Successful socialization requires the ability of understanding of others’ mental states. This ability called as mentalization (Theory of Mind) may become deficient and contribute to everyday life difficulties in multiple sclerosis. We aimed to explore the impact of brain pathology on mentalization performance in multiple sclerosis. Mentalization performance of 49 patients with multiple sclerosis was compared to 24 age- and gender matched healthy controls. T1- and T2-weighted three-dimensional brain MRI images were acquired at 3Tesla from patients with multiple sclerosis and 18 gender- and age matched healthy controls. We assessed overall brain cortical thickness in patients with multiple sclerosis and the scanned healthy controls, and measured the total and regional T1 and T2 white matter lesion volumes in patients with multiple sclerosis. Performances in tests of recognition of mental states and emotions from facial expressions and eye gazes correlated with both total T1-lesion load and regional T1-lesion load of association fiber tracts interconnecting cortical regions related to visual and emotion processing . Both of these tests showed correlations with specific cortical areas involved in emotion recognition from facial expressions , processing of emotions  and socially relevant information . Thus, both disconnection mechanism due to white matter lesions and cortical thinning of specific brain areas may result in cognitive deficit in multiple sclerosis affecting emotion and mental state processing from facial expressions and contributing to everyday and social life difficulties of these patients. Cognitive decline is a common clinical manifestation of multiple sclerosis (MS) with an estimated prevalence between 40–65% Deficits of general intellectual abilities significantly interfere with quality of life by impacting everyday life activities and decision-making Such functional limitations may also relate to deficits of social cognition. Social cognition is a human ability of making inferences about mental states of other people. An important aspect of social cognition is the capacity to interpret and predict mental states of other people in terms of thoughts, intentions, desires, and beliefs known as theory of mind (ToM), also referred as mentalizing. ToM is enabled by decoding nonverbal cues, such as facial expression, eye gaze, body gestures; and complex abstract reasoning about verbal information The neural basis of ToM abilities has been widely investigated using advanced neuroimaging methods in healthy subjects and in clinical conditions showing social cognitive impairment, particularly in high-functioning autism and in schizophrenia Previously, two functional MRI studies have investigated the brain activation during recognition of emotional face expressions in MS Here, using quantitative MRI methods in patients with MS we investigated (i) the impact of WM lesion load, and cortical atrophy on ToM performance; (ii) we identified the topography of these pathologies to explore neuroanatomic substrates, which if damaged, impact mentalization ability.The Research Ethical Committee of the Clinical Centre of the University of Pecs approved the study, and written informed consent was obtained from all participants. All patients with MS included in the study had complete clinical and neurological exam, during which capacity and ability to give consent for participation in the study had been evaluated. All of the included patients had been established to be fully able to give informed consent, and there was no need to involve guardians due to compromised capacity to consent. Healthy control volunteers had been recruited from university students and staff of the Medical School of Pecs, University of Pecs, and during an initial short interview capacity to be fully able to give informed consent had been established. All healthy participants gave written informed consent.Forty-nine white patients with MS according to revised McDonald criteria Two cohorts of healthy controls were used. For neuropsychological testing, we recruited 24 gender- and age-matched healthy volunteers with no previous history of neurological dysfunction, and normal neurological examination Table 1.All tests were administered and have been validated in Hungarian.Computerized version of the Faces test Faces test: The Faces test consists of 20 photographs of the face of the same white actress expressing different mental states Eyes test: The Eyes test consists of 36 photographs depicting only the eye region of the face of different white individuals expressing various mental states Faux pas test Correct comprehension scored one point. A control task was used to assess basic story comprehension.Perspective taking and reasoning about mental and emotional states of others was evaluated with the Depression was assessed with the Beck Depression Inventory (DBI) a validated screening tool for depressive symptoms 2, matrix size = 240×256, bandwidth = 240 Hz/pixel, scanning time ∼5 minutes); (2) single-slab 3D fast spin-echo Fluid-attenuated inversion recovery (FLAIR) .MRI was performed on a 3T Magnetom TIM Trio whole-body MRI scanner  with a 12-channel phased array head coil. The following sequences were performed: (1) T1-weighted three-dimensional (3D) Magnetization Prepared Rapid Gradient Echo (MPRAGE) . The method relies on user-guided specification of the intensity threshold range, which is manually adjustable during lesion segmentation. The algorithm labels all pixels with signal intensity within the set threshold range in the lesion area identified manually by the user . For parcellation of the WM lesions the International Consortium for Brain Mapping (ICBM) DTI-81 Atlas 3/voxel) using a two-step process. First, brain-extracted MPRAGE images were registered to the MNI152 standard brain image  using the FMRIB Linear Image Registration Tool (FLIRT) Table S2). All volume measures were performed in patients’ native space.Cortical thickness measures: We measured the thickness of the neocortex throughout the whole brain on 3D MPRAGE images using the automated reconstruction algorithm of the software FreeSurfer, version 4.5 (http://surfer.nmr.mgh.harvard.edu) 2 were manually outlined (region of interest [ROI]), and automatically mapped back on the brain surface of each patient with MS and participant of the healthy control group recruited for MRI examination. The average thickness of ROIs was calculated for each participant.Statistical analyses were performed using SPSS software version 18.0 . Distribution of the standardized residuals of the dependent variables resulting from the applied regression models of interest were analyzed by the Shapiro-Wilk test. In cases of non-normal distribution, square root and logarithmic transformations of the dependent variable data were performed, and distributions of the standardized residuals were examined with the same statistical method. Dependent variables showing non-normal distribution were dichotomized by the median.Mentalization performance of patients with MS and healthy controls were compared using ANOVA for Eyes test, and binary logistic regression for Faces test and Faux pas test accounting for confounding factors .Relationships between mentalization performance of patients with MS and total T1- and T2-weighted WM lesion volumes , regional T1- and T2LVs , and cortical thickness of the ROIs accounting for confounding factors of gender, EDSS, anxiety, and depression were analyzed with ANCOVA. Correlations between Faux pas test and tT1- and tT2LV accounting for the confounding factors were analyzed by binary logistic regression. Multiple comparisons were corrected with Bonferroni method.Cortical thicknesses of the ROIs correlating with the Eyes test were compared between control group recruited for MRI examination and total MS cohort, and MS subgroups with and without social cognitive impairment using ANCOVA accounting for age. We defined impaired social cognitive performance among patients with MS as test performance 1 standard deviation below the mean of the MRI control group’s performance.To assess the contribution of regional WM lesion volumes and cortical thicknesses of ROIs to the variance of Eyes test performance, ANCOVA analyses were performed corrected for gender, EDSS, anxiety, and depression.All statistical testing were performed at Type I error = 0.05 for single tests or family-wise Type I error = 0.05 for multiple testing.2 = 0.276), but not in Faux pas test compared to the group of 24 healthy controls.Results of social cognitive testing and psychometric assessment are presented in We examined MRI correlations with all three mentalization tests  even though performance in Faux pas test was not deficient.2 = 0.315, p = 0.034, R2 = 0.230, respectively), but not with tT2LV. No relationship was found between Faux pas test performance and any of the two total WM lesion measures.Descriptive data of the total lesion load measures are presented in Table S2). After correction for confounding variables and using Bonferroni adjustment  the Faces test performance showed significant linear negative correlation with rT1LV of the genu of corpus callosum (GCC) , splenium of corpus callosum (SCC) , left anterior corona radiata (ACR) , right sagittal stratum including the inferior longitudinal fasciculus (ILF) and inferior fronto-occipital fasciculus (IFOF) , and left and right uncinate fasciculus (UF)  (Table S1). Faces test performance also showed correlation with rT2LV of the GCC  and left Fornix (cres)/Stria terminalis  (Table S2). The Eyes test performance correlated with rT1LV of the splenium of corpus callosum (SCC)  (Table S1), and did not show correlation with rT2LV of any fiber tract (Table S2). The Faux pas test performance did not correlate with rT1LV or rT2LV of any fiber tract (Table S2).We investigated the impact of regional T1-, and T2-lesions located within the inter-connecting fiber bundles on mentalization performance of patients with MS. Only tracts containing lesions in a considerable number of patients  were included in the statistical analysis: for T1-lesions 21, and for T2-lesions 37 out of 50 fiber bundles fulfilled these criteria . After correction for the confounding factors and using a Bonferroni adjustment (p<0.01), ROIs at the left temporal pole in the anterior inferior temporal gyrus (Brodmann area [BA] 20), left FFA of the fusiform gyrus (BA19), and right caudal middle frontal gyrus  remained significant  of the fusiform gyrus (BA19), and an area in the right entorhinal cortex (BA28). After correction for anxiety, depression, gender, and EDSS and using a Bonferroni adjustment (p<0.017), all ROIs invariably remained significant Table 3.2 = 0.082)  Table 2.Multivariate regression analysis of the contribution of rT1LVs and cortical ROIs showing significant correlation with the Faces test performance showed that the rT1LV of the left UF independently predicted the Faces performance (p = 0.031) and accounted for a 65.1% variance of the performance. Regional T1LV of the SCC (p = 0.046), cortical thickness of ROIs in left FFA (p = 0.024), and left temporal pole (p = 0.002) independently predicted the Eyes test performance, and accounted for 59.1% of the variance of the performance. The right premotor area did not remain significant (p = 0.088).The present MRI study investigated the impact of WM lesion burden and cortical thinning on mentalization ability of patients with MS. Exploration of brain areas, which structural damage associate with impaired mentalization performance in MS may point to neurophysiologic mechanisms involved in processing of information of social relevance. In contrast to autism Mentalization ability was evaluated by tests of emotion recognition from facial expressions (Faces test), mental state decoding from eye gazes (Eyes test), and reasoning about mental states from verbal stories (Faux pas test). After correction for confounding effects of anxiety, depression, and gender, patients with MS performed significantly poorer in the Faces test and Eyes test compared to healthy subjects. These results obtained with an MS cohort different from our previous study confirmed those findings Poorer emotion and mental state recognition performances from facial expressions and eye gazes showed relationship with both WM and cortical gray matter damages measured by MRI in patients with MS. The multivariate regression analysis has demonstrated that both regional T1LV and cortical thinning of certain focal brain areas may independently compromise the mentalization performance, and as such both a disconnection mechanism due to WM damage and the cortical gray matter damage may contribute to impaired cognitive performance in MS.After correction for confounding effects of gender, EDSS, anxiety, and depression, both poorer Faces test and Eyes test performance showed correlation with higher tT1LV. To examine whether these relationships are specifically associated with damages of specific WM fiber tracts, we measured the WM lesion volumes in each major fiber bundle anatomically identified by a WM fiber tract atlas With the exceptions of the relationships between Faces test performance and rT2LV of the GCC and fornix/stria terminalis, nor total and regional T2LV showed correlations with performances of emotion and mental state recognition from facial expressions. This may presumably be explained by the finding that T1-hypointense lesions reflect more directly the severity of myelin and/or axonal loss Cortical gray matter thinning was also found to relate to mentalization performance of patients with MS. Secondary neurodegeneration resulted from axonal injury in WM lesions, cortical lesion formation, and primary neurodegenerative processes are presumed to play a role in cortical atrophy developing in MS All cortical areas identified in our study have been implicated as neural nodes of large-scale neural networks involved in facial expression and emotion processing, and in social cognition Correlation between cortical thickness of the right premotor FEF (BA8) and Eye test performance Table 3 Emotion decoding and mental state attribution from facial expressions are processed by large-scale neuronal networks specialized for information with social relevance Recent studies have shown relationship between facial expression recognition deficit and other cognitive domains characteristically impaired in MS Except of the temporal pole, we did not find correlations between mentalization performance and brain areas, which have been previously implicated in neural circuits for making inferences about mental states. This may be due to task-specific reasons: the Eyes test and Faces test does not need the processing of contextual social cues, which would engage these centers.Faux pas performance was not different between patients with MS and healthy controls. Sensitivity of the Faux pas test to detect verbal mentalization deficit in MS has been found low As a limitation of our study the healthy control group recruited for MRI scanning was not tested for anxiety and depression. Therefore we could not include mood state indicators as confounding factors into the statistical analysis and the model can only explain 8.2% of the variation of the Eyes test performance. Finally, we are aware that further studies with higher number of participants are needed to reproduce the findings and corroborate the results.a priori hypothesis about possible anatomic substrates relating to mentalization performance have provided evidence for the significance of specific WM tracts and cortical brain areas associating with emotion and mental state decoding from facial expressions and mentalization function. Disconnection mechanism related to WM lesions and gray matter damage in cortical hubs constituting neural networks may independently compromise cognitive function in MS.Our results demonstrated impaired emotion and mental state recognition ability from facial expressions in patients with MS. Cortical thickness measure throughout of the whole brain on a vertex-to-vertex basis and lesion content assessment of each major fiber bundle in patients with MS without Table S1Regional T1-lesion content of the white matter tracts from the ICBM DTI-81 Atlas, and correlations between mentalization test performance and regional T1-lesion loads in patients with multiple sclerosis. The volumes of each individual fiber bundle were assessed, and then their relative lesion content was calculated. Correlation between regional T1-lesion volumes and the social cognition test performances were analyzed with ANCOVA controlling for gender, EDSS, depression, and anxiety. P-value after Bonferroni correction was p<0.0024. Significant correlations are indicated with bold format.(DOCX)Click here for additional data file.Table S2Regional T2-lesion content of the white matter tracts from the ICBM DTI-81 Atlas, and correlations between mentalization test performance and regional T2-lesion loads in patients with multiple sclerosis. The volumes of each individual fiber bundle were assessed, and then their relative lesion content was calculated. Correlation between regional T2-lesion volumes and social cognition test performances were analyzed with ANCOVA controlling for gender, EDSS, depression, and anxiety. P-value after Bonferroni correction was p<0.0014. Significant correlations are indicated with bold format.(DOCX)Click here for additional data file.Text S1Example from the Faux pas test. The presented story contains a faux pas situation.(DOCX)Click here for additional data file.


Arabidopsis and rice using computational prediction, experimental validation by overexpression in transgenic plants, and by degradome or PARE  sequencing. However, miRNA targets mostly remain unknown in soybean (Glycine max). More specifically miRNA mediated gene regulation at different seed developmental stages in soybean is largely unexplored. In order to dissect miRNA guided gene regulation in soybean developing seeds, we performed a transcriptome-wide experimental method using degradome sequencing to directly detect cleaved miRNA targets.MicroRNAs (miRNAs) regulate the expression of target genes by mediating gene silencing in both plants and animals. The miRNA targets have been extensively investigated in In this study, degradome libraries were separately prepared from immature soybean cotyledons representing three stages of development and from seed coats of two stages. Sequencing and analysis of 10 to 40 million reads from each library resulted in identification of 183 different targets for 53 known soybean miRNAs. Among these, some were found only in the cotyledons representing cleavage by 25 miRNAs and others were found only in the seed coats reflecting cleavage by 12 miRNAs. A large number of targets for 16 miRNAs families were identified in both tissues irrespective of the stage. Interestingly, we identified more miRNA targets in the desiccating cotyledons of late seed maturation than in immature seed. We validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-5’RACE). Gene Ontology (GO) analysis indicated the involvement of miRNA target genes in various cellular processes during seed development.The miRNA targets in both the cotyledons and seed coats of several stages of soybean seed development have been elucidated by experimental evidence from comprehensive, high throughput sequencing of the enriched fragments resulting from miRNA-guided cleavage of messenger RNAs. Nearly 50% of the miRNA targets were transcription factors in pathways that are likely important in setting or maintaining the developmental program leading to high quality soybean seeds that are one of the dominant sources of protein and oil in world markets. MicroRNAs (miRNAs) are endogenous noncoding small RNAs which play significant roles in the regulation of gene expression. Post-transcriptional gene regulation by miRNAs constitutes one of the most conserved and well characterized gene regulatory mechanisms. It is important for growth, development, stress responses and numerous other biological processes in eukaryotes -5. ThereMost plant miRNAs facilitate the degradation of their mRNA targets by slicing precisely between the tenth and eleventh nucleotides (nt) from the 5’ end of the miRNA. As a result, the 3’ fragment of the target mRNA possesses a monophosphate at its 5’ end. This important property has been used to validate miRNA targets . IsolatiArabidopsis or rice. High-throughput sequencing technologies such as massively parallel signature sequencing (MPSS), 454 and sequencing-by-synthesis (SBS) have enabled the identification of miRNAs in soybean. The extent of miRNA-directed post-transcriptional gene regulation in any organism can only be fully realized by identifying not only the miRNA component but also the set of their RNA targets.Soybean is one of the most important crops cultivated all over the world. It is a good source of vegetable protein and oil. However, the role of miRNAs in soybean seed development is mostly unknown. So it is important to identify the seed developmental stage-specific and tissues-specific miRNAs and their potential target genes. Identification of the consequences of miRNA-guided target degradation that occurs in a developmental and tissue specific manner could help to elucidate how lipid and protein metabolic pathways operated during seed development. The soybean genome (cv. Williams82) was decoded a year ago , and thiRecently, miRNA targets have been reported for one of the many stages of soybean seed development, namely very early at 15 days after flowering, and without dissection of the maternal seed coats from the cotyledons which develop from the zygote . To compIn higher plants, most miRNAs regulate their targets via cleavage, which normally occurs between the tenth and eleventh nucleotides of the complementary region between the miRNA and the mRNA target . The 3’ Five different degradome libraries, which capture the cleaved mRNAs, were constructed from cotyledons and seed coats from different seed developmental stages. These represented early maturation seed  and mid-maturation , the stages when the biosynthetic capacity of the seed is maximal and proteins and oils are accumulated at a high rate. In addition, we constructed a library from the yellow cotyledons (300–400 mg fresh weight) that are undergoing dehydration and desiccation.SBS sequencing of these libraries produced raw reads from 10 million to 45 million  that plot the abundance of the signatures relative to their position in the transcript . RepreseThe location of the cleavage site within the target gene is another important aspect of miRNA-mediated gene silencing. In soybean, the cleavage site of the miRNA was usually located in the CDS (coding sequence) of the target genes Tables 2. Since tThe full complement of targets found in each of the five degradome libraries is presented in Tissue specific miRNA and target identification is very important for understanding the regulation of gene expression in a spatial manner. In this study, we constructed cotyledon and seed coat libraries separately to identify miRNA targets both at younger (25–50 mg fresh weight seed) and older stages (100–200 mg and 300–400 mg fresh weight seed) of soybean seed development. Tissue specific siRNAs generated from a cluster of inverted repeat chalcone synthase (CHS) genes that downregulate CHS mRNAs and lead to lack of pigment on soybean seed coats have been described ,28, but Glyma12g08110.1, Glyma12g29720.1, Glyma14g33730.1 and Glyma11g20490.1, were also verified by RLM-5’RACE to be subjected to cleavage guided by gma-miR160  degradome library (Table 00 mg degThe identified targets for miRNAs in the three cotyledon degradome libraries were classified by their gene ontology (GO) using the AgriGO toolkit  Figure . Higher Arabidopsis and rice miRNA targets have been widely studied by high throughput sequencing [Arabidopsis and rice. The number of identified miRNAs and their potential targets in soybean is limited. To date, degradome sequencing has been reported for only one soybean tissue, namely the very young whole seed extracted 15 days after flowering from the cultivar Heinong44 [Regulation of gene expression by miRNAs has been comprehensively investigated in animals and plants ,22,30,31quencing ,32-34. Seinong44 . In ordeAfter computational analysis using the Cleaveland pipeline , we idenComparison of the data in our report with the first soybean degradome data reported by Song et al.  from a vArabidopsis, rice and in some other plants, miR156 regulates leaf development by targeting Squamosa-Promoter Binding protein-like (SBP) transcription factors [The 183 identified targets from our degradome analyses belonged to 57 different annotation groupings  . Compari2 factors ,39. The Arabidopsis, miR172 has been reported to be involved in the regulation of flowering time and floral development [Arabidopsis flower development [Our analyses of the early, mid and late maturation developmental stages of soybean show a number of targets similar to those found by Song et al.,  in the velopment . Alternaelopment . Nuclearelopment . We deteTo obtain a deeper understanding of soybean seed development, we investigated tissue specific miRNA target identification in the cotyledons and seed coats at different seed developmental stages. Based on the degradome data, we identified some miRNAs that may act differentially in the cotyledons versus the seed coats to degrade their targets Tables  and 4. F2Arabidopsis, miR160 and miR167 are involved in auxin signaling via regulation of ARF genes [Glyma12g08110.1Glyma12g29720.1Glyma14g33730.1 and Glyma11g20490.1, were validated by RLM-5’RACE showing precise cleavage as expected  plants were grown in a greenhouse and seeds were collected at different developmental stages including early maturation, green 25–50 mg fresh weight seed, mid- maturation green 100–200 mg, and late maturation yellow 300–400 mg fresh weight seed. Immediately, cotyledons and seed coats were separated by dissecting whole seeds and then frozen in liquid nitrogen. Subsequently the tissue was freeze dried and stored at −80°C.Soybean  was performed using the FirstChoice RLM-RACE Kit (Ambion). Total RNA was isolated from cotyledons and seed coats separately at different soybean seed developmental stages. Poly (A) + RNAs were purified using Oligotex mRNA mini kit (Qiagen). A 5’ RNA adaptor  was ligated to approximately 100 ng of mRNA using T4 RNA ligase. The ligated mRNAs were then reverse transcribed using oligo(dT) primer via M-MLV reverse transcriptase (Ambion). Two rounds of 5’ RACE reactions were performed with two nested primers  and two gene-specific outer and inner primers (Additional file The authors declare that they have no competing interests.MS designed experiments, performed RNA extractions, constructed degradome libraries, analyzed degradome data and drafted the manuscript. LOV designed initial approach, obtained funding, led and coordinated the project, and edited the manuscript. All authors have read and approved the final manuscript.Department of Crop Sciences, University of Illinois, Urbana, Illinois, 61801, USA.Complete list of identified miRNA targets identified in soybean seed coats and cotyledons during seed development.click here for fileGO analysis of miRNA targets identified in seed coats in different soybean seed developmental stages.Click here for fileGene Specific primer sequences for RLM-5’RACE.click here for file


Background. Current treatment of chronic hepatitis C with pegylated interferon and ribavirin has the ability to eliminate viral infection in about half of the patients treated. Therapeutic options, for those with remaining chronic hepatitis, will remain limited untilnovel antivirals become available in the future. Consensus interferon is currently available and has demonstrated clinical efficacy with superior invitro antiviral activity, but the maximum tolerated dose is not defined. Methods. We assessed the efficacy of daily high-dose (24 ug) consensus interferon with weight-based (1000–1200 mg daily) ribavirin in HCV genotype 1-infected non-responder patients. Results. Six adverse events were documented in five patients, and the trial was terminated with no subject achieving viral clearance. Conclusions. The occurrence of serious adverse events effectively defined the upper limit of acceptable dose, while also revealing that this dose did not offer enhanced sustained viral clearance.  The most commonly employed treatment of patients with hepatitis C virus (HCV) infection who are treatment-naïve consists of pegylated interferon-alfa and ribavirin with sustained viral response (SVR) rates in genotype 1 patients of up to 52% . Consensinvitro activity compared to other interferons [μg followed by 9 μg for 40 weeks did not result in a higher SVR compared to 9 μg for 48 weeks, presumably due to dose modifications in subjects receiving the induction dose of CIFN. When the subgroup of patients receiving the higher dose of CIFN, without early dose modification, was evaluated, 40% achieved SVR [μg or 15 μg with weight-based ribavirin administration in the treatment of patients unresponsive to prior pegylated interferon and ribavirin treatment. Viral clearance was achieved in a dose-dependent manner with greatest loss of HCV in the 15 μg group [CIFN has more potent antiviral erferons , 5. Currerferons –8. Cornbμg) with weight-based ribavirin dosage given with maximal support of patient adherence including blood cell growth factors. Previous clinical trials did not evaluate this high dose of daily CIFN in the treatment of non-responders to pegylated interferon and ribavirin beyond an 8-week induction period [The aim of our clinical trial was to evaluate the efficacy, safety and tolerability of high-dose daily CIFN  from baseline at 12 weeks of therapy or the presence of detectable HCV RNA at 24 weeks of therapy. All subjects must have previously received a weight-based dosage of ribavirin. At least one liver biopsy, consistent with chronic hepatitis C without significant alternative liver disease, must have been performed prior to screening for enrollment into the trial. All subjects, prior to participation, signed a consent form approved by each institutional review board.μg dose of CIFN  given subcutaneously and a daily weight-based dose of ribavirin (1000–1200 mg per day). All patients were evaluated at weeks one, two, and four of therapy and at subsequent four week intervals unless additional urgent clinical evaluation was needed. At each visit, subjects underwent physical examination and clinical history detailing adverse events and completion of the Beck Depression Inventory second edition (BDI-II). Treatment was discontinued and the subject considered a treatment failure if the patient failed to achieve early virologic response (EVR) defined as less than a 2-log drop of HCV RNA from baseline at Week 12 of therapy, or had detectable HCV RNA at or after Week 24 of therapy. Sustained viral response (SVR) was defined as an undetectable HCV RNA level using Quest Diagnostics  Heptimax and TMA serum assays 24 weeks after completion of 48 weeks of treatment.This study was an open-label pilot trial. All study subjects were treated with a daily 24 Potential subjects for this clinical trial were recruited from three hepatology practices in New York. Compliance with the experimental protocol was assessed by the subjects' self-reported administration of ribavirin and the return of used CIFN vials. In addition, the trial had a data safety monitor (DSM), who assessed the safety of this trial at specified time points during the study. The DSM was an independent physician with extensive clinical experience with treatment of patients with hepatitis C infection. t-test, chi-square and Fisher's exact tests. Response rates between partial and null responders were compared using chi-square and Fisher's exact tests.The subject's baseline demographics were compared using Student's Thirteen subjects (8 men) with a mean age of 55-years were enrolled in this study. The median baseline HCV RNA level was 3,300,000 IU/mL with only two subjects with levels less than 1,000,000 IU/mL. Ten patients had advanced fibrosis . During n = 6) in five patients. These serious adverse events included abdominal wall abscess requiring hospitalization, severe dehydration, and hypotension resulting in metabolic derangements, loss of consciousness, anemia, and ventricular tachycardia. In addition, one subject discontinued therapy due to disabling fatigue. The trial had initially been planned to enroll thirty subjects at this interferon dose. The study termination decision was made by the Principal Investigator on the advice of the DSM after assessing the frequency of serious toxicity while no patient had achieved a sustained loss of HCV RNA. The severity and frequency, of serious adverse events, are evidence that the high-dose aggressive treatment regimen of this study defined an upper dose limit of tolerability for CIFN-ribavirin therapy. The trial was terminated early due to the occurrence of serious adverse events  had greater than a 2-log drop from baseline HCV RNA while on treatment. One of these had transient loss of HCV but subsequently experienced a viral breakthrough. The four subjects who received 24 weeks of therapy all had partial viral response after 12 weeks of therapy, but had detectable hepatitis C virus at week 24 and therapy was discontinued as they were deemed non-responders .Three subjects with partial viral response prematurely discontinued therapy due to serious adverse events. There were three subjects still on therapy when the trial was halted. One subject had greater than a 2-log drop in HCV RNA level from baseline by week 8 of therapy, but the HCV RNA level at the time of termination had markedly increased, and there was no longer a 2-log drop in the HCV RNA level from baseline at that point. The second subject did have a greater than 2-log decrease from baseline HCV RNA level after 16 weeks of therapy, but by 20 weeks the hepatitis C viral level had increased over 1-log from the nadir level. The third subject did not have a 2-log decline from baseline viral level after 12 weeks of therapy and was deemed a non-responder. The final three subjects were non-responders to the study medications at the time of discontinuation of therapy due to adverse events.P < .04).There were three subjects with greater than a 2-log drop in viral level by four weeks of therapy. One of these subjects could not tolerate therapy and did not complete 12 weeks of therapy. Another was a nonresponder at 24 weeks of therapy. The third subject was still on study medication when the clinical trial was halted, however, while on therapy there was marked increase in HCV RNA level. All three subjects who had greater than a 2-log drop from the baseline viral level after four weeks of therapy were partial responders to previous pegylated interferon and ribavirin therapy compared to none of the prior null responders (n = 12) and flu-like symptoms (n = 12), anorexia (n = 10), anxiety/depression (n = 7), and insomnia (n = 7). The mean decrease in body weight during the experimental protocol was 8.1 ± 5.8 kilograms. The median increase in BDI-II score was 9 ± 10.1. Serious adverse events (n = 6) occurred in five patients and included abdominal wall abscess, severe dehydration, hypotension, and ventricular tachycardia all requiring hospitalization and loss of consciousness and anemia. These events occurred within the first six to 16 weeks of therapy. Two subjects (15%) had their dose of CIFN reduced during the trial from 24 μg to 15 μg daily. Five subjects (38.5%) had a dose reduction of ribavirin and one of the five subjects had ribavirin discontinued. The mean decrease in hemoglobin level from baseline to nadir was 3.6 ± 1.6 g/dL with eight subjects receiving epoetin alpha injections. The most common adverse events were fatigue  to lower-dose treatment  in those patients who had previously failed treatment with interferon and ribavirin therapy. The week 12 and 24 responses were superior in the high- versus low-dose group (P < .05), but the SVR rates were identical at 26%. This suggests that the initial positive effect of the 24 weeks of high-dose daily induction was lost after dose reduction to three times per week treatment [Previous studies demonstrate promising SVR rates of 23% to 37% using daily CIFN and ribavirin in the retreatment of non-responders to the combination of standard or pegylated interferon and ribavirin –8. HCV cμg for four weeks followed by 18 μg for eight weeks versus 18 μg for 12 weeks in 120 patients infected with HCV who were previous non-responders to pegylated interferon and weight-based ribavirin [Kaiser etal. from Germany evaluated an induction dose of daily CIFN of 27 ibavirin . No ribaμg for the first 8-weeks of treatment resulted in an enhanced first-phase HCV-RNA decay suggesting higher antiviral efficacy of a higher dose of CIFN, but this did not translate to a better SVR, presumably due to dose modifications [Another study by Cornberg and colleagues investigated the efficacy of CIFN plus ribavirin in HCV patients who were non-responders to standard interferon and ribavirin . ConsensPrevious partial responders to pegylated interferon and weight-based ribavirin as compared to null responders had better viral kinetic responses with all three partial responders having greater than a 2-log drop in HCV RNA from baseline viral level by week four. This response, however, did not translate into a higher overall efficacy since these subjects could not tolerate continued therapy and viral response did not decline to undetectable levels.μg daily dose when used in combination with weight-based ribavirin. This observation is important since the efficacy results of the prior studies left open the possibility of greater efficacy of high-dose CIFN.This trial used a sustained high daily dose (24 mg) of CIFN, a treatment regimen which showed promising early results in achieving a decline in HCV RNA levels in a population of subjects resistant to prior therapy. The major factor leading to termination of this study was the occurrence of serious adverse events. Although each event was not unique in patients receiving interferon ribavirin treatment, the occurrence of frequent serious adverse events in a small patient population raised significant concern. The abdominal wall abscess was related to injection site infection. The development of dehydration and hypotension was related to anorexia and severe lassitude. The episode of loss of consciousness may have been related to anorexia and anemia. The observed adverse events, even including cardiac arrhythmias, have been reported previously in patients receiving interferon alfa and ribavirin. The frequency and severity of adverse events, however, effectively determined the upper limit of tolerability of daily consensus interferon to be less than the 24 Despite use of blood cell growth factors and maximal support of experienced investigators, this high-dose of daily CIFN therapy in combination with a weight-based ribavirin dose was not tolerated due to serious adverse events. Most of the subjects required epoetin alfa injections for therapy-associated anemia. Even in the subjects who could tolerate high-dose CIFN therapy with weight-based ribavirin, sustained loss of HCV was not achieved by 24 weeks. μg dose of CIFN in combination with weight-based ribavirin. Our study effectively defines the upper limit of tolerability of daily CIFN dosage when used in combination with ribavirin. Furthermore, this high dosage of CIFN and weight-based ribavirin combination treatment was ineffective in eradicating hepatitis C virus in patients who failed previous pegylated interferon and ribavirin therapy.We cannot recommend this higher daily 24


Physicians’ shortage in many countries and demands of high-quality and affordable care make physician-nurse substitution an appealing workforce strategy. The objective of this study is to conduct a systematic review and meta-analysis of randomised controlled trials (RCTs) assessing the impact of physician-nurse substitution in primary care on clinical parameters.We systematically searched OVID Medline and Embase, The Cochrane Library and CINAHL, up to August 2012; selected peer-reviewed RCTs comparing physician-led care with nurse-led care on changes in clinical parameters. Study selection and data extraction were performed in duplicate by independent reviewers. We assessed the individual study risk of bias; calculated the study-specific and pooled relative risks (RR) or weighted mean differences (WMD); and performed fixed-effects meta-analyses.11 RCTs  were included; most were from Europe, generally small with higher risk of bias. In all studies, nurses provided care for complex conditions including HIV, hypertension, heart failure, cerebrovascular diseases, diabetes, asthma, Parkinson’s disease and incontinence. Meta-analyses showed greater reductions in systolic blood pressure (SBP) in favour of nurse-led care  but no statistically significant differences between groups in the reduction of diastolic blood pressure (DBP) , total cholesterol (TC)  or glycosylated haemoglobin . Of other 32 clinical parameters identified, less than a fifth favoured nurse-led care while 25 showed no significant differences between groups.disease-specific interventions from a small selection of healthcare systems, insufficient quantity and quality of studies, many different parameters.trained nurses appeared to be better than physicians at lowering SBP but similar at lowering DBP, TC or HbA1c. There is insufficient evidence that nurse-led care leads to better outcomes of other clinical parameters than physician-led care. A WHO Report showed the global number of health care providers, namely physicians, nurses and midwives, remains lower than required per 1,000 population We developed a protocol prior to the commencement of the review and followed the PRISMA guidelines We included peer reviewed randomised controlled trials (RCTs) published in English from any country that examined physician-nurse substitution. The studies had to focus on patients of all ages seeking first contact or undergoing care for all conditions including mental health and addiction restricted to primary care; and had to compare care from nurses to care from physicians . We further limited the inclusion criteria to studies: in which the intervention or follow-up care had taken place in general practices, community or ambulatory care settings regardless of the recruitment sources; and which reported on clinical parameters that detected changes in the clinical status and/or physiological capability of patients in relation to various forms of disease, e.g. blood pressure for hypertension or cardiovascular disease risk. Based on a published framework We comprehensively searched OVID Medline, Embase, The Cochrane Library of Systematic Reviews, CINHAL and the Cochrane Effective Practice and Organisation of Care Group (EPOC) from all available dates until August 2012. The searches - not age-, date- or country-specific - included ‘primary care’, ‘skill mix’, ‘doctor’-‘nurse’ substitution’ . We alsoTwo authors independently screened titles and abstracts and assessed the full-texts of potentially eligible publications for inclusion, resolving differences through consensus.a-priori, and resolved differences through consensus. Data from more than one control group of interest (e.g. family physicians and paediatricians) were combined and compared as one to the intervention group.Two authors independently extracted both qualitative data  and numeric data (dichotomous and continuous format) using standardised data collection forms designed and developed Two authors independently assessed the risk of bias of individual trials following established criteria 2 statistic: values of <25% represent low heterogeneity and ≥50% represent high heterogeneity We performed meta-analyses when at least three trials reported appropriate data for the same outcome using the generic inverse variance fixed-effects method in Review Manager (Version 5.1) A total of 4,133 original records were identified . Forty-fEleven trials - eight RCTs of parallel design and three cluster RCTs - were conducted in the UK (n = 2), The Netherlands (n = 4), USA (n = 2), South Africa (n = 2) and Russia (n = 1) . Median The number of nurses delivering care was reported in eight trials. It ranged from 1 to 10 in six trials. In two other trials, 31 clinics were randomised with 103 nurses. Six trials reported the number of physicians delivering care, which ranged from 5 to 28 in five trials, and another employed 108. Physicians’ resources, location of practices  and social settings were scarcely reported. Nurses’ years of experience were not reported in any of the trials but in most of them nurses were already enrolled as staff or took specific courses for delivering care. Nurses’ roles were described under various terminologies and their qualifications and skills varied from practice nurses with or without extra training  to middle nurse managers, registered or licensed nurses.In all studies nurses were the main figure of care and performed tasks for complex conditions that required specialised skills including cerebrovascular disease, hypertension, heart failure, diabetes mellitus, asthma, incontinence, Parkinson’s disease and HIV . Nurses’All RCTs 2 = 0%, p = 0.53; DBP: I2 = 38%, p = 0.19).Five trials provided sufficient quantitative continuous data for meta-analysis . Compare2 = 0%)  at follow up, with no significant heterogeneity between trials  . Individ2 = 0%)  at follow up, with no significant heterogeneity between trials  . SimilarIndividual trial estimates showed no significant differences between groups in various parameters of lung function including measurements of peak flow at six months Compared to physician-led care, there were significantly more patients with nurse-led care who had a decrease or regression in the levels of functional exercise capacity, N-terminal pro-brain natriuretic peptide or in the left ventricular end-diastolic volume index Individual trial estimates showed no significant differences between groups in the frequency (number and volume) or volume (number of pads) of incontinent episodes at either 6 or 12 months follow-up Individual trial estimates showed no significant differences between groups in the fractures sustained during study or in the results from the mobility stand-up test at 24 months follow-up In one trial, CD4 cell-counts were used as indication for ART initiation (Cohort 1) and ART continuation and management of regimens (Cohort 2) at 12–18 months follow-up We systematically evaluated the published evidence for the effects of physician-nurse substitution on clinical parameters in 11 RCTs involving more than 30,000 patients with various conditions. The first important and surprising finding of our review is that the number of studies in this area is increasing slowly and studies continue to be of poor quality despite evidence reports published ten years ago The evidence represents interventions for which nurses trained to provide care in various settings and for a wide range of complex conditions. In most cases, (82%), this required specialised skills and the use of guidelines. This suggests that the level of skills may be critical for the success of disease management when physician-nurse substitution takes place. The level of substitution did not seem consistent across studies however. Trials employed nurses of various qualifications and the tasks performed varied regardless of the level of training. Moreover, despite possessing some level of advanced skills, nurses required support or communication with the physicians for various tasks. Thus it was difficult to explore and identify patterns of potential influences of this criterion on the outcomes. On one hand, all studies included in the meta-analyses employed nurse practitioners (with or without further degrees), and the direction of effect remained after systematic exclusion of each trial. On the other hand, studies for which data could not be pooled involved licensed nurses, registered nurses, or nurse practitioners (with or without further degrees). Perhaps the development of nurse-led clinics may be a more appealing strategy to allow nurses to establish full clinical autonomy. The reporting of other clinicians’ characteristics  also remains insufficient despite previous findings There are gaps in the current evidence which merit consideration in further studies. Nurses’ roles and the level of experience required to qualify for substitution need a better definition of boundaries and task allocation in clinical practice. In addition, future research should generate more methodologically sound studies. Consistent and complete reporting of the aforementioned characteristics could improve the understanding and identification of the optimal benefits of this strategy. In spite of these limitations and heterogeneity, our meta-analyses demonstrate a statistically significant systolic blood pressure-reducing effect of nurse-led care (delivered by nurse practitioners) compared to physician-led care but no significant differences in reducing diastolic blood pressure, total cholesterol and glycosylated haemoglobin. Results from the other 32 individual trial estimates reported in nine of the trials suggest that nurse-led care (by nurses with various titles) may be similarly (26 estimates) or more (6 estimates) effective than physicians at managing the variety of clinical parameters evaluated in our review.To our knowledge this is the first systematic review with a focus on clinical parameters in patients undergoing care with the implementation of physician-nurse substitution in primary care. It benefits from a thorough assessment and critical appraisal of RCTs which are at lower risk of bias Trained nurses appeared to be better than physicians at lowering systolic blood pressure but similar at lowering diastolic blood pressure, total cholesterol or glycosylated haemoglobin. While only a few individual trial estimates of 32 clinical parameters (e.g. kidney and lung function) favoured nurse-led care, there is insufficient evidence to conclude that nurse-led care leads to better outcomes of clinical parameters than physician-led care. The main limiting factor is the insufficient quantity of studies using good quality methods and reporting the same outcome. The current evidence also shows disease specific interventions from a small selection of healthcare systems. In order to provide more general conclusions, far more good quality trials in larger numbers of patients need to be carried out. Furthermore, additional studies should map clinicians’ characteristics, including the wider range of nurse care and tasks provided in many countries and the various levels of training and clinical autonomy.Table S1Search strategy in Ovid Medline*. *Similar search strategies were performed and run in EMBASE, The Cochrane Library of Systematic Reviews and CINAHL. All included specific search filters for RCTs.(DOCX)Click here for additional data file.Table S2Studies excluded from the review based on appraisal of full-text articles.(DOCX)Click here for additional data file.Table S3Participants, interventions and outcomes, in the included studies. Studies are listed by year (y) of publication, in decreasing order. Abbreviations: US = United States; NL = The Netherlands; UK = United Kingdom; ZA = South Africa; RU = Russia; RCT = Randomised Controlled Trial; cRCT = Cluster Randomised Controlled Trial; NR = Not Reported; ART = Antiretroviral Therapy; HbA1c = Haemoglobin; BP = Blood Pressure; TC = Total Cholesterol; GH = Glycosylated Haemoglobin; CD4 = t-cell surface glycoprotein CD4; HDL = High Density Lipoprotein levels; LDL = Low Density Lipoprotein; PD20 = provocative dose of methacholine causing a 20% fall in forced expiratory volume in one second (FEV1); FENO = Fraction of Exhaled Nitric Oxide. * start and end year when studies were conducted.(DOCX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation  Human blood cells all originate from a common precursor, the hematopoietic stem cell. Telomerase, the enzyme responsible for adding telomere repeats to chromosome ends, is active in human hematopoietic stem cells but appears unable to maintain a constant telomere length with age. We first document the telomere length of different blood cell subsets from 835 healthy individuals between birth and 100 years, to delineate the normal rate of telomere attrition with age. Telomere lengths of blood cells were found to be slightly longer in women than in men, from birth and throughout life. We then compared this reference data to the telomere length in similar blood cell subsets from individuals with reduced telomerase activity as a result of a mutation in one of the genes encoding telomerase and from their direct relatives. Strikingly short telomeres were found in telomerase-deficient individuals, consistent with their cellular pathology and disease susceptibility, and somewhat shorter telomeres than expected were found in cells of relatives with normal telomerase maintenance. Our data can be used as a reference for blood cell telomere length in studies of normal and accelerated aging. At least a few hundred nucleotides of telomere repeats must “cap” each chromosome end in order to suppress DNA damage signals and avoid the activation of DNA repair pathways Despite increasing evidence that telomere homeostasis is important in human aging, cancer and disease states, detailed and comparative information regarding the telomere length in different human cell subtypes of healthy individuals in relation to their age is surprisingly modest. Apart from being technically challenging In most somatic cells the telomere length declines with age and with cell division in culture, albeit at different rates hTERC) or the telomerase reverse transcriptase gene (hTERT) can present with a wide spectrum of diseases The precise role of telomerase in hematopoietic stem and progenitor cells and in lymphocytes remains poorly understood. Telomerase expression is readily detected in hematopoietic cells hTERC or hTERT. The telomere length in leukocytes from healthy individuals was found to vary over a broad range at any given age and the rate of telomere attrition also varied with age and with cell type. Strikingly, the telomeres in cells from individuals with telomerase deficiency were found to be very short in all cell types, and this deficit was found to be comparable for most cell subtypes for hTERC or hTERT deficiency. The largest (age adjusted) differences in telomere length deficits between hTERC or hTERT were seen in “naïve” T cells for hTERC deficient individuals and in NK/differentiated T cells for hTERT deficient individuals. These results demonstrate that normal telomerase levels are essential to maintain normal telomere homeostasis in HSC and lymphocytes. Our results provide valuable reference data for further studies of telomere biology in health and disease and point to a crucial rate-limiting role for telomerase in HSC and immune cells.Here we report our data on the median telomere length (MTL) in five distinct leukocyte subpopulations of over 800 healthy individuals between birth and 100 years of age as well as 60 individuals that are heterozygous for one of the telomerase genes, http://www.cdc.gov/growthcharts/clinical_charts.htm). A second arbitrary cut-off was set at 18 years of age because the decline in telomere length in all leukocytes appeared to drop notably after puberty. Telomere length data within the three selected age segments: below 1 year (yr), 1–18 yrs and 19 yr and higher are shown in We measured the telomere length in lymphocytes and granulocytes of 835 healthy individuals using automated multicolor flow FISH . On averth, 90th, 10th and 1st percentile of the telomere length distribution in each age segment for each blood cell subset in healthy individuals. The rate of telomere length decline varied amongst the different lymphocyte subsets analyzed. The telomere length decline with age in B lymphocyte subset (CD45RA+ CD20+) was comparable to that in granulocytes. Memory T (CD45RA−CD20−) and mature NK/T (CD45RA+CD57+) lymphocyte subsets showed the sharpest decline in telomere length with age, particularly during childhood with slopes of −144 and −155 bp per year respectively. The CD45RA+CD20− T lymphocyte subset enriched for “naïve” T cells and the CD45RA+CD57+ mature NK/T lymphocyte subset displayed the widest distributions, 2.80 and 2.89 kilobase (kb) respectively between the 10th and 90th percentile of the normal distribution, throughout the age ranges. Unlike other subsets CD45RA+CD20− T lymphocytes showed only a modest difference in the telomere attrition rate between childhood and adulthood: 89 and 51 bp per year respectively. In contrast, the memory T lymphocyte subset (CD45RA−CD20−) displayed the narrowest range of telomere length distribution . Overall, the shortest telomere lengths were measured in memory T and mature NK/T lymphocytes from older individuals.Telomere length measurements versus age in granulocytes and lymphocyte subpopulations were used to determine the regression lines for telomere attrition in the three selected age ranges : F = 5.7; P = 0.0002, hTERT or hTERC (n = 60) in comparison to non-carrier relatives (n = 37). The results, plotted on the telomere length versus age distribution curves derived from healthy individuals  at all ages and for all blood cell subsets tested  and a slightly increased effect (difference of 0.2 Kb) on the CD45RA+CD20− “naive” lymphocyte subset for hTERC deficient individuals (n = 23).To study the role of telomerase in hematopoietic cells, we analyzed the telomere length in leukocyte subpopulations of individuals carrying a mutation in either ividuals  are showsubtypes . ExceptihTERT and hTERC considered together), despite having intact telomerase genes, also showed somewhat shorter median telomere lengths in all leukocytes compared to the control population. The largest difference was measured in the granulocyte subset of non-carrier relatives considered together, with Δtel: 0.9 kb, which may be indicative of HSC deficit and warrants further investigation  and BC Cancer Agency Research Ethics Board. All samples from patients outside Vancouver were obtained with informed consent and approval of local ethical review boards in accordance with the Declaration of Helsinki.Anonymous cord blood samples were obtained from healthy full term births with parental informed consent. Since no associate information was available for these samples, gender testing was performed by FISH as described below.Anonymous peripheral blood samples were obtained from 835 healthy individuals between the ages of 6 months to 102 years of age screened for clotting disorders; samples where no clotting disorders were found were made available for study; only gender and age information were provided.hTERT) or the RNA template (hTERC) gene and their 37 (non-carrier) relatives were included in our analysis and were described previously, , denatured in formamide at 87°C, and hybridized with a fluorescein-conjugated (CCCTAA)3 peptide nucleic acid (PNA) probe specific for telomere repeats and counterstained with LDS751 DNA dye. The fluorescence intensity in, granulocytes, total lymphocytes and lymphocyte subsets defined by labeled antibodies specific for CD20, CD45RA and CD57 relative to internal control cells and unstained controls was measured on a FACSCalibur instrument (Becton Dickinson) to calculate the median telomere length from duplicate measurements. Further details regarding telomere length measurements and data sets are described in Online Supplementary Material as well as depicted in Telomere length measurements using automated multicolor flow-fluorescence in situ hybridization (flow FISH) was performed as described th to 90th percentile range, the two sets were merged and analyzed together for curve fitting models and reference comparisons.Some of the total 835 healthy subjects did not have sufficient cells for analyses of one or more of the cell subsets tested: granulocytes, B lymphocytes, or mature NK/T cells. Specific improvements were developed during the 8 year of the healthy donor study allowing for the testing of additional cell subsets  that were representative for this segment of distribution. To compare the telomere lengths of one population against another, the ANOVA function in the R language was employed to test if the data was from the same or 2 different model fits Analyses were performed using Microsoft Excel (Microsoft Office 2007), GraphPad Prism (version 4) and R ; t-Tests were two-tailed and performed on data with a normal distribution (KS test). Linear modeling (lm function in the R language) was used to carry out the regression analysis and estimate the piecewise linear curves with breakpoints hinged at 1 and 18 years of age. We found that this gave the best fit with the least mean square error and more consistent error distribution across the age ranges as compared to using a number of polynomial fits . The 99Figure S1Comparison between leukocyte telomere length data sets and between statistical models over the study period. A. In the first study period, median lymphocyte and granulocyte telomere length measurements determined by flow FISH in 391 healthy individuals from birth  to 102 years of age (blue dots) with reference curves derived from a quadratic function which provided a better fit than linear regression models over the entire age-range. Quadratic model distribution curves represent the projected modeled percentiles of the data: 99th (red), 90th (green top), 50th (green centre), 10th (green bottom) and 1st (blue) percentile of telomere range distribution per age. B. Median lymphocyte and granulocyte telomere length measurements in 391 consecutive individuals after 96 well format red cell lysis  and 444 other individuals  after large volume red cell lysis. C. In the combined data set, median lymphocyte and granulocyte telomere length measurements from the combined data sets with piece-wise linear model percentile estimates: 99th (red), 90th (dashed green top), 50th (green centre), 10th (dashed green bottom) and 1st (blue) percentile of the telomere range distribution at any given age. Three linear regression segments were connected at respectively one year and eighteen years of age, to give better representation of the data at younger and older age ranges; insets represent magnifications of the predicted telomere length distribution between birth and five years of age.(EPS)Click here for additional data file.Figure S2Comparison of median telomere length (MTL) in CD45RA+CD20− “Naïve” T lymphocytes and other nucleated blood cell types from the same individual over four distinct age groups. This table complements (EPS)Click here for additional data file.Figure S3Gender differences in telomere length between leukocyte subpopulations. Median telomere length values in leukocyte subpopulations from 835 healthy females and males ranging from newborns  to hundred years of age determined by flow FISH. The following white blood cell subtypes were tested: lymphocytes, granulocytes, B lymphocytes (CD20+), “naïve” T lymphocytes (CD45RA−pos CD20+), memory T lymphocytes (CD45RA−) and mature NK/T cells (CD57+). Curves representing the 50th percentile curve of the modeled median telomere length per age for females (red) versus males (blue) are shown. Three linear segments connected at one year and eighteen years of age were used. ANOVA test P values for the difference between females and males for each gender specific subset were: lymphocytes P = 1.56×10−5, granulocytes P = 0.37, B lymphocytes P = 0.0018, “naïve” T lymphocytes P = 3.7×10−7, memory T lymphocytes P = 0.0012, mature NK/T cells P = 0.0028 respectively. Click here for additional data file.Figure S4Leukocyte telomere lengths of non-carrier siblings or parents of telomerase deficient individuals. The telomere length distribution in healthy individuals  was used(EPS)Click here for additional data file.Table S1Complete telomere length data sets from healthy individuals. Leukocyte median telomere length data  were collected over an eight year period and analyzed on two occasions . For the first set of data, freshly isolated nucleated blood cells were used whereas for the second set, nucleated cells were frozen prior to flow FISH Click here for additional data file.Table S2Telomere length cell subset correlations at different age ranges. This table complements (DOC)Click here for additional data file.Table S3ANOVA results summary table on the effect of gender on cord blood telomere length measurements. Effect of gender test of cord blood samples, in reference to (DOC)Click here for additional data file.Table S4ANOVA post test results summary table on the effect of gender on cord blood telomere length measurements. One way ANOVA with Tukey's multiple comparison test results of the effect of gender on cord blood telomere length measurements.(DOC)Click here for additional data file.Table S5TERT or TERC for heterozygous individuals or relatives; effect of family relationship to heterozygous individual (sibling or parent). A linear model was applied to fit the telomere length variation with age to a piece-wise linear function with two age breakpoints (age 1and age 18). An ANOVA (F-test) of the model fit against a model that also takes into consideration the gender of the individual and was applied and showed that there was a significant difference in gender on telomere length (P = 3.7e-07). The ANOVA compares the two linear models (an estimate of the entire population (one linear fit) and an estimate where gender (or another factor) was taken into consideration (two linear fits to the data)) and showed a significance difference  With reference to the ANOVA tables, the first line shows the Residual degree of freedom (Res.Df) and Residual sum of squares (RSS) for the first model (no difference in gender). The second line (difference in gender model) shows in addition, the degree of freedom for the model, sum of squares, the F-value and the probability of the F-statistics (Pr (>F). If this value is less than 0.05, then there is a good chance that gender (for example) has an effect on telomere length.ANOVA results summary table on the effects of gender on telomere length measurements. This table summarizes the results comparing the two linear models (an estimate of the entire population) and an estimate of where factor “X” (gender for example) was taken into consideration and showed a significant difference. The following factors were tested: effect of gender ; effect of telomerase deficiency; effect for relatives of telomerase deficient individuals; effect of gene affected (DOC)Click here for additional data file.


Mttp-IKO).Gut derived lipid factors have been implicated in systemic injury and inflammation but the precise pathways involved are unknown. In addition, dietary fat intake and obesity are independent risk factors for the development of colorectal cancer. Here we studied the severity of experimental colitis and the development of colitis associated cancer (CAC) in mice with an inducible block in chylomicron secretion and fat malabsorption, following intestine-specific deletion of microsomal triglyceride transfer protein  induced colitis, compared to <20% in controls. Intestinal permeability was increased in Mttp-IKO mice compared to controls, both at baseline and after DSS administration, in association with increased circulating levels of TNFα. DSS treatment increased colonic mRNA expression of IL-1β and IL-17A as well as inflammasome expression in both genotypes, but the abundance of TNFα was selectively increased in DSS treated Mttp-IKO mice. There was a 2-fold increase in colonic tumor burden in Mttp-IKO mice following azoxymethane/DSS treatment, which was associated with increased colonic inflammation as well as alterations in cytokine expression. To examine the pathways by which alterations in fatty acid abundance might interact with cytokine signaling to regulate colonic epithelial growth, we used primary murine myofibroblasts to demonstrate that palmitate induced expression of amphiregulin and epiregulin and augmented the increase in both of these growth mediators when added to IL-1βor to TNFα.Mttp-IKO mice, despite absorbing virtually no dietary fat, exhibit augmented fatty acid dependent signaling that in turn exacerbates colonic injury and increases tumor formation.These studies demonstrate that  The continued epidemic of obesity and its associated comorbidities has fueled interest in understanding the pathways and mediators involved, particularly in relation to inflammation and cancer. Obesity and its related metabolic complications have been demonstrated to play a role in a number of cancers, including colorectal cancer (CRC) and consequently dietary modification and weight control are viewed as key modifiable risk factors In addition to the role for obesity and fat intake in modulating inflammation and CRC risk, there is emerging information suggesting that the small intestine plays an important role as a mediator in the inflammatory response to systemic sepsis Mttp-IKO) develop virtually complete block of intestinal absorption Pseudomonas aeruginosa, the most common cause of gram negative pneumonia Mttp-IKO mice developed worse colonic injury and increased tumor burden. Furthermore, we found that altered fatty acid signaling may play a key role in promoting these phenotypes.In this study, we have examined the role of intestinal chylomicron secretion in order to understand the role of intestinal lipid transport in the pathogenesis of colonic inflammation and colitis associate cancer (CAC). We recently demonstrated that mice with conditional intestine-specific deletion of microsomal triglyceride transfer protein (flox/flox villin-Cre-ERT2 (Mttp-IKO) mice  were used for these studies, in a background of ∼75% C57BL/6 and ∼25% 129/SvJ. Cre recombinase expression in villus epithelial cells was induced by five daily intraperitoneal injections of 1 mg tamoxifen (Sigma), as described previously th(2%), 26th (2.5%) and 46th (2.5%) day after AOM injection, minor modifications of the protocol used by others MttpSamples of small intestine and colon were fixed and embedded for sectioning and hematoxylin and eosin (H&E) staining. Where indicated, intestinal sections were stained for lipid droplets using osmium tetroxide as described Mttp-IKO mice were gavaged with FD-4 (400 mg/Kg body weight), prior to and 7 days after 2.5% DSS treatment and sera were collected 4 hour later. Serum FITC was measured on a fluorimeter  at excitation 485/20, emission 528/20. For LPS determinations, sera were diluted 1∶10 and heated at 70°C for 15 minutes to inactive inhibitors. LPS was then measured using the LAL chromogenic endotoxin quantitation kit according the manufacturer's instructions . Serum TNFα and IL-1β were quantitated by ELISA using kits purchased from BD Biosciences  or R&D systems  following the manufacturers' instructions.Intestinal permeability was determined following injection of FITC labeled dextran . Control and Total RNA was isolated from intestinal tissues using Trizol either under standard protocols or for samples treated with DSS, using the modification described by Kerr and colleagues Total lipids were extracted from feces as well as from proximal colon, as previously described 5 cells per well were seeded in 6-well plates and cultured overnight. The phenotype of these cells was verified by positive staining for smooth muscle actin and vimentin and negative staining for desmin and cytokeratin Myofibroblasts were isolated from C57BL/6 wild type mice as described Multiple group comparisons of continuous data sets were performed using one-way analysis of variance followed by post-hoc t-test using Prism 4.0  and Microsoft Excel. Data are reported as means ± SEM. Survival studies were analyzed via the log-rank test. A p value <0.05 was considered to be statistically significant.Mttp-IKO mice exhibit accelerated death and markedly reduced survival (1/10) compared to control mice (9/11) Mttp-IKO mice was associated with greater weight loss in shorter (7 days) experiments  since previous studies have shown that this strategy attenuates the UPR in metabolic stress conditions in mice Mttp-IKO mice and if anything led to accelerated death in these animals  and integrated stress response pathways in the small intestine and colon of both genotypes and the impact of DSS exposure. Those findings demonstrated increased baseline expression of Grp78 and Chop in the small intestine of IKO mice , as prevIKO mice . By contenotypes . These f animals . These fMttp-IKO mice compared to controls  colitis and leads to increased tumor burden in a model of CAC. The mechanisms and pathways mediating these phenotypes include alterations in intestinal barrier function with increased permeability and increased serum levels of TNFα, coupled with activation of myofibroblast secretion of growth factors mediated at least in part through altered fatty acid signaling. Together the findings link aspects of intestinal lipid metabolism with intestinal inflammation and also to pathways that promote development of colorectal cancer. Some of these core observations merit more detailed discussion.Mttp-IKO mice. By contrast, we found elevated serum levels of TNFα in Mttp-IKO mice, both at baseline and following DSS treatment, suggesting that blocking chylomicron formation was associated with increased production or altered turnover of this proinflammatory cytokine in the setting of acute chemical colitis. Further work will be required to resolve the cellular origins of TNFα and to address the important questions of production and clearance rates of this cytokine in DSS mediated colonic injury. Nevertheless, our findings are consistent with other work that has implicated lipid-dependent alterations in intestinal delivery of dietary antigens that modulate systemic responses, including the release of inflammatory mediators from adipose tissue Previous studies have implicated lipid-derived factors secreted from the small intestine as key components in mediating distal organ failure in systemic sepsis Mttp-IKO mice were protected against pneumonia-associated sepsis induced intestinal injury and exhibited decreased mortality Mttp-IKO mice have a far worse outcome when challenged in a model of chemical (DSS) colitis. The apparent discordance in these outcomes lead us to suggest that blocking chylomicron assembly and secretion may protect animals from the secondary manifestations of intestinal injury only when the source of sepsis involves extraintestinal tissues (such as the lung). In the setting of pneumonia-associated sepsis, for example, Mttp-IKO mice exhibit a compensatory increase in hepatic HDL production Our recent findings demonstrated that MTTP gene is defective) have been shown to exhibit defective CD1 function as a result of impaired lipid antigen loading Mttp deletion was protective against experimental oxazalone-induced colitis, which is dependent on the presumptively haptenated antigen being presented via invariant NKT cells Mttp deletion, but rather used an Mx-1 (interferon-inducible) Cre Mttp deletion (as in the current study) exhibits the same protective phenotype as that observed using the interferon inducible system employed in those earlier studies will await clarification.Another consideration in attempting to understand the pathways by which blocking chylomicron secretion exaggerates the inflammation associated with colonic injury is that the genetic pathways involved in Mttp-dependent lipid delivery into the endoplasmic reticulum Mttp-IKO mice resulted from the unabsorbed lipid rather than from increased dietary fat intake or from obesity (since Mttp-IKO mice are lean), or from increased colonic accumulation of neutral lipid. In this regard, we validated fecal FFA content in control, chow-fed mice (∼60 µmol/g, Mttp-IKO mice.The current findings also raise the question of how dietary fat and intestinal lipid metabolism might influence inflammation and pathways relevant to the pathogenesis of colorectal cancer. It has been shown for example that mice chronically fed high fat diets exhibit increased mortality in some models of sepsis Mttp deletion beyond lipid malabsorption. Pharmacologic compounds that block Mttp activity are currently in clinical trials, including some that are by design intestine-specific Finally, our findings raise the possibility that there may be other adverse effects of


Respiratory epithelium integrity impairment caused by intensive exercise may lead to exercise-induced bronchoconstriction. Clara cell protein (CC16) has anti-inflammatory properties and its serum level reflects changes in epithelium integrity and airway inflammation. This study aimed to investigate serum CC16 in elite athletes and to seek associations of CC16 with asthma or allergy, respiratory tract infections (RTIs) and immune response to respiratory pathogens.The study was performed in 203 Olympic athletes. Control groups comprised 53 healthy subjects and 49 mild allergic asthmatics. Serum levels of CC16 and IgG against respiratory viruses and Mycoplasma pneumoniae were assessed. Allergy questionnaire for athletes was used to determine symptoms and exercise pattern. Current versions of ARIA and GINA guidelines were used when diagnosing allergic rhinitis and asthma, respectively.Asthma was diagnosed in 13.3% athletes, of whom 55.6% had concomitant allergic rhinitis. Allergic rhinitis without asthma was diagnosed in 14.8% of athletes. Mean CC16 concentration was significantly lower in athletes versus healthy controls and mild asthmatics. Athletes reporting frequent RTIs had significantly lower serum CC16 and the risk of frequent RTIs was more than 2-fold higher in athletes with low serum CC16 . Athletes had significantly higher anti-adenovirus IgG than healthy controls while only non-atopic athletes had anti-parainfluenza virus IgG significantly lower than controls. In all athletes weak correlation of serum CC16 and anti-parainfluenza virus IgG was present . In atopic athletes a weak positive correlations of CC16 with IgG specific for respiratory syncytial virus , parainfluenza virus  and adenovirus  were seen as well.Regular high-load exercise is associated with decrease in serum CC16 levels. Athletes with decreased CC16 are more susceptible to respiratory infections. Atopy may be an additional factor modifying susceptibility to infections in subjects performing regular high-load exercise. Strenuous exercise characteristic of endurance sports is believed to have a detrimental effect on the integrity of the structure of the respiratory epithelium. Such damage may lead to increased bronchial hyperresponsiveness to non-specific stimuli and contribute to the increased prevalence of exercise-induced bronchoconsriction observed in elite endurance athletes -4. EndurVarious proteins have been assessed as potentially useful tools in monitoring airway inflammation and epithelial damage -9. ClaraChanges in serum CC16 levels were observed after acute, short-lasting exposure to exercise or provocative or irritating stimuli. Non-specific challenges with exercise and eucapnic voluntary hyperpnea (EVH) resulted in increased urinary CC16 levels ,13. In aChronic airway inflammation may also be associated with shifts in local and systemic presence of CC16. During pollen season, CC16 in nasal lavage fluid is decreased in subjects with birch pollen-induced allergic rhinitis . DecreasThe aim of this study was to investigate the airway epithelium inflammation and potential damage in competitive athletes, as reflected by changes in serum CC16 levels. Secondly, we sought to identify possible associations of serum CC16 level with pattern of exercise, asthma or allergy, frequent respiratory infections and the immune response to respiratory pathogens reflected by pathogen-specific IgG levels in the serum.2POLO) which was a part of the multicentre Olympic Study performed within the framework of GA2LEN . One hundred and forty-six (71.9%) of the recruited athletes were eventually included into the Polish Olympic Team for the Beijing games. The athletes represented 21 sport disciplines which, according to the predominant pattern of exercise, were divided into endurance (80 athletes) and non-endurance (123 athletes)  preparing for the Olympic Games in Beijing in 2008 who were included into Allergy and Asthma in Polish Olympic Athletes study  and Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines ,19.Two control groups of subjects who had never performed sport at a competitive level were recruited as a reference for serum concentrations of lung specific protein. The first group consisted of 53 healthy subjects without history of allergy or any pulmonary condition whereas the second control group comprised 49 allergic non-smoking mild asthmatics with well controlled symptoms, diagnosed according to GINA  and receThe following subgroups of athletes were distinguished for the purpose of further analysis: athletes diagnosed with allergic rhinitis (n = 30); athletes diagnosed with allergic asthma with or without concomitant rhinitis (n = 27); and athletes without asthma or allergy (n = 146), basing on the diagnosis established during clinical evaluation of athletes. The data from an Allergy Questionnaire for Athletes (AQUA) were analysed separately.Data regarding type of sport, allergy symptoms and history of respiratory infections, as well as demographical data, were acquired from the Allergy Questionnaire in Athletes (AQUA©) which was filled by each athlete entering the study. The AQUA questionnaire, developed and validated by Bonini et al  in ItaliSpirometry was performed according to criteria defined by a joint ERS/ATS task force  using a g for 10 minutes and serum was transferred to cryotubes and frozen at –80°C for further analyses.Serum samples were obtained from 86% (n = 119) non-endurance and 94% (n = 75) endurance athletes. The remaining athletes were not available for blood sampling due to training schedules. Blood was drawn into Monovette tubes for serum . Each sample was left to clot at room temperature for ca. 30 minutes. Subsequently, samples were centrifuged at 700 CC16 concentration was determined using Human Clara Cell Protein ELISA kits  in accordance with instructions provided by the manufacturer. Samples were run in duplicate at a 25-fold dilution, following the manufacturer’s recommendations. The lower CC16 detection limit is 20 pg/ml.The serum concentration of IgG antibodies against parainfluenza virus 1,2 and 3 (PIV) was determined by commercially available ELISA kit . The threshold value for positive anti-PIV IgG testing was 10 U/ml.Mycoplasma pneumoniae were determined by commercially available semi-quantitative ELISA kits  The threshold value for positive testing was 25 Enzyme Immunoassay Units (EIU).The serum concentrations of IgG against respiratory syncytial virus (RSV), adenovirus and Concentrations of serum IgG’s against repiratory pathogens were determined in athletes and healthy control groups only.U test or Kruskal-Wallis test with Dunn’s post hoc test were used where applicable. Correlations of age, total IgE, lung function parameters, reversibility challenge results and levels of IgG against respiratory pathogens with serum CC16 level were assessed with Spearman’s rank correlation test.Statistical analyses were performed using Statistica  and GraphPad Prism  software. The Kolmogorov-Smirnov test with Lilliefors correction was used to check for normality of values’ distribution. To identify differences between groups, the Mann-Whitney M. pneumoniae.The chi-square test with Yates’ correction was used for between-the-group comparisons of percentage of positive tests for IgG against the presence of PIV, RSV, adenovirus or Relationships between serum CC16 level and AQUA score value with the presence of asthma/allergy symptoms and frequent respiratory infections were analysed using logistic regression.P value less than 0.05 was considered statistically significant in all analyses.A Upon clinical evaluation, asthma was diagnosed in 27 athletes (13.3%) of whom 15 (55.6%) had concomitant allergic rhinitis, whereas allergic rhinitis (without concomitant asthma) - in 30 subjects (14.8%).χ2 test).An affirmative answer to the question “Do you frequently suffer from upper respiratory infections  or fever?” was given by 39 athletes (19.2%). No differences in the prevalence of self-reported frequent infections were seen between atopic and non atopic athletes  and any of the control groups – Table U test).Serum CC16 concentration was measured in 75 endurance and 123 non-endurance athletes and in two control groups of non-athletes. The mean serum CC16 concentration was significantly lower in the “all-athletes” group than in healthy controls and asthmatic controls – Figure U test) – Figure Levels of serum CC16 did not differ significantly between atopic and non-atopic athletes . No association was found between CC16 levels and self-reported asthma symptoms or between serum CC16 and physician-diagnosed asthma or allergic rhinitis. However, in athletes reporting frequent upper respiratory infections, the median serum CC16 levels were significantly lower than in the remaining athletes  as an independent variable showed that the risk of frequent URTIs, as reported in the AQUA questionnaire, was significantly higher in athletes with low serum CC16 . Also, the risk of declared nasal symptoms, not identical with physician-diagnosed rhinitis, was higher in athletes with serum CC16 ≤ 4.99 ng/ml .No association was found between a serum CC16 level lower than 4.99 ng/ml and diagnosis of allergic rhinitis, diagnosis of asthma, declared cough and/or dyspnoea without physician-confirmed asthma, presence of atopy or positive serological test for any respiratory pathogen. Logistic regression analysis using ‘serum CC16 ≤ 4.99 ng/ml’ as a dependent variable did not show decreased serum CC16 levels to be determined either by predominant endurance exercise nor by length of the single training session.M.pneumoniae (but not for antiadenovirus) antibody, was significantly lower among athletes than among healthy subjects. The mean serum concentration of Anti-PIV IgG levels was significantly lower, whereas concentration of anti-AdV IgG was significantly higher in professional athletes - Table The prevalence of positive serological testing for anti-PIV, anti-RSV or and anti- The percentage of positive serological tests and median serum levels of IgG against respiratory pathogens did not differ between athletes declaring frequent URTIs in AQUA questionnaire and the remaining group - Table In all athletes, a weak yet significant positive correlation of serum CC16 and IgG concentration was observed only for PIV  but not for the remaining pathogens. In atopic athletes, positive correlations were observed between the levels of serum CC16 and IgG against RSV , PIV  and Adenovirus . No significant correlations were observed in healthy controls.Mycoplasma pneumoniae IgG levels (data not shown).Higher anti-adenovirus IgG titres were found in both atopic and non-atopic athletes as compared to HC  - Figure .5-64.8 aM. pneumoniae – data not shown.When the results of the positive serological testing were analysed, a significantly lower percentage of subjects with positive anti-RSV serology was observed in non atopic athletes (60.8%) than in healthy controls  and atopic athletes . The presence of a positive RSV serology test was significantly associated with atopy . Such associations were not observed regarding serological testing for IgG against PIV, adenovirus and This study documents that serum CC16 concentrations are significantly lower in top athletes than in control subjects from the general population. Decreased serum CC16 concentrations were reported in infants and children regularly visiting indoor pools ,23 but oCC16 is a protein of multifaceted characteristics. It may be considered both as a marker of the disruption of airway epithelial barrier integrity and as a co-participant in anti-inflammatory response. Degree of CC16 involvement and significance in the aforementioned processes is still a matter of research and dispute.per se that stimulates airway inflammation, irrespective of training intensity. Reduced presence of CC16, an anti-inflammatory factor, may additionally contribute to airway inflammation and hyperresponsiveness.Our results suggest, that it is an intensive and regularly performed exercise In addition, the control non-athletes asthmatic patients were found to have CC16 levels similar to those in healthy controls, which contrasts with the results of other report, documenting a significant decrease of serum CC16 in asthma . Our datAlthough recent data published by Jacobs et al  show thaAnother novel observation is that athletes declaring frequent upper respiratory tract infections had significantly lower serum CC16 concentrations than those without frequent infections - Figure The common belief that moderate exercise is beneficial to one’s health whereas excessive physical training may considerably increase the susceptibility to infection was confirmed by the concept of the U-shaped relationship between physical activity and resistance to disease . Our stuM. pneumoniae in professional athletes compared to controls may be interpreted as indirect evidence for impaired immunocompetence resulting from continuous high exercise load. In a mouse model, CC16 was shown to influence the lymphocyte response of the airways after infection with RSV. Mice lacking CC16 demonstrated significantly higher concentrations of Th2 cytokines IL-5 and IL-13 in BALF for several days following the infection [Less frequent positive serological test results against PIV, RSV and nfection . The stiper se influences the immune response to respiratory viruses.The frequency of positive immune response to RSV was comparable between atopic athletes and healthy control subjects, but it was significantly lower in non-atopic athletes. In addition, correlations between serum CC16 level and intensity of immune response to respiratory viruses  were only significant in atopic athletes. Given that serum CC16 levels in athletes were not seen to be modified by atopy status, it may suggest, that atopy Our study has several limitations. The study group was quite heterogeneous in terms of predominant pattern of exercise and training environment not allowing for identification of possible effects of any particular sport on CC16 or immune response against respiratory pathogens. Accordingly, no standardised and comparable measures of exercise load/intensity could be included into analysis. Furthermore, the AQUA question concerning respiratory infections does not determine their actual frequency, allowing only for the subjective assessment of whether the URTIs are frequent or not.Another limitation of our study results from the inter-subject variability of CC16 levels. Overlaps in the serum CC16 are observed between the groups and it could be argued that outliers influence the overall result. Significant differences were shown through non-parametric tests which makes possibility of such influence less likely. However, this issue should be considered possible confounder and addressed in next studies on CC16 in larger populations.For the purpose of logistic regression analysis, 5.0 ng/ml was set arbitrarily as the CC16 threshold value, based on the reference value given by the manufacturers of the ELISA kits. However, the CC16 levels in healthy subjects are variable and the accepted reference values for CC16 are actually lacking, which substantially hinders such analysis and signals the need for further assessment of CC16 levels in various clinical models ,24,28.CC16 gene polymporphism with airway hyperresponsiveness and asthma phenotype [Bronchial hyperreactivity was assessed only in few patients (due to their training schedules and low accessibility) and could not be analysed in this study. However, of the role of CC16 in the context of bronchial hyperresponsiveness may be important for understanding the pathogenesis of EIB, especially in view of the data showing association of henotype .Our study demonstrated that regular high-load exercise training is associated with a decrease in serum CC16 protein levels and lower CC16 levels in competitive athletes are associated with increased prevalence of reported respiratory infections. Further studies are necessary to elucidate the mechanism of association between increased susceptibility to respiratory infections in top athletes and epithelial cell injury markers such as CC16.The authors declare they have no competing interests regarding this article.MK and MLK participated in conception and design of the study, in analysis and interpretation of data, and in manuscript preparation; MK, JJ and HK participated in subjects’ recruitment and clinical assessment; MJ and SM performed laboratory measurements; JSM recruited and assessed subjects from control groups. All authors read and approved the final manuscript.