.09 ( dcm acetone , 6:4 v / v ) . ms ( apci ) m / z 283.4 [ ( mh ) , 100% ] . hrms ( es ) calcd for c11h15n4o5 , 283.1037 ( mh ) ; found , 283.1038 . 6-o - methylinosine 20 ( 80 mg , 0.264 mmol ) was dissolved in triethylphosphate ( 1 ml ) by heating with a heatgun . the resulting colorless solution was cooled to 0 c , and water ( 2 l ) was added followed by pocl3 ( 0.1 ml , 1.056 mmol ) . it was stirred at 0 c until disappearance of starting material and formation of a single peak as shown by hplc . after 1 h , the reaction mixture was quenched by addition of ice / water ( 15 ml ) and stirred for 15 min at 0 c , after which it was warmed up to rt . triethylphosphate was extracted with etoac ( 6 6 ml ) , and the aqueous phase was neutralized with 2 m naoh . it was then applied to a reverse phase gradifrac column eluted with a gradient of 565% mecn in 0.05 m teab . the appropriate fractions were collected and lyophilized overnight to afford the desired monophosphate as its triethylammonium salt . h ( 270 mhz , d2o ) 9.01 ( s , 1h , h-8 ) , 8.51 ( s , 1h , h-2 ) , 6.14 ( d , 1h , j1,2 = 3.7 , h-1 ) , 4.63 ( app t , 1h , j2,1 = j2,3 = 4.2 , h-2 ) , 4.414.37 ( m , 1h , h-3 ) , 4.31 ( br s , 1h , h-3 ) , 4.224.15 ( m , 1h , h-5a ) and 4.11 ( m , 4h , och3 and h-5b ) . c ( 68 mhz , cdcl3 ) 159.5 ( c-6 ) , 153.6 ( c-2 ) , 149.4 ( c-4 ) , 129.6 ( c-8 ) , 115.7 ( c-5 ) , 89.6 ( c-1 ) , 83.7 ( c-4 , j = 8.7 ) , 74.7 ( c-2 ) , 69.5 ( c-3 ) , 64.2 ( c-5 ) and 55.9 ( och3 ) . ms : ( es ) m / z 361.5 [ ( m h ) , 100% ] . hrms ( es ) calcd for c11h14n4o8p , 361.0555 [ ( m h ) ] ; found , 361.0558 . 6-o - me - imp 21 ( 120 mg , 0.331 mmol ) was dissolved in dry dmso ( 2 ml ) and coevaporated with dry dmf ( 5 3 ml ) . the residue was dissolved in dmso ( 1 ml ) to which was added morpholine ( 150 l , 1.724 mmol ) , dipyridyldisulfide ( 182 mg , 0.827 mmol ) , and triphenylphosphine ( 217 mg , 0.827 mmol ) , at which point the solution became bright yellow . it was stirred for 1 h at rt , after which hplc analysis showed formation of a new peak . precipitation of the product occurred by dropwise addition of a solution of nai in acetone ( 0.1 m ) . the resulting precipitate was filtered and washed with acetone to yield the desired product as a pale - yellow solid , which was used in the next step without further purification . 6-o - me - imp morpholidate ( 100 mg , 0.232 mmol ) , -nmn ( 85 mg , 0.253 mmol ) , and mgso4 ( 54 mg , 0.464 mmol ) were dissolved in a 0.2 m solution of mncl2 in formamide ( 1.7 ml ) and stirred at rt for 16 h , after which hplc analysis showed completion of the reaction ( rt ( -nmn ) = 2.1 min and rt ( 6-o - me - nhd ) = 3.8 min ) . mecn was added to precipitate the product , which was filtered , dissolved in milli - q , and applied to a reverse phase gradifrac column eluted with a gradient of 565% mecn in 0.05 m teab . further treatment with chelex 100 to remove any paramagnetic particles afforded the desired product as the sodium salt ( 18 mg , 8% ) . h ( 400 mhz , d2o ) 9.21 ( s , 1h , hn2 ) , 9.07 ( d , 1h , j6,5 = 6.3 , hn6 ) , 8.67 ( d , 1h , j4,5 = 8.2 , hn4 ) , 8.39 ( s , 1h , h-8 ) , 8.27 ( s , 1h , h-2 ) , 8.098.06 ( m , 1h , hn5 ) , 5.96 ( d , 1h , j1,2 = 5.9 , h-1 ) , 5.94 ( d , 1h , j1,2 = 5.5 , h-1 ) , 4.62 ( app t , 1h , j2,1 = j2,3 = 5.5 , h-2 ) and 4.384.06 ( m , 9h , hsugar ) . c ( 100 mhz , d2o ) 165.1 ( c = o ) , 160.7 ( c-6 ) , 151.1 ( c-8 ) , 151.0 ( c-4 ) , 145.7 ( cn4 ) , 142.4 ( cn6 ) , 141.5 ( c-2 ) , 139.8 ( cn2 ) , 133.6 ( cn3 ) , 128.6 ( cn5 ) , 120.4 ( c-5 ) , 99.9 ( c-1 ) , 87.0 ( c-1 ) , 86.8 ( c-4 , d , j = 9.2 ) , 83.7 ( c-4 , d , j = 9.2 ) , 77.4 ( c-2 ) , 74.0 ( c-2 ) , 70.4 ( c-3 ) , 70.2 ( c-3 ) , 64.8 , 63.3 ( 2 ch2 ) and 54.9 ( ch3 ) . p ( 109 mhz , d2o ) 11.4 ( d , j = 20.7 ) and 11.7 ( d , j = 20.7 ) . ms ( es ) m / z 678.2 [ ( m h ) , 100% ] . hrms ( es ) calcd for c22h28n6o15p2 , 678.1093 [ ( m h ) ] ; found , 678.1088 . 6-o - me - nhd sodium salt 23 ( 17.3 mg , 25.5 mol ) was incubated with aplysia cyclase ( 40 l ) in a 25 mm hepes buffer ( 35 ml , ph 7.4 ) at rt . after 4 h at rt , hplc analysis showed completion of the reaction ( rt ( nicotinamide ) = 1.7 min and rt ( product ) = 15.9 min ) . the mixture was then applied to a q - sepharose ion exchange column eluted with 1 m teab buffer . the appropriate fractions were collected and evaporated under vacuum , and the residue was coevaporated with meoh to afford the hydrolyzed product 6-o - me - idpr as a triethylammonium salt . h ( 270 mhz , d2o ) 8.58 ( s , 1h , h-2 ) , 8.45 ( s , 1h , h-8 ) , 6.15 ( d , 1h , j1,2 = 5.6 , h-1 ) , 5.26 ( d , 0.5 h , j1,2 = 4.2 , h-1 ) , 5.16 ( d , 0.5 h , j1,2 = 2.2 , h-1 ) , 4.78 ( 1h , hidden under hdo peak ) , 4.484.47 ( m , 1h ) , 4.34 ( br s , 1h ) , 4.274.17 ( m , 3h ) , 4.12 ( s , 3h , ome ) , 4.083.92 ( m , 3h ) and 3.843.82 ( m , 1h ) . p ( 109 mhz , d2o ) 10.2 ( d , j = 21.1 ) and 10.6 ( d , j = 21.1 ) . ms : ( es ) m / z 573.4 [ ( m h ) , 80% ] . hrms ( es ) calcd for c16h23n4o15p2 , 573.0641 [ ( m h ) ] ; found , 573.0646 . 2-deoxy - nad32 ( 22 mol ) was reacted with nadase under general protocol b to yield the desired hydrolyzed product ( 18.7 mol , 85% ) . h ( 270 mhz , d2o ) 8.41 ( s , 1h , h-2 ) , 8.17 ( s , 1h , h-8 ) , 6.486.43 ( m , 1h , h-1 ) , 5.26 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.15 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.71 ( m , 1h , partially hidden under hdo peak , h-2 ) , 4.273.87 ( m , 8h , h-3 , h-4 , h-5 , h-3 , h-4 and h-5 ) , 2.832.78 ( m , 1h , h-2a ) and 2.55 ( ddd , 1h , j2b,2a = 14.0 , j2b,1 = 6.3 and j2b,3 = 3.3 , h-2b ) . p ( decoupled , 109 mhz , d2o ) 10.4 ( br s ) , 10.5 ( br s ) . hrms ( es ) calcd for c15h22n5o13p2 , 542.0695 ( m h ) ; found , 542.0681 . uv ( h2o , ph 7.4 ) max 259 nm ( 15400 ) . to a solution of 3-deoxy - nad42 ( 16 mol ) in tris buffer ( 100 mm , ph 7.3 , 5 ml ) was added nadase ( 200 l ) . the reaction was left for 2 h at 37 c , after which hplc analysis showed no remaining starting material . the volatiles were evaporated under reduced pressure , and the residue was applied to a semipreparative c18 column developed with a linear gradient of 0.1 m teab against mecn . the appropriate fractions were evaporated , and excess tea salt was removed by coevaporation with meoh to leave the desired adpr analogue ( 2.6 mol , 20% ) as a glassy solid tea salt . h ( 400 mhz , d2o ) 8.37 ( s , 1h , h-8 ) , 8.16 ( s , 1h , h-2 ) , 6.03 ( d , 1h , j1,2 = 5.0 , h-1 ) , 5.20 ( d , 1h , j1,2 = 4.1 , h-1 ) , 5.10 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.714.63 ( m , 2h , h - sugar ) , 4.233.85 ( m , 7h , h - sugar ) , 2.35 ( dd , 1h , j3a,3b = 10.0 and j3a,4 = 5.8 , h-3a ) and 2.172.12 ( m , 1h , h-3b ) . p ( decoupled , 162 mhz , d2o ) 11.1 ( br m ) . hrms ( es ) calcd for c15h22n5o13p2 , 542.3090 ( m h ) ; found , 542.3098 . 9-(4-hydroxybutyl)adenine 27 ( 80 mg , 0.386 mmol ) was dissolved in trimethylphosphate ( 1.3 ml ) by heating with a heatgun . phosphorus oxychloride ( 144 l , 1.545 mmol ) and water ( 2 l ) were added at 0 c , and the resulting solution was stirred at rt for 3 h. ice / water ( 15 ml ) was then added , and the mixture was stirred for further 15 min , after which it was extracted with etoac ( 6 ) . the aqueous layer was neutralized with 5 n naoh and applied to a reverse phase column and the product eluted with a gradient of 0.05 m teab against mecn . the residue obtained was coevaporated with meoh to remove excess tea salt , leaving the desired monophosphate as its triethylammonium salt ( 92 mg , 72% ) . h ( 270 mhz , dmso - d6 ) 8.13 ( s , 1h , h-2 or h-8 ) , 7.92 ( s , 1h , h-8 or h-2 ) , 7.88 ( br s , 2h , nh2 ) , 4.03 ( t , 2h , j = 7.2 , ch2-n ) , 3.85 ( q , 2h , j = 7.2 , ch2-o ) , 1.801.75 ( m , 2h , o - ch2-ch2 ) and 1.561.49 ( m , 2h , o - ch2-ch2 ) . 9-(4-acetoxybutyl)adenine-5-monophosphate1tea 28 ( 92 mg , 0.277 mmol ) was dissolved in dry dmso ( 1 ml ) and coevaporated with dry dmf ( 5 3 ml ) . the residue was dissolved in dmso ( 400 l ) to which was added morpholine ( 106 l , 1.233 mmol ) , dipyridyldisulfide ( 130 mg , 0.592 mmol ) , and triphenylphosphine ( 155 mg , 0.592 mmol ) , at which point the solution became bright yellow . it was stirred for 2 h at rt , after which hplc analysis showed completion of the reaction . precipitation of the product occurred by dropwise addition of a solution of nai in acetone ( 0.1 m , 20 ml ) . the resulting precipitate was filtered , washed with acetone , and dried ( p : = 6.7 ppm ) . it was then reacted with -nmn ( 84 mg , 0.250 mmol ) and mgso4 ( 53 mg , 0.454 mmol ) in a 0.2 m solution of mncl2 in formamide ( 1.5 ml ) at rt overnigh , t after which hplc analysis showed completion of the reaction ( rt = 2.9 min ) . the precipitate was filtered , dissolved in milli - q , and applied to a reverse phase column eluted with a 565% gradient of mecn in 0.05 m teab . further treatment with chelex 100 to remove any paramagnetic particles afforded the desired dinucleotide as its sodium salt . nicotinamide-9-(4-acetoxybutyl)adenine-5-dinucleotide 29 ( 10 mol ) was treated with nadase under general procedure b to leave the desired acyclic - adpr ( 8.1 mol , 81% ) . h ( 270 mhz , d2o ) 8.09 ( s , 2h , h-2 and h-8 ) , 5.25 ( d , 0.4h , j1,2 = 3.8 , h-1 ) , 5.17 ( d , 0.6h , j1,2 = 1.9 , h-1 ) , 4.204.02 ( m , 3h , h-2 and ch2-n ) , 3.973.90 ( m , 5h , h-3 , h-4 , h-5 and ch2-o ) , 1.901.83 ( m , 2h , o - ch2ch2 ) and 1.621.55 ( m , 2h , o - ch2ch2 ) . . hrms ( es ) calcd for c14h22n5o11p2 , 498.0795 ( m h ) ; found , 498.0786 . uv ( h2o , ph 7.2 ) max 261 nm ( 16000 ) . a solution of catpr 46 ( 5 mol ) in tris hcl ( 100 mm , ph 7 ) was heated to 100 c for 1 h , after which hplc analysis showed conversion to a new product . the solution was applied to a reverse phase column eluted with a 565% gradient of mecn in 0.05 m teab . the appropriate fractions were collected and evaporated to afford the desired nucleotide as its triethylammonium salt ( 2.7 mol , 54% ) . h ( 270 mhz , do ) 8.54 ( s , 1h , h-2 ) , 8.26 ( s , 1h , h-8 ) , 6.11 ( d , 1h , j1,2 = 5.8 , h-1 ) , 5.31 ( d , 0.4h , j1,2 = 4.1 , h-1 ) , 5.15 ( d , 0.6h , j1,2 = 2.3 , h-1 ) , 4.554.52 ( m , 1h , h-2 ) and 4.373.96 ( m , 9h , h-3 , h-4 , h-5 , h-2 , h-3 , h-4 and h-5 ) . p ( decoupled , 109 mhz , d2o ) 11.6 ( br s ) , 23.4 ( br s , o - p - o ) . hrms ( es ) calcd for c15h23n5o17p2 , 638.0307 ( m h ) ; found , 638.0331 . uv ( h2o , ph 7.2 ) max 259 nm ( 17180 ) . 10% pd / c ( 110 mg ) was added to a solution of 2,3-o - isopropylidene-5-azido-5-deoxyadenosine ( 1.0 g , 3.01 mmol ) in etoh . the mixture was stirred for 16 h under a hydrogen atmosphere , after which the palladium was filtered and the solvent was removed under vacuum , yielding the desired compound as a white solid ( 0.9 g , 95% ) . h ( 400 mhz , dmso - d6 ) 8.34 ( s , 1h , h-8 ) , 8.14 ( s , 1h , h-2 ) , 7.29 ( br s , 2h , nh2 ) , 6.11 ( d , 1h , j1,2 = 3.0 , h-1 ) , 5.42 ( dd , 1h , j2,3 = 6.2 and j2,1 = 3.0 , h-2 ) , 5.01 ( dd , 1h , j3,2 = 6.2 and j3,4 = 2.9 , h-3 ) , 4.204.16 ( m , 1h , h-4 ) , 2.912.81 ( m , 2h , 2 h-5 ) , 1.52 ( s , 3h , ch3 ) and 1.30 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.7 ( c-8 ) , 148.8 ( c-4 ) , 140.0 ( c-2 ) , 119.2 ( c-5 ) , 113.3 ( c ) , 89.2 ( c-1 ) , 878.0 ( c-4 ) , 82.7 ( c-2 ) , 81.6 ( c-3 ) , 43.7 ( c-5 ) , 27.0 and 25.2 ( 2 ch3 ) . hrms ( es ) calcd for c13h19n6o3 , 307.1513 ( mh ) ; found , 307.1511 . to a solution of 1-o - methyl-2,3-o - isopropylidene--d - ribofuranose 49 ( 0.61 g , 2.989 mmol ) in dry pyridine ( 1 ml ) , externally cooled with ice , was added p - toluenesulfonyl chloride ( 0.7 g , 3.668 mmol ) and a catalytic amount of dmap . the reaction mixture was stirred at rt under nitrogen for 5 h. water ( 0.3 ml ) was added and stirring continued for 30 min . this mixture was extracted with chloroform ( 3 10 ml ) and the combined organic phases washed sequentially with cuso4 ( 10% w / v , aq satd ) , nahco3 ( aq satd ) and water and then dried over anhydrous sodium sulfate . the solvent was evaporated , and the residue was purified on an isco chromatographic system ( petrol etoac , 7:3 v / v ) to yield the desired compound as a white solid ( 0.92 g , 81% ) . h ( 400 mhz , cdcl3 ) 7.71 ( d , 2h , j = 8.7 2 ar h ) , 7.26 ( d , 2h , j = 8.0 , ar h ) , 4.83 ( s , 1h , h-1 ) , 4.51 ( dd , 1h , j3,2 = 6.0 and j3,4 = 0.6 , h-3 ) , 4.44 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.21 ( dt , 1h , j4,5 = 7.1 and j4,3 = 0.6 , h-4 ) , 3.933.91 ( m , 2h , h-5 ) , 3.14 ( s , 3h , ome ) , 2.36 ( s , 3h , ch3 ) , 1.35 ( s , 3h , ch3 ) and 1.19 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 144.9 ( c - so2 ) , 132.8 ( c - me ) , 129.8 ( 2c ) , 127.9 ( 2c ) ( all ch ) , 112.6 ( c ) , 109.4 ( c-1 ) , 84.8 ( c-4 ) , 83.5 ( c-2 ) , 81.3 ( c-3 ) , 69.1 ( c-5 ) , 54.9 ( ome ) , 26.2 , 24.8 ( 2 ch3 ) and 21.5 ( ch3-ph ) . hrms ( es ) calcd for c16h22nao7s , 381.0978 ( mh ) ; found , 381.0969 . to a solution of 1-o - methyl-2,3-o - isopropylidene-5-o - p - toluenesulfonyl--d - ribofuranose 50 ( 2.4 g , 6.7 mmol ) in dmf ( 20 ml ) was added nan3 ( 5.2 g , 80.4 mmol ) , and the reaction mixture was stirred at 120 c for 16 h. after cooling to rt , acetone ( 20 ml ) was added and the solid was removed by filtration . the solvents were evaporated under reduced pressure , and the residue was dissolved in dcm ( 50 ml ) and washed successively with water ( 50 ml ) , satd aq nahco3 ( 50 ml ) , and water ( 50 ml ) . the organic layer was dried ( na2so4 ) , filtered , and evaporated to leave an oil which was purified on an isco chromatographic system ( petrol etoac , 1:1 v / v ) , yielding the title compound as a colorless oil ( 1.4 g , 91% ) . h ( 400 mhz , cdcl3 ) 4.90 ( s , 1h , h-1 ) , 4.50 ( s , 2h , h-2 and h-3 ) , 4.19 ( ddd , 1h , j4,5a = 7.6 , j4,5b = 6.8 and j4,3 = 0.6 , h-4 ) , 3.35 ( dd , 1h , j5a,5b = 12.5 and j5a,4 = 7.6 , h-5a ) , 3.28 ( s , 3h , ome ) , 3.17 ( dd , 1h , j5b,5a = 12.5 and j5b,4 = 6.8 , h-5b ) , 1.39 ( s , 3h , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.6 ( c ) , 109.8 ( c-1 ) , 85.3 ( c-4 ) , 85.1 ( c-2 ) , 82.0 ( c-3 ) , 55.1 ( ome ) , 53.7 ( c-5 ) , 26.4 and 24.9 ( 2 ch3 ) . hrms ( es ) calcd for c9h15n3nao4 , 252.0955 ( mh ) ; found , 252.0949 . pph3 ( 1.95 g , 7.45 mmol ) was added to a solution of 1-o - methyl-2,3-o - isopropylidene-5-azido-5-deoxy--d - ribofuranose 51 ( 1.4 g , 6.11 mmol ) in thf ( 7 ml ) . the reaction mixture was stirred at rt for 16 h , after which water ( 7 ml ) was added and it was stirred for further 7 h. evaporation of the solvents followed by purification on an isco chromatographic system ( petrol etoac , 1:1 v / v ) gave the title compound as a colorless oil ( 1.04 g , 85% ) . h ( 400 mhz , cdcl3 ) 4.84 ( s , 1h , h-1 ) , 4.494.46 ( s , 2h , h-2 and h-3 ) , 4.054.01 ( m , 1h , h-4 ) , 3.24 ( s , 3h , ome ) , 2.712.62 ( m , 2h , 2 h-5 ) , 1.36 ( s , 3h , ch3 ) and 1.19 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.2 ( c ) , 109.5 ( c-1 ) , 88.8 ( c-4 ) , 85.4 ( c-2 ) , 82.1 ( c-3 ) , 54.9 ( ome ) , 45.4 ( c-5 ) , 26.4 and 24.8 ( 2 ch3 ) . hrms ( es ) calcd for c9h18no4 , 204.1230 ( mh ) ; found , 204.1226 . 1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose 52 ( 90 mg , 0.443 mmol ) , dipea ( 42 l , 0.239 mmol ) , and diethylsquarate ( 72 l , 0.487 mmol ) were reacted under the general protocol c to yield the desired product as a white foam ( 137 mg , 95% ) . h ( 400 mhz , cdcl3 ) 4.91 ( s , 1h , h-1 ) , 4.66 ( q , 4h , j = 6.9 , ch2 ) , 4.534.49 ( m , 2h , h-2 and h-3 ) , 4.29 ( app t , 1h , j = 5.6 , h-4 ) , 3.733.71 ( br m , 1h , h-5a ) , 3.513.49 ( br m , 1h , h-5b , 3.31 ( s , 3h , ome ) , 1.38 ( s , 3h , ch3 ) , 1.37 ( t , 3h , j = 6.9 , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 189.4 ( c = o ) , 184.3 ( c = o ) , 172.6 ( 2 c = c ) , 112.8 ( c ) , 109.9 ( c-1 ) , 85.8 ( c-4 ) , 85.2 ( c-2 ) , 81.5 ( c-3 ) , 69.7 ( ch2 ) , 55.5 ( ome ) , 46.4 ( c-5 ) , 26.3 , 24.8 ( 2 ch3 isopropyl ) and 15.7 ( ch3 ) . hrms ( es ) calcd for c15h22no7 , 328.1391 ( mh ) ; found , 328.1408 . to a solution of 3-(1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose)-4-ethoxycyclobut-3-ene-1,2-dione ( 91 mg , 0.305 mmol ) and dipea ( 26 l , 0.152 mmol ) in etoh ( 2 ml ) was added 2,3-isopropylidene-5-amino-5-deoxyadenosine ( 98 mg , 0.320 mmol ) . the reaction was stirred at rt for 1 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 106 mg , 60% ) . h ( 400 mhz , dmso - d6 ) 8.30 ( s , 1h , h-2 ) , 8.16 ( s , 1h , h-8 ) , 7.29 ( br s , 2h , nh2 ) , 6.18 ( d , 1h , j1,2 = 2.5 , h-1 ) , 5.42 ( dd , 1h , j2,3 = 6.3 and j2,1 = 2.5 , h-2 ) , 5.0 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.5 , h-3 ) , 4.91 ( s , 1h , h-1 ) , 4.63 ( d , 1h , j3,2 = 6.0 , h-3 ) , 4.55 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.264.22 ( m , 1h , h-4 ) , 4.12 ( app t , 1h , j4,5 = 7.0 , h-4 ) , 3.91 ( br s , 1h , h-5a ) , 3.753.68 ( m , 1h , h-5b ) , 3.64 ( br s , 1h , h-5a ) , 3.493.47 ( m , 1h , h-5b ) , 3.27 ( s , 3h , ome ) , 1.52 ( s , 3h , ch3 ) , 1.34 ( s , 3h , ch3 ) , 1.30 ( s , 3h , ch3 ) and 1.22 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 , 182.6 ( 2 c = o ) , 167.6 ( 2 c = c ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.2(c-5 ) , 113.7 , 111.6 ( 2 c ) , 108.8 ( c-1 ) , 88.7 ( c-1 ) , 85.5 ( c-4 ) , 85.1 ( c-4 ) , 84.5 ( c-2 ) , 83.2 ( c-2 ) , 81.2 ( c-3 ) , 81.1 ( c-3 ) , 54.4 ( och3 ) , 46.4 ( c-5 ) , 45.1 ( c-5 ) , 27.0 , 26.2 , 25.3 , and 24.7 ( 4 ch3 ) . hrms ( es ) calcd for c26h34n7o9 , 588.2413 ( mh ) ; found , 588.2429 . 3-(2,3-isopropylidene-5-amino-5-deoxyadenosine)-4-(1-o - methyl-2,3-o - isopropylidene-5-amino-5-deoxy--d - ribofuranose ) cyclobut-3-ene-1,2-dione 60 ( 40 mg , 0.07 mmol ) was deprotected by stirring in 0.1 m h2so4 for 16 h at 80 c to yield the desired compound as a white solid ( 15 mg , 45% ) . h ( 400 mhz , dmso - d6 ) 8.31 ( s , 1h , h-2 ) , 8.16 ( s , 1h , h-8 ) , 7.27 ( br s , 2h , nh2 ) , 6.17 ( d , 1h , j1,2 = 2.5 , h-1 ) , 4.91 ( s , 1h , h-1 ) , 4.354.24 ( m , 4h , h-2 , h-2 , h-3 , h-3 ) , 4.224.18 ( m , 1h , h-4 ) , 4.12 ( app t , 1h , j4,5 = 7.0 , h-4 ) , 3.92 ( br s , 1h , h-5a ) , 3.753.68 ( m , 1h , h-5b ) , 3.65 ( br s , 1h , h-5a ) , 3.493.47 ( m , 1h , h-5b ) . c ( 100 mhz , dmso - d6 ) 182.8 , 182.6 ( 2 c = o ) , 167.7 ( 2 c = c ) , 156.2 ( c-6 ) , 152.8 ( c-2 ) , 148.9 ( c-4 ) , 139.9 ( c-8 ) , 119.2(c-5 ) , 108.8 ( c-1 ) , 88.7 ( c-1 ) , 85.5 ( c-4 ) , 85.1 ( c-4 ) , 84.5 ( c-2 ) , 83.2 ( c-2 ) , 81.2 ( c-3 ) , 81.1 ( c-3 ) , 46.4 ( c-5 ) , 45.1 ( c-5 ) . hrms ( es ) calcd for c19h23n7o9 , 493.1557 ( mh ) ; found , 493.1564 . cyclopentylamine ( 104 l , 0.863 mmol ) , dipea ( 99 l , 0.570 mmol ) , and diethylsquarate ( 172 l , 1.162 mmol ) were reacted under the general protocol c to yield the desired product as a white foam ( 137 mg , 95% ) . h ( 400 mhz , cdcl3 ) 7.04 ( br s , 1h , nh ) , 4.754.73 ( m , 2h , och2 ) , 4.084.03 ( m , 1h , ch ) , 1.991.96 ( m , 2h ) , 1.741.72 ( m , 2h ) , 1.591.56 ( m , 4h ) ( 4 ch2 ) and 1.41 ( t , 3h , j = 6.6 , ch3 ) . c ( 100 mhz , cdcl3 ) 189.6 ( c-2 ) , 182.6 ( c-1 ) , 177.3 ( c-3 ) , 171.8 ( c-4 ) , 69.4 ( ch2 ) , 56.4 ( ch ) , 33.8 ( ch2 ) , 23.5 ( ch2 ) and 15.7 ( ch3 ) . hrms ( es ) calcd for c11h15no3 , 210.1130 ( mh ) ; found , 211.1127 . rf = 0.3 ( petrol etoac , 6:4 v / v ) . to a solution of 3-cyclopentylamino-4-ethoxycyclobut-3-ene-1,2-dione 55 ( 110 mg , 0.526 mmol ) and dipea ( 50 l , 0.284 mmol ) in etoh ( 3 ml ) the reaction mixture was stirred at rt for 18 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white solid ( 106 mg , 43% ) . h ( 400 mhz , meoh - d4 ) 8.32 ( s , 1h , h-8 ) , 8.29 ( s , 1h , h-2 ) , 6.25 ( d , 1h , j1,2 = 2.6 , h-1 ) , 5.55 ( dd , 1h , j2,3 = 6.4 and j2,1 = 2.6 , h-2 ) , 5.16 ( dd , 1h , j3,2 = 6.4 and j3,4 = 3.8 , h-3 ) , 4.444.40 ( m , 1h , h-4 ) , 4.07 ( dd , 1h , j5a,5b = 14.1 and j5a,4 = 4.4 , h-5a ) , 3.93 ( dd , 1h , j5ba,5a = 14.1 and j5b,4 = 6.7 , h-5b ) , 3.78 ( sept , 1h , j = 6.6 , ch ) , 1.722.07 ( m , 8h , 4 ch2 ) , 1.64 ( s , 3h , ch3 ) and 1.43 ( s , 3h , ch3 ) . c ( 100 mhz , meoh - d4 ) 183.9 ( c-2 ) , 183.5 ( c-1 ) , 169.4 ( c-3 ) , 169.0 ( c-4 ) , 157.4 ( c-6 ) , 154.2 ( c-2 ) , 150.3 ( c-4 ) , 141.9 ( c-8 ) , 120.7 ( c-5 ) , 115.9 ( c ) , 91.4 ( c-1 ) , 87.0 ( c-4 ) , 85.2 ( c-2 ) , 82.9 ( c-3 ) , 55.9 ( ch ) , 46.6 ( c-5 ) , 43.8 , 35.1 ( 4 ch2 ) , 27.5 and 25.6 ( 2 ch3 ) . hrms ( es ) calcd for c22h28n7o5 , 470.2146 ( mh ) ; found , 470.2158 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-(cyclopentylamino)-cyclobut-3-ene-1,2dione 61 ( 50 mg , 0.11 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid ( 40 mg , 85% ) . h ( 400 mhz , dmso - d6 ) 9.32 , 9.17 ( 2 nh ) , 8.39 ( s , 1h , h-8 ) , 8.30 ( s , 1h , h-2 ) , 7.74 ( br s , 2h , nh2 ) , 5.94 ( d , 1h , j = 5.9 , h-1 ) , 4.744.63 ( m , 4h , 2 ch2 ) , 4.254.14 ( m , 2h , h-2 , h-3 ) , 3.993.96 ( m , 1h , h-4 ) , 3.843.80 ( m , 1h , h-5a ) , 3.753.67 ( m , 2h , ch , h-5b ) , 1.42 ( t , 2h , j = 6.8 ) and 1.33 ( t , 2h , j = 6.8)(2 ch2 ) . c ( 100 , dmso - d6 ) 189.2 ( c = o ) , 182.3 ( c = o ) , 176.8 ( c = c ) , 172.8 ( c = c ) , 155.3 ( c-6 ) , 151.3 ( c-2 ) , 148.8 ( c-4 ) , 140.7 ( c-2 ) , 119.5 ( c-5 ) , 88.3 ( c-1 ) , 83.5 ( c-2 ) , 72.5 ( c-3 ) , 70.8 ( c-4 ) , 68.8 ( 2c , 2 ch2 ) , 45.8 ( c-5 ) , 15.5 ( 2c , 2 ch2 ) . hrms ( es ) calcd for c19h24n7o5 , 430.1833 ( mh ) ; found , 418.1838 . butylamine ( 135 l , 1.367 mmol ) , dipea ( 141 l , 0.811 mmol ) , and diethylsquarate 53 ( 222 l , 1.503 mmol ) were reacted under general protocol c to leave the desired product as a white foam ( 230 mg , 91% ) . h ( 400 mhz , cdcl3 ) 4.77 ( q , 2h , j = 7.2 , och2-me ) , 3.66 ( t , 1h , j = 7.0 , chh ) , 3.48 ( t , 1h , j = 7.0 , chh ) , 1.691.62 ( m , 2h , ch2 ) , 1.531.40 ( br m , 5h , ch2 and ch3 ) and 1.01 ( t , 3h , j = 7.2 , ch3 ) . c ( 100 mhz , cdcl3 ) 189.9 ( c-2 ) , 184.5 ( c-1 ) , 177.6 ( c-3 ) , 174.7 ( c-4 ) , 70.7 , 45.3 , 33.7 , 20.6 ( 4 ch2 ) , 16.2 ( et : ch3 ) and 14.0 ( bu : ch3 ) . hrms ( es ) calcd for c10h16no3 , 198.1125 ( mh ) ; found , 198.1124 . rf = 0.5 ( petrol etoac , 6:4 v / v ) . to a solution of 3-butylamino-4-ethoxycyclobut-3-ene-1,2-dione 56 ( 200 mg , 1.081 mmol ) and dipea ( 92 l , 0.531 mmol ) in etoh ( 5 ml ) the reaction mixture was stirred at rt for 24 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 364 mg , 81% ) . h ( 400 mhz , dmso - d6 ) 8.38 ( s , 1h , h-8 ) , 8.23 ( s , 1h , h-2 ) , 7.39 ( br s , 2h , nh2 ) , 6.25 ( d , 1h , j1,2 = 2.4 , h-1 ) , 5.50 ( dd , 1h , j2,3 = 6.3 and j2,1 = 2.4 , h-2 ) , 5.04 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.5 , h-3 ) , 4.334 .. 29 ( m , 1h , h-4 ) , 3.95 ( br , 1h , h-5a ) , 3.813.75 ( br , 1h , h-5b ) , 3.51 ( br , 2h , ch2 ) , 3.383.34 ( m , 1h , chh ) , 1.60 ( s , 3h , ch3 ) , 1.51 ( br , 1h , chh ) , 1.32 ( s , 3h , ch3 ) , 1.32 ( q , 2h , j = 7.3 , ch2-me ) and 0.91 ( t , 3h , j = 7.3 , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 ( c = o ) , 182.2 ( c = o ) , 167.7 ( c = c ) , 167.1 ( c = c ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.1 ( c-5 ) , 113.7 ( c ) , 88.7 ( c-1 ) , 85.1 ( c-4 ) , 83.6 ( c-2 ) , 81.2 ( c-3 ) , 45.0 ( ch2 ) , 42.9 ( c-5 ) , 32.7 ( ch2 ) , 27.0 , 25.2 ( 2 ch3 ) , 18.9 ( ch2 ) and 13.4 ( ch3 ) . hrms ( es ) calcd for c21h28n7o5 , 458.2146 ( mh ) ; found , 458.2142 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-butylamino - cyclobut-3-ene-1,2-dione 62 ( 50 mg , 0.109 mmol ) was deprotected under general protocol d to give the desired compound as a white solid ( 41 mg , 91% ) . h ( 400 mhz , dmso - d6 ) 8.40 ( s , 1h , h-8 ) , 8.26 ( s , 1h , h-2 ) , 7.567.45 ( br m , 4h , nh2 and 2 nh ) , 5.98 ( d , 1h , j1,2 = 5.8 , h-1 ) , 4.73 ( app t , 1h , j2,3 = j2,1 = 5.8 , h-2 ) , 4.23 ( app t , 1h , j3,2 = j3,4 = 5.8 , h-3 ) , 4.104.06 ( m , 1h , h-4 ) , 3.863.79 ( m , 2h , 2 h-5 ) , 3.535.52 ( m , 2h , ch2 ) , 1.511.49 ( m , 2h , ch2 ) , 1.34 ( q , 2h , j = 7.3 ) and 0.92 ( t , 3h , j = 7.3 ) . c ( 100 mhz , dmso - d6 ) 182.2 ( 2 c = o ) , 167.6 ( c-3 ) , 167.2 ( c-4 ) , 154.9 ( c-6 ) , 151.2 ( c-2 ) , 149.2 ( c-4 ) , 140.4 ( c-8 ) , 119.2 ( c-5 ) , 87.8 ( c-1 ) , 83.7 ( c-4 ) , 72.9 ( c-2 ) , 70.8 ( c-3 ) , 45.2 ( c-5 ) , 33.4 , 24.8 , 23.8 ( 3 ch2 ) and 15.8 ( ch3 ) . hrms ( es ) calcd for c18h24n7o5 , 418.1833 ( mh ) ; found , 418.1834 . hexylamine ( 130 l , 0.988 mmol ) , dipea ( 92 l , 0.533 mmol ) , and diethylsquarate 53 ( 161 l , 1.087 mmol ) were reacted under general protocol c to yield the desired product as a white foam ( 200 mg , 95% ) . h ( 400 mhz , cdcl3 ) 4.804.75 ( m , 2h , ch2-me ) , 3.65 ( t , 1h , j = 7.0 , chh - nh ) , 3.48 ( t , 1h , j = 7.0 , chh - nh ) , 1.691.64 ( m , 2h , ch2 ) , 1.531.44 ( m , 9h , 3 ch2 and ch3 ) and 0.990.96 ( m , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 189.9 ( c-2 ) , 184.5 ( c-1 ) , 177.5 ( c-3 ) , 174.8 ( c-4 ) , 70.7 ( et : ch2 ) , 45.6 , 32.5 , 31.6 , 27.1 , 23.6 ( 5 ch2 ) , 16.2 ( et : ch3 ) and 14.5 ( hex : ch3 ) . hrms ( es ) calcd for c12h20no3 , 226.1438 ( mh ) ; found , 226.1443 . rf = 0.62 ( petrol etoac , 6:4 v / v ) . to a solution of 3-hexylamino-4-ethoxycyclobut-3-ene-1,2-dione 57 ( 190 mg , 0.892 mmol ) and dipea ( 76 l , 0.438 mmol ) in etoh ( 5 ml ) was added 2,3-o - isopropylidene-5-amino-5-deoxyadenosine 59 ( 248 mg , 0.811 mmol ) . the reaction was stirred at rt for 20 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 291 mg , 74% ) . h ( 400 mhz , dmso - d6 ) 8.37 ( s , 1h , h-8 ) , 8.23 ( s , 1h , h-2 ) , 7.38 ( br s , 4h , nh2 and 2 nh ) , 6.26 ( d , 1h , j1,2 = 2.4 , h-1 ) , 5.49 ( dd , 1h , j2,3 = 6.2 and j2,1 = 2.4 , h-2 ) , 5.07 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.334.29 ( m , 1h , h-4 ) , 3.96 ( br , 1h , h-5a ) , 3.813.74 ( br , 1h , h-5b ) , 3.50 ( br , 2h , ch2 ) , 1.60 ( s , 3h , ch3 ) , 1.51 ( br , 1h , chh ) , 1.38 ( s , 3h , ch3 ) , 1.30 ( br , 7h , 3 ch2 and chh ) and 0.910.88 ( m , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 182.7 ( c-2 ) , 182.2 ( c-1 ) , 167.9 ( c-3 ) , 168.5 ( c-4 ) , 156.1 ( c-6 ) , 152.8 ( c-2 ) , 148.8 ( c-4 ) , 139.9 ( c-8 ) , 119.2 ( c-5 ) , 113.7 ( c ) , 88.7 ( c-1 ) , 85.2 ( c-4 ) , 83.1 ( c-2 ) , 81.2 ( c-3 ) , 45.1 ( ch2 ) , 43.2 ( c-5 ) , 30.7 , 30.6 ( 2 ch2 ) , 27.0 ( ch3 ) , 25.4 ( ch2 ) , 25.3 ( ch3 ) 21.9 ( ch2 ) and 13.8 ( ch3 ) . hrms ( es ) calcd for c23h32n7o5 , 486.2459 ( mh ) ; found , 486.2475 . 3-(2,3-o - isopropylidene-5-amino-5-deoxyadenosine)-4-(hexylamino)cyclobut-3-ene-1,2-dione 63 ( 50 mg , 0.102 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid ( 41 mg , 91% ) . h ( 400 mhz , dmso - d6 ) 8.42 ( s , 1h , h-8 ) , 8.27 ( s , 1h , h-2 ) , 7.65 ( br s , 2h , nh2 ) , 7.44 ( br s , 2h , 2 nh ) , 5.98 ( d , 1h , j1,2 = 5.7 , h-1 ) , 4.72 ( app t , 1h , j = 5.0 , h-2 ) , 4.22 ( app t , 1h , j = 4.3 , h-3 ) , 4.104.06 ( m , 2h , h-4 and h-5a ) , 3.853.79 ( m , 1h , h-5b ) , 3.51 ( br s , 2h , ch2-nh ) , 1.511.29 ( m , 8h , 4 ch2 ) and 0.910.88 c ( 100 mhz , dmso - d6 ) 182.6 ( c-2 ) , 182.3 ( c-1 ) , 167.9 ( 2 c = c ) , 155.2 ( c-6 ) , 151.5 ( c-2 ) , 149.2 ( c-4 ) , 140.2 ( c-8 ) , 119.2 ( c-5 ) , 87.6 ( c-1 ) , 83.7 ( c-4 ) , 72.9 ( c-2 ) , 70.8 ( c-3 ) , 45.5 ( ch2 ) , 43.2 ( c-5 ) , 30.7 , 30.6 , 25.4 , 21.9 ( 4 ch2 ) and 13.8 ( ch3 ) . hrms ( es ) calcd for c20h28n7o5 , 446.2146 ( mh ) ; found , 446.2157 . a solution of 1-o - methyl-2,3-o - isopropylidene--d - ribofuranose 49 ( 600 mg , 2.94 mmol ) in dmf ( 40 ml ) was cooled to 0 c , and nah ( 156 mg , 3.91 mmol , 60% in mineral oil ) was added . the mixture was stirred at 0 c for 30 min , after which tbai ( 65 mg , 0.176 mmol ) and propargyl chloride ( 0.25 ml , 3.528 mmol ) were added . the reaction mixture was stirred at rt for 16 h , the solvent was removed under reduced pressure , and the residue was purified by column chromatography using an isco chromatographic system ( petrol etoac , 1:0 0:1 v / v ) . the product was obtained as a colorless liquid ( 512 mg , 72% ) . h ( 400 mhz , cdcl3 ) 4.95 ( s , 1h , h-1 ) , 4.65 ( d , 1h , j2,3 = 6.0 , h-2 ) , 4.55 ( d , 1h , j3,2 = 6.0 , h-3 ) , 4.334.30 ( m , 1h , h-4 ) , 4.17 ( d , 2h , j = 2.4 , ch2 ) , 3.58 ( dd , 1h , j5a,5b = 9.5 and j5a,4 = 6.5 , h-5a ) , 3.523.47 ( m , 1h , h-5b ) , 3.32 ( s , 3h , ome ) , 2.42 ( t , 1h , j = 6.3 hz , ch ) , 1.46 ( s , 3h , ch3 ) and 1.30 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 112.3 ( c ) , 109.2 ( c-1 ) , 85.0 ( c-4 ) , 84.8 ( c-2 ) , 81.9 ( c-3 ) , 79.3 ( c ) , 74.6 ( hc ) , 70.5 ( c-5 ) , 58.3 ( ch2 ) , 54.8 ( och3 ) , 26.4 and 24.9 ( 2 ch3 ) . hrms ( es ) calcd for c12h18nao5 , 265.1046 ( mh ) ; found , 265.1042 . 2,3-o - isopropylidene-5-azido-5-deoxyadenosine 68 ( 100 mg , 0.30 mmol ) was taken up in naoac buffer ( ph 4 , 1 m , 15 ml ) and br2 ( 12 l , 0.45 mmol ) added . the resulting solution was stirred in the dark for 24 h and then a solution of nahso3 ( 4 m , aq ) added until the solution was colorless . all solvents were evaporated and the residue purified by column chromatography using an isco chromatographic system ( dcm acetone , 6:4 v / v ) . the title compound was obtained as an off - white solid ( 123 mg , 99% ) . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 6.20 ( d , 1h , j1,2 = 1.8 hz , h-1 ) , 5.99 ( br s , 2h , nh2 ) , 5.68 ( dd , 1h , j2,3 = 6.3 and j2,1 = 1.8 , h-2 ) , 5.15 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.6 , h-4 ) , 4.364.31 ( m , 1h , h-4 ) , 3.543.43 ( m , 2h , 2 h-5 ) , 1.61 ( s , 3h , ch3 ) and 1.39 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 154.4 ( c-6 ) , 152.8 ( c-2 ) , 150.3 ( c-4 ) , 127.5 ( c-8 ) , 120.1 ( c-5 ) , 114.5 ( c ) , 91.2 ( c-1 ) , 86.4 ( c-4 ) , 83.4 ( c-2 ) , 82.4 ( c-3 ) , 52.1 ( c-5 ) , 27.0 and 25.3 ( 2 ch3 ) . hrms ( es ) calcd for c13h16n8o3br , 411.0523 ( mh ) ; found , 411.0532 ; and calcd for c13h16n8o3br , 413.0503 ( mh ) ; found , 413.0522 . rf = 0.58 ( dcm acetone , 3:2 v / v ) . to a solution of 2,3-o - isopropylidene-5-deoxy-5-azido-8-bromoadenosine 69 ( 123 mg , 0.30 mmol ) in a mixture of buoh - h2o ( 1:1 v / v , 6 ml ) was added cuso45h2o ( 2 mg , 0.015 mmol ) , sodium ascorbate ( 6 mg , 0.03 mmol ) , and 1-o - methyl-2,3-o - isopropylidene-5-o - propargyl--d - ribofuranose 70 ( 73 mg , 0.30 mmol ) . the reaction mixture was stirred at rt for 16 h , the solvents were removed under vacuum , and the residue was purified on an isco chromatographic system ( dcm acetone , 6:4 v / v ) to yield the product as a pale - yellow solid ( 140 mg , 71% ) . h ( 400 mhz , cdcl3 ) 8.22 ( s , 1h , h-2 ) , 7.32 ( s , 1h , ch - triazole ) , 6.29 ( br s , 2h , nh2 ) , 6.17 ( d , 1h , j1,2 = 1.7 hz , h-1 ) , 5.55 ( dd , 1h , j2,3 = 6.3 and j2,1 = 1.7 , h-2 ) , 5.22 ( dd , 1h , j3,2 = 6.3 and j3,4 = 3.9 , h-3 ) , 4.88 ( s , 1h , h-1 ) , 4.74 ( dd , 1h , j5a,5b = 13.9 and j5a,4 = 4.1 , h-5a ) , 4.624.48 ( m , 6h , h-4 , h-2 , h-3 , h-5b and ch2-triazole ) , 4.254.21 ( m , 1h , h-4 ) , 3.503.39 ( m , 2h , 2 h-5 ) , 3.20 ( s , 3h , ome ) , 1.55 ( s , 3h , ch3 ) , 1.40 ( s , 3h , ch3 ) , 1.33 ( s , 3h , ch3 ) and 1.21 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 154.6 ( c-6 ) , 153.1 ( c-2 ) , 150.1 ( c-4 ) , 144.9 ( c - triazole ) , 127.0 ( c-8 ) , 123.5 ( ch - triazole ) , 120.1 ( c-5 ) , 114.7 , 112.3 ( 2 c ) , 109.2 ( c-1 ) , 90.9 ( c-1 ) , 85.9 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.9 ( c-2 ) , 81.9 ( 2c , c-3 and c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.5 ( c-5 ) , 27.0 , 26.3 , 25.3 , and 24.9 ( 4 ch3 ) . hrms ( es ) calcd for c25h33n8nao8br , 675.1497 ( mh ) ; found , 675.1469 ; calcd for c25h33n8nao8br , 677.1476 ( mh ) ; found , 677.1451 . rf = 0.58 ( dcm acetone , 3:2 v / v ) . to 1-(2,3-o - isopropylidene-5-deoxy-8-bromoadenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 71 ( 140 mg , 0.21 mmol ) , na2cl4pd ( 3 mg , 5 mol % ) , phb(oh2 ) ( 32 mg , 0.27 mmol ) , tppts ( 30 mg , 25 mol % ) , and na2co3 ( 68 mg , 0.64 mmol ) was added degassed mecn h2o ( 3 ml , 1:2 v / v ) and the resulting solution stirred at 80 c for 1 h. all solvents were evaporated and the residue purified by column chromatography using an isco chromatography system ( dcm acetone , 6:4 v / v ) to yield the product ( 30 mg , 21% ) . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 7.707.48 ( m , 5h , ph ) , 7.35 ( s , 1h , ch - triazole ) , 6.90 ( br s , 2h , nh2 ) , 6.04 ( d , 1h , j1,2 = 1.6 , h-1 ) , 5.57 ( dd , 1h , j2,3 = 6.2 and j2,1 = 1.6 , h-2 ) , 5.28 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.87 ( s , 1h , h-1 ) , 4.82 ( dd , 1h , j5a,5b = 14.2 and j5a,4 = 4.7 , h-5a ) , 4.71 ( dd , 1h , j5b,5a = 14.2 and j5b,4 = 8.0 , h-5a ) , 4.594.46 ( m , 5h , h-4 , h-2 , h-3 and ch2-triazole ) , 4.234.20 ( m , 1h , h-4 ) , 3.47 ( dd , 1h , j5a,5b = 9.7 and j5a,4 = 6.5 , h-5a ) , 3.433.40 ( m , 1h , h-5b ) , 3.18 ( s , 3h , ome ) , 1.45 ( s , 3h , ch3 ) , 1.38 ( s , 3h , ch3 ) , 1.29 ( s , 3h , ch3 ) and 1.21 ( s , 3h , ch3 ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 114.3 , 112.2 ( 2 c ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.8 ( c-5 ) , 26.9 , 26.3 , 25.2 , and 24.9 ( 4 ch3 ) . hrms ( es ) calcd for c31h38n8nao8 , 673.2705 ( mh ) ; found , 673.2678 . 1-(2,3-o - isopropylidene-5-deoxy-8-phenyladenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 72 ( 30 mg , 0.046 mmol ) was deprotected by stirring in 0.1 m h2so4 for 16 h at 80 c to yield the desired compound ( 6 mg , 24% ) as a white solid . h ( 400 mhz , cdcl3 ) 8.27 ( s , 1h , h-2 ) , 7.707.48 ( m , 5h , ph ) , 7.35 ( s , 1h , ch - triazole ) , 6.90 ( br s , 2h , nh2 ) , 6.04 ( d , 1h , j1,2 = 1.6 , h-1 ) , 5.57 ( dd , 1h , j2,3 = 6.2 and j2,1 = 1.6 , h-2 ) , 5.28 ( dd , 1h , j3,2 = 6.2 and j3,4 = 3.5 , h-3 ) , 4.87 ( s , 1h , h-1 ) , 4.82 ( dd , 1h , j5a,5b = 14.2 and j5a,4 = 4.7 , h-5a ) , 4.71 ( dd , 1h , j5b,5a = 14.2 and j5b,4 = 8.0 , h-5a ) , 4.594.46 ( m , 5h , h-4 , h-2 , h-3 and ch2-triazole ) , 4.234.20 ( m , 1h , h-4 ) , 3.47 ( dd , 1h , j5a,5b = 9.7 and j5a,4 = 6.5 , h-5a ) and 3.433.40 ( m , 1h , h-5b ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) and 51.8 ( c-5 ) . hrms ( es ) calcd for c24h29n8o8 , 557.2103 ( mh ) ; found , 557.2097 . to a solution of tetrazole ( 81 mg , 1.16 mmol ) and diisopropyl - dibenzylphosphoramidite ( 300 mg , 0.871 mmol ) in dcm ( 10 ml ) was added cyclopentanol 77 ( 50 mg , 0.58 mmol ) . the reaction mixture was stirred at rt for 20 min , after which tlc analysis showed total conversion of starting material to a single phosphite ( petrol etoac , 6:4 v / v , rf = 0.32 ) . the solution was cooled to 0 c , and mcpba ( 200 mg , 1.16 mmol ) was added in one portion . the mixture was warmed up to rt , diluted with etoac ( 20 ml ) , and washed with 10% na2so3 ( 20 ml ) , satd aq nahco3 ( 20 ml ) , and brine ( 20 ml ) . the organic phase was collected , dried ( na2so4 ) , filtered , and evaporated to dryness . the residue was purified with an isco chromatographic system ( petrol etoac , 7:3 v / v ) to yield the title compound as a colorless oil ( 173 mg , 86% ) . h ( 400 mhz , cdcl3 ) 7.367.20 ( m , 10h , h - benzyl ) , 5.064.97 ( m , 4h , 2 ch2 ) , 4.904.85 ( m , 1h , ch p ( 161 mhz , cdcl3 , decoupled ) 1.6 ( s ) . the above material ( 78 , 173 mg , 0.5 mmol ) was dissolved in a mixture of meoh h2o cyclohexane ( 10:1:5 v / v / v , 16 ml ) to which was added pd(oh)2/c ( 20% ) . the solution was heated to 80 c for 2 h , after which the palladium was removed by filtration through celite and the filtrate was evaporated under reduced pressure , leaving a residue which was used directly in the next step . ampna salt ( 190 mg , 0.547 mmol ) was passed through a small dowex column ( tea form ) and eluted with milli - q water . the solvent was evaporated to leave a residue , which was dissolved in dmso and coevaporated with dmf ( 3 3 ml ) . the residue obtained was dissolved in dmso ( 3 ml ) and morpholine ( 0.25 ml , 2.845 mmol ) , dipyridyldisulfide ( 301 mg , 1.367 mmol ) , and triphenylphosphine ( 358 mg , 1.367 mmol ) were added in this order . the resulting yellow solution was stirred for 90 min , after which a 0.1 m solution of nai in acetone was added . the precipitate obtained was collected by filtration and used directly in the next step . to a solution of amp - morpholidate ( 154 mg , 0.350 mmol ) and cyclopentane monophosphate 79 ( 64 mg , 0.380 mmol ) in 0.2 n mncl2 in formamide ( 2 ml ) was added mgso4 ( 82 mg , 0.70 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . precipitation of the product occurred on addition of mecn and purification on rp-18 afforded ( after treatment with chelex 100 ) the desired dinucleotide as a glassy solid ( 55 mol , 14% over 2 steps ) . h ( 400 mhz , d2o ) 8.44 ( s , 1h , h-2 ) , 8.19 ( s , 1h , h-8 ) , 6.07 ( s , 1h , h-1 ) , 4.734.71 ( m , 1h , ch o ) , 4.66 ( br s , 1h , h-2 ) , 4.48 ( br s , 1h , h-3 ) , 4.32 ( br s , 1h , h-4 ) , 4.15 ( br s , 2h , 2 h-5 ) , 1.621.60 ( m , 4h ) , 1.521.50 ( m , 2h ) and 1.361.34 ( m , 2h ) ( 4 ch2 ) . c ( 100 mhz , d2o ) 158.2 ( c-6 ) , 153.0 ( c-8 ) , 149.3 ( c-4 ) , 140.0 ( c-2 ) , 113.3 ( c-5 ) , 87.0 ( c-1 ) , 84.0 ( c-4 ) , 79.9 ( ch ) , 74.3 ( c-2 ) , 70.5 ( c-3 ) , 65.2 ( c-5 ) , 33.4 and 22.7 ( 2 ch2 ) . a flask containing 8-bromo-2,3-o - isopropylidene - adenosine 81 ( 200 mg , 0.519 mmol ) , na2cl4pd ( 5 mol % ) , tppts ( 25 mol % ) , phb(oh)2 ( 190 mg , 1.562 mmol ) , and na2co3 ( 165 mg , 1.557 mmol ) was purged with argon , and a degassed mixture of mecn h2o ( 1:1 v / v , 6 ml ) was added . the resulting mixture was refluxed for 1 h , then water ( 6 ml ) was added and the solution neutralized with 1 m hcl . the white precipitate obtained was collected by filtration and dried under vacuum ( 161 mg , 81% ) . h ( 400 mhz , dmso - d6 ) 8.16 ( s , 1h , h-2 ) , 7.737.71 ( m , 2h , ar h ) , 7.44 ( s , 2h , nh2 ) , 5.84 ( d , 1h , j1,2 = 3.4 , h-1 ) , 5.56 ( dd , 1h , j2,3 = 6.1 and j2,1 = 3.4 , h-2 ) , 5.385.36 ( m , 1h , 5oh ) , 5.03 ( dd , 1h , j3,2 = 6.1 and j3,4 = 2.5 , h-3 ) , 4.17 ( dd , 1h , j4,5 = 5.1 and j4,3 = 2.5 , h-4 ) , 3.63 ( dd , 1h , j5a,5b = 11.5 and j5a,4 = 5.1 , h-5a ) , 3.563.51 ( m , 1h , h-5b ) , 1.41 ( s , 3h , ch3 ) and 1.28 ( s , 3h , ch3 ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.4 ( c-2 ) , 150.1 ( c-4 ) , 149.6 ( c - ph ) , 130.3 , 129.6 ( 2 ch ) , 129.1 ( c-8 ) , 128.8 ( ch ) , 118.8 ( c-5 ) , 113.0 ( c ) , 90.4 ( c-1 ) , 86.5 ( c-4 ) , 81.9 ( c-2 ) , 81.8 ( c-3 ) , 61.9 ( c-5 ) , 27.0 and 25.2 ( 2 ch3 ) . hrms ( es ) calcd for c19h21n5o4 , 384.1672 ( mh ) ; found , 384.1686 . 8-phenyl-2,3-o - isopropylidene - adenosine ( 82 , 150 mg , 0.39 mmol ) was deprotected under general protocol d to yield the desired compound as a white solid which was used directly in the next step . 8-phenyl - adenosine ( 0.39 mmol ) was dissolved in triethylphosphate ( 1 ml ) by heating with a heatgun . the resulting colorless solution was cooled to 0 c , and water ( 2 l ) was added followed by pocl3 ( 0.15 ml , 1.56 mmol ) , then stirred at 0 c until disappearance of the starting material and formation of a single peak was observed as shown by hplc . water ( 15 ml ) and the mixture was stirred for 15 min at 0 c , after which it was warmed to rt . triethylphosphate was extracted with etoac ( 6 6 ml ) , and the aqueous phase was neutralized with 2 n naoh . it was then applied to a reverse phase gradifrac column eluted with a 565% gradient of mecn in 0.05 m teab . the appropriate fractions were collected and lyophilized to afford the desired monophosphate as its triethylammonium salt . h ( 400 mhz , dmso - d6 ) 8.16 ( s , 1h , h-2 ) , 7.737.71 ( m , 2h , ar h ) , 7.44 ( s , 2h , nh2 ) , 5.84 ( d , 1h , j1,2 = 3.4 , h-1 ) , 5.56 ( dd , 1h , j2,3 = 6.1 and j2,1 = 3.4 , h-2 ) , 5.03 ( dd , 1h , j3,2 = 6.1 and j3,4 = 2.5 , h-3 ) , 4.17 ( dd , 1h , j4,5 = 5.1 and j4,3 = 2.5 , h-4 ) , 3.63 ( dd , 1h , j5a,5b = 11.5 and j5a,4 = 5.1 , h-5a ) and 3.563.51 ( m , 1h , h-5b ) . c ( 100 mhz , dmso - d6 ) 156.1 ( c-6 ) , 152.4 ( c-2 ) , 150.1 ( c-4 ) , 149.6 ( c - ph ) , 130.3 , 129.6 ( 2 ch ) , 129.1 ( c-8 ) , 128.8 ( ch ) , 118.8 ( c-5 ) , 113.0 ( c ) , 90.4 ( c-1 ) , 86.5 ( c-4 , d , j = 8.3 hz ) , 81.9 ( c-2 ) , 81.8 ( c-3 ) , 61.9 ( c-5 , d , j = 8.8 hz ) . 8-ph - ampna salt ( 83 , 53 mg , 0.092 mmol ) was passed through a small dowex column ( tea form ) and eluted with milli - q water . the solvent was evaporated to leave a residue , which was dissolved in dmso and coevaporated with dmf ( 3 3 ml ) . the residue obtained was dissolved in dmso ( 90 l ) and morpholine ( 42 l , 0.478 mmol ) , dipyridyldisulfide ( 51 mg , 0.23 mmol ) , and triphenylphosphine ( 60 mg , 0.23 mmol ) were added in this order . the resulting yellow solution was stirred for 90 min , after which a 0.1 m solution of nai in acetone was added . the precipitate obtained was collected by filtration and used directly in the next step . to a solution of 8-ph - amp - morpholidate ( 31 mg , 0.060 mmol ) and cyclopentane monophosphate 79 ( 11 mg , 0.066 mmol ) in 0.2 n mncl2 in formamide ( 0.5 ml ) was added mgso4 ( 14 mg , 0.12 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . precipitation of the product occurred on addition of mecn and purification on rp-18 afforded ( after treatment with chelex 100 ) the desired dinucleotide as a glassy solid ( 11 mol , 12% over 2 steps ) . h ( 400 mhz , d2o ) 8.21 ( s , 1h , h-2 ) , 7.67 ( d , 1h , j = 6.2 ) , 7.607.56 ( m , 2h ) , 7.28 ( d , 2h , j = 8.1 ) ( 5 arh ) , 5.82 ( d , 1h , j = 6.4 , h-1 ) , 5.15 ( app t , 1h , h-2 ) , 4.31 ( dd , 1h , j = 6.4 , 4.5 , h-3 ) , 4.083.98 ( m , 4h , h-4 , 2 h-5 , ch o ) , 1.621.60 ( m , 4h ) , 1.521.50 ( m , 2h ) and 1.361.34 ( m , 2h ) ( 4 ch2 ) . c ( 100 mhz , d2o ) 158.3 ( c-6 ) , 152.1 ( c-8 ) , 149.3 ( c-4 ) , 140.1 ( c-2 ) , 132.4 ( 2c ) , 129.7 ( 2c ) , 128.6 ( 5 arch ) , 113.2 ( c-5 ) , 87.2 ( c-1 ) , 83.8 ( c-4 ) , 79.7 ( ch ) , 73.9 ( c-2 ) , 70.4 ( c-3 ) , 65.3 ( c-5 ) , 33.6 and 22.9 ( 2 ch2 ) . hrms ( es ) calcd for c21h27n5o10p2 , 570.1155 ( m h ) ; found , 570.1149 . uv ( h2o , ph 7.4 ) max 276 nm ( 17600 ) . 8-phenyl-2-deoxy - cadpr 85 ( 13 mol ) was incubated in kh2po4 buffer ( 100 mm , ph 7.4 ) at 70 c for 2.5 h , after which hplc analysis showed a new peak at rt = 28 min . the volatiles were removed under reduced pressure , and the residue was applied to a c18 semipreparative column eluted with a gradient of 0.1 m teab in mecn . the appropriate fractions were combined and evaporated to leave the desired product as a glassy solid in its triethylammonium form ( 5.06 mol , 39% ) . h ( 500 mhz , d2o ) 8.21 ( s , 1h , h-2 ) , 7.657.55 ( m , 5h , ar - h ) , 6.296.27 ( m , 1h , h-1 ) , 5.21 ( d , 1h , j1,2 = 4.5 , h-1 ) , 5.10 ( d , 1h , j1,2 = 2.2 , h-1 ) , 4.543.81 ( m , 9h , h - ribose ) , 3.223.16 ( m , 1h , h-2a ) and 2.202.13 ( m , 1h , h-2b ) . p ( decoupled , 109 mhz , d2o ) 11.1 ( br s ) . c ( 100 m , d2o ) 153.0 ( c-6 ) , 152.6 ( c-8 ) , 150.3 ( c-2 ) , 148.4 ( c-4 ) , 131.1 , 129.7 , 129.0 , 128.5 ( 5 ch phenyl ) , 101.2 ( c-1 ) , 91.1 ( c-1 ) , 86.6 ( c-4/c-4 ) , 75.2 ( c-2 ) , 74.8 ( c-2 ) , 70.9 ( c-3 ) , 70.4 ( c-3 ) , 65.4 ( c-5 ) and 62.8 ( c-5 ) . hrms ( es ) calcd for c21h26n5o13p2 , 618.1008 ( m h ) ; found , 618.1013 . bsa ( bovine serum albumin ) , fura-2/am , nacl , egta , nmdg , tris base , and histopaque-1119 were purchased from sigma aldrich ( mnchen , germany ) . kcl , mgso4 , mgcl2 , cacl2 , nah2po4 , d - glucose , l - ascorbic acid , tween , ionomycin , and edta were obtained from merck ( darmstadt , germany ) . fibronectin , dmem , and penicillin / streptomycin were supplied by invitrogen ( darmstadt , germany ) . fbs ( fetal bovine serum ) and g418 sulfate were purchased from biochrom ( berlin , germany ) . the anti - trpm2 antibody was procured from novus biologicals ( littleton , usa ) . the goat antirabbit antiserum was purchased from dianova ( hamburg , germany ) . percoll and ecl western blotting detection reagents were supplied by ge healthcare ( uppsala , sweden ) . hek293 cells were maintained in dmem medium containing glutamax i complemented with 10% fbs , 100 units / ml penicillin , and 100 g / ml streptomycin at 37 c in the presence of 5% co2 . hek293 clones expressing trpm2/egfp ( or egfp for control ) were cultured under the same conditions , while the medium was supplemented with 400 g / ml g418 sulfate . hek293 wild - type cells were transfected with two different expression vectors coding either for human trpm2 and egfp ( pires2-egfp - trpm2 ) or for egfp alone ( pires2-egfp ) as described previously . cells carrying the expression constructs were selected by addition of 400 g / ml g418 sulfate ( biochrom ) to the culture medium . cells were seeded at low density the day before use . during the experiments , cells were kept at room temperature in bath solution ( 1 mm cacl2 , 140 mm nmdg , 5 mm kcl , 3.3 mm mgcl2 , 1 mm cacl2 , 5 mm d - glucose , 10 mm hepes , ph 7.4 ) . patch pipets with resistances of 1.73.5 m were pulled from 1.5 mm diameter borosilicate glass capillaries and filled with pipet solution ( 120 mm kcl , 8 mm nacl , 1 mm mgcl2 , 10 mm hepes , 10 mm egta , 5.6 mm cacl2 ) , resulting in 200 nm free [ ca ] as calculated by cabuf software ( g. droogmans , formerly available from ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip ) . data were acquired with an epc10 amplifier and patchmaster software ( heka elektronik , germany ) and were compensated for fast and slow capacity transients . the cells were held at 50 mv and current was measured during 140 ms voltage ramps from 85 to 20 mv every 5 s over a period of 450 s. series resistance was compensated by 70% . for activation of trpm2 , adpr was added to the pipet solution at a concentration of 100 m . antagonist activity of the adpr analogues was tested by adding them at different concentrations to a pipet solution with 100 m adpr . during some experiments , the pipet solution contained 0.1% dmso because stock solutions of the more lipophilic adpr analogues were prepared in dmso . fresh blood with edta as anticoagulant was obtained from indiscriminately selected volunteers . neutrophils were isolated as described elsewhere . in brief , the blood was fractionated with a histopaque-1119 density gradient and subsequently neutrophils were further purified by the use of percoll layers ranging from 65 to 85% in density . after a final washing step , the cells were resuspended in ca measurement buffer ( 140 mm nacl , 5 mm kcl , 1 mm mgso4 , 1 mm cacl2 , 1 mm nah2po4 , 4 mm glucose , 20 mm hepes , ph 7.4 ) and kept on ice until use . to avoid premature activation of the neutrophils , all buffers used during the isolation were supplemented with 2 mm edta , cell concentrations exceeding 5 10 cells / ml were avoided , and only endotoxin free materials and solutions were used . neutrophils were incubated with 4 m fura2/am for 30 min at 37 c in the dark , washed twice , and resuspended in ca measurement buffer ( see above ) at a concentration of 1 10 cells / ml . for each measurement , 5 10 cells were transferred to a small chamber consisting of a rubber o - ring fixed with silicon grease on a glass coverslip coated subsequently with 25 ng of bsa and 250 ng of fibronectin . the cells were incubated for 15 min at ambient temperature in the presence of 10 mm l - ascorbic acid ( ph 7.4 ) and , if applicable , varying concentrations of 8-phenyl - adpr . the loaded coverslip was mounted on the stage of a perkinelmer / imrovision imaging system built around a leica dm ire 2 fluorescence microscope . approximately 70 s after the beginning of the measurement , the cells were stimulated by addition of fmlp ( final concentration 1 m ) or a5 peptide ( final concentration 10 m ) . the migration of neutrophils was observed microscopically in microfluidic devices ( -slide chemotaxis , ibidi , martinsried , germany ) . first the -slides were coated with 50 g / ml fibronectin for 30 min at ambient temperature before washing three times and drying . isolated neutrophils were resuspended to a concentration of 3 10 cells / ml in ca measurement buffer supplemented with 10% ( v / v ) plasma obtained from the same donor . if applicable , 8-phenyl - adpr or egta was added and the whole slide was loaded according to the manufacturer s instructions and incubated at room temperature for 15 min . adding 18 l of fmlp ( 125 nm ) to the upper reservoir resulted in a chemotactical gradient from 0 50 nm fmlp across the observation chamber . the slide was mounted on the stage of the imaging system , and the main chamber observed at 10 times magnification in bright - field mode . after a 5 min resting period , greyscale images with a resolution of 672 510 pixels were recorded every 30 s for 1 h using openlab software 4.0.4 . cell migration was tracked manually with a 5 5 pixel maximum intensity centering correction using the manual - tracking plugin for imagej ( 1.45e ) . migrational parameters were calculated from the movement paths using the chemotaxis and migration tool software ( v2.0 , ibidi gmbh ) .

adenosine 5-diphosphoribose ( adpr ) activates trpm2 , a ca2 + , na+ , and k+ permeable cation channel . activation is induced by adpr binding to the cytosolic c - terminal nudt9-homology domain . to generate the first structure activity relationship , systematically modified adpr analogues were designed , synthesized , and evaluated as antagonists using patch - clamp experiments in hek293 cells overexpressing human trpm2 . compounds with a purine c8 substituent show antagonist activity , and an 8-phenyl substitution ( 8-ph - adpr , 5 ) is very effective . modification of the terminal ribose results in a weak antagonist , whereas its removal abolishes activity . an antagonist based upon a hybrid structure , 8-phenyl-2-deoxy - adpr ( 86 , ic50 = 3 m ) , is more potent than 8-ph - adpr ( 5 ) . initial bioisosteric replacement of the pyrophosphate linkage abolishes activity , but replacement of the pyrophosphate and the terminal ribose by a sulfamate - based group leads to a weak antagonist , a lead to more drug - like analogues . 8-ph - adpr ( 5 ) inhibits ca2 + signalling and chemotaxis in human neutrophils , illustrating the potential for pharmacological intervention at trpm2 .

Experimental Section Synthesis of 8-Modified ADPR Analogues Synthesis of Purine Modified ADPR Analogues Synthesis of Adenosine Ribose Modified ADPR Analogues Synthesis of Pyrophosphate Modified ADPR Analogues Synthesis of Squarate Analogues: Adenosine Squaryl ( Synthesis of Click Analogue: 8-Phenyladenosine-1,4-Triazole Ribose (8-Ph-ATrR) Synthesis of Terminal Ribose Modification: Synthesis of Cyclopentyl-ADP Synthesis of Cyclopentyl-8-Phenyl-ADP Synthesis of 8-Phenyl-2-Deoxy-ADPR Pharmacology

cesium carbonate ( 0.24 mmol , 2.9 equiv ) was added in one portion to a stirred solution of the corresponding boronic acid ( 0.103 mmol , 1.2 equiv ) , palladium acetate ( 0.004 mmol , 0.05 equiv ) , tppts ( 0.02 mmol , 0.24 equiv ) , and 8-br - adpr ( tea salt , 0.0823 mmol ) in degassed mecn h2o ( 1:2 v / v ; 2.4 ml ) under an argon atmosphere . the reaction mixture was cooled to room temperature , quadrapure tu ( 100 mg ) added , and the mixture stirred for 16 h. the mixture was filtered and evaporated under reduced pressure to leave a crude product that was purified by ion - exchange ( q - sepharose ) chromatography eluted with a gradient ( 040% ) of teab ( 1.0 m ) in milli - q water followed by reverse phase ( rp-18 ) column chromatography , eluted with 020% mecn in teab ( 0.05 m ) to isolate the desired 8-substituted adpr product . the protected compound ( 0.1 mmol ) was stirred in a 75% aq tfa solution ( 5 ml ) at rt for 1 h. the solvents were evaporated under reduced pressure , and the residue was coevaporated with meoh to remove any residual tfa . the morpholidate was dissolved in a solution of mncl2 in formamide ( 1 ml , 0.2 m ) , mgso4 ( 48 mg , 0.4 mmol ) and -nmn ( 67 mmol , 0.2 mmol ) were added , and the mixture was stirred for 2 days . after 4 h , all of the starting material had been consumed , the reaction mixture was diluted with water until the conductivity < 200 s / cm , and the product purified by ion - exchange ( q - sepaharose ) chromatography eluting with a gradient ( 050% ) of teab ( 1.0 m ) in milli - q . 6-o - me - imp morpholidate ( 100 mg , 0.232 mmol ) , -nmn ( 85 mg , 0.253 mmol ) , and mgso4 ( 54 mg , 0.464 mmol ) were dissolved in a 0.2 m solution of mncl2 in formamide ( 1.7 ml ) and stirred at rt for 16 h , after which hplc analysis showed completion of the reaction ( rt ( -nmn ) = 2.1 min and rt ( 6-o - me - nhd ) = 3.8 min ) . the volatiles were evaporated under reduced pressure , and the residue was applied to a semipreparative c18 column developed with a linear gradient of 0.1 m teab against mecn . to a solution of 1-o - methyl-2,3-o - isopropylidene-5-o - p - toluenesulfonyl--d - ribofuranose 50 ( 2.4 g , 6.7 mmol ) in dmf ( 20 ml ) was added nan3 ( 5.2 g , 80.4 mmol ) , and the reaction mixture was stirred at 120 c for 16 h. after cooling to rt , acetone ( 20 ml ) was added and the solid was removed by filtration . h ( 400 mhz , meoh - d4 ) 8.32 ( s , 1h , h-8 ) , 8.29 ( s , 1h , h-2 ) , 6.25 ( d , 1h , j1,2 = 2.6 , h-1 ) , 5.55 ( dd , 1h , j2,3 = 6.4 and j2,1 = 2.6 , h-2 ) , 5.16 ( dd , 1h , j3,2 = 6.4 and j3,4 = 3.8 , h-3 ) , 4.444.40 ( m , 1h , h-4 ) , 4.07 ( dd , 1h , j5a,5b = 14.1 and j5a,4 = 4.4 , h-5a ) , 3.93 ( dd , 1h , j5ba,5a = 14.1 and j5b,4 = 6.7 , h-5b ) , 3.78 ( sept , 1h , j = 6.6 , ch ) , 1.722.07 ( m , 8h , 4 ch2 ) , 1.64 ( s , 3h , ch3 ) and 1.43 ( s , 3h , ch3 ) . to a solution of 3-butylamino-4-ethoxycyclobut-3-ene-1,2-dione 56 ( 200 mg , 1.081 mmol ) and dipea ( 92 l , 0.531 mmol ) in etoh ( 5 ml ) the reaction mixture was stirred at rt for 24 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 364 mg , 81% ) . to a solution of 3-hexylamino-4-ethoxycyclobut-3-ene-1,2-dione 57 ( 190 mg , 0.892 mmol ) and dipea ( 76 l , 0.438 mmol ) in etoh ( 5 ml ) the reaction was stirred at rt for 20 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 291 mg , 74% ) . to a solution of 2,3-o - isopropylidene-5-deoxy-5-azido-8-bromoadenosine 69 ( 123 mg , 0.30 mmol ) in a mixture of buoh - h2o ( 1:1 v / v , 6 ml ) was added cuso45h2o ( 2 mg , 0.015 mmol ) , sodium ascorbate ( 6 mg , 0.03 mmol ) , and 1-o - methyl-2,3-o - isopropylidene-5-o - propargyl--d - ribofuranose 70 ( 73 mg , 0.30 mmol ) . c ( 100 mhz , cdcl3 ) 154.6 ( c-6 ) , 153.1 ( c-2 ) , 150.1 ( c-4 ) , 144.9 ( c - triazole ) , 127.0 ( c-8 ) , 123.5 ( ch - triazole ) , 120.1 ( c-5 ) , 114.7 , 112.3 ( 2 c ) , 109.2 ( c-1 ) , 90.9 ( c-1 ) , 85.9 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.9 ( c-2 ) , 81.9 ( 2c , c-3 and c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.5 ( c-5 ) , 27.0 , 26.3 , 25.3 , and 24.9 ( 4 ch3 ) . to 1-(2,3-o - isopropylidene-5-deoxy-8-bromoadenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 71 ( 140 mg , 0.21 mmol ) , na2cl4pd ( 3 mg , 5 mol % ) , phb(oh2 ) ( 32 mg , 0.27 mmol ) , tppts ( 30 mg , 25 mol % ) , and na2co3 ( 68 mg , 0.64 mmol ) was added degassed mecn h2o ( 3 ml , 1:2 v / v ) and the resulting solution stirred at 80 c for 1 h. all solvents were evaporated and the residue purified by column chromatography using an isco chromatography system ( dcm acetone , 6:4 v / v ) to yield the product ( 30 mg , 21% ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 114.3 , 112.2 ( 2 c ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.8 ( c-5 ) , 26.9 , 26.3 , 25.2 , and 24.9 ( 4 ch3 ) . a flask containing 8-bromo-2,3-o - isopropylidene - adenosine 81 ( 200 mg , 0.519 mmol ) , na2cl4pd ( 5 mol % ) , tppts ( 25 mol % ) , phb(oh)2 ( 190 mg , 1.562 mmol ) , and na2co3 ( 165 mg , 1.557 mmol ) was purged with argon , and a degassed mixture of mecn h2o ( 1:1 v / v , 6 ml ) was added . to a solution of 8-ph - amp - morpholidate ( 31 mg , 0.060 mmol ) and cyclopentane monophosphate 79 ( 11 mg , 0.066 mmol ) in 0.2 n mncl2 in formamide ( 0.5 ml ) was added mgso4 ( 14 mg , 0.12 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . h ( 400 mhz , d2o ) 8.21 ( s , 1h , h-2 ) , 7.67 ( d , 1h , j = 6.2 ) , 7.607.56 ( m , 2h ) , 7.28 ( d , 2h , j = 8.1 ) ( 5 arh ) , 5.82 ( d , 1h , j = 6.4 , h-1 ) , 5.15 ( app t , 1h , h-2 ) , 4.31 ( dd , 1h , j = 6.4 , 4.5 , h-3 ) , 4.083.98 ( m , 4h , h-4 , 2 h-5 , ch o ) , 1.621.60 ( m , 4h ) , 1.521.50 ( m , 2h ) and 1.361.34 ( m , 2h ) ( 4 ch2 ) . the volatiles were removed under reduced pressure , and the residue was applied to a c18 semipreparative column eluted with a gradient of 0.1 m teab in mecn . antagonist activity of the adpr analogues was tested by adding them at different concentrations to a pipet solution with 100 m adpr . the morpholidate was dissolved in a solution of mncl2 in formamide ( 1 ml , 0.2 m ) , mgso4 ( 48 mg , 0.4 mmol ) and -nmn ( 67 mmol , 0.2 mmol ) were added , and the mixture was stirred for 2 days . after 4 h , all of the starting material had been consumed , the reaction mixture was diluted with water until the conductivity < 200 s / cm , and the product purified by ion - exchange ( q - sepaharose ) chromatography eluting with a gradient ( 050% ) of teab ( 1.0 m ) in milli - q . 6-o - me - imp morpholidate ( 100 mg , 0.232 mmol ) , -nmn ( 85 mg , 0.253 mmol ) , and mgso4 ( 54 mg , 0.464 mmol ) were dissolved in a 0.2 m solution of mncl2 in formamide ( 1.7 ml ) and stirred at rt for 16 h , after which hplc analysis showed completion of the reaction ( rt ( -nmn ) = 2.1 min and rt ( 6-o - me - nhd ) = 3.8 min ) . the volatiles were evaporated under reduced pressure , and the residue was applied to a semipreparative c18 column developed with a linear gradient of 0.1 m teab against mecn . to a solution of 1-o - methyl-2,3-o - isopropylidene-5-o - p - toluenesulfonyl--d - ribofuranose 50 ( 2.4 g , 6.7 mmol ) in dmf ( 20 ml ) was added nan3 ( 5.2 g , 80.4 mmol ) , and the reaction mixture was stirred at 120 c for 16 h. after cooling to rt , acetone ( 20 ml ) was added and the solid was removed by filtration . the solvents were evaporated under reduced pressure , and the residue was dissolved in dcm ( 50 ml ) and washed successively with water ( 50 ml ) , satd aq nahco3 ( 50 ml ) , and water ( 50 ml ) . to a solution of 3-butylamino-4-ethoxycyclobut-3-ene-1,2-dione 56 ( 200 mg , 1.081 mmol ) and dipea ( 92 l , 0.531 mmol ) in etoh ( 5 ml ) the reaction mixture was stirred at rt for 24 h. the solvent was removed under reduced pressure , and the residue was purified on an isco chromatographic system ( dcm meoh , 8:2 v / v ) to yield the desired product as a white foam ( 364 mg , 81% ) . to a solution of 2,3-o - isopropylidene-5-deoxy-5-azido-8-bromoadenosine 69 ( 123 mg , 0.30 mmol ) in a mixture of buoh - h2o ( 1:1 v / v , 6 ml ) was added cuso45h2o ( 2 mg , 0.015 mmol ) , sodium ascorbate ( 6 mg , 0.03 mmol ) , and 1-o - methyl-2,3-o - isopropylidene-5-o - propargyl--d - ribofuranose 70 ( 73 mg , 0.30 mmol ) . to 1-(2,3-o - isopropylidene-5-deoxy-8-bromoadenosine)-4-(2,3-o - isopropylidene-5-o - methylribosyl)-1,2,3-triazole 71 ( 140 mg , 0.21 mmol ) , na2cl4pd ( 3 mg , 5 mol % ) , phb(oh2 ) ( 32 mg , 0.27 mmol ) , tppts ( 30 mg , 25 mol % ) , and na2co3 ( 68 mg , 0.64 mmol ) was added degassed mecn h2o ( 3 ml , 1:2 v / v ) and the resulting solution stirred at 80 c for 1 h. all solvents were evaporated and the residue purified by column chromatography using an isco chromatography system ( dcm acetone , 6:4 v / v ) to yield the product ( 30 mg , 21% ) . c ( 100 mhz , cdcl3 ) 155.6 ( c-6 ) , 152.6 ( c-2 ) , 151.5 ( c-8 ) , 150.0 ( c-4 ) , 144.8 ( c - triazole ) , 130.5 ( 2c ) , 129.6 ( 2c ) , 128.9 ( 5 ch - phenyl ) , 128.6 ( c - phenyl ) , 123.5 ( ch - triazole ) , 119.4 ( c-5 ) , 114.3 , 112.2 ( 2 c ) , 109.1 ( c-1 ) , 90.4 ( c-1 ) , 86.1 ( c-4 ) , 85.0 ( c-4 ) , 84.9 ( c-2 ) , 83.8 ( c-2 ) , 82.5 ( c-3 ) , 81.9 ( c-3 ) , 71.3 ( c-5 ) , 64.6 ( och2-tr ) , 54.6 ( och3 ) , 51.8 ( c-5 ) , 26.9 , 26.3 , 25.2 , and 24.9 ( 4 ch3 ) . to a solution of 8-ph - amp - morpholidate ( 31 mg , 0.060 mmol ) and cyclopentane monophosphate 79 ( 11 mg , 0.066 mmol ) in 0.2 n mncl2 in formamide ( 0.5 ml ) was added mgso4 ( 14 mg , 0.12 mmol ) , and it was stirred for 16 h at rt , after which hplc analysis showed product formation . the volatiles were removed under reduced pressure , and the residue was applied to a c18 semipreparative column eluted with a gradient of 0.1 m teab in mecn . antagonist activity of the adpr analogues was tested by adding them at different concentrations to a pipet solution with 100 m adpr .

one of the most important challenges many societies are currently facing is the supply of fuel for their increasing energy needs . the extensive use of fossil fuels during the past century is reaching its limits , mostly because of the deleterious effects their combustion is causing to the environment . in this context , the search for alternative energy sources is of critical significance . among various solutions , solar light is very important , because of its accessibility in most parts of the globe and the power it delivers on the surface of the earth . it is , however , only available during a certain portion of the day and in some places during certain times of the year . an optimal use of this energy source therefore requires its efficient storage . a very promising energy carrier that is able to fulfill this role is hydrogen . a tremendous amount of work has been carried out in the past decades to develop systems capable of sustainably producing hydrogen from water electrolysis . a class of compounds that meets both the technical and economical requirements of large - scale hydrogen production is molybdenum sulfides ( mosx ) . these inorganic materials have been extensively studied and used as natural gas hydrodesulfurization ( hds ) catalysts . more recently , they have been shown to catalyze the hydrogen evolution reaction ( her ) in their molecular , nanoparticulate and amorphous forms . in particular , some of us have reported on the preparation of electrodeposited amorphous mosx thin films that are catalytically active for the reduction of protons into hydrogen in acidic water . these films have been extensively studied by x - ray photoelectron spectroscopy ( xps ) to determine their electronic structure . although very informative , this technique only probes the very first nanometers of a surface and is very difficult to perform under functional conditions . in the current paper , we investigate the structure of an amorphous , her - active mosx film observed under catalytic conditions using in situ x - ray spectroscopic techniques . an in situ electrochemical cell was developed and applied in the tender x - ray energy region ( 23 kev ) to the study of an mosx her catalyst under functional conditions . this method allowed us to probe the local and electronic structure of the molybdenum centers and sulfur ligands of the film under precatalytic and catalytic conditions . by comparing the data obtained on the catalytic film poised at constant potentials with those of reference compounds ( see table 1 ) , we propose a structural model for the catalyst film as prepared and under functional conditions in its precatalytic and catalytic states . the results provide evidence for the presence of terminal disulfide units in the mosx film and demonstrate their involvement in the catalytic cycle . although the role of disulfide units in the reduction of protons into dihydrogen by mosx materials has often been suggested , it is , to the best of our knowledge , the first time that this chemical motif is experimentally observed during the her . we also suggest on a spectroscopic basis that the rate - limiting step of the reaction is the reductive breaking of the s s bond . these mechanistic insights will be important in designing hydrogen - evolving catalysts with better performances . the structures of [ mo3s4(oh2)9 ] and mos2 have been determined previously by xrd . only one structural model out of four possible is shown here . for more structural models hno3 ( 90% ) , mos2 ( 99% ) , mos4(nh4)2 ( > 99.9% ) , and naclo4 ( > 98% ) were purchased from aldrich , and mos32h2o was purchased from alfa - aesar . prior to electrodeposition of the mosx catalyst film , a 500 nm thick silicon nitride ( si3n4 , purchased from silson ltd . ) membrane was sputter coated with a 150 nm layer of indium - doped tin oxide ( ito ) to produce a suitable x - ray transparent conductive surface . the ito surface was connected from the front with copper tape to ensure electrical contact . the ito electrode was immersed into a 2 mm solution of ( nh4)2mos4 in 0.1 m naclo4 in water ( 8 ml ) . consecutive cyclic voltammograms ( typically 25 ) were carried out using an ivium stat potentiostat ( ivium technologies ) with a saturated silver / silver chloride reference electrode ( separated by a porous vycor tip ) and a titanium wire counter electrode . the cyclic voltammograms were performed between + 0.7 and 0.4 v vs reversible hydrogen electrode ( rhe ) and a scan rate of 0.05 v / s was employed . x - ray absorption data were collected at the stanford synchrotron radiation lightsource ( ssrl ) on beamline 7 - 3 ( mo k - edge ) and 4 - 3 ( s k - edge and mo l - edge ) at an electron energy of 3.0 gev with an average current of 300 ma . at beamline 7 - 3 , the intensity of the incident x - ray was monitored by an ar - filled ion chamber ( i0 ) in front of the sample . solid samples were diluted in boron nitride ( 1% w / w ) and placed in an aluminum sample holder sealed with kapton tape . data were collected as fluorescence excitation spectra with a ge 30 element detector ( canberra ) . energy was calibrated by the first peak maximum of the first derivative of a molybdenum foil ( 20003.9 ev ) , placed between two ar - filled ionization chambers ( i1 and i2 ) after the sample . , the incoming x - rays were monochromatized by a si(111 ) double - crystal monochromator . the intensity of the incident x - rays was monitored by a he - filled ion chamber ( i0 ) in front of the sample . solid sample spectra were collected from a thin layer of the sample smeared on sulfur - free tape . data were collected as fluorescence excitation spectra with a vortex 4 element silicon drift detector ( sii nanotechnology ) . monochromator energy was calibrated to the first peak of thiosulfate reference sample , which was assigned at 2470.8 ev . the sample environment was kept under he gas atmosphere with an he - filled bag to reduce air absorption of incoming x - rays and fluorescence signals . additional data on reference samples were also collected at the lucia beam line at soleil , at an electron energy of 2.7 gev and an average ring current of 430 ma . the incoming photons were selected with a si ( 111 ) double crystal monochromator . samples were compressed as pellets in 1:10 ( w / w ) mixture with cellulose and fixed on a copper sample holder with conductive carbon tape . the s k - edge and mo l - edges were measured as fluorescence spectra with a grazing angle ( < 2 ) for the outgoing photons in order to avoid self - absorption phenomena . energy ranges for the first moment calculation are as follow : s k - edge : 24652475 ev ; mo l3-edge : 25202535 ev ; mo l2-edge : 26232640 ev . a glass electrochemical cell consisting of two compartments separated by a porous frit the working compartment has flat walls ( 1.5 cm wide ) with a single circular hole of 0.8 cm in diameter . an mosx - coated ito / si3n4 membrane as described above was in contact with a slip of copper tape and fixed with epoxy glue to the exterior of the wall of the cell , over the 0.8 cm hole , with the mosx layer facing inward . a nitric acid solution adjusted to ph 2 the cell was connected to a potentiostat by making electrical contact to the copper tape slip that protruded from the side of the working compartment . a teflon cap fitted with a reference electrode ( ag / agcl ) was used to cover the working compartment and to ensure a fixed distance between working and reference electrodes for all experiments . a platinum gauze was placed in the second compartment separated by a frit from the first one and used as a counter electrode . x - ray absorption spectra were recorded at different positions on the electrode to check the materials for homogeneity . the same electrodes were used to measure the mo k - edge ( bl 7 - 3 ) and l - edges ( bl 4 - 3 ) and the s k - edge ( bl 4 - 3 ) spectra . for mo k - edge measurements , the spectra were recorded in air . for s k - edge and mo l - edge measurements , the electrochemical cell was placed in a he - filled bag with a polypropylene membrane placed as close as possible to the si3n4 membrane to maximize the penetration of x - rays into and from the mosx film . spectra were recorded on the dry electrodeposited mosx films at first and then in ph 2 nitric acid solution at 0.3 , 0.1 , 0.1 , and 0.3 v vs rhe . the experiments were conducted at ph = 2 since the ito layer deposited on the si3n4 membrane could not withstand lower ph values . the open - circuit potential measured prior to applying a potential was 0.8 v. at each potential , 10 scans were recorded for the mo k - edge and 4 scans for the s k - edge and mo l - edge . after each potential change , the system was allowed to equilibrate for 10 min before recording a spectrum . no noticeable change was observed between the first and last spectra recorded at a given potential . the current density measured for the potentials of interest ( see figure s1 ) was similar to those recorded on a regular ito electrode under identical conditions ( room temperature , ph 2 in nitric acid ) . a polarization curve of the film on a rotating disk carbon electrode at ph = 2 is also shown in figure s2 . data reduction of the mo k - edge extended x - ray absorption fine structure ( exafs ) spectra was performed using exafspak ( drs . pre - edge and post - edge backgrounds were subtracted from the xas spectra , and the results were normalized with respect to edge height . background removal in k - space was achieved through a five - domain cubic spline . curve fitting was performed with artemis and ifeffit software using ab initio calculated phases and amplitudes from the program feff 8.2 . for the s k - edge spectra , an e0 value of 2470.8 ev was used , and normalization was performed over the range 2488.82510.8 ev . for the mo l3-edge and l2-edge , e0 values of 2523.6 and 2627.0 ev were used , and normalization was performed over the ranges 2545.32591.6 ev and 2648.72666.7 ev , respectively . we use the notation precatalytic and catalytic states for the mosx film poised at 0.3 and 0.3 v in a nitric acid solution at ph = 2 , respectively . these states were measured in situ , while the as prepared film was measured ex situ . the deposition and characterization of mosx films under various conditions were described in a recent publication . the as prepared mosx film studied in this work corresponds to the mos3-cv film before activation in ref ( 19 ) . the films deposited did not show any x - ray diffraction ( xrd ) signal , which indicates that they are amorphous . we therefore used x - ray absorption spectroscopy to study the structure of these films . the mo k - edge x - ray absorption near edge spectroscopy ( xanes ) spectra were recorded ex situ on the mosx film as prepared and in situ in the precatalytic and catalytic states ( figure 1a ) using a custom - made x - ray spectroelectrochemical cell described in the electrodeposition of mosx electrocatalyst section . for comparison , the xanes spectra of mos3 , mos2 , and [ mo3s4(h2o)9]cl4 are shown in figure 1b . no clear trend reflecting the formal oxidation state of mo is observed in the mo rising edge energy of the reference compounds , which is largely due to the charge delocalization between the molybdenum ion and the sulfur ligands . for the mosx film , changes at the peak top are observed , while the rising edge energy remains identical when changing the potential applied to the film . none of these spectra present a good match with those of the reference compounds , suggesting a mixed composition for the mosx film . the mo k - edge exafs spectra were recorded ex situ on the mosx film ( as prepared ) and in situ in the precatalytic and catalytic states ( figure 1c ) . for comparison , the exafs spectra of the mos3 , mos2 , and [ mo3s4(h2o)9]cl4 are shown in figure 1d . the exafs spectrum of as prepared film shows two peaks at r = 1.2 and 1.9 ( r being the apparent distance and r the actual distance ) , corresponding to mo o and mo s interactions , respectively ( vide infra ) . when poised at 0.3 v , the short - distance peak ( r = 1.2 ) disappears , and the spectrum of the precatalytic state shows similarities with that of mos3 , with a high intensity peak at ca . r = 2.5 . under catalytic conditions , the main peak shifts to a lower apparent distance ( r = 1.86 ) , suggesting a shortening of the mo s bond . in addition , the small intensity peak at r = 2.5 disappears , suggesting the breaking of the mo ( a ) mo k - edge xanes spectra of the mosx film as prepared and in the precatalytic and catalytic states . ( b ) mo k - edge xanes spectra of mo3s4 , mos2 and mos3 . ( c ) mo k - edge fourier transform exafs ( k - weighted ) of the mosx film as prepared and in the precatalytic and catalytic states . ( d ) mo k - edge fourier transform exafs ( k - weighted ) of mo3s4 , mos2 , and mos3 . prior to fitting the exafs spectra of mosx films , the exafs spectra of three model compounds were fit using structural data derived from their crystal structures to extract the exafs fitting parameters . the final fitting parameters are listed in table 2 . in the case of mos3 , two distances are required to fit the spectrum , one accounting for 6 mo two competing structural models have been proposed for mos3 by hibble et al . and weber et al . the hibble structure for mos3 consists of chains of molybdenum ions alternatively bridged by three sulfides ( - s ) or one sulfide and one disulfide ( -: s2 ) ligands ( see table 1 ) . several authors have shown that the exafs spectrum of mos3 is indeed best fit with a single mo mo interaction because mo ions only interact with neighboring mo through the short bridge containing a disulfide unit but not through the long one containing sulfide groups only . the weber structure consists of a random arrangement of four isostructural mo3s9 clusters , which are linked to each other by one or two - sulfide bridge(s ) . each cluster contains three sulfide and three disulfide ligands , which can be either bridging or terminal . the fitting parameters are in agreement with the literature for both proposed structures , and we can not discriminate one from the other . in the weber structure , each molybdenum center is surrounded by 5 , 6 , or 7 sulfur ligands . this is compatible with the quite large debye waller factor ( 0.010 ) found for the mo the spectrum of mos2 ( which is microcrystalline ) shows two well - separated peaks at ca . r = 1.95 and r = 2.80 that can be fit with mo s and mo mo interactions at 2.41 and 3.17 with n values of 6 for each interaction . these values are in very good agreement with the xrd distances reported for mos2 ( 2.40 and 3.15 ) . the low r peak was fit with mo o and mo s interactions at 2.13 and 2.23 , respectively , with n values of 3 for both interactions . these values are in very good agreement with the xrd structure of [ mo3s4(h2o)9]cl4 . the exafs curve fitting results of the mosx film as prepared and in the precatalytic and catalytic states are summarized in table 2 , and the k - space curves are displayed in figure s4 . for the as prepared film , an mo o interaction with a distance of r = 1.74 and an n value of 1.2 had to be included to model the peak at short r value . s interaction ( r = 2.44 , n = 3.0 ) , while the low intensity one at r = 2.61 was fit with an mo mo interaction ( r = 2.76 , n = 0.8 ) . for the film in the precatalytic state ( poised at 0.3 v ) , the best fit was obtained with an mo s interaction at 2.40 with an n value close to 6 and an mo mo interaction at 2.74 with an n value of 1.1 . under these conditions , the parameters of the mo s and mo mo interaction are in good agreement with those of mos3 . we therefore considered the film in the precatalytic state to be predominantly composed of mos3 . when the potential was lowered to 0.3 v ( catalytic state ) , the mo fitting the data with a single shell ( fit # 1 ) , yields an mo s interaction with r = 2.36 and n = 6 . this distance is slightly shorter ( 0.05 ) than that of the film poised at 0.3 v. we also considered the presence of an mo mo second shell ( fit # 2 ) . in this fit , the n value for the mo mo interaction dropped to 0.2 , while it did not show a significant improvement in the fit quality . mo bond is significantly diminished in the mosx film in the catalytic state . r is the apparent distance in from the central atom to the scatterer , k is the wave vector describing the trajectory of the scattered photoelectron , n is the number of scatterers , r is the debye fitting range : 3.10 k ( / ) 11.5 and 1.57 r ( ) 2.82 . fitting range : 3.10 k ( / ) 11.5 and 1.15 r fitting range : 3.10 k ( / ) 11.5 and 1.15 r ( ) 2.82 . fitting range : 3.10 k ( / ) 10.5 and 1.15 r ( ) 2.82 . 2472 ev ) is just below that of the molybdenum l - edges ( ca . 2526 and 2631 ev for the l3- and l2-edges , respectively ) , we recorded spectra with both edges in one single scan ( see figure s5 ) . figure 2a shows the s k - edge spectra collected on the mosx film as prepared and in the precatalytic and catalytic states as well as the reference compounds listed in table 1 . the spectrum of mo3s4 shows several features , with a main peak at 2471.6 ev ( measured at peak top ) , a shoulder at 2472.8 ev , and an intense low energy peak at 2469.8 ev . in mos2 , a single , sharp peak is observed at ca . the s k - edge spectra of the mosx film poised at 0.3 , 0.1 , 0.1 , and 0.3 v are shown in figure 2b , and the corresponding first moment energies are listed in table 3 . in the as prepared mosx film , the sulfur k - edge main peak energy ( 2472.1 ev ) and shape are similar to that of mos3 , although a shoulder is present at low energy ( 2469.7 ev ) as in mo3s4 , but with a much smaller intensity . when the sample is set in a ph 2 nitric acid solution and the potential is poised to 0.3 v , the first moment energy of the main peak remains at 2472.0 ev , while the shoulder at lower energy ( 2469.8 ev ) increases and the one at higher energy ( 2472.4 ev ) decreases . when the potential is gradually decreased to 0.3 v , the first moment energy of the main peak shifts to a slightly higher energy ( from 2472.0 to 2472.3 ev ) , which indicates an overall oxidation of the sulfur ligands . figure 2c shows the molybdenum l3-edge spectra recorded on the mosx film as prepared and in the precatalytic and catalytic states as well as the reference compounds listed in table 1 . the mo l3-edge spectrum of mo3s4 shows one main , broad feature , centered at 2525.1 ev ( measured at peak top ) , with a shoulder at 2523.25 ev . the spectrum of mos2 shows a single feature at 2524.7 ev , while that of mos3 has a main peak at 2525.8 ev with a shoulder at higher energy ( 2528.4 ev ) . the spectrum recorded on the as prepared mosx film is very similar to that of mos3 . for instance , it has a shoulder at 2528.7 ev , which is a strong component of the mos3 spectrum . this is even more visible in the l2-edge spectrum ( see figure 2e ) , where the as prepared spectrum shows the double peak feature ( 2629.3 and 2629.6 ev ) that is also present in mos3 . the mo l3-edge spectra of the mosx film poised at 0.3 , 0.1 , 0.1 , and 0.3 v are shown in figure 2d , and the corresponding first moment energies ( as well as those of the mo l2-edge spectra ) are reported in table 3 . under precatalytic conditions ( ph 2 nitric acid , 0.3 v ) , the l3-edge peak first moment energy shifts to a lower energy by ca . 0.4 ev as compared to the as prepared film , and the higher energy component ( 2528.4 ev ) characteristic of mos3 disappears . when progressively decreasing the potential to 0.3 v , the first moment energy of the mo l3-edge peak shifts to lower values by ca . the same trend is observed for the l2-edge part of the spectra ( see figure 2d ) with an even more pronounced shift of the first moment to lower energies ( by ca . top : sulfur k - edge spectra of ( a ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 and mos3 reference spectra , and ( b ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . middle : molybdenum l3-edge spectra of ( c ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 , and mos3 and ( d ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . bottom : molybdenum l2-edge spectra of ( e ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 , and mos3 and ( f ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . the mosx films studied in this paper were prepared by electrochemical cycling of an ito electrode in an [ nh4]2mos4 aqueous solution . this procedure was shown to involve anodic and cathodic deposition processes , as well as cathodic corrosion . eventually , the final composition of the film was estimated from xps and electrochemical quartz crystal microbalance ( eqcm ) analysis as a mixture of mos2 and mos3 phases . in line with this first analysis , the x - ray spectroscopic data of the as prepared sample presented in this paper show features associated with several compounds , clearly indicating that the mosx electrocatalyst is a mixture of species . table 4 summarizes the spectroscopic information collected on the as prepared , precatalytic and catalytic states . it also indicates the structural and electronic changes observed when the potential is gradually decreased from 0.3 ( precatalytic state ) to 0.3 v ( catalytic state ) . in the mo k - edge exafs fourier transform spectrum of the as prepared sample , the peak observed at low r values ( 1.05 ) indicates the presence of an mo the mo o distance obtained from the fits ( r = ca . 1.75 ) is much shorter than usually observed in most moxoysz oxysulfide compounds ( typically 2.0 r 2.2 ) or in mo3s4(oh2)9 ( vide supra ) , and is longer than the mo the presence of oxygen has also been observed previously in the xps spectra recorded ex situ on the as prepared mosx film . given that the latter technique is only sensitive to the surface , we estimated that a layer of molybdenum oxide moox covers the bulk mos3 material in the as prepared film , as a result of air oxidation during transfer from the synthesis laboratory to the synchrotron facility . since the film is highly porous , the actual surface exposed to air and converted into an oxide could be significant . all parameters corresponding to an mos3 structure , except for the lower number of mo s vectors . in mos3 , the molybdenum ion is described as mo(v ) in the hibble model and as mo(iv ) in the weber model . this deviation can be explained by the presence of the outer - shell moox layer in the as prepared film that decreases the average mo s bonds per mo center . this analysis is consistent with the previous xps observations showing the presence of mo(iv ) and of s and s2 ligands . the presence of peaks characteristic of mos3 at 2528.7 and 2633.5 ev in the mo l3- and l2-edge spectra , respectively , confirms an mos3 structure . the small intensity feature at 2470 ev that is characteristic of mo3s4 suggests the presence of similar chemical motifs in the as prepared material . indeed , the mo3s9 trinuclear units proposed by weber as constituents of mos3 are closely related to mo3s4 , since they share the same geometrical arrangement as well as - and - bridging s ligands . it is , however , very difficult to distinguish small contributions of mo3s4 within the mos3 k - edge exafs , since the mo s and mo mo distances of these two materials are almost identical ( see table 2 ) . the starting material is thus best described as an amorphous film of mos3 in the bulk , with a surface layer of moox . the oxidation state of molybdenum centers is predominantly + iv , while the sulfur ligands are present as bridging sulfides ( - and - s ) and disulfides ( -: s2 ) . when the mosx film is set in a nitric acid solution at ph = 2 and poised at 0.3 v , the mo o interaction in the mo k - edge exafs spectrum of the as prepared material disappears , indicating that the molybdenum oxide fraction is hydrolyzed . this phenomenon was already observed by xps on the surface of a similar system . the current data extend these structural considerations to the bulk of the material . under precatalytic conditions , s interaction at r = 2.40 and 1 mo mo interactions at r = 2.74 , which correspond to the fitting parameters of mos3 . however , the mo l2,3-edge and s k - edge xas show that the material formed can not be considered as pure mos3 . in the s k - edge spectrum , the shoulder at 2470 ev characteristic of mo3s4 is more intense than in the as prepared material , which suggests a larger fraction of trinuclear mo3sx units as compared to the as prepared material . hence , the overall s k - edge first moment energy is shifted to lower energy by 0.15 ev , indicating a reduction of the disulfide units originally present in the as prepared material . the features characteristic of mos3 in the mo l3- and l2-edge spectra ( at 2528.7 and 2633.5 ev , respectively ) disappeared , and the first moment of these peaks are shifted to lower energy by ca . these data indicate that , when placed in an acidic media , the as prepared material is reduced , both from the sulfur and the molybdenum side , into an amorphous material where molybdenum and sulfur are in the + iv and ii oxidation state , respectively . although mos2 fits best with this description , the spectral features of the s k and mo l2,3-edge xas suggest that a fraction of mos3 and/or mo3s4 is still present . it is likely that the outer shell , which is accessible to the solvent , is converted into mos2 . we suggest that the reduction under acidic conditions of the disulfide ligands in mos3 produces sulfide ligands , which in turn displaces the oxygen ligands in moox to yield mos2 . we note that the strong peak at r = 3.15 corresponding to the mo mo interaction in microcrystalline mos2 is not observed , which can be explained by the amorphous nature of the mos2 layer . the disappearance of this peak , corresponding to mo mo interactions in mo3s4 and mos3 , can be interpreted as the formation of mos2 from mos3 and the corresponding disappearance of the bridging disulfide units in the bulk of the film . s distance at 0.3 v , which is longer than that in mo3s4 and matches the one found in mos2 . as the potential is decreased from 0.3 to 0.3 v , the s k - edge xanes spectra of the mosx film shows a progressive increase in their first moment energy ( figure 2b ) , which indicates an overall oxidation of the sulfur ligands . the difference in peak position between the highest and lowest potentials is only of 0.27 ev ( compared to the 1.1 ev shift observed in the sulfur oxidation state change from mos2 to mos3 ) , indicating a partial fraction of sulfur being oxidized in the sample . this small shift can be explained by a modification of the material only at the surface in contact with the electrolyte . since the mo mo interaction disappears in the mo k - edge exafs , the increase in the oxidation state of the sulfur ligands is likely due to the presence of terminal disulfide ligands ( s2 ) rather than bridging ones ( -: s2 ) . when the potential is decreased from 0.3 to 0.3 v , the mo l - edge first moment concomitantly shifts to lower energies ( 0.06 and 0.2 ev for the mo l3- and l2-edges , respectively ) , which indicates that the molybdenum centers are reduced to a lower oxidation state . as for the s k - edge xanes , the energy shift is limited as compared to what is observed for pure , bulk compounds ( 0.48 and 0.55 ev from mos3 to mos2 for the l3- and l2-edges , respectively ) . namely , the s k - edge and mo l - edge shifts are correlated to each other , and it suggests a change in oxidation state for the molybdenum centers located only at the solid liquid interface . in summary , the material s bulk structure at 0.3 v is modified from amorphous mos3 to amorphous mos2+x , in which bridging disulfides are absent and the predominant oxidation state of molybdenum is + iv . as mentioned before , the small changes observed at the s k- and mo l - edges strongly suggest that these changes occur at the solid liquid interface only , where the mosx material is in contact with the acidic media . at 0.3 v , this interface presents a significant amount of oxidized sulfur ligands ( as terminal disulfide units ) as well as reduced molybdenum ( formally mo(iii ) ) centers . these conclusions are in line with the previous xps and electrochemical quartz crystal microbalance ( eqcm ) analysis of the same film after the first reductive voltametric scan . according to these techniques , which probe the surface and mass composition of the film , the surface of the material was converted upon immersion in acid from mos3 to a material very similar to mos2 , with a slight excess of sulfur ligands . the material was thus formulated as mos2+x , which corresponds to a loss of sulfur ligands . we did not , however , observe any decrease in the sulfur x - ray fluorescence intensity when the potential was reduced from + 0.3 to 0.3 v. this indicates that the loss of sulfur occurs before setting the potential to the precatalytic conditions . scheme 1 shows the proposed phase changes of the mosx film under the various conditions studied in this paper , and proposes catalytic intermediates for the proton reduction reaction . the cat species is the one predominantly observed at 0.3 v , while cat it is important to note that the species observed spectroscopically at a given potential represent the kinetically most populated state under these conditions . therefore , in the case of a catalytic reaction , it also probes the rate - limiting step of the process , which involves the disappearance of the species observed spectroscopically . the cat h species is putative and has not been observed experimentally . in the mosx film , the interfacial mo centers are in the + iv oxidation state when the potential is poised to 0.3 v , and the sulfur is protonated due to the highly acidic conditions . since the potential is maintained at a quite high value , no hydrogen evolution is observed . when the potential is decreased , the driving force for the reduction of mos3 into cat increases , leading to the reduction of the molybdenum centers from + iv to + iii , the release of dihydrogen and the formation of terminal disulfide units . although the cat species may not be stable in the long - term under such acidic and reductive conditions ( the terminal disulfides are likely to be reduced and/or protonated ) , it is longer - lived than cat h , which is consumed as soon as it is reformed . consequently , the rate - limiting step under her conditions appears to be the protonation and reduction of the interfacial mo(s2 ) units formed together with the release of h2 . h is therefore favored ( and thus faster ) than it is to cat , which accumulates and can be observed spectroscopically . have also proposed that dimeric , bis--hydrosulfido mo2(iii ) complexes can eliminate h2 when reacting with alkenes or alkynes in 2 + 2 additions . this type of reaction parallels the her studied here , where electrons are provided by the electrode in place of the unsaturated olefins . by analogy with this addition elimination mechanism and on the basis of our own observations , we suggest that the last intermediate before the release of h2 is an mo(iii ) species with two terminal hydrosulfide ligands , generated by the facile addition of a hydrogen atom to the mo(s)(sh ) species in cat h . by combining in situ x - ray spectroscopies with electrochemistry , we have investigated the structural changes that occur in an mosx hydrogen - evolving electrocatalyst under functional conditions . the starting material before catalysis is identified as predominantly mos3 . when set in the acidic conditions used for catalysis , as the potential is decreased to induce the production of h2 , a progressive reduction of the molybdenum centers is observed concomitantly with the oxidation of the sulfur ligands . we therefore suggest that mo(iii ) units with terminal disulfide ligands ( s2 ) are transiently formed at the interface with the solution when h2 is released . as a corollary , the catalytic species prior to the release of h2 is very similar to mos2 , with terminal hydrosulfide ligands at the interface with the liquid . following this analysis , we propose that the rate - limiting step under her conditions is the protonation and reduction of interfacial mo(s2 ) sites . these results show experimental evidence for the direct involvement of a disulfide unit in molybdenum sulfide - based her and proposes new hypotheses for the mechanism of hydrogen evolution in these materials . further investigations are currently underway to understand in more detail the structure of these films under functional conditions and their reaction mechanism .

the reduction of protons into dihydrogen is important because of its potential use in a wide range of energy applications . the preparation of efficient and cheap catalysts for this reaction is one of the issues that need to be tackled to allow the widespread use of hydrogen as an energy carrier . in this paper , we report the study of an amorphous molybdenum sulfide ( mosx ) proton reducing electrocatalyst under functional conditions , using in situ x - ray absorption spectroscopy . we probed the local and electronic structures of both the molybdenum and sulfur elements for the as prepared material as well as the precatalytic and catalytic states . the as prepared material is very similar to mos3 and remains unmodified under functional conditions ( ph = 2 aqueous hno3 ) in the precatalytic state ( + 0.3 v vs rhe ) . in its catalytic state ( 0.3 v vs rhe ) , the film is reduced to an amorphous form of mos2 and shows spectroscopic features that indicate the presence of terminal disulfide units . these units are formed concomitantly with the release of hydrogen , and we suggest that the rate - limiting step of the her is the reduction and protonation of these disulfide units . these results show the implication of terminal disulfide chemical motifs into her driven by transition - metal sulfides and provide insight into their reaction mechanism .

Introduction Experimental Section Results Discussion Conclusion

among various solutions , solar light is very important , because of its accessibility in most parts of the globe and the power it delivers on the surface of the earth . in particular , some of us have reported on the preparation of electrodeposited amorphous mosx thin films that are catalytically active for the reduction of protons into hydrogen in acidic water . in the current paper , we investigate the structure of an amorphous , her - active mosx film observed under catalytic conditions using in situ x - ray spectroscopic techniques . an in situ electrochemical cell was developed and applied in the tender x - ray energy region ( 23 kev ) to the study of an mosx her catalyst under functional conditions . this method allowed us to probe the local and electronic structure of the molybdenum centers and sulfur ligands of the film under precatalytic and catalytic conditions . by comparing the data obtained on the catalytic film poised at constant potentials with those of reference compounds ( see table 1 ) , we propose a structural model for the catalyst film as prepared and under functional conditions in its precatalytic and catalytic states . the results provide evidence for the presence of terminal disulfide units in the mosx film and demonstrate their involvement in the catalytic cycle . although the role of disulfide units in the reduction of protons into dihydrogen by mosx materials has often been suggested , it is , to the best of our knowledge , the first time that this chemical motif is experimentally observed during the her . we also suggest on a spectroscopic basis that the rate - limiting step of the reaction is the reductive breaking of the s s bond . at beamline 7 - 3 , the intensity of the incident x - ray was monitored by an ar - filled ion chamber ( i0 ) in front of the sample . spectra were recorded on the dry electrodeposited mosx films at first and then in ph 2 nitric acid solution at 0.3 , 0.1 , 0.1 , and 0.3 v vs rhe . a polarization curve of the film on a rotating disk carbon electrode at ph = 2 is also shown in figure s2 . data reduction of the mo k - edge extended x - ray absorption fine structure ( exafs ) spectra was performed using exafspak ( drs . we use the notation precatalytic and catalytic states for the mosx film poised at 0.3 and 0.3 v in a nitric acid solution at ph = 2 , respectively . we therefore used x - ray absorption spectroscopy to study the structure of these films . the mo k - edge x - ray absorption near edge spectroscopy ( xanes ) spectra were recorded ex situ on the mosx film as prepared and in situ in the precatalytic and catalytic states ( figure 1a ) using a custom - made x - ray spectroelectrochemical cell described in the electrodeposition of mosx electrocatalyst section . the mo k - edge exafs spectra were recorded ex situ on the mosx film ( as prepared ) and in situ in the precatalytic and catalytic states ( figure 1c ) . when poised at 0.3 v , the short - distance peak ( r = 1.2 ) disappears , and the spectrum of the precatalytic state shows similarities with that of mos3 , with a high intensity peak at ca . in addition , the small intensity peak at r = 2.5 disappears , suggesting the breaking of the mo ( a ) mo k - edge xanes spectra of the mosx film as prepared and in the precatalytic and catalytic states . ( c ) mo k - edge fourier transform exafs ( k - weighted ) of the mosx film as prepared and in the precatalytic and catalytic states . this is compatible with the quite large debye waller factor ( 0.010 ) found for the mo the spectrum of mos2 ( which is microcrystalline ) shows two well - separated peaks at ca . the exafs curve fitting results of the mosx film as prepared and in the precatalytic and catalytic states are summarized in table 2 , and the k - space curves are displayed in figure s4 . for the as prepared film , an mo o interaction with a distance of r = 1.74 and an n value of 1.2 had to be included to model the peak at short r value . for the film in the precatalytic state ( poised at 0.3 v ) , the best fit was obtained with an mo s interaction at 2.40 with an n value close to 6 and an mo mo interaction at 2.74 with an n value of 1.1 . we therefore considered the film in the precatalytic state to be predominantly composed of mos3 . when the potential was lowered to 0.3 v ( catalytic state ) , the mo fitting the data with a single shell ( fit # 1 ) , yields an mo s interaction with r = 2.36 and n = 6 . this distance is slightly shorter ( 0.05 ) than that of the film poised at 0.3 v. we also considered the presence of an mo mo second shell ( fit # 2 ) . in this fit , the n value for the mo mo interaction dropped to 0.2 , while it did not show a significant improvement in the fit quality . figure 2a shows the s k - edge spectra collected on the mosx film as prepared and in the precatalytic and catalytic states as well as the reference compounds listed in table 1 . the s k - edge spectra of the mosx film poised at 0.3 , 0.1 , 0.1 , and 0.3 v are shown in figure 2b , and the corresponding first moment energies are listed in table 3 . in the as prepared mosx film , the sulfur k - edge main peak energy ( 2472.1 ev ) and shape are similar to that of mos3 , although a shoulder is present at low energy ( 2469.7 ev ) as in mo3s4 , but with a much smaller intensity . when the sample is set in a ph 2 nitric acid solution and the potential is poised to 0.3 v , the first moment energy of the main peak remains at 2472.0 ev , while the shoulder at lower energy ( 2469.8 ev ) increases and the one at higher energy ( 2472.4 ev ) decreases . when the potential is gradually decreased to 0.3 v , the first moment energy of the main peak shifts to a slightly higher energy ( from 2472.0 to 2472.3 ev ) , which indicates an overall oxidation of the sulfur ligands . figure 2c shows the molybdenum l3-edge spectra recorded on the mosx film as prepared and in the precatalytic and catalytic states as well as the reference compounds listed in table 1 . the spectrum recorded on the as prepared mosx film is very similar to that of mos3 . this is even more visible in the l2-edge spectrum ( see figure 2e ) , where the as prepared spectrum shows the double peak feature ( 2629.3 and 2629.6 ev ) that is also present in mos3 . the mo l3-edge spectra of the mosx film poised at 0.3 , 0.1 , 0.1 , and 0.3 v are shown in figure 2d , and the corresponding first moment energies ( as well as those of the mo l2-edge spectra ) are reported in table 3 . under precatalytic conditions ( ph 2 nitric acid , 0.3 v ) , the l3-edge peak first moment energy shifts to a lower energy by ca . when progressively decreasing the potential to 0.3 v , the first moment energy of the mo l3-edge peak shifts to lower values by ca . top : sulfur k - edge spectra of ( a ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 and mos3 reference spectra , and ( b ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . middle : molybdenum l3-edge spectra of ( c ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 , and mos3 and ( d ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . bottom : molybdenum l2-edge spectra of ( e ) the mosx film as prepared , in the precatalytic and catalytic states , together with mo3s4 , mos2 , and mos3 and ( f ) the mosx film poised at 0.3 ( black ) , 0.1 ( blue ) , 0.1 ( red ) , and 0.3 v ( green ) in nitric acid at ph = 2 . eventually , the final composition of the film was estimated from xps and electrochemical quartz crystal microbalance ( eqcm ) analysis as a mixture of mos2 and mos3 phases . in line with this first analysis , the x - ray spectroscopic data of the as prepared sample presented in this paper show features associated with several compounds , clearly indicating that the mosx electrocatalyst is a mixture of species . table 4 summarizes the spectroscopic information collected on the as prepared , precatalytic and catalytic states . it also indicates the structural and electronic changes observed when the potential is gradually decreased from 0.3 ( precatalytic state ) to 0.3 v ( catalytic state ) . in the mo k - edge exafs fourier transform spectrum of the as prepared sample , the peak observed at low r values ( 1.05 ) indicates the presence of an mo the mo o distance obtained from the fits ( r = ca . 1.75 ) is much shorter than usually observed in most moxoysz oxysulfide compounds ( typically 2.0 r 2.2 ) or in mo3s4(oh2)9 ( vide supra ) , and is longer than the mo the presence of oxygen has also been observed previously in the xps spectra recorded ex situ on the as prepared mosx film . given that the latter technique is only sensitive to the surface , we estimated that a layer of molybdenum oxide moox covers the bulk mos3 material in the as prepared film , as a result of air oxidation during transfer from the synthesis laboratory to the synchrotron facility . in mos3 , the molybdenum ion is described as mo(v ) in the hibble model and as mo(iv ) in the weber model . this deviation can be explained by the presence of the outer - shell moox layer in the as prepared film that decreases the average mo s bonds per mo center . the small intensity feature at 2470 ev that is characteristic of mo3s4 suggests the presence of similar chemical motifs in the as prepared material . when the mosx film is set in a nitric acid solution at ph = 2 and poised at 0.3 v , the mo o interaction in the mo k - edge exafs spectrum of the as prepared material disappears , indicating that the molybdenum oxide fraction is hydrolyzed . in the s k - edge spectrum , the shoulder at 2470 ev characteristic of mo3s4 is more intense than in the as prepared material , which suggests a larger fraction of trinuclear mo3sx units as compared to the as prepared material . hence , the overall s k - edge first moment energy is shifted to lower energy by 0.15 ev , indicating a reduction of the disulfide units originally present in the as prepared material . these data indicate that , when placed in an acidic media , the as prepared material is reduced , both from the sulfur and the molybdenum side , into an amorphous material where molybdenum and sulfur are in the + iv and ii oxidation state , respectively . we suggest that the reduction under acidic conditions of the disulfide ligands in mos3 produces sulfide ligands , which in turn displaces the oxygen ligands in moox to yield mos2 . the disappearance of this peak , corresponding to mo mo interactions in mo3s4 and mos3 , can be interpreted as the formation of mos2 from mos3 and the corresponding disappearance of the bridging disulfide units in the bulk of the film . as the potential is decreased from 0.3 to 0.3 v , the s k - edge xanes spectra of the mosx film shows a progressive increase in their first moment energy ( figure 2b ) , which indicates an overall oxidation of the sulfur ligands . since the mo mo interaction disappears in the mo k - edge exafs , the increase in the oxidation state of the sulfur ligands is likely due to the presence of terminal disulfide ligands ( s2 ) rather than bridging ones ( -: s2 ) . when the potential is decreased from 0.3 to 0.3 v , the mo l - edge first moment concomitantly shifts to lower energies ( 0.06 and 0.2 ev for the mo l3- and l2-edges , respectively ) , which indicates that the molybdenum centers are reduced to a lower oxidation state . namely , the s k - edge and mo l - edge shifts are correlated to each other , and it suggests a change in oxidation state for the molybdenum centers located only at the solid liquid interface . as mentioned before , the small changes observed at the s k- and mo l - edges strongly suggest that these changes occur at the solid liquid interface only , where the mosx material is in contact with the acidic media . at 0.3 v , this interface presents a significant amount of oxidized sulfur ligands ( as terminal disulfide units ) as well as reduced molybdenum ( formally mo(iii ) ) centers . according to these techniques , which probe the surface and mass composition of the film , the surface of the material was converted upon immersion in acid from mos3 to a material very similar to mos2 , with a slight excess of sulfur ligands . we did not , however , observe any decrease in the sulfur x - ray fluorescence intensity when the potential was reduced from + 0.3 to 0.3 v. this indicates that the loss of sulfur occurs before setting the potential to the precatalytic conditions . scheme 1 shows the proposed phase changes of the mosx film under the various conditions studied in this paper , and proposes catalytic intermediates for the proton reduction reaction . the cat species is the one predominantly observed at 0.3 v , while cat it is important to note that the species observed spectroscopically at a given potential represent the kinetically most populated state under these conditions . therefore , in the case of a catalytic reaction , it also probes the rate - limiting step of the process , which involves the disappearance of the species observed spectroscopically . in the mosx film , the interfacial mo centers are in the + iv oxidation state when the potential is poised to 0.3 v , and the sulfur is protonated due to the highly acidic conditions . when the potential is decreased , the driving force for the reduction of mos3 into cat increases , leading to the reduction of the molybdenum centers from + iv to + iii , the release of dihydrogen and the formation of terminal disulfide units . consequently , the rate - limiting step under her conditions appears to be the protonation and reduction of the interfacial mo(s2 ) units formed together with the release of h2 . by analogy with this addition elimination mechanism and on the basis of our own observations , we suggest that the last intermediate before the release of h2 is an mo(iii ) species with two terminal hydrosulfide ligands , generated by the facile addition of a hydrogen atom to the mo(s)(sh ) species in cat h . by combining in situ x - ray spectroscopies with electrochemistry , we have investigated the structural changes that occur in an mosx hydrogen - evolving electrocatalyst under functional conditions . when set in the acidic conditions used for catalysis , as the potential is decreased to induce the production of h2 , a progressive reduction of the molybdenum centers is observed concomitantly with the oxidation of the sulfur ligands . as a corollary , the catalytic species prior to the release of h2 is very similar to mos2 , with terminal hydrosulfide ligands at the interface with the liquid . following this analysis , we propose that the rate - limiting step under her conditions is the protonation and reduction of interfacial mo(s2 ) sites . these results show experimental evidence for the direct involvement of a disulfide unit in molybdenum sulfide - based her and proposes new hypotheses for the mechanism of hydrogen evolution in these materials . further investigations are currently underway to understand in more detail the structure of these films under functional conditions and their reaction mechanism .

atherothrombosis subsequent to plaque rupture or erosion with prominent features of inflammation constitutes the underlying pathophysiology in patients with acute coronary syndromes ( acs ) . however , its triggers remain poorly understood and not all rupture - prone plaques culminate in atherothrombosis . we have previously identified monocytes / macrophages as the most abundant inflammatory cell type of innate immunity in coronary atherothrombosis , co - expressing toll - like receptor ( tlr)-4 . although less abundant in number , t cells orchestrate the antigen - specific ( adaptive ) immune response in atherogenesis with prominent effects mediated by distinct t cell subsets throughout the stages of atherosclerosis , including acs . among t cell subsets , regulatory t cells ( treg ) exert atheroprotective effects and constitute an inherent anti - inflammatory component of adaptive immunity . identifying a novel mechanistic link between treg immunity and lipid metabolism , we recently demonstrated that selective depletion of treg ( foxp3 ) impacted on lipid metabolism mediating hypercholesterolaemia and increased atherosclerotic lesions . furthermore , we and others showed that atheroprotective treg can be induced in response to immunization with antigen specificity for the apolipoprotein b-100 peptide used in the vaccine . in humans , treg were found in increased numbers in lipid - rich , advanced plaques , whereas reduced numbers of circulating treg were found in patients with acs . from a clinical perspective , treg may be an attractive therapeutic target in atherosclerosis due to their anti - inflammatory effects . each cell expresses several copies of a single antigen receptor with a unique antigen - binding site . the great variety of antigen specificities in the t cell receptor ( tcr ) repertoire is generated in the thymus by random recombination of separate inherited tcr / gene segments termed v(d)j which encode the variable parts of the heterodimeric receptor . t cell receptor diversity is confined to the complementarity determining regions ( cdrs ) as the binding site for a peptide presented on the major histocompatibility complex by an antigen - presenting cell ( i.e. dendritic cell ) . recent advances in multiplex assay technology made it possible to measure usage frequencies of gene segments v(d)j and enable detection and tracking of specific tcr sequences expressed by clonally restricted t cells . compared with spectratyping which employs only a limited set of v and j primers , this method enables a more comprehensive analysis that avoids bias due to transcriptional regulation . evidence for an antigen - specific local immune response carried by t cells in unstable atherosclerotic plaque mandates clonal restriction of t cells when compared with peripheral blood . initial reports on t cells isolated from human atherosclerotic plaques showed a high tcr diversity indicating a polyclonal origin of t cells . in circulating cd4 cd28 t cells from patients with unstable angina compared with patients with stable coronary artery disease , clonal restriction of t cells clonal restriction of t cells was demonstrated using spectratyping of atherectomy specimens from coronary plaque compared with peripheral blood mononuclear cells ( pbmcs ) from patients with acs and only to a lesser extent in patients with stable coronary artery disease . similarly , clonal restriction was demonstrated when comparing carotid plaques with pbmc from asymptomatic and symptomatic patients with > 90% carotid artery stenosis . however , so far t cell diversity in thrombi of acs patients has not been investigated . the aims of this study , therefore , were ( i ) to compare treg counts in coronary thrombi with pbmc from patients with acs , ( ii ) to compare tcr diversity in coronary thrombi with pbmc from patients with acs , ( iii ) to compare tcr diversity in pbmc from age - matched healthy subjects with patients with acs . sixteen patients referred to the university hospital zurich , switzerland , for coronary angiography with the diagnosis of acs between 08/2010 and 11/2011 were enrolled as part of a larger cohort study ( spum - acs , clinical trial number nct01000701 ) . all patients aged 18 years and older presenting within 72 h after pain onset with the main diagnosis of st - elevation myocardial infarction ( stemi ) or non - st elevation myocardial infarction ( nstemi ) were included in the study . patients had symptoms compatible with angina pectoris ( chest pain , dyspnoea ) and at least one of the following inclusion criteria : ( i ) persistent st - segment elevation or depression , t inversion or dynamic ecg changes , new lbbb ; ( ii ) evidence of positive troponin based on high sensitive tnt measurement ( 14 ng / l ) with rise and/or fall in serial tnt levels . exclusion criteria comprised documented active autoimmune disease or neoplasm ; stent thrombosis ; inability to comprehend study , less than 1 year of life - expectancy ( for non - cardiac reasons ) . the gensini score was calculated by two interventional cardiology fellows ( inter - observer variability = 17.4 ) for each of the 16 patients using the formula severity score segment location multiplying factor collateral adjustment factor to determine the extent and severity of coronary artery disease in the epicardial coronary arteries . in seven patients , the gensini score differed by more than 10 points based on a different interpretation of collateral flow / total occlusion and an expert opinion was consulted from a third experienced senior interventional cardiologist . samples from 16 healthy subjects were obtained from a larger cohort study ( susa study , afssaps clinical trial number 2010- a00428 - 31 , immunid ) that were recruited between between 07/2010 and 12/2011 at optimed clinical research , lyon , france . individuals were included following a detailed medical history , physical exam with vital signs , and blood draw . exclusion criteria comprised autoimmune disease or neoplasm , inability to comprehend study , treatment with corticosteroids or other immunomodulatory therapy , or vaccination in the three preceding months . all individuals were older than 18 years of age with birth control in place for at least 2 months prior to inclusion in women of childbearing age in the absence of pregnancy or breast feeding . informed consent was obtained from all individuals with the approval by the kantonale ethik - kommission zurich , switzerland ( ek-1688 ) , for patients with acs and the comit de protection des personnes cpp sud - est iii , france ( numero eudract : ol039/susa ) , for healthy subjects . individual estimated glomerular filtration rate was calculated using the ckd - epi formula based on serum creatinine , gender , and ethnicity . based on the published trials , coronary thrombi were aspirated using an export catheter ( medtronic , tolochenaz , switzerland ) at the site of coronary occlusion from patients with acs undergoing primary percutaneous coronary intervention ( pci ) and were immediately immersed in phosphate - buffered saline ( pbs)-containing vials . peripheral blood was sampled from the inguinal arterial sheath from the same patient and stored in citrate vials . thrombi and the corresponding peripheral blood were treated with 50 l tissue plasminogen activator ( 0.001% actilyse ; boehringer ingelheim pharma gmbh & co. , ingelheim , germany ) to remove fibrin in 1 ml 1% accutase ( paa laboratories , pasching , austria ) , and rotated for 2 h at 37c . thrombi and blood were then gently dissociated through a 40 m pore - size cell strainer ( bd falcon ) with a rubber syringe plunger and rinsing with ice - cold pbs . half of the cell suspensions were separated for ficoll - paque centrifugation and subsequent immuntracker analysis ( immunid , grenoble , france ) , whereas remaining cells were analysed by flow cytometry . in healthy subjects , pbmcs were separated from whole blood ( 5 ml edta ) with a density gradient tube ( uni - sep , novamed , jerusalem , israel ) . peripheral blood mononuclear cells were prepared and stabilized in easy'id stabilization solution provided by immunid , grenoble following a standard operating procedure . human tcr diversity was measured using immuntracker tests ( immunid technologies , grenoble , france ) and performed as described . genomic dna was extracted using standard techniques and multi - n - plex pcr was performed using an upstream primer specic for all functional members of a given t cell receptor v segment and a downstream primer specic for a given t cell receptor j segment ( international immunogenetics information system , www.imgt.org ) encoding for the cdr 3 in the human tcr chain . this assay allows the simultaneous exhaustive detection of v(d)j rearrangements in the same reaction expressed as percentage based on the detected v(d)j combinatorial diversity divided by the expected maximal diversity ( 276 gene segments ) which corresponds to the theoretical combinatorial diversity obtained with all rearrangements of all functional v(d)j genes within the genome . each vx j1 , j2 , j3 , j4 , jn product was separated as a function of its size . constel'id software ( immunid technologies ) was used for further analytical studies , including generation of three - dimensional repertoire illustration . numeration and diversity of t cells is presented together through ndl scoring which enables to determine a correlation between lymphocyte counts and t cell diversity . a decreased percentage ( i.e. a decreased tcr diversity ) defines a state of divpenia ( www.divpenia.com ) . normal values for tcr -chain combinatorial diversity were defined based on a cohort of 16 healthy volunteers extracted from a larger study ( susa study , afssaps clinical trial number 2010- a00428 - 31 , immunid ; age range , 42.874.9 years ; 6 females and 10 males ) . for flow cytometry , thrombus and peripheral blood - derived cell suspensions obtained from patients with acs were centrifuged at 200 g or 10 min and resuspended in 1 ml of ice - cold facs buffer ( pbs , 1% foetal calf serum , 0.05% edta ) with fcr block ( human trustain fcx , biolegend , san diego , ca , usa ) . cells were labelled for 1 h at 4c with monoclonal antibodies for extracellular staining of cd3 , cd4 , cd8 , cd28 , and a three - color reagent ( cd4 , cd25 , cd127 ) for identification of treg ( all from bd biosciences , san jose , ca , usa ) and analysed on a facscalibur ( bd biosciences ) . subpopulation analyses were performed using ssc / fsc scatters , differentiating lymphocyte , monocytes , and granulocyte gates ( quadrant analysis ) . mouse igg1 was used as isotype control and the proportion of positive cells per lymphocyte gate were determined . for comparisons of continuous and categorical data , we used the non - parametric mann intra - individual comparisons of distributions of combinatorial diversity and percentages of cell counts are the result of wilcoxon signed rank test for paired data . unless otherwise indicated , data are shown as mean sem or relative and absolute frequencies . pearson 's correlation coefficient was determined for the gensini score ( mean ) and tcr diversity in pbmc and thrombi , respectively . sixteen patients referred to the university hospital zurich , switzerland , for coronary angiography with the diagnosis of acs between 08/2010 and 11/2011 were enrolled as part of a larger cohort study ( spum - acs , clinical trial number nct01000701 ) . all patients aged 18 years and older presenting within 72 h after pain onset with the main diagnosis of st - elevation myocardial infarction ( stemi ) or non - st elevation myocardial infarction ( nstemi ) were included in the study . patients had symptoms compatible with angina pectoris ( chest pain , dyspnoea ) and at least one of the following inclusion criteria : ( i ) persistent st - segment elevation or depression , t inversion or dynamic ecg changes , new lbbb ; ( ii ) evidence of positive troponin based on high sensitive tnt measurement ( 14 ng / l ) with rise and/or fall in serial tnt levels . exclusion criteria comprised documented active autoimmune disease or neoplasm ; stent thrombosis ; inability to comprehend study , less than 1 year of life - expectancy ( for non - cardiac reasons ) . the gensini score was calculated by two interventional cardiology fellows ( inter - observer variability = 17.4 ) for each of the 16 patients using the formula severity score segment location multiplying factor collateral adjustment factor to determine the extent and severity of coronary artery disease in the epicardial coronary arteries . in seven patients , the gensini score differed by more than 10 points based on a different interpretation of collateral flow / total occlusion and an expert opinion was consulted from a third experienced senior interventional cardiologist . samples from 16 healthy subjects were obtained from a larger cohort study ( susa study , afssaps clinical trial number 2010- a00428 - 31 , immunid ) that were recruited between between 07/2010 and 12/2011 at optimed clinical research , lyon , france . individuals were included following a detailed medical history , physical exam with vital signs , and blood draw . exclusion criteria comprised autoimmune disease or neoplasm , inability to comprehend study , treatment with corticosteroids or other immunomodulatory therapy , or vaccination in the three preceding months . all individuals were older than 18 years of age with birth control in place for at least 2 months prior to inclusion in women of childbearing age in the absence of pregnancy or breast feeding . informed consent was obtained from all individuals with the approval by the kantonale ethik - kommission zurich , switzerland ( ek-1688 ) , for patients with acs and the comit de protection des personnes cpp sud - est iii , france ( numero eudract : ol039/susa ) , for healthy subjects . individual estimated glomerular filtration rate was calculated using the ckd - epi formula based on serum creatinine , gender , and ethnicity . based on the published trials , coronary thrombi were aspirated using an export catheter ( medtronic , tolochenaz , switzerland ) at the site of coronary occlusion from patients with acs undergoing primary percutaneous coronary intervention ( pci ) and were immediately immersed in phosphate - buffered saline ( pbs)-containing vials . peripheral blood was sampled from the inguinal arterial sheath from the same patient and stored in citrate vials . thrombi and the corresponding peripheral blood were treated with 50 l tissue plasminogen activator ( 0.001% actilyse ; boehringer ingelheim pharma gmbh & co. , ingelheim , germany ) to remove fibrin in 1 ml 1% accutase ( paa laboratories , pasching , austria ) , and rotated for 2 h at 37c . thrombi and blood were then gently dissociated through a 40 m pore - size cell strainer ( bd falcon ) with a rubber syringe plunger and rinsing with ice - cold pbs . half of the cell suspensions were separated for ficoll - paque centrifugation and subsequent immuntracker analysis ( immunid , grenoble , france ) , whereas remaining cells were analysed by flow cytometry . in healthy subjects , pbmcs were separated from whole blood ( 5 ml edta ) with a density gradient tube ( uni - sep , novamed , jerusalem , israel ) . peripheral blood mononuclear cells were prepared and stabilized in easy'id stabilization solution provided by immunid , grenoble following a standard operating procedure . human tcr diversity was measured using immuntracker tests ( immunid technologies , grenoble , france ) and performed as described . peripheral blood mononuclear cells and thrombus samples were frozen and analysed by immunid laboratories . genomic dna was extracted using standard techniques and multi - n - plex pcr was performed using an upstream primer specic for all functional members of a given t cell receptor v segment and a downstream primer specic for a given t cell receptor j segment ( international immunogenetics information system , www.imgt.org ) encoding for the cdr 3 in the human tcr chain . this assay allows the simultaneous exhaustive detection of v(d)j rearrangements in the same reaction expressed as percentage based on the detected v(d)j combinatorial diversity divided by the expected maximal diversity ( 276 gene segments ) which corresponds to the theoretical combinatorial diversity obtained with all rearrangements of all functional v(d)j genes within the genome . each vx j1 , j2 , j3 , j4 , jn product was separated as a function of its size . constel'id software ( immunid technologies ) was used for further analytical studies , including generation of three - dimensional repertoire illustration . numeration and diversity of t cells is presented together through ndl scoring which enables to determine a correlation between lymphocyte counts and t cell diversity . a decreased percentage ( i.e. a decreased tcr diversity ) defines a state of divpenia ( www.divpenia.com ) . normal values for tcr -chain combinatorial diversity were defined based on a cohort of 16 healthy volunteers extracted from a larger study ( susa study , afssaps clinical trial number 2010- a00428 - 31 , immunid ; age range , 42.874.9 years ; 6 females and 10 males ) . for flow cytometry , thrombus and peripheral blood - derived cell suspensions obtained from patients with acs were centrifuged at 200 g or 10 min and resuspended in 1 ml of ice - cold facs buffer ( pbs , 1% foetal calf serum , 0.05% edta ) with fcr block ( human trustain fcx , biolegend , san diego , ca , usa ) . cells were labelled for 1 h at 4c with monoclonal antibodies for extracellular staining of cd3 , cd4 , cd8 , cd28 , and a three - color reagent ( cd4 , cd25 , cd127 ) for identification of treg ( all from bd biosciences , san jose , ca , usa ) and analysed on a facscalibur ( bd biosciences ) . subpopulation analyses were performed using ssc / fsc scatters , differentiating lymphocyte , monocytes , and granulocyte gates ( quadrant analysis ) . mouse igg1 was used as isotype control and the proportion of positive cells per lymphocyte gate were determined . for comparisons of continuous and categorical data , we used the non - parametric mann intra - individual comparisons of distributions of combinatorial diversity and percentages of cell counts are the result of wilcoxon signed rank test for paired data . unless otherwise indicated , data are shown as mean sem or relative and absolute frequencies . pearson 's correlation coefficient was determined for the gensini score ( mean ) and tcr diversity in pbmc and thrombi , respectively . a two - sided p - value of < 0.05 was considered significant . pairs of coronary thrombi and peripheral blood were obtained from 16 patients with acs using an aspiration catheter as part of the pci for a native coronary culprit lesion . among these , 12 patients were diagnosed with stemi , 4 patients with nstemi , 11 patients were symptomatic for < 24 h , 4 patients had symptoms for 2448 h and 1 patient for 4872 h. for the evaluation of tcr chain diversity in peripheral blood , 16 patients with acs were compared with 16 healthy age - matched subjects . the clinical , demographic , and laboratory characteristics of patients with acs and matched healthy subjects are summarized in table 1 . compared with healthy subjects , patients with acs had increased leucocyte and platelet counts , elevated glucose concentration in peripheral blood , presence of coronary artery disease , cardiovascular risk factors , and received medications . table 1clinical , demographic , and laboratory characteristics of patients and healthy subjectsacshealthyp - valueage ( years)58.9 2.358.7 2.3n.s.male gender ( % ) 15 ( 94)10 ( 63)n.scaucasian ethnicity ( % ) 16 ( 100)16 ( 100)n.s.leucocyte count ( 10/l)11.6 1.55.8 0.3p 0.001lymphocyte count ( 10/l)3.2 1.21.6 0.08n.s.erythrocytes ( 10/l)4.3 0.24.5 0.1n.s.platelets ( 10/l)279 25219 8p < 0.05total cholesterol ( mmol / l)5.1 0.45.6 0.3n.s.baseline glucose ( mmol / l)9.0 1.75.2 0.08p 0.01serum creatinine ( mol / l)79.9 6.372.1 2.7n.s.egfr ( ml / min)90.1 4.992.3 2.4n.s.bmi ( kg / m)28.4 2.024.6 1.1n.s.gensini score39.7 5.70p 0.001cardiovascular risk factorshypercholesterolaemia8 ( 50)0p 0.01hypertension ( % ) 4 ( 25)0p < 0.05diabetes ( % ) 2 ( 13)0n.s.smoking ( current , % ) 7 ( 44)0p 0.01previous medical historyprevious mi3 ( 19)0n.s.previous pci2 ( 13)0n.s.medicationsaspirin ( % ) 16 ( 100)0p 0.001statins ( % ) 4 ( 25)0p < 0.05ace - i / arb ( % ) 2 ( 13)0n.s.-blockers ( % ) 3 ( 19)0n.s.nitrates ( % ) 15 ( 94)0p 0.001calcium antagonists ( % ) 00diuretics ( % ) 1 ( 6)0n.s.values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively.n = 1216 ; n.s . non - significant . ace - i / arb , angiotensin converting enzyme - inhibitor / angiotensin receptor blocker ; aspirin comprises oral and intravenous formulation ; bmi , body mass index ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; pci , percutaneous coronary intervention . clinical , demographic , and laboratory characteristics of patients and healthy subjects values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . ace - i / arb , angiotensin converting enzyme - inhibitor / angiotensin receptor blocker ; aspirin comprises oral and intravenous formulation ; bmi , body mass index ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; pci , percutaneous coronary intervention . to evaluate t cell subset distribution , flow cytometry was performed for phenotypic characterization of coronary thrombi compared with pbmc from patients with acs . overall cd3 t cell content was unaltered and no difference was detected for t helper cells ( cd4 cd3 cd8 ) , but for a trend to numerical reduction vs. peripheral blood ( figure 1a and b ) . in contrast , treg ( cd4 cd25 cd127 ) were significantly increased by 2.2-fold in coronary thrombi ( figure 1c ) . cytotoxic t cells ( cd8 cd3 cd4 ) , cd4 cd3 cd28 t cells and double negative ( cd4 cd8 cd3 ) t cells were found in similar numbers in coronary thrombi and pbmc ( figure 1d f ) . supplementary material online , figures s1s6 show representative graphs on how facs analysis for individual cell subsets was performed . supplementary material online , figure s7 shows a representative immunostaining of cd3 t cells . figure 1treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . double - staining for t cell subtypes ( cd3 with cd8 or cd4 , respectively ) . the bottom and top of the box represent the first and third quartiles ( q1 , q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3q1 ) from the box , with individual outliers shown beyond the whisker ; n = 912 ; * p = 0.0098 . treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . double - staining for t cell subtypes ( cd3 with cd8 or cd4 , respectively ) . the bottom and top of the box represent the first and third quartiles ( q1 , q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3q1 ) from the box , with individual outliers shown beyond the whisker ; n = 912 ; * p = 0.0098 . pairwise intra - individual comparisons of coronary thrombi demonstrated a markedly reduced tcr diversity in coronary thrombi vs. pbmc ( reduction by 0.28-fold ) , shown in figure 2a c . among v(d)j gene segments identified in coronary thrombi from 16 patients with acs , 8 rearrangements were found in common in at least 6 thrombi from individual patients ( table 2 ) . these gene rearrangements are vbeta18-j2.3 , which is the most frequent ( 8/16 ) ; vbeta05-j2.7 and vbeta25-j2.6 , both found in common in 7/16 samples ; vbeta27-j2.7 , vbeta24-j2.3 , vbeta24-j2.5 , vbeta15-j2.4 , and vbeta19-j2.5 were found in common in 6/16 samples ( imgt nomenclature ) . in turn , in peripheral blood from patients with acs , vbeta05-j2.7 was the most frequently detected gene rearrangement ( 13/16 ) . interestingly , vbeta05-j2.7 was less common in coronary thrombi compared with pbmc ( table 2 ) . t cell receptor diversity was only weakly correlated with the extent and severity of epicardial coronary artery disease as assessed by the gensini score ( supplementary material online , figure s8 ) . pearson 's correlation coefficient for the comparison of the gensini score ( mean ) with tcr diversity in pbmc was 0.418 and 0.058 for the comparison of the score with tcr diversity in coronary thrombus , respectively . table 2 t cell receptor gene segment rearrangements in patients with an acute coronary syndromev - j rearrangementpbmcthrombusp - valuevbeta18-j2.37 ( 44)8 ( 50)n.s.vbeta05-j2.713 ( 81)7 ( 44)p = 0.028vbeta25-j2.63 ( 19)7 ( 44)n.s.vbeta27-j2.711 ( 69)6 ( 38)n.s.vbeta24-j2.310 ( 63)6 ( 38)n.s.vbeta24-j2.58 ( 50)6 ( 38)n.s.vbeta15-j2.46 ( 38)6 ( 38)n.s.vbeta19-j2.53 ( 19)6 ( 38)n.s.vbeta04-j2.39 ( 56)5 ( 31)n.s.vbeta05-j2.58 ( 50)5 ( 31)n.s.vbeta24-j2.47 ( 44)5 ( 31)n.s.vbeta20-j2.63 ( 19)5 ( 31)n.s.all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . figure 2reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . representative 3-d plots of human tcr chain diversity in an individual patient with acs derived from pbmc ( b ) and coronary thrombus ( c ) . t cell receptor gene segment rearrangements in patients with an acute coronary syndrome all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . representative 3-d plots of human tcr chain diversity in an individual patient with acs derived from pbmc ( b ) and coronary thrombus ( c ) . t cell receptor diversity was measured using genomic dna isolated from pbmcs from 16 healthy subjects and pbmcs from 16 patients with acs . compared with matched healthy subjects , patients with acs had a profoundly reduced tcr diversity in peripheral blood ( reduction by 0.36-fold ) , shown in figure 3a c . importantly , reduced tcr diversity did not correlate with overall lymphocyte cell number in peripheral blood from patients with acs and healthy subjects ( figure 3d ) . figure 3reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . human tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) measured in pbmcs from healthy subjects and patients with acs ( a ) . the bottom and top of the box are the first and third quartiles ( q1,q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3 q1 ) from the box . representative 3-d plots of htrb diversity derived from pbmc from a healthy subject ( b ) and a patient with acs ( c ) . graph linking peripheral lymphocyte count to human tcr diversity in the human tcr chain ( d ) . pbmc from healthy subjects ( open squares ) and patients with acs ( grey circles ) . reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . human tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) measured in pbmcs from healthy subjects and patients with acs ( a ) . the bottom and top of the box are the first and third quartiles ( q1,q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3 q1 ) from the box . representative 3-d plots of htrb diversity derived from pbmc from a healthy subject ( b ) and a patient with acs ( c ) . graph linking peripheral lymphocyte count to human tcr diversity in the human tcr chain ( d ) . pbmc from healthy subjects ( open squares ) and patients with acs ( grey circles ) . pairs of coronary thrombi and peripheral blood were obtained from 16 patients with acs using an aspiration catheter as part of the pci for a native coronary culprit lesion . among these , 12 patients were diagnosed with stemi , 4 patients with nstemi , 11 patients were symptomatic for < 24 h , 4 patients had symptoms for 2448 h and 1 patient for 4872 h. for the evaluation of tcr chain diversity in peripheral blood , 16 patients with acs were compared with 16 healthy age - matched subjects . the clinical , demographic , and laboratory characteristics of patients with acs and matched healthy subjects are summarized in table 1 . compared with healthy subjects , patients with acs had increased leucocyte and platelet counts , elevated glucose concentration in peripheral blood , presence of coronary artery disease , cardiovascular risk factors , and received medications . table 1clinical , demographic , and laboratory characteristics of patients and healthy subjectsacshealthyp - valueage ( years)58.9 2.358.7 2.3n.s.male gender ( % ) 15 ( 94)10 ( 63)n.scaucasian ethnicity ( % ) 16 ( 100)16 ( 100)n.s.leucocyte count ( 10/l)11.6 1.55.8 0.3p 0.001lymphocyte count ( 10/l)3.2 1.21.6 0.08n.s.erythrocytes ( 10/l)4.3 0.24.5 0.1n.s.platelets ( 10/l)279 25219 8p < 0.05total cholesterol ( mmol / l)5.1 0.45.6 0.3n.s.baseline glucose ( mmol / l)9.0 1.75.2 0.08p 0.01serum creatinine ( mol / l)79.9 6.372.1 2.7n.s.egfr ( ml / min)90.1 4.992.3 2.4n.s.bmi ( kg / m)28.4 2.024.6 1.1n.s.gensini score39.7 5.70p 0.001cardiovascular risk factorshypercholesterolaemia8 ( 50)0p 0.01hypertension ( % ) 4 ( 25)0p < 0.05diabetes ( % ) 2 ( 13)0n.s.smoking ( current , % ) 7 ( 44)0p 0.01previous medical historyprevious mi3 ( 19)0n.s.previous pci2 ( 13)0n.s.medicationsaspirin ( % ) 16 ( 100)0p 0.001statins ( % ) 4 ( 25)0p < 0.05ace - i / arb ( % ) 2 ( 13)0n.s.-blockers ( % ) 3 ( 19)0n.s.nitrates ( % ) 15 ( 94)0p 0.001calcium antagonists ( % ) 00diuretics ( % ) 1 ( 6)0n.s.values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively.n = 1216 ; n.s . non - significant . ace - i / arb , angiotensin converting enzyme - inhibitor / angiotensin receptor blocker ; aspirin comprises oral and intravenous formulation ; bmi , body mass index ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; pci , percutaneous coronary intervention . clinical , demographic , and laboratory characteristics of patients and healthy subjects values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . ace - i / arb , angiotensin converting enzyme - inhibitor / angiotensin receptor blocker ; aspirin comprises oral and intravenous formulation ; bmi , body mass index ; egfr , estimated glomerular filtration rate ; mi , myocardial infarction ; pci , percutaneous coronary intervention . to evaluate t cell subset distribution , flow cytometry was performed for phenotypic characterization of coronary thrombi compared with pbmc from patients with acs . overall cd3 t cell content was unaltered and no difference was detected for t helper cells ( cd4 cd3 cd8 ) , but for a trend to numerical reduction vs. peripheral blood ( figure 1a and b ) . in contrast , treg ( cd4 cd25 cd127 ) were significantly increased by 2.2-fold in coronary thrombi ( figure 1c ) . cytotoxic t cells ( cd8 cd3 cd4 ) , cd4 cd3 cd28 t cells and double negative ( cd4 cd8 cd3 ) t cells were found in similar numbers in coronary thrombi and pbmc ( figure 1d f ) . supplementary material online , figures s1s6 show representative graphs on how facs analysis for individual cell subsets was performed . supplementary material online , figure s7 shows a representative immunostaining of cd3 t cells . figure 1treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . double - staining for t cell subtypes ( cd3 with cd8 or cd4 , respectively ) . the bottom and top of the box represent the first and third quartiles ( q1 , q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3q1 ) from the box , with individual outliers shown beyond the whisker ; n = 912 ; * p = 0.0098 . treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . double - staining for t cell subtypes ( cd3 with cd8 or cd4 , respectively ) . the bottom and top of the box represent the first and third quartiles ( q1 , q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3q1 ) from the box , with individual outliers shown beyond the whisker ; n = 912 ; * p = 0.0098 . pairwise intra - individual comparisons of coronary thrombi demonstrated a markedly reduced tcr diversity in coronary thrombi vs. pbmc ( reduction by 0.28-fold ) , shown in figure 2a c . among v(d)j gene segments identified in coronary thrombi from 16 patients with acs , 8 rearrangements were found in common in at least 6 thrombi from individual patients ( table 2 ) . these gene rearrangements are vbeta18-j2.3 , which is the most frequent ( 8/16 ) ; vbeta05-j2.7 and vbeta25-j2.6 , both found in common in 7/16 samples ; vbeta27-j2.7 , vbeta24-j2.3 , vbeta24-j2.5 , vbeta15-j2.4 , and vbeta19-j2.5 were found in common in 6/16 samples ( imgt nomenclature ) . in turn , in peripheral blood from patients with acs , vbeta05-j2.7 was the most frequently detected gene rearrangement ( 13/16 ) . interestingly , vbeta05-j2.7 was less common in coronary thrombi compared with pbmc ( table 2 ) . t cell receptor diversity was only weakly correlated with the extent and severity of epicardial coronary artery disease as assessed by the gensini score ( supplementary material online , figure s8 ) . pearson 's correlation coefficient for the comparison of the gensini score ( mean ) with tcr diversity in pbmc was 0.418 and 0.058 for the comparison of the score with tcr diversity in coronary thrombus , respectively . table 2 t cell receptor gene segment rearrangements in patients with an acute coronary syndromev - j rearrangementpbmcthrombusp - valuevbeta18-j2.37 ( 44)8 ( 50)n.s.vbeta05-j2.713 ( 81)7 ( 44)p = 0.028vbeta25-j2.63 ( 19)7 ( 44)n.s.vbeta27-j2.711 ( 69)6 ( 38)n.s.vbeta24-j2.310 ( 63)6 ( 38)n.s.vbeta24-j2.58 ( 50)6 ( 38)n.s.vbeta15-j2.46 ( 38)6 ( 38)n.s.vbeta19-j2.53 ( 19)6 ( 38)n.s.vbeta04-j2.39 ( 56)5 ( 31)n.s.vbeta05-j2.58 ( 50)5 ( 31)n.s.vbeta24-j2.47 ( 44)5 ( 31)n.s.vbeta20-j2.63 ( 19)5 ( 31)n.s.all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . n.s . figure 2reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . representative 3-d plots of human tcr chain diversity in an individual patient with acs derived from pbmc ( b ) and coronary thrombus ( c ) . t cell receptor gene segment rearrangements in patients with an acute coronary syndrome all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . representative 3-d plots of human tcr chain diversity in an individual patient with acs derived from pbmc ( b ) and coronary thrombus ( c ) . t cell receptor diversity was measured using genomic dna isolated from pbmcs from 16 healthy subjects and pbmcs from 16 patients with acs . compared with matched healthy subjects , patients with acs had a profoundly reduced tcr diversity in peripheral blood ( reduction by 0.36-fold ) , shown in figure 3a c . importantly , reduced tcr diversity did not correlate with overall lymphocyte cell number in peripheral blood from patients with acs and healthy subjects ( figure 3d ) . figure 3reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . human tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) measured in pbmcs from healthy subjects and patients with acs ( a ) . the bottom and top of the box are the first and third quartiles ( q1,q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3 q1 ) from the box . representative 3-d plots of htrb diversity derived from pbmc from a healthy subject ( b ) and a patient with acs ( c ) . graph linking peripheral lymphocyte count to human tcr diversity in the human tcr chain ( d ) . pbmc from healthy subjects ( open squares ) and patients with acs ( grey circles ) . reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . human tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) measured in pbmcs from healthy subjects and patients with acs ( a ) . the bottom and top of the box are the first and third quartiles ( q1,q3 ) , and the band inside the box is the median . the whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range ( iqr = q3 q1 ) from the box . representative 3-d plots of htrb diversity derived from pbmc from a healthy subject ( b ) and a patient with acs ( c ) . graph linking peripheral lymphocyte count to human tcr diversity in the human tcr chain ( d ) . pbmc from healthy subjects ( open squares ) and patients with acs ( grey circles ) . the present study confers the following key findings : first , we identified treg as the major t cell subset in aspirated coronary thrombus adjacent to the culprit lesion in patients with acs . second , we found a restricted tcr diversity within coronary thrombi compared with peripheral blood of patients with acs . third , we show a markedly reduced tcr diversity also in peripheral blood of patients with acs compared with healthy age - matched subjects . we previously showed an increased immune response at the site of coronary occlusion in patients with acs compared with peripheral blood , demonstrating elevated levels of serum amyloid a , interleukin ( il)-6 , myeloid - related protein 8/14 , and expression of tlr-4 on monocytes as part of innate immunity . analysis of adaptive immunity carried by t cell subsets in patients with acs was performed by comparing coronary thrombi with peripheral blood intra - individually and peripheral blood from healthy subjects . treg were reported in increased numbers in lipid - rich , advanced plaques , whereas reduced numbers of circulating treg were found in patients with an acs . this decrease in the circulating treg pool is currently unclear as it may be due to either a global treg defect or increased redistribution between the blood and local sites of inflammation . interestingly , the latter study found reduced circulating treg in patients with unstable angina , but increased circulating treg in patients with acute myocardial infarction ( ami ) . this may be attributable to insufficient treg recruitment to the site of the culprit lesion in patients with ami unlike in unstable angina where sufficient influx of treg is maintained to control the inflammatory reaction in the lesion . in contrast , we herein demonstrated a 2.2-fold increase in treg counts in coronary thrombus adjacent to the ruptured plaque compared with peripheral blood , suggesting efficient redistribution of the circulating treg pool to inflammatory sites in patients with acs . furthermore , we found 8 trb vj rearrangements common in at least 6 of the 16 analysed thrombus samples . treg accumulation in non - lymphoid tissues ( coronary thrombus in our study ) is shaped by several mechanisms including migration and retention of circulating treg as well as expansion of treg clones specific for tissue - specific antigens . the local chemokine and cytokine milieu and expression of antigens specific for the tissue promote chemotaxis and clonal expansion of treg . temporal changes in these factors may impact on the amount of treg detected by flow cytometry in our study . it is possible that the increase in treg in coronary thrombi found in the current study reflects a local compensatory response to attenuate inflammation in the surrounding pro - inflammatory milieu characterized by elevated concentrations of pro - inflammatory cytokines in coronary blood distal to the occluding coronary thrombus . however , intra - individual comparison of tcr diversity in coronary thrombus vs. peripheral blood was reduced in all but three patients in the present study ( figure 2a ) , demonstrating a consistent pattern of clonal restriction in thrombus - resident t cells , the majority of which are likely treg . t cell receptor diversity both in peripheral blood and coronary thrombi , respectively , was not associated with the extent and severity of coronary artery disease in our study as assessed by the gensini score . this finding suggests that the observed restricted tcr diversity in coronary thrombi may reflect differential trapping of antigen - primed t cells from the circulating t cell pool , unrelated to the burden of underlying atherosclerotic disease . to provide definitive cues to the origin of treg in coronary thrombi , future work should analyse treg in atherothrombosis using a suitable experimental model such as the dereg mouse model to track labelled treg in secondary lymphoid organs , atherosclerotic lesions , and thrombus , respectively . along those lines , our data are in favour of a post hoc alteration of t cell subsets as a consequence of the acs rather than a predisposing factor reflecting an a priori immune imbalance . the concept of expanding antigen - specific treg to diminish vascular inflammation and prevent atherothrombotic clinical events by immunotherapy is appealing . using an immunization strategy , we and others identified antigen - specific treg as a critical component of atheroprotection in mice . however , more data on the origin , recruitment , and kinetics of treg during myocardial infarction are needed before such an approach can be evaluated in a clinical trial . the recently completed phase ii glacier study ( nct01258907 ) reported no change in inflammatory activity in an index arterial vessel after 12 weeks of treatment with a monoclonal antibody targeting oxidized forms of ldl compared with controls , as measured by fdg - pet / ct imaging ( [ 18f]-2-deoxyglucose positron emission - tomography / computed tomography but showed a good safety profile . pending full publication of the data , the choice of primary endpoint , treatment duration , and patients appear pivotal for future trials to address efficacy of immunotherapy in atherosclerotic disease . we could not perform cell - sorting and subsequent repertoire analysis for tcr diversity in treg only as coronary thrombi are very small with very few t cells present . our study is the first to report a restricted tcr diversity in a markedly increased cell pool of leucocytes in peripheral blood from patients with acs when compared with age - matched healthy subjects . together with the profound reduction in tcr diversity identified in coronary thrombi , these findings imply an antigen - specific immune response carried by t cells in patients with acs . interestingly , patients with rheumatoid arthritis also show a reduced tcr diversity in circulating t cells compared with healthy individuals . our finding adds to the shared features between rheumatoid arthritis and clinical atherosclerosis , suggesting similar autoimmune features in the pathogenesis of atherothrombosis . in conclusion , we demonstrate a perturbed t cell compartment characterized by clonal restriction of t cells in both peripheral blood and coronary thrombi of patients with acs . our data provide novel evidence for antigen - specific adaptive immunity in atherothrombosis with treg as the prominent t cell subset . the authors received support by the swiss national science foundation ( sonderprogramm universitre medizin spum 33cm30 - 124112 and nr . 310030 - 118353 to t.f.l . ) ; the swiss heart foundation ; the fondation leducq and the zurich heart house foundation for cardiovascular research , zurich . funding to pay the open access publication charges for this article was provided by zurich heart house . funding to pay the open access publication charges for this article

aimsregulatory t cells ( treg ) exert anti - inflammatory and atheroprotective effects in experimental atherosclerosis . treg can be induced against specific antigens using immunization strategies associated with clonal restriction . no data exist on treg in combination with clonal restriction of t cells in patients with acute coronary syndromes ( acs).methods and resultsamong t cell subsets characterized by flow cytometry , treg ( cd4 + cd25 + cd127low ) were twice as frequent in coronary thrombi compared with peripheral blood . treg prevailed among t cell subsets identified in coronary thrombi . to evaluate clonal restriction , genomic dna was extracted from coronary thrombi and peripheral blood in order to evaluate t cell receptor ( tcr ) chain diversity by means of multi - n - plex pcr using a primer specic for all tcr v gene segments and another primer specic for tcr j gene segments . t cell receptor diversity was reduced in thrombi compared with peripheral blood ( intra - individual comparisons in 16 patients ) with 8 gene rearrangements in the tcr common in at least 6 out of 16 analysed coronary thrombi . compared with age - matched healthy controls ( n = 16 ) , tcr diversity was also reduced in peripheral blood of patients with acs ; these findings were independent of peripheral t cell numbers.conclusionwe provide novel evidence for a perturbed t cell compartment characterized by clonal restriction in peripheral blood and coronary thrombi from patients with acs . our findings warrant further studies on treg as novel therapeutic targets aimed at enhancing this anti - inflammatory component of adaptive immunity in human atherothrombosis .

Introduction Methods Characteristics of patients and healthy subjects Analyses of blood and coronary thrombi Analysis of T cell receptor diversity Flow cytometry Statistical analysis Results Clinical characteristics of patients with acute coronary syndromes and healthy subjects Increased Treg counts in coronary thrombi compared with peripheral blood in patients with acute coronary syndromes Decreased T cell receptor diversity in coronary thrombi compared with peripheral blood in patients with acute coronary syndromes Reduced T cell receptor diversity in circulating T cells of patients with acute coronary syndromes compared with healthy subjects Discussion Limitations Summary and conclusions Supplementary material Funding

among t cell subsets , regulatory t cells ( treg ) exert atheroprotective effects and constitute an inherent anti - inflammatory component of adaptive immunity . the great variety of antigen specificities in the t cell receptor ( tcr ) repertoire is generated in the thymus by random recombination of separate inherited tcr / gene segments termed v(d)j which encode the variable parts of the heterodimeric receptor . evidence for an antigen - specific local immune response carried by t cells in unstable atherosclerotic plaque mandates clonal restriction of t cells when compared with peripheral blood . in circulating cd4 cd28 t cells from patients with unstable angina compared with patients with stable coronary artery disease , clonal restriction of t cells clonal restriction of t cells was demonstrated using spectratyping of atherectomy specimens from coronary plaque compared with peripheral blood mononuclear cells ( pbmcs ) from patients with acs and only to a lesser extent in patients with stable coronary artery disease . the aims of this study , therefore , were ( i ) to compare treg counts in coronary thrombi with pbmc from patients with acs , ( ii ) to compare tcr diversity in coronary thrombi with pbmc from patients with acs , ( iii ) to compare tcr diversity in pbmc from age - matched healthy subjects with patients with acs . genomic dna was extracted using standard techniques and multi - n - plex pcr was performed using an upstream primer specic for all functional members of a given t cell receptor v segment and a downstream primer specic for a given t cell receptor j segment ( international immunogenetics information system , www.imgt.org ) encoding for the cdr 3 in the human tcr chain . for flow cytometry , thrombus and peripheral blood - derived cell suspensions obtained from patients with acs were centrifuged at 200 g or 10 min and resuspended in 1 ml of ice - cold facs buffer ( pbs , 1% foetal calf serum , 0.05% edta ) with fcr block ( human trustain fcx , biolegend , san diego , ca , usa ) . genomic dna was extracted using standard techniques and multi - n - plex pcr was performed using an upstream primer specic for all functional members of a given t cell receptor v segment and a downstream primer specic for a given t cell receptor j segment ( international immunogenetics information system , www.imgt.org ) encoding for the cdr 3 in the human tcr chain . for flow cytometry , thrombus and peripheral blood - derived cell suspensions obtained from patients with acs were centrifuged at 200 g or 10 min and resuspended in 1 ml of ice - cold facs buffer ( pbs , 1% foetal calf serum , 0.05% edta ) with fcr block ( human trustain fcx , biolegend , san diego , ca , usa ) . pairs of coronary thrombi and peripheral blood were obtained from 16 patients with acs using an aspiration catheter as part of the pci for a native coronary culprit lesion . among these , 12 patients were diagnosed with stemi , 4 patients with nstemi , 11 patients were symptomatic for < 24 h , 4 patients had symptoms for 2448 h and 1 patient for 4872 h. for the evaluation of tcr chain diversity in peripheral blood , 16 patients with acs were compared with 16 healthy age - matched subjects . table 1clinical , demographic , and laboratory characteristics of patients and healthy subjectsacshealthyp - valueage ( years)58.9 2.358.7 2.3n.s.male gender ( % ) 15 ( 94)10 ( 63)n.scaucasian ethnicity ( % ) 16 ( 100)16 ( 100)n.s.leucocyte count ( 10/l)11.6 1.55.8 0.3p 0.001lymphocyte count ( 10/l)3.2 1.21.6 0.08n.s.erythrocytes ( 10/l)4.3 0.24.5 0.1n.s.platelets ( 10/l)279 25219 8p < 0.05total cholesterol ( mmol / l)5.1 0.45.6 0.3n.s.baseline glucose ( mmol / l)9.0 1.75.2 0.08p 0.01serum creatinine ( mol / l)79.9 6.372.1 2.7n.s.egfr ( ml / min)90.1 4.992.3 2.4n.s.bmi ( kg / m)28.4 2.024.6 1.1n.s.gensini score39.7 5.70p 0.001cardiovascular risk factorshypercholesterolaemia8 ( 50)0p 0.01hypertension ( % ) 4 ( 25)0p < 0.05diabetes ( % ) 2 ( 13)0n.s.smoking ( current , % ) 7 ( 44)0p 0.01previous medical historyprevious mi3 ( 19)0n.s.previous pci2 ( 13)0n.s.medicationsaspirin ( % ) 16 ( 100)0p 0.001statins ( % ) 4 ( 25)0p < 0.05ace - i / arb ( % ) 2 ( 13)0n.s.-blockers ( % ) 3 ( 19)0n.s.nitrates ( % ) 15 ( 94)0p 0.001calcium antagonists ( % ) 00diuretics ( % ) 1 ( 6)0n.s.values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively.n = 1216 ; n.s . to evaluate t cell subset distribution , flow cytometry was performed for phenotypic characterization of coronary thrombi compared with pbmc from patients with acs . overall cd3 t cell content was unaltered and no difference was detected for t helper cells ( cd4 cd3 cd8 ) , but for a trend to numerical reduction vs. peripheral blood ( figure 1a and b ) . cytotoxic t cells ( cd8 cd3 cd4 ) , cd4 cd3 cd28 t cells and double negative ( cd4 cd8 cd3 ) t cells were found in similar numbers in coronary thrombi and pbmc ( figure 1d f ) . figure 1treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . pairwise intra - individual comparisons of coronary thrombi demonstrated a markedly reduced tcr diversity in coronary thrombi vs. pbmc ( reduction by 0.28-fold ) , shown in figure 2a c . among v(d)j gene segments identified in coronary thrombi from 16 patients with acs , 8 rearrangements were found in common in at least 6 thrombi from individual patients ( table 2 ) . table 2 t cell receptor gene segment rearrangements in patients with an acute coronary syndromev - j rearrangementpbmcthrombusp - valuevbeta18-j2.37 ( 44)8 ( 50)n.s.vbeta05-j2.713 ( 81)7 ( 44)p = 0.028vbeta25-j2.63 ( 19)7 ( 44)n.s.vbeta27-j2.711 ( 69)6 ( 38)n.s.vbeta24-j2.310 ( 63)6 ( 38)n.s.vbeta24-j2.58 ( 50)6 ( 38)n.s.vbeta15-j2.46 ( 38)6 ( 38)n.s.vbeta19-j2.53 ( 19)6 ( 38)n.s.vbeta04-j2.39 ( 56)5 ( 31)n.s.vbeta05-j2.58 ( 50)5 ( 31)n.s.vbeta24-j2.47 ( 44)5 ( 31)n.s.vbeta20-j2.63 ( 19)5 ( 31)n.s.all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . figure 2reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . t cell receptor gene segment rearrangements in patients with an acute coronary syndrome all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . t cell receptor diversity was measured using genomic dna isolated from pbmcs from 16 healthy subjects and pbmcs from 16 patients with acs . compared with matched healthy subjects , patients with acs had a profoundly reduced tcr diversity in peripheral blood ( reduction by 0.36-fold ) , shown in figure 3a c . figure 3reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . pairs of coronary thrombi and peripheral blood were obtained from 16 patients with acs using an aspiration catheter as part of the pci for a native coronary culprit lesion . among these , 12 patients were diagnosed with stemi , 4 patients with nstemi , 11 patients were symptomatic for < 24 h , 4 patients had symptoms for 2448 h and 1 patient for 4872 h. for the evaluation of tcr chain diversity in peripheral blood , 16 patients with acs were compared with 16 healthy age - matched subjects . table 1clinical , demographic , and laboratory characteristics of patients and healthy subjectsacshealthyp - valueage ( years)58.9 2.358.7 2.3n.s.male gender ( % ) 15 ( 94)10 ( 63)n.scaucasian ethnicity ( % ) 16 ( 100)16 ( 100)n.s.leucocyte count ( 10/l)11.6 1.55.8 0.3p 0.001lymphocyte count ( 10/l)3.2 1.21.6 0.08n.s.erythrocytes ( 10/l)4.3 0.24.5 0.1n.s.platelets ( 10/l)279 25219 8p < 0.05total cholesterol ( mmol / l)5.1 0.45.6 0.3n.s.baseline glucose ( mmol / l)9.0 1.75.2 0.08p 0.01serum creatinine ( mol / l)79.9 6.372.1 2.7n.s.egfr ( ml / min)90.1 4.992.3 2.4n.s.bmi ( kg / m)28.4 2.024.6 1.1n.s.gensini score39.7 5.70p 0.001cardiovascular risk factorshypercholesterolaemia8 ( 50)0p 0.01hypertension ( % ) 4 ( 25)0p < 0.05diabetes ( % ) 2 ( 13)0n.s.smoking ( current , % ) 7 ( 44)0p 0.01previous medical historyprevious mi3 ( 19)0n.s.previous pci2 ( 13)0n.s.medicationsaspirin ( % ) 16 ( 100)0p 0.001statins ( % ) 4 ( 25)0p < 0.05ace - i / arb ( % ) 2 ( 13)0n.s.-blockers ( % ) 3 ( 19)0n.s.nitrates ( % ) 15 ( 94)0p 0.001calcium antagonists ( % ) 00diuretics ( % ) 1 ( 6)0n.s.values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively.n = 1216 ; n.s . clinical , demographic , and laboratory characteristics of patients and healthy subjects values are shown as mean sem or absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . to evaluate t cell subset distribution , flow cytometry was performed for phenotypic characterization of coronary thrombi compared with pbmc from patients with acs . overall cd3 t cell content was unaltered and no difference was detected for t helper cells ( cd4 cd3 cd8 ) , but for a trend to numerical reduction vs. peripheral blood ( figure 1a and b ) . cytotoxic t cells ( cd8 cd3 cd4 ) , cd4 cd3 cd28 t cells and double negative ( cd4 cd8 cd3 ) t cells were found in similar numbers in coronary thrombi and pbmc ( figure 1d f ) . figure 1treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . treg as prominent t cell subset in coronary thrombi vs. peripheral blood from patients with acs . pairwise intra - individual comparisons of coronary thrombi demonstrated a markedly reduced tcr diversity in coronary thrombi vs. pbmc ( reduction by 0.28-fold ) , shown in figure 2a c . among v(d)j gene segments identified in coronary thrombi from 16 patients with acs , 8 rearrangements were found in common in at least 6 thrombi from individual patients ( table 2 ) . table 2 t cell receptor gene segment rearrangements in patients with an acute coronary syndromev - j rearrangementpbmcthrombusp - valuevbeta18-j2.37 ( 44)8 ( 50)n.s.vbeta05-j2.713 ( 81)7 ( 44)p = 0.028vbeta25-j2.63 ( 19)7 ( 44)n.s.vbeta27-j2.711 ( 69)6 ( 38)n.s.vbeta24-j2.310 ( 63)6 ( 38)n.s.vbeta24-j2.58 ( 50)6 ( 38)n.s.vbeta15-j2.46 ( 38)6 ( 38)n.s.vbeta19-j2.53 ( 19)6 ( 38)n.s.vbeta04-j2.39 ( 56)5 ( 31)n.s.vbeta05-j2.58 ( 50)5 ( 31)n.s.vbeta24-j2.47 ( 44)5 ( 31)n.s.vbeta20-j2.63 ( 19)5 ( 31)n.s.all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . figure 2reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . t cell receptor gene segment rearrangements in patients with an acute coronary syndrome all values are shown as absolute and relative frequencies ( in brackets with respect to n = 16 patients ) , respectively . reduced t cell receptor diversity in coronary thrombus vs. peripheral blood from patients with acs . intra - individual comparisons of tcr diversity expressed as percentage of 276 possible v(d)j gene segment rearrangements in the human tcr chain ( htrb ) in coronary thrombus vs. pbmc ( a ) . t cell receptor diversity was measured using genomic dna isolated from pbmcs from 16 healthy subjects and pbmcs from 16 patients with acs . compared with matched healthy subjects , patients with acs had a profoundly reduced tcr diversity in peripheral blood ( reduction by 0.36-fold ) , shown in figure 3a c . figure 3reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . reduced t cell receptor diversity in peripheral blood from patients with acs vs. healthy subjects . second , we found a restricted tcr diversity within coronary thrombi compared with peripheral blood of patients with acs . third , we show a markedly reduced tcr diversity also in peripheral blood of patients with acs compared with healthy age - matched subjects . analysis of adaptive immunity carried by t cell subsets in patients with acs was performed by comparing coronary thrombi with peripheral blood intra - individually and peripheral blood from healthy subjects . in contrast , we herein demonstrated a 2.2-fold increase in treg counts in coronary thrombus adjacent to the ruptured plaque compared with peripheral blood , suggesting efficient redistribution of the circulating treg pool to inflammatory sites in patients with acs . it is possible that the increase in treg in coronary thrombi found in the current study reflects a local compensatory response to attenuate inflammation in the surrounding pro - inflammatory milieu characterized by elevated concentrations of pro - inflammatory cytokines in coronary blood distal to the occluding coronary thrombus . however , intra - individual comparison of tcr diversity in coronary thrombus vs. peripheral blood was reduced in all but three patients in the present study ( figure 2a ) , demonstrating a consistent pattern of clonal restriction in thrombus - resident t cells , the majority of which are likely treg . t cell receptor diversity both in peripheral blood and coronary thrombi , respectively , was not associated with the extent and severity of coronary artery disease in our study as assessed by the gensini score . our study is the first to report a restricted tcr diversity in a markedly increased cell pool of leucocytes in peripheral blood from patients with acs when compared with age - matched healthy subjects . together with the profound reduction in tcr diversity identified in coronary thrombi , these findings imply an antigen - specific immune response carried by t cells in patients with acs . in conclusion , we demonstrate a perturbed t cell compartment characterized by clonal restriction of t cells in both peripheral blood and coronary thrombi of patients with acs . our data provide novel evidence for antigen - specific adaptive immunity in atherothrombosis with treg as the prominent t cell subset .

amphotericin b is commonly the treatment of choice in invasive fungal infections ( ifis ) . clinicians may choose among amphotericin b deoxycholate ( conventional amphotericin b [ cab ] ) or a number of lipid - based formulations ( lf - amb ) , such as liposomal amphotericin b and amphotericin b lipid complex . many factors influence the treatment decision , including the patient s current clinical condition and the potential to experience and/or tolerate adverse effects , cost of the drugs , and formulary specifications . the indications for lf - amb , in part , include patients who are refractory to or intolerant of cab therapy.1,2 this potential for toxicity associated with cab including infusion - related reactions acutely , and nephrotoxicity associated with chronic use and the lower risks associated with lf - amb are well documented.310 however , in real world clinical practice , hospitals and/or physicians may reserve lf - amb for the sickest patients and chance administering cab to only the lower acuity patients perceived to be at low risk for adverse consequences.11 these underlying differences in patients clinical characteristics are likely to affect outcomes , potentially skewing the results of effectiveness research efforts . few real - world data are available on the use of cab and lf - amb and associated outcomes among patients with conditions precluding the use of cab . alvarez - lerma et al conducted a subanalysis on 49 critically ill patients with elevated serum creatinine ( greater than 1.5 mg / dl ) at initiation of treatment in an observational study of liposomal amb.12 there was minimal effect on renal function , though overall in - hospital mortality was 67.3% . the aims of the current study were to examine the use and outcome of cab and lf - amb therapies in patients with known renal disease or other potential contraindications to cab and to determine factors associated with lf - amb initiation vs ( versus ) cab , using a large , multicenter database . this was a retrospective cohort study using data collected from hospitals in the health facts electronic health record ( ehr ) database ( cerner corporation , kansas city , mo , usa ) . cerner corporation develops , implements , and supports ehr software for hospitals and health systems globally . us - based institutions using cerner s comprehensive suite of solutions can opt to contribute their ehr data to a database for use in research and quality improvement initiatives . health facts contains a comprehensive clinical record for each encounter and includes pharmacy , clinical and microbiology laboratory , admission , and billing information from affiliated patient care locations . clinical information is date- and time - stamped , providing a temporal relationship between clinical information relating to the drugs dispensed and the results of diagnostic laboratory testing . cerner corporation has established health insurance portability and accountability act - compliant operating policies to establish deidentification for health facts . patients were selected if they were hospitalized between january 2001 and june 2010 , aged 18 years or older upon admission and had orders for lf - amb or for cab on 2 or more calendar days . additional requirements to capture patients with conditions that may constitute a relative contraindication to the use of cab were the presence of at least one of the following : evidence of renal insufficiency or other conditions and characteristics such as a history of organ transplant or advanced age ( appendix a ) , exposure to nephrotoxic agents during the index encounter ( appendix b ) , or cab exposure within 90 days prior to the admission date ( suggesting a cab - refractory infection ) . finally evidence of infection with aspergillus , candida , and/or cryptococcus during the index encounter or within 90 days prior to the index encounter was required as indicated by a positive blood culture and/or relevant international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) codes as a discharge diagnosis . for patients with multiple eligible encounters in health facts , all patients had exposure to amphotericin b. the two study groups were defined by having a first amphotericin b order for cab or for lf - amb and were required to have an active order for this first formulation on at least 2 calendar days . patients could have subsequent orders for the alternate amphotericin b formulation or for other antifungal agents . patient clinical characteristics and comorbidities of interest were derived from administrative ( eg , icd-9-cm codes ) and clinical ( eg , pharmacy , laboratory ) records of encounters within the previous 12 months , including the current encounter . the diagnosis - related group ( drg ) classified the patient as surgical or medical . evidence of impaired immune function comprised medications ( eg , systemic corticosteroids , chemotherapy ) and discharge diagnoses ( eg , autoimmune diseases , certain malignancies ) . organ dysfunction was identified within a 48-hour window surrounding the time of admission using measures modeled after and intended to equate to a sepsis - related organ failure assessment score 2.13 critical care exposure was defined as having two or more orders from an intensive care unit 12 or more hours apart , mechanical ventilation , or orders for vasopressors . to determine the predictors most strongly associated with initial exposure to lf - amb vs cab , we used a multilevel ( ie , hierarchical ) mixed - effects logistic regression model structure with random intercepts at the hospital level to allow for the fact that the choice of drugs given to patients within each hospital ( but not between hospitals ) may not be independent ( eg , influenced by hospital formulary).14 to avoid including potential complications of amphotericin b use , we limited the candidate variables to chronic comorbidities and events that occurred prior to amphotericin b initiation . the final model was chosen based on a stepwise bootstrapping procedure15 combined with an analysis of the bayesian information criterion among nested models.16 the influence of hospital on treatment choice was assessed using a likelihood ratio test for the significance of random intercepts . outcomes of interest were length of stay ( los ) following the first order for amphotericin b ( post - amphotericin b los ) among survivors , and in - hospital mortality . bivariate differences by amphotericin b type were assessed with either a chi - square test or a t - test , with p values < 0.05 being considered statistically significant . a logistic regression model that adjusted for serial correlation at the hospital level was used to generate a propensity score with the outcome of initiation on cab vs lf - amb . namely , patient demographics , comorbid conditions , encounter events , microbiology results , laboratory values prior to initiation of amphotericin b , and pre - amphotericin b los were included in the propensity score . variables that showed colinearity were removed ( and this was only true for hepatic organ dysfunction , which was collinear with laboratory measures of bilirubin or aspartate aminotransferase ) . baseline total bilirubin and aspartate aminotransferase were defined as binary variables ( normal vs abnormal ) and missing values in these variables were assumed to be normal . for the small number of patients with missing values for mechanical ventilation ( 7.9% ) or organ dysfunction ( 1.2% ) , ventilation or organ dysfunction was also assumed to be absent . for baseline serum creatinine , a univariate imputation sampling method was used which predicted missing values based on all other predictor variables used in the propensity score . the primary matching algorithm was kernel matching , a one - to - many approach in which patients with smaller propensity score differences were weighted more heavily in deriving matched estimates . two sensitivity analyses were then done.17 the first used the propensity scores based only on predictors that entered the model with a p value < 0.25 after a stepwise regression procedure . in the second sensitivity analysis , a 5:1 greedy matching algorithm was applied to the non - parse- and stepwise - regression - based propensity scores . to explore which categories of variables most explained differences in mortality between the cab and lf - amb initiator groups , we created a series of multilevel ( ie , hierarchical ) mixed - effects logistic regression models of increasing size . namely , the variable order was demographics , chronic comorbidities , surgical vs medical drg , laboratory values , and finally clinical variables indicating acuity . for each model , we present the odds ratio ( or ) related to treatment choice ( lf - amb vs cab ) and its adjusted p value with 95% confidence intervals ( cis ) . this was a retrospective cohort study using data collected from hospitals in the health facts electronic health record ( ehr ) database ( cerner corporation , kansas city , mo , usa ) . cerner corporation develops , implements , and supports ehr software for hospitals and health systems globally . us - based institutions using cerner s comprehensive suite of solutions can opt to contribute their ehr data to a database for use in research and quality improvement initiatives . health facts contains a comprehensive clinical record for each encounter and includes pharmacy , clinical and microbiology laboratory , admission , and billing information from affiliated patient care locations . clinical information is date- and time - stamped , providing a temporal relationship between clinical information relating to the drugs dispensed and the results of diagnostic laboratory testing . cerner corporation has established health insurance portability and accountability act - compliant operating policies to establish deidentification for health facts . patients were selected if they were hospitalized between january 2001 and june 2010 , aged 18 years or older upon admission and had orders for lf - amb or for cab on 2 or more calendar days . additional requirements to capture patients with conditions that may constitute a relative contraindication to the use of cab were the presence of at least one of the following : evidence of renal insufficiency or other conditions and characteristics such as a history of organ transplant or advanced age ( appendix a ) , exposure to nephrotoxic agents during the index encounter ( appendix b ) , or cab exposure within 90 days prior to the admission date ( suggesting a cab - refractory infection ) . finally evidence of infection with aspergillus , candida , and/or cryptococcus during the index encounter or within 90 days prior to the index encounter was required as indicated by a positive blood culture and/or relevant international classification of diseases , ninth revision , clinical modification ( icd-9-cm ) codes as a discharge diagnosis . for patients with multiple eligible encounters in health facts , all patients had exposure to amphotericin b. the two study groups were defined by having a first amphotericin b order for cab or for lf - amb and were required to have an active order for this first formulation on at least 2 calendar days . patients could have subsequent orders for the alternate amphotericin b formulation or for other antifungal agents . patient clinical characteristics and comorbidities of interest were derived from administrative ( eg , icd-9-cm codes ) and clinical ( eg , pharmacy , laboratory ) records of encounters within the previous 12 months , including the current encounter . the diagnosis - related group ( drg ) classified the patient as surgical or medical . evidence of impaired immune function comprised medications ( eg , systemic corticosteroids , chemotherapy ) and discharge diagnoses ( eg , autoimmune diseases , certain malignancies ) . organ dysfunction was identified within a 48-hour window surrounding the time of admission using measures modeled after and intended to equate to a sepsis - related organ failure assessment score 2.13 critical care exposure was defined as having two or more orders from an intensive care unit 12 or more hours apart , mechanical ventilation , or orders for vasopressors . to determine the predictors most strongly associated with initial exposure to lf - amb vs cab , we used a multilevel ( ie , hierarchical ) mixed - effects logistic regression model structure with random intercepts at the hospital level to allow for the fact that the choice of drugs given to patients within each hospital ( but not between hospitals ) may not be independent ( eg , influenced by hospital formulary).14 to avoid including potential complications of amphotericin b use , we limited the candidate variables to chronic comorbidities and events that occurred prior to amphotericin b initiation . the final model was chosen based on a stepwise bootstrapping procedure15 combined with an analysis of the bayesian information criterion among nested models.16 the influence of hospital on treatment choice was assessed using a likelihood ratio test for the significance of random intercepts . outcomes of interest were length of stay ( los ) following the first order for amphotericin b ( post - amphotericin b los ) among survivors , and in - hospital mortality . bivariate differences by amphotericin b type were assessed with either a chi - square test or a t - test , with p values < 0.05 being considered statistically significant . the primary analysis was performed using propensity score matching . a logistic regression model that adjusted for serial correlation at the hospital level was used to generate a propensity score with the outcome of initiation on cab vs lf - amb . namely , patient demographics , comorbid conditions , encounter events , microbiology results , laboratory values prior to initiation of amphotericin b , and pre - amphotericin b los were included in the propensity score . variables that showed colinearity were removed ( and this was only true for hepatic organ dysfunction , which was collinear with laboratory measures of bilirubin or aspartate aminotransferase ) . baseline total bilirubin and aspartate aminotransferase were defined as binary variables ( normal vs abnormal ) and missing values in these variables were assumed to be normal . for the small number of patients with missing values for mechanical ventilation ( 7.9% ) or organ dysfunction ( 1.2% ) , ventilation or organ dysfunction was also assumed to be absent . for baseline serum creatinine , a univariate imputation sampling method was used which predicted missing values based on all other predictor variables used in the propensity score . the primary matching algorithm was kernel matching , a one - to - many approach in which patients with smaller propensity score differences were weighted more heavily in deriving matched estimates . two sensitivity analyses were then done.17 the first used the propensity scores based only on predictors that entered the model with a p value < 0.25 after a stepwise regression procedure . in the second sensitivity analysis , a 5:1 greedy matching algorithm was applied to the non - parse- and stepwise - regression - based propensity scores . to explore which categories of variables most explained differences in mortality between the cab and lf - amb initiator groups , we created a series of multilevel ( ie , hierarchical ) mixed - effects logistic regression models of increasing size . namely , the variable order was demographics , chronic comorbidities , surgical vs medical drg , laboratory values , and finally clinical variables indicating acuity . for each model , we present the odds ratio ( or ) related to treatment choice ( lf - amb vs cab ) and its adjusted p value with 95% confidence intervals ( cis ) . a total of 655 patients from 53 hospitals were identified ; 333 patients first amphotericin b order was for lf - amb and 322 were initiated on cab . of these , 81% and 70% , respectively , also received another systemic antifungal agent during their hospitalization . fifty - three percent of patients were identified during the first half of the study period . clinically , the cohorts were heterogeneous : lf - amb patients were younger and more likely to be male , and had greater underlying disease severity ( table 1 ) . mean charlson comorbidity index score was higher among lf - amb initiators , as was the frequency of several individual comorbidities : hematologic malignancy , solid tumor , human immunodeficiency syndrome or acquired immunodeficiency syndrome , and history of solid organ transplant . lf - amb patients were far more likely to have any evidence of impaired immune function . during the hospital encounter itself , multiple measures of patient acuity were more common among lf - amb initiators : organ dysfunction upon admission , bacteremia , diagnosis of sepsis , critical care use , and use of systemic corticosteroids or chemotherapy . more cab initiators had an icd-9-cm discharge diagnosis of candidiasis during the encounter , while more lf - amb patients were diagnosed with aspergillosis . cab initiators were more likely to have a history of diabetes , hypertension , or heart failure and to have higher mean baseline serum creatinine values . we found several clinical factors that were significantly associated with starting lf - amb rather than cab ( table 2 ) . patients with critical care exposure prior to amphotericin b , impaired immune function , liver dysfunction upon admission , or aplastic anemia / pancytopenia were more likely to be started on lf - amb . diabetes , cryptococcosis , candidiasis , and history of stem cell transplant were associated with starting on cab . the specific hospital had a strong influence , perhaps driven by formulary guidelines , on the choice of amphotericin b formulation , as allowing each hospital to have its own intercept significantly improved model fit ( p < 0.001 ) . crude analysis of in - hospital mortality favored initiation of cab , with an or of 1.53 for initiating lf - amb ( p = 0.02 ) ( table 3 ) . among survivors , the observed pointestimate of post - amphotericin b los was nonsignificantly shorter in the cab group ( difference in los = 2.5 days shorter for cab group , 95% ci : 6.11.1 ; p = 0.17 ) . the primary propensity score model and the less parse , secondary model based on stepwise regression both exhibited good calibration ( hosmer lemeshow statistics were either borderline significant [ p = 0.03 ] for the primary model or nonsignificant [ p = 0.16 ] for the stepwise model ) . the mean propensity scores for the cab and lf - amb groups were 0.35 and 0.66 , respectively . while not a goal of the propensity score modeling , we did observe relatively strong discrimination ( high area under the receiver - operating characteristic curve values ) of 0.83 and 0.82 for the primary and secondary models , respectively . matching on the propensity to receive lf - amb eliminated the significant differences in odds of mortality ( or = 1.05 , 95% ci : 0.621.77 ; p = 0.85 ) and reversed the directionality of observed differences in post - amphotericin b los ( difference in los = 2.6 days longer in the cab group , 95% ci : 2.67.9 ; p = 0.32 ) . sensitivity analyses using the secondary propensity score based on stepwise regression and the alternative matching procedure based on greedy matching produced similar findings ( results not shown ) . sequentially adding covariates into our models for hospital mortality explained the effect of initial treatment ( table 4 ) . addition of variables for demographics and baseline clinical status to the models had little impact , but addition of acuity variables eliminated differences in the odds of mortality across the treatment groups . a total of 655 patients from 53 hospitals were identified ; 333 patients first amphotericin b order was for lf - amb and 322 were initiated on cab . of these , 81% and 70% , respectively , also received another systemic antifungal agent during their hospitalization . fifty - three percent of patients were identified during the first half of the study period . clinically , the cohorts were heterogeneous : lf - amb patients were younger and more likely to be male , and had greater underlying disease severity ( table 1 ) . mean charlson comorbidity index score was higher among lf - amb initiators , as was the frequency of several individual comorbidities : hematologic malignancy , solid tumor , human immunodeficiency syndrome or acquired immunodeficiency syndrome , and history of solid organ transplant . lf - amb patients were far more likely to have any evidence of impaired immune function . during the hospital encounter itself , multiple measures of patient acuity were more common among lf - amb initiators : organ dysfunction upon admission , bacteremia , diagnosis of sepsis , critical care use , and use of systemic corticosteroids or chemotherapy . more cab initiators had an icd-9-cm discharge diagnosis of candidiasis during the encounter , while more lf - amb patients were diagnosed with aspergillosis . cab initiators were more likely to have a history of diabetes , hypertension , or heart failure and to have higher mean baseline serum creatinine values . we found several clinical factors that were significantly associated with starting lf - amb rather than cab ( table 2 ) . patients with critical care exposure prior to amphotericin b , impaired immune function , liver dysfunction upon admission , or aplastic anemia / pancytopenia were more likely to be started on lf - amb . diabetes , cryptococcosis , candidiasis , and history of stem cell transplant were associated with starting on cab . the specific hospital had a strong influence , perhaps driven by formulary guidelines , on the choice of amphotericin b formulation , as allowing each hospital to have its own intercept significantly improved model fit ( p < 0.001 ) . crude analysis of in - hospital mortality favored initiation of cab , with an or of 1.53 for initiating lf - amb ( p = 0.02 ) ( table 3 ) . among survivors , the observed pointestimate of post - amphotericin b los was nonsignificantly shorter in the cab group ( difference in los = 2.5 days shorter for cab group , 95% ci : 6.11.1 ; p = 0.17 ) . the primary propensity score model and the less parse , secondary model based on stepwise regression both exhibited good calibration ( hosmer lemeshow statistics were either borderline significant [ p = 0.03 ] for the primary model or nonsignificant [ p = 0.16 ] for the stepwise model ) . the mean propensity scores for the cab and lf - amb groups were 0.35 and 0.66 , respectively . while not a goal of the propensity score modeling , we did observe relatively strong discrimination ( high area under the receiver - operating characteristic curve values ) of 0.83 and 0.82 for the primary and secondary models , respectively . matching on the propensity to receive lf - amb eliminated the significant differences in odds of mortality ( or = 1.05 , 95% ci : 0.621.77 ; p = 0.85 ) and reversed the directionality of observed differences in post - amphotericin b los ( difference in los = 2.6 days longer in the cab group , 95% ci : 2.67.9 ; p = 0.32 ) . sensitivity analyses using the secondary propensity score based on stepwise regression and the alternative matching procedure based on greedy matching produced similar findings ( results not shown ) . sequentially adding covariates into our models for hospital mortality explained the effect of initial treatment ( table 4 ) . addition of variables for demographics and baseline clinical status to the models had little impact , but addition of acuity variables eliminated differences in the odds of mortality across the treatment groups . to our knowledge , this is the first study to use real - world data to compare los and mortality in patients with ifis initiated on cab vs lf - amb with clinical conditions warranting the use of lf - amb . in evaluating 10 years of data on hospitalized patients with ifis and evidence of renal impairment or other comorbidities or exposures that might put them at risk for cab - associated toxicity when physicians believe amphotericin b treatment is needed for patients with serious fungal infections , they have a choice between cab and lf - amb . the risk of nephrotoxicity associated with cab is well documented,4,18 and thus , particularly in patients with renal compromise or otherwise at risk for toxic effects , lf - amb may be more appropriate.19,20 preliminary descriptive analysis demonstrated that patients initiated on lf - amb were generally sicker than those initiated on cab . the multivariate model predicting lf - amb initiation ( vs cab ) extended this , and showed that critical care exposure and impaired immune function both doubled the odds of receiving lf - amb . based on the inclusion criteria ( eg , history of kidney disease ) , all patients in the study were at risk for adverse effects of cab . absent these restrictions , we would have expected to see even more pronounced differences between the treatment groups , with greater acuity and comorbidity burden particularly related to renal disease among lf - amb patients . we observed no clear trend toward certain types of variables ( eg , higher baseline serum creatinine or a diagnosis of end - stage renal disease ) being associated with lf - amb . most striking was the clustering of amphotericin b formulation by hospital , which may indicate that formulary requirements or other institutional practices are important drivers of amphotericin b choice . this implies that the clinical drivers of choice were very powerful to have emerged in the setting of apparently common administrative constraints . as many of the patients included here were from early in the study period , further research should examine trends in choice of formulation over time . raw hospital mortality was significantly higher and observed post - amphotericin b los nonsignificantly longer among patients initiated on lf - amb prior to adjustment . absent further analysis , a nave interpretation would be that lf - amb was inferior , with increased mortality potentially due to toxicity or to limited effectiveness in treating the fungal infection . propensity score matching eliminated the differences in both acuity and effect , indicating that outcomes were driven by factors affecting choice of therapy . this was confirmed by the nested model exercise , which demonstrated that acuity variables accounted for the differences in mortality between the groups . these findings suggest that , contrary to the impression given by nave analyses , the real - world effectiveness of the treatments is consistent with that found in randomized clinical trials.9,2124 a strength of our analysis is that our ehr - based data source includes clinical characteristics that are not available in administrative claims - based data , such as laboratory results . we attempted to minimize confounding in our multivariate adjustment by including numerous demographic characteristics , comorbidities , and encounter events in the propensity score and regression models . , we did not collect data on anti - fungal exposure subsequent to the amphotericin b orders , which may have provided further insight into patients course of illness and , indirectly , severity of the fungal infection . future analyses should investigate use of additional antifungals to better understand treatment sequencing and duration following initiation of amphotericin b. information on specific institutional formulary policies would have been useful in controlling for a patient s potential to be treated with cab vs lf - amb , but was not available in health facts at the time this study was conducted . however , we observed in our analyses that a given hospital influenced its patients starting formulations , and accounted for this in our outcomes analysis . in an ehr database of patients with ifis and contraindications to use of cab , clinical factors appeared to drive therapeutic decisions regarding initiation of lf - amb or cab . real - world outcomes may initially appear to contradict what has been demonstrated in clinical trials . proper adjustment for underlying patient acuity is needed to accurately estimate comparative effectiveness between cab and lf - amb .

backgroundlipid - based formulations of amphotericin b ( lf - amb ) are indicated for treatment of invasive fungal infections in patients intolerant to conventional amphotericin b ( cab ) or with refractory infections . physicians still may choose to administer cab to such patients . we described the use of cab and lf - amb in this population and quantified differences in post - amphotericin b length of stay ( los ) among survivors and hospital mortality in matched patients.methodsdata were extracted from health facts ( cerner corporation , kansas city , mo , usa ) for a retrospective cohort analysis . inpatients aged 18 years with evidence of fungal infection and with orders for lf - amb or cab on 2 days from january 2001 to june 2010 were identified . patients were required to have renal insufficiency or other relative contraindications to use of cab , exposure to nephrotoxic agents , or evidence of a cab - refractory infection . multilevel ( hierarchical ) mixed - effects logistic regression was used to determine factors associated with initial exposure to lf - amb versus cab . multivariate adjustment of outcomes was done using propensity score matching.results655 patients were identified : 322 patients initiated therapy with cab and 333 initiated treatment with lf - amb . compared to those initiating cab , patients initiating lf - amb had greater acuity and underlying disease severity . in unadjusted analyses , hospital mortality was significantly higher in the lf - amb group ( 32.2% versus 23.7% ; p = 0.02 ) . after propensity score matching and covariate adjustment , mortality equalized and observed differences in los after amphotericin b initiation decreased.conclusionamong patients at risk for amphotericin b toxicity , differences between cab and lf - amb seen in crude outcomes analyses relate to channeling of sicker patients to initiate treatment with lf - amb . failing to account for differences among patients that drive clinical decision - making will result in inaccurate conclusions about the real - world effectiveness of different amphotericin b formulations .

Introduction and objectives Methods Study design and data source Population selection Study group definitions and other measures Predicting initial exposure Outcomes analysis Nested variable analysis of mortality Results Patient and clinical characteristics Predictors of LF-AMB initiation Outcomes Nested model analysis Discussion Conclusion

amphotericin b is commonly the treatment of choice in invasive fungal infections ( ifis ) . clinicians may choose among amphotericin b deoxycholate ( conventional amphotericin b [ cab ] ) or a number of lipid - based formulations ( lf - amb ) , such as liposomal amphotericin b and amphotericin b lipid complex . the indications for lf - amb , in part , include patients who are refractory to or intolerant of cab therapy.1,2 this potential for toxicity associated with cab including infusion - related reactions acutely , and nephrotoxicity associated with chronic use and the lower risks associated with lf - amb are well documented.310 however , in real world clinical practice , hospitals and/or physicians may reserve lf - amb for the sickest patients and chance administering cab to only the lower acuity patients perceived to be at low risk for adverse consequences.11 these underlying differences in patients clinical characteristics are likely to affect outcomes , potentially skewing the results of effectiveness research efforts . few real - world data are available on the use of cab and lf - amb and associated outcomes among patients with conditions precluding the use of cab . alvarez - lerma et al conducted a subanalysis on 49 critically ill patients with elevated serum creatinine ( greater than 1.5 mg / dl ) at initiation of treatment in an observational study of liposomal amb.12 there was minimal effect on renal function , though overall in - hospital mortality was 67.3% . the aims of the current study were to examine the use and outcome of cab and lf - amb therapies in patients with known renal disease or other potential contraindications to cab and to determine factors associated with lf - amb initiation vs ( versus ) cab , using a large , multicenter database . this was a retrospective cohort study using data collected from hospitals in the health facts electronic health record ( ehr ) database ( cerner corporation , kansas city , mo , usa ) . cerner corporation has established health insurance portability and accountability act - compliant operating policies to establish deidentification for health facts . patients were selected if they were hospitalized between january 2001 and june 2010 , aged 18 years or older upon admission and had orders for lf - amb or for cab on 2 or more calendar days . additional requirements to capture patients with conditions that may constitute a relative contraindication to the use of cab were the presence of at least one of the following : evidence of renal insufficiency or other conditions and characteristics such as a history of organ transplant or advanced age ( appendix a ) , exposure to nephrotoxic agents during the index encounter ( appendix b ) , or cab exposure within 90 days prior to the admission date ( suggesting a cab - refractory infection ) . for patients with multiple eligible encounters in health facts , all patients had exposure to amphotericin b. the two study groups were defined by having a first amphotericin b order for cab or for lf - amb and were required to have an active order for this first formulation on at least 2 calendar days . patients could have subsequent orders for the alternate amphotericin b formulation or for other antifungal agents . evidence of impaired immune function comprised medications ( eg , systemic corticosteroids , chemotherapy ) and discharge diagnoses ( eg , autoimmune diseases , certain malignancies ) . organ dysfunction was identified within a 48-hour window surrounding the time of admission using measures modeled after and intended to equate to a sepsis - related organ failure assessment score 2.13 critical care exposure was defined as having two or more orders from an intensive care unit 12 or more hours apart , mechanical ventilation , or orders for vasopressors . to determine the predictors most strongly associated with initial exposure to lf - amb vs cab , we used a multilevel ( ie , hierarchical ) mixed - effects logistic regression model structure with random intercepts at the hospital level to allow for the fact that the choice of drugs given to patients within each hospital ( but not between hospitals ) may not be independent ( eg , influenced by hospital formulary).14 to avoid including potential complications of amphotericin b use , we limited the candidate variables to chronic comorbidities and events that occurred prior to amphotericin b initiation . outcomes of interest were length of stay ( los ) following the first order for amphotericin b ( post - amphotericin b los ) among survivors , and in - hospital mortality . a logistic regression model that adjusted for serial correlation at the hospital level was used to generate a propensity score with the outcome of initiation on cab vs lf - amb . namely , patient demographics , comorbid conditions , encounter events , microbiology results , laboratory values prior to initiation of amphotericin b , and pre - amphotericin b los were included in the propensity score . for baseline serum creatinine , a univariate imputation sampling method was used which predicted missing values based on all other predictor variables used in the propensity score . in the second sensitivity analysis , a 5:1 greedy matching algorithm was applied to the non - parse- and stepwise - regression - based propensity scores . to explore which categories of variables most explained differences in mortality between the cab and lf - amb initiator groups , we created a series of multilevel ( ie , hierarchical ) mixed - effects logistic regression models of increasing size . for each model , we present the odds ratio ( or ) related to treatment choice ( lf - amb vs cab ) and its adjusted p value with 95% confidence intervals ( cis ) . this was a retrospective cohort study using data collected from hospitals in the health facts electronic health record ( ehr ) database ( cerner corporation , kansas city , mo , usa ) . cerner corporation has established health insurance portability and accountability act - compliant operating policies to establish deidentification for health facts . patients were selected if they were hospitalized between january 2001 and june 2010 , aged 18 years or older upon admission and had orders for lf - amb or for cab on 2 or more calendar days . additional requirements to capture patients with conditions that may constitute a relative contraindication to the use of cab were the presence of at least one of the following : evidence of renal insufficiency or other conditions and characteristics such as a history of organ transplant or advanced age ( appendix a ) , exposure to nephrotoxic agents during the index encounter ( appendix b ) , or cab exposure within 90 days prior to the admission date ( suggesting a cab - refractory infection ) . for patients with multiple eligible encounters in health facts , all patients had exposure to amphotericin b. the two study groups were defined by having a first amphotericin b order for cab or for lf - amb and were required to have an active order for this first formulation on at least 2 calendar days . patients could have subsequent orders for the alternate amphotericin b formulation or for other antifungal agents . organ dysfunction was identified within a 48-hour window surrounding the time of admission using measures modeled after and intended to equate to a sepsis - related organ failure assessment score 2.13 critical care exposure was defined as having two or more orders from an intensive care unit 12 or more hours apart , mechanical ventilation , or orders for vasopressors . to determine the predictors most strongly associated with initial exposure to lf - amb vs cab , we used a multilevel ( ie , hierarchical ) mixed - effects logistic regression model structure with random intercepts at the hospital level to allow for the fact that the choice of drugs given to patients within each hospital ( but not between hospitals ) may not be independent ( eg , influenced by hospital formulary).14 to avoid including potential complications of amphotericin b use , we limited the candidate variables to chronic comorbidities and events that occurred prior to amphotericin b initiation . outcomes of interest were length of stay ( los ) following the first order for amphotericin b ( post - amphotericin b los ) among survivors , and in - hospital mortality . the primary analysis was performed using propensity score matching . a logistic regression model that adjusted for serial correlation at the hospital level was used to generate a propensity score with the outcome of initiation on cab vs lf - amb . namely , patient demographics , comorbid conditions , encounter events , microbiology results , laboratory values prior to initiation of amphotericin b , and pre - amphotericin b los were included in the propensity score . for baseline serum creatinine , a univariate imputation sampling method was used which predicted missing values based on all other predictor variables used in the propensity score . in the second sensitivity analysis , a 5:1 greedy matching algorithm was applied to the non - parse- and stepwise - regression - based propensity scores . to explore which categories of variables most explained differences in mortality between the cab and lf - amb initiator groups , we created a series of multilevel ( ie , hierarchical ) mixed - effects logistic regression models of increasing size . for each model , we present the odds ratio ( or ) related to treatment choice ( lf - amb vs cab ) and its adjusted p value with 95% confidence intervals ( cis ) . a total of 655 patients from 53 hospitals were identified ; 333 patients first amphotericin b order was for lf - amb and 322 were initiated on cab . fifty - three percent of patients were identified during the first half of the study period . clinically , the cohorts were heterogeneous : lf - amb patients were younger and more likely to be male , and had greater underlying disease severity ( table 1 ) . mean charlson comorbidity index score was higher among lf - amb initiators , as was the frequency of several individual comorbidities : hematologic malignancy , solid tumor , human immunodeficiency syndrome or acquired immunodeficiency syndrome , and history of solid organ transplant . lf - amb patients were far more likely to have any evidence of impaired immune function . during the hospital encounter itself , multiple measures of patient acuity were more common among lf - amb initiators : organ dysfunction upon admission , bacteremia , diagnosis of sepsis , critical care use , and use of systemic corticosteroids or chemotherapy . more cab initiators had an icd-9-cm discharge diagnosis of candidiasis during the encounter , while more lf - amb patients were diagnosed with aspergillosis . cab initiators were more likely to have a history of diabetes , hypertension , or heart failure and to have higher mean baseline serum creatinine values . we found several clinical factors that were significantly associated with starting lf - amb rather than cab ( table 2 ) . patients with critical care exposure prior to amphotericin b , impaired immune function , liver dysfunction upon admission , or aplastic anemia / pancytopenia were more likely to be started on lf - amb . the specific hospital had a strong influence , perhaps driven by formulary guidelines , on the choice of amphotericin b formulation , as allowing each hospital to have its own intercept significantly improved model fit ( p < 0.001 ) . crude analysis of in - hospital mortality favored initiation of cab , with an or of 1.53 for initiating lf - amb ( p = 0.02 ) ( table 3 ) . among survivors , the observed pointestimate of post - amphotericin b los was nonsignificantly shorter in the cab group ( difference in los = 2.5 days shorter for cab group , 95% ci : 6.11.1 ; p = 0.17 ) . the primary propensity score model and the less parse , secondary model based on stepwise regression both exhibited good calibration ( hosmer lemeshow statistics were either borderline significant [ p = 0.03 ] for the primary model or nonsignificant [ p = 0.16 ] for the stepwise model ) . the mean propensity scores for the cab and lf - amb groups were 0.35 and 0.66 , respectively . matching on the propensity to receive lf - amb eliminated the significant differences in odds of mortality ( or = 1.05 , 95% ci : 0.621.77 ; p = 0.85 ) and reversed the directionality of observed differences in post - amphotericin b los ( difference in los = 2.6 days longer in the cab group , 95% ci : 2.67.9 ; p = 0.32 ) . addition of variables for demographics and baseline clinical status to the models had little impact , but addition of acuity variables eliminated differences in the odds of mortality across the treatment groups . a total of 655 patients from 53 hospitals were identified ; 333 patients first amphotericin b order was for lf - amb and 322 were initiated on cab . fifty - three percent of patients were identified during the first half of the study period . clinically , the cohorts were heterogeneous : lf - amb patients were younger and more likely to be male , and had greater underlying disease severity ( table 1 ) . mean charlson comorbidity index score was higher among lf - amb initiators , as was the frequency of several individual comorbidities : hematologic malignancy , solid tumor , human immunodeficiency syndrome or acquired immunodeficiency syndrome , and history of solid organ transplant . lf - amb patients were far more likely to have any evidence of impaired immune function . during the hospital encounter itself , multiple measures of patient acuity were more common among lf - amb initiators : organ dysfunction upon admission , bacteremia , diagnosis of sepsis , critical care use , and use of systemic corticosteroids or chemotherapy . more cab initiators had an icd-9-cm discharge diagnosis of candidiasis during the encounter , while more lf - amb patients were diagnosed with aspergillosis . cab initiators were more likely to have a history of diabetes , hypertension , or heart failure and to have higher mean baseline serum creatinine values . we found several clinical factors that were significantly associated with starting lf - amb rather than cab ( table 2 ) . patients with critical care exposure prior to amphotericin b , impaired immune function , liver dysfunction upon admission , or aplastic anemia / pancytopenia were more likely to be started on lf - amb . the specific hospital had a strong influence , perhaps driven by formulary guidelines , on the choice of amphotericin b formulation , as allowing each hospital to have its own intercept significantly improved model fit ( p < 0.001 ) . crude analysis of in - hospital mortality favored initiation of cab , with an or of 1.53 for initiating lf - amb ( p = 0.02 ) ( table 3 ) . among survivors , the observed pointestimate of post - amphotericin b los was nonsignificantly shorter in the cab group ( difference in los = 2.5 days shorter for cab group , 95% ci : 6.11.1 ; p = 0.17 ) . the primary propensity score model and the less parse , secondary model based on stepwise regression both exhibited good calibration ( hosmer lemeshow statistics were either borderline significant [ p = 0.03 ] for the primary model or nonsignificant [ p = 0.16 ] for the stepwise model ) . the mean propensity scores for the cab and lf - amb groups were 0.35 and 0.66 , respectively . matching on the propensity to receive lf - amb eliminated the significant differences in odds of mortality ( or = 1.05 , 95% ci : 0.621.77 ; p = 0.85 ) and reversed the directionality of observed differences in post - amphotericin b los ( difference in los = 2.6 days longer in the cab group , 95% ci : 2.67.9 ; p = 0.32 ) . addition of variables for demographics and baseline clinical status to the models had little impact , but addition of acuity variables eliminated differences in the odds of mortality across the treatment groups . to our knowledge , this is the first study to use real - world data to compare los and mortality in patients with ifis initiated on cab vs lf - amb with clinical conditions warranting the use of lf - amb . in evaluating 10 years of data on hospitalized patients with ifis and evidence of renal impairment or other comorbidities or exposures that might put them at risk for cab - associated toxicity when physicians believe amphotericin b treatment is needed for patients with serious fungal infections , they have a choice between cab and lf - amb . the risk of nephrotoxicity associated with cab is well documented,4,18 and thus , particularly in patients with renal compromise or otherwise at risk for toxic effects , lf - amb may be more appropriate.19,20 preliminary descriptive analysis demonstrated that patients initiated on lf - amb were generally sicker than those initiated on cab . the multivariate model predicting lf - amb initiation ( vs cab ) extended this , and showed that critical care exposure and impaired immune function both doubled the odds of receiving lf - amb . based on the inclusion criteria ( eg , history of kidney disease ) , all patients in the study were at risk for adverse effects of cab . absent these restrictions , we would have expected to see even more pronounced differences between the treatment groups , with greater acuity and comorbidity burden particularly related to renal disease among lf - amb patients . we observed no clear trend toward certain types of variables ( eg , higher baseline serum creatinine or a diagnosis of end - stage renal disease ) being associated with lf - amb . most striking was the clustering of amphotericin b formulation by hospital , which may indicate that formulary requirements or other institutional practices are important drivers of amphotericin b choice . this implies that the clinical drivers of choice were very powerful to have emerged in the setting of apparently common administrative constraints . raw hospital mortality was significantly higher and observed post - amphotericin b los nonsignificantly longer among patients initiated on lf - amb prior to adjustment . absent further analysis , a nave interpretation would be that lf - amb was inferior , with increased mortality potentially due to toxicity or to limited effectiveness in treating the fungal infection . propensity score matching eliminated the differences in both acuity and effect , indicating that outcomes were driven by factors affecting choice of therapy . these findings suggest that , contrary to the impression given by nave analyses , the real - world effectiveness of the treatments is consistent with that found in randomized clinical trials.9,2124 a strength of our analysis is that our ehr - based data source includes clinical characteristics that are not available in administrative claims - based data , such as laboratory results . we attempted to minimize confounding in our multivariate adjustment by including numerous demographic characteristics , comorbidities , and encounter events in the propensity score and regression models . , we did not collect data on anti - fungal exposure subsequent to the amphotericin b orders , which may have provided further insight into patients course of illness and , indirectly , severity of the fungal infection . future analyses should investigate use of additional antifungals to better understand treatment sequencing and duration following initiation of amphotericin b. information on specific institutional formulary policies would have been useful in controlling for a patient s potential to be treated with cab vs lf - amb , but was not available in health facts at the time this study was conducted . in an ehr database of patients with ifis and contraindications to use of cab , clinical factors appeared to drive therapeutic decisions regarding initiation of lf - amb or cab . real - world outcomes may initially appear to contradict what has been demonstrated in clinical trials . proper adjustment for underlying patient acuity is needed to accurately estimate comparative effectiveness between cab and lf - amb .

this study included a total of 77 patients with vd who were recruited at the department of vascular surgery , oslo university hospital , aker , oslo , norway . they were scheduled for vascular surgery , including abdominal aortic aneurysm repair and carotid or femoral arterial endarterectomy . clinical oral examinations were performed at the hospital ward to determine the periodontal status of the patient . the patients were categorized into two groups according to their clinical periodontal status . the first group ( vd with cp , n=30 ) were patients undergoing treatment for vd and who were diagnosed with cp . the second group ( vd without cp , n=47 ) were patients undergoing treatment for vd and who were assessed without periodontitis . questions on ethnicity and smoking behavior were self - reported . written informed consent was obtained from all participants . this study was approved by the regional ethical committee ( rek sr , no . 08/322b ) and was in accordance with the helsinki declaration of 1975 , as revised in 1983 . the patients in both groups were diagnosed and treated according to standard procedures at the department of vascular surgery , oslo university hospital , aker . diagnosis of cp was based on the classification system of the american academy of periodontology , established in 1999 at the international workshop for classification of periodontal diseases and conditions ( 22 ) . the measurements were done at the mesial , buccal , distal , lingual , and palatinal surfaces of all teeth . subjects who had at least four sites with a probing depth 5 mm and bleeding on probing were categorized as having cp . no subject had received periodontal treatment within the last 6 months or taken antibiotics within the past month . vascular tissue biopsies and subgingival plaque samples were collected for extraction of genomic dna and checkerboard dna dna hybridization analysis , respectively . the vascular biopsies were collected under surgical treatment from the walls of aneurysms and during excision of intravascular plaques in carotid or common femoral arteries . the biopsies were immediately transferred from bedside to a sterilized container that was brought on ice to the laboratory and stored at 80c . the collection of the biopsies and further processing in the laboratory were performed under strict aseptic conditions . extraction of genomic dna was done by using the masterpure complete dna purification kit ( epicentre biotechnologies , madison , wi ) , according to the manufacturer 's extraction protocol for tissue samples with some modifications . in order to make a representable selection of the biopsies for dna extraction and after the samples were treated with rnase a ( 5 g ) and protein - precipitated , they were kept in 0.5 m nacl . further , they were purified with ( 1:1 v / v ) phenol chloroform ( vwr international as , oslo , norway ) and centrifuged in phase lock gel tubes ( 5prime , gaithersburg , md ) . the aqueous phase was treated twice with chloroform isoamyl alcohol ( 1:1 v / v ) ( applichem gmbh , darmstadt , germany ) . dna was precipitated with 100% ethanol ( kemetyl , vestby , norway ) in 0.3 m sodium acetate ( applichem gmbh ) . the pellet was washed twice with 75% ethanol , resuspended in 1te buffer ( 10 mm tris , 1 mm edta , ph 8.0 ) and stored at 20c . negative and positive control reactions were performed for the tissue homogenization and dna extraction methods . the subgingival plaque samples were collected from four periodontal sites for each subject . in patients with cp , the deepest pockets with bleeding on probing were chosen , while in patients without cp , four sites were chosen randomly . after removal of supragingival plaque , isolation , and drying of sample sites with cotton rolls , subgingival plaque was collected by using sterile gracey curettes . universal bacterial primers were used for 16s rdna amplification under standardized conditions with forward primer : e334f 5-ccagactcctacgggaggcagc-3 and reverse primer : e939r 5-cttgtgcgggcccccgtcaattc-3 ( 23 ) . these primers cover the hypervariable region v3-v5 of the 16s rrna gene , have high specificity to bacterial sequences , and low match to eukarya and archaea sequences ( 23 ) . pcrs were performed with accuprime supermix ii ( invitrogen , carlsbad , ca ) at an annealing temperature of 69c and a total of 32 cycles . species - specific pcr assays and fima genotyping were applied for the identification of the periodontopathogen p. gingivalis in vascular biopsies from patients with cp . the previously reported primers for p. gingivalis 16s rrna and fima gene ( 24 , 25 ) were used ( table 1 ) . pcrs were performed with onetaq 2x master mix with standard buffer ( new england biolabs , beverly , ma ) and annealing temperatures as given in table 1 . porphyromonas gingivalis 16s rdna and fima gene - specific primers used in this study each set of experiments included negative controls with sterile molecular grade water instead of template dna and positive controls containing purified dna from veillonella dispar , p. gingivalis strains for fima type i ( atcc 33277 t ) and fima type ii ( a7a1 - 28 ) . the pcr products were detected by 1% agarose gel electrophoresis . gels were stained with ethidium bromide , using 1 kb plus dna ladder ( invitrogen ) . the universal 16s rdna pcr amplicons ( primers e334/e939 ) were ligated into the pcr4-topo vector using the topo ta cloning kit ( invitrogen ) and transformed into competent escherichia coli top10 cells according to the manufacturer 's instructions . ninety - six colonies were collected from each sample to make a representative library of the pcr products , which were stored in the te buffer at 20c until further processing . after pcr amplification of the inserts with m13 forward primer , 5-gta aaa cga cgg cca g-3 , and m13 reverse primer , 5-cag gaa aca gct atg ac-3 , the pcr products were purified by exosap - it ( affymetrix , santa clara , ca ) according to the manufacturer 's protocol . purified amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , foster city , ca ) and the m13 forward primer . the p. gingivalis - specific pcr amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit according to the manufacturer 's instructions and the respective forward primers ( table 1 ) . sequence reactions were run on an abi prism 3,730 dna analyzer ( applied biosystems ) . sequence trimming and quality check were performed with the sequencher 5.0 program ( gene codes corp . , ann arbor , mi ) . the uchime program ( 26 ) at the mothur platform after the elimination of suspected chimeric sequences , the 16s rdna sequences ( ~500 bp ) were used to determine bacterial identity . the sequences were aligned by using the molecular evolutionary genetics analysis ( mega ) software version 5 ( 28 ) . for identification of closest relatives , the consensus sequences were compared with known sequences in the genbank databases using the ncbi blast search tool ( http://www.ncbi.nlm.nih.gov/blast/ ) and human oral microbiome database ( homd ; http://www.homd.org/ ) ( 29 ) . ten randomly selected subgingival plaque samples from vd patients with cp were assessed for the presences of red complex bacteria ( rcb ) using checkerboard dna dna hybridization . naoh ( 100 l of 0.5 m ) was added to 100 l of subgingival plaque samples in te buffer and vortexed . after boiling for 10 min the dna was then applied into the extended slots of a minislot-30 apparatus ( immunetics , cambridge , ma ) with a positively charged nylon membrane ( boehringer mannheim , indianapolis , in ) . the membrane had two lanes with dna standards equivalent to 1010 cells per target species tested . the membranes were hybridized with 20 digoxigenin labeled whole genome probes of species significant for the subgingival microbiota , including rcb ( table 2 ) ( 30 ) . dna hybridization ( checkerboard analysis ) in subgingival dental plaque samples from patients with atherosclerotic and abdominal aortic aneurysmal vascular disease and chronic periodontitis ten randomly selected vascular biopsies from vd patients with periodontitis were selected at random for sem . tissue blocks were fixed in 2.5% glutaraldehyde or 0.1 mol / l sorensen phosphate buffer , and stored at 4c until processing . after dehydration in ethanol , the blocks were critically point - dried with carbon dioxide . the specimens were finally sputter - coated with gold or palladium in a vacuum evaporator and examined in a scanning electron microscope ( xl 30 esem ; philips , eidenhoven , the netherlands ) ( 31 ) . the statistical analyses were performed using the spss software ( ibm spss statistic version19 ) . independent samples t - tests were used for comparisons of species diversity and bacterial load between the two groups . for comparisons of demographic data between the two groups , independent t - test and fisher 's exact test this study included a total of 77 patients with vd who were recruited at the department of vascular surgery , oslo university hospital , aker , oslo , norway . they were scheduled for vascular surgery , including abdominal aortic aneurysm repair and carotid or femoral arterial endarterectomy . clinical oral examinations were performed at the hospital ward to determine the periodontal status of the patient . the patients were categorized into two groups according to their clinical periodontal status . the first group ( vd with cp , n=30 ) were patients undergoing treatment for vd and who were diagnosed with cp . the second group ( vd without cp , n=47 ) were patients undergoing treatment for vd and who were assessed without periodontitis . questions on ethnicity and smoking behavior were self - reported . written informed consent was obtained from all participants . this study was approved by the regional ethical committee ( rek sr , no . 08/322b ) and was in accordance with the helsinki declaration of 1975 , as revised in 1983 . the patients in both groups were diagnosed and treated according to standard procedures at the department of vascular surgery , oslo university hospital , aker . diagnosis of cp was based on the classification system of the american academy of periodontology , established in 1999 at the international workshop for classification of periodontal diseases and conditions ( 22 ) . the measurements were done at the mesial , buccal , distal , lingual , and palatinal surfaces of all teeth . subjects who had at least four sites with a probing depth 5 mm and bleeding on probing were categorized as having cp . no subject had received periodontal treatment within the last 6 months or taken antibiotics within the past month . vascular tissue biopsies and subgingival plaque samples were collected for extraction of genomic dna and checkerboard dna dna hybridization analysis , respectively . the vascular biopsies were collected under surgical treatment from the walls of aneurysms and during excision of intravascular plaques in carotid or common femoral arteries . the biopsies were immediately transferred from bedside to a sterilized container that was brought on ice to the laboratory and stored at 80c . the collection of the biopsies and further processing in the laboratory were performed under strict aseptic conditions . extraction of genomic dna was done by using the masterpure complete dna purification kit ( epicentre biotechnologies , madison , wi ) , according to the manufacturer 's extraction protocol for tissue samples with some modifications . in order to make a representable selection of the biopsies for dna extraction and after the samples were treated with rnase a ( 5 g ) and protein - precipitated , they were kept in 0.5 m nacl . further , they were purified with ( 1:1 v / v ) phenol chloroform ( vwr international as , oslo , norway ) and centrifuged in phase lock gel tubes ( 5prime , gaithersburg , md ) . the aqueous phase was treated twice with chloroform isoamyl alcohol ( 1:1 v / v ) ( applichem gmbh , darmstadt , germany ) . dna was precipitated with 100% ethanol ( kemetyl , vestby , norway ) in 0.3 m sodium acetate ( applichem gmbh ) . the pellet was washed twice with 75% ethanol , resuspended in 1te buffer ( 10 mm tris , 1 mm edta , ph 8.0 ) and stored at 20c . negative and positive control reactions were performed for the tissue homogenization and dna extraction methods . the subgingival plaque samples were collected from four periodontal sites for each subject . in patients with cp , the deepest pockets with bleeding on probing were chosen , while in patients without cp , four sites were chosen randomly . after removal of supragingival plaque , isolation , and drying of sample sites with cotton rolls , subgingival plaque was collected by using sterile gracey curettes . universal bacterial primers were used for 16s rdna amplification under standardized conditions with forward primer : e334f 5-ccagactcctacgggaggcagc-3 and reverse primer : e939r 5-cttgtgcgggcccccgtcaattc-3 ( 23 ) . these primers cover the hypervariable region v3-v5 of the 16s rrna gene , have high specificity to bacterial sequences , and low match to eukarya and archaea sequences ( 23 ) . pcrs were performed with accuprime supermix ii ( invitrogen , carlsbad , ca ) at an annealing temperature of 69c and a total of 32 cycles . species - specific pcr assays and fima genotyping were applied for the identification of the periodontopathogen p. gingivalis in vascular biopsies from patients with cp . the previously reported primers for p. gingivalis 16s rrna and fima gene ( 24 , 25 ) were used ( table 1 ) . pcrs were performed with onetaq 2x master mix with standard buffer ( new england biolabs , beverly , ma ) and annealing temperatures as given in table 1 . porphyromonas gingivalis 16s rdna and fima gene - specific primers used in this study each set of experiments included negative controls with sterile molecular grade water instead of template dna and positive controls containing purified dna from veillonella dispar , p. gingivalis strains for fima type i ( atcc 33277 t ) and fima type ii ( a7a1 - 28 ) . gels were stained with ethidium bromide , using 1 kb plus dna ladder ( invitrogen ) . the universal 16s rdna pcr amplicons ( primers e334/e939 ) were ligated into the pcr4-topo vector using the topo ta cloning kit ( invitrogen ) and transformed into competent escherichia coli top10 cells according to the manufacturer 's instructions . ninety - six colonies were collected from each sample to make a representative library of the pcr products , which were stored in the te buffer at 20c until further processing . after pcr amplification of the inserts with m13 forward primer , 5-gta aaa cga cgg cca g-3 , and m13 reverse primer , 5-cag gaa aca gct atg ac-3 , the pcr products were purified by exosap - it ( affymetrix , santa clara , ca ) according to the manufacturer 's protocol . purified amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , foster city , ca ) and the m13 forward primer . the p. gingivalis - specific pcr amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit according to the manufacturer 's instructions and the respective forward primers ( table 1 ) . sequence reactions were run on an abi prism 3,730 dna analyzer ( applied biosystems ) . sequence trimming and quality check were performed with the sequencher 5.0 program ( gene codes corp . , ann arbor , mi ) . the uchime program ( 26 ) at the mothur platform after the elimination of suspected chimeric sequences , the 16s rdna sequences ( ~500 bp ) were used to determine bacterial identity . the sequences were aligned by using the molecular evolutionary genetics analysis ( mega ) software version 5 ( 28 ) . for identification of closest relatives , the consensus sequences were compared with known sequences in the genbank databases using the ncbi blast search tool ( http://www.ncbi.nlm.nih.gov/blast/ ) and human oral microbiome database ( homd ; http://www.homd.org/ ) ( 29 ) . ten randomly selected subgingival plaque samples from vd patients with cp were assessed for the presences of red complex bacteria ( rcb ) using checkerboard dna dna hybridization . naoh ( 100 l of 0.5 m ) was added to 100 l of subgingival plaque samples in te buffer and vortexed . after boiling for 10 min , the dna was then applied into the extended slots of a minislot-30 apparatus ( immunetics , cambridge , ma ) with a positively charged nylon membrane ( boehringer mannheim , indianapolis , in ) . the membrane had two lanes with dna standards equivalent to 1010 cells per target species tested . the membranes were hybridized with 20 digoxigenin labeled whole genome probes of species significant for the subgingival microbiota , including rcb ( table 2 ) ( 30 ) . bacterial species detected by dna dna hybridization ( checkerboard analysis ) in subgingival dental plaque samples from patients with atherosclerotic and abdominal aortic aneurysmal vascular disease and chronic periodontitis ten randomly selected vascular biopsies from vd patients with periodontitis were selected at random for sem . tissue blocks were fixed in 2.5% glutaraldehyde or 0.1 mol / l sorensen phosphate buffer , and stored at 4c until processing . after dehydration in ethanol , the blocks were critically point - dried with carbon dioxide . the specimens were finally sputter - coated with gold or palladium in a vacuum evaporator and examined in a scanning electron microscope ( xl 30 esem ; philips , eidenhoven , the netherlands ) ( 31 ) . the statistical analyses were performed using the spss software ( ibm spss statistic version19 ) . independent samples t - tests were used for comparisons of species diversity and bacterial load between the two groups . for comparisons of demographic data between the two groups , independent t - test and fisher 's exact test the first group ( vd with cp ) were patients undergoing treatment for vd and who were diagnosed with cp ( n=30 , mean age=66.9 , standard deviation ( sd)=7.9 , range : 51.385.0 , male=25/30 ( 83% ) . the second group ( vd without cp ) were patients undergoing treatment for vd and who were assessed without periodontitis ( n=47 , mean age=70.9 , sd= 9.7 , range : 45.784.8 , male=37/47 ( 79% ) . vascular biopsies from all 30 vd patients with cp and 10 randomly selected vd patients without cp were further analyzed for detection of 16s rdna sequences . in total , 27 biopsies from abdominal aortic aneurysmal walls , 9 from carotid , and 4 from common femoral atherosclerotic plaque were examined . the patients with cp had significantly higher number of periodontal pockets with probing depth 5 mm ( p<0.01 ) . there was no significant difference in terms of age , gender , diabetes , body mass index , and number of teeth between the two groups . demographic data of the study groups with atherosclerotic and abdominal aortic aneurysmal vascular disease ( vd ) , with and without chronic periodontitis ( cp ) based on a fisher 's exact test . after discarding incomplete sequences , a total of 2,638 sequences from vd patients with cp and 859 sequences from vd patients without cp were analyzed . a sequence similarity threshold of 97 and 99% were applied for identification at the genus and species level , respectively ( 32 ) . sequences with similarity lower than 97% were ranked as unclassified and were excluded ( listed according to the closest relative in supplementary file sa ) . after optimization of the pcr , weak pcr bands were detected in the negative pcr controls ( ~50% ) and were subsequently sequenced . these background sequences ( supplementary file sb ) as well as eukaryote and chimeric sequences were also discarded from the sequences obtained from the vascular samples and further analyzed . a final total of 38 out of 40 ( 95% ) vascular samples with bacterial dna were subjected to further analysis . after discarding unwanted sequences , the final fraction of bacterial sequences , reflecting overall bacterial load , obtained from the vascular biopsies in patients with cp was in average 6823% ( range : 098% ) at an individual level . in patients without cp , this fraction was significantly lower with an average of 2526% ( range : 084% ; p<0.01 ) . no significant difference in bacterial load was found comparing intravascular plaque versus aneurysmal vascular biopsies in any group . eighty - four different bacterial taxa were detected from a total of 1,808 sequences from the vd patients with cp ( table 4 ) . from the vd patients without cp , 18 different taxa were identified from a total of 210 sequences ( table 5 ) . the relative abundance of the different taxa detected in vascular biopsies from patients with and without cp is presented in tables 4 and 5 , respectively . the mean diversity of bacterial taxa detected from the vascular biopsies was significantly higher in patients with cp ( n=30 ) , average of 7.13.2 ( range : 012 ) , compared to those without cp ( n=10 ) , average of 2.81.6 ( range : 05 ; p<0.01 ) . no significant difference in mean bacterial diversity was found comparing intravascular plaque versus aneurysmal vascular biopsies in any group . bacterial taxa identified in atherosclerotic plaque and abdominal aortic aneurysmal wall biopsies from patients with chronic periodontitis the different taxa were identified by ncbi blast analysis using a similarity threshold of 97% for identification possible overlap with bacterial taxa identified in vascular biopsies from vd patients without cp at bacterial taxa underlined are listed among the human oral microbial taxa in human oral microbiome database ( homd ) . bacterial taxa identified in atherosclerotic plaque and abdominal aortic aneurysmal wall biopsies from patients without chronic periodontitis the different taxa were identified by ncbi blast analysis using a similarity threshold of 97% for identification . possible overlap with bacterial taxa identified in vascular biopsies from vd patients with cp is highlighted in bold . bacterial taxa underlined are listed among the human oral microbial taxa in human oral microbiome database ( homd ) . patients with cp displayed a wide variety of bacteria identified at the genus level from their vascular biopsies , including some known oral bacterial taxa ( e.g. streptococcus spp . were also detected in both groups and were among the most prevalent species in the vd group with cp . in total , 15% and 4.3% of the sequences were identified as not - yet - cultured bacterial spp . in the vd group with and without cp , respectively . using universal bacterial 16s rdna pcr , p. gingivalis was detected in the vascular biopsy from only one vd patients with cp . no other members of the rcb were detected from the vascular biopsies in either group . none of the 30 vascular biopsies from vd patients with cp were positive for p. gingivalis using the specific primers listed in table 1 . weak pcr bands were detected in the vascular samples using the fima ii primer set . however , checkerboard analysis revealed presence of one to three members of the rcb species in 7 out of 10 subgingival plaque samples from the cp patients ( table 2 ) . cocci and rod - shaped bacterial cells of different sizes were seen in all biopsies ( fig . bacteria were observed at the surface of the intravascular plaque lining the arterial lumen entangled in a meshwork of delicate fibers and plaque remnants . several preparations from each biopsy were inspected in order to visualize bacterial cells . most often , the bacterial cells were found coaggregated as micro - colonies at a distance from each other . apparent bacterial cell division with invagination of the cell membrane was seen in some of the sem images . ( a ) area of aneurysmal wall with bacteria entangled in meshwork of delicate fibers . ( b ) area of aneurysmal wall with bacteria entangled in meshwork of delicate fibers and remnants of intravascular plaque on aneurysmal wall . ( c ) microorganisms ( rods ) with remnants of intravascular plaque on aneurysmal wall . ( d ) area of aneurysmal wall with bacteria entangled in meshwork of delicate fibers and remnants of intravascular plaque on aneurysmal wall . ( e , f ) area of aneurysmal wall with coccus - shaped bacteria entangled in meshwork of delicate fibers and remnants of intravascular plaque on aneurysmal wall . bacterial dna was detected in 95% of the vascular biopsies , and most biopsies contained dna from multiple bacterial species , pointing to a less specific relationship between single species and vd . these findings are supported by recent studies suggesting multiple bacterial species being involved in cardiovascular diseases ( 5 , 21 ) . we detected a significantly higher mean diversity and a higher load of bacteria in the vascular biopsies from the cp group as compared to the group without cp . in patients with cp , the defective epithelial barrier of the periodontal pockets together with a high load and diversity of bacteria in the subgingival plaque make it conceivable that the oral bacterial translocation and transient bacteremia are caused by dental procedures , oral infections , and may even follow tooth brushing and chewing ( 10 ) . , favorable conditions may allow colonization at a given site , as seen in cases of infective endocarditis ( 33 ) . even though rcbs were detected at high frequencies in the subgingival plaque samples , only p. gingivalis was identified from only one vascular biopsy from a patient with cp . in addition , we used p. gingivalis - specific primers ; however , none of these primers yielded positive detection of p. gingivalis in our vd biopsies . similarly , several other studies , using species - specific pcr , nested pcr and hybridization detection techniques , also reported no detection of rcb in atherosclerotic carotid lesions , despite the presence of one or more periodontopathogens in the patients subgingival plaque samples ( 3436 ) . a possible explanation to the negative or sparse detection of rcb in vd biopsies may be that , in contrast to commensal bacteria , more antigenic microbes such as the rcb are readily recognized by the immune system and eliminated from the circulation . in a study by nakano et al . ( 37 ) , streptococcus mutans strains serotype k survived in blood for a longer time due to lower antigenicity as compared to more cariogenic strains . in addition , a variation in ability to invade cardiovascular cells has been demonstrated among different p. gingivalis strains ( 38 ) . similar invasive heterogeneity is reported for aggregatibacter actinomycetemcomitans and s. mutans strains ( 39 , 40 ) . individual differences in harboring invasive strains may be an alternative explanation of the discrepancies in the detection of rcb in vascular tissue reported in the literature ( 11 ) . nevertheless , many studies have reported the presence of periodontopathogens in atherosclerotic lesions from aorta , coronary , carotid , and femoral arteries ( 14 , 16 , 17 , 41 ) , although with variable frequency ( 11 , 36 ) . these variations may be due to methodological differences , e.g. origin of specimens , dna extraction techniques , the use of specific or universal primers , and pcr conditions ( 14 , 35 ) . however , even by using similar pcr - based techniques with the same specific primers , various detection rates for rcb ( 032% for p. gingivalis ) in peripheral atherosclerotic biopsies from patient with cp have been reported ( 41 , 42 ) . with the p. gingivalis fima ii primer set previously used for p. gingivalis detection in cardiovascular specimen ( 43 ) , these findings together with the human sequences identified , using bacterial - specific 16s rdna primers , emphasize the importance of identifying pcr products by sequencing in studies involving specimens where bacterial dna is sparse . using nested pcr and sequencing , fiehn et al . ( 44 ) reported a detection rate of 4.17% for p. gingivalis in carotid and femoral atherosclerotic biopsies . ( 19 ) found an overall high bacterial diversity with more than 50 bacterial species in coronary atherosclerotic tissue using 16s rdna clone libraries ; however , they did not detect any members of rcb . by using 454 high - throughput sequencing , koren et al . ( 45 ) also could not confirm the presence of dna from periodontopathogens in carotid artery biopsies . sparse finding of rcb in abdominal aneurysmal samples ( tannerella forsythia in 1 out of 10 samples ) was also reported in a study by marques da silva et al . using 16s rrna pcr and sequencing ( 31 ) . dna from none of the other rcb however , the dna of periodontopathogens was detected in the atheromatous plaques from coronary arteries in a chinese population with cp ( 46 ) . the prevalence of t. forsythia , fusobacterium nucleatum , prevotella intermedia , and p. gingivalis was 31 , 12 , 18 , and 33% , respectively . the same specific primers for t. forsythia , f. nucleatum , and p. intermedia were used by cairo et al . they were not able to detect dna from these species , even though dental plaque samples were positive in 79 , 63 , and 53% of the cases , respectively . although we were able to detect bacterial dna in most vd biopsies , the bacterial load was generally sparse as reflected by the amount of pcr products obtained . also , with sem several preparations from each biopsy had to be inspected in order to visualize bacterial cells . most often the bacterial cells were found coaggregated in intravascular plaque lining the arterial lumen . due to scant amounts of pcr products , several rounds of cloning and sequencing had to be performed to obtain 96 clone libraries with sequences of adequate quality and size , especially when processing vd biopsies from patients without periodontitis . in these patients , the final fraction of bacterial sequences averaged 25% , and the majority of the excluded sequences were background sequences and eukaryote sequences ( supplementary file sb ) . thus , the number of vascular biopsies examined in this group was limited to 10 randomly selected biopsies . ( 47 ) using 16s rdna sequencing , reported the average number of different bacterial sequences from atherosclerotic lesions in patients without signs of clinical infections to be 2.21.2 , which is comparable to our patients without cp ( 2.81.6 ) . in subjects with cp , several common oral taxa were detected in the vascular biopsies , i.e. streptococcus , prevotella , capnocytophaga , veillonella , and porphyromonas spp . , while in patients without cp , only one common oral taxon was found ( streptococcus sp . ) . these species and other possible oral taxa detected in the vascular biopsies are highlighted in tables 4 and 5 . although these taxa represent only a minor proportion of the oral bacterial microbiota , the data suggest that the oral cavity may be a potential source for bacterial dissemination to vascular tissue . due to the near absence of rcb in vascular biopsies , we wanted to confirm their presence in the subgingival plaque in cp patients by using checkerboard dna dna hybridization analysis . although this method can not be compared to 16s rdna sequencing , it is an efficient and sensitive detection method for this purpose ( 30 ) . the checkerboard analysis revealed presence of one to three members of the rcb in 7 out of 10 subgingival plaque samples from the cp patients ( table 2 ) . further , only 3 out of 19 bacterial spp . found in the subgingival plaque by checkerboard analysis were detected in the vascular biopsies in the cp group ( i.e. p. gingivalis , actinomyces sp . , and streptococcus spp . ) , suggesting dissimilar bacterial composition in the subgingival plaque and vascular biopsies . in a previous study , 16s rdna - based pcr was positive for bacterial detection , whereas checkerboard dna dna hybridization methods were negative . this indicated that the number of rcb - positive subgingival plaque samples in our study could possibly be even higher ( 48 ) . ( 49 ) showed that the bacterial composition in dental plaque was different from what was found in cardiovascular specimens . the presence of periodontopathogens and other oral bacterial species were sparse in cardiovascular tissue , and only a few species , including s. mutans , may have originated from the oral cavity . a relatively high percentage ( 15.4% ) of the sequences detected in vascular biopsies from patients with cp belonged to the enterobacteriaceae family , i.e. serratia sp . and klebsiella sp . these and other enteric species have been isolated from subgingival sites ( 50 ) . in addition , enteric bacteria are known to be important nosocomial pathogens , and it can not be excluded that they were transmitted to the patients through the hospital environment ( 51 ) . however , the highest load of enterobacteriaceae is found as part of the gut microbiota . the intestines harbor the largest number of bacterial cells in the human body , making it likely that a major part of the bacterial taxa detected in our vascular biopsies had originated from the intestines . several mechanisms for dissemination of gut bacteria into the blood stream have been described ( 52 ) . koren et al . ( 45 ) found several operational taxonomic units shared between the gut and atherosclerotic plaque suggesting that bacteria present in the atherosclerotic plaque could have been derived from the gut . ( 53 ) , systemic inflammation induced by intravenously administrated e. coli lipopolysaccharide increased the intestinal permeability which may facilitate bacterial translocation . animal studies have shown inflammation to induce and sustain increased intestinal permeability ( 54 , 55 ) . we may hypothesize that low - grade chronic systemic inflammation caused by periodontal disease ( 56 ) may affect intestinal permeability and thereby translocation of enteric bacteria , dissemination to the bloodstream , and further colonization of vascular lesions , as seen in our vascular biopsies from patients with cp . much of the human microbiota is not yet cultivated ( 57 ) , and the presence of different not - yet - cultured bacterial taxa is to be expected . several environmental bacterial species with undefined pathogenicity were detected in the vd biopsies , such as members of the families chitinophagaceae and rhodobacteraceae , geobacter sp . , and alishewanella sp . through the skin and mucous membrane linings of the respiratory , gastrointestinal , and genitourinary tracts , some of the environmental bacteria detected in the vd biopsies may be part of our transient microbiota . the inclusion of negative controls and the high level of interindividual differences in detection of environmental species make it unlikely that these bacterial taxa are introduced by technical contamination . several other studies have reported the presence of environmental species in vascular lesions and clinical samples ( 47 , 58 ) ; however , the pathogenicity and clinical relevance of most environmental bacterial taxa in patient samples are yet to be determined . bacterial dna belonging to pseudomonas sp . and propionibacterium acnes were frequently detected in both groups of patients . pseudomonas is an opportunistic human pathogen of clinical relevance , and has been considered by some to be associated with periodontal disease ( 59 ) . p. acnes is part of the commensal microbiota of skin and respiratory and digestive mucosa . this species is a common blood contaminant and can be the cause of postoperative infections , and has been associated with different cases of apical periodontitis and endocarditis ( 60 , 61 ) . acinetobacter sp . was found in both groups and was among the most prevalent in the group with cp . acinetobacter has been isolated from periodontal pockets ( 50 ) , and is known to be a frequent cause of nosocomial infections , especially pneumonias , as well as bloodstream and urinary tract infections ( 62 , 63 ) . are part of the commensal microbiota of the mouth , skin , intestines , and upper respiratory tract . however , they can cause serious conditions such as bacteremia , meningitis , and pneumonia , and have been associated with endocarditis ( 64 ) . , such as s. epidermidis and s. aureus , is part of the commensal skin and respiratory tract microflora . similar to streptococci they can cause cardiovascular conditions such as endocarditis ( 64 ) . by sem , bacterial cells with different morphology these findings emphasize the presence of bacterial cells , not only bacterial dna , in the vd biopsies . viable invasive bacteria such as p. gingivalis and a. actinomycetemcomitans have been cultivated from human atherosclerotic plaque ( 65 , 66 ) . others have suggested that bacteria from the circulation might get caught in the vascular lesions as however , animal studies have shown that bacterial taxa such as chlamydia pneumonia and p. gingivalis may invade atherosclerotic plaque , alter inflammatory pathways , and cause molecular mimicry , and thereby may contribute to the progression of atherogenesis ( 6 , 13 ) . in conclusion , our results indicate that the cumulative effect of different infectious agents may be associated with the pathophysiology of atherosclerotic and abdominal aortic aneurysmal vascular diseases . however , periodontopathogens were rarely identified in the vascular biopsies , whilst gut bacteria were detected more frequently . a possible association between chronic periodontal disease , systemic inflammation , intestinal permeability , and enterobacterial colonization of atherosclerotic and aneurysmal tissue should be studied further .

backgroundseveral studies have reported an association between chronic periodontitis ( cp ) and cardiovascular diseases . detection of periodontopathogens , including red complex bacteria ( rcb ) , in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms.objectivein this study , we investigate bacteria and their dna in vascular biopsies from patients with vascular diseases ( vd ; i.e. abdominal aortic aneurysms , atherosclerotic carotid , and common femoral arteries ) , with and without cp.methodsdna was extracted from vascular biopsies selected from 40 vd patients : 30 with cp and 10 without cp . the v3-v5 region of the 16s rdna ( v3-v5 ) was polymerase chain reaction ( pcr)-amplified , and the amplicons were cloned into escherichia coli , sequenced , and classified ( genbank and the human oral microbiome database ) . species - specific primers were used for the detection of porphyromonas gingivalis . in addition , 10 randomly selected vascular biopsies from the cp group were subjected to scanning electron microscopy ( sem ) for visualization of bacteria . checkerboard dna dna hybridization was performed to assess the presence of rcb in 10 randomly selected subgingival plaque samples from cp patients.resultsa higher load and mean diversity of bacteria were detected in vascular biopsies from vd patients with cp compared to those without cp . enterobacteriaceae were frequently detected in vascular biopsies together with cultivable , commensal oral , and not - yet - cultured bacterial species . while 70% of the subgingival plaque samples from cp patients showed presence of rcb , only p. gingivalis was detected in one vascular biopsy . bacterial cells were seen in all 10 vascular biopsies examined by sem.conclusionsa higher bacterial load and more diverse colonization were detected in vd lesions of cp patients as compared to patients without cp . this indicated that a multitude of bacterial species both from the gut and the oral cavity , rather than exclusively periodontopathogens , may be involved as additional risk factors in the pathogenesis of vd .

Material and methods Participants Inclusion criteria Sample collection and DNA extraction PCR amplification of bacterial DNA Cloning and sequencing Data analysis of the sequences Checkerboard DNADNA hybridization Scanning electron microscopy Statistical analyses Results Discussion Conflict of interest and funding

vascular tissue biopsies and subgingival plaque samples were collected for extraction of genomic dna and checkerboard dna dna hybridization analysis , respectively . the vascular biopsies were collected under surgical treatment from the walls of aneurysms and during excision of intravascular plaques in carotid or common femoral arteries . in patients with cp , the deepest pockets with bleeding on probing were chosen , while in patients without cp , four sites were chosen randomly . universal bacterial primers were used for 16s rdna amplification under standardized conditions with forward primer : e334f 5-ccagactcctacgggaggcagc-3 and reverse primer : e939r 5-cttgtgcgggcccccgtcaattc-3 ( 23 ) . these primers cover the hypervariable region v3-v5 of the 16s rrna gene , have high specificity to bacterial sequences , and low match to eukarya and archaea sequences ( 23 ) . species - specific pcr assays and fima genotyping were applied for the identification of the periodontopathogen p. gingivalis in vascular biopsies from patients with cp . porphyromonas gingivalis 16s rdna and fima gene - specific primers used in this study each set of experiments included negative controls with sterile molecular grade water instead of template dna and positive controls containing purified dna from veillonella dispar , p. gingivalis strains for fima type i ( atcc 33277 t ) and fima type ii ( a7a1 - 28 ) . the universal 16s rdna pcr amplicons ( primers e334/e939 ) were ligated into the pcr4-topo vector using the topo ta cloning kit ( invitrogen ) and transformed into competent escherichia coli top10 cells according to the manufacturer 's instructions . purified amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit ( applied biosystems , foster city , ca ) and the m13 forward primer . the p. gingivalis - specific pcr amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit according to the manufacturer 's instructions and the respective forward primers ( table 1 ) . the uchime program ( 26 ) at the mothur platform after the elimination of suspected chimeric sequences , the 16s rdna sequences ( ~500 bp ) were used to determine bacterial identity . for identification of closest relatives , the consensus sequences were compared with known sequences in the genbank databases using the ncbi blast search tool ( http://www.ncbi.nlm.nih.gov/blast/ ) and human oral microbiome database ( homd ; http://www.homd.org/ ) ( 29 ) . ten randomly selected subgingival plaque samples from vd patients with cp were assessed for the presences of red complex bacteria ( rcb ) using checkerboard dna dna hybridization . naoh ( 100 l of 0.5 m ) was added to 100 l of subgingival plaque samples in te buffer and vortexed . dna hybridization ( checkerboard analysis ) in subgingival dental plaque samples from patients with atherosclerotic and abdominal aortic aneurysmal vascular disease and chronic periodontitis ten randomly selected vascular biopsies from vd patients with periodontitis were selected at random for sem . vascular tissue biopsies and subgingival plaque samples were collected for extraction of genomic dna and checkerboard dna dna hybridization analysis , respectively . the vascular biopsies were collected under surgical treatment from the walls of aneurysms and during excision of intravascular plaques in carotid or common femoral arteries . in patients with cp , the deepest pockets with bleeding on probing were chosen , while in patients without cp , four sites were chosen randomly . universal bacterial primers were used for 16s rdna amplification under standardized conditions with forward primer : e334f 5-ccagactcctacgggaggcagc-3 and reverse primer : e939r 5-cttgtgcgggcccccgtcaattc-3 ( 23 ) . these primers cover the hypervariable region v3-v5 of the 16s rrna gene , have high specificity to bacterial sequences , and low match to eukarya and archaea sequences ( 23 ) . species - specific pcr assays and fima genotyping were applied for the identification of the periodontopathogen p. gingivalis in vascular biopsies from patients with cp . porphyromonas gingivalis 16s rdna and fima gene - specific primers used in this study each set of experiments included negative controls with sterile molecular grade water instead of template dna and positive controls containing purified dna from veillonella dispar , p. gingivalis strains for fima type i ( atcc 33277 t ) and fima type ii ( a7a1 - 28 ) . the p. gingivalis - specific pcr amplicons were sequenced using the bigdye terminator v1.1 cycle sequencing kit according to the manufacturer 's instructions and the respective forward primers ( table 1 ) . for identification of closest relatives , the consensus sequences were compared with known sequences in the genbank databases using the ncbi blast search tool ( http://www.ncbi.nlm.nih.gov/blast/ ) and human oral microbiome database ( homd ; http://www.homd.org/ ) ( 29 ) . ten randomly selected subgingival plaque samples from vd patients with cp were assessed for the presences of red complex bacteria ( rcb ) using checkerboard dna dna hybridization . naoh ( 100 l of 0.5 m ) was added to 100 l of subgingival plaque samples in te buffer and vortexed . bacterial species detected by dna dna hybridization ( checkerboard analysis ) in subgingival dental plaque samples from patients with atherosclerotic and abdominal aortic aneurysmal vascular disease and chronic periodontitis ten randomly selected vascular biopsies from vd patients with periodontitis were selected at random for sem . independent samples t - tests were used for comparisons of species diversity and bacterial load between the two groups . the second group ( vd without cp ) were patients undergoing treatment for vd and who were assessed without periodontitis ( n=47 , mean age=70.9 , sd= 9.7 , range : 45.784.8 , male=37/47 ( 79% ) . vascular biopsies from all 30 vd patients with cp and 10 randomly selected vd patients without cp were further analyzed for detection of 16s rdna sequences . in total , 27 biopsies from abdominal aortic aneurysmal walls , 9 from carotid , and 4 from common femoral atherosclerotic plaque were examined . demographic data of the study groups with atherosclerotic and abdominal aortic aneurysmal vascular disease ( vd ) , with and without chronic periodontitis ( cp ) based on a fisher 's exact test . after discarding incomplete sequences , a total of 2,638 sequences from vd patients with cp and 859 sequences from vd patients without cp were analyzed . after optimization of the pcr , weak pcr bands were detected in the negative pcr controls ( ~50% ) and were subsequently sequenced . after discarding unwanted sequences , the final fraction of bacterial sequences , reflecting overall bacterial load , obtained from the vascular biopsies in patients with cp was in average 6823% ( range : 098% ) at an individual level . eighty - four different bacterial taxa were detected from a total of 1,808 sequences from the vd patients with cp ( table 4 ) . from the vd patients without cp , 18 different taxa were identified from a total of 210 sequences ( table 5 ) . the relative abundance of the different taxa detected in vascular biopsies from patients with and without cp is presented in tables 4 and 5 , respectively . the mean diversity of bacterial taxa detected from the vascular biopsies was significantly higher in patients with cp ( n=30 ) , average of 7.13.2 ( range : 012 ) , compared to those without cp ( n=10 ) , average of 2.81.6 ( range : 05 ; p<0.01 ) . bacterial taxa identified in atherosclerotic plaque and abdominal aortic aneurysmal wall biopsies from patients with chronic periodontitis the different taxa were identified by ncbi blast analysis using a similarity threshold of 97% for identification possible overlap with bacterial taxa identified in vascular biopsies from vd patients without cp at bacterial taxa underlined are listed among the human oral microbial taxa in human oral microbiome database ( homd ) . bacterial taxa identified in atherosclerotic plaque and abdominal aortic aneurysmal wall biopsies from patients without chronic periodontitis the different taxa were identified by ncbi blast analysis using a similarity threshold of 97% for identification . possible overlap with bacterial taxa identified in vascular biopsies from vd patients with cp is highlighted in bold . bacterial taxa underlined are listed among the human oral microbial taxa in human oral microbiome database ( homd ) . patients with cp displayed a wide variety of bacteria identified at the genus level from their vascular biopsies , including some known oral bacterial taxa ( e.g. in total , 15% and 4.3% of the sequences were identified as not - yet - cultured bacterial spp . in the vd group with and without cp , respectively . using universal bacterial 16s rdna pcr , p. gingivalis was detected in the vascular biopsy from only one vd patients with cp . no other members of the rcb were detected from the vascular biopsies in either group . none of the 30 vascular biopsies from vd patients with cp were positive for p. gingivalis using the specific primers listed in table 1 . however , checkerboard analysis revealed presence of one to three members of the rcb species in 7 out of 10 subgingival plaque samples from the cp patients ( table 2 ) . cocci and rod - shaped bacterial cells of different sizes were seen in all biopsies ( fig . bacterial dna was detected in 95% of the vascular biopsies , and most biopsies contained dna from multiple bacterial species , pointing to a less specific relationship between single species and vd . these findings are supported by recent studies suggesting multiple bacterial species being involved in cardiovascular diseases ( 5 , 21 ) . we detected a significantly higher mean diversity and a higher load of bacteria in the vascular biopsies from the cp group as compared to the group without cp . in patients with cp , the defective epithelial barrier of the periodontal pockets together with a high load and diversity of bacteria in the subgingival plaque make it conceivable that the oral bacterial translocation and transient bacteremia are caused by dental procedures , oral infections , and may even follow tooth brushing and chewing ( 10 ) . even though rcbs were detected at high frequencies in the subgingival plaque samples , only p. gingivalis was identified from only one vascular biopsy from a patient with cp . in addition , we used p. gingivalis - specific primers ; however , none of these primers yielded positive detection of p. gingivalis in our vd biopsies . similarly , several other studies , using species - specific pcr , nested pcr and hybridization detection techniques , also reported no detection of rcb in atherosclerotic carotid lesions , despite the presence of one or more periodontopathogens in the patients subgingival plaque samples ( 3436 ) . a possible explanation to the negative or sparse detection of rcb in vd biopsies may be that , in contrast to commensal bacteria , more antigenic microbes such as the rcb are readily recognized by the immune system and eliminated from the circulation . ( 37 ) , streptococcus mutans strains serotype k survived in blood for a longer time due to lower antigenicity as compared to more cariogenic strains . in addition , a variation in ability to invade cardiovascular cells has been demonstrated among different p. gingivalis strains ( 38 ) . individual differences in harboring invasive strains may be an alternative explanation of the discrepancies in the detection of rcb in vascular tissue reported in the literature ( 11 ) . nevertheless , many studies have reported the presence of periodontopathogens in atherosclerotic lesions from aorta , coronary , carotid , and femoral arteries ( 14 , 16 , 17 , 41 ) , although with variable frequency ( 11 , 36 ) . however , even by using similar pcr - based techniques with the same specific primers , various detection rates for rcb ( 032% for p. gingivalis ) in peripheral atherosclerotic biopsies from patient with cp have been reported ( 41 , 42 ) . with the p. gingivalis fima ii primer set previously used for p. gingivalis detection in cardiovascular specimen ( 43 ) , these findings together with the human sequences identified , using bacterial - specific 16s rdna primers , emphasize the importance of identifying pcr products by sequencing in studies involving specimens where bacterial dna is sparse . dna from none of the other rcb however , the dna of periodontopathogens was detected in the atheromatous plaques from coronary arteries in a chinese population with cp ( 46 ) . the prevalence of t. forsythia , fusobacterium nucleatum , prevotella intermedia , and p. gingivalis was 31 , 12 , 18 , and 33% , respectively . the same specific primers for t. forsythia , f. nucleatum , and p. intermedia were used by cairo et al . due to scant amounts of pcr products , several rounds of cloning and sequencing had to be performed to obtain 96 clone libraries with sequences of adequate quality and size , especially when processing vd biopsies from patients without periodontitis . in these patients , the final fraction of bacterial sequences averaged 25% , and the majority of the excluded sequences were background sequences and eukaryote sequences ( supplementary file sb ) . thus , the number of vascular biopsies examined in this group was limited to 10 randomly selected biopsies . ( 47 ) using 16s rdna sequencing , reported the average number of different bacterial sequences from atherosclerotic lesions in patients without signs of clinical infections to be 2.21.2 , which is comparable to our patients without cp ( 2.81.6 ) . in subjects with cp , several common oral taxa were detected in the vascular biopsies , i.e. , while in patients without cp , only one common oral taxon was found ( streptococcus sp . ) these species and other possible oral taxa detected in the vascular biopsies are highlighted in tables 4 and 5 . although these taxa represent only a minor proportion of the oral bacterial microbiota , the data suggest that the oral cavity may be a potential source for bacterial dissemination to vascular tissue . due to the near absence of rcb in vascular biopsies , we wanted to confirm their presence in the subgingival plaque in cp patients by using checkerboard dna dna hybridization analysis . the checkerboard analysis revealed presence of one to three members of the rcb in 7 out of 10 subgingival plaque samples from the cp patients ( table 2 ) . found in the subgingival plaque by checkerboard analysis were detected in the vascular biopsies in the cp group ( i.e. , suggesting dissimilar bacterial composition in the subgingival plaque and vascular biopsies . in a previous study , 16s rdna - based pcr was positive for bacterial detection , whereas checkerboard dna dna hybridization methods were negative . this indicated that the number of rcb - positive subgingival plaque samples in our study could possibly be even higher ( 48 ) . the presence of periodontopathogens and other oral bacterial species were sparse in cardiovascular tissue , and only a few species , including s. mutans , may have originated from the oral cavity . a relatively high percentage ( 15.4% ) of the sequences detected in vascular biopsies from patients with cp belonged to the enterobacteriaceae family , i.e. in addition , enteric bacteria are known to be important nosocomial pathogens , and it can not be excluded that they were transmitted to the patients through the hospital environment ( 51 ) . the intestines harbor the largest number of bacterial cells in the human body , making it likely that a major part of the bacterial taxa detected in our vascular biopsies had originated from the intestines . ( 45 ) found several operational taxonomic units shared between the gut and atherosclerotic plaque suggesting that bacteria present in the atherosclerotic plaque could have been derived from the gut . we may hypothesize that low - grade chronic systemic inflammation caused by periodontal disease ( 56 ) may affect intestinal permeability and thereby translocation of enteric bacteria , dissemination to the bloodstream , and further colonization of vascular lesions , as seen in our vascular biopsies from patients with cp . much of the human microbiota is not yet cultivated ( 57 ) , and the presence of different not - yet - cultured bacterial taxa is to be expected . several environmental bacterial species with undefined pathogenicity were detected in the vd biopsies , such as members of the families chitinophagaceae and rhodobacteraceae , geobacter sp . through the skin and mucous membrane linings of the respiratory , gastrointestinal , and genitourinary tracts , some of the environmental bacteria detected in the vd biopsies may be part of our transient microbiota . several other studies have reported the presence of environmental species in vascular lesions and clinical samples ( 47 , 58 ) ; however , the pathogenicity and clinical relevance of most environmental bacterial taxa in patient samples are yet to be determined . acinetobacter has been isolated from periodontal pockets ( 50 ) , and is known to be a frequent cause of nosocomial infections , especially pneumonias , as well as bloodstream and urinary tract infections ( 62 , 63 ) . by sem , bacterial cells with different morphology these findings emphasize the presence of bacterial cells , not only bacterial dna , in the vd biopsies . others have suggested that bacteria from the circulation might get caught in the vascular lesions as however , animal studies have shown that bacterial taxa such as chlamydia pneumonia and p. gingivalis may invade atherosclerotic plaque , alter inflammatory pathways , and cause molecular mimicry , and thereby may contribute to the progression of atherogenesis ( 6 , 13 ) . in conclusion , our results indicate that the cumulative effect of different infectious agents may be associated with the pathophysiology of atherosclerotic and abdominal aortic aneurysmal vascular diseases . however , periodontopathogens were rarely identified in the vascular biopsies , whilst gut bacteria were detected more frequently . a possible association between chronic periodontal disease , systemic inflammation , intestinal permeability , and enterobacterial colonization of atherosclerotic and aneurysmal tissue should be studied further .

although many nongenetic factors , including age , concomitant therapy , drug interactions , and the nature of the disease , may influence the outcomes of medications , there are many inter - individual differences in drug response owing to the sequence variants of genes that encode drug - metabolizing enzymes , drug transporters , or drug targets . most of the drug - metabolizing enzymes and transporters have a broad range of genetic polymorphisms , which may cause inter - individual variability with different concentrations of drugs . in addition , anticancer therapies are known to have a narrow therapeutic range ; a high concentration in a patient s body increases the toxicity , and a low concentration decreases the effect of the drug . some of the single - nucleotide polymorphisms ( snps ) have already been correlated with substantial changes in the metabolism or efficacy of the drug , and some are being utilized to predict clinical outcomes . docetaxel has an effective antitumor activity against many cancers , such as locally advanced or metabolic breast cancer , non - small - cell lung cancer ( nsclc ) , and androgen - independent prostate cancer , and can induce response rates between 20% and 40% . however , despite its widespread use , an inter - individual variability in the toxicities of docetaxel has been a major challenge in clinical practice . dose - limiting toxicities of docetaxel are considered to be neutropenia , anemia , nausea , asthenia , and skin toxicity . among the severe toxicities , neutropenia is one of the dose - limiting adverse reactions and is often observed at a frequency of approximately 36% . the other side effects include alopecia , asthenia , dermatologic reactions , fluid retention , hypersensitivity reactions , and stomatitis . one of the most important factors that limit the use of docetaxel use is its unpredictability of inter - individual variation in toxicity . the potential causes of variability include pathogenesis and disease severity , occurrence of unintended drug interactions , and impairment of hepatic and/or renal functions . the elimination of docetaxel occurs mainly through a metabolic conversion by cyp3a4 and cyp3a5 , which results in a formation of metabolites with reduced cytotoxic activity . the biotransformation of docetaxel at the hepatic level leads to the inactivation of the molecule and reduction of its therapeutic effect . biotransformation is the main route for elimination of docetaxel , which makes it an interesting drug for the investigation of the genetic polymorphism in cyp450 enzymes . moreover , the correlation between high cyp3a4 mrna expression and low chemical susceptibility of docetaxel in breast cancer tissues has been shown by real - time polymerase chain reaction and immunohistochemistry . a high cyp3a4 mrna expression in tumor tissues can be predicted to accelerate the speed of docetaxel metabolism , and thus , results in resistance . the elimination pathway is widely known to be mediated by the drug efflux abc transporter , abcb1 ( also known as p - glycoprotein or mdr1 ) . moreover , the expression of these genes is regulated by the nuclear hormone receptors pregnane x receptor and constitutive androstane receptor encoded by nr1i2 and nr1i3 , respectively . other transporters , abcc2 ( mrp2 ) and slco1b3 ( otap1b3 or oatp8 ) , are responsible for the efflux and uptake of docetaxel in vitro . consequently , these genes are considered to be candidates that may affect the toxicity of docetaxel . there have been a few reports concerning the association between the toxicity of docetaxel and genetic variants of cyp3a4 , cyp3a5 , and abcb1 , and there were no reports on nr1i2 , nr1i3 , abcc2 , and slco1b3 . in this study , we investigated the role of cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 polymorphisms on docetaxel toxicity . we recruited a total of 92 patients , who were treated with docetaxel as a single agent or combination therapy between 2009 and 2011 ; clinical characteristics of patients are shown in table 1 . ethical permission for this study was obtained from the institutional review boards of seoul st . mary s hospital provided written informed consent , in accordance to the declaration of helsinki . other eligibility criteria included age ( 18 years or older ) , normal liver function , and performance status of less than 3 in accordance to the eastern cooperative oncology group criteria . ineligibility criteria include cytotoxic chemotherapy in the previous 4 weeks and corticoid treatment in the past 2 weeks . the demographics of age and gender , as well as indications for docetaxel therapy , additional medical problems , and concurrent medications were also recorded during the clinic visit . the dose of docetaxel was 60 mg / m or less in 12 patients and 60 - 80 mg / m in 80 patients . patients had lung , stomach , head and neck , as well as esophagus cancer , and who received docetaxel concomitantly with capecitabine , doxorubicin , cisplatin , cisplatin - cetuximab , and ifosfamide . we obtained hematologic toxicities , such as neutropenia , leukopenia , anemia , and thrombocytopenia , and also collected non - hematologic toxicities , including stomatitis , neuropathy , alopecia , diarrhea , and anorexia at the baseline of the pretreatment and nadir ( 10 - 14 days post - treatment ) . furthermore , clinical data , such as white blood cell count , neutrophil , and platelet counts , as well as hemoglobin values , were collected as the first baseline before chemotherapy and again at 10 - 14 days after the first chemotherapy cycle , if and when available . the polymorphisms for the cyp3a4 ( cyp3a41b , cyp3a418 , and cyp3a43 ) , cyp3a5 ( cyp3a52 and cyp3a53 ) , abcb1 ( c1236 t , g2677g / t , and c3435 t ) , slco1b3 ( rs11045585 ) , and abcc2 ( rs12762549 ) genes were analyzed ( http://www.ncbi.nlm.gov/ ) . for each sample , the genomic dna was isolated from the whole blood , using the qiaamp dna blood mini kit ( qiagen , germantown , md ) in accordance with the supplier s instructions . polymerase chain reaction ( pcr ) was performed using a hot start ace taq dna polymerase kit ( genenmed , seoul , korea ) . all the primers for pcr amplification and dna sequencing were designed using a primer3 software ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3 ) ; the sequences are available upon request . pcr reaction was carried out in a final volume of 25 l containing 10 buffer , 1.5 mmol / l mgcl2 , 20 mol / l dntp , 0.5 mol / l of each primer , 10 ng of genomic dna as template , and 0.5 u polymerase . each pcr product was purified and subjected to dna sequencing by using bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca ) and the abi prism 3730 genetic analyzer ( applied biosystems ) after confirming the purity and mobility of each pcr product by agarose gel electrophoresis . all statistical analyses were conducted using a statistical program , spss ver . 10.0 ( spss inc . , s exact test was used to determine the associations between different side effects after docetaxel treatment and the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes in accordance with suitable conditions . the odds ratio ( or ) and 95% confidence intervals ( ci ) of the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes for different side effects were estimated using a logistic regression . we recruited a total of 92 patients , who were treated with docetaxel as a single agent or combination therapy between 2009 and 2011 ; clinical characteristics of patients are shown in table 1 . ethical permission for this study was obtained from the institutional review boards of seoul st . mary s hospital provided written informed consent , in accordance to the declaration of helsinki . other eligibility criteria included age ( 18 years or older ) , normal liver function , and performance status of less than 3 in accordance to the eastern cooperative oncology group criteria . ineligibility criteria include cytotoxic chemotherapy in the previous 4 weeks and corticoid treatment in the past 2 weeks . the demographics of age and gender , as well as indications for docetaxel therapy , additional medical problems , and concurrent medications were also recorded during the clinic visit . the dose of docetaxel was 60 mg / m or less in 12 patients and 60 - 80 mg / m in 80 patients . patients had lung , stomach , head and neck , as well as esophagus cancer , and who received docetaxel concomitantly with capecitabine , doxorubicin , cisplatin , cisplatin - cetuximab , and ifosfamide . we obtained hematologic toxicities , such as neutropenia , leukopenia , anemia , and thrombocytopenia , and also collected non - hematologic toxicities , including stomatitis , neuropathy , alopecia , diarrhea , and anorexia at the baseline of the pretreatment and nadir ( 10 - 14 days post - treatment ) . furthermore , clinical data , such as white blood cell count , neutrophil , and platelet counts , as well as hemoglobin values , were collected as the first baseline before chemotherapy and again at 10 - 14 days after the first chemotherapy cycle , if and when available . the polymorphisms for the cyp3a4 ( cyp3a41b , cyp3a418 , and cyp3a43 ) , cyp3a5 ( cyp3a52 and cyp3a53 ) , abcb1 ( c1236 t , g2677g / t , and c3435 t ) , slco1b3 ( rs11045585 ) , and abcc2 ( rs12762549 ) genes were analyzed ( http://www.ncbi.nlm.gov/ ) . for each sample , the genomic dna was isolated from the whole blood , using the qiaamp dna blood mini kit ( qiagen , germantown , md ) in accordance with the supplier s instructions . polymerase chain reaction ( pcr ) was performed using a hot start ace taq dna polymerase kit ( genenmed , seoul , korea ) . all the primers for pcr amplification and dna sequencing were designed using a primer3 software ( http://frodo.wi.mit.edu/cgi-bin/primer3/primer3 ) ; the sequences are available upon request . pcr reaction was carried out in a final volume of 25 l containing 10 buffer , 1.5 mmol / l mgcl2 , 20 mol / l dntp , 0.5 mol / l of each primer , 10 ng of genomic dna as template , and 0.5 u polymerase . each pcr product was purified and subjected to dna sequencing by using bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca ) and the abi prism 3730 genetic analyzer ( applied biosystems ) after confirming the purity and mobility of each pcr product by agarose gel electrophoresis . all statistical analyses were conducted using a statistical program , spss ver . 10.0 ( spss inc . , chicago , il ) . the chi - square test or fisher s exact test was used to determine the associations between different side effects after docetaxel treatment and the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes in accordance with suitable conditions . the odds ratio ( or ) and 95% confidence intervals ( ci ) of the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes for different side effects were estimated using a logistic regression . table 1 describes the general characteristics of the eligible study subjects . we included 92 cancer patients ( 71 men and 21 women ) with a median age of 58.4 years ( range , 28 to 78 years ) . in brief , most of the cases had a single site ( right or left ) tumor , and 59.8% of the cases were diagnosed with lung cancer . the clinical stages of nearly 90% of the cases were stage iii and stage iv . the dose of docetaxel was 60 mg / m in 12 patients ( 13.0% ) , and 60 - 80 mg / m in 80 patients ( 87.0% ) . the occurrence frequencies of the side effects , including hematologic toxicities , such as neutropenia , leucopenia , anemia , and thrombocytopenia , after docetaxel treatment in patients were investigated ( data not shown ) . we analyzed all grades of toxicities , as well as grade 2 or higher . at nadir , 10 - 14 days after the first chemotherapy cycle , 74 out of the 92 patients were evaluated with regard to the occurrence of neutropenia , the main adverse effect . twenty - five patients ( 33.8% ) had grade 3 neutropenia and 45 ( 60.8% ) had grade 4 neutropenia . fifty - eight patients ( 85.3% ) had grades 1 and 2 anemia , and 38 ( 41.3% ) had non - hematologic toxicities , such as alopecia ( appendix 1 ) . the frequencies of the side effects , such as neutropenia , leucopenia , anemia , and thrombopenia , after docetaxel treatment in patients carrying various snps of cyp3a5 ( cyp3a53 ) , abcb1 ( 1236c > t , 2677g > t / a , and 3435c > t ) , abcc2 ( rs12762549 ) , and slco1b3 ( rs11045585 ) genes are shown in table 2 . the associations between the different snps of the abcb1 and abcc2 genes and the side effects after docetaxel treatment were analyzed . t / a and patients with leucopenia ( p=0.025 ) , as well as the abcb1 3435c > t snp and patients with neutropenia ( p=0.029 ) and anemia ( p=0.044 ) . however , we found no statistical significance between the frequencies of neutropenia and cyp3a53 , as well as abcb1 1236c > t and slco1b3 rs11045585 snps . these results are shown in table 2 . additionally , we estimated the or and 95% ci of the abcb1 and abcc2 snps , showing significant results . based on the results shown in table 2 , we conducted a multiple analysis to estimate the relative contributions of age , gender , tumor stage , dose of docetaxel , chemotherapy regimen , and two genetic polymorphisms to the docetaxel - induced leucopenia . leucopenia was dominant in the abcb1 2677g > t / a snp t / a allele carriers with or of 6.48-fold ( 95% ci , 1.92 to 21.94 ; p=0.003 ) . there were no significant associations between the side - effect occurrences and clinical factors , such as age , gender , tumor stage , dose of docetaxel , and chemotherapy regimen . the or ( 95% ci ) of leucopenia was 2.77 ( range , 0.65 to 11.92 ) for the dose of docetaxel ; however , there was no significant association . the frequencies of estimated tumor response , including complete response , partial response , stable disease , and progressive disease , after docetaxel treatment in patients carrying various snps of cyp3a4 ( 3 different sites ) , cyp3a5 ( 2 different sites ) , abcb1 ( 3 different sites ) , abcc2 ( rs12762549 ) , and slco1b ( rs11045585 ) genes are shown in table 4 . there was a significant difference between the partial disease and patients with the abcb1 2677tt / gt / ga / aa / ta genotype ( p=0.020 ) . in addition , we found a marginal association between tumor response and patients with the slco1b ( rs11045585 ) gg genotype , although without statistical significance ( table 4 ) . in a subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a ( or , 4.54 ) in abcb1 and slco1b3 ( or , 9.44 ) a multivariate analysis was performed to estimate the relative contributions of gender , tumor stage , chemotherapy regimens , and two genetic polymorphisms to the inter - individual variations of docetaxel - induced toxicity . the associations between the snps of abcb1 genes and docetaxel - induced toxicity were observed , and there was a significant difference between docetaxel - induced toxicity and patients with the slco1b3 rs11045585 gg genotype ( p=0.022 ) . in addition , we found an association between toxicity and chemotherapy regimen ( table 5 ) . table 1 describes the general characteristics of the eligible study subjects . we included 92 cancer patients ( 71 men and 21 women ) with a median age of 58.4 years ( range , 28 to 78 years ) . in brief , most of the cases had a single site ( right or left ) tumor , and 59.8% of the cases were diagnosed with lung cancer . the clinical stages of nearly 90% of the cases were stage iii and stage iv . the dose of docetaxel was 60 mg / m in 12 patients ( 13.0% ) , and 60 - 80 mg / m in 80 patients ( 87.0% ) . the occurrence frequencies of the side effects , including hematologic toxicities , such as neutropenia , leucopenia , anemia , and thrombocytopenia , after docetaxel treatment in patients were investigated ( data not shown ) . we analyzed all grades of toxicities , as well as grade 2 or higher . at nadir , 10 - 14 days after the first chemotherapy cycle , 74 out of the 92 patients were evaluated with regard to the occurrence of neutropenia , the main adverse effect . twenty - five patients ( 33.8% ) had grade 3 neutropenia and 45 ( 60.8% ) had grade 4 neutropenia . fifty - eight patients ( 85.3% ) had grades 1 and 2 anemia , and 38 ( 41.3% ) had non - hematologic toxicities , such as alopecia ( appendix 1 ) . the frequencies of the side effects , such as neutropenia , leucopenia , anemia , and thrombopenia , after docetaxel treatment in patients carrying various snps of cyp3a5 ( cyp3a53 ) , abcb1 ( 1236c > t , 2677g > t / a , and 3435c > t ) , abcc2 ( rs12762549 ) , and slco1b3 ( rs11045585 ) genes are shown in table 2 . the associations between the different snps of the abcb1 and abcc2 genes and the side effects after docetaxel treatment were analyzed . t / a and patients with leucopenia ( p=0.025 ) , as well as the abcb1 3435c > t snp and patients with neutropenia ( p=0.029 ) and anemia ( p=0.044 ) . however , we found no statistical significance between the frequencies of neutropenia and cyp3a53 , as well as abcb1 1236c > t and slco1b3 rs11045585 snps . additionally , we estimated the or and 95% ci of the abcb1 and abcc2 snps , showing significant results . based on the results shown in table 2 , we conducted a multiple analysis to estimate the relative contributions of age , gender , tumor stage , dose of docetaxel , chemotherapy regimen , and two genetic polymorphisms to the docetaxel - induced leucopenia . leucopenia was dominant in the abcb1 2677g > t / a snp t / a allele carriers with or of 6.48-fold ( 95% ci , 1.92 to 21.94 ; p=0.003 ) . there were no significant associations between the side - effect occurrences and clinical factors , such as age , gender , tumor stage , dose of docetaxel , and chemotherapy regimen . the or ( 95% ci ) of leucopenia was 2.77 ( range , 0.65 to 11.92 ) for the dose of docetaxel ; however , there was no significant association . the frequencies of estimated tumor response , including complete response , partial response , stable disease , and progressive disease , after docetaxel treatment in patients carrying various snps of cyp3a4 ( 3 different sites ) , cyp3a5 ( 2 different sites ) , abcb1 ( 3 different sites ) , abcc2 ( rs12762549 ) , and slco1b ( rs11045585 ) genes are shown in table 4 . there was a significant difference between the partial disease and patients with the abcb1 2677tt / gt / ga / aa / ta genotype ( p=0.020 ) . in addition , we found a marginal association between tumor response and patients with the slco1b ( rs11045585 ) gg genotype , although without statistical significance ( table 4 ) . in a subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a ( or , 4.54 ) in abcb1 and slco1b3 ( or , 9.44 ) a multivariate analysis was performed to estimate the relative contributions of gender , tumor stage , chemotherapy regimens , and two genetic polymorphisms to the inter - individual variations of docetaxel - induced toxicity . the associations between the snps of abcb1 genes and docetaxel - induced toxicity were observed , and there was a significant difference between docetaxel - induced toxicity and patients with the slco1b3 rs11045585 gg genotype ( p=0.022 ) . in addition , we found an association between toxicity and chemotherapy regimen ( table 5 ) . although several studies have shown an association between the polymorphisms and toxicity of anticancer drugs , such as docetaxel , the genetic determinants of the adverse reaction of docetaxel has not been elucidated . in this study , we genotyped 10 polymorphisms located in 5 candidate genes involved in the metabolism ( cyp3a4 and cyp3a5 ) and transport ( abcb1 , abcc2 , and slco1b3 ) of docetaxel to identify the genes related to the docetaxel - induced toxicity . we determined that 2677g / t in abcb1 and rs12762549 in abcc2 are significantly associated with severe docetaxel - induced leucopenia . in the subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a in abcb1 and rs11045585 in slco1b3 were also observed , suggesting that abcb1 and slco1b3 may be largely involved in the transport of docetaxel . most of the genes that encode the enzymes involved in the activation and detoxification pathways are considered to be highly polymorphic . in cancer treatment , inter - individual variations in a drug - metabolizing capacity and drug response can complicate the outcome of the same treatment even in patients who have been diagnosed with the same disease . in the present study , we found no association between the genotypes of the cyp3a4 and cyp3a5 genes and an increase in docetaxel toxicity . the disposition of docetaxel is mediated by cyp3a4 and cyp3a5 enzymes and transporters , such as abcb1 , abcc2 , and slco1b3 . the estimated quantitative contribution of cyp3a4/5 to the metabolism of docetaxel in the human liver is 64%-93% , making it the major cyp450 subfamily in docetaxel elimination . previously , several studies on patients with the cyp3a41a/1b ( rs2740574 ) and cyp3a51/3 ( rs776746 ) haplotypes showed an association with an increased clearance and/or decreased exposure to docetaxel . both rs2710574 and cyp3a51 alleles have shown to be in a linkage disequilibrium in caucasians and african americans . previous studies have shown that cyp3a4-cyp3a5 haplotypes may be correlated to certain diseases , impaired drug clearance , or undesirable adverse drug events . in asian cancer patients , however , another study observed no association between the clearance and cyp3a53 genotype status in cyp3a51/1 , cyp3a51/3 , and cyp3a53/3 patients . generally , variants of the coding regions in cyp3a4 have 5% allele frequencies as heterozygous with the wild - type allele . due to low allele frequencies , these coding variants are not likely to be the major cause of inter - individual differences in cyp3a - dependent clearance and limited alterations in enzyme expression or catalytic function . the most common variant , cyp3a41b , an a-392 g transition in the 5-flanking region , has an allele frequency that ranges from 0% ( chinese and japanese ) to 45% ( african americans ) . in our population in contrast , a linkage disequilibrium between cyp3a41b and another cyp3a allele ( cyp3a51 ) may be the cause of clinical phenotype . cyp3a5 is polymorphically expressed in adults : approximately 10%-20% in caucasians , 33% in japanese , and 55% in african - americans . however , it is unlikely that cyp3a4 and cyp3a5 polymorphisms will be of clinical benefit in influencing docetaxel disposition in our patients . drug transporters consist of uptake and efflux transporters , indicating intracellular or extracellular transport directions . these transporters play an essential role as the defense mechanisms against penetration of xenobiotics or transmembrane transportation of various endogenous compounds . in our study , 2677g / t in abcb1 and rs12762549 in abcc2 are significantly associated with severe docetaxel - induced leucopenia . among the abc proteins , some members , such as abcb1 , abcc1 , abcc2 , and abcg2 , recently , genetic polymorphisms in the abcb1 gene and their association with the p - gp level have been investigated . some studies examined 15 snps , including 6 in the coding region of healthy caucasians and found a significant association of polymorphism in exon 26 ( c3435 t ) of abcb1 with the expression levels and function of abcb1 . in a japanese population , 8 snps have been identified in the region of abcb1 , and among these snps , 2410t > c , 1910t > c , and 692t > c are in perfect linkage disequilibrium . in addition , several studies have shown that the inter - individual variations in activity and expression levels of abcb1 and abcc2 may have an effect on drug response and response to toxic agents the allelic frequencies of the non - synonymous variants 334t > g ( ser112ala ; rs4149117 ) and 699g > a ( met233lle ; rs7311358 ) display a great degree of heterogeneity across diverse ethnic populations , ranging from 41% in african - americans to approximately 71%-90% in caucasians and chinese . however , we estimate that the allelic frequency of slco1b3 ( g2677t / a ; rs11045585 ) is almost 1% in this population compared to that of the previous studies . this study shows a significant association of tumor response with g2677t / a and slco1b3 in a subgroup of lung cancer patients . this study highlights the importance of slco1b3 polymorphic variations in influencing docetaxel disposition in cancer patients . an important limitation associated with docetaxel use is the unpredictability of inter - individual efficacy and toxicity . in spite of the potential importance of the clinical variables in determining the drug effects , it is considered that inherited differences in metabolism and excretion several studies have attempted to identify the genetic polymorphisms in genes encoding drug metabolizing enzymes and transporters accounting for the remarkable inter - individual variation . for example , some researchers reported an increase in docetaxel clearance in carriers of cyp3a4 polymorphisms ( rs2740574 ) , a finding that was not supported by others . differences in the sample size , various tumor types , different treatment regimens ( single agent vs. combination of drugs interacting with docetaxel ) and various ethnic groups might be an explanation for these conflicting findings . additionally , most of these results were obtained from only a few snps in genes involved in docetaxel disposition . recently , maemondo et al . suggested that carboplatin and paclitaxel ( cp ) regimen achieved a longer progression - free survival with less toxicity , excluding moderate anemia and thrombocytopenia than single - agent docetaxel . considering the results of this phase ii and ifct-0501 trials , they selected the cp regimen as a candidate for a future phase iii trial in elderly patients with advanced nsclc . although monotherapy with the third - generation agents has been regarded as the preferred treatment option for elderly patients with nsclc , quoix et al . recently reported the results of ifct-0501 , a phase iii study comparing a similar cp regimen with monotherapy with either vinorelbine or gemcitabine in an elderly population . they demonstrated a significant superiority of the cp regimen in terms of the efficacy ( overall survival ) ; however , severe toxicity in the cp arm , including a treatment - related death rate of 4.4% , was observed . in this study , we found an association between toxicity and chemotherapy regimen as the previous studies in a multivariate logistic regression analysis , and docetaxel monotherapy might be more toxic than the combination therapy . our data suggest that abcb1 ( 2677g / t ) and slco1b3 ( rs11055585 ) may be major genetic predictors of docetaxel - related adverse events in patients receiving docetaxel chemotherapy .

purposedose - limiting toxicities of docetaxel are widely considered to be neutropenia , anemia , skin toxicity , and nausea . one of the factors that limit the use of docetaxel is its unpredictability of inter - individual variation in toxicity.materials and methodsin order to identify the genetic factors that affect the risk of docetaxel - induced toxicities , we recruited patients who received docetaxel chemotherapy . we genotyped 92 patients with single - nucleotide polymorphisms ( snps ) in 5 genes : cyp3a4 ( cyp3a4 * 1b , cyp3a4 * 18 , and cyp3a4 * 3 ) , cyp3a5 ( cyp3a5 * 2 and cyp3a5 * 3 ) , abcb1 ( c1236 t , g2677g / t , and c3435 t ) , slco1b3 ( rs11045585 ) , and abcc2 ( rs12762549).resultsout of 92 patients , 70 had grade 3 or 4 neutropenia ; 4 had grade 1 or 2 ; and 18 had no toxicity ( 76.1% , 4.3% , and 19.6% , respectively ) . the findings of the snp analysis showed that patients with tt genotype of abcb1 3435c > t polymorphism showed significantly higher risk of neutropenia and anemia ( p=0.029 and p=0.044 , respectively ) . there were significant associations between docetaxel - induced leucopenia and 2677g / t of abcb1 and rs12762549 of abcc2 ( p=0.025 and p=0.028 , respectively ) . in a multivariate analysis , we observed that patients carrying 2677g > t in abcb1might be associated with higher risk of chemo - resistance when treated with docetaxel ( odds ratio [ or ] , 6.48 ; confidence interval , 1.92 to 21.94 ; p=0.003 ) . in a subgroup analysis of non - small cell lung cancer patients , a significant association of tumor response with g2677t / a ( or , 4.54 ) in abcb1 and slco1b3 ( or , 9.44 ) was observed.conclusionour data suggest that abcb1 ( 2677g / t ) and slco1b3 ( rs11055585 ) might be major genetic predictors of docetaxel - related toxicities in patients receiving docetaxel chemotherapy .

Introduction Materials and Methods 1. Study subjects 2. Analysis of the 3. Statistical analysis Results 1. Study subjects clinical characteristics 2. Association between polymorphisms of 3. Risks of toxicity between 4. Associations between genetic polymorphisms and tumor response in the 92 cancer patients 5. Multivariate analysis to estimate the relative contributions of clinical factors to docetaxel toxicity Discussion Conclusion

although many nongenetic factors , including age , concomitant therapy , drug interactions , and the nature of the disease , may influence the outcomes of medications , there are many inter - individual differences in drug response owing to the sequence variants of genes that encode drug - metabolizing enzymes , drug transporters , or drug targets . in addition , anticancer therapies are known to have a narrow therapeutic range ; a high concentration in a patient s body increases the toxicity , and a low concentration decreases the effect of the drug . some of the single - nucleotide polymorphisms ( snps ) have already been correlated with substantial changes in the metabolism or efficacy of the drug , and some are being utilized to predict clinical outcomes . docetaxel has an effective antitumor activity against many cancers , such as locally advanced or metabolic breast cancer , non - small - cell lung cancer ( nsclc ) , and androgen - independent prostate cancer , and can induce response rates between 20% and 40% . however , despite its widespread use , an inter - individual variability in the toxicities of docetaxel has been a major challenge in clinical practice . dose - limiting toxicities of docetaxel are considered to be neutropenia , anemia , nausea , asthenia , and skin toxicity . among the severe toxicities , neutropenia is one of the dose - limiting adverse reactions and is often observed at a frequency of approximately 36% . one of the most important factors that limit the use of docetaxel use is its unpredictability of inter - individual variation in toxicity . the elimination of docetaxel occurs mainly through a metabolic conversion by cyp3a4 and cyp3a5 , which results in a formation of metabolites with reduced cytotoxic activity . other transporters , abcc2 ( mrp2 ) and slco1b3 ( otap1b3 or oatp8 ) , are responsible for the efflux and uptake of docetaxel in vitro . consequently , these genes are considered to be candidates that may affect the toxicity of docetaxel . there have been a few reports concerning the association between the toxicity of docetaxel and genetic variants of cyp3a4 , cyp3a5 , and abcb1 , and there were no reports on nr1i2 , nr1i3 , abcc2 , and slco1b3 . in this study , we investigated the role of cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 polymorphisms on docetaxel toxicity . we recruited a total of 92 patients , who were treated with docetaxel as a single agent or combination therapy between 2009 and 2011 ; clinical characteristics of patients are shown in table 1 . patients had lung , stomach , head and neck , as well as esophagus cancer , and who received docetaxel concomitantly with capecitabine , doxorubicin , cisplatin , cisplatin - cetuximab , and ifosfamide . we obtained hematologic toxicities , such as neutropenia , leukopenia , anemia , and thrombocytopenia , and also collected non - hematologic toxicities , including stomatitis , neuropathy , alopecia , diarrhea , and anorexia at the baseline of the pretreatment and nadir ( 10 - 14 days post - treatment ) . the polymorphisms for the cyp3a4 ( cyp3a41b , cyp3a418 , and cyp3a43 ) , cyp3a5 ( cyp3a52 and cyp3a53 ) , abcb1 ( c1236 t , g2677g / t , and c3435 t ) , slco1b3 ( rs11045585 ) , and abcc2 ( rs12762549 ) genes were analyzed ( http://www.ncbi.nlm.gov/ ) . pcr reaction was carried out in a final volume of 25 l containing 10 buffer , 1.5 mmol / l mgcl2 , 20 mol / l dntp , 0.5 mol / l of each primer , 10 ng of genomic dna as template , and 0.5 u polymerase . , s exact test was used to determine the associations between different side effects after docetaxel treatment and the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes in accordance with suitable conditions . the odds ratio ( or ) and 95% confidence intervals ( ci ) of the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes for different side effects were estimated using a logistic regression . we recruited a total of 92 patients , who were treated with docetaxel as a single agent or combination therapy between 2009 and 2011 ; clinical characteristics of patients are shown in table 1 . we obtained hematologic toxicities , such as neutropenia , leukopenia , anemia , and thrombocytopenia , and also collected non - hematologic toxicities , including stomatitis , neuropathy , alopecia , diarrhea , and anorexia at the baseline of the pretreatment and nadir ( 10 - 14 days post - treatment ) . the polymorphisms for the cyp3a4 ( cyp3a41b , cyp3a418 , and cyp3a43 ) , cyp3a5 ( cyp3a52 and cyp3a53 ) , abcb1 ( c1236 t , g2677g / t , and c3435 t ) , slco1b3 ( rs11045585 ) , and abcc2 ( rs12762549 ) genes were analyzed ( http://www.ncbi.nlm.gov/ ) . pcr reaction was carried out in a final volume of 25 l containing 10 buffer , 1.5 mmol / l mgcl2 , 20 mol / l dntp , 0.5 mol / l of each primer , 10 ng of genomic dna as template , and 0.5 u polymerase . the chi - square test or fisher s exact test was used to determine the associations between different side effects after docetaxel treatment and the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes in accordance with suitable conditions . the odds ratio ( or ) and 95% confidence intervals ( ci ) of the polymorphisms of the cyp3a4 , cyp3a5 , abcb1 , slco1b3 , and abcc2 genes for different side effects were estimated using a logistic regression . the dose of docetaxel was 60 mg / m in 12 patients ( 13.0% ) , and 60 - 80 mg / m in 80 patients ( 87.0% ) . the occurrence frequencies of the side effects , including hematologic toxicities , such as neutropenia , leucopenia , anemia , and thrombocytopenia , after docetaxel treatment in patients were investigated ( data not shown ) . at nadir , 10 - 14 days after the first chemotherapy cycle , 74 out of the 92 patients were evaluated with regard to the occurrence of neutropenia , the main adverse effect . twenty - five patients ( 33.8% ) had grade 3 neutropenia and 45 ( 60.8% ) had grade 4 neutropenia . fifty - eight patients ( 85.3% ) had grades 1 and 2 anemia , and 38 ( 41.3% ) had non - hematologic toxicities , such as alopecia ( appendix 1 ) . the frequencies of the side effects , such as neutropenia , leucopenia , anemia , and thrombopenia , after docetaxel treatment in patients carrying various snps of cyp3a5 ( cyp3a53 ) , abcb1 ( 1236c > t , 2677g > t / a , and 3435c > t ) , abcc2 ( rs12762549 ) , and slco1b3 ( rs11045585 ) genes are shown in table 2 . the associations between the different snps of the abcb1 and abcc2 genes and the side effects after docetaxel treatment were analyzed . t / a and patients with leucopenia ( p=0.025 ) , as well as the abcb1 3435c > t snp and patients with neutropenia ( p=0.029 ) and anemia ( p=0.044 ) . however , we found no statistical significance between the frequencies of neutropenia and cyp3a53 , as well as abcb1 1236c > t and slco1b3 rs11045585 snps . additionally , we estimated the or and 95% ci of the abcb1 and abcc2 snps , showing significant results . based on the results shown in table 2 , we conducted a multiple analysis to estimate the relative contributions of age , gender , tumor stage , dose of docetaxel , chemotherapy regimen , and two genetic polymorphisms to the docetaxel - induced leucopenia . leucopenia was dominant in the abcb1 2677g > t / a snp t / a allele carriers with or of 6.48-fold ( 95% ci , 1.92 to 21.94 ; p=0.003 ) . there were no significant associations between the side - effect occurrences and clinical factors , such as age , gender , tumor stage , dose of docetaxel , and chemotherapy regimen . the frequencies of estimated tumor response , including complete response , partial response , stable disease , and progressive disease , after docetaxel treatment in patients carrying various snps of cyp3a4 ( 3 different sites ) , cyp3a5 ( 2 different sites ) , abcb1 ( 3 different sites ) , abcc2 ( rs12762549 ) , and slco1b ( rs11045585 ) genes are shown in table 4 . in addition , we found a marginal association between tumor response and patients with the slco1b ( rs11045585 ) gg genotype , although without statistical significance ( table 4 ) . in a subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a ( or , 4.54 ) in abcb1 and slco1b3 ( or , 9.44 ) a multivariate analysis was performed to estimate the relative contributions of gender , tumor stage , chemotherapy regimens , and two genetic polymorphisms to the inter - individual variations of docetaxel - induced toxicity . the associations between the snps of abcb1 genes and docetaxel - induced toxicity were observed , and there was a significant difference between docetaxel - induced toxicity and patients with the slco1b3 rs11045585 gg genotype ( p=0.022 ) . in brief , most of the cases had a single site ( right or left ) tumor , and 59.8% of the cases were diagnosed with lung cancer . the occurrence frequencies of the side effects , including hematologic toxicities , such as neutropenia , leucopenia , anemia , and thrombocytopenia , after docetaxel treatment in patients were investigated ( data not shown ) . at nadir , 10 - 14 days after the first chemotherapy cycle , 74 out of the 92 patients were evaluated with regard to the occurrence of neutropenia , the main adverse effect . twenty - five patients ( 33.8% ) had grade 3 neutropenia and 45 ( 60.8% ) had grade 4 neutropenia . fifty - eight patients ( 85.3% ) had grades 1 and 2 anemia , and 38 ( 41.3% ) had non - hematologic toxicities , such as alopecia ( appendix 1 ) . the frequencies of the side effects , such as neutropenia , leucopenia , anemia , and thrombopenia , after docetaxel treatment in patients carrying various snps of cyp3a5 ( cyp3a53 ) , abcb1 ( 1236c > t , 2677g > t / a , and 3435c > t ) , abcc2 ( rs12762549 ) , and slco1b3 ( rs11045585 ) genes are shown in table 2 . the associations between the different snps of the abcb1 and abcc2 genes and the side effects after docetaxel treatment were analyzed . t / a and patients with leucopenia ( p=0.025 ) , as well as the abcb1 3435c > t snp and patients with neutropenia ( p=0.029 ) and anemia ( p=0.044 ) . however , we found no statistical significance between the frequencies of neutropenia and cyp3a53 , as well as abcb1 1236c > t and slco1b3 rs11045585 snps . additionally , we estimated the or and 95% ci of the abcb1 and abcc2 snps , showing significant results . based on the results shown in table 2 , we conducted a multiple analysis to estimate the relative contributions of age , gender , tumor stage , dose of docetaxel , chemotherapy regimen , and two genetic polymorphisms to the docetaxel - induced leucopenia . leucopenia was dominant in the abcb1 2677g > t / a snp t / a allele carriers with or of 6.48-fold ( 95% ci , 1.92 to 21.94 ; p=0.003 ) . there were no significant associations between the side - effect occurrences and clinical factors , such as age , gender , tumor stage , dose of docetaxel , and chemotherapy regimen . the frequencies of estimated tumor response , including complete response , partial response , stable disease , and progressive disease , after docetaxel treatment in patients carrying various snps of cyp3a4 ( 3 different sites ) , cyp3a5 ( 2 different sites ) , abcb1 ( 3 different sites ) , abcc2 ( rs12762549 ) , and slco1b ( rs11045585 ) genes are shown in table 4 . in addition , we found a marginal association between tumor response and patients with the slco1b ( rs11045585 ) gg genotype , although without statistical significance ( table 4 ) . in a subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a ( or , 4.54 ) in abcb1 and slco1b3 ( or , 9.44 ) a multivariate analysis was performed to estimate the relative contributions of gender , tumor stage , chemotherapy regimens , and two genetic polymorphisms to the inter - individual variations of docetaxel - induced toxicity . the associations between the snps of abcb1 genes and docetaxel - induced toxicity were observed , and there was a significant difference between docetaxel - induced toxicity and patients with the slco1b3 rs11045585 gg genotype ( p=0.022 ) . although several studies have shown an association between the polymorphisms and toxicity of anticancer drugs , such as docetaxel , the genetic determinants of the adverse reaction of docetaxel has not been elucidated . in this study , we genotyped 10 polymorphisms located in 5 candidate genes involved in the metabolism ( cyp3a4 and cyp3a5 ) and transport ( abcb1 , abcc2 , and slco1b3 ) of docetaxel to identify the genes related to the docetaxel - induced toxicity . we determined that 2677g / t in abcb1 and rs12762549 in abcc2 are significantly associated with severe docetaxel - induced leucopenia . in the subgroup analysis of nsclc patients , significant associations of tumor response and g2677t / a in abcb1 and rs11045585 in slco1b3 were also observed , suggesting that abcb1 and slco1b3 may be largely involved in the transport of docetaxel . in cancer treatment , inter - individual variations in a drug - metabolizing capacity and drug response can complicate the outcome of the same treatment even in patients who have been diagnosed with the same disease . in the present study , we found no association between the genotypes of the cyp3a4 and cyp3a5 genes and an increase in docetaxel toxicity . the disposition of docetaxel is mediated by cyp3a4 and cyp3a5 enzymes and transporters , such as abcb1 , abcc2 , and slco1b3 . due to low allele frequencies , these coding variants are not likely to be the major cause of inter - individual differences in cyp3a - dependent clearance and limited alterations in enzyme expression or catalytic function . in our study , 2677g / t in abcb1 and rs12762549 in abcc2 are significantly associated with severe docetaxel - induced leucopenia . some studies examined 15 snps , including 6 in the coding region of healthy caucasians and found a significant association of polymorphism in exon 26 ( c3435 t ) of abcb1 with the expression levels and function of abcb1 . in addition , several studies have shown that the inter - individual variations in activity and expression levels of abcb1 and abcc2 may have an effect on drug response and response to toxic agents the allelic frequencies of the non - synonymous variants 334t > g ( ser112ala ; rs4149117 ) and 699g > a ( met233lle ; rs7311358 ) display a great degree of heterogeneity across diverse ethnic populations , ranging from 41% in african - americans to approximately 71%-90% in caucasians and chinese . however , we estimate that the allelic frequency of slco1b3 ( g2677t / a ; rs11045585 ) is almost 1% in this population compared to that of the previous studies . this study shows a significant association of tumor response with g2677t / a and slco1b3 in a subgroup of lung cancer patients . an important limitation associated with docetaxel use is the unpredictability of inter - individual efficacy and toxicity . in spite of the potential importance of the clinical variables in determining the drug effects , it is considered that inherited differences in metabolism and excretion several studies have attempted to identify the genetic polymorphisms in genes encoding drug metabolizing enzymes and transporters accounting for the remarkable inter - individual variation . they demonstrated a significant superiority of the cp regimen in terms of the efficacy ( overall survival ) ; however , severe toxicity in the cp arm , including a treatment - related death rate of 4.4% , was observed . in this study , we found an association between toxicity and chemotherapy regimen as the previous studies in a multivariate logistic regression analysis , and docetaxel monotherapy might be more toxic than the combination therapy . our data suggest that abcb1 ( 2677g / t ) and slco1b3 ( rs11055585 ) may be major genetic predictors of docetaxel - related adverse events in patients receiving docetaxel chemotherapy .

thoracic ossification of the posterior longitudinal ligament ( t - opll ) causes severe myelopathy and may lead to permanent confinement to a wheelchair . surgery is required in most cases and while many surgical procedures have been reported,1 2 3 4 5 6 7 8 9 10 11 all carry a high risk of postoperative paralysis . decompression and instrumented fusion surgery for t - opll have recently been shown to have a favorable outcome.9 12 13 we have performed posterior decompression and dekyphotic corrective fusion with instrumentation since 1999 , especially for beak - type t - opll . we reported on the first 20 patients in 2009 , most of whom experienced an improvement in preoperative symptoms after the surgery.14 patients whose condition did not improve or whose preoperative symptoms were aggravated , subsequently underwent an anterior decompression of the spinal cord with resection of the t - opll from the posterior side . this approach , first reported by ohtsuka et al,15 has the major drawback of postoperative aggravation of motor function . therefore , we performed a modified ohtsuka procedure with pediculectomy , resection of the transverse process , spinal root sacrifice , and concomitant costotransversectomy as needed under intraoperative neurophysiological monitoring ( ionm ) . we refer to this procedure as resection at an anterior site of the spinal cord from a posterior approach , we describe the surgical procedures , ionm , and outcomes as a technical note in three cases treated with raspa after paralysis resulting from posterior decompression and dekyphotic corrective fusion with instrumentation for beak - type t - opll . fifty - eight consecutive patients ( 28 males and 30 females , average age 54 years old ) with beak - type t - opll underwent posterior decompression and dekyphotic corrective fusion with instrumentation at our institute . among these patients , 3 ( 5% ) underwent raspa surgery due to a lack of improvement after the initial surgery , or due to the aggravation of motor paralysis postoperatively . the mean follow - up period was 4.3 years ( range , 2 to 7 years ) . the disease duration , pre- and postoperative ambulatory status with mmt , operative time , estimated blood loss , decompressed and fused area of the thoracic spine , change in cobb angle of the thoracic spine from before to after surgery , intraoperative ultrasonography findings , ionm findings ( transcranial motor evoked potentials [ 32 channels ] ) , intraoperative and postoperative complications , and outcomes after the first and second surgery were prospectively evaluated . outcomes were assessed using the modified japanese orthopaedic association ( joa ) score , based on a possible total of 11 points ( excluding the cervical score from the original joa score).16 17 an unpaired t test and a fisher exact test were used to compare preoperative joa scores and surgical outcomes for patients who did and who did not undergo raspa . posterior decompression and dekyphotic corrective fusion with instrumentation was beak - type opll with severe anterior spinal cord compression . we described this surgical procedure in 2009.14 briefly , after pedicle screws were gently inserted , a temporary rod ( titanium alloy or cobalt - chromium alloy rod of 6 mm in diameter , which was the most rigid rod available at that time ) was placed on either side ; a laminectomy was then performed using an air drill . in order to achieve further decompression of the spinal cord , rigid bilateral rods for kyphosis reduction were placed using the cantilever technique ; this was done with the aid of spinal cord monitoring . , we informed the patients and families about the possible need for a second decompression surgery using raspa if there was no improvement or if motor paralysis was exacerbated after the first surgery.18 the raspa surgery proceeded as follows . first , a unilateral rod and several pedicle screws were removed at the level of the opll resection . because a laminectomy had already been performed in the first surgery , a resection of the transverse process and pedicle was necessary ( fig . next , a posterior partial osteotomy of vertebra adjacent to beak - type t - opll levels and an opll resection were performed with an air drill from the posterolateral direction , avoiding spinal cord compression ( fig . if these surgical devices were obstructed by a posterior rib , a partial resection of the rib ( so - called costotransversectomy ) was performed . we leaned the air drill as laterally as possible so that we could resect the opll of the anterior spinal cord ( fig . after resetting the temporary rod , we performed the same procedure on the contralateral side according to opll extension ( fig . while total resection of the opll is ideal , a residual thin opll may help avoid cerebrospinal fluid leakage . a thin opll does not prevent spinal cord decompression with postoperative dural sac enlargement after floating surgery for cervical opll.19 20 in our study , the cerebrospinal fluid pressure resulted in the gradual anterior migration of the floating opll after about 6 weeks . we confirmed the amount of spinal cord decompression and central opll resection with ultrasonography , as well as with intraoperative computed tomography ( ct ) scan when available ( fig . importantly , we did not retract or rotate the vulnerable spinal cord in beak - type t - opll because this movement can lead to severe spinal cord damage and permanent motor paralysis after surgery . if the ionm amplitude was decreased , the surgical procedure was suspended , the surgical field was filled with warm saline , and the blood pressure and body temperature were increased until normal amplitude was restored . a posterior interbody fusion with local bone graft was also performed at the opll resection level . this procedure was selected because diskectomy is typically easy , although in some cases ossification of the anterior longitudinal ligament ( oall ) and fusion of the thoracic spine may occur spontaneously after surgery ( even if interbody fusion was not achieved ) . surgical procedures in resection at an anterior site of the spinal cord from a posterior approach ( raspa ) surgery . resection of the transverse process and pedicle with spinal root sacrifice at the ossification of the posterior longitudinal ligament ( opll ) resection level . ( b ) posterior partial osteotomy of vertebra and opll resection with an air drill from the posterolateral direction , with costotransversectomy if needed . ( c ) we did not retract or rotate the vulnerable thoracic spinal cord to avoid spinal cord injury . ( d ) same procedure on the contralateral side according to opll extension after resetting the temporary rod . ( e ) spinal cord decompression was achieved with confirmation of the subarachnoid space in intraoperative ultrasonography . ( f ) after removal of the opll , additional dekyphosis and compression maneuver with a bilateral rod were performed , which gave indirect decompression at other opll levels with spinal cord compression and spinal cord shortening . local bone graft was performed at the opll resection level because diskectomy is usually straightforward . postoperatively , patients were prescribed several days of bed rest with drainage tubes in order to avoid a hematoma . therapy at this stage included a lower extremity range of motion ( rom ) exercise and tilt table standing . fifty - eight consecutive patients ( 28 males and 30 females , average age 54 years old ) with beak - type t - opll underwent posterior decompression and dekyphotic corrective fusion with instrumentation at our institute . among these patients , 3 ( 5% ) underwent raspa surgery due to a lack of improvement after the initial surgery , or due to the aggravation of motor paralysis postoperatively . the mean follow - up period was 4.3 years ( range , 2 to 7 years ) . the disease duration , pre- and postoperative ambulatory status with mmt , operative time , estimated blood loss , decompressed and fused area of the thoracic spine , change in cobb angle of the thoracic spine from before to after surgery , intraoperative ultrasonography findings , ionm findings ( transcranial motor evoked potentials [ 32 channels ] ) , intraoperative and postoperative complications , and outcomes after the first and second surgery were prospectively evaluated . outcomes were assessed using the modified japanese orthopaedic association ( joa ) score , based on a possible total of 11 points ( excluding the cervical score from the original joa score).16 17 an unpaired t test and a fisher exact test were used to compare preoperative joa scores and surgical outcomes for patients who did and who did not undergo raspa . the indication for the first surgery posterior decompression and dekyphotic corrective fusion with instrumentation was beak - type opll with severe anterior spinal cord compression . we described this surgical procedure in 2009.14 briefly , after pedicle screws were gently inserted , a temporary rod ( titanium alloy or cobalt - chromium alloy rod of 6 mm in diameter , which was the most rigid rod available at that time ) was placed on either side ; a laminectomy was then performed using an air drill . in order to achieve further decompression of the spinal cord , rigid bilateral rods for kyphosis reduction were placed using the cantilever technique ; this was done with the aid of spinal cord monitoring . , we informed the patients and families about the possible need for a second decompression surgery using raspa if there was no improvement or if motor paralysis was exacerbated after the first surgery.18 the raspa surgery proceeded as follows . first , a unilateral rod and several pedicle screws were removed at the level of the opll resection . because a laminectomy had already been performed in the first surgery , a resection of the transverse process and pedicle was necessary ( fig . next , a posterior partial osteotomy of vertebra adjacent to beak - type t - opll levels and an opll resection were performed with an air drill from the posterolateral direction , avoiding spinal cord compression ( fig . if these surgical devices were obstructed by a posterior rib , a partial resection of the rib ( so - called costotransversectomy ) was performed . we leaned the air drill as laterally as possible so that we could resect the opll of the anterior spinal cord ( fig . after resetting the temporary rod , we performed the same procedure on the contralateral side according to opll extension ( fig . while total resection of the opll is ideal , a residual thin opll may help avoid cerebrospinal fluid leakage . a thin opll does not prevent spinal cord decompression with postoperative dural sac enlargement after floating surgery for cervical opll.19 20 in our study , the cerebrospinal fluid pressure resulted in the gradual anterior migration of the floating opll after about 6 weeks . we confirmed the amount of spinal cord decompression and central opll resection with ultrasonography , as well as with intraoperative computed tomography ( ct ) scan when available ( fig . importantly , we did not retract or rotate the vulnerable spinal cord in beak - type t - opll because this movement can lead to severe spinal cord damage and permanent motor paralysis after surgery . if the ionm amplitude was decreased , the surgical procedure was suspended , the surgical field was filled with warm saline , and the blood pressure and body temperature were increased until normal amplitude was restored . a posterior interbody fusion with local bone graft was also performed at the opll resection level . this procedure was selected because diskectomy is typically easy , although in some cases ossification of the anterior longitudinal ligament ( oall ) and fusion of the thoracic spine may occur spontaneously after surgery ( even if interbody fusion was not achieved ) . surgical procedures in resection at an anterior site of the spinal cord from a posterior approach ( raspa ) surgery . resection of the transverse process and pedicle with spinal root sacrifice at the ossification of the posterior longitudinal ligament ( opll ) resection level . ( b ) posterior partial osteotomy of vertebra and opll resection with an air drill from the posterolateral direction , with costotransversectomy if needed . ( c ) we did not retract or rotate the vulnerable thoracic spinal cord to avoid spinal cord injury . ( d ) same procedure on the contralateral side according to opll extension after resetting the temporary rod . ( e ) spinal cord decompression was achieved with confirmation of the subarachnoid space in intraoperative ultrasonography . ( f ) after removal of the opll , additional dekyphosis and compression maneuver with a bilateral rod were performed , which gave indirect decompression at other opll levels with spinal cord compression and spinal cord shortening . local bone graft was performed at the opll resection level because diskectomy is usually straightforward . postoperatively , patients were prescribed several days of bed rest with drainage tubes in order to avoid a hematoma . therapy at this stage included a lower extremity range of motion ( rom ) exercise and tilt table standing . all of the patients had four levels of beak - type t - opll involving ossification of the ligamentum flavum , with severe motor paralysis that prevented walking . as shown in table 2 , in the first operation , all of the patients underwent concomitant cervical decompression surgery and five to eight levels of spinal fusion with thoracic dekyphosis . the rate of concomitant cervical laminoplasty and the number of levels of laminectomy and spine fusion did not differ significantly between patients who underwent raspa and those who did not . in the first surgery , ionm findings deteriorated in all cases , but there were no other intraoperative complications . none of the three patients developed a hematoma or experienced implant failure after the first surgery , but two reoperations were required due to hematoma in patients who did not undergo raspa . after the first surgery , no motor recovery was found in any of the three patients after several weeks of bed rest . abbreviations : bmi , body mass index ; joa score , japanese orthopaedic association score ; mmt , manual muscle test ; olf , ossification of the ligamentum flavum ; opll , ossification of the posterior longitudinal ligament . abbreviations : ebl , estimated blood loss ; ionm , intraoperative neurophysiological monitoring ; mmt , manual muscle test . all three patients underwent raspa as a second surgery , which was performed 3 weeks after the first surgery . the intraoperative findings in the second surgery are shown in table 3 . in the raspa surgery , one to three levels of the opll were resected from the posterior side with two to six pediculectomy and one to four roots sacrificed . per ultrasound measurement , spinal cord floating two of the three patients had an intraoperative pinhole dural tear , both of which were covered with fibrin glue ( beriplast p , csl behring , tokyo , japan ) after dural suture . each of the three patients experienced an improvement in motor function ( beginning on postoperative days 1 , 2 , and 17 , respectively ) with a gradual reduction in motor paralysis over several months . 2 ) and were able to walk with a cane , representing a better ambulatory status than before the first surgery . preoperative and postoperative course of manual muscle test ( mmt ) scores for the lower extremity . after the second surgery ( resection at an anterior site of the spinal cord from a posterior approach ) , the three cases achieved almost full mmt scores in 3 days , 4.5 months , and 7 months , respectively ( shown in table 4 ) . abbreviations : bil . , bilateral ; ebl , estimated blood loss ; lt . , left ; opll , ossification of the posterior longitudinal ligament ; raspa , resection at an anterior site of the spinal cord from a posterior approach ; ionm , intraoperative neurophysiological monitoring . abbreviations : joa score , japanese orthopaedic association score ; mmt , manual muscle test ; raspa , resection at an anterior site of the spinal cord from a posterior approach . total resection of beak - type t - opll and more dekyphosis was achieved after raspa surgery ( fig . 3d , e ) , with gradual improvement of the paralyzed lower extremity . at 4 years after raspa surgery , ct images showed stability and spinal fusion with oall extension ( fig . 3f ) ; at final follow - up the patient 's ambulatory status had improved to walking with a single cane . ( b , c ) lateral plain radiograph and ct sagittal image after posterior decompression and instrumented fusion ( first surgery ) . ( d , e ) postoperative lateral plain radiograph and ct sagittal image after resection at an anterior site of the spinal cord from a posterior approach ( raspa ; second surgery , arrow ) . ( f ) at final follow - up , the ossification of the anterior longitudinal ligament was also extended and fused , with stabilization of the thoracic spine ( arrow ) . while there are several approaches for t - opll surgery , the optimal method is unclear because of the high rates of postoperative motor disturbance and complications.2 3 4 5 6 7 8 9 10 11 14 posterior decompression and fusion surgery for t - opll typically results in good surgical outcomes12 14 21 ; this surgical procedure also has the advantage of concomitant cervical laminoplasty . all three patients who underwent raspa surgery had concomitant cervical laminoplasty . because this had no impact on the outcome of the first thoracic surgery there is no need to avoid one - stage combined posterior surgery for cervical and thoracic opll . however , some patients experience motor paralysis or severe myelopathy postoperatively . therefore , we informed all patients prior to the first surgery of the option of additional anterior decompression with a posterior approach if symptoms show no improvement at about 3 weeks after the first surgery or if symptoms worsen . the two - stage posterior decompression with dekyphosis and anterior decompression surgery proposed by kawahara et al involve decompression along the circumferential spinal cord with spinal fusion ( the ideal decompression).3 kawahara et al also suggested a need for additional anterior decompression from an anterior approach at 3 weeks after the posterior surgery . we also believe that a second surgery of anterior decompression of the spinal cord with opll resection is required if there is no improvement or if symptoms are aggravated at 3 weeks after surgery . however , because improvement is gradual , the second surgery may not be necessary if there is even a slight improvement in symptoms after the first surgery . anterior decompression of the spinal cord with opll resection can be performed using an anterior or a posterior approach . t - opll resection with an anterior approach has been reported as being a good surgical procedure with solid outcomes.10 22 the two - stage posterior and anterior combined approach is also a reasonable strategy.3 4 6 7 as the anterior approach is technically challenging , it should be performed by surgeons who are familiar with the procedure . the surgeon must have sufficient skills so as to avoid complications such as massive bleeding , postoperative persistent cerebrospinal fluid leakage in the thoracic cage , and pneumocephalus.23 24 it is for the following reasons that we recommend the posterior approach for the second surgery after posterior decompression and fusion surgery . first , the dura mater and spinal cord are visible during the surgical procedure , which helps avoid injury to the spinal cord . second , and more importantly , it is possible to perform additional dekyphosis after t - opll resection . for multilevel beak - type opll , total multilevel resection of the opll is ideal but is an invasive surgery with a longer operative time , greater estimated blood loss , and a higher complication rate . the treating surgeon must determine whether a total resection of the opll is possible according to the surgical invasiveness and the condition of patients . therefore , in these cases , we first conduct one or two levels of opll resection at the most severe site of spinal cord compression . following this procedure in cases without a massive oall the more flexible thoracic spine after opll resection allows more dekyphosis ; the result is greater decompression at the remaining opll levels . third , spinal cord shortening is achieved with dekyphosis and compression , and more blood flow is supplied to the spinal cord . utilizing a dog model , kawahara et al found increased blood flow to the spinal cord with spinal shortening.25 this slight spinal cord shortening effect achieved with dekyphosis may also contribute to improvement after raspa surgery , as well as direct mechanical decompression by opll resection . our raspa procedure is a modified ohtsuka method . while the original ohtsuka method is an excellent procedure that enables resection of the total opll posteriorly,15 it also risks spinal cord damage and postoperative paralysis . we note that raspa surgery may be better described as a new treatment approach , rather than a new operation , because it is based on the ohtsuka method . however , the resection procedure of beak - type opll from a posterior approach has a high risk of intraoperative spinal cord injury , which is why this procedure is not preferred for all cases of beak - type opll . we emphasize the importance of this modification in raspa surgery to achieve a good surgical outcome without intraoperative spinal cord injury for severely paralyzed patients . raspa surgery is safer due to the wide working space after pediculectomy , total resection of the transverse process , partial resection of the posterior vertebra , costotransversectomy , root sacrifice , and performance of the procedure under ionm . total pediculectomy at the opll resection level , total resection of the transverse process , and partial resection of the posterior vertebra with an air drill and rongeur enable an approach to the central anterior spinal cord without spinal cord retraction . costotransversectomy is advantageous in thin patients without thick soft tissue because it is possible to position the surgical device to approach the central portion while avoiding the spinal cord . in order to obtain sufficient working space during raspa surgery it is necessary to sacrifice the nerve roots . murakami et al reported that the sacrifice of up to three pairs of thoracic nerve roots , even at the level of the artery of adamkiewicz , did not result in ischemic neurologic deterioration in total spondylectomy.26 in a dog model , the ligation of bilateral segmental arteries at four or more consecutive thoracic levels carries a risk of ischemic spinal cord dysfunction.27 kato et al demonstrated the safety of three or fewer levels of t - opll resection with sacrifice of nerve roots from a posterior approach with total pediculectomy.28 based on this data , we are comfortable performing up to three levels of t - opll resection with the sacrifice of nerve roots . for patients with severe myelopathy , we do not retract and rotate the thoracic spinal cord during raspa surgery as it may cause spinal cord damage in beak - type t - opll . kato et al recommended dural sac rotation with traction of the sacrificed roots as a novel procedure that enables viewing of the opll at the anterior side of the spinal cord from a posterior approach.28 however , we do not recommend nerve root traction in beak - type t - opll surgery because ionm has revealed a deterioration in amplitude . we believe that performing the procedure with root traction is more dangerous because it may result in spinal cord retraction and damage . while it is true that the spinal cord should never be retracted directly , the heat and vibration of the air drill itself may damage the thoracic spinal cord . in the raspa procedure , this can be addressed by using ionm . as it is often difficult to obtain an ionm wave in patients with severe preoperative motor paralysis , we have utilized multichannel transcranial motor evoked potentials ( 32 channels ) to try and observe a wave in at least one muscle.29 30 multimodal spinal cord monitoring , including somatosensory evoked potential and d ( direct)-wave monitoring is also recommended . vigilance while utilizing ionm is required in order to prevent intraoperative severe spinal cord damage , with appropriate management that may include suspension of the surgical procedure . case 1 had a postoperative mmt score of 4 , which then deteriorated during rehabilitation . this occurred even after thoracic fusion surgery , and despite the absence of hematoma , implant loosening , or implant breakage . there was no recovery during a few weeks of rest before raspa surgery , but motor recovery after raspa started after postoperative day 1 . even after instrumented fixation , rod bending may occur upon standing until bony union of the fixed area is achieved . even a slight bending of the rod with occult slight hematoma during this period may induce micromotion at the beak - type opll level and motor functional deterioration with accompanying spinal cord injury . most of the thoracic spinal cord is vulnerable to compression , with massive beak - type opll and residual spinal cord compression likely to occur in the area of fixation . for these reasons , we recommend a full segmental pedicle screw construct using the most rigid , large diameter rod possible . this study is limited to three cases because not only is surgery for beak - type t - opll relatively uncommon , but it is rare that someone requires a second surgery for opll resection after initial t - opll surgery . however , there are cases with postoperative aggravation of symptoms or a poor surgical outcome after initial t - opll surgery . given the gravity of paralysis , the findings emanating from this salvage surgery are vital for these patients . one - stage posterior decompression and dekyphotic corrective fusion with instrumentation and raspa is ideal for spinal cord decompression , but this procedure is particularly invasive . thus , we believe that such a procedure should be utilized at least for single- or double - level beak - type t - opll at the point of prevention of increasing the surgical invasiveness . our two - stage strategy may still be appropriate , given the invasiveness of concomitant cervical laminoplasty , posterior decompression and fusion of the thoracic spine , and raspa surgery . further studies are required to determine the indication for raspa surgery before initial beak - type t - opll surgery and the timing for raspa surgery after the initial surgery . in conclusion , raspa as a second surgery for beak - type t - opll after posterior decompression and fusion surgery contributes to a good functional outcome and improved ambulatory status for paralyzed patients up to 3 weeks after initial surgery . the advantages of raspa include a wide working space , no retraction of the spinal cord , management based on ionm findings , more decompression at levels without opll resection , and spinal cord shortening at the most severe level after additional dekyphosis and compression.this modified surgical procedure gives favorable outcomes with use of ionm for avoidance of postoperative permanent paralysis .

study design prospective clinical study . objective posterior decompression and fusion surgery for beak - type thoracic ossification of the posterior longitudinal ligament ( t - opll ) generally has a favorable outcome . however , some patients require additional surgery for postoperative severe paralysis , a condition that is inadequately discussed in the literature . the objective of this study was to describe the efficacy of a procedure we refer to as resection at an anterior site of the spinal cord from a posterior approach ( raspa ) for severely paralyzed patients after posterior decompression and fusion surgery for beak - type t - opll . methods among 58 consecutive patients who underwent posterior decompression and fusion surgery for beak - type t - opll since 1999 , 3 with postoperative paralysis ( 5% ) underwent raspa in our institute . clinical records , the japanese orthopaedic association score , gait status , intraoperative neurophysiological monitoring ( ionm ) findings , and complications were evaluated in these cases . results all three patients experienced a postoperative decline in manual muscle test ( mmt ) scores of 0 to 2 after the first surgery . raspa was performed 3 weeks after the first surgery . all patients showed gradual improvements in mmt scores for the lower extremity and in ambulatory status ; all could walk with a cane at an average of 4 months following raspa surgery . there were no postoperative complications . conclusions raspa surgery for beak - type t - opll after posterior decompression and fusion surgery resulted in good functional outcomes as a salvage surgery for patients with severe paralysis . advantages of raspa include a wide working space , no spinal cord retraction , and additional decompression at levels without t - opll resection and spinal cord shortening after additional dekyphosis and compression maneuvers . when used with ionm , this procedure may help avoid permanent postoperative paralysis .

Introduction Materials and Methods Patients and Outcomes Surgical Indications and Procedures Results Discussion

thoracic ossification of the posterior longitudinal ligament ( t - opll ) causes severe myelopathy and may lead to permanent confinement to a wheelchair . decompression and instrumented fusion surgery for t - opll have recently been shown to have a favorable outcome.9 12 13 we have performed posterior decompression and dekyphotic corrective fusion with instrumentation since 1999 , especially for beak - type t - opll . we reported on the first 20 patients in 2009 , most of whom experienced an improvement in preoperative symptoms after the surgery.14 patients whose condition did not improve or whose preoperative symptoms were aggravated , subsequently underwent an anterior decompression of the spinal cord with resection of the t - opll from the posterior side . therefore , we performed a modified ohtsuka procedure with pediculectomy , resection of the transverse process , spinal root sacrifice , and concomitant costotransversectomy as needed under intraoperative neurophysiological monitoring ( ionm ) . we refer to this procedure as resection at an anterior site of the spinal cord from a posterior approach , we describe the surgical procedures , ionm , and outcomes as a technical note in three cases treated with raspa after paralysis resulting from posterior decompression and dekyphotic corrective fusion with instrumentation for beak - type t - opll . fifty - eight consecutive patients ( 28 males and 30 females , average age 54 years old ) with beak - type t - opll underwent posterior decompression and dekyphotic corrective fusion with instrumentation at our institute . among these patients , 3 ( 5% ) underwent raspa surgery due to a lack of improvement after the initial surgery , or due to the aggravation of motor paralysis postoperatively . the disease duration , pre- and postoperative ambulatory status with mmt , operative time , estimated blood loss , decompressed and fused area of the thoracic spine , change in cobb angle of the thoracic spine from before to after surgery , intraoperative ultrasonography findings , ionm findings ( transcranial motor evoked potentials [ 32 channels ] ) , intraoperative and postoperative complications , and outcomes after the first and second surgery were prospectively evaluated . outcomes were assessed using the modified japanese orthopaedic association ( joa ) score , based on a possible total of 11 points ( excluding the cervical score from the original joa score).16 17 an unpaired t test and a fisher exact test were used to compare preoperative joa scores and surgical outcomes for patients who did and who did not undergo raspa . posterior decompression and dekyphotic corrective fusion with instrumentation was beak - type opll with severe anterior spinal cord compression . , we informed the patients and families about the possible need for a second decompression surgery using raspa if there was no improvement or if motor paralysis was exacerbated after the first surgery.18 the raspa surgery proceeded as follows . because a laminectomy had already been performed in the first surgery , a resection of the transverse process and pedicle was necessary ( fig . next , a posterior partial osteotomy of vertebra adjacent to beak - type t - opll levels and an opll resection were performed with an air drill from the posterolateral direction , avoiding spinal cord compression ( fig . if these surgical devices were obstructed by a posterior rib , a partial resection of the rib ( so - called costotransversectomy ) was performed . while total resection of the opll is ideal , a residual thin opll may help avoid cerebrospinal fluid leakage . a thin opll does not prevent spinal cord decompression with postoperative dural sac enlargement after floating surgery for cervical opll.19 20 in our study , the cerebrospinal fluid pressure resulted in the gradual anterior migration of the floating opll after about 6 weeks . importantly , we did not retract or rotate the vulnerable spinal cord in beak - type t - opll because this movement can lead to severe spinal cord damage and permanent motor paralysis after surgery . this procedure was selected because diskectomy is typically easy , although in some cases ossification of the anterior longitudinal ligament ( oall ) and fusion of the thoracic spine may occur spontaneously after surgery ( even if interbody fusion was not achieved ) . surgical procedures in resection at an anterior site of the spinal cord from a posterior approach ( raspa ) surgery . resection of the transverse process and pedicle with spinal root sacrifice at the ossification of the posterior longitudinal ligament ( opll ) resection level . ( f ) after removal of the opll , additional dekyphosis and compression maneuver with a bilateral rod were performed , which gave indirect decompression at other opll levels with spinal cord compression and spinal cord shortening . fifty - eight consecutive patients ( 28 males and 30 females , average age 54 years old ) with beak - type t - opll underwent posterior decompression and dekyphotic corrective fusion with instrumentation at our institute . among these patients , 3 ( 5% ) underwent raspa surgery due to a lack of improvement after the initial surgery , or due to the aggravation of motor paralysis postoperatively . the disease duration , pre- and postoperative ambulatory status with mmt , operative time , estimated blood loss , decompressed and fused area of the thoracic spine , change in cobb angle of the thoracic spine from before to after surgery , intraoperative ultrasonography findings , ionm findings ( transcranial motor evoked potentials [ 32 channels ] ) , intraoperative and postoperative complications , and outcomes after the first and second surgery were prospectively evaluated . outcomes were assessed using the modified japanese orthopaedic association ( joa ) score , based on a possible total of 11 points ( excluding the cervical score from the original joa score).16 17 an unpaired t test and a fisher exact test were used to compare preoperative joa scores and surgical outcomes for patients who did and who did not undergo raspa . the indication for the first surgery posterior decompression and dekyphotic corrective fusion with instrumentation was beak - type opll with severe anterior spinal cord compression . in order to achieve further decompression of the spinal cord , rigid bilateral rods for kyphosis reduction were placed using the cantilever technique ; this was done with the aid of spinal cord monitoring . , we informed the patients and families about the possible need for a second decompression surgery using raspa if there was no improvement or if motor paralysis was exacerbated after the first surgery.18 the raspa surgery proceeded as follows . because a laminectomy had already been performed in the first surgery , a resection of the transverse process and pedicle was necessary ( fig . next , a posterior partial osteotomy of vertebra adjacent to beak - type t - opll levels and an opll resection were performed with an air drill from the posterolateral direction , avoiding spinal cord compression ( fig . if these surgical devices were obstructed by a posterior rib , a partial resection of the rib ( so - called costotransversectomy ) was performed . while total resection of the opll is ideal , a residual thin opll may help avoid cerebrospinal fluid leakage . a thin opll does not prevent spinal cord decompression with postoperative dural sac enlargement after floating surgery for cervical opll.19 20 in our study , the cerebrospinal fluid pressure resulted in the gradual anterior migration of the floating opll after about 6 weeks . importantly , we did not retract or rotate the vulnerable spinal cord in beak - type t - opll because this movement can lead to severe spinal cord damage and permanent motor paralysis after surgery . this procedure was selected because diskectomy is typically easy , although in some cases ossification of the anterior longitudinal ligament ( oall ) and fusion of the thoracic spine may occur spontaneously after surgery ( even if interbody fusion was not achieved ) . surgical procedures in resection at an anterior site of the spinal cord from a posterior approach ( raspa ) surgery . resection of the transverse process and pedicle with spinal root sacrifice at the ossification of the posterior longitudinal ligament ( opll ) resection level . ( f ) after removal of the opll , additional dekyphosis and compression maneuver with a bilateral rod were performed , which gave indirect decompression at other opll levels with spinal cord compression and spinal cord shortening . all of the patients had four levels of beak - type t - opll involving ossification of the ligamentum flavum , with severe motor paralysis that prevented walking . in the first surgery , ionm findings deteriorated in all cases , but there were no other intraoperative complications . none of the three patients developed a hematoma or experienced implant failure after the first surgery , but two reoperations were required due to hematoma in patients who did not undergo raspa . after the first surgery , no motor recovery was found in any of the three patients after several weeks of bed rest . abbreviations : bmi , body mass index ; joa score , japanese orthopaedic association score ; mmt , manual muscle test ; olf , ossification of the ligamentum flavum ; opll , ossification of the posterior longitudinal ligament . abbreviations : ebl , estimated blood loss ; ionm , intraoperative neurophysiological monitoring ; mmt , manual muscle test . all three patients underwent raspa as a second surgery , which was performed 3 weeks after the first surgery . in the raspa surgery , one to three levels of the opll were resected from the posterior side with two to six pediculectomy and one to four roots sacrificed . each of the three patients experienced an improvement in motor function ( beginning on postoperative days 1 , 2 , and 17 , respectively ) with a gradual reduction in motor paralysis over several months . 2 ) and were able to walk with a cane , representing a better ambulatory status than before the first surgery . preoperative and postoperative course of manual muscle test ( mmt ) scores for the lower extremity . after the second surgery ( resection at an anterior site of the spinal cord from a posterior approach ) , the three cases achieved almost full mmt scores in 3 days , 4.5 months , and 7 months , respectively ( shown in table 4 ) . , left ; opll , ossification of the posterior longitudinal ligament ; raspa , resection at an anterior site of the spinal cord from a posterior approach ; ionm , intraoperative neurophysiological monitoring . abbreviations : joa score , japanese orthopaedic association score ; mmt , manual muscle test ; raspa , resection at an anterior site of the spinal cord from a posterior approach . total resection of beak - type t - opll and more dekyphosis was achieved after raspa surgery ( fig . ( b , c ) lateral plain radiograph and ct sagittal image after posterior decompression and instrumented fusion ( first surgery ) . ( d , e ) postoperative lateral plain radiograph and ct sagittal image after resection at an anterior site of the spinal cord from a posterior approach ( raspa ; second surgery , arrow ) . ( f ) at final follow - up , the ossification of the anterior longitudinal ligament was also extended and fused , with stabilization of the thoracic spine ( arrow ) . while there are several approaches for t - opll surgery , the optimal method is unclear because of the high rates of postoperative motor disturbance and complications.2 3 4 5 6 7 8 9 10 11 14 posterior decompression and fusion surgery for t - opll typically results in good surgical outcomes12 14 21 ; this surgical procedure also has the advantage of concomitant cervical laminoplasty . all three patients who underwent raspa surgery had concomitant cervical laminoplasty . because this had no impact on the outcome of the first thoracic surgery there is no need to avoid one - stage combined posterior surgery for cervical and thoracic opll . therefore , we informed all patients prior to the first surgery of the option of additional anterior decompression with a posterior approach if symptoms show no improvement at about 3 weeks after the first surgery or if symptoms worsen . the two - stage posterior decompression with dekyphosis and anterior decompression surgery proposed by kawahara et al involve decompression along the circumferential spinal cord with spinal fusion ( the ideal decompression).3 kawahara et al also suggested a need for additional anterior decompression from an anterior approach at 3 weeks after the posterior surgery . we also believe that a second surgery of anterior decompression of the spinal cord with opll resection is required if there is no improvement or if symptoms are aggravated at 3 weeks after surgery . however , because improvement is gradual , the second surgery may not be necessary if there is even a slight improvement in symptoms after the first surgery . anterior decompression of the spinal cord with opll resection can be performed using an anterior or a posterior approach . t - opll resection with an anterior approach has been reported as being a good surgical procedure with solid outcomes.10 22 the two - stage posterior and anterior combined approach is also a reasonable strategy.3 4 6 7 as the anterior approach is technically challenging , it should be performed by surgeons who are familiar with the procedure . the surgeon must have sufficient skills so as to avoid complications such as massive bleeding , postoperative persistent cerebrospinal fluid leakage in the thoracic cage , and pneumocephalus.23 24 it is for the following reasons that we recommend the posterior approach for the second surgery after posterior decompression and fusion surgery . first , the dura mater and spinal cord are visible during the surgical procedure , which helps avoid injury to the spinal cord . second , and more importantly , it is possible to perform additional dekyphosis after t - opll resection . for multilevel beak - type opll , total multilevel resection of the opll is ideal but is an invasive surgery with a longer operative time , greater estimated blood loss , and a higher complication rate . therefore , in these cases , we first conduct one or two levels of opll resection at the most severe site of spinal cord compression . third , spinal cord shortening is achieved with dekyphosis and compression , and more blood flow is supplied to the spinal cord . utilizing a dog model , kawahara et al found increased blood flow to the spinal cord with spinal shortening.25 this slight spinal cord shortening effect achieved with dekyphosis may also contribute to improvement after raspa surgery , as well as direct mechanical decompression by opll resection . however , the resection procedure of beak - type opll from a posterior approach has a high risk of intraoperative spinal cord injury , which is why this procedure is not preferred for all cases of beak - type opll . we emphasize the importance of this modification in raspa surgery to achieve a good surgical outcome without intraoperative spinal cord injury for severely paralyzed patients . raspa surgery is safer due to the wide working space after pediculectomy , total resection of the transverse process , partial resection of the posterior vertebra , costotransversectomy , root sacrifice , and performance of the procedure under ionm . total pediculectomy at the opll resection level , total resection of the transverse process , and partial resection of the posterior vertebra with an air drill and rongeur enable an approach to the central anterior spinal cord without spinal cord retraction . murakami et al reported that the sacrifice of up to three pairs of thoracic nerve roots , even at the level of the artery of adamkiewicz , did not result in ischemic neurologic deterioration in total spondylectomy.26 in a dog model , the ligation of bilateral segmental arteries at four or more consecutive thoracic levels carries a risk of ischemic spinal cord dysfunction.27 kato et al demonstrated the safety of three or fewer levels of t - opll resection with sacrifice of nerve roots from a posterior approach with total pediculectomy.28 based on this data , we are comfortable performing up to three levels of t - opll resection with the sacrifice of nerve roots . for patients with severe myelopathy , we do not retract and rotate the thoracic spinal cord during raspa surgery as it may cause spinal cord damage in beak - type t - opll . kato et al recommended dural sac rotation with traction of the sacrificed roots as a novel procedure that enables viewing of the opll at the anterior side of the spinal cord from a posterior approach.28 however , we do not recommend nerve root traction in beak - type t - opll surgery because ionm has revealed a deterioration in amplitude . while it is true that the spinal cord should never be retracted directly , the heat and vibration of the air drill itself may damage the thoracic spinal cord . as it is often difficult to obtain an ionm wave in patients with severe preoperative motor paralysis , we have utilized multichannel transcranial motor evoked potentials ( 32 channels ) to try and observe a wave in at least one muscle.29 30 multimodal spinal cord monitoring , including somatosensory evoked potential and d ( direct)-wave monitoring is also recommended . even a slight bending of the rod with occult slight hematoma during this period may induce micromotion at the beak - type opll level and motor functional deterioration with accompanying spinal cord injury . most of the thoracic spinal cord is vulnerable to compression , with massive beak - type opll and residual spinal cord compression likely to occur in the area of fixation . this study is limited to three cases because not only is surgery for beak - type t - opll relatively uncommon , but it is rare that someone requires a second surgery for opll resection after initial t - opll surgery . however , there are cases with postoperative aggravation of symptoms or a poor surgical outcome after initial t - opll surgery . one - stage posterior decompression and dekyphotic corrective fusion with instrumentation and raspa is ideal for spinal cord decompression , but this procedure is particularly invasive . thus , we believe that such a procedure should be utilized at least for single- or double - level beak - type t - opll at the point of prevention of increasing the surgical invasiveness . our two - stage strategy may still be appropriate , given the invasiveness of concomitant cervical laminoplasty , posterior decompression and fusion of the thoracic spine , and raspa surgery . further studies are required to determine the indication for raspa surgery before initial beak - type t - opll surgery and the timing for raspa surgery after the initial surgery . in conclusion , raspa as a second surgery for beak - type t - opll after posterior decompression and fusion surgery contributes to a good functional outcome and improved ambulatory status for paralyzed patients up to 3 weeks after initial surgery . the advantages of raspa include a wide working space , no retraction of the spinal cord , management based on ionm findings , more decompression at levels without opll resection , and spinal cord shortening at the most severe level after additional dekyphosis and compression.this modified surgical procedure gives favorable outcomes with use of ionm for avoidance of postoperative permanent paralysis .

the association of childhood excess weight or childhood obesity with dyslipidemia was well documented ( 1 ) . it is suggested that some metabolic disorders start early in life and can be further agrevated by other factors in the external environment . researchers believe that genetic factors are important determinants of plasma lipid levels in adults ; although , it is less clear in children and adolescents . one of the main proteins involved in lipoprotein metabolism is cholesterol ester transfer protein ( cetp ) . however , according to evidence cetp has atherogenic roles naturally , which function depending on the metabolic settings . thus , plasma cetp acts as an important protein in the lipid and lipoprotein metabolism and its polymorphisms may affect dyslipidemia and cvd ( 2 ) . several single nucleotide polymorphisms ( snp ) have been identified within cetp ; they influence enzymatic activity or gene expression level . two common polymorphisms of cetp gene are taq1b ( rs708272 ) and a373p ( rs5880 ) . taq1b is a polymorphism at 277 nucleotide in the first intron of cetp and has a restriction site for the endonuclease taq1 . taq1b polymorphism may affect the plasma cetp activity and its concentrations , as well as hdl - c levels . a373p polymorphism is a guanine to cytosine mutation at codon 373 in exon 12 of cetp gene that is one of the most common snps , found in 15 % of asians and europeans . it is shown that these polymorphisms are associated with lipid and lipoprotein metabolism in different populations ( 3 ) . however , to our knowledge , no data exist on the interaction of cetp polymorphisms and weight or birth weight and the risk of dyslipidemia in children and adolescents . given the tracking of atherosclerotic cvd risk factors including dyslipidemia from childhood to adulthood , it is important to determine the predisposing factors of these risk factors in early life . this study aims to investigate joint association between cetp polymorphisms and bmi or birth weight with the risk of dyslipidemia in iranian children and adolescents . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . the main survey included 5528 students aged 1018 years who were recruited by multistage random cluster sampling from urban and rural areas of 27 provincial counties in iran . details of data collection and sampling were published previously ( 4 ) . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . the current survey was approved by the ethical committee of isfahan university of medical sciences . written informed consent was obtained from parents and oral assent from children and adolescents . bmi was calculated as the weight ( kg ) divided by the height squared ( m ) . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . waist circumference was measured at the smallest point of circumference between the iliac crest and the rib cage . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the pattern of physical activity , three indicators were used including : hours of physical education at school , hours of screening time , and hours spent on sports club training . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . of note for parental occupational status and level of education , i.e. questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . for assessing dietary habits , we used questions about the type of bread ( i.e. white or whole grain ) and the type of fat consumed in meals at home . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) , as well as salty / high fat snacks and sweets / candies . dna was extracted using the qiaamp dna blood mini kit ( qiagen , germany ) according to the manufacturer s protocol from peripheral blood . real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . primers were designed by beacon designer 7.91 to flank the genomic regions ( premier biosoft international , usa ) and were synthesized by tib molbiol ( germany ) . amplicons from all genes were generated under the following conditions using the type - it hrm kit ( qiagen , germany ) : one cycle at 95c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle at 95 c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle of 95 c for 1 sec , 72 c for 90 sec and a melt from 70 to 95 c rising at 0.1 c per second . the amplification mixture of a total volume of 25 l included 12.5 l of hrm pcr master mix , 1.75 l of 10 m primer mix , 2 l of genomic dna as template , and 8.25 l of rnase - free water . for each genotype reaction , we included sequence - proven major and minor allele homozygote and heterozygote controls . the hrm analysis was performed by instrument software , which allows clustering of the samples into groups based on a difference plot obtained by analyzing the differences in melting curve shape between known controls and samples . primer sequence used for cetp taqib rs708272 was f : gtatagggattt - gtgtttgtct , r : cctaacctggctcagatc and for cetp a373p rs5880 it was f : tctccccaggatatc - gtgactac , r : gcagcacatactggaaatccaaga . the statistical analyses were performed using statistical package for the social sciences ( ver 20.0 ) software ( chicago , il , usa ) . the student s t - test or mann - whitney test , as appropriate , were used to determine differences in continuous variables . categorical variables are presented as percentage . the pearson s chi - square test or fisher s exact test , as appropriate , were used to determine the differences in categorical variables . the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. , physical activity , fasting blood sugar , diastolic blood pressure , and systolic blood pressure ) . exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . the main survey included 5528 students aged 1018 years who were recruited by multistage random cluster sampling from urban and rural areas of 27 provincial counties in iran . details of data collection and sampling were published previously ( 4 ) . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . the current survey was approved by the ethical committee of isfahan university of medical sciences . written informed consent was obtained from parents and oral assent from children and adolescents . bmi was calculated as the weight ( kg ) divided by the height squared ( m ) . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . waist circumference was measured at the smallest point of circumference between the iliac crest and the rib cage . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the pattern of physical activity , three indicators were used including : hours of physical education at school , hours of screening time , and hours spent on sports club training . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . of note for parental occupational status and level of education , i.e. questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . for assessing dietary habits , we used questions about the type of bread ( i.e. white or whole grain ) and the type of fat consumed in meals at home . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) , as well as salty / high fat snacks and sweets / candies . dna was extracted using the qiaamp dna blood mini kit ( qiagen , germany ) according to the manufacturer s protocol from peripheral blood . real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . primers were designed by beacon designer 7.91 to flank the genomic regions ( premier biosoft international , usa ) and were synthesized by tib molbiol ( germany ) . amplicons from all genes were generated under the following conditions using the type - it hrm kit ( qiagen , germany ) : one cycle at 95c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle at 95 c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle of 95 c for 1 sec , 72 c for 90 sec and a melt from 70 to 95 c rising at 0.1 c per second . the amplification mixture of a total volume of 25 l included 12.5 l of hrm pcr master mix , 1.75 l of 10 m primer mix , 2 l of genomic dna as template , and 8.25 l of rnase - free water . for each genotype reaction , we included sequence - proven major and minor allele homozygote and heterozygote controls . the hrm analysis was performed by instrument software , which allows clustering of the samples into groups based on a difference plot obtained by analyzing the differences in melting curve shape between known controls and samples . primer sequence used for cetp taqib rs708272 was f : gtatagggattt - gtgtttgtct , r : cctaacctggctcagatc and for cetp a373p rs5880 it was f : tctccccaggatatc - gtgactac , r : gcagcacatactggaaatccaaga . the statistical analyses were performed using statistical package for the social sciences ( ver 20.0 ) software ( chicago , il , usa ) . the student s t - test or mann - whitney test , as appropriate , were used to determine differences in continuous variables . categorical variables are presented as percentage . the pearson s chi - square test or fisher s exact test , as appropriate , were used to determine the differences in categorical variables . the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. , physical activity , fasting blood sugar , diastolic blood pressure , and systolic blood pressure ) . exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . the main survey included 5528 students aged 1018 years who were recruited by multistage random cluster sampling from urban and rural areas of 27 provincial counties in iran . details of data collection and sampling were published previously ( 4 ) . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . the current survey was approved by the ethical committee of isfahan university of medical sciences . written informed consent was obtained from parents and oral assent from children and adolescents . bmi was calculated as the weight ( kg ) divided by the height squared ( m ) . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . waist circumference was measured at the smallest point of circumference between the iliac crest and the rib cage . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the pattern of physical activity , three indicators were used including : hours of physical education at school , hours of screening time , and hours spent on sports club training . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . of note for parental occupational status and level of education , i.e. questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . for assessing dietary habits , we used questions about the type of bread ( i.e. white or whole grain ) and the type of fat consumed in meals at home . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) , as well as salty / high fat snacks and sweets / candies . dna was extracted using the qiaamp dna blood mini kit ( qiagen , germany ) according to the manufacturer s protocol from peripheral blood . real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . primers were designed by beacon designer 7.91 to flank the genomic regions ( premier biosoft international , usa ) and were synthesized by tib molbiol ( germany ) . amplicons from all genes were generated under the following conditions using the type - it hrm kit ( qiagen , germany ) : one cycle at 95c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle at 95 c for 15 min ; 40 cycles at 95 c for 15 sec , 60.0 c for 15 sec , 72 c for 15 sec ; one cycle of 95 c for 1 sec , 72 c for 90 sec and a melt from 70 to 95 c rising at 0.1 c per second . the amplification mixture of a total volume of 25 l included 12.5 l of hrm pcr master mix , 1.75 l of 10 m primer mix , 2 l of genomic dna as template , and 8.25 l of rnase - free water . for each genotype reaction , we included sequence - proven major and minor allele homozygote and heterozygote controls . the hrm analysis was performed by instrument software , which allows clustering of the samples into groups based on a difference plot obtained by analyzing the differences in melting curve shape between known controls and samples . primer sequence used for cetp taqib rs708272 was f : gtatagggattt - gtgtttgtct , r : cctaacctggctcagatc and for cetp a373p rs5880 it was f : tctccccaggatatc - gtgactac , r : gcagcacatactggaaatccaaga . the statistical analyses were performed using statistical package for the social sciences ( ver 20.0 ) software ( chicago , il , usa ) . the data for continuous variables is expressed as meansd . the student s t - test or mann - whitney test , as appropriate , categorical variables are presented as percentage . the pearson s chi - square test or fisher s exact test , as appropriate , the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. , physical activity , fasting blood sugar , diastolic blood pressure , and systolic blood pressure ) . exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . p - value of hardy - weinberg expectations was 0.073 for taq1b polymorphism and 0.75 for a373p polymorphism . characteristics of the study population across the cholesterol ester transfer protein gene polymorphisms : the caspian - iii study wc = waist circumference , fbs = fasting blood sugar , dbp = diastolic blood pressure , sbp = systolic blood pressure , ldl - c = low - density lipoprotein , hdl - c = high - density lipoprotein , tg = triglyceride , bmi = body mass index , bw = birth weight the multivariate logistic regression analysis showed a protective effect of ct / tt genotype on dyslipidemia in the crude and adjusted models . g allele of a373p polymorphism increased the risk of dyslipidemia with an or of 4.12 ( 95% ci : 2.57 - 6.62 , p - value < 0.001 ) in the crude and adjusted models ( table 2 ) . multivariate logistic regression analysis of cholesterol ester transfer protein gene polymorphisms associated with dyslipidemia model 1 : adjusted with age , sex , physical activity , bmi , waist circumference model 2 : adjusted with model 1 and fasting blood sugar , diastolic blood pressure , systolic blood pressure we evaluated the joint effect of the taq1b polymorphism or a373p polymorphism and bmi for the risk of dyslipidemia ( table 3 ) . we observed interactive effects of cetp gene polymorphisms and bmi on dyslipidemia ( p - interaction < 0.05 ) . on joint analysis , combination of carrying the t allele with bmi was inversely associated with dyslipidemia . in the crude analysis , we observed a decreased risk of dyslipidemia for the subjects with ct / tt genotypes of the taq1b polymorphism as well as persons with a bmi in underweight or normal categories , with an or of 0.13 ( 95% ci : 0.06 - 0.27 ) and 0.18 ( 95% ci : 0.09 - 0.33 ) , respectively . adjustment for the covariates did not change the statistical significance ( p - value<0.05 ) . among individuals with a bmi in underweight or normal categories , carrying cg / gg was positively associated with dyslipidemia and carrying the g allele increased the risk of dyslipidemia ( or=3.27 , 95% ci : 1.29 - 8.29 and or=5.65 , 95% ci : 2.90 , 11.02 , respectively ) . adjustment for the covariates did not change the statistical significance ( p - value<0.05 ) . combined association of cetp polymorphisms and bmi or birth weight with dyslipidemia cetp= cholesterol ester transfer protein , bmi= body mass index , bw= birth weight adjusted for age , sex , physical activity , waist circumference , birth weight , socioeconomic status , healthy and unhealthy food intake adjusted for age , sex , physical activity , waist circumference , bmi , socioeconomic status , healthy and unhealthy food intake the results for the joint effects of the taq1b polymorphism or a373p polymorphism with birth weight on dyslipidemia and p - interaction are presented in table 3 . we observed interactive effects of cetp gene polymorphisms and birth weight on dyslipidemia ( p - interaction < 0.05 ) . in crude and adjusted analysis , exposure to both the ct / tt genotypes for the taq1b polymorphism and the birth weight in each category posed a decreased risk for the dyslipidemia ( p - value < 0.05 ) . in the crude analysis , among persons with normal birth weight , carrying cg / gg for a373p polymorphism was positively associated with dyslipidemia and carrying the g allele increased the risk of dyslipidemia ( or=2.58 , 95% ci : 1.27 , 5.24 ) . although the risk of dyslipidemia in younger populations has been increasing over the last few decades , much less effort has been made in understanding the gene - environmental interaction in lipid metabolism in younger populations , especially in the young iranian population . the additive effect of the interaction between genetic and environmental factors is greater than the contribution of either risk factor ( 7 ) . the present study suggested the association between the cetp polymorphisms and dyslipidemia in iranian children and adolescents . taq1b polymorphism had protective effect on dyslipidemia . however , a373p polymorphism had adverse effect on dyslipidemia . furthermore , combined effects were observed between the cetp polymorphisms and bmi or birth weight on the risk of dyslipidemia . genome wide association studies ( gwas ) identified strong associations between cetp and plasma lipid concentrations in adults ( 8) . however , the role of genetic factors in the plasma lipid levels is less clear in children ( 2 ) . there is still a huge controversy over the association of taq1b polymorphism with risk of cvd . our results showed taq1b polymorphism improved serum lipid levels and decreased the risk of dyslipidemia . although various previous studies ( 9 , 10 ) have shown a lower cetp activity , higher hdl - c levels , lower postprandial triglyceride , lower risk of cvd and atheroprotective effect in t allele carriers in taq1b polymorphism . others suggested that the t allele had no protective effect or even increased cvd risk in adults ( 11 , 12 ) . in addition , the association of the t allele with higher hdl - c levels was observed in children ( 2 ) . taq1b polymorphism changes the activity of cetp and affects hdl - c concentrations and probably does not affect protein folding . this association may be population specific and is influenced by environmental factors , such as bmi ( 13 ) . our findings showed the a373p polymorphism had deleterious effects on lipid profile levels and approximately increased fourfold the risk of dyslipidemia in children and adolescents . agerholm - larsen et al ( 14 ) reported the a373p polymorphism in cetp associated with decrease in hdl - c levels in both genders . two studies ( 15 , 16 ) found a373p polymorphisms were associated with increasing relative risk of subclinical cardiovascular disease ( relative risk = 1.22 , p = 0.018 ) and 12.2% higher triglyceride concentration ( p = 0.03 ) . they showed that the a373p polymorphism was associated with higher cetp activity and concentration , lower hdl - c levels and atherogenic effects . the copenhagen city heart study reported that a373p polymorphism reduced levels of hdl - c ( 14 ) . however , when 1,236 french and irish subjects were examined , there was no change in cetp activity and hdl - c levels in a373p polymorphism ( 17 ) . the substitution of c for g at amino acid 373 leads to the a373p polymorphism and increases in mass . adverse effect of the a373p polymorphism can be explained by increasing plasma cetp activity and its concentration . effect of this snp on serum lipids is due to chance or indicates a functional effect on cetp gene expression and needs to be assessed by further studies ( 18 ) . gene - environment interactions are common in the pathogenesis of prevalent complex disorders such as dyslipidemia . in our study combined association analyses showed that the taq1b polymorphism had a protective effect and the a373p polymorphism had an adverse effect on dyslipidemia only in underweight and normal weight subjects in both crude and adjusted models . findings showed the relationship between the taq1b polymorphism and hdl - c levels was modified by environmental factors such as obesity ( 19 ) . it is reported that the association between taq1b genotype and hdl - c levels was attenuated by obesity ( 20 ) . however , vohl et al ( 22 ) showed taq1b polymorphism was not associated with higher hdl - c levels in men with hyperinsulinemia and obesity . in this study , we observed that dyslipidemia was not associated with the taq1b or a373p polymorphisms in obese subjects . this has been supported by others who found no association between some lipid profiles such as hdl - c concentrations and taq1b among subjects in the highest bmi tertile ( 26.240.4 kg / m ) . however , they reported this association in the lowest bmi tertile similar to our findings ( 20 , 23 ) . study showed that obesity was able to decrease the protective cardiovascular effect of the t allele in taq1b polymorphism ( 24 ) . findings showed higher cetp activity in obese subjects significantly in comparison with controls ( 25 ) . a similar finding our findings showed that taq1b polymorphism had a protective effect on dyslipidemia in all categories of birth weight , and the a373p polymorphism only in normal birth weight category had a significantly adverse effect on dyslipidemia . according to previous studies low birth weight correlated with higher risk for metabolic syndrome and lipid metabolism disorders in young adults . however , the evidence on the correlation between birth weight and the lipid levels is less in agreement ( 27 ) . british birth cohort showed inverse associations between birth weight and total cholesterol , ldl - c and triglyceride levels ( 28 ) . researchers supposed genetic factors and their interactions could potentially affect associations between birth weight and lipid levels ( 28 , 29 ) . intrauterine environment , including nutritional status and fetal exposure to stress may lead to impaired fetal growth , structural changes in the liver and permanent changes in lipid metabolism ( 27 ) . the main limitation of this study is the cross - sectional nature of the associations . the strengths of the present study are its novelty in the pediatric age group and a relatively large number of population - based samples . the present study showed that taq1b polymorphism had a protective effect and a373p polymorphism had a deleterious effect on dyslipidemia in iranian children and adolescents . more investigation is needed to assess the gene - environment interaction in children and adolescents .

objective(s):this study aims to investigate joint association between cholesterol ester transfer protein ( cetp ) polymorphisms and body mass index ( bmi ) or birth weight with the risk of dyslipidemia in iranian children and adolescents.materials and methods : this study was conducted as a sub - study of the school - based nationwide health survey ( caspian - iii ) . we randomly selected 750 samples from the whole blood samples . real - time pcr and high resolution melt ( hrm ) analysis were performed to determine taq1b ( rs708272 ) and a373p ( rs5880 ) polymorphisms.results:taq1b polymorphism increased hdl - c , and total cholesterol ( tc ) as well as decreased triglyceride and ldl - c concentrations . ldl - c and triglyceride levels were significantly higher and hdl - c and tc levels were significantly lower among those with a373p polymorphism . ct / tt genotype in taq1b polymorphism showed a protective effect on dyslipidemia ( or= 0.12 , 95% ci : 0.07 - 0.20 ) . g allele of a373p polymorphism increased the risk of dyslipidemia ( or=4.10 , 95% ci : 2.14 , 7.83 ) after adjusting the confounders . we observed interactive effects of cetp gene polymorphisms and bmi or birth weight on dyslipidemia.conclusion:findings showed taq1b polymorphism might have a protective effect and a373p polymorphism had deleterious effect on dyslipidemia in iranian children and adolescents . these associations interacted with bmi and birth weight .

Introduction Materials and Methods None Study population Physical examination and biochemical measurements Assessment of physical activity, diet and socioeconomic status DNA extraction Statistical analysis Results Discussion Conclusion

researchers believe that genetic factors are important determinants of plasma lipid levels in adults ; although , it is less clear in children and adolescents . one of the main proteins involved in lipoprotein metabolism is cholesterol ester transfer protein ( cetp ) . two common polymorphisms of cetp gene are taq1b ( rs708272 ) and a373p ( rs5880 ) . taq1b polymorphism may affect the plasma cetp activity and its concentrations , as well as hdl - c levels . a373p polymorphism is a guanine to cytosine mutation at codon 373 in exon 12 of cetp gene that is one of the most common snps , found in 15 % of asians and europeans . however , to our knowledge , no data exist on the interaction of cetp polymorphisms and weight or birth weight and the risk of dyslipidemia in children and adolescents . this study aims to investigate joint association between cetp polymorphisms and bmi or birth weight with the risk of dyslipidemia in iranian children and adolescents . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . written informed consent was obtained from parents and oral assent from children and adolescents . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) , as well as salty / high fat snacks and sweets / candies . real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . written informed consent was obtained from parents and oral assent from children and adolescents . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) , as well as salty / high fat snacks and sweets / candies . real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . this study was conducted as a sub - study of the school - based nationwide health survey , which was the third survey of the school - based surveillance system entitled childhood and adolescence surveillance and prevention of adult non communicable disease ( caspian - iii ) study . for the current study , we randomly selected 750 samples from the whole blood samples kept frozen at -70 c . written informed consent was obtained from parents and oral assent from children and adolescents . world health organization ( who ) defines , normal weight as bmi - z score between -2 and 1 , wasting as bmi - z score between -2 and -3 , risk of overweight as bmi - z score between 1 and 2 , overweight as bmi - z score between 2 and 3 , and obesity as bmi - z score more than 3 ( 5 ) . birth weight was categorized as low birth weight ( less than 2500 g ) , normal ( 25004000 g ) and high birth weight ( more than 4000 g ) . for assessing lipid profile , fasting venous blood was taken and lipid profile variables including total cholesterol ( tc ) , hdl - c , ldl - c , and triglyceride ( tg ) were examined . cut - off points for abnormal levels of lipids included : tc 200 mg / dl , ldl - c 130 mg / dl , hdl - c < 35 mg / dl , and tg 130 mg / dl ( 6 ) . we used the questionnaire of the world health organization - global school - based student health survey ( who - gshs ) to assess physical activity . to evaluate the family s socioeconomic status ( ses ) , we included questions about the following socioeconomic indicators : ( i ) parental level of education ( illiterate : score 1 , less than high school : score 2 , high school graduate : score 3 , academic education : score 4 ) ; ( ii ) parental occupational status ( unemployed : score 1 , worker / farmer : score 2 , governmental employee : score 3 , self - employed : score 4 ) ; and ( iii ) number of household members , and ( iv ) possessing a family private car ( yes / no ) . questions ( i ) and ( ii ) , only data on the parent with the higher occupational status or education was considered . food items were grouped into the following categories : carbohydrates ( rice , bread , pasta , and potato ) , vegetables ( potato and french fries not included ) , fruit ( fresh , dried , juice ) , dairy products ( milk , cheese , yogurt ) , proteins , including both animal ( red meat , poultry , fish , egg ) and plant ( beans , soy , nuts ) , fast foods ( pizza , hamburgers , sausages , etc . ) real - time pcr and high resolution melt ( hrm ) analysis were performed in the corbett rotor - gene 6000 instrument ( corbett research pty ltd , sydney , australia ) . the pearson s chi - square test or fisher s exact test , as appropriate , the multivariate logistic regression analyses was performed to determine how the association of cetp gene polymorphisms with the risk of dyslipidemia after controlling for blocks of covariates related to patient demographics ( age , sex ) , anthropometric characteristics ( bmi and waist circumference ) , and the patient s usual physical characteristics ( e.g. exploratory interaction analyses based on cetp polymorphisms and bmi / birth weight were also run using generalized linear model , with and without confounder adjustment ( age , sex , physical activity , waist circumference , socioeconomic status , healthy , and unhealthy food intake ) . p - value of hardy - weinberg expectations was 0.073 for taq1b polymorphism and 0.75 for a373p polymorphism . characteristics of the study population across the cholesterol ester transfer protein gene polymorphisms : the caspian - iii study wc = waist circumference , fbs = fasting blood sugar , dbp = diastolic blood pressure , sbp = systolic blood pressure , ldl - c = low - density lipoprotein , hdl - c = high - density lipoprotein , tg = triglyceride , bmi = body mass index , bw = birth weight the multivariate logistic regression analysis showed a protective effect of ct / tt genotype on dyslipidemia in the crude and adjusted models . g allele of a373p polymorphism increased the risk of dyslipidemia with an or of 4.12 ( 95% ci : 2.57 - 6.62 , p - value < 0.001 ) in the crude and adjusted models ( table 2 ) . multivariate logistic regression analysis of cholesterol ester transfer protein gene polymorphisms associated with dyslipidemia model 1 : adjusted with age , sex , physical activity , bmi , waist circumference model 2 : adjusted with model 1 and fasting blood sugar , diastolic blood pressure , systolic blood pressure we evaluated the joint effect of the taq1b polymorphism or a373p polymorphism and bmi for the risk of dyslipidemia ( table 3 ) . we observed interactive effects of cetp gene polymorphisms and bmi on dyslipidemia ( p - interaction < 0.05 ) . in the crude analysis , we observed a decreased risk of dyslipidemia for the subjects with ct / tt genotypes of the taq1b polymorphism as well as persons with a bmi in underweight or normal categories , with an or of 0.13 ( 95% ci : 0.06 - 0.27 ) and 0.18 ( 95% ci : 0.09 - 0.33 ) , respectively . among individuals with a bmi in underweight or normal categories , carrying cg / gg was positively associated with dyslipidemia and carrying the g allele increased the risk of dyslipidemia ( or=3.27 , 95% ci : 1.29 - 8.29 and or=5.65 , 95% ci : 2.90 , 11.02 , respectively ) . combined association of cetp polymorphisms and bmi or birth weight with dyslipidemia cetp= cholesterol ester transfer protein , bmi= body mass index , bw= birth weight adjusted for age , sex , physical activity , waist circumference , birth weight , socioeconomic status , healthy and unhealthy food intake adjusted for age , sex , physical activity , waist circumference , bmi , socioeconomic status , healthy and unhealthy food intake the results for the joint effects of the taq1b polymorphism or a373p polymorphism with birth weight on dyslipidemia and p - interaction are presented in table 3 . we observed interactive effects of cetp gene polymorphisms and birth weight on dyslipidemia ( p - interaction < 0.05 ) . in crude and adjusted analysis , exposure to both the ct / tt genotypes for the taq1b polymorphism and the birth weight in each category posed a decreased risk for the dyslipidemia ( p - value < 0.05 ) . in the crude analysis , among persons with normal birth weight , carrying cg / gg for a373p polymorphism was positively associated with dyslipidemia and carrying the g allele increased the risk of dyslipidemia ( or=2.58 , 95% ci : 1.27 , 5.24 ) . although the risk of dyslipidemia in younger populations has been increasing over the last few decades , much less effort has been made in understanding the gene - environmental interaction in lipid metabolism in younger populations , especially in the young iranian population . the present study suggested the association between the cetp polymorphisms and dyslipidemia in iranian children and adolescents . taq1b polymorphism had protective effect on dyslipidemia . however , a373p polymorphism had adverse effect on dyslipidemia . furthermore , combined effects were observed between the cetp polymorphisms and bmi or birth weight on the risk of dyslipidemia . our results showed taq1b polymorphism improved serum lipid levels and decreased the risk of dyslipidemia . although various previous studies ( 9 , 10 ) have shown a lower cetp activity , higher hdl - c levels , lower postprandial triglyceride , lower risk of cvd and atheroprotective effect in t allele carriers in taq1b polymorphism . in addition , the association of the t allele with higher hdl - c levels was observed in children ( 2 ) . taq1b polymorphism changes the activity of cetp and affects hdl - c concentrations and probably does not affect protein folding . our findings showed the a373p polymorphism had deleterious effects on lipid profile levels and approximately increased fourfold the risk of dyslipidemia in children and adolescents . agerholm - larsen et al ( 14 ) reported the a373p polymorphism in cetp associated with decrease in hdl - c levels in both genders . two studies ( 15 , 16 ) found a373p polymorphisms were associated with increasing relative risk of subclinical cardiovascular disease ( relative risk = 1.22 , p = 0.018 ) and 12.2% higher triglyceride concentration ( p = 0.03 ) . they showed that the a373p polymorphism was associated with higher cetp activity and concentration , lower hdl - c levels and atherogenic effects . the copenhagen city heart study reported that a373p polymorphism reduced levels of hdl - c ( 14 ) . however , when 1,236 french and irish subjects were examined , there was no change in cetp activity and hdl - c levels in a373p polymorphism ( 17 ) . in our study combined association analyses showed that the taq1b polymorphism had a protective effect and the a373p polymorphism had an adverse effect on dyslipidemia only in underweight and normal weight subjects in both crude and adjusted models . findings showed the relationship between the taq1b polymorphism and hdl - c levels was modified by environmental factors such as obesity ( 19 ) . it is reported that the association between taq1b genotype and hdl - c levels was attenuated by obesity ( 20 ) . however , vohl et al ( 22 ) showed taq1b polymorphism was not associated with higher hdl - c levels in men with hyperinsulinemia and obesity . in this study , we observed that dyslipidemia was not associated with the taq1b or a373p polymorphisms in obese subjects . this has been supported by others who found no association between some lipid profiles such as hdl - c concentrations and taq1b among subjects in the highest bmi tertile ( 26.240.4 kg / m ) . study showed that obesity was able to decrease the protective cardiovascular effect of the t allele in taq1b polymorphism ( 24 ) . a similar finding our findings showed that taq1b polymorphism had a protective effect on dyslipidemia in all categories of birth weight , and the a373p polymorphism only in normal birth weight category had a significantly adverse effect on dyslipidemia . british birth cohort showed inverse associations between birth weight and total cholesterol , ldl - c and triglyceride levels ( 28 ) . the main limitation of this study is the cross - sectional nature of the associations . the strengths of the present study are its novelty in the pediatric age group and a relatively large number of population - based samples . the present study showed that taq1b polymorphism had a protective effect and a373p polymorphism had a deleterious effect on dyslipidemia in iranian children and adolescents . more investigation is needed to assess the gene - environment interaction in children and adolescents .

recent advances in next - generation sequencing technologies facilitated genome - wide discoveries such as variant analysis , expression quantification , and copy number alteration ( cna ) analysis . cnas are variations in the genome that result in either gain or loss of one or more copies of the dna segment . the cnas range from 1 kilobase ( kb ) to several megabases and are one of the essential constituents of genomic diversity.1 in humans , cnas have been reported to account for approximately 12% of genomic dna.2 while some cnas do not have any observable effects on phenotype , some have been linked to diseases such as autism,3 to susceptibility to hiv,4 and to cancers such as non small - cell lung cancer5 and acute myeloid leukemia.6 wide ranges of computational approaches have been developed to identify cna events in whole - genome sequencing data . as previously described by liu et al , there are three common steps in these algorithms : data preprocessing , data segmentation , and data interpretation.7 data preprocessing starts with normalization of read depths or counts that are considered the most common input . then , log2 ratios of those counts are calculated and compared to a selected reference value , which typically is a matched control . tools such as segseq,8 readdepth,9 hmmcopy,10 bayesian information criterion sequencing ( bic - seq),11 patchwork,12 varscan2,13 control - freec14 use this approach . some of the algorithms also include steps to handle systematic biases such as genomic mappability ( control - freec , readdepth , and hmmcopy ) and gc - content ( control - freec , patchwork , and hmm - copy ) . moreover , some of them also incorporate b allele frequency information to improve cna detection ( patchwork ) . circular binary segmentation ( cbs ) and hidden markov model ( hmm ) are the most commonly adopted algorithms to implement this procedure . in addition , the hmm - based approaches simultaneously assign copy number status to each region during this segmentation step . the final step in the cna detection algorithms is interpretation of data in order to determine the copy number state . typically , an empirical cutoff is applied on each segment to identify copy number changes ( control - freec , bic - seq , varscan2 ) . some algorithms optimize these cutoff values to a desired sensitivity and specificity ( patchwork , segseq ) . once the next - generation high - throughput sequencing experiments generate millions of short ( 36 bp100 bp ) sequence reads , mapping tools such as bwa,15 bowtie,16 or soap217 align those reads to the reference genome . the uniqueness of the reference genome sequence plays a major role during this alignment stage and is one of the limitations of current cna detections tools . lack of sequence uniqueness ( mappability ) might lead to low complexity regions on the genome and therefore even the best mapping tools can not align all reads , despite using the highest quality sequence reads . therefore , with the technology in hand , it is not possible to thoroughly sequence the entire genome . for instance , only 86% of the human genome can be securely mapped by using 100-bp sequence reads ( table 1 ) . the current standard in a typical copy number analysis is to compare the tumor ( case ) sample with the matching normal sample from the same individual . however , in some cases , this might not be possible due to technical problems , availability of samples , or cost . the third issue with the current cna detection tools is the size of the detected cnas . typically , these tools detect alterations of around or larger than 100 kb with confidence . by tweaking parameters however , several hundred to thousand candidate cnas would be generated , suggesting very high false - positive rates . we believe that it is important to understand these characteristics and limitations of the genome as well as cnas detection tools and address them when developing new methods . in this paper , we describe a simple yet effective method that addresses these issues . our method detects medium - sized cnas from whole - genome sequencing data in the absence of a matching control sample by using a pool of normal samples as a baseline . in addition , our method employs median filtering to evaluate shape of the coverage around the candidate cnas and effectively eliminates false positives . mappability tracks can be generated computationally based on the level of sequence uniqueness of the reference genomes . as the sequence reads get longer , the mappability increases significantly.18 the university of california santa cruz ( ucsc ) genome browser provides mappability tracks from encode project for different sequence read lengths ( eg , 100 mer , 75 mers , 50 mers ) . these tracks are generated by gem ( genome multitool ) mappability tool and regions are scored from 0 to 1 based on the sequence uniqueness , and therefore , the higher the mappability score , the more unique is the mapping position . we downloaded mappability tracks for mouse ( mm9 ) reference genome for sequence reads of 100 , 75 , 50 , 40 , and 36 bp . then , we combined the close by regions that had mappability scores of less than 1 and defined them as unmappable regions of the genome . we observed that about 28% , 26% , 23% , 18% , and 16% of the mouse genome can not be mapped with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively . size of unmappable regions could be as short as a few base pairs or as long as several million base pairs . for example , median width of unmappable regions with 75-bp sequence reads is 21 bp and 81% of its unmappable regions are shorter than 100 bp . to better understand the size of unmappable regions across genome , we extended unmappable regions by 1 kb , if they span more than so that bordering large unmappable regions will be consolidated . this approach suggests that practically about 55% , 52% , 46% , 33% , 27% of the mouse genome is unmappable with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively ( table 1 ) . all animal studies were performed under approved protocols following the ohio state university institutional animal care and use committee . we analyzed bone marrow mononuclear cells of 11 samples obtained from two healthy wild - type control mice and nine transgenic mice with mll - partial tandem duplication ( ptd ) deficiencies that have been associated with acute myeloid leukemia.19 three of the transgenic mice were mll ( ptd / wt ) : flt3 ( itd / itd ) double knock - in mouse , three were nonleukemic mll ( ptd / wt ) mouse , and the remaining three were flt3 ( itd / wt ) single - knock - in mouse ( unpublished data ) . all transgenic mice were genotyped and six mice were validated to have ptd in mll1 gene on chromosome 9 . the whole - genome sequencing of all mouse samples was performed by the beijing genomics institute using illumina hiseq 2000 platform . the sequence reads were mapped to mouse ( mm9 ) reference genome by bwa.15 the mapping quality filter was applied to remove potential optical and polymerase chain reaction duplicate reads , nonspecific reads , and improper read pairs . on average , 638 million , 90-bp paired - end sequence reads per sample were aligned to mm9 reference genome providing 43x average coverage ( 41x minimum , 44x maximum ) . in this study , we were specifically interested in identifying medium - sized ( ~1 kb30 kb ) cna events . we started with computing the total number of sequence reads per kilobase of genome ( read counts ) for each sample using bedtools.20 next , these read counts were normalized to the total number of sequence reads per sample . therefore , we eliminated 1-kb intervals with unmappable bases . since our data consisted of 90-bp paired - end reads with slight quality drop toward the end of the reads , we chose to use mappability tracks with 75-bp reads . then , we generated pairwise log2 ratios between case and control samples using normalized read counts for the remaining 1-kb intervals . following the calculation of log2 ratios of the coverage between case and control samples , we used r dnacopy library from bioconductor21 to combine intervals into segments . this library initially implemented for the analysis of array - based dna copy number data using a cbs algorithm.21 this algorithm consolidates neighboring intervals with similar ratios into segments and reports mean ratio for the segment and total number of intervals that support this outcome . first , we applied a cutoff on the mean ratio to account for 25% loss or gain . second , since we eliminated intervals with unmappable bases at the beginning , identified regions might span through unmappable regions . therefore , we re - evaluated mappability of the identified regions and their surroundings ( including w upstream and w downstream , where w is the width of the candidate cna ) as a whole and kept the regions with 90% or more mappable bases . finally , we applied one - dimensional median filter on pairwise ratios and calculated adjusted ratio as a mean of differences before and after the median filtering . median filtering is a nonlinear filtering technique generally used to reduce noise in a signal . for the log2 ratio data over a region , r = r1r2 rn , median filtering replaces each ri with the median of [ ri d/2 , , ri , , ri + d/2 ] , where 1 < i < n and d + 1 is the sliding window size . at this step , we zoomed in the data and calculated log2 ratios at 100-bp resolution for cna candidates . then , median filtering was applied around each candidate cna staring from 4w upstream to 4w downstream , with a sliding window size of 3w , where w is the width of the candidate cna . average difference between original log2 ratios and median filtered log2 ratios over each candidate region is reported as median filter adjusted ratio . finally , cnas with 25% loss or gain after median filter adjustment and common to both wt comparisons were reported as final cnas . mappability tracks can be generated computationally based on the level of sequence uniqueness of the reference genomes . as the sequence reads get longer , the mappability increases significantly.18 the university of california santa cruz ( ucsc ) genome browser provides mappability tracks from encode project for different sequence read lengths ( eg , 100 mer , 75 mers , 50 mers ) . these tracks are generated by gem ( genome multitool ) mappability tool and regions are scored from 0 to 1 based on the sequence uniqueness , and therefore , the higher the mappability score , the more unique is the mapping position . we downloaded mappability tracks for mouse ( mm9 ) reference genome for sequence reads of 100 , 75 , 50 , 40 , and 36 bp . then , we combined the close by regions that had mappability scores of less than 1 and defined them as unmappable regions of the genome . we observed that about 28% , 26% , 23% , 18% , and 16% of the mouse genome can not be mapped with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively . size of unmappable regions could be as short as a few base pairs or as long as several million base pairs . for example , median width of unmappable regions with 75-bp sequence reads is 21 bp and 81% of its unmappable regions are shorter than 100 bp . to better understand the size of unmappable regions across genome , we extended unmappable regions by 1 kb , if they span more than so that bordering large unmappable regions will be consolidated . this approach suggests that practically about 55% , 52% , 46% , 33% , 27% of the mouse genome is unmappable with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively ( table 1 ) . all animal studies were performed under approved protocols following the ohio state university institutional animal care and use committee . we analyzed bone marrow mononuclear cells of 11 samples obtained from two healthy wild - type control mice and nine transgenic mice with mll - partial tandem duplication ( ptd ) deficiencies that have been associated with acute myeloid leukemia.19 three of the transgenic mice were mll ( ptd / wt ) : flt3 ( itd / itd ) double knock - in mouse , three were nonleukemic mll ( ptd / wt ) mouse , and the remaining three were flt3 ( itd / wt ) single - knock - in mouse ( unpublished data ) . all transgenic mice were genotyped and six mice were validated to have ptd in mll1 gene on chromosome 9 . the whole - genome sequencing of all mouse samples was performed by the beijing genomics institute using illumina hiseq 2000 platform . the sequence reads were mapped to mouse ( mm9 ) reference genome by bwa.15 the mapping quality filter was applied to remove potential optical and polymerase chain reaction duplicate reads , nonspecific reads , and improper read pairs . on average , 638 million , 90-bp paired - end sequence reads per sample were aligned to mm9 reference genome providing 43x average coverage ( 41x minimum , 44x maximum ) . in this study , we were specifically interested in identifying medium - sized ( ~1 kb30 kb ) cna events . we started with computing the total number of sequence reads per kilobase of genome ( read counts ) for each sample using bedtools.20 next , these read counts were normalized to the total number of sequence reads per sample . since our data consisted of 90-bp paired - end reads with slight quality drop toward the end of the reads , we chose to use mappability tracks with 75-bp reads . then , we generated pairwise log2 ratios between case and control samples using normalized read counts for the remaining 1-kb intervals . following the calculation of log2 ratios of the coverage between case and control samples , we used r dnacopy library from bioconductor21 to combine intervals into segments . this library initially implemented for the analysis of array - based dna copy number data using a cbs algorithm.21 this algorithm consolidates neighboring intervals with similar ratios into segments and reports mean ratio for the segment and total number of intervals that support this outcome . first , we applied a cutoff on the mean ratio to account for 25% loss or gain . second , since we eliminated intervals with unmappable bases at the beginning , identified regions might span through unmappable regions . therefore , we re - evaluated mappability of the identified regions and their surroundings ( including w upstream and w downstream , where w is the width of the candidate cna ) as a whole and kept the regions with 90% or more mappable bases . finally , we applied one - dimensional median filter on pairwise ratios and calculated adjusted ratio as a mean of differences before and after the median filtering . median filtering is a nonlinear filtering technique generally used to reduce noise in a signal . for the log2 ratio data over a region , r = r1r2 rn , median filtering replaces each ri with the median of [ ri d/2 , , ri , , ri + d/2 ] , where 1 < i < n and d + 1 is the sliding window size . at this step , we zoomed in the data and calculated log2 ratios at 100-bp resolution for cna candidates . then , median filtering was applied around each candidate cna staring from 4w upstream to 4w downstream , with a sliding window size of 3w , where w is the width of the candidate cna . average difference between original log2 ratios and median filtered log2 ratios over each candidate region is reported as median filter adjusted ratio . finally , cnas with 25% loss or gain after median filter adjustment and common to both wt comparisons were reported as final cnas . since it is not possible to thoroughly and accurately sequence the entire genome with short sequence reads generated by current high - throughput sequencing technology , the genome mappability was the first issue we aimed to address in this paper . a large portion of this region ( about 3 million bases ) is mostly unmappable ( gem score < 1 ) with 75-bp sequence reads . this region also contains segmental duplications ( duplications of > 1000 bases that are not masked by repeatmasker ) . it is not possible to identify medium - sized cnas within this kind of regions . as shown in table 1 , approximately 33% of mouse genome is unmappable with 75-bp reads . our approach handles this issue by eliminating intervals with low mappability at the beginning of the analysis . intervals with unmappable bases ( marked with green ) were eliminated prior to running the cbs algorithm on pairwise log2 ratios between ptd / itd samples versus wt samples . mean log2 ratio between the first ptd / itd sample and the first wt sample over this 3000-bp region was 0.79 , suggesting 75% loss and 94% mappable bases including the surrounding region ( 3000 bp upstream and 3000 bp downstream ) . after applying median filtering on log2 ratios in 100-bp resolution , average difference between original ratios and median filtered ratios within this region was 0.89 , reporting 85% loss in this region . this 3000-bp cna made the final list since it was reported in comparisons to both wt samples with more than 25% loss . the transgenic mice samples evaluated in this study were genotyped and six mice were experimentally validated to have ptd in mll1 gene . sequencing showed the presence of multiple copies in the mll1 gene and therefore the alignment of sequence reads to the reference genome resulted in a larger number of reads being mapped to this region . this led to larger coverage in this duplicated region compared to the rest of the gene . figure 4 shows a slight enrichment ( 30% gain ) around ptd region in ptd / itd and ptd samples coverage tracks around mll1 gene . our method was able to successfully detect this enrichment , because our approach allows application of low cutoffs with confidence , while median filter adjustment allows easy distinction between true positives and false positives . figure 5 shows a 3000-bp candidate cna on chromosome 1 , with a mean log2 ratio of 0.57 , suggesting 49% loss in that region . median filter - adjusted ratio would be 0.20 , suggesting only 15% loss . ptd / itd 2 seems to have a slightly lower coverage across this region compared to wt 2 sample . however , this is only a slight fluctuation in coverage and median filter adjustment easily picks up this kind of fluctuations and eliminates such false positives . although , 25% loss or gain cutoff is a very loose cutoff and results in a lot of false positives , median filter adjustment helps us to eliminate these false positives effectively and allows us to report loss or gain events confidently . when evaluating coverage tracks of ptd / itd mouse samples following the elimination of intervals with low mappability , cbs algorithm detected about 292 cna candidates per ptd / itd sample compared to wt . finally , selection of cnas common to both control comparisons resulted in 16% of the initially reported candidates , which is 47 cnas per ptd / itd sample with a median size of 3000 bases ( average size of 10,300 bases ) . however , if we had applied that filter without median filter adjustment , we would end up with 30% of the initially reported candidates , which is 88 cnas per ptd / itd sample . therefore , median filter adjustment is clearly a crucial step in this approach , as we would have reported twice as much cna candidates without this adjustment . the next generation sequencing ( ngs ) data allow us to investigate genome - wide discoveries such as single - nucleotide polymorphisms , expression data , dna s structural variations , and cnas . designing tools that facilitate efficient detection of those discoveries is the next step that brings us closer to personalized medicine . currently , tools designed to detect cna face challenges such as a lack of sequence uniqueness in the genome , dependence on matching control samples for comparison and inability to detect smaller size cnas . in this paper , we propose a simple yet effective approach that takes into account these limitations of current technology and specifically targets to identify medium - sized ( 130 kb ) cnas . first , our method addresses the issue of mappability by eliminating regions of the genome with low mappability at the beginning of the analysis . this resulted in elimination of about 33% of the mouse genome , leaving us with the remaining 67% for analyses . although mappability - associated fluctuations in the coverage smoothen out and thus do not pose difficulties when identifying large aberrations ( > 500 kb ) in the genome , such fluctuations become the signal when detecting the medium - sized cnas . therefore , unmappable regions of the genome should be masked , for the sake of avoiding high false - positive rates . when testing for cnas , the usual approach is to compare the tumor sample with the control sample from the same individual . in some cases , similarly , in our study , we tested 11 samples , 9 of which were from transgenic mice , and therefore these samples did not have their matched controls . so , in accordance with previous studies,22 we used the samples from the control mice that represented a pool of control samples . median filtering is applied around each candidate cna and window size is directly determined by cna size . the difference between the median filtered ratios and original ratios suggests that candidate cna indeed has a different coverage profile compared to its surrounding and it can be reported confidently . if median filtered ratios are not much different than the original ratios , adjusted ratios would suggest much smaller gain or loss , thus a false positive . median filtering was used by lee et al.23 with a constant window size to smooth the coverage signal as part of the preprocessing before cna detection . we employ median filtering with variable window size to adjust copy number change rate and cna ratio . we also tested the bic - seq algorithm , which is one of the well - established tools for the detection of cnas from whole - genome sequencing data,11 with our data . the bic - seq is a nonparametric model that segments the genome into small bins and iteratively combines adjacent bins with the same copy number . due to its nonparametric nature , however , one of its limitations is that it relies on comparison of case samples with matched control samples . this limitation confines the utilization of this algorithm to analyses that only have matched control samples . despite not having the matched control in our samples , we still tested this algorithm using our wt samples as the control . we ran the bic - seq algorithm using ptd / itd samples as case and each wt as a control . the bic - seq has two main adjustable parameters : starting bin and lambda , which is used for tuning the smoothness of the cnv profile . the average number of detected cnas decreases with increasing bin size and lambda ; however , the average size of cnas increases . we tested three combinations of bin sizes and lambda values and observed that in all the cases only about 5.5% of cnas were coming from securely mappable regions , while the rest was from unmappable regions of the genome ( table 2 ) . in comparison to our method , which detected 159 cnas from 40% of securely mappable regions of the genome in all ptd / itd samples , bic - seq falls short in identifying cnas from mappable regions , while reporting high rate of cnas , most of which might be false positives . another method we tested was control - freec , which automatically calculates the copy number profiles from the ngs data.14 one advantage of control - freec is that the use of matched normal samples is optional . this approach also allows exclusion of regions with low mappability from the analysis by using provided mappability tracks . however , unmappable regions are not eliminated entirely but are rather skipped during the evaluation . in other words , control - freec algorithm considers an unmappable region as a gap . when we tested our samples with the control - freec method , it detected approximately 160 cnas per sample with an average cna size of 500 kb . only about 4% of those cnas were in completely mappable areas . given the large sizes of the detected cnas , it was not a surprise that control - freec identifies cnas in unmappable regions . this algorithm identified fewer cnas from mappable regions compared to both the bic - seq and our method . most of the cna detection tools allow adjustments in the size of the detected cna by modifying some parameters . however , one of the most common problems is that as the cna size gets smaller , the number of detected cnas gets larger . unfortunately , most of these detected cnas are false positives , because these approaches can not manage fluctuation in coverage when the bin size gets smaller . in the larger bin sizes , alas , there is no reliable and effective algorithm for detection of medium - sized cna that takes these problems into consideration . in this paper , we discussed potential issues in medium - sized cna detection using whole - genome sequencing data and why existing approaches are not suitable for the detection of medium - sized cnas . we tested an approach to demonstrate that by addressing underlying issues , we can effectively identify medium - sized cnas . we understand that our approach requires further modifications ; nevertheless , the fact that our method successfully detects cnas from mappable regions of the genome is a proof of concept . this approach can be improved by implementing a sliding window approach to better identify cna start and end sites ( rather than bin start and end ) . we will also work on a methodology to eliminate false positives due to nonuniform coverage in control samples .

the genome - wide discoveries such as detection of copy number alterations ( cna ) from high - throughput whole - genome sequencing data enabled new developments in personalized medicine . the cnas have been reported to be associated with various diseases and cancers including acute myeloid leukemia . however , there are multiple challenges to the use of current cna detection tools that lead to high false - positive rates and thus impede widespread use of such tools in cancer research . in this paper , we discuss these issues and propose possible solutions . first , since the entire genome can not be mapped due to some regions lacking sequence uniqueness , current methods can not be appropriately adjusted to handle these regions in the analyses . thus , detection of medium - sized cnas is also being directly affected by these mappability problems . the requirement for matching control samples is also an important limitation because acquiring matching controls might not be possible or might not be cost efficient . here we present an approach that addresses these issues and detects medium - sized cnas in cancer genomes by ( 1 ) masking unmappable regions during the initial cna detection phase , ( 2 ) using pool of a few normal samples as control , and ( 3 ) employing median filtering to adjust cna ratios to its surrounding coverage and eliminate false positives .

Introduction Methods Genome mappability Whole-genome sequencing data Detection of CNAs Results Discussion

recent advances in next - generation sequencing technologies facilitated genome - wide discoveries such as variant analysis , expression quantification , and copy number alteration ( cna ) analysis . cnas are variations in the genome that result in either gain or loss of one or more copies of the dna segment . the cnas range from 1 kilobase ( kb ) to several megabases and are one of the essential constituents of genomic diversity.1 in humans , cnas have been reported to account for approximately 12% of genomic dna.2 while some cnas do not have any observable effects on phenotype , some have been linked to diseases such as autism,3 to susceptibility to hiv,4 and to cancers such as non small - cell lung cancer5 and acute myeloid leukemia.6 wide ranges of computational approaches have been developed to identify cna events in whole - genome sequencing data . as previously described by liu et al , there are three common steps in these algorithms : data preprocessing , data segmentation , and data interpretation.7 data preprocessing starts with normalization of read depths or counts that are considered the most common input . some of the algorithms also include steps to handle systematic biases such as genomic mappability ( control - freec , readdepth , and hmmcopy ) and gc - content ( control - freec , patchwork , and hmm - copy ) . moreover , some of them also incorporate b allele frequency information to improve cna detection ( patchwork ) . in addition , the hmm - based approaches simultaneously assign copy number status to each region during this segmentation step . the final step in the cna detection algorithms is interpretation of data in order to determine the copy number state . typically , an empirical cutoff is applied on each segment to identify copy number changes ( control - freec , bic - seq , varscan2 ) . once the next - generation high - throughput sequencing experiments generate millions of short ( 36 bp100 bp ) sequence reads , mapping tools such as bwa,15 bowtie,16 or soap217 align those reads to the reference genome . the uniqueness of the reference genome sequence plays a major role during this alignment stage and is one of the limitations of current cna detections tools . lack of sequence uniqueness ( mappability ) might lead to low complexity regions on the genome and therefore even the best mapping tools can not align all reads , despite using the highest quality sequence reads . therefore , with the technology in hand , it is not possible to thoroughly sequence the entire genome . for instance , only 86% of the human genome can be securely mapped by using 100-bp sequence reads ( table 1 ) . the current standard in a typical copy number analysis is to compare the tumor ( case ) sample with the matching normal sample from the same individual . however , in some cases , this might not be possible due to technical problems , availability of samples , or cost . the third issue with the current cna detection tools is the size of the detected cnas . by tweaking parameters however , several hundred to thousand candidate cnas would be generated , suggesting very high false - positive rates . we believe that it is important to understand these characteristics and limitations of the genome as well as cnas detection tools and address them when developing new methods . in this paper , we describe a simple yet effective method that addresses these issues . our method detects medium - sized cnas from whole - genome sequencing data in the absence of a matching control sample by using a pool of normal samples as a baseline . in addition , our method employs median filtering to evaluate shape of the coverage around the candidate cnas and effectively eliminates false positives . these tracks are generated by gem ( genome multitool ) mappability tool and regions are scored from 0 to 1 based on the sequence uniqueness , and therefore , the higher the mappability score , the more unique is the mapping position . we downloaded mappability tracks for mouse ( mm9 ) reference genome for sequence reads of 100 , 75 , 50 , 40 , and 36 bp . then , we combined the close by regions that had mappability scores of less than 1 and defined them as unmappable regions of the genome . we observed that about 28% , 26% , 23% , 18% , and 16% of the mouse genome can not be mapped with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively . size of unmappable regions could be as short as a few base pairs or as long as several million base pairs . for example , median width of unmappable regions with 75-bp sequence reads is 21 bp and 81% of its unmappable regions are shorter than 100 bp . to better understand the size of unmappable regions across genome , we extended unmappable regions by 1 kb , if they span more than so that bordering large unmappable regions will be consolidated . this approach suggests that practically about 55% , 52% , 46% , 33% , 27% of the mouse genome is unmappable with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively ( table 1 ) . we analyzed bone marrow mononuclear cells of 11 samples obtained from two healthy wild - type control mice and nine transgenic mice with mll - partial tandem duplication ( ptd ) deficiencies that have been associated with acute myeloid leukemia.19 three of the transgenic mice were mll ( ptd / wt ) : flt3 ( itd / itd ) double knock - in mouse , three were nonleukemic mll ( ptd / wt ) mouse , and the remaining three were flt3 ( itd / wt ) single - knock - in mouse ( unpublished data ) . the whole - genome sequencing of all mouse samples was performed by the beijing genomics institute using illumina hiseq 2000 platform . the sequence reads were mapped to mouse ( mm9 ) reference genome by bwa.15 the mapping quality filter was applied to remove potential optical and polymerase chain reaction duplicate reads , nonspecific reads , and improper read pairs . in this study , we were specifically interested in identifying medium - sized ( ~1 kb30 kb ) cna events . we started with computing the total number of sequence reads per kilobase of genome ( read counts ) for each sample using bedtools.20 next , these read counts were normalized to the total number of sequence reads per sample . therefore , we eliminated 1-kb intervals with unmappable bases . since our data consisted of 90-bp paired - end reads with slight quality drop toward the end of the reads , we chose to use mappability tracks with 75-bp reads . then , we generated pairwise log2 ratios between case and control samples using normalized read counts for the remaining 1-kb intervals . following the calculation of log2 ratios of the coverage between case and control samples , we used r dnacopy library from bioconductor21 to combine intervals into segments . this library initially implemented for the analysis of array - based dna copy number data using a cbs algorithm.21 this algorithm consolidates neighboring intervals with similar ratios into segments and reports mean ratio for the segment and total number of intervals that support this outcome . first , we applied a cutoff on the mean ratio to account for 25% loss or gain . second , since we eliminated intervals with unmappable bases at the beginning , identified regions might span through unmappable regions . therefore , we re - evaluated mappability of the identified regions and their surroundings ( including w upstream and w downstream , where w is the width of the candidate cna ) as a whole and kept the regions with 90% or more mappable bases . finally , we applied one - dimensional median filter on pairwise ratios and calculated adjusted ratio as a mean of differences before and after the median filtering . median filtering is a nonlinear filtering technique generally used to reduce noise in a signal . for the log2 ratio data over a region , r = r1r2 rn , median filtering replaces each ri with the median of [ ri d/2 , , ri , , ri + d/2 ] , where 1 < i < n and d + 1 is the sliding window size . at this step , we zoomed in the data and calculated log2 ratios at 100-bp resolution for cna candidates . then , median filtering was applied around each candidate cna staring from 4w upstream to 4w downstream , with a sliding window size of 3w , where w is the width of the candidate cna . mappability tracks can be generated computationally based on the level of sequence uniqueness of the reference genomes . these tracks are generated by gem ( genome multitool ) mappability tool and regions are scored from 0 to 1 based on the sequence uniqueness , and therefore , the higher the mappability score , the more unique is the mapping position . we downloaded mappability tracks for mouse ( mm9 ) reference genome for sequence reads of 100 , 75 , 50 , 40 , and 36 bp . then , we combined the close by regions that had mappability scores of less than 1 and defined them as unmappable regions of the genome . we observed that about 28% , 26% , 23% , 18% , and 16% of the mouse genome can not be mapped with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively . size of unmappable regions could be as short as a few base pairs or as long as several million base pairs . for example , median width of unmappable regions with 75-bp sequence reads is 21 bp and 81% of its unmappable regions are shorter than 100 bp . to better understand the size of unmappable regions across genome , we extended unmappable regions by 1 kb , if they span more than so that bordering large unmappable regions will be consolidated . this approach suggests that practically about 55% , 52% , 46% , 33% , 27% of the mouse genome is unmappable with 36 , 40 , 50 , 75 , and 100-bp sequence reads , respectively ( table 1 ) . we analyzed bone marrow mononuclear cells of 11 samples obtained from two healthy wild - type control mice and nine transgenic mice with mll - partial tandem duplication ( ptd ) deficiencies that have been associated with acute myeloid leukemia.19 three of the transgenic mice were mll ( ptd / wt ) : flt3 ( itd / itd ) double knock - in mouse , three were nonleukemic mll ( ptd / wt ) mouse , and the remaining three were flt3 ( itd / wt ) single - knock - in mouse ( unpublished data ) . the whole - genome sequencing of all mouse samples was performed by the beijing genomics institute using illumina hiseq 2000 platform . the sequence reads were mapped to mouse ( mm9 ) reference genome by bwa.15 the mapping quality filter was applied to remove potential optical and polymerase chain reaction duplicate reads , nonspecific reads , and improper read pairs . in this study , we were specifically interested in identifying medium - sized ( ~1 kb30 kb ) cna events . we started with computing the total number of sequence reads per kilobase of genome ( read counts ) for each sample using bedtools.20 next , these read counts were normalized to the total number of sequence reads per sample . since our data consisted of 90-bp paired - end reads with slight quality drop toward the end of the reads , we chose to use mappability tracks with 75-bp reads . then , we generated pairwise log2 ratios between case and control samples using normalized read counts for the remaining 1-kb intervals . following the calculation of log2 ratios of the coverage between case and control samples , we used r dnacopy library from bioconductor21 to combine intervals into segments . this library initially implemented for the analysis of array - based dna copy number data using a cbs algorithm.21 this algorithm consolidates neighboring intervals with similar ratios into segments and reports mean ratio for the segment and total number of intervals that support this outcome . first , we applied a cutoff on the mean ratio to account for 25% loss or gain . second , since we eliminated intervals with unmappable bases at the beginning , identified regions might span through unmappable regions . therefore , we re - evaluated mappability of the identified regions and their surroundings ( including w upstream and w downstream , where w is the width of the candidate cna ) as a whole and kept the regions with 90% or more mappable bases . finally , we applied one - dimensional median filter on pairwise ratios and calculated adjusted ratio as a mean of differences before and after the median filtering . median filtering is a nonlinear filtering technique generally used to reduce noise in a signal . for the log2 ratio data over a region , r = r1r2 rn , median filtering replaces each ri with the median of [ ri d/2 , , ri , , ri + d/2 ] , where 1 < i < n and d + 1 is the sliding window size . at this step , we zoomed in the data and calculated log2 ratios at 100-bp resolution for cna candidates . then , median filtering was applied around each candidate cna staring from 4w upstream to 4w downstream , with a sliding window size of 3w , where w is the width of the candidate cna . since it is not possible to thoroughly and accurately sequence the entire genome with short sequence reads generated by current high - throughput sequencing technology , the genome mappability was the first issue we aimed to address in this paper . a large portion of this region ( about 3 million bases ) is mostly unmappable ( gem score < 1 ) with 75-bp sequence reads . it is not possible to identify medium - sized cnas within this kind of regions . after applying median filtering on log2 ratios in 100-bp resolution , average difference between original ratios and median filtered ratios within this region was 0.89 , reporting 85% loss in this region . the transgenic mice samples evaluated in this study were genotyped and six mice were experimentally validated to have ptd in mll1 gene . sequencing showed the presence of multiple copies in the mll1 gene and therefore the alignment of sequence reads to the reference genome resulted in a larger number of reads being mapped to this region . this led to larger coverage in this duplicated region compared to the rest of the gene . however , this is only a slight fluctuation in coverage and median filter adjustment easily picks up this kind of fluctuations and eliminates such false positives . however , if we had applied that filter without median filter adjustment , we would end up with 30% of the initially reported candidates , which is 88 cnas per ptd / itd sample . therefore , median filter adjustment is clearly a crucial step in this approach , as we would have reported twice as much cna candidates without this adjustment . the next generation sequencing ( ngs ) data allow us to investigate genome - wide discoveries such as single - nucleotide polymorphisms , expression data , dna s structural variations , and cnas . designing tools that facilitate efficient detection of those discoveries is the next step that brings us closer to personalized medicine . currently , tools designed to detect cna face challenges such as a lack of sequence uniqueness in the genome , dependence on matching control samples for comparison and inability to detect smaller size cnas . in this paper , we propose a simple yet effective approach that takes into account these limitations of current technology and specifically targets to identify medium - sized ( 130 kb ) cnas . first , our method addresses the issue of mappability by eliminating regions of the genome with low mappability at the beginning of the analysis . although mappability - associated fluctuations in the coverage smoothen out and thus do not pose difficulties when identifying large aberrations ( > 500 kb ) in the genome , such fluctuations become the signal when detecting the medium - sized cnas . therefore , unmappable regions of the genome should be masked , for the sake of avoiding high false - positive rates . in some cases , similarly , in our study , we tested 11 samples , 9 of which were from transgenic mice , and therefore these samples did not have their matched controls . so , in accordance with previous studies,22 we used the samples from the control mice that represented a pool of control samples . median filtering is applied around each candidate cna and window size is directly determined by cna size . the difference between the median filtered ratios and original ratios suggests that candidate cna indeed has a different coverage profile compared to its surrounding and it can be reported confidently . median filtering was used by lee et al.23 with a constant window size to smooth the coverage signal as part of the preprocessing before cna detection . we employ median filtering with variable window size to adjust copy number change rate and cna ratio . we also tested the bic - seq algorithm , which is one of the well - established tools for the detection of cnas from whole - genome sequencing data,11 with our data . the bic - seq is a nonparametric model that segments the genome into small bins and iteratively combines adjacent bins with the same copy number . due to its nonparametric nature , however , one of its limitations is that it relies on comparison of case samples with matched control samples . this limitation confines the utilization of this algorithm to analyses that only have matched control samples . despite not having the matched control in our samples , we still tested this algorithm using our wt samples as the control . we ran the bic - seq algorithm using ptd / itd samples as case and each wt as a control . the average number of detected cnas decreases with increasing bin size and lambda ; however , the average size of cnas increases . we tested three combinations of bin sizes and lambda values and observed that in all the cases only about 5.5% of cnas were coming from securely mappable regions , while the rest was from unmappable regions of the genome ( table 2 ) . in comparison to our method , which detected 159 cnas from 40% of securely mappable regions of the genome in all ptd / itd samples , bic - seq falls short in identifying cnas from mappable regions , while reporting high rate of cnas , most of which might be false positives . another method we tested was control - freec , which automatically calculates the copy number profiles from the ngs data.14 one advantage of control - freec is that the use of matched normal samples is optional . however , unmappable regions are not eliminated entirely but are rather skipped during the evaluation . given the large sizes of the detected cnas , it was not a surprise that control - freec identifies cnas in unmappable regions . most of the cna detection tools allow adjustments in the size of the detected cna by modifying some parameters . however , one of the most common problems is that as the cna size gets smaller , the number of detected cnas gets larger . unfortunately , most of these detected cnas are false positives , because these approaches can not manage fluctuation in coverage when the bin size gets smaller . in the larger bin sizes , alas , there is no reliable and effective algorithm for detection of medium - sized cna that takes these problems into consideration . in this paper , we discussed potential issues in medium - sized cna detection using whole - genome sequencing data and why existing approaches are not suitable for the detection of medium - sized cnas . we tested an approach to demonstrate that by addressing underlying issues , we can effectively identify medium - sized cnas . we understand that our approach requires further modifications ; nevertheless , the fact that our method successfully detects cnas from mappable regions of the genome is a proof of concept . we will also work on a methodology to eliminate false positives due to nonuniform coverage in control samples .

stroke is the leading cause of adult disability in the developed world.1 , 2 in ischemic stroke , a blood clot ( thrombus ) leads to an area of brain experiencing a significant drop in cerebral blood flow , which can ultimately result in death of this hypoperfused tissue . collateral blood flow gives rise to the ischemic penumbra , which is defined as an area of hypoperfused tissue that is at risk of death ( infarction ) if hypoperfusion persists , and is separate from tissue that has already died ( or programmed to die ) soon after stroke onset ( core).3 the concept of the ischemic penumbra led to the notion that opening an occluded blood vessel in order to re - establish blood flow to the ischemic area ( reperfusion ) would rescue the ischemic penumbra from death3,4 ( figure 1 ) . reperfusion can be achieved through the use of systemic thrombolytic drugs , although thrombolytic agents may theoretically be more effective if delivered directly into the clot ( intra - arterial delivery ) or if assisted with ultrasound insonation of the clot ( sono - thrombolysis ) . a thrombus can originate from several different causes , which may alter its susceptibility to thrombolytic drugs . the main causes of thrombus formation in ischemic stroke are atherosclerosis ( in - situ thrombus or thromboembolic ) or forming in the heart ( cardioembolic ) . it was originally hypothesized that the source of thrombi had a large determining influence on clot composition and therefore potentially upon susceptibility to thrombolytic treatment as thromboembolic clots tend to be platelet and fibrin rich and were formed in high flow areas ( often referred to white clots ) , whilst cardioembolic can contain tissue debris such as fat , air or bacteria interwoven inside the platelet and fibrin mesh which were formed in low flow areas ( often referred to as red clots).5,6 however recent histological studies analyzing the thrombi extracted from patients using intra - arterial clot retrieval showed thrombi composition was similar between cardiac and arterial origins.7 atherosclerotic plaque rupture or cardio - embolic thrombosis ( typically due to atrial fibrillation ) leads to activation of the coagulation cascade as well as platelet activation . in the cascade , zymogens ( free floating inactive coagulation factor precursors ) are converted to an activated coagulation factor by interaction with the atherosclerotic plaque / thrombus.8 each active zymogene is able to activate nearby zymogens , leading to a large localized reaction of coagulation factors . activated platelets then catalyze an interaction between activated coagulation factors ( zymogens ) to aid in the generation of thrombin by conversion of the soluble protein fibrinogen to insoluble fibrin , forming a blood clot . there are two blood coagulation cascades ( intrinsic and extrinsic pathways ) , that have separate initial pathways but converge on a common pathway9 ( figure 2).10 thrombolytic drugs dissolve ( lyse ) thrombi in the vascular bed by activating plasminogen to form plasmin . plasmin is a proteolytic enzyme that breaks the crosslinks between fibrin molecules to destabilise the structural integrity of blood clots . the main types of thrombolytic drugs used in ischemic stroke to activate plasminogen are urokinase / streptokinase and tissue plasminogen activators ( eg , alteplase ) . thrombolytic drug development has undergone at least three generations with the aim of enhancing fibrin specificity or reducing inhibition of thrombolysis by plasminogen activator inhibitor type 1 ( table 1 ) . tissue plasminogen activator ( tpa ) is a serine protease found on the endothelial cells lining blood vessels and is involved in the breakdown of blood clots ( thrombus ) . a thrombus is composed of fibrin monomers that are cross - linked through lysine side chains which tpa binds too . lysine binding of tpa results in a activation of plasminogen only around a thrombus , which minimises activation of circulating plasminogen . the lysine side chains have a high affinity for binding with plasminogen , making the thrombus plasminogen rich . the tpa enzyme binds to fibrin components of a thrombus and catalyses plasminogen conversion to plasmin by cleavage of the arginine - valine bond at positions 560 and 561 to break down a clot by degrading the fibrin matrix of a thrombus . plasmin then breaks the thrombus down into fibrin degradation products due to the plasmin 's protease action in dissolving the thrombus.11 however , on fibrin bound plasmin , the inhibitory effect of alpha 2-antiplasmin and type 1 plasminogen activeator inhibitor restricts lysine binding.12 streptokinase is a first generation thrombolytic agent . therefore streptokinase is not a protease but binds to plasminogen for the generation of plasmin and is not restricted at the site of thrombus formation . because of its non lysine specificity , streptokinase , produces more fibrin degradation products ( fibrinogensis ) as a result of widespread lytic action in the body . fibrinogensis occurs when there are high levels of fibrin in the blood and can cause thrombosis , haemorrhage or tissue oedema . although much less expensive , this makes streptokinase a less attractive agent for acute treatments in ischemic stroke than tpa products . studies of streptokinase in acute stroke were stopped due to an increase in mortality compared to placebo due to increased haemorrhage rates.13,14 the specificity of tpa drugs ( alteplase , retaplase , tenecteplase and desmoteplase ) for plasminogen bound fibrin means that conversion of plasminogen to plasma occurs in clots with minimal circulating plasma . the 1999 prolyse in acute cerebral thromboembolism ii trial ( proact ii ) tested the benefit of intra arterial delivery of 9 mg of recombinant prourokinasea ( r - prouk ) in 180 patients . despite an increased rate of symptomatic intracranial haemorrhage the study found a significant benefit in patients treated with r - prouk at 90 days.15 to date , the proact ii study is the only positive phase iii intra - arterial trial . some centres use r - prouk intravenously due to its low cost compared to other thrombolytic drugs . the second generation thrombolytic drug alteplase , is a recombinant form of human rtpa and has undergone the most study in the clinical stroke setting . recombinant techniques mean that molecular cloning is used to bring genetic material together from multiple sources that would not otherwise be found together , to create new dna sequences , which can be used to manufacture drugs . alteplase is a purified glycoprotein ( a protein with sugar chains covalently attached to polypeptide side chains ) of 527 amino acids that is synthesised from the complementary dna ( cdna ) of natural human tissue - type plasminogen activator found in human melanoma cells and is made up of five structural components : a protease , epidermal growth factor ( egf ) and two kringle domains ( figure 3 ) . the lysine binding sites of alteplase are on the kringle 2 domain and are why alteplase has a high binding affinity to thrombi . in terms of pharmacodynamics , rtpa has a short half life of around 5 min , and therefore requires an infusion after a bolus injection in the acute ischemic stroke setting . the majority of ischemic stroke clinical research has revolved around alteplase , as it is the only food and drug administration ( fda ) approved drug for ischemic stroke . fda approval for alteplase was licensed for the management of acute myocardial infarction in 1987 , acute massive pulmonary embolism in 1990 and acute ischemic stroke in 1996.16 the total dose of alteplase for acute ischemic stroke is divided into a 10% bolus and 90% as an infusion over 60 min . small dose - range studies for alteplase in human stroke found increasing neurological benefit up to a dose of 0.85 mg / kg , but an increased rate of haemorrhage with a dose of 0.95 mg / kg.17 the ground - breaking ninds - rtpa study was the first positive phase iii acute stroke trial . this trial showed that early treatment ( within 3 hours ) of an ischemic stroke with alteplase substantially improved clinical outcomes . an alteplase dose of 0.9 mg / kg was used in the ninds trial.18 and found no significant increase in the risk of haemorrhage.19 the ideal dose of alteplase is still under debate with the original alteplase dose - ranging studies being quite small . it is felt that asian people may be more prone to haemorrhage from tpa , although this is not based on level one evidence . to this point , the standard dose of alteplase in japan in 0.6 mg / kg based on a single japanese study.20 on the other hand , there is justifiable concern that a reduced dose of alteplase will not successfully lyse larger clots , typically in more proximal locations . it has been reliably demonstrated that long clots on non contrast computed tomography ( ncct ) show a reduced rate of reperfusion and subsequent clinical improvement.21 indeed , a recent study of 130 patients using non - contrast ct to measure occlusion length identified that the tpa dose was independently predicative of thrombus resolution , with complete resolution in only 8% of patients . the average occlusion reduced by 20% , while 15% of patients had an increase in clot length following tpa administration . therefore tpa dose was associated with thrombus resolution and improved clinical outcome.22 following the ninds study , which showed that early treatment ( within 3 hours ) was the key to improving ischemic stroke outcome , there have been several studies investigating extending this treatment time window . the european cooperative acute stroke study ( ecass ) i and ii looked at administering alteplase between 0 and 6 hours after ischemic stroke onset and did not find an overall benefit , with an increased rate of haemorrhage in ecass i. however ecass i and ii did not use advanced imaging and may have had a high rate of patients with very little penumbra to salvage , especially in the 3 - 6 hours time window . ecass iii subsequently demonstrated evidence of good outcome following treatment with 0.9 mg / kg alteplase between 3 and 4.5 hours in patients < 80 years after symptom onset using only ncct to exclude patients with haemorrhage or oedema . as a result of ecass iii , many countries have amended the license for stroke treatment with rtpa to 4.5 hours following ncct assessment . there has been much interest in using advanced imaging to visualise the volume of the penumbra and infarct core , particularly with a view to extending the treatment window for thrombolysis . the echoplanar imaging thrombolytic evaluation trial ( epithet ) randomised ischemic stroke patients 3 - 6 hours after symptom onset to alteplase or placebo and used acute magnetic resonance imaging ( mri ) to define the core and penumbra ( ' diffusion - perfusion mismatch ' ) . the primary hypothesis of this phase ii trial was that patients with perfusion - diffusion mismatch would have less infarct growth with alteplase than with placebo . although the primary outcome was negative ( mean geometric infarct growth ) all secondary outcomes were positive and confirmed that alteplase attenuated infarct growth in mismatch patients . other analyses from the epithet dataset have shown that the diffusion infarct core is the strongest predictor of response to thrombolysis , with a chance of good outcome dropping if the core > 25 ml and there being virtually no benefit > 70 ml.23,24 pooled analysis from epithet and another trial of alteplase 3 - 6 hours after ischemic stroke , the diffusion and perfusion imaging evaluation for understanding stroke evolution study ( defuse ) , showed that patients in the 3 - 6 hours time window with a favourable imaging profile , i.e. a large penumbra , and small infarct core.25 as stroke occurs predominantly in the aged population , with > 45% of patients receiving alteplase being over 70 years old,26 there is a concern about decreasing benefit from thrombolysis with age . the main concerns are over the possibility of aged related renal and hepatic impairments affecting the metabolising and clearance of alteplase as well as the possibility of an increased risk of haemorrhagic transformation of the infarct following alteplase administration . there is also concern that the elderly tend to have subtle pre - morbid cognitive dysfunction and also may have less capacity for brain plasticity and recovery after stroke . however , the international stroke trial 3 ( ist3 ) , recently found there was no direct association between alteplase administration and haemorrhage rate in elderly patients selected for alteplase treatment using ncct imaging.27 this was the largest stroke thrombolytic trial ever , and randomised 0 - 6 hours patients to 0.9 mg / kg alteplase or placebo ( without using advanced imaging selection ) . although the primary outcome was negative , ist3 confirmed that patients treated within 3 hours of symptom onset clearly benefited from alteplase , and the elderly had as much benefit as did younger patients . tenecteplase ( tnk - tpa ) is a third generation point mutation tissue plasminogen activator created by recombinant dna technology from a mammalian cell line . like alterphase tenecteplase has modifications at three sites of the protein structure on the complementary dna template that differentiate tnk from alterphase , such as substitution of threonine 103 with asparagine , and a substitution of asparagine 117 with glutamine , both within the kringle 1 domain , and a tetra - alanine substitution at amino acids 296 - 299 in the protease domain ( figure 4 ) . these structural and functional changes to the cdna mean that tenecteplase has a longer half life and greater binding affinity for fibrin and better resistance to inactivation by endogenous inhibitor ( pai-1 ) compared to alteplase . the amino acids that were replaced at the three positions are called t , n , and k according to the one letter code for amino acids , giving the drug the name tnk . tenecteplase is expressed with carbohydrate side chains linked to the glycosylation sites of the polypeptide . the carbohydrate side chains enlarge the molecule , reducing its elimination and prolonging its plasma half life . tenecteplase can be administered by iv bolus , without the need for follow - up infusion , with a half life of 17 minutes . tenecteplase has been approved for acute myocardial infarction , and was demonstrated to be superior to alteplase.28,29 in ischemic stroke , the tenecteplase dose used for myocardial infarction ( 0.5 mg / kg ) initially did not show benefit when using standard clinical criteria to select patients for treatment , with an increase in symptomatic haemorrhage.30 however when patients were selected for treatment based on acute computed tomography perfusion imaging to identify the volume of the infarct core and penumbra within 3 - 6 hours of symptom onset , treatment with tenecteplase at low dose ( 0.1 mg / kg ) appeared to be superior to alteplase in a non - randomised study.31 a subsequent randomised phase iib study using multimodal ct to select patients ( vessel occlusion , small core , large penumbra ) found that moderate ( 0.25 mg / kg ) and low ( 0.1 mg / kg ) doses did lead to significantly better patient outcomes than standard dose alteplase ( 0.9 mg / kg ) . tenecteplase was associated with significantly increased reperfusion , early neurological improvement and improved 3 month functional outcome32 with a strong dose - dependent relationship , with a 0.25 mg / kg dose achieving significant reperfusion and neurological improvement compared to 0.1 mg / kg . furthermore , despite the enhanced efficacy of tenecteplase for the larger proximal clots , there was not an increase in ich ( in fact a trend towards lower rates ) . it was noted in 1932 that vampire bat saliva interfered with the haemostatic mechanisms of the host.33 the dna sequencing of four plasminogen activators in vampire bat saliva were completed in 1991 , with activator alpha 1 ( rdspa1 , desmoteplase ) being the most active34 and shows a 72% homology to human t - pa.35 desmoteplase is structurally similar to alteplase , but is missing the plasmin sensitive cleavage site and lysine binding kringle 2 domain . this results in desmoteplase being more selective for fibrin ( 2increase in catalytic activity ) , and having no known effect on the blood brain barrier . desmoteplase has a half life of 4 hours ( compared to alteplase 5 minutes , reteplase 13 minutes , tenecteplase 17 minutes ) . a small phase three trial of desmoteplase in acute ischemic stroke ( dias 2 ) has been completed and but demonstrated no benefit in patients selected for treatment biased on acute advanced neuroimaging findings.17,36 the trial randomised patients to desmoteplase or placebo 3 - 9 hours after stroke onset using imaging selection with ct perfusion or mri , assessed visually for mismatch between core and penumbra volume of > 20% by the local investigator . this patient selection paradigm followed the promising phase 2 studies , dias and dedas , which demonstrated increased reperfusion and strong trends to improved outcome with desmoteplase compared to placebo . apart from a very small sample size for a phase iii study , it is thought the predominant reason for lack of benefit seen with desmoteplase was the lack of standardisation of the advanced imaging assessment . additionally , dias 2 did not have a minimum or maximum volume of infarct or penumbra volume as inclusion criteria , as such , many patients had small perfusion lesions with a favourable natural history that would have made detecting a difference with desmoteplase treatment challenging even with a larger sample size . subsequent analyses of pooled data from dias and dias-2 identified that patients with a proximal cerebral vessel occlusion or high grade stenosis had much greater mismatch tissue volumes and a positive response to desmoteplase compared to placebo.37 the rate of early ( effective ) recanalization using alteplase is 25% in patients with a proximal middle cerebral artery occlusion and 10% in patients with an internal carotid occlusion.38 additionally , the rate of re - occlusion , a situation where a thrombus moves down a vascular tree but does not dissolve and re - occludes a distal blood vessel , is as high as 30% with alteplase.39 therefore , methods to enhance recanalization are required . intra arterial ( ia ) thrombolysis and more recently clot retrieval have become increasingly popular acute stroke treatments , albeit without strong evidence . the proact ii trial was the first ia thrombolysis trial to show benefit ( compared to placebo ) . subsequent phase ii trials such as interventional management of stroke ( ims ) i and ii suggested benefit of a ' bridging ' iv / ia approach when compared to iv rtpa alone . with evolution of technology , there has been an increased interest in mechanically removing clots.40 however , three recent ia clot retrieval tials using first generation endovascular devices have recently been published including , ims iii , synthesis and mr rescue , all failed to show benefit compared to iv rtpa . this mostly likely relates to delays in revascularisation compared to iv treatment , and also to poor patient selection . for example , mr rescue not only had a very late time to endovascular treatment ( median > 6 hours ) , but the pre - treatment baseline infarct core volume was exceptionally large ( median 60 ml).41,42 these trials with the first generation clot retrieval devices ( merci and penumbra ) had quite poor recanalization rates , which may be another reason why they failed to show improved clinical outcomes compared to iv tpa alone . however , there is unbridled enthusiasm for the newer ' thrombectomy ' devices such as solitaire43 and trevo,44 with phase ii studies showing much better recanalization than with the first generation devices . however , it is unlikely any device will prove superiority to iv tpa unless clot retrieval can occur at a similar time point to iv treatment . however there is still a trend towards increased haemorrhage in patients undergoing ia treatments regardless of time to treatment . sono enhanced thrombolysis has been shown in vitro and in vivo to accelerate the enzymatic activity of thrombolysis.45 one proposed method of action , is that ultrasound pops the microbubbles in a targeted blood vessel to generate micro - streaming of blood to the occlusion as a delivery mechanism for tpa . additionally , ultrasound causes enlargement of the fibrin mesh to allow better binding and penetration of tpa into a thrombus.46 phase 2 clinical trials have shown that continuous transcranial ultrasound at 2 mhz significantly increased the rate of early recanalisation in patients treated with alteplase.47 however these studies did not use the gold standard of angiography to assess recanalisation , rather they relied on transcranial ultrasound changes , which are less well validated . there has also been interest in administering intravenous micro - bubbles as a contrast agent in order to increase the available volume of micro - bubbles for ultrasound to affect , however this approach may increase risk of ich.48 glycoprotein iib / iiia inhibitors may prevent platelet activation induced by thrombolysis and promote a more complete and rapid action of thrombus breakup.49 glycoprotein inhibitors are used to prevent platelet activation to stop new blood clots forming to avoid re - occlusion . platelet activation by adenosine diphosphate ( adp , stored in blood platelets and is released on platelet activation which can be blocked by clopidogrel ) leads to a conformational change in platelet gpiib / iiia receptors that induces binding to fibrinogen . however recent myocardial studies have shown that gpiib / iiia inhibitors lead to more effective reperfusion , but also increased the rate of intracranial haemorrhage.50 for ischemic stroke there is currently little clinical data , with only small case series indicating a low rate of symptomatic intracranial haemorrhage after duel alteplase and gpiib / iiia administration.51,52 nanotherapeutics aim to increase tpa delivery to the vessel occlusion . the current concept of thrombolysis is to inject a thrombus dissolving drug into the blood stream and hope that it reaches the occlusion site in a high enough concentration to totally dissolve the thrombus . the chosen doses of clot - lysing drugs that can be administered are a trade off between the potential risk of bleeding and the required dosage to have an effect . however , the more distal an occlusion is , the less likely there is to be effective drug delivery regardless of dose . one recent approach to guide tpa delivery is through the use of shear - activated nanotherapeutics ( sa - nts).53 however nanoparticles are only in phase one clinical trials . treatment of ischemic stroke with thrombolysis is a trade off between the risk of haemorrhage and the possible benefit . clinical trials have shown that treatment with tpa in selected patients results in highly clinically significant improvement . however the criteria on how we best identify these highly treatmentresponsive patients are still undergoing investigation . there is much interest in the use of neuroimaging to enhance our ability to identify these responders but this approach is yet to be proven in a phase iii clinical trial.54 while better thrombolytics and drug delivery mechanisms such as sono - thrombolysis may increase the breakdown of a thrombus , there is still the major risk of haemorrhage brought about by damaged to the blood brain barrier by the stroke . it is unknown if a resulting haemorrhage is caused by interactions of thrombolytic drugs and the blood brain barrier , or extensive brain damage brought on by an established infarct . therefore it is unknown if better drugs or mechanisms will reduce the risk of haemorrhage after stroke , or if appropriately selecting patients with a large penumbra and without a large core using imaging reduces the rate of post lysis haemorrhage . the recent failure of the endovascular trials to show benefit compared to iv alteplase demonstrate that we should focus on early treatment with lytics and concentrate on either enhancing their delivery , and/or on better agents such as tenecteplase .

the cornerstone of acute ischemic stroke treatment relies on rapid clearance of an offending thrombus in the cerebrovascular system . there are various drugs and different methods of assessment to select patients more likely to respond to treatment . current clinical guidelines recommend the administration of intravenous alteplase ( following a brain noncontract ct to exclude hemorrhage ) within 4.5 hours of stroke onset . because of the short therapeutic time window , the risk of hemorrhage , and relatively limited efficacy of alteplase for large clot burden , research is ongoing to find more effective and safer reperfusion therapy , as well as focussing on refinement of patient selection for acute reperfusion treatment . studies using advanced imaging ( incorporating perfusion ct or diffusion / perfusion mri ) may allow us to use thrombolytics , or possibly endovascular therapy , in an extended time window . recent clinical trials have suggested that tenecteplase , used in conjunction with advanced imaging selection , resulted in more effective reperfusion than alteplase , which translated into increased clinical benefit . studies using desmoteplase have suggested its potential benefit in a sub - group of patients with large artery occlusion and salveageable tissue , in an extended time window . other ways to improve acute reperfusion approaches are being actively explored , including endovascular therapy , and the enhancement of thrombolysis by ultrasound insonation of the clot ( sono - thrombolysis ) .

Introduction Thrombus formation and fibrinolysis Thrombolytic drugs Enhancing thrombolysis delivery

stroke is the leading cause of adult disability in the developed world.1 , 2 in ischemic stroke , a blood clot ( thrombus ) leads to an area of brain experiencing a significant drop in cerebral blood flow , which can ultimately result in death of this hypoperfused tissue . collateral blood flow gives rise to the ischemic penumbra , which is defined as an area of hypoperfused tissue that is at risk of death ( infarction ) if hypoperfusion persists , and is separate from tissue that has already died ( or programmed to die ) soon after stroke onset ( core).3 the concept of the ischemic penumbra led to the notion that opening an occluded blood vessel in order to re - establish blood flow to the ischemic area ( reperfusion ) would rescue the ischemic penumbra from death3,4 ( figure 1 ) . reperfusion can be achieved through the use of systemic thrombolytic drugs , although thrombolytic agents may theoretically be more effective if delivered directly into the clot ( intra - arterial delivery ) or if assisted with ultrasound insonation of the clot ( sono - thrombolysis ) . a thrombus can originate from several different causes , which may alter its susceptibility to thrombolytic drugs . the main causes of thrombus formation in ischemic stroke are atherosclerosis ( in - situ thrombus or thromboembolic ) or forming in the heart ( cardioembolic ) . it was originally hypothesized that the source of thrombi had a large determining influence on clot composition and therefore potentially upon susceptibility to thrombolytic treatment as thromboembolic clots tend to be platelet and fibrin rich and were formed in high flow areas ( often referred to white clots ) , whilst cardioembolic can contain tissue debris such as fat , air or bacteria interwoven inside the platelet and fibrin mesh which were formed in low flow areas ( often referred to as red clots).5,6 however recent histological studies analyzing the thrombi extracted from patients using intra - arterial clot retrieval showed thrombi composition was similar between cardiac and arterial origins.7 atherosclerotic plaque rupture or cardio - embolic thrombosis ( typically due to atrial fibrillation ) leads to activation of the coagulation cascade as well as platelet activation . in the cascade , zymogens ( free floating inactive coagulation factor precursors ) are converted to an activated coagulation factor by interaction with the atherosclerotic plaque / thrombus.8 each active zymogene is able to activate nearby zymogens , leading to a large localized reaction of coagulation factors . activated platelets then catalyze an interaction between activated coagulation factors ( zymogens ) to aid in the generation of thrombin by conversion of the soluble protein fibrinogen to insoluble fibrin , forming a blood clot . there are two blood coagulation cascades ( intrinsic and extrinsic pathways ) , that have separate initial pathways but converge on a common pathway9 ( figure 2).10 thrombolytic drugs dissolve ( lyse ) thrombi in the vascular bed by activating plasminogen to form plasmin . the main types of thrombolytic drugs used in ischemic stroke to activate plasminogen are urokinase / streptokinase and tissue plasminogen activators ( eg , alteplase ) . thrombolytic drug development has undergone at least three generations with the aim of enhancing fibrin specificity or reducing inhibition of thrombolysis by plasminogen activator inhibitor type 1 ( table 1 ) . tissue plasminogen activator ( tpa ) is a serine protease found on the endothelial cells lining blood vessels and is involved in the breakdown of blood clots ( thrombus ) . lysine binding of tpa results in a activation of plasminogen only around a thrombus , which minimises activation of circulating plasminogen . plasmin then breaks the thrombus down into fibrin degradation products due to the plasmin 's protease action in dissolving the thrombus.11 however , on fibrin bound plasmin , the inhibitory effect of alpha 2-antiplasmin and type 1 plasminogen activeator inhibitor restricts lysine binding.12 streptokinase is a first generation thrombolytic agent . because of its non lysine specificity , streptokinase , produces more fibrin degradation products ( fibrinogensis ) as a result of widespread lytic action in the body . fibrinogensis occurs when there are high levels of fibrin in the blood and can cause thrombosis , haemorrhage or tissue oedema . although much less expensive , this makes streptokinase a less attractive agent for acute treatments in ischemic stroke than tpa products . despite an increased rate of symptomatic intracranial haemorrhage the study found a significant benefit in patients treated with r - prouk at 90 days.15 to date , the proact ii study is the only positive phase iii intra - arterial trial . the second generation thrombolytic drug alteplase , is a recombinant form of human rtpa and has undergone the most study in the clinical stroke setting . recombinant techniques mean that molecular cloning is used to bring genetic material together from multiple sources that would not otherwise be found together , to create new dna sequences , which can be used to manufacture drugs . in terms of pharmacodynamics , rtpa has a short half life of around 5 min , and therefore requires an infusion after a bolus injection in the acute ischemic stroke setting . the majority of ischemic stroke clinical research has revolved around alteplase , as it is the only food and drug administration ( fda ) approved drug for ischemic stroke . fda approval for alteplase was licensed for the management of acute myocardial infarction in 1987 , acute massive pulmonary embolism in 1990 and acute ischemic stroke in 1996.16 the total dose of alteplase for acute ischemic stroke is divided into a 10% bolus and 90% as an infusion over 60 min . this trial showed that early treatment ( within 3 hours ) of an ischemic stroke with alteplase substantially improved clinical outcomes . an alteplase dose of 0.9 mg / kg was used in the ninds trial.18 and found no significant increase in the risk of haemorrhage.19 the ideal dose of alteplase is still under debate with the original alteplase dose - ranging studies being quite small . to this point , the standard dose of alteplase in japan in 0.6 mg / kg based on a single japanese study.20 on the other hand , there is justifiable concern that a reduced dose of alteplase will not successfully lyse larger clots , typically in more proximal locations . it has been reliably demonstrated that long clots on non contrast computed tomography ( ncct ) show a reduced rate of reperfusion and subsequent clinical improvement.21 indeed , a recent study of 130 patients using non - contrast ct to measure occlusion length identified that the tpa dose was independently predicative of thrombus resolution , with complete resolution in only 8% of patients . therefore tpa dose was associated with thrombus resolution and improved clinical outcome.22 following the ninds study , which showed that early treatment ( within 3 hours ) was the key to improving ischemic stroke outcome , there have been several studies investigating extending this treatment time window . the european cooperative acute stroke study ( ecass ) i and ii looked at administering alteplase between 0 and 6 hours after ischemic stroke onset and did not find an overall benefit , with an increased rate of haemorrhage in ecass i. however ecass i and ii did not use advanced imaging and may have had a high rate of patients with very little penumbra to salvage , especially in the 3 - 6 hours time window . ecass iii subsequently demonstrated evidence of good outcome following treatment with 0.9 mg / kg alteplase between 3 and 4.5 hours in patients < 80 years after symptom onset using only ncct to exclude patients with haemorrhage or oedema . as a result of ecass iii , many countries have amended the license for stroke treatment with rtpa to 4.5 hours following ncct assessment . there has been much interest in using advanced imaging to visualise the volume of the penumbra and infarct core , particularly with a view to extending the treatment window for thrombolysis . the echoplanar imaging thrombolytic evaluation trial ( epithet ) randomised ischemic stroke patients 3 - 6 hours after symptom onset to alteplase or placebo and used acute magnetic resonance imaging ( mri ) to define the core and penumbra ( ' diffusion - perfusion mismatch ' ) . the primary hypothesis of this phase ii trial was that patients with perfusion - diffusion mismatch would have less infarct growth with alteplase than with placebo . other analyses from the epithet dataset have shown that the diffusion infarct core is the strongest predictor of response to thrombolysis , with a chance of good outcome dropping if the core > 25 ml and there being virtually no benefit > 70 ml.23,24 pooled analysis from epithet and another trial of alteplase 3 - 6 hours after ischemic stroke , the diffusion and perfusion imaging evaluation for understanding stroke evolution study ( defuse ) , showed that patients in the 3 - 6 hours time window with a favourable imaging profile , i.e. a large penumbra , and small infarct core.25 as stroke occurs predominantly in the aged population , with > 45% of patients receiving alteplase being over 70 years old,26 there is a concern about decreasing benefit from thrombolysis with age . the main concerns are over the possibility of aged related renal and hepatic impairments affecting the metabolising and clearance of alteplase as well as the possibility of an increased risk of haemorrhagic transformation of the infarct following alteplase administration . however , the international stroke trial 3 ( ist3 ) , recently found there was no direct association between alteplase administration and haemorrhage rate in elderly patients selected for alteplase treatment using ncct imaging.27 this was the largest stroke thrombolytic trial ever , and randomised 0 - 6 hours patients to 0.9 mg / kg alteplase or placebo ( without using advanced imaging selection ) . although the primary outcome was negative , ist3 confirmed that patients treated within 3 hours of symptom onset clearly benefited from alteplase , and the elderly had as much benefit as did younger patients . like alterphase tenecteplase has modifications at three sites of the protein structure on the complementary dna template that differentiate tnk from alterphase , such as substitution of threonine 103 with asparagine , and a substitution of asparagine 117 with glutamine , both within the kringle 1 domain , and a tetra - alanine substitution at amino acids 296 - 299 in the protease domain ( figure 4 ) . tenecteplase is expressed with carbohydrate side chains linked to the glycosylation sites of the polypeptide . tenecteplase has been approved for acute myocardial infarction , and was demonstrated to be superior to alteplase.28,29 in ischemic stroke , the tenecteplase dose used for myocardial infarction ( 0.5 mg / kg ) initially did not show benefit when using standard clinical criteria to select patients for treatment , with an increase in symptomatic haemorrhage.30 however when patients were selected for treatment based on acute computed tomography perfusion imaging to identify the volume of the infarct core and penumbra within 3 - 6 hours of symptom onset , treatment with tenecteplase at low dose ( 0.1 mg / kg ) appeared to be superior to alteplase in a non - randomised study.31 a subsequent randomised phase iib study using multimodal ct to select patients ( vessel occlusion , small core , large penumbra ) found that moderate ( 0.25 mg / kg ) and low ( 0.1 mg / kg ) doses did lead to significantly better patient outcomes than standard dose alteplase ( 0.9 mg / kg ) . furthermore , despite the enhanced efficacy of tenecteplase for the larger proximal clots , there was not an increase in ich ( in fact a trend towards lower rates ) . it was noted in 1932 that vampire bat saliva interfered with the haemostatic mechanisms of the host.33 the dna sequencing of four plasminogen activators in vampire bat saliva were completed in 1991 , with activator alpha 1 ( rdspa1 , desmoteplase ) being the most active34 and shows a 72% homology to human t - pa.35 desmoteplase is structurally similar to alteplase , but is missing the plasmin sensitive cleavage site and lysine binding kringle 2 domain . this results in desmoteplase being more selective for fibrin ( 2increase in catalytic activity ) , and having no known effect on the blood brain barrier . a small phase three trial of desmoteplase in acute ischemic stroke ( dias 2 ) has been completed and but demonstrated no benefit in patients selected for treatment biased on acute advanced neuroimaging findings.17,36 the trial randomised patients to desmoteplase or placebo 3 - 9 hours after stroke onset using imaging selection with ct perfusion or mri , assessed visually for mismatch between core and penumbra volume of > 20% by the local investigator . this patient selection paradigm followed the promising phase 2 studies , dias and dedas , which demonstrated increased reperfusion and strong trends to improved outcome with desmoteplase compared to placebo . apart from a very small sample size for a phase iii study , it is thought the predominant reason for lack of benefit seen with desmoteplase was the lack of standardisation of the advanced imaging assessment . subsequent analyses of pooled data from dias and dias-2 identified that patients with a proximal cerebral vessel occlusion or high grade stenosis had much greater mismatch tissue volumes and a positive response to desmoteplase compared to placebo.37 the rate of early ( effective ) recanalization using alteplase is 25% in patients with a proximal middle cerebral artery occlusion and 10% in patients with an internal carotid occlusion.38 additionally , the rate of re - occlusion , a situation where a thrombus moves down a vascular tree but does not dissolve and re - occludes a distal blood vessel , is as high as 30% with alteplase.39 therefore , methods to enhance recanalization are required . subsequent phase ii trials such as interventional management of stroke ( ims ) i and ii suggested benefit of a ' bridging ' iv / ia approach when compared to iv rtpa alone . this mostly likely relates to delays in revascularisation compared to iv treatment , and also to poor patient selection . for example , mr rescue not only had a very late time to endovascular treatment ( median > 6 hours ) , but the pre - treatment baseline infarct core volume was exceptionally large ( median 60 ml).41,42 these trials with the first generation clot retrieval devices ( merci and penumbra ) had quite poor recanalization rates , which may be another reason why they failed to show improved clinical outcomes compared to iv tpa alone . sono enhanced thrombolysis has been shown in vitro and in vivo to accelerate the enzymatic activity of thrombolysis.45 one proposed method of action , is that ultrasound pops the microbubbles in a targeted blood vessel to generate micro - streaming of blood to the occlusion as a delivery mechanism for tpa . additionally , ultrasound causes enlargement of the fibrin mesh to allow better binding and penetration of tpa into a thrombus.46 phase 2 clinical trials have shown that continuous transcranial ultrasound at 2 mhz significantly increased the rate of early recanalisation in patients treated with alteplase.47 however these studies did not use the gold standard of angiography to assess recanalisation , rather they relied on transcranial ultrasound changes , which are less well validated . there has also been interest in administering intravenous micro - bubbles as a contrast agent in order to increase the available volume of micro - bubbles for ultrasound to affect , however this approach may increase risk of ich.48 glycoprotein iib / iiia inhibitors may prevent platelet activation induced by thrombolysis and promote a more complete and rapid action of thrombus breakup.49 glycoprotein inhibitors are used to prevent platelet activation to stop new blood clots forming to avoid re - occlusion . however recent myocardial studies have shown that gpiib / iiia inhibitors lead to more effective reperfusion , but also increased the rate of intracranial haemorrhage.50 for ischemic stroke there is currently little clinical data , with only small case series indicating a low rate of symptomatic intracranial haemorrhage after duel alteplase and gpiib / iiia administration.51,52 nanotherapeutics aim to increase tpa delivery to the vessel occlusion . the current concept of thrombolysis is to inject a thrombus dissolving drug into the blood stream and hope that it reaches the occlusion site in a high enough concentration to totally dissolve the thrombus . the chosen doses of clot - lysing drugs that can be administered are a trade off between the potential risk of bleeding and the required dosage to have an effect . one recent approach to guide tpa delivery is through the use of shear - activated nanotherapeutics ( sa - nts).53 however nanoparticles are only in phase one clinical trials . treatment of ischemic stroke with thrombolysis is a trade off between the risk of haemorrhage and the possible benefit . clinical trials have shown that treatment with tpa in selected patients results in highly clinically significant improvement . there is much interest in the use of neuroimaging to enhance our ability to identify these responders but this approach is yet to be proven in a phase iii clinical trial.54 while better thrombolytics and drug delivery mechanisms such as sono - thrombolysis may increase the breakdown of a thrombus , there is still the major risk of haemorrhage brought about by damaged to the blood brain barrier by the stroke . it is unknown if a resulting haemorrhage is caused by interactions of thrombolytic drugs and the blood brain barrier , or extensive brain damage brought on by an established infarct . therefore it is unknown if better drugs or mechanisms will reduce the risk of haemorrhage after stroke , or if appropriately selecting patients with a large penumbra and without a large core using imaging reduces the rate of post lysis haemorrhage .

it is required to tolerate intubation and other icu related procedures , to lie down in the same position for a long time , to prevent ventilator dysynchrony , for optimization of oxygenation and for patients safety . attaining an optimal level of sedation is a challenging act for the icu clinician . both inadequate sedation and oversedation compromise patient 's recovery and may prolong icu stay along with associated complications and increased cost . many of the currently used agents have specific drawbacks that limit their practical utility along the full spectrum of patients and clinical situations that intensivists face every day . the discovery that clonidine has an opioid sparing property and attenuated withdrawal symptoms , sparked further interest in the use of alpha - 2 ( 2 ) agonists as intravenous ( iv ) sedatives . a resurgence in the research of 2 agonists for sedation developed after the approval of dexmedetomidine for icu sedation . unlike most other sedative drugs , 2 agonists produce both sedation and analgesia with minimal respiratory depression . we therefore planned this study to compare sedative , analgesic and cardiovascular effects and safety profile of two 2 agonists , clonidine , and dexmedetomidine for patients requiring short - term sedation in icu . after approval from the institutional review board and informed written consent , 70 adult patients of either sex were enrolled for this study . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the patients were predominantly postsurgical who were operated for major abdominal , gynecological or urological procedures under general anesthesia on an elective basis . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . epidural or spinal technique was not used in any patient . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . dexmedetomidine was supplied in 2 ml ampoules that contained 100 g / ml diluted with normal saline to a concentration of 4 g / ml . physical examination , baseline vitals , electrocardiogram and central venous pressure ( cvp ) was noted on admission to the icu . hematological ( complete blood count , coagulation profile ) and biochemical profile ( electrolytes , glucose , urea , creatinine , and liver function test ) were obtained prior to the administration of sedatives and 24 h after the study period . patients were ventilated with oxygen enriched air to obtain acceptable arterial blood gas ( abg ) levels . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . if systolic bp reduced below 80 mmhg or increased above 180 mmhg , diastolic bp reduced below 50 mmhg or increased above 100 mmhg or heart rate was below 50 or above 120 bpm , they were labeled as adverse cardiovascular events . any change > 30% from the baseline in bp and heart rate were also considered as adverse cardiovascular event . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . rss was also assessed prior to and 10 min after any titration in the study drug infusion rate or the use of additional sedation . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . assessment of pain was by direct communication of the patient and fentanyl was given prior to anticipate noxious stimulus . inadequate analgesia was treated with iv bolus of 20 g of fentanyl or infusion if pain persisted . a sample size of minimum 32 patients / group was expected to have an 80% power to detect a 30% reduction in additional sedation requirements ( primary endpoint ) with a significance level of 5% . all data were recorded and noted on observation charts and were analyzed at the end of the study . data were expressed as mean standard deviation ( sd ) or as median and interquartile range ( iqr ) and comparisons made using the unpaired t - test . medians were quoted for skewed data and were compared using the mann - whitney u - test . analysis was carried out using the spss 18.0 software ( ibm ( pasw statistics 18 ) ) . after approval from the institutional review board and informed written consent , 70 adult patients of either sex were enrolled for this study . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the patients were predominantly postsurgical who were operated for major abdominal , gynecological or urological procedures under general anesthesia on an elective basis . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . epidural or spinal technique was not used in any patient . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . dexmedetomidine was supplied in 2 ml ampoules that contained 100 g / ml diluted with normal saline to a concentration of 4 g / ml . physical examination , baseline vitals , electrocardiogram and central venous pressure ( cvp ) was noted on admission to the icu . hematological ( complete blood count , coagulation profile ) and biochemical profile ( electrolytes , glucose , urea , creatinine , and liver function test ) were obtained prior to the administration of sedatives and 24 h after the study period . patients were ventilated with oxygen enriched air to obtain acceptable arterial blood gas ( abg ) levels . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . if systolic bp reduced below 80 mmhg or increased above 180 mmhg , diastolic bp reduced below 50 mmhg or increased above 100 mmhg or heart rate was below 50 or above 120 bpm , they were labeled as adverse cardiovascular events . any change > 30% from the baseline in bp and heart rate were also considered as adverse cardiovascular event . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . rss was also assessed prior to and 10 min after any titration in the study drug infusion rate or the use of additional sedation . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . assessment of pain was by direct communication of the patient and fentanyl was given prior to anticipate noxious stimulus . inadequate analgesia was treated with iv bolus of 20 g of fentanyl or infusion if pain persisted . a sample size of minimum 32 patients / group was expected to have an 80% power to detect a 30% reduction in additional sedation requirements ( primary endpoint ) with a significance level of 5% . all data were recorded and noted on observation charts and were analyzed at the end of the study . data were expressed as mean standard deviation ( sd ) or as median and interquartile range ( iqr ) and comparisons made using the unpaired t - test . medians were quoted for skewed data and were compared using the mann - whitney u - test . analysis was carried out using the spss 18.0 software ( ibm ( pasw statistics 18 ) ) . after approval from the institutional review board and informed written consent , 70 adult patients of either sex were enrolled for this study . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the patients were predominantly postsurgical who were operated for major abdominal , gynecological or urological procedures under general anesthesia on an elective basis . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . epidural or spinal technique was not used in any patient . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . dexmedetomidine was supplied in 2 ml ampoules that contained 100 g / ml diluted with normal saline to a concentration of 4 g / ml . physical examination , baseline vitals , electrocardiogram and central venous pressure ( cvp ) was noted on admission to the icu . hematological ( complete blood count , coagulation profile ) and biochemical profile ( electrolytes , glucose , urea , creatinine , and liver function test ) were obtained prior to the administration of sedatives and 24 h after the study period . patients were ventilated with oxygen enriched air to obtain acceptable arterial blood gas ( abg ) levels . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . if systolic bp reduced below 80 mmhg or increased above 180 mmhg , diastolic bp reduced below 50 mmhg or increased above 100 mmhg or heart rate was below 50 or above 120 bpm , they were labeled as adverse cardiovascular events . any change > 30% from the baseline in bp and heart rate were also considered as adverse cardiovascular event . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . rss was also assessed prior to and 10 min after any titration in the study drug infusion rate or the use of additional sedation . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . assessment of pain was by direct communication of the patient and fentanyl was given prior to anticipate noxious stimulus . inadequate analgesia was treated with iv bolus of 20 g of fentanyl or infusion if pain persisted . a sample size of minimum 32 patients / group was expected to have an 80% power to detect a 30% reduction in additional sedation requirements ( primary endpoint ) with a significance level of 5% . all data were recorded and noted on observation charts and were analyzed at the end of the study . data were expressed as mean standard deviation ( sd ) or as median and interquartile range ( iqr ) and comparisons made using the unpaired t - test . medians were quoted for skewed data and were compared using the mann - whitney u - test . analysis was carried out using the spss 18.0 software ( ibm ( pasw statistics 18 ) ) . over a period of 18 months , 70 patients were enrolled in the study to receive sedation with either dexmedetomidine ( n = 35 ) or clonidine ( n = 35 ) . these included 59 postsurgical , 7 medical and 4 polytrauma patients evenly distributed in each group [ table 1 ] . demographic data and intraoperative details such as operative time , fentanyl requirements , apache ii scores , and duration of sedative infusions in the icu were comparable [ table 1 ] . demographic and intraoperative details : median ( iqr ) or number additional sedation with diazepam ( primary endpoint ) was needed by eight patients in dexmedetomidine treated and by 14 patients in clonidine treated patients ( p = 0.034 ) . of these patients , three patients of group d and 11 of group c could not attain target sedation due to development of significant hypotension on increasing infusion rate . median dose of diazepam required in group c was significantly higher compared to group d ( 15 mg , iqr : 5 - 22 mg in group c and 8.5 mg , iqr : 2 - 10 mg in group d , p = 0.043 ) . need for additional sedation was about 43% less in group d. the mean sd maintenance infusion dose was 0.47 0.27 g / kg / h for dexmedetomidine and 1.67 8.6 g / kg / h for clonidine . median infusion dose was 0.4 g / kg / h ( group d ) and 1.4 g / kg / h ( group c ) . a total of 373 observations of rss were obtained for group c , of which 235 ( 62% ) observations were in the target sedation range ( rss : 3 - 4 ) . in group d , a total of 403 observations were obtained , of which 347 ( 86% ) were in the target sedation range . the proportion of time spent in the target sedation range was greater in group d ( p = 0.04 ) . a score 1 - 2 was observed on 86 ( 23% ) occasions in group c and 36 ( 9% ) occasions in group d ( p = 0.047 ) . rss : 5 - 6 was achieved in 52 ( 14% ) observations in group c and 20 ( 5% ) observations in group d ( p = 0.048 ) . the baseline hemodynamic parameters were comparable in both groups . a significant reduction in systolic and diastolic bp from the baseline ( p < 0.05 ) occurred after bolus infusion in group d but in none of the patients fall was > 30% from baseline . thereafter , mean values remained well within range throughout study period [ figures 1 and 2 ] . mean heart rate also decreased from baseline 2 h after commencement of sedative infusion in group d , but at none of the observation times fall was significant ( p = 0.079 ) [ figure 3 ] . in group c significant fall from baseline values in bp were noted 2 and 4 h after sedative infusion was started ; but thereafter , it showed minimal change figures [ 1 and 2 ] . patients receiving clonidine ( group c ) had significantly lower heart rates from baseline ( p < 0.05 ) [ figure 3 ] . on comparison , the hemodynamic parameters were comparable between the two groups during the study period ( p > 0.05 ) . systolic blood pressure ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation diastolic blood pressure ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation heart rate ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation bradycardia occurred in 3 of the 35 patients in group c and 4 of the 35 patients in group d ( p = 0.64 ) . hypotension occurred in 11 of the 35 patients in group c ( 31% ) and 3 of the 35 patients in group d ( 9% ) ( p = 0.01 ) . about 50% of the hypotensive episodes occurred within 2 - 4 h in group c and at 2 h in group d. no patient experienced hypotension after 14 h in group c and after 6 h in group d. sustained increase in systolic and diastolic pressure and heart rate occurred after cessation of infusion in group d , but there were no clinically significant rebound phenomena in any patient . in group c , rebound hypertension was seen in four patients after cessation of clonidine infusion . in two patients , it increased above 180 mmhg about 2 h after discontinuation of clonidine . the median 24 h fentanyl requirement was 162 g ( range : 105 - 175 ) for group c and 171 g ( range : 110 - 185 ) for group d ( p = 0.73 ) . a median of 3 ( range : 2 - 5 ) boli of fentanyl was required in both groups over the study period . mean time for extubation was similar in both groups , being 19 h ( range : 14 - 30 h ) in group d patients and 18 h ( range : 16 - 32 h ) in group c. there were no adverse respiratory events after extubation in any patient in either group . biochemical and hematological parameters were not different between two groups at arrival in icu and 24 h after admission . the chief results of this study showed that target sedation was achieved in more number of patients receiving dexmedetomidine with lesser need for additional sedation . this study and many previous studies have documented dexmedetomidine to be a safe and effective agent for icu sedation of postsurgical patients . although mean cumulative sedation scores over the study period were not significantly different in two groups ( 3.37 + 1.37 vs. 3.20 + 0.75 in groups c and d , respectively ) , percentage of patients who attained target sedation was significantly higher in group d compared with group c ( 86 vs. 62% in groups d and c , respectively , p = 0.04 ) . dexmedetomidine is 8 times more specific for 2 receptors than clonidine and the improved specificity for the 2 adrenoreceptors , especially for the 2a subtype may make it to be a much more effective sedative than clonidine . however , our findings are in contrast with those of riker et al . who suggested that dexmedetomidine attained target sedation less frequently . a rss of 1 - 2 or 5 - 6 occurred in more number of observations in group c than in group d. the short distribution half - life of dexmedetomidine ( 6 min ) makes it an ideal drug for iv titration . this could be the reason for the rapid titration to target sedation and the lesser number of observations pertaining to inadequate sedation in group d. although more than 60% patients of both groups attained acceptable sedation , significantly more number of patients in clonidine treated group required additional sedation by diazepam on account of fall of bp on increasing infusion rate to maximum set level . requirement of additional sedation in this group was about 43% more than dexmedetomidine treated group . in a retrospective analysis of patients receiving clonidine for icu sedation , gillison et al . have reported that clonidine reduces requirement of additional sedation and analgesia , but at the cost of higher than routinely prescribed dose . only 8/35 patients ( 23% ) patients in group d needed additional sedation which agrees with findings of martin et al . there is no consensus on appropriate dose regimen of clonidine during icu sedation and is extremely variable when given by continuous infusion . however , the usual dose is in the order of 100 g / h . we used an initial dose of 1 g / kg / h of clonidine for infusion titrated to 2 g / kg / h as the maximum dose . the dose of dexmedetomidine for icu sedation varies greatly ranging between 0.2 and 2.5 g / kg / h . in our study , we used a loading dose of 0.7 g / kg followed by 0.2 - 0.7 g / kg / h . a meta - analysis by tan and ho ( 2010 ) observed that incidence of bradycardia requiring intervention increased in studies that used both a loading dose and maintenance doses of dexmedetomidine in excess of 0.7 g / kg / h . transient hypertensive responses have also been observed with higher doses due to initial stimulation of 2b receptors present in vascular smooth muscles . baseline heart rates which were high in both groups settled to an optimal range over the study period . hypotension was more commonly seen in group c compared with group d. 50% of the hypotensive episodes occurred within 2 - 4 h in group c and after bolus infusion and within 2 h after maintenance infusion in group d , as the steady state plasma concentration of the drugs are achieved at this time duration , causing vasodilatation and hypotension . in general , hemodynamic stability was preserved in most patients receiving dexmedetomidine , a finding in agreement with many previous studies . eleven out of 14 patients in group c requiring additional sedation to achieve target sedation experienced hypotension on increasing the dose from 1 up to 2 g / kg / h . this observation was consistent with previous studies of clonidine where adverse hemodynamic effects occurred at doses required for sedation . previous studies of icu sedation with dexmedetomidine have found no or minimal increase in heart rate and bp following abrupt cessation , the finding similar to this study . in group c , a small study group containing patients mostly postsurgical precluded an extensive study on a heterogeneous icu population . a short study period was considered as dexmedetomidine has been approved by food and drug administration as a sedative in the icu for patients undergoing mechanical ventilation of < 24 h duration . being an open - label study , there is an inherent potential for observer bias . due to the unavailability of bispectral index ( bis ) at our center we restricted our assessment of the degree of sedation to rss . it is a highly reliable and well - validated sedation scale for use in icu and has also been shown to have a good correlation with bis . both dexmedetomidine and clonidine can be used as sedative agents for short term icu sedation of postsurgical patients . on the basis of our study data further trials with both drugs may define their exact role for sedation of icu patients .

background and objectives : patients on mechanical ventilation in intensive care unit ( icu ) are often uncomfortable because of anxiety , pain , and endotracheal intubation ; therefore , require sedation . alpha-2 agonists are known to produce sedation . we compared clonidine and dexmedetomidine as sole agents for sedation.study design : prospective , randomized , controlled open - label study.materials and methods : a total of 70 patients requiring a minimum of 12 h of mechanical ventilation with concomitant sedation , were randomly allocated into two groups . group c ( n = 35 ) received intravenous ( iv ) clonidine ( 1 g / kg / h titrated up to 2 g / kg / h to attain target sedation ) , and group d ( n = 35 ) received iv dexmedetomidine for sedation ( loading 0.7 g / kg and maintenance 0.2 g / kg / h titrated up to 0.7 g / kg / h to achieve target sedation ) . a ramsay sedation score of 3 - 4 was considered as target sedation . additional sedation with diazepam was given when required to achieve target sedation . the quality of sedation , hemodynamic changes and adverse effects were noted and compared between the two groups.results:target sedation was achieved in 86% observations in group d and 62% in group c ( p = 0.04 ) . additional sedation was needed by more patients in group c compared with group d ( 14 and 8 in groups c and d , respectively , p = 0.034 ) , mainly due to concomitant hypotension on increasing the dose of clonidine . hypotension was the most common side - effect in group c , occurring in 11/35 patients of group c and 3/35 patients of group d ( p = 0.02 ) . rebound hypertension was seen in four patients receiving clonidine , but none in receiving dexmedetomidine.conclusion:both clonidine and dexmedetomidine produced effective sedation ; however , the hemodynamic stability provided by dexmedetomidine gives it an edge over clonidine for short - term sedation of icu patients .

Introduction Materials and Methods None Patients Study drugs Monitoring Protocol for sedation and analgesia Statistical analysis Results Discussion Conclusion

attaining an optimal level of sedation is a challenging act for the icu clinician . the discovery that clonidine has an opioid sparing property and attenuated withdrawal symptoms , sparked further interest in the use of alpha - 2 ( 2 ) agonists as intravenous ( iv ) sedatives . a resurgence in the research of 2 agonists for sedation developed after the approval of dexmedetomidine for icu sedation . we therefore planned this study to compare sedative , analgesic and cardiovascular effects and safety profile of two 2 agonists , clonidine , and dexmedetomidine for patients requiring short - term sedation in icu . after approval from the institutional review board and informed written consent , 70 adult patients of either sex were enrolled for this study . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . dexmedetomidine was supplied in 2 ml ampoules that contained 100 g / ml diluted with normal saline to a concentration of 4 g / ml . hematological ( complete blood count , coagulation profile ) and biochemical profile ( electrolytes , glucose , urea , creatinine , and liver function test ) were obtained prior to the administration of sedatives and 24 h after the study period . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . any change > 30% from the baseline in bp and heart rate were also considered as adverse cardiovascular event . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . rss was also assessed prior to and 10 min after any titration in the study drug infusion rate or the use of additional sedation . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . a sample size of minimum 32 patients / group was expected to have an 80% power to detect a 30% reduction in additional sedation requirements ( primary endpoint ) with a significance level of 5% . after approval from the institutional review board and informed written consent , 70 adult patients of either sex were enrolled for this study . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . any change > 30% from the baseline in bp and heart rate were also considered as adverse cardiovascular event . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . a sample size of minimum 32 patients / group was expected to have an 80% power to detect a 30% reduction in additional sedation requirements ( primary endpoint ) with a significance level of 5% . the main inclusion criteria were age > 18 years , mechanical ventilation with endotracheal intubation and clinical need for light or moderate sedation for < 24 h. we excluded pregnant females , patients with a neurological condition , central nervous system trauma , asthma or chronic obstructive pulmonary disease , hemodynamically unstable patients , known cases of conduction defects , cardiac failure , those with a creatinine clearance < 30 ml / min , and those requiring neuromuscular blockade and prior use of 2 agonists . the anesthetic technique was individualized by the anesthetist in - charge ; however , fentanyl alone was used for intraoperative analgesia and the dose was recorded . on arrival to the icu , patients were randomly allocated into two groups , group c and d , based on computer generated random number tables . clonidine was supplied in 1 ml ampoules , containing 150 g / ml and diluted with normal saline to a concentration of 3 g / ml . heart rate , cvp , noninvasive blood pressure ( bp ) , respiratory rate , and oxygen saturation ( measured by pulse oximetry ) were monitored continuously over 24 h. hemodynamic parameters were recorded at 10 min , 30 min after the commencement of sedative infusions and then 2 hourly for the study period . the degree of sedation was assessed by ramsay sedation score ( rss ) ( 1 : patient anxious , agitated or restless , 2 : cooperative , oriented and tranquil , 3 : responds to commands only , 4 : exhibits brisk response to light glabellar tap or loud auditory stimulus , 5 : sluggish response to light glabellar tap or loud auditory sound , 6 : no response ) obtained on arrival in the icu , at 10 and 30 min after commencement of the infusion and 2 hourly thereafter for the study period . rss of 3 - 4 was considered as target sedation and the infusion rates were titrated within their respective range until target sedation was achieved . rss was also assessed prior to and 10 min after any titration in the study drug infusion rate or the use of additional sedation . group c patients were administered an iv infusion of clonidine 1 g / kg / h and titration was achieved with dosage increments up to 2 g / kg / h . patients in group d received dexmedetomidine as a loading dose of 0.7 g / kg over a period of 10 min followed by maintenance of 0.2 g / kg / h with dosage increments titrated up to 0.7 g / kg / h . additional sedation with iv diazepam bolus of 0.1 mg / kg was given if the patient did not achieve target sedation on titrating the sedative to the maximum selected dose ( 2 g / kg / h for clonidine and 0.7 g / kg / h for dexmedetomidine ) or if the patient experienced side - effects ( hypotension ) with the drugs . assessment of pain was by direct communication of the patient and fentanyl was given prior to anticipate noxious stimulus . over a period of 18 months , 70 patients were enrolled in the study to receive sedation with either dexmedetomidine ( n = 35 ) or clonidine ( n = 35 ) . demographic and intraoperative details : median ( iqr ) or number additional sedation with diazepam ( primary endpoint ) was needed by eight patients in dexmedetomidine treated and by 14 patients in clonidine treated patients ( p = 0.034 ) . of these patients , three patients of group d and 11 of group c could not attain target sedation due to development of significant hypotension on increasing infusion rate . median dose of diazepam required in group c was significantly higher compared to group d ( 15 mg , iqr : 5 - 22 mg in group c and 8.5 mg , iqr : 2 - 10 mg in group d , p = 0.043 ) . need for additional sedation was about 43% less in group d. the mean sd maintenance infusion dose was 0.47 0.27 g / kg / h for dexmedetomidine and 1.67 8.6 g / kg / h for clonidine . median infusion dose was 0.4 g / kg / h ( group d ) and 1.4 g / kg / h ( group c ) . a total of 373 observations of rss were obtained for group c , of which 235 ( 62% ) observations were in the target sedation range ( rss : 3 - 4 ) . in group d , a total of 403 observations were obtained , of which 347 ( 86% ) were in the target sedation range . the proportion of time spent in the target sedation range was greater in group d ( p = 0.04 ) . a score 1 - 2 was observed on 86 ( 23% ) occasions in group c and 36 ( 9% ) occasions in group d ( p = 0.047 ) . rss : 5 - 6 was achieved in 52 ( 14% ) observations in group c and 20 ( 5% ) observations in group d ( p = 0.048 ) . a significant reduction in systolic and diastolic bp from the baseline ( p < 0.05 ) occurred after bolus infusion in group d but in none of the patients fall was > 30% from baseline . mean heart rate also decreased from baseline 2 h after commencement of sedative infusion in group d , but at none of the observation times fall was significant ( p = 0.079 ) [ figure 3 ] . in group c significant fall from baseline values in bp were noted 2 and 4 h after sedative infusion was started ; but thereafter , it showed minimal change figures [ 1 and 2 ] . patients receiving clonidine ( group c ) had significantly lower heart rates from baseline ( p < 0.05 ) [ figure 3 ] . on comparison , the hemodynamic parameters were comparable between the two groups during the study period ( p > 0.05 ) . systolic blood pressure ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation diastolic blood pressure ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation heart rate ( mean standard error of the mean ) during dexmedetomidine and clonidine infusion and after discontinuation bradycardia occurred in 3 of the 35 patients in group c and 4 of the 35 patients in group d ( p = 0.64 ) . hypotension occurred in 11 of the 35 patients in group c ( 31% ) and 3 of the 35 patients in group d ( 9% ) ( p = 0.01 ) . about 50% of the hypotensive episodes occurred within 2 - 4 h in group c and at 2 h in group d. no patient experienced hypotension after 14 h in group c and after 6 h in group d. sustained increase in systolic and diastolic pressure and heart rate occurred after cessation of infusion in group d , but there were no clinically significant rebound phenomena in any patient . in group c , rebound hypertension was seen in four patients after cessation of clonidine infusion . the median 24 h fentanyl requirement was 162 g ( range : 105 - 175 ) for group c and 171 g ( range : 110 - 185 ) for group d ( p = 0.73 ) . mean time for extubation was similar in both groups , being 19 h ( range : 14 - 30 h ) in group d patients and 18 h ( range : 16 - 32 h ) in group c. there were no adverse respiratory events after extubation in any patient in either group . the chief results of this study showed that target sedation was achieved in more number of patients receiving dexmedetomidine with lesser need for additional sedation . although mean cumulative sedation scores over the study period were not significantly different in two groups ( 3.37 + 1.37 vs. 3.20 + 0.75 in groups c and d , respectively ) , percentage of patients who attained target sedation was significantly higher in group d compared with group c ( 86 vs. 62% in groups d and c , respectively , p = 0.04 ) . dexmedetomidine is 8 times more specific for 2 receptors than clonidine and the improved specificity for the 2 adrenoreceptors , especially for the 2a subtype may make it to be a much more effective sedative than clonidine . however , our findings are in contrast with those of riker et al . who suggested that dexmedetomidine attained target sedation less frequently . a rss of 1 - 2 or 5 - 6 occurred in more number of observations in group c than in group d. the short distribution half - life of dexmedetomidine ( 6 min ) makes it an ideal drug for iv titration . this could be the reason for the rapid titration to target sedation and the lesser number of observations pertaining to inadequate sedation in group d. although more than 60% patients of both groups attained acceptable sedation , significantly more number of patients in clonidine treated group required additional sedation by diazepam on account of fall of bp on increasing infusion rate to maximum set level . requirement of additional sedation in this group was about 43% more than dexmedetomidine treated group . in a retrospective analysis of patients receiving clonidine for icu sedation , gillison et al . have reported that clonidine reduces requirement of additional sedation and analgesia , but at the cost of higher than routinely prescribed dose . only 8/35 patients ( 23% ) patients in group d needed additional sedation which agrees with findings of martin et al . however , the usual dose is in the order of 100 g / h . we used an initial dose of 1 g / kg / h of clonidine for infusion titrated to 2 g / kg / h as the maximum dose . the dose of dexmedetomidine for icu sedation varies greatly ranging between 0.2 and 2.5 g / kg / h . in our study , we used a loading dose of 0.7 g / kg followed by 0.2 - 0.7 g / kg / h . a meta - analysis by tan and ho ( 2010 ) observed that incidence of bradycardia requiring intervention increased in studies that used both a loading dose and maintenance doses of dexmedetomidine in excess of 0.7 g / kg / h . hypotension was more commonly seen in group c compared with group d. 50% of the hypotensive episodes occurred within 2 - 4 h in group c and after bolus infusion and within 2 h after maintenance infusion in group d , as the steady state plasma concentration of the drugs are achieved at this time duration , causing vasodilatation and hypotension . in general , hemodynamic stability was preserved in most patients receiving dexmedetomidine , a finding in agreement with many previous studies . eleven out of 14 patients in group c requiring additional sedation to achieve target sedation experienced hypotension on increasing the dose from 1 up to 2 g / kg / h . this observation was consistent with previous studies of clonidine where adverse hemodynamic effects occurred at doses required for sedation . previous studies of icu sedation with dexmedetomidine have found no or minimal increase in heart rate and bp following abrupt cessation , the finding similar to this study . in group c , a small study group containing patients mostly postsurgical precluded an extensive study on a heterogeneous icu population . a short study period was considered as dexmedetomidine has been approved by food and drug administration as a sedative in the icu for patients undergoing mechanical ventilation of < 24 h duration . being an open - label study , there is an inherent potential for observer bias . due to the unavailability of bispectral index ( bis ) at our center we restricted our assessment of the degree of sedation to rss . both dexmedetomidine and clonidine can be used as sedative agents for short term icu sedation of postsurgical patients . on the basis of our study data further trials with both drugs may define their exact role for sedation of icu patients .

kawasaki disease ( kd ) is an acute systemic vasculitis occurring predominantly in children aged < 5 years.1 the main complication of this disease is the development of coronary artery aneurysm ( caa ) . caa develops in 15% to 25% of untreated patients.2 kd is the leading cause of acquired heart disease in developed countries.3 standard treatment consists of a single administration of highdose intravenous immunoglobulins ( ivig ) and oral aspirin for 6 to 8 weeks . this has been shown to reduce the risk of caa to < 10% when treated within 10 days.4 both the etiology and pathophysiology of kd as well as the working mechanism of ivig have remained unclear to date . it has been hypothesized that kd represents a systemic vasculitis that , apart from the absence or presence of caa , results in increased cardiovascular disease risk at a later age . although controversial , this hypothesis was supported by abnormal myocardial perfusion , as shown by nuclear scintigraphy , even when echocardiography of the arteries was unremarkable.5 dysfunctional vasculature , however , may not be limited to the heart , and cardiovascular disease may be more widespread than just to the coronary arteries , as shown by increased flowmediated dilatation of the brachial artery after kd.6 in addition , a study by kato et al followed 594 patients from the acute kd phase up to 20 years afterward.7 in 2.2% of the patients , they found more widespread disease with extracardiac vascular lesions , although this study was performed at a time when ivig infusions were not routine . carotid intimamedia thickness ( cimt ) is a wellvalidated noninvasive surrogate marker for cardiovascular disease risk in multiple populations.8 , 9 , 10 in several studies , cimt was compared between participants with a history of kd and unaffected controls.11 , 12 , 13 some found increased cimt in all kd patients or in caapositive patients compared with controls , whereas others did not find any difference ; therefore , results were conflicting , and often the studies showed methodological limitations.6 furthermore , the included studies had a crosssectional design or were cohort studies with short followup ( < 6 months ) and were not able to show the course of the cimt change over time . in our own crosssectional study , we observed that children with kd had significantly increased cimt following early disease compared with siblings.14 in a large number of patients , 2 cimt measurements were obtained . having collected these cimt data over a period of almost 15 years , we investigated whether kd patients had increased cimt in a data set with longterm followup and whether these data supported our previous conclusions from our initial crosssectional study data . the study was conducted between october 2001 and december 2014 at the emma children 's hospital , a tertiary referral center . participants with a history of kd , based on criteria of the american heart association , were recruited consecutively during followup as outpatients.3 patients in the acute or subacute ( specified as within 6 months after the disease ) phase of kd were excluded to minimize the potential confounding influence of acute or subacute inflammation . unaffected siblings of children with kd and other unaffected persons ( family of staff members and staff of our hospital ) without a history of kd were eligible as controls if they did not take any cardiovascular medication . all participants and/or their parents gave informed consent , as approved by the institution 's research ethics board . using data from the fifth dutch growth study performed in 2009 in 20 867 children in the netherlands , standard deviation scores for body mass index ( bmi ) were calculated based on the age and sex of each participant ( http://groeiweb.pgdata.nl/calculator.asp [ in dutch ] ) . the medical records of the kd patients were retrospectively reviewed to collect clinical details : age at onset of disease , treatment with ivig , and presence of caa . caas were specified by worstever z scores : z scores adjusted for basal surface area.15 , 16 we defined the caas by their worstever scores because arteries may be damaged even when the lumen of a previously affected coronary artery has returned to its normal size , indicating more severe initial systemic vasculitis compared with children who never had enlargement . caa was defined as a coronary z score 2.5 , and a giant aneurysm was defined as a z score 10 or a diameter of 8 mm . following kd , after an overnight fast , a venous blood sample was taken to measure total cholesterol , high and lowdensity lipoprotein cholesterol , and triglycerides . we measured cimt in the participants with a history of kd when they visited the outpatient clinic from the age of 5 years onward . all participants were scanned by 2 experienced and certified sonographers . over the course of 14 years , 3 ultrasound machines were used : an acuson 128xp ( october 2001 to june 2006 ) , an acuson aspen ( june 2006 to january 2008 ) , and an acuson sequoia 512 ( january 2008 to january 2015 ) . l7 linear array vascular transducers were used on the acuson 128 xp and aspen , and an 8l5 transducer was used on the acuson sequoia ( siemens ag ) . all scans were performed according to a validated and standardized scanning and image analysis protocol . briefly , in all participants , the right and left common carotid , carotid bulb , and internal carotid arterial wall segments were visualized . for each segment , a 5second cineloop of 25 full frames per second was temporarily saved in the memory of the ultrasound equipment . because cimt is known to change slightly ( 68% ) during the cardiac cycle , cimt measurement at a fixed point in the arterial cycle is preferred.17 all ultrasound instruments were equipped with a dedicated carotid scan protocol in which a highpersistence scan setting was used . because of rapid arterial wall movement , this highpersistence setting provides a slight movement artifact on the averaged image when the artery is in systole , at which , at the relative resting phase of the diastole , the averaged image of the arterial wall is crisp . from the cineloop , the sonographer was trained and certified to select and save the highest quality and crispest image frame of the segment as a 22cm highresolution dicom still . the scan protocol is described in full elsewhere.18 the image analyses were done offline . the mean combined cimt per participant was calculated as follows : ( [ mean of the left and right common carotid arteries]+[mean of the left and right carotid bulb]+[mean of the left and right internal carotid arteries])/3 . for participants in whom 1 of the segments failed , the cimt of the same segment of the opposite carotid artery was taken as the mean of both carotid arteries . one image analyst performed all cimt measurements manually and was blinded to the patients ' case and caa status . the intraclass correlation coefficient was 0.92 ( 95% ci 0.750.97 ) for the mean cimt . the acuson sequioa , compared with the less advanced and technically similar xp and aspen , has improved hardware , software , and transducer properties regarding signaltonoise ratio , image display / pixel density , and image file size and format . to allow for comparison of cimt data of different machines , normalization of measurements is required ; therefore , we created a correction factor for the scans provided by the different instruments . in 10 volunteers on the same day , the most reliable artery segment the common carotid artery far walls was scanned on the acuson aspen and sequoia . subsequently , we evaluated cimt data for comparable age groups of all kd study participants by ultrasound instruments . this statistical evaluation of cimt participant data revealed the same and systematic differences in cimt between instruments as those of the volunteer scans . based on both calculations ( mean cimt and measurement differences within the cohort ) , a correction factor was applied with the most advanced ultrasound instrument , the acuson sequoia , as the reference . we evaluated differences in age and sex between patients and controls at the time of the first cimt measurement by using mann differences in the remaining demographics ( length , weight , mean arterial pressure , and bmi standard deviation score ) between patients with kd and controls were assessed by linear regression analysis , taking family bonds into account by creating a random term . differences in demographics between kd subgroups were evaluated by anova for parameters with normal distribution , by kruskal wallis test for parameters with a nonnormal distribution , and by chisquare test for binary parameters . a multilevel , repeatedmeasures , linear mixedeffects model was used to evaluate the association between kd and cimt . in the first model , all kd patients were compared with controls ; in the second model , 4 groups were compared : controls , caanegative patients , patients with small medium aneurysms , and patients with giant aneurysms . the analyses were adjusted for potentially confounding variables ( age , sex , mean arterial pressure , and bmi standard deviation score ) , which were entered as fixed effects . in addition , family relations were taken into account and were adjusted for by creating a random term . because measurements started at age 5 in patients , we calculated the intercept at this age . to evaluate whether ivig treatment , total cholesterol , lowdensity lipoprotein cholesterol , and triglycerides were of significant influence on cimt , we also performed in patients only a linear mixedeffect analysis including these variables . multiple imputation was performed for missing blood pressure ( 30% ) and missing segment ( 4.7% ) values . for each missing value , 5 imputations were performed based on age , weight , height , bmi standard deviation score , and the remaining segments that did not fail . a figure was created using r statistics version 3.0.1 ( r foundation for statistical computing ) . the study was conducted between october 2001 and december 2014 at the emma children 's hospital , a tertiary referral center . participants with a history of kd , based on criteria of the american heart association , were recruited consecutively during followup as outpatients.3 patients in the acute or subacute ( specified as within 6 months after the disease ) phase of kd were excluded to minimize the potential confounding influence of acute or subacute inflammation . unaffected siblings of children with kd and other unaffected persons ( family of staff members and staff of our hospital ) without a history of kd were eligible as controls if they did not take any cardiovascular medication . all participants and/or their parents gave informed consent , as approved by the institution 's research ethics board . using data from the fifth dutch growth study performed in 2009 in 20 867 children in the netherlands , standard deviation scores for body mass index ( bmi ) were calculated based on the age and sex of each participant ( http://groeiweb.pgdata.nl/calculator.asp [ in dutch ] ) . the medical records of the kd patients were retrospectively reviewed to collect clinical details : age at onset of disease , treatment with ivig , and presence of caa . caas were specified by worstever z scores : z scores adjusted for basal surface area.15 , 16 we defined the caas by their worstever scores because arteries may be damaged even when the lumen of a previously affected coronary artery has returned to its normal size , indicating more severe initial systemic vasculitis compared with children who never had enlargement . caa was defined as a coronary z score 2.5 , and a giant aneurysm was defined as a z score 10 or a diameter of 8 mm . following kd , after an overnight fast , a venous blood sample was taken to measure total cholesterol , high and lowdensity lipoprotein cholesterol , and triglycerides . we measured cimt in the participants with a history of kd when they visited the outpatient clinic from the age of 5 years onward . all participants were scanned by 2 experienced and certified sonographers . over the course of 14 years , 3 ultrasound machines were used : an acuson 128xp ( october 2001 to june 2006 ) , an acuson aspen ( june 2006 to january 2008 ) , and an acuson sequoia 512 ( january 2008 to january 2015 ) . l7 linear array vascular transducers were used on the acuson 128 xp and aspen , and an 8l5 transducer was used on the acuson sequoia ( siemens ag ) . all scans were performed according to a validated and standardized scanning and image analysis protocol . briefly , in all participants , the right and left common carotid , carotid bulb , and internal carotid arterial wall segments were visualized . for each segment , a 5second cineloop of 25 full frames per second was temporarily saved in the memory of the ultrasound equipment . because cimt is known to change slightly ( 68% ) during the cardiac cycle , cimt measurement at a fixed point in the arterial cycle is preferred.17 all ultrasound instruments were equipped with a dedicated carotid scan protocol in which a highpersistence scan setting was used . because of rapid arterial wall movement , this highpersistence setting provides a slight movement artifact on the averaged image when the artery is in systole , at which , at the relative resting phase of the diastole , the averaged image of the arterial wall is crisp . from the cineloop , the sonographer was trained and certified to select and save the highest quality and crispest image frame of the segment as a 22cm highresolution dicom still . the scan protocol is described in full elsewhere.18 the image analyses were done offline . the mean combined cimt per participant was calculated as follows : ( [ mean of the left and right common carotid arteries]+[mean of the left and right carotid bulb]+[mean of the left and right internal carotid arteries])/3 . for participants in whom 1 of the segments failed , the cimt of the same segment of the opposite carotid artery was taken as the mean of both carotid arteries . when both sides failed , the segment was considered missing . one image analyst performed all cimt measurements manually and was blinded to the patients ' case and caa status . the intraclass correlation coefficient was 0.92 ( 95% ci 0.750.97 ) for the mean cimt . the acuson sequioa , compared with the less advanced and technically similar xp and aspen , has improved hardware , software , and transducer properties regarding signaltonoise ratio , image display / pixel density , and image file size and format . to allow for comparison of cimt data of different machines , normalization of measurements is required ; therefore , we created a correction factor for the scans provided by the different instruments . in 10 volunteers on the same day , the most reliable artery segment the common carotid artery far walls was scanned on the acuson aspen and sequoia . subsequently , we evaluated cimt data for comparable age groups of all kd study participants by ultrasound instruments . this statistical evaluation of cimt participant data revealed the same and systematic differences in cimt between instruments as those of the volunteer scans . based on both calculations ( mean cimt and measurement differences within the cohort ) , a correction factor was applied with the most advanced ultrasound instrument , the acuson sequoia , as the reference . we evaluated differences in age and sex between patients and controls at the time of the first cimt measurement by using mann differences in the remaining demographics ( length , weight , mean arterial pressure , and bmi standard deviation score ) between patients with kd and controls were assessed by linear regression analysis , taking family bonds into account by creating a random term . differences in demographics between kd subgroups were evaluated by anova for parameters with normal distribution , by kruskal wallis test for parameters with a nonnormal distribution , and by chisquare test for binary parameters . a multilevel , repeatedmeasures , linear mixedeffects model was used to evaluate the association between kd and cimt . in the first model , all kd patients were compared with controls ; in the second model , 4 groups were compared : controls , caanegative patients , patients with small medium aneurysms , and patients with giant aneurysms . the analyses were adjusted for potentially confounding variables ( age , sex , mean arterial pressure , and bmi standard deviation score ) , which were entered as fixed effects . in addition , family relations were taken into account and were adjusted for by creating a random term . because measurements started at age 5 in patients , we calculated the intercept at this age . to evaluate whether ivig treatment , total cholesterol , lowdensity lipoprotein cholesterol , and triglycerides were of significant influence on cimt , we also performed in patients only a linear mixedeffect analysis including these variables . multiple imputation was performed for missing blood pressure ( 30% ) and missing segment ( 4.7% ) values . for each missing value , 5 imputations were performed based on age , weight , height , bmi standard deviation score , and the remaining segments that did not fail . a figure was created using r statistics version 3.0.1 ( r foundation for statistical computing ) . a total of 319 participants with a history of kd were included , with a median age of 8.1 years during their first cimt measurement ( range 5.043.3 years ) . in these patients , a total of 150 controls ( 130 siblings and 20 unrelated participants ) were included with a median age of 12.5 years ( range 7.031.1 years ) . the demographic characteristics of patients and controls during the first cimt measurement are shown in table 1 . demographics of controls and kawasaki disease subgroups at the time of their first cimt measurement bmi indicates body mass index ; caa , coronary artery aneurysm ; cimt , carotid intimamedia thickness . these variables were adjusted for in the model . mainly consisting of individuals of indosurinamese and asian descent . number of cimt measurements per subgroup caa indicates coronary artery aneurysm ; cimt , carotid intimamedia thickness . of the 319 participants with a history of kd , 241 ( 75.5% ) had a worstever coronary artery z score < 2.5 . the other participants had caa , of whom 51 ( 16.0% ) had caa with a z score of 2.5 to 10 and 27 ( 8.5% ) had giant aneurysms , with a z score of 10 and/or a diameter of 8 mm . there was no significant difference in sex between patients and controls , but there were significantly more male participants in the small medium caa group ( p=0.02 ) and in the giant caa group ( p<0.001 ) compared with the caanegative patients . the clinical characteristics of kd patients and the kd subgroups are shown in table 3 . patients with small medium caa were significantly younger during their disease compared with patients without caa . there was no significant difference in age at kd between patients with giant aneurysms and caanegative patients . median followup time between first and last carotid intimamedia thickness measurement of patients with > 1 measurement ( total n=148 ; caanegative , n=98 ; small medium caa , n=31 ; giant caa , n=19 ) . patients with a history of kd had a significantly higher estimated marginal mean compared with controls ( 0.375 mm [ 95% ci 0.3720.378 mm ] versus 0.363 mm [ 95% ci 0.3580.368 mm ] ; p=0<0.001 ) . the model for the longitudinal cimt data analysis over time shows that patients with a history of kd started with a higher cimt ( intercept + 0.0145 mm [ 95% ci : 0.00420.0248 mm ; p=0.006 ] at age 5 compared with controls ) . there was no difference in increase per ageyear ( 0.0004 mm [ 95% ci 0.0014 to 0.0007 mm ; p=0.490 ] increase per year compared with controls ) . in comparing the different groups based on caa status in our kd cohort , we found that the estimated marginal mean of caanegative patients was 0.373 mm ( 95% ci 0.3690.376 mm ; p<0.01 compared with controls ) , of patients with small medium caa was 0.374 mm ( 95% ci 0.3670.382 mm ; p<0.05 compared with controls ) , and of patients with giant aneurysms of 0.381 mm ( 95% ci 0.3700.392 mm ; p<0.01 compared with controls ) . the complete model shown in table 4 indicates that , compared with controls , caanegative patients started with a significantly higher cimt at the age of 5 years ( + 0.0193 mm [ 95% ci 0.00890.0297 mm ] ; p<0.001 ) , but this difference decreased per year ( 0.0014 mm per year [ 95% ci 0.0025 to 0.0003 mm ] ; p=0.012 ) . there was no significant difference between controls and patients with small medium caa either in cimt at the age of 5 years or in cimt progression per year . compared with controls , patients with giant caa had a higher but nonsignificant cimt level at age 5 years and a trend toward increased cimt progression per year ( 0.0013 mm per year , [ 95% ci 0.0000 to 0.0027 mm ] ; p=0.058 ) . association between cimt and kawasaki disease subgroups and controls bmi indicates body mass index ; caa , coronary artery aneurysm ; cimt , carotid intimamedia thickness . the estimated in millimeters per year was based on the model in which controls were reference , and the p value of age and the estimated of the other covariates including the p values were identical in both models . in comparing caanegative patients with patients with small medium and giant caa , both groups had comparable intercepts at age 5 years but had significantly increased progression ( 0.0015 mm per year [ 95% ci 0.00010.0030 mm ] ; p=0.038 ; and 0.0027 mm per year [ 95% ci 0.00150.0039 mm ] ; p<0.001 , respectively ) . figures 1 and 2 show the regression lines ( 95% ci ) for cimt against age , corrected for sex , bmi z score , mean arterial pressure , and family relations for controls and patients and for controls and the different patient groups based on caa worstever z score . the mean carotid imt regression line ( 95% ci ) of patients and controls against age . the mean regression line is represented by the continuous line , and the 95% cis are indicated by dashed lines , after adjusting for sex , body mass index z score , mean arterial pressure , and family relations . imt indicates intimamedia thickness . the mean carotid imt regression line ( 95% cis ) of the different patient groups based on caa worstever z score and controls against age . the mean regression line is represented by the continuous line , and the 95% cis are indicated by dashed lines after adjusting for sex , body mass index z score , mean arterial pressure , and family relations . ivig , total cholesterol , lowdensity lipoprotein cholesterol , and triglycerides were not significantly associated with cimt in the multivariable model . because not all of our patients obtained multiple measurements , we also analyzed the data including only the measurements of patients with followup data . patients with small medium and giant caa had comparable intercepts at age 5 compared with caanegative patients ( p=0.131 and p=0.071 , respectively ) , but progression was significantly increased in patients with giant caa ( 0.0035 mm [ 95% ci 0.00190.0051 mm ] ; p<0.001 ) ; progression was not significantly increased in patients with small medium caa ( 0.0019 mm [ 0.0002 to 0.0039 mm ] ; p=0.081 ) . after obtaining the results of the increased progression in patients with giant aneurysms but not in patients with small medium aneurysms ( z scores 2.510 ) , we performed a post hoc analysis to evaluate whether the participants with giant aneurysms composed a different group as such or represented the more extreme phenotype of a spectrum of the disease . children with mediumsized aneurysms ( z scores 510 , n=15 , 29 cimt measurements ) had an estimated marginal mean of 0.381 mm ( 95% ci 0.3680.394 ) . in evaluating the model , these patients showed a trend toward an increased intercept ( 0.0192 mm , [ 0.0008 to 0.0392 mm ] ; p=0.060 ) with comparable progression in cimt parallel to the control curves ( p=0.833 ) , indicating that normalization toward the healthy siblings in participants without caa was absent in those with medium caa . patients with a history of kd had a significantly higher estimated marginal mean compared with controls ( 0.375 mm [ 95% ci 0.3720.378 mm ] versus 0.363 mm [ 95% ci 0.3580.368 mm ] ; p=0<0.001 ) . the model for the longitudinal cimt data analysis over time shows that patients with a history of kd started with a higher cimt ( intercept + 0.0145 mm [ 95% ci : 0.00420.0248 mm ; p=0.006 ] at age 5 compared with controls ) . there was no difference in increase per ageyear ( 0.0004 mm [ 95% ci 0.0014 to 0.0007 mm ; p=0.490 ] increase per year compared with controls ) . in comparing the different groups based on caa status in our kd cohort , we found that the estimated marginal mean of caanegative patients was 0.373 mm ( 95% ci 0.3690.376 mm ; p<0.01 compared with controls ) , of patients with small medium caa was 0.374 mm ( 95% ci 0.3670.382 mm ; p<0.05 compared with controls ) , and of patients with giant aneurysms of 0.381 mm ( 95% ci 0.3700.392 mm ; p<0.01 compared with controls ) . the complete model shown in table 4 indicates that , compared with controls , caanegative patients started with a significantly higher cimt at the age of 5 years ( + 0.0193 mm [ 95% ci 0.00890.0297 mm ] ; p<0.001 ) , but this difference decreased per year ( 0.0014 mm per year [ 95% ci 0.0025 to 0.0003 mm ] ; p=0.012 ) . there was no significant difference between controls and patients with small medium caa either in cimt at the age of 5 years or in cimt progression per year . compared with controls , patients with giant caa had a higher but nonsignificant cimt level at age 5 years and a trend toward increased cimt progression per year ( 0.0013 mm per year , [ 95% ci 0.0000 to 0.0027 mm ] ; p=0.058 ) . association between cimt and kawasaki disease subgroups and controls bmi indicates body mass index ; caa , coronary artery aneurysm ; cimt , carotid intimamedia thickness . the estimated in millimeters per year was based on the model in which controls were reference , and the p value of age and the estimated of the other covariates including the p values were identical in both models . in comparing caanegative patients with patients with small medium and giant caa , both groups had comparable intercepts at age 5 years but had significantly increased progression ( 0.0015 mm per year [ 95% ci 0.00010.0030 mm ] ; p=0.038 ; and 0.0027 mm per year [ 95% ci 0.00150.0039 mm ] ; p<0.001 , respectively ) . figures 1 and 2 show the regression lines ( 95% ci ) for cimt against age , corrected for sex , bmi z score , mean arterial pressure , and family relations for controls and patients and for controls and the different patient groups based on caa worstever z score . the mean carotid imt regression line ( 95% ci ) of patients and controls against age . the mean regression line is represented by the continuous line , and the 95% cis are indicated by dashed lines , after adjusting for sex , body mass index z score , mean arterial pressure , and family relations . imt indicates intimamedia thickness . the mean carotid imt regression line ( 95% cis ) of the different patient groups based on caa worstever z score and controls against age . the mean regression line is represented by the continuous line , and the 95% cis are indicated by dashed lines after adjusting for sex , body mass index z score , mean arterial pressure , and family relations . ivig , total cholesterol , lowdensity lipoprotein cholesterol , and triglycerides were not significantly associated with cimt in the multivariable model . because not all of our patients obtained multiple measurements , we also analyzed the data including only the measurements of patients with followup data . patients with small medium and giant caa had comparable intercepts at age 5 compared with caanegative patients ( p=0.131 and p=0.071 , respectively ) , but progression was significantly increased in patients with giant caa ( 0.0035 mm [ 95% ci 0.00190.0051 mm ] ; p<0.001 ) ; progression was not significantly increased in patients with small medium caa ( 0.0019 mm [ 0.0002 to 0.0039 mm ] ; p=0.081 ) . after obtaining the results of the increased progression in patients with giant aneurysms but not in patients with small medium aneurysms ( z scores 2.510 ) , we performed a post hoc analysis to evaluate whether the participants with giant aneurysms composed a different group as such or represented the more extreme phenotype of a spectrum of the disease . children with mediumsized aneurysms ( z scores 510 , n=15 , 29 cimt measurements ) had an estimated marginal mean of 0.381 mm ( 95% ci 0.3680.394 ) . in evaluating the model , these patients showed a trend toward an increased intercept ( 0.0192 mm , [ 0.0008 to 0.0392 mm ] ; p=0.060 ) with comparable progression in cimt parallel to the control curves ( p=0.833 ) , indicating that normalization toward the healthy siblings in participants without caa was absent in those with medium caa . our longitudinal cohort study in kd demonstrated that the severity of the coronary arteritis at the acute stage of the disease is associated with cimt of the extracardiac vasculature . the estimated marginal means of the cimt of the carotid artery increased with increasing severity of the original vasculitis , as reflected by the extent and diameter of caa . the cimt of caanegative patients was observed to be initially increased but normalized over time . patients with the most severe caa at the initial stage of the disease showed a trend toward significantly increased cimt progression over time compared with controls . in comparing patients with small medium and giant caa with patients without caa , together , these data suggest a spectrum of disease , not only of the coronary arteries but also of the peripheral vasculature . some studies found significantly increased cimt in kd patients compared with controls,19 whereas others did not.12 in comparing caapositive patients with controls , some studies found significantly increased cimt,11 , 20 and again others did not report any difference13 , 21 ; however , most of these studies were small , used variable study designs , or lacked a sufficient number of caapositive patients , and none of the studies had a followup of > 6 months.6 our study is the first longitudinal cimt study in kd and demonstrates an apparently gradual increase in cimt in kd patients with larger caa . the cimt of caanegative patients showed an increased cimt following complete convalescence of the acute disease , which normalized over time . patients with small caa showed increased ( but nonsignificant ) initial cimt but comparable cimt progression parallel to the curves of controls . patients with mediumsized caa showed a trend toward increased initial cimt with cimt progression comparable to that of controls . patients with giant caa showed a trend toward increased cimt progression over time . in evaluating this model , patients with giant caa and , to a much lesser degree , patients with mediumsized caa showed a trend toward continuously increasing cimt compared with controls . patients with giant caa are most severely affected by the original vasculitis of the coronary vasculature . our study suggests that these patients should be followed up for broader cardiovascular assessment beyond the heart . although these findings need to be confirmed in additional prospective cohort studies , our study also suggests that patients without any enlargement at any point of the disease may not need lifelong followup . several factors such as age , sex , bmi , blood pressure , lipids , and lifestyle influence cimt.22 for the latter , a suitable control group is vital ; therefore , we included siblings as controls , having the same environmental and genetic factors . there were significantly more male participants in the small medium and giant caa group compared with the caanegative patients . it should be emphasized that in the model used , sex , bmi , age , and blood pressure were adjusted for , and lipids were not significantly associated with cimt in our patient group . cimt is strongly correlated with cardiovascular events.9 a systematic review by lorenz et al calculated a relative risk of 1.15 ( 95% ci 1.121.17 ) per 0.10mm cimt difference for myocardial infarction and a relative risk of 1.18 ( 95% ci 1.161.21 ) per 0.10 mm cimt difference for stroke from studies mainly investigating older populations . eikendal et al found a hazard ratio of 1.4 per standard deviation increase in cimt for myocardial infarction or stroke in adults aged < 45 years.10 increased cimt is seen in children and adolescents with known cardiovascular risk factors such as familial hypercholesterolemia or obesity.23 , 24 , 25 increased cimt progression was found to be significantly related to the incidence of stroke by polak et al.26 in contrast , a metaanalysis of individual patient data from longitudinal studies in 2012 showed no significant association between cimt progression and cardiovascular events in mainly middleaged to older adults.27 this could be explained by the different methods of measuring cimt at the different institutes . another systematic review of randomized controlled trials measuring cimt change over time found a statistically significant association between mean change in cimt over time and the likelihood of developing nonfatal myocardial infarction ( p=0.018 ) and the combined end point of myocardial infarction and death ( p=0.021).28 in younger participants , the bogalusa heart study found a significant association between some cimt segment progression and multiple cardiovascular risk factors such as waist circumference , waist / height ratio , mean arterial pressure , cholesterol , and smoking.29 the young finns study showed that young adult cimt progression was associated with risk factors in childhood such as bmi , physical activity , and fruit consumption.30 although the exact risk prediction of ( increased ) cimt in children and young adults is still unknown , cimt is clearly correlated with cardiovascular risk factors in a younger population . although cimt is often considered a surrogate marker for clinical or subclinical atherosclerosis , it is unlikely that this is also the case in kd patients . first , the cimt course in caanegative patients does not seem to be concordant with atherosclerosis because one would expect the cimt values to worsen over time instead of normalize . this suggests that the increased cimt in kd patients originates from a different type of vasculopathy and is supported by earlier postmortem ( histology ) reports that show no accumulation of lipid in the intima or other features consistent with atherosclerosis in coronary arteries.31 , 32 the etiology and consequences of this kd vasculopathy have yet to be determined . most cimt studies in adults relate increased cimt to the extent of atherosclerosis ; therefore , the cardiovascular risks derived from these studies can not be directly adopted for the kd population . moreover , lorenz et al found a relative risk of myocardial infarction of 1.15 per 0.10mm cimt increase , whereas our study shows a difference of 0.012 mm between patients and controls.9 although much smaller than in adult atherosclerosis , in patients with giant caa , this difference might increase each year , potentially leading to relevant peripheral vasculature changes over time . the increase in cimt at the extreme end of the spectrum is not completely unexpected . suda et al described 76 patients with giant aneurysms ( > 8 mm ) in a retrospective cohort with a median followup of 19 years and found that 7 patients died during followup.33 they calculated 10 , 20 , and 30year survival rates of 95% , 88% , and 88% , respectively , and 5 , 15 , and 25year cumulative coronary intervention rates of 28% , 43% , and 59% , respectively , indicating that the coronary arteries are still remodeling years after acute disease . first , although the cimt protocol did not change throughout the years , different ultrasound machines were used . second , blood pressure data at the time of cimt was missing in 30% of children . by imputing the missing data based on many of the known variables , we were able to correct for mean arterial pressure in our model . because blood pressure did not seem to influence the cimt ( almost all of the children were normotensive ) , the missing data were unlikely to have influenced the results . third , patients were stratified based on their worstever z score . because the study was conducted in a tertiary referral center , most of the echocardiograms in the acute phase were not performed at our own center but rather by pediatric cardiologists at other centers . this may have led to misclassification of some patients in the caa subgroups . moreover , our study was conducted at a tertiary referral center , with inevitable referral bias . in our case , the relatively large number of patients with caa , and especially with giant caa , helped identify that these patients had the most abnormal response at the peripheral noncardiac vasculature . finally , our controls and a proportion of our patients did not undergo > 1 cimt measurement ; however , by creating a multilevel , repeatedmeasures , linear mixedeffects model , we could use all cimt measurements to create a large study group . even though this is the largest study of cimt in patients after kd the arterial walls of the young encompass 0.4 mm , whereas cimt as measured by bmode ultrasound has an axial resolution of 0.04 to 0.05 mm ; therefore , to detect submillimeter differences , large group sizes are required . we observed a trend toward significance in both intercept and progression in different subgroups ; however , because significance is dependent on the sample size , and because in both groups the mean was indeed significantly increased , it is likely that a significant finding will be present in larger groups . consequently , our data need confirmation in a ( very ) large number of participants with longitudinal followup , in particular in the smaller subgroups of caapositive patients . although the cimt protocol did not change throughout the years , different ultrasound machines were used . second , blood pressure data at the time of cimt was missing in 30% of children . by imputing the missing data based on many of the known variables , we were able to correct for mean arterial pressure in our model . because blood pressure did not seem to influence the cimt ( almost all of the children were normotensive ) , the missing data were unlikely to have influenced the results . third , patients were stratified based on their worstever z score . because the study was conducted in a tertiary referral center , most of the echocardiograms in the acute phase were not performed at our own center but rather by pediatric cardiologists at other centers . this may have led to misclassification of some patients in the caa subgroups . moreover , our study was conducted at a tertiary referral center , with inevitable referral bias . in our case , the relatively large number of patients with caa , and especially with giant caa , helped identify that these patients had the most abnormal response at the peripheral noncardiac vasculature . finally , our controls and a proportion of our patients did not undergo > 1 cimt measurement ; however , by creating a multilevel , repeatedmeasures , linear mixedeffects model , we could use all cimt measurements to create a large study group . even though this is the largest study of cimt in patients after kd the arterial walls of the young encompass 0.4 mm , whereas cimt as measured by bmode ultrasound has an axial resolution of 0.04 to 0.05 mm ; therefore , to detect submillimeter differences , large group sizes are required . we observed a trend toward significance in both intercept and progression in different subgroups ; however , because significance is dependent on the sample size , and because in both groups the mean was indeed significantly increased , it is likely that a significant finding will be present in larger groups . consequently , our data need confirmation in a ( very ) large number of participants with longitudinal followup , in particular in the smaller subgroups of caapositive patients . although the cimt of caanegative children is initially increased , the values normalize at a later age , suggesting vascular repair of a generalized vasculopathy distinctive from atherosclerosis . patients with a history of kd complicated by giant and , to a lesser degree , mediumsized caa have a trend toward continued increased cimt , suggesting more severe impact on the arterial wall . until more data become available , these patients need cardiovascular counseling and followup beyond the heart . jeroen g. noordzij md phd , reinier de graaf hospital , delft , the netherlands . lieke rozendaal md , leids university medical center , willem alexander children 's hospital , leiden , the netherlands . this work was supported by the stinafo foundation ( the hague , the netherlands ) . the sponsor had no role in the study design , the data collection and analysis , the writing of the report , or the decision to submit the manuscript for publication .

backgroundkawasaki disease ( kd ) is a pediatric vasculitis with coronary artery aneurysm ( caa ) as a major complication . controversy exists about cardiovascular risk later in life . the aim of our study was to evaluate whether kd patients are at increased risk , as assessed by carotid intimamedia thickness ( cimt).methods and resultswe measured cimt over 15 years by bmode ultrasonography in kd patients during followup and in unaffected controls ( mostly siblings ) . a multilevel , repeatedmeasures , linear mixedeffects model was used to evaluate the association between kd and cimt . a total of 319 patients with 528 measurements were compared with 150 controls . in kd patients , the mean cimt was increased compared with controls ( 0.375 mm [ 95% ci 0.3720.378 mm ] versus 0.363 mm [ 95% ci 0.3580.368 mm ] ; p<0.001 ) . furthermore , mean cimt of caanegative patients was 0.373 mm ( p<0.01 compared with controls ) , of patients with small medium caa was 0.374 mm ( p<0.05 compared with controls ) , and of patients with giant caa was 0.381 mm ( p<0.01 compared with controls ) . compared with controls , caanegative participants started with an increased cimt ( + 0.01930.0053 mm , p<0.001 ) but showed slower progression ( 0.00140.0006 mm / year , p=0.012 ) . patients with giant caa showed a trend toward increased cimt progression ( 0.00130.0007 mm / year , p=0.058).conclusionswe observed a positive correlation between cimt and kd severity of coronary arteritis at the acute stage . although initially increased , the cimt in caanegative patients normalized at a later age . in contrast , patients with a history of kd complicated by giant caa showed a trend toward persistently increased cimt . these patients may need cardiovascular counseling and followup beyond the heart .

Introduction Methods Participants Study Protocol Measurements of cIMT Statistics Results Carotid IntimaMedia Thickness Additional and Post Hoc Analyses Discussion Study Limitations Conclusion Appendix: Dutch Kawasaki Study Group Sources of Funding Disclosures

kawasaki disease ( kd ) is an acute systemic vasculitis occurring predominantly in children aged < 5 years.1 the main complication of this disease is the development of coronary artery aneurysm ( caa ) . although controversial , this hypothesis was supported by abnormal myocardial perfusion , as shown by nuclear scintigraphy , even when echocardiography of the arteries was unremarkable.5 dysfunctional vasculature , however , may not be limited to the heart , and cardiovascular disease may be more widespread than just to the coronary arteries , as shown by increased flowmediated dilatation of the brachial artery after kd.6 in addition , a study by kato et al followed 594 patients from the acute kd phase up to 20 years afterward.7 in 2.2% of the patients , they found more widespread disease with extracardiac vascular lesions , although this study was performed at a time when ivig infusions were not routine . carotid intimamedia thickness ( cimt ) is a wellvalidated noninvasive surrogate marker for cardiovascular disease risk in multiple populations.8 , 9 , 10 in several studies , cimt was compared between participants with a history of kd and unaffected controls.11 , 12 , 13 some found increased cimt in all kd patients or in caapositive patients compared with controls , whereas others did not find any difference ; therefore , results were conflicting , and often the studies showed methodological limitations.6 furthermore , the included studies had a crosssectional design or were cohort studies with short followup ( < 6 months ) and were not able to show the course of the cimt change over time . having collected these cimt data over a period of almost 15 years , we investigated whether kd patients had increased cimt in a data set with longterm followup and whether these data supported our previous conclusions from our initial crosssectional study data . participants with a history of kd , based on criteria of the american heart association , were recruited consecutively during followup as outpatients.3 patients in the acute or subacute ( specified as within 6 months after the disease ) phase of kd were excluded to minimize the potential confounding influence of acute or subacute inflammation . we measured cimt in the participants with a history of kd when they visited the outpatient clinic from the age of 5 years onward . we evaluated differences in age and sex between patients and controls at the time of the first cimt measurement by using mann differences in the remaining demographics ( length , weight , mean arterial pressure , and bmi standard deviation score ) between patients with kd and controls were assessed by linear regression analysis , taking family bonds into account by creating a random term . a multilevel , repeatedmeasures , linear mixedeffects model was used to evaluate the association between kd and cimt . in the first model , all kd patients were compared with controls ; in the second model , 4 groups were compared : controls , caanegative patients , patients with small medium aneurysms , and patients with giant aneurysms . participants with a history of kd , based on criteria of the american heart association , were recruited consecutively during followup as outpatients.3 patients in the acute or subacute ( specified as within 6 months after the disease ) phase of kd were excluded to minimize the potential confounding influence of acute or subacute inflammation . we measured cimt in the participants with a history of kd when they visited the outpatient clinic from the age of 5 years onward . we evaluated differences in age and sex between patients and controls at the time of the first cimt measurement by using mann differences in the remaining demographics ( length , weight , mean arterial pressure , and bmi standard deviation score ) between patients with kd and controls were assessed by linear regression analysis , taking family bonds into account by creating a random term . a multilevel , repeatedmeasures , linear mixedeffects model was used to evaluate the association between kd and cimt . in the first model , all kd patients were compared with controls ; in the second model , 4 groups were compared : controls , caanegative patients , patients with small medium aneurysms , and patients with giant aneurysms . a total of 319 participants with a history of kd were included , with a median age of 8.1 years during their first cimt measurement ( range 5.043.3 years ) . in these patients , a total of 150 controls ( 130 siblings and 20 unrelated participants ) were included with a median age of 12.5 years ( range 7.031.1 years ) . there was no significant difference in sex between patients and controls , but there were significantly more male participants in the small medium caa group ( p=0.02 ) and in the giant caa group ( p<0.001 ) compared with the caanegative patients . median followup time between first and last carotid intimamedia thickness measurement of patients with > 1 measurement ( total n=148 ; caanegative , n=98 ; small medium caa , n=31 ; giant caa , n=19 ) . patients with a history of kd had a significantly higher estimated marginal mean compared with controls ( 0.375 mm [ 95% ci 0.3720.378 mm ] versus 0.363 mm [ 95% ci 0.3580.368 mm ] ; p=0<0.001 ) . the model for the longitudinal cimt data analysis over time shows that patients with a history of kd started with a higher cimt ( intercept + 0.0145 mm [ 95% ci : 0.00420.0248 mm ; p=0.006 ] at age 5 compared with controls ) . there was no difference in increase per ageyear ( 0.0004 mm [ 95% ci 0.0014 to 0.0007 mm ; p=0.490 ] increase per year compared with controls ) . in comparing the different groups based on caa status in our kd cohort , we found that the estimated marginal mean of caanegative patients was 0.373 mm ( 95% ci 0.3690.376 mm ; p<0.01 compared with controls ) , of patients with small medium caa was 0.374 mm ( 95% ci 0.3670.382 mm ; p<0.05 compared with controls ) , and of patients with giant aneurysms of 0.381 mm ( 95% ci 0.3700.392 mm ; p<0.01 compared with controls ) . the complete model shown in table 4 indicates that , compared with controls , caanegative patients started with a significantly higher cimt at the age of 5 years ( + 0.0193 mm [ 95% ci 0.00890.0297 mm ] ; p<0.001 ) , but this difference decreased per year ( 0.0014 mm per year [ 95% ci 0.0025 to 0.0003 mm ] ; p=0.012 ) . there was no significant difference between controls and patients with small medium caa either in cimt at the age of 5 years or in cimt progression per year . compared with controls , patients with giant caa had a higher but nonsignificant cimt level at age 5 years and a trend toward increased cimt progression per year ( 0.0013 mm per year , [ 95% ci 0.0000 to 0.0027 mm ] ; p=0.058 ) . association between cimt and kawasaki disease subgroups and controls bmi indicates body mass index ; caa , coronary artery aneurysm ; cimt , carotid intimamedia thickness . in comparing caanegative patients with patients with small medium and giant caa , both groups had comparable intercepts at age 5 years but had significantly increased progression ( 0.0015 mm per year [ 95% ci 0.00010.0030 mm ] ; p=0.038 ; and 0.0027 mm per year [ 95% ci 0.00150.0039 mm ] ; p<0.001 , respectively ) . patients with small medium and giant caa had comparable intercepts at age 5 compared with caanegative patients ( p=0.131 and p=0.071 , respectively ) , but progression was significantly increased in patients with giant caa ( 0.0035 mm [ 95% ci 0.00190.0051 mm ] ; p<0.001 ) ; progression was not significantly increased in patients with small medium caa ( 0.0019 mm [ 0.0002 to 0.0039 mm ] ; p=0.081 ) . after obtaining the results of the increased progression in patients with giant aneurysms but not in patients with small medium aneurysms ( z scores 2.510 ) , we performed a post hoc analysis to evaluate whether the participants with giant aneurysms composed a different group as such or represented the more extreme phenotype of a spectrum of the disease . in evaluating the model , these patients showed a trend toward an increased intercept ( 0.0192 mm , [ 0.0008 to 0.0392 mm ] ; p=0.060 ) with comparable progression in cimt parallel to the control curves ( p=0.833 ) , indicating that normalization toward the healthy siblings in participants without caa was absent in those with medium caa . patients with a history of kd had a significantly higher estimated marginal mean compared with controls ( 0.375 mm [ 95% ci 0.3720.378 mm ] versus 0.363 mm [ 95% ci 0.3580.368 mm ] ; p=0<0.001 ) . the model for the longitudinal cimt data analysis over time shows that patients with a history of kd started with a higher cimt ( intercept + 0.0145 mm [ 95% ci : 0.00420.0248 mm ; p=0.006 ] at age 5 compared with controls ) . there was no difference in increase per ageyear ( 0.0004 mm [ 95% ci 0.0014 to 0.0007 mm ; p=0.490 ] increase per year compared with controls ) . in comparing the different groups based on caa status in our kd cohort , we found that the estimated marginal mean of caanegative patients was 0.373 mm ( 95% ci 0.3690.376 mm ; p<0.01 compared with controls ) , of patients with small medium caa was 0.374 mm ( 95% ci 0.3670.382 mm ; p<0.05 compared with controls ) , and of patients with giant aneurysms of 0.381 mm ( 95% ci 0.3700.392 mm ; p<0.01 compared with controls ) . the complete model shown in table 4 indicates that , compared with controls , caanegative patients started with a significantly higher cimt at the age of 5 years ( + 0.0193 mm [ 95% ci 0.00890.0297 mm ] ; p<0.001 ) , but this difference decreased per year ( 0.0014 mm per year [ 95% ci 0.0025 to 0.0003 mm ] ; p=0.012 ) . there was no significant difference between controls and patients with small medium caa either in cimt at the age of 5 years or in cimt progression per year . compared with controls , patients with giant caa had a higher but nonsignificant cimt level at age 5 years and a trend toward increased cimt progression per year ( 0.0013 mm per year , [ 95% ci 0.0000 to 0.0027 mm ] ; p=0.058 ) . association between cimt and kawasaki disease subgroups and controls bmi indicates body mass index ; caa , coronary artery aneurysm ; cimt , carotid intimamedia thickness . in comparing caanegative patients with patients with small medium and giant caa , both groups had comparable intercepts at age 5 years but had significantly increased progression ( 0.0015 mm per year [ 95% ci 0.00010.0030 mm ] ; p=0.038 ; and 0.0027 mm per year [ 95% ci 0.00150.0039 mm ] ; p<0.001 , respectively ) . patients with small medium and giant caa had comparable intercepts at age 5 compared with caanegative patients ( p=0.131 and p=0.071 , respectively ) , but progression was significantly increased in patients with giant caa ( 0.0035 mm [ 95% ci 0.00190.0051 mm ] ; p<0.001 ) ; progression was not significantly increased in patients with small medium caa ( 0.0019 mm [ 0.0002 to 0.0039 mm ] ; p=0.081 ) . after obtaining the results of the increased progression in patients with giant aneurysms but not in patients with small medium aneurysms ( z scores 2.510 ) , we performed a post hoc analysis to evaluate whether the participants with giant aneurysms composed a different group as such or represented the more extreme phenotype of a spectrum of the disease . in evaluating the model , these patients showed a trend toward an increased intercept ( 0.0192 mm , [ 0.0008 to 0.0392 mm ] ; p=0.060 ) with comparable progression in cimt parallel to the control curves ( p=0.833 ) , indicating that normalization toward the healthy siblings in participants without caa was absent in those with medium caa . our longitudinal cohort study in kd demonstrated that the severity of the coronary arteritis at the acute stage of the disease is associated with cimt of the extracardiac vasculature . the cimt of caanegative patients was observed to be initially increased but normalized over time . patients with the most severe caa at the initial stage of the disease showed a trend toward significantly increased cimt progression over time compared with controls . some studies found significantly increased cimt in kd patients compared with controls,19 whereas others did not.12 in comparing caapositive patients with controls , some studies found significantly increased cimt,11 , 20 and again others did not report any difference13 , 21 ; however , most of these studies were small , used variable study designs , or lacked a sufficient number of caapositive patients , and none of the studies had a followup of > 6 months.6 our study is the first longitudinal cimt study in kd and demonstrates an apparently gradual increase in cimt in kd patients with larger caa . the cimt of caanegative patients showed an increased cimt following complete convalescence of the acute disease , which normalized over time . patients with mediumsized caa showed a trend toward increased initial cimt with cimt progression comparable to that of controls . patients with giant caa showed a trend toward increased cimt progression over time . in evaluating this model , patients with giant caa and , to a much lesser degree , patients with mediumsized caa showed a trend toward continuously increasing cimt compared with controls . eikendal et al found a hazard ratio of 1.4 per standard deviation increase in cimt for myocardial infarction or stroke in adults aged < 45 years.10 increased cimt is seen in children and adolescents with known cardiovascular risk factors such as familial hypercholesterolemia or obesity.23 , 24 , 25 increased cimt progression was found to be significantly related to the incidence of stroke by polak et al.26 in contrast , a metaanalysis of individual patient data from longitudinal studies in 2012 showed no significant association between cimt progression and cardiovascular events in mainly middleaged to older adults.27 this could be explained by the different methods of measuring cimt at the different institutes . another systematic review of randomized controlled trials measuring cimt change over time found a statistically significant association between mean change in cimt over time and the likelihood of developing nonfatal myocardial infarction ( p=0.018 ) and the combined end point of myocardial infarction and death ( p=0.021).28 in younger participants , the bogalusa heart study found a significant association between some cimt segment progression and multiple cardiovascular risk factors such as waist circumference , waist / height ratio , mean arterial pressure , cholesterol , and smoking.29 the young finns study showed that young adult cimt progression was associated with risk factors in childhood such as bmi , physical activity , and fruit consumption.30 although the exact risk prediction of ( increased ) cimt in children and young adults is still unknown , cimt is clearly correlated with cardiovascular risk factors in a younger population . in our case , the relatively large number of patients with caa , and especially with giant caa , helped identify that these patients had the most abnormal response at the peripheral noncardiac vasculature . finally , our controls and a proportion of our patients did not undergo > 1 cimt measurement ; however , by creating a multilevel , repeatedmeasures , linear mixedeffects model , we could use all cimt measurements to create a large study group . we observed a trend toward significance in both intercept and progression in different subgroups ; however , because significance is dependent on the sample size , and because in both groups the mean was indeed significantly increased , it is likely that a significant finding will be present in larger groups . in our case , the relatively large number of patients with caa , and especially with giant caa , helped identify that these patients had the most abnormal response at the peripheral noncardiac vasculature . finally , our controls and a proportion of our patients did not undergo > 1 cimt measurement ; however , by creating a multilevel , repeatedmeasures , linear mixedeffects model , we could use all cimt measurements to create a large study group . we observed a trend toward significance in both intercept and progression in different subgroups ; however , because significance is dependent on the sample size , and because in both groups the mean was indeed significantly increased , it is likely that a significant finding will be present in larger groups . although the cimt of caanegative children is initially increased , the values normalize at a later age , suggesting vascular repair of a generalized vasculopathy distinctive from atherosclerosis . patients with a history of kd complicated by giant and , to a lesser degree , mediumsized caa have a trend toward continued increased cimt , suggesting more severe impact on the arterial wall . until more data become available , these patients need cardiovascular counseling and followup beyond the heart .

cone - beam computed tomography ( cbct ) has rapidly become a huge success in the dental radiographic world . the first cbct was installed in sweden in 2002 , and in 2007 the first device was installed in norway . a multinational research project , sedentexct , supported by the seventh framework programme of the european atomic energy community ( euratom ) published guidelines for the use of cbct in 2012 . in europe , not all countries have incorporated these guidelines into their national regulations . as a result , in countries like sweden , where the eu guidelines have not been incorporated yet , the general radiation protection regulations and regulations regarding for specialist radiographic equipment and medical ct should be applied in to the use of cbct . according to the regulations in sweden and in norway , all cbct units have to be registered and supervised by a medical physicist responsible for performing quality assurance ( qa ) , including dose measurements . a medical or a dento - maxillofacial radiologist has to be responsible for the clinical use of the cbct unit , including interpretation of the results from the examinations . in norway , the radiologist may delegate cbct image interpretation to another competent dentist when imaging the dento - alveolar region with scan volumes of 8 x 8 cm or smaller . the norwegian radiation protection authority ( nrpa ) published guidelines for the use of cbct in dental practices in 2010 , and excluding the mandatory demand of a responsible radiologist and physicist , the guidelines are adjusted to , though not identical to the basic principles in the eu guidelines described by the sedentexct project . both countries have high gross domestic products and governmental spending per capital , and low population densities . the population of sweden is approximately twice that of norway ( 9.6 and 5.1 million ) . according to swedish statistics from 2012 and norway in 2014 , the number of inhabitants per active dentist were almost identical ( 1.235 in sweden and 1.153 in norway ) , as were the number inhabitants per active specialist ( 10.842 in sweden 2007 and 11.161 in norway 2008 ) . in both countries there were slightly more female dentists ( 54 and 52% respectively ) . however , one major difference was the number of general practice dentists in private care , 46% in sweden whilst 69% in norway . given all these statistics the amount of registered cbct units in each country ( 75 in sweden in december 2013 and 39 in norway december 2012 ) were comparable in relation to both population and numbers of dentists / specialists . in december 2012 , a questionnaire was sent with a wide range of questions , to all cbct clinics registered nationally with the nrpa in norway . the study focused on clinically related questions , including the actual workflow with the cbct . due to the relatively early use of cbct in sweden without explicit regulations , it might be reasonable to expect a difference in the use of cbct compared to norway . in addition , there should also be many similarities according to the analogy of the two countries , for example both countries have an acknowledged speciality in dental and maxillofacial radiology , and to engage a medical physicist is mandatory for the use of cbct in both countries . previous studies in turkey have concluded that there is a difference in knowledge about cbct technique among the dental students , and that digital techniques and specific knowledge about cbct and its usefulness in the clinic should be highlighted . today most dentists and dental staff in norway and sweden are familiar with the use of service agreements , support and back - up routines using intraoral digital radiography . the use of technical parameters to improve image quality and reduce image dose , such as tube current ( ma ) and tube voltage ( kv ) , support devices for patient positioning , field of view ( fov ) and scout images , however , is a new challenge for the dental staff with regard to cbct examinations . to our knowledge , there is no literature regarding dental staff and their use of the more advanced technical parameters used with cbct . thus , the aim of this study was to compare the outcome of an investigation in the use of cone - beam computed tomography in sweden with previous responses made in norway , with regard specifically to technical issues , not previously reported . in november 2013 , 76 questionnaire forms were sent to all dental clinics with cbct equipment , registered by the swedish radiation safety authority ( srsa , www.ssm.se ) in september 2013 , and another six to additional cbct clinics known by the authors , but not registered by srsa by the time the copy of the registry was achieved . one of the clinics that was added by authors overlapped with one registered by srsa but had been renamed and was thus excluded . furthermore , five respondents reported that their cbct units were discontinued or scrapped , and another clinic was yet to install their cbct machine . these respondents were also excluded , which meant that the response rate was calculated based on the remaining 75 questionnaires . the swedish questionnaire was sent to the contact person registered by srsa or to the clinic . a cover letter accompanying the swedish questionnaire informed the respondents that their answers would be treated anonymously . a coded and stamped envelope was supplied for the return of the questionnaire , and two reminders were sent out . the questionnaire contained a total of 45 questions , including 35 identical or comparable questions to the norwegian questionnaire , which had been sent to the norwegian clinics one year earlier . the present study was based on inter - comparison of the results of the swedish questionnaire and the acquired data from the norwegian questionnaire , focusing on questions related to technical issues of the cbct . in the previous study , the questionnaire comprised questions regarding the characteristics of the respondents , such as gender and age , as well as the formal competence of the staff in the clinic , the radiographic equipment and its use . the questionnaire also included a question about perceived radiation dose in cbct , as well as about the most common clinical indications for using cbct , qa program , image processing , image quality , installation , radiation protection and technical support . data in both the swedish and the norwegian questionnaires were imported into microsoft excel 2013 ( microsoft corporation , redmond , wa , usa ) . pivot tables were used to sort , count and relate the parameters of the different questions . some parameters were also checked for correlation by statistical hypothesis testing . the hypothesis that one numerical parameter was a function of another was evaluated by calculating the coefficient of determination , r which indicated how well data was consistent with a statistical model . correlations between numerical and non - numerical parameters were tested with one - way analysis of variance ( anova ) which was automated in microsoft excel 2013 and provided a measure of significance . the respondents were asked to rank the commonness of each indication with a commonness rank , r , ranging from 1 to 5 . in the swedish questionnaire a commonness rank of 1 demonstrated that the indication was the most common , whereas 5 showed the most common rank in the norwegian study . for statistical reasons the swedish commonness factors were reversed so that they corresponded to the norwegian and indications , which were not ranked by a respondent , were treated as r = 0 . to assess the frequency of the indications , rather than just the most common indication , the mean value of the commonness rank xwas calculated for each indication according to the following formula of weighted arithmetic mean value , where xr was the number of respondents who had stated rank r for a certain indication , and n the total number of respondents : x=r=05xrrn responses from the swedish questionnaire were 53 out of 75 ( 71% ) , and were received from 50 clinics , including hospital and university clinics . in the norwegian study , 29 out of 39 ( 74% ) responded the questionnaire . due to the fact that two swedish clinics owned more than one cbct unit , these questionnaires represented the same clinic but different cbct units , and thus were answered identically except for the technical details of the equipment the respondents were almost exclusively male in norway ( 93% ) , whereas only 65% of the swedish respondents were male . the age distribution of the respondents differed between the countries ( table 1 ) . in sweden , 50% of the respondents were 55 years or older , compared to 28% in norway . in norway , 83% of the clinics had at least one dental specialist , other than oral and maxillofacial radiologist , in addition to one radiologist required by regulations , compared to only 54% of the swedish clinics . on the other hand , 9% of the swedish clinics had more than one radiologist , whereas none of the norwegian respondents reported more than one radiologist in their clinics . the respondents age distribution in percent the cbct units in sweden came from ten different manufacturers , in norway seven , all of which were represented in sweden . in sweden , j. morita corporation ( fushimi - ku , kyoto , japan ) was the dominating manufacturer ( 40% ) , whilst in norway , sirona unit ( sirona dental systems , inc . long island city , ny , usa ) ( 31% ) was most commonly represented . the cbct units were installed during the last two decades , gradually increasing with a clear peak in purchase at the beginning of this decade ( figure 1 ) . about half of the clinics needed to rebuild before installation of cbct ( 45% norway , 54% sweden ) and the most common issue was to expand the radiation protection . distribution of purchase year for cone - beam computed tomography ( cbct ) in sweden and norway . most clinics had service agreements for their equipment ( sweden 88% , norway 97% ) . the service agreements included upgrades of hardware ( sweden 63% , norway 55% ) , firmware ( sweden 65% , norway 67% ) and software ( sweden 88% , norway 96% ) , as well as technical support ( sweden 93% , norway 96% ) . some ( 7 - 29% ) respondents in both countries did not know if all of these options were included in the service agreement . almost every respondent claimed to be satisfied with the support and none claimed to be dissatisfied . in both countries , more than 96% of the respondents reported to performing regular back - ups . cone - beam computed tomography scanning in 60% of the swedish clinics , the same person performed all cbct examinations compared to 86 % in norway . in sweden , most examinations were performed by dental nurses in contrast to norway , where they were performed by specialists . in both countries , in both countries patients in the standing position was most common ( table 2 ) . at least three different support devises were used by 58% of the swedish respondents compared to 83% of the norwegian respondents . frontal head and chin support were common , whereas neck support was not commonly used in any of the countries ( table 3 ) . patient positioning in the cone - beam computed tomography unit frequency of respondents using patient support in both countries , more than two - thirds of the cbct units had a scout image function , which was regularly used ( 79% in sweden and 75% in norway ) . among examination parameters , field of view ( fov ) was by far the most common parameter to modify . other common parameters were size of voxel , tube current and tube voltage ( table 4 ) . frequency of respondents altering the technical parameters the most common fov , defined as the product of width and height , was plotted against the smallest fov ( figure 2 ) . the hypothesis that the smallest fov was most commonly used would be modelled by a linear graph with slope one in the plot . the hypothesis was tested by calculating the coefficients of determination ( r ) of the model to the data . the same calculation was made for the most common fov plotted against the largest fov ( figure 3 ) with the hypothesis that the largest fov was the most commonly used . the most commonly used field of view ( fov ) as a function of the smallest selectable fov measured in cm as the product of height and diameter of the radiation field in isocenter . the most commonly used field of view ( fov ) as a function of the largest selectable fov measured in cm as the product of height and diameter of the radiation field in isocenter . the tests supported the hypothesis that the smallest fov was most commonly used ( r = 0.75 in sweden , r = 0.7 in norway ) rather than the largest fov ( r = 0.41 in sweden , r = 0.32 in norway ) . indications and use of images in both countries , the most common indication for performing cbct examinations was implant treatment planning ( 76% sweden , 34% norway ) . following implant treatment planning the most common indications were impacted teeth , jaw pathology and pain - related problems in both countries . other indications were only common in few clinics , although 14% of the swedish clinics ( 3% in norway ) stated that other indications , such as sinus or root anatomy , were the most common . correlations between most common indication and most commonly used fov were tested with anova , but no significant correlation was found . a majority of the swedish respondents also reviewed the images , 96% , while 78% of the norwegian respondents evaluated the images . four percent of the swedish respondents and 41% of the norwegian respondents did not wait for the report before initiating treatment . demonstrating the images to the patients was very common in both countries ( 93% in sweden and 85% in norway ) . cone - beam computed tomography dose level the conception of cbct dose level , compared to intraoral imaging was similar between the countries , with a tendency towards larger number of periapical images ( table 5 ) . correlations between conception of cbct dose levels and other parameters were tested with anova , but no significant correlation was found . the parameters tested were age , sex and education of the respondent , education of the operator , cbct manufacturer and trade name , variable exposure parameters and year of cbct purchase as well as most common indications , frequency of examinations and most common fov . the frequency of the respondents who estimated the smallest field of view , 4 x 4 cm , cone - beam computed tomography radiation dose , correlated to the numbers of periapical images the first and principal purpose of the current study was to compare the use of cbct devices between the two countries , sweden and norway . furthermore the use of the devices within each country was registered , as well as the technical parameters related to the use of the cbct . the similarities in cbct use were obvious in terms of how the respondents worked according to national regulations that exist , even though in sweden one must interpret the rules of cbct in the context of medical x - ray machines . however , the differences when the regulations were applied in practice might be of interest to notice . these differences could be explained by various interpretations of the regulations due to lack of information and/or knowledge , no clear definition of responsibilities to everyone in the team working with cbct , and/or no qa program defined , including continuous follow - up courses from both technical and diagnostic aspects . it was not mandatory and there were no rules that prescribed attending radiological courses in any of the countries , although there is the obvious need for knowledge concerning continuous optimization of image quality and to maintain high diagnostic accuracy . , the questionnaire forms were sent to all 76 clinics , registered by the srsa and to six additional clinics not registered at the time of sending the survey to respondents . the swedish questionnaire was sent out a year later than the norwegian questionnaire in a time of rapid increase in the use of cbct . due to the fact that there are certain well known difficulties connected with questionnaire surveys , and among them maybe the most important is to get answers from the respondents , a response rate of 70 - 80% is considered to be acceptable . our questionnaire was sent out as a printed copy , with two reminders , whilst in norway it was sent both in a digital and analogue form with four reminders , resulting in a response rate of 74% . surprisingly , there were some difficulties to get response with the questionnaire which was sent digitally . therefore , in sweden it was chosen to send the investigation only by postal mail . another recognized problem with questionnaire surveys is that the actual respondent is not known . in our study the questionnaire was sent to the person registered as owner of the cbct device , which allowed either the owner or the user , if not the same person , to answer the questions . the problem was the same in the norwegian study and thus the results were comparable . the answers received in the swedish survey represented all kind of clinics , as general dentists , specialists , hospitals and universities . the clinics in sweden that chose not to answer included all type of clinics and therefore none obvious bias can be detected . the difference in kind of responses from clinics between the countries , might have affected the results of the answers , especially concerning the workflow and questions about indication for cbct examinations . in the swedish survey , six answers were registered , where most common indications were examinations of sinuses and root investigations . these answers might indicate that the examinations were performed and interpreted by a specialist clinic in dento - maxillofacial radiology or endodontics . implant treatment planning , impacted teeth , jaw pathology and pain - related problems were the most common in both countries , as expected . another difference at the time of the norwegian survey was that only dento - maxillofacial radiologists , radiographers and specially trained dentists were allowed to perform the actual cbct scans and not dental nurses as in sweden . this might have influenced some of the answers of the surveys , since the education in radiology , both about radiological techniques and diagnostics , was on a higher level in norway , than for dental nurses in sweden . considering the indication for a cbct examination in relation to choosing exposure parameters and fov , one hypothesis could be that larger fov than necessary would be chosen , to ensure enough volume for interpretation , when knowledge about the radiological technique and image quality optimisation would be expected to be lower . the answers did not result in any statistically significant differences related to the level of education for the study participants between sweden and norway . a notable difference between the countries was the use of patient head support , where the use of at least three different head supports were commonly used in norway during cbct exposure , in contrast to sweden , where only two head supports were generally used . this difference was substantial even when the same models of cbct were compared . in a previous study simulation showed that only chin and forehead support were inadequate to prevent risk of head rotations , which can cause clearly visible loss in image quality and the importance of using multiple supports have been advised by the european academy of dental and maxillofacial radiology ( eadmr ) . the results in this study could be related to different brand of cbct machine with enclosed alternative head supports . it is therefore , important to be aware when invest in a new cbct , the head supports included should be clearly documented . another difference was how the respondents handled the report from the specialist in dento - maxillofacial radiology , after the cbct examination in relation to start of treatment . in sweden around ten times more respondents waited for the specialist report before starting treatment than in norway . this might be explained by the fact that in norway most of the responsible users were specialists in dentistry , other than in dento - maxillofacial radiology . far more were general dentists using cbct in sweden and thus maybe less experienced in analysing advanced radiological examinations . to our knowledge it remains to be determined , if there would be any changes in treatment , if the dentist had read the report in advance of start of treatment . in both countries fov an important fact , worth considering , was that the size of the smallest fov depended on the cbct model . the ratio between the cbct with largest and smallest areas for the smallest selectable fov was found 16 in sweden and 11 in norway . this indicated that the choice of equipment could have had an obvious impact on the radiation dose , which might not be compensated by optimisation of the scanning parameters . in our study it was shown that a scout - image was available on most cbct devices in order to be able to focus the correct position of volume of interest before exposing the patient . this function was not used in 20 - 25% of the examinations , regardless of which country and size of fov . the consequence of not using this function , amongst others , is that it will increase the risk of re - exposure , due to failure of positioning the volume correctly , and it might be especially difficult when positioning small volumes . this remarkable negligence of using the scout - image function could thus lead to the risk of a higher dose to the patient , a possible necessity of retaking the volume , or maybe less image quality if accepting sub - optimal images . the scout - image function should be regarded as part of a qa program in every clinic where this function is available on the cbct device . the two scout - images are acquired using a very low x - ray tube current and short exposure times and will thus contribute only marginally to the total dose to the patient from the whole cbct examination . the conception of cbct dose level , compared to intraoral imaging in both sweden and norway , ranged from less than five intraoral exposures to more than 40 . this more than eight - fold range had no significant correlation to the cbct equipment , fov , indication or any other reasonable parameter . considering that several clinics in both countries used the same kind of equipment and fov , plus the fact that the same indications were the most common , this showed a great need for further education , such as more knowledge on cbct doses , and elaboration of some standard qa methods . the qa methods could be used for cbct owners and users , other than those expected from the responsible specialist in radiology or the medical physicist . previous studies have investigated the dose to the patient in cbct devices in relation to image quality [ 17 - 19 ] . it is clear that the dose to patients differed a lot and it was difficult to express an adequate dose from the cbct examination due to the many different parameters that could and are used . hence , the risk that the dose might be raised , without concomitant benefit for the patient when interpreting the examination , is obvious . these facts , about different possibilities in the use of cbct , emphasise the importance of knowledge in qa optimisation , in order to work according to the alara principle . in this context the relevant question also arises if it is suitable to express dose as effective dose , if the irradiated volume is very small [ 20 - 24 ] . this is emphasized by the facts that the tissue types included in the icrp 103 weight factor table and which are irradiated in a cbct examination ( red bone marrow , parotid gland , oral mucosa , thyroid gland , skin , bone surfaces , brain , extrathoracic tissue and lymph nodes ) either have a low weight factor or are irradiated only to a small fraction ( or both ) . the effective dose will therefore be orders of magnitude smaller than the local absorbed dose in the irradiated volume . furthermore , due to the steep dose gradients around a cbct volume and variations in human anatomy and placement of the cbct field of view between individuals , the effective dose will exhibit very large variations between individuals . any value of effective dose compiled from phantom measurements should therefore be used with great caution , if at all . a thought that strikes when you add up all the results is that there is a strong need for continuous upgrade of knowledge to everyone in the team working with cbct , as well as to clarify and define the different roles and responsibilities within the team involved using cbct devices . this study compared the use of cone - beam computed tomography for dento - maxillofacial purposes in sweden and norway . the bilateral comparison showed an overall similarity between the two countries , probably due to the fact that the national regulations concerning radiation safety and protection corresponded very well . the two countries also have the same kind of specialist training in dento - maxillofacial radiology . the knowledge of how cone - beam computed tomography was used in dental practices is very important to survey , since radiation dose to the patient could vary a lot for the same kind of radiographic examination . thus , it is essential to establish quality assurance protocols with defined responsibilities in the team in order to maintain high diagnostic accuracy for all examinations when using cone - beam computed tomography for patient examinations .

abstractobjectivescone - beam computed tomography in dentistry can be used in some countries by other dentists than specialists in radiology . the frequency of buying cone - beam computed tomography to examine patients is rapidly growing , thus knowledge of how to use it is very important . the aim was to compare the outcome of an investigation on the use of cone - beam computed tomography in sweden with a previous norwegian study , regarding specifically technical aspects.material and methodsthe questionnaire contained 45 questions , including 35 comparable questions to norwegian clinics one year previous . results were based on inter - comparison of the outcome from each of the two questionnaire studies.resultsresponses rate was 71% in sweden . there , most of cone - beam computed tomography ( cbct ) examinations performed by dental nurses , while in norway by specialists . more than two - thirds of the cbct units had a scout image function , regularly used in both sweden ( 79% ) and norway ( 75% ) . in sweden 4% and in norway 41% of the respondents did not wait for the report from the radiographic specialist before initiating treatment.conclusionsthe bilateral comparison showed an overall similarity between the two countries . the survey gave explicit and important knowledge of the need for education and training of the whole team , since radiation dose to the patient could vary a lot for the same kind of radiographic examination . it is essential to establish quality assurance protocols with defined responsibilities in the team in order to maintain high diagnostic accuracy for all examinations when using cone - beam computed tomography for patient examinations .

INTRODUCTION MATERIAL AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS

cone - beam computed tomography ( cbct ) has rapidly become a huge success in the dental radiographic world . the first cbct was installed in sweden in 2002 , and in 2007 the first device was installed in norway . a multinational research project , sedentexct , supported by the seventh framework programme of the european atomic energy community ( euratom ) published guidelines for the use of cbct in 2012 . as a result , in countries like sweden , where the eu guidelines have not been incorporated yet , the general radiation protection regulations and regulations regarding for specialist radiographic equipment and medical ct should be applied in to the use of cbct . according to the regulations in sweden and in norway , all cbct units have to be registered and supervised by a medical physicist responsible for performing quality assurance ( qa ) , including dose measurements . a medical or a dento - maxillofacial radiologist has to be responsible for the clinical use of the cbct unit , including interpretation of the results from the examinations . in norway , the radiologist may delegate cbct image interpretation to another competent dentist when imaging the dento - alveolar region with scan volumes of 8 x 8 cm or smaller . the norwegian radiation protection authority ( nrpa ) published guidelines for the use of cbct in dental practices in 2010 , and excluding the mandatory demand of a responsible radiologist and physicist , the guidelines are adjusted to , though not identical to the basic principles in the eu guidelines described by the sedentexct project . according to swedish statistics from 2012 and norway in 2014 , the number of inhabitants per active dentist were almost identical ( 1.235 in sweden and 1.153 in norway ) , as were the number inhabitants per active specialist ( 10.842 in sweden 2007 and 11.161 in norway 2008 ) . however , one major difference was the number of general practice dentists in private care , 46% in sweden whilst 69% in norway . given all these statistics the amount of registered cbct units in each country ( 75 in sweden in december 2013 and 39 in norway december 2012 ) were comparable in relation to both population and numbers of dentists / specialists . in december 2012 , a questionnaire was sent with a wide range of questions , to all cbct clinics registered nationally with the nrpa in norway . the study focused on clinically related questions , including the actual workflow with the cbct . due to the relatively early use of cbct in sweden without explicit regulations , it might be reasonable to expect a difference in the use of cbct compared to norway . in addition , there should also be many similarities according to the analogy of the two countries , for example both countries have an acknowledged speciality in dental and maxillofacial radiology , and to engage a medical physicist is mandatory for the use of cbct in both countries . today most dentists and dental staff in norway and sweden are familiar with the use of service agreements , support and back - up routines using intraoral digital radiography . the use of technical parameters to improve image quality and reduce image dose , such as tube current ( ma ) and tube voltage ( kv ) , support devices for patient positioning , field of view ( fov ) and scout images , however , is a new challenge for the dental staff with regard to cbct examinations . to our knowledge , there is no literature regarding dental staff and their use of the more advanced technical parameters used with cbct . thus , the aim of this study was to compare the outcome of an investigation in the use of cone - beam computed tomography in sweden with previous responses made in norway , with regard specifically to technical issues , not previously reported . in november 2013 , 76 questionnaire forms were sent to all dental clinics with cbct equipment , registered by the swedish radiation safety authority ( srsa , www.ssm.se ) in september 2013 , and another six to additional cbct clinics known by the authors , but not registered by srsa by the time the copy of the registry was achieved . furthermore , five respondents reported that their cbct units were discontinued or scrapped , and another clinic was yet to install their cbct machine . these respondents were also excluded , which meant that the response rate was calculated based on the remaining 75 questionnaires . the swedish questionnaire was sent to the contact person registered by srsa or to the clinic . a cover letter accompanying the swedish questionnaire informed the respondents that their answers would be treated anonymously . a coded and stamped envelope was supplied for the return of the questionnaire , and two reminders were sent out . the questionnaire contained a total of 45 questions , including 35 identical or comparable questions to the norwegian questionnaire , which had been sent to the norwegian clinics one year earlier . the present study was based on inter - comparison of the results of the swedish questionnaire and the acquired data from the norwegian questionnaire , focusing on questions related to technical issues of the cbct . in the previous study , the questionnaire comprised questions regarding the characteristics of the respondents , such as gender and age , as well as the formal competence of the staff in the clinic , the radiographic equipment and its use . the questionnaire also included a question about perceived radiation dose in cbct , as well as about the most common clinical indications for using cbct , qa program , image processing , image quality , installation , radiation protection and technical support . pivot tables were used to sort , count and relate the parameters of the different questions . the hypothesis that one numerical parameter was a function of another was evaluated by calculating the coefficient of determination , r which indicated how well data was consistent with a statistical model . the respondents were asked to rank the commonness of each indication with a commonness rank , r , ranging from 1 to 5 . in the swedish questionnaire a commonness rank of 1 demonstrated that the indication was the most common , whereas 5 showed the most common rank in the norwegian study . to assess the frequency of the indications , rather than just the most common indication , the mean value of the commonness rank xwas calculated for each indication according to the following formula of weighted arithmetic mean value , where xr was the number of respondents who had stated rank r for a certain indication , and n the total number of respondents : x=r=05xrrn responses from the swedish questionnaire were 53 out of 75 ( 71% ) , and were received from 50 clinics , including hospital and university clinics . in the norwegian study , 29 out of 39 ( 74% ) responded the questionnaire . due to the fact that two swedish clinics owned more than one cbct unit , these questionnaires represented the same clinic but different cbct units , and thus were answered identically except for the technical details of the equipment the respondents were almost exclusively male in norway ( 93% ) , whereas only 65% of the swedish respondents were male . the age distribution of the respondents differed between the countries ( table 1 ) . in sweden , 50% of the respondents were 55 years or older , compared to 28% in norway . in norway , 83% of the clinics had at least one dental specialist , other than oral and maxillofacial radiologist , in addition to one radiologist required by regulations , compared to only 54% of the swedish clinics . on the other hand , 9% of the swedish clinics had more than one radiologist , whereas none of the norwegian respondents reported more than one radiologist in their clinics . the respondents age distribution in percent the cbct units in sweden came from ten different manufacturers , in norway seven , all of which were represented in sweden . in sweden , j. morita corporation ( fushimi - ku , kyoto , japan ) was the dominating manufacturer ( 40% ) , whilst in norway , sirona unit ( sirona dental systems , inc . the cbct units were installed during the last two decades , gradually increasing with a clear peak in purchase at the beginning of this decade ( figure 1 ) . about half of the clinics needed to rebuild before installation of cbct ( 45% norway , 54% sweden ) and the most common issue was to expand the radiation protection . distribution of purchase year for cone - beam computed tomography ( cbct ) in sweden and norway . some ( 7 - 29% ) respondents in both countries did not know if all of these options were included in the service agreement . in both countries , more than 96% of the respondents reported to performing regular back - ups . cone - beam computed tomography scanning in 60% of the swedish clinics , the same person performed all cbct examinations compared to 86 % in norway . in sweden , most examinations were performed by dental nurses in contrast to norway , where they were performed by specialists . in both countries , in both countries patients in the standing position was most common ( table 2 ) . frontal head and chin support were common , whereas neck support was not commonly used in any of the countries ( table 3 ) . patient positioning in the cone - beam computed tomography unit frequency of respondents using patient support in both countries , more than two - thirds of the cbct units had a scout image function , which was regularly used ( 79% in sweden and 75% in norway ) . frequency of respondents altering the technical parameters the most common fov , defined as the product of width and height , was plotted against the smallest fov ( figure 2 ) . the hypothesis was tested by calculating the coefficients of determination ( r ) of the model to the data . the same calculation was made for the most common fov plotted against the largest fov ( figure 3 ) with the hypothesis that the largest fov was the most commonly used . the most commonly used field of view ( fov ) as a function of the largest selectable fov measured in cm as the product of height and diameter of the radiation field in isocenter . the tests supported the hypothesis that the smallest fov was most commonly used ( r = 0.75 in sweden , r = 0.7 in norway ) rather than the largest fov ( r = 0.41 in sweden , r = 0.32 in norway ) . indications and use of images in both countries , the most common indication for performing cbct examinations was implant treatment planning ( 76% sweden , 34% norway ) . other indications were only common in few clinics , although 14% of the swedish clinics ( 3% in norway ) stated that other indications , such as sinus or root anatomy , were the most common . a majority of the swedish respondents also reviewed the images , 96% , while 78% of the norwegian respondents evaluated the images . four percent of the swedish respondents and 41% of the norwegian respondents did not wait for the report before initiating treatment . demonstrating the images to the patients was very common in both countries ( 93% in sweden and 85% in norway ) . cone - beam computed tomography dose level the conception of cbct dose level , compared to intraoral imaging was similar between the countries , with a tendency towards larger number of periapical images ( table 5 ) . the parameters tested were age , sex and education of the respondent , education of the operator , cbct manufacturer and trade name , variable exposure parameters and year of cbct purchase as well as most common indications , frequency of examinations and most common fov . the frequency of the respondents who estimated the smallest field of view , 4 x 4 cm , cone - beam computed tomography radiation dose , correlated to the numbers of periapical images the first and principal purpose of the current study was to compare the use of cbct devices between the two countries , sweden and norway . furthermore the use of the devices within each country was registered , as well as the technical parameters related to the use of the cbct . the similarities in cbct use were obvious in terms of how the respondents worked according to national regulations that exist , even though in sweden one must interpret the rules of cbct in the context of medical x - ray machines . these differences could be explained by various interpretations of the regulations due to lack of information and/or knowledge , no clear definition of responsibilities to everyone in the team working with cbct , and/or no qa program defined , including continuous follow - up courses from both technical and diagnostic aspects . it was not mandatory and there were no rules that prescribed attending radiological courses in any of the countries , although there is the obvious need for knowledge concerning continuous optimization of image quality and to maintain high diagnostic accuracy . , the questionnaire forms were sent to all 76 clinics , registered by the srsa and to six additional clinics not registered at the time of sending the survey to respondents . the swedish questionnaire was sent out a year later than the norwegian questionnaire in a time of rapid increase in the use of cbct . due to the fact that there are certain well known difficulties connected with questionnaire surveys , and among them maybe the most important is to get answers from the respondents , a response rate of 70 - 80% is considered to be acceptable . in our study the questionnaire was sent to the person registered as owner of the cbct device , which allowed either the owner or the user , if not the same person , to answer the questions . the problem was the same in the norwegian study and thus the results were comparable . the answers received in the swedish survey represented all kind of clinics , as general dentists , specialists , hospitals and universities . the clinics in sweden that chose not to answer included all type of clinics and therefore none obvious bias can be detected . the difference in kind of responses from clinics between the countries , might have affected the results of the answers , especially concerning the workflow and questions about indication for cbct examinations . another difference at the time of the norwegian survey was that only dento - maxillofacial radiologists , radiographers and specially trained dentists were allowed to perform the actual cbct scans and not dental nurses as in sweden . this might have influenced some of the answers of the surveys , since the education in radiology , both about radiological techniques and diagnostics , was on a higher level in norway , than for dental nurses in sweden . the answers did not result in any statistically significant differences related to the level of education for the study participants between sweden and norway . a notable difference between the countries was the use of patient head support , where the use of at least three different head supports were commonly used in norway during cbct exposure , in contrast to sweden , where only two head supports were generally used . this difference was substantial even when the same models of cbct were compared . it is therefore , important to be aware when invest in a new cbct , the head supports included should be clearly documented . another difference was how the respondents handled the report from the specialist in dento - maxillofacial radiology , after the cbct examination in relation to start of treatment . in sweden around ten times more respondents waited for the specialist report before starting treatment than in norway . this might be explained by the fact that in norway most of the responsible users were specialists in dentistry , other than in dento - maxillofacial radiology . in both countries fov an important fact , worth considering , was that the size of the smallest fov depended on the cbct model . the ratio between the cbct with largest and smallest areas for the smallest selectable fov was found 16 in sweden and 11 in norway . this indicated that the choice of equipment could have had an obvious impact on the radiation dose , which might not be compensated by optimisation of the scanning parameters . in our study it was shown that a scout - image was available on most cbct devices in order to be able to focus the correct position of volume of interest before exposing the patient . this function was not used in 20 - 25% of the examinations , regardless of which country and size of fov . this remarkable negligence of using the scout - image function could thus lead to the risk of a higher dose to the patient , a possible necessity of retaking the volume , or maybe less image quality if accepting sub - optimal images . the scout - image function should be regarded as part of a qa program in every clinic where this function is available on the cbct device . the two scout - images are acquired using a very low x - ray tube current and short exposure times and will thus contribute only marginally to the total dose to the patient from the whole cbct examination . the conception of cbct dose level , compared to intraoral imaging in both sweden and norway , ranged from less than five intraoral exposures to more than 40 . this more than eight - fold range had no significant correlation to the cbct equipment , fov , indication or any other reasonable parameter . considering that several clinics in both countries used the same kind of equipment and fov , plus the fact that the same indications were the most common , this showed a great need for further education , such as more knowledge on cbct doses , and elaboration of some standard qa methods . the qa methods could be used for cbct owners and users , other than those expected from the responsible specialist in radiology or the medical physicist . previous studies have investigated the dose to the patient in cbct devices in relation to image quality [ 17 - 19 ] . it is clear that the dose to patients differed a lot and it was difficult to express an adequate dose from the cbct examination due to the many different parameters that could and are used . hence , the risk that the dose might be raised , without concomitant benefit for the patient when interpreting the examination , is obvious . these facts , about different possibilities in the use of cbct , emphasise the importance of knowledge in qa optimisation , in order to work according to the alara principle . in this context the relevant question also arises if it is suitable to express dose as effective dose , if the irradiated volume is very small [ 20 - 24 ] . the effective dose will therefore be orders of magnitude smaller than the local absorbed dose in the irradiated volume . furthermore , due to the steep dose gradients around a cbct volume and variations in human anatomy and placement of the cbct field of view between individuals , the effective dose will exhibit very large variations between individuals . a thought that strikes when you add up all the results is that there is a strong need for continuous upgrade of knowledge to everyone in the team working with cbct , as well as to clarify and define the different roles and responsibilities within the team involved using cbct devices . this study compared the use of cone - beam computed tomography for dento - maxillofacial purposes in sweden and norway . the bilateral comparison showed an overall similarity between the two countries , probably due to the fact that the national regulations concerning radiation safety and protection corresponded very well . the two countries also have the same kind of specialist training in dento - maxillofacial radiology . the knowledge of how cone - beam computed tomography was used in dental practices is very important to survey , since radiation dose to the patient could vary a lot for the same kind of radiographic examination . thus , it is essential to establish quality assurance protocols with defined responsibilities in the team in order to maintain high diagnostic accuracy for all examinations when using cone - beam computed tomography for patient examinations .