Alcohol has been implicated in the high mortality in Central and Eastern Europe but the magnitude of its effect, and whether it is due to regular high intake or episodic binge drinking remain unclear. The aim of this paper was to estimate the contribution of alcohol to mortality in four Central and Eastern European countries. We used data from the Health, Alcohol and Psychosocial factors in Eastern Europe is a prospective multi-centre cohort study in Novosibirsk (Russia), Krakow (Poland), Kaunas (Lithuania) and six Czech towns. Random population samples of 34,304 men and women aged 45–69 years in 2002–2005 were followed up for a median 7 years. Drinking volume, frequency and pattern were estimated from the graduated frequency questionnaire. Deaths were ascertained using mortality registers. In 230,246 person-years of follow-up, 2895 participants died from all causes, 1222 from cardiovascular diseases (CVD), 672 from coronary heart disease (CHD) and 489 from pre-defined alcohol-related causes (ARD). In fully-adjusted models, abstainers had 30–50 % increased mortality risk compared to light-to-moderate drinkers. Adjusted hazard ratios (HR) in men drinking on average ≥60 g of ethanol/day (3 % of men) were 1.23 (95 % CI 0.95–1.59) for all-cause, 1.38 (0.95–2.02) for CVD, 1.64 (1.02–2.64) for CHD and 2.03 (1.28–3.23) for ARD mortality. Corresponding HRs in women drinking on average ≥20 g/day (2 % of women) were 1.92 (1.25–2.93), 1.74 (0.76–3.99), 1.39 (0.34–5.76) and 3.00 (1.26–7.10). Binge drinking increased ARD mortality in men only. Mortality was associated with high average alcohol intake but not binge drinking, except for ARD in men.The online version of this article (doi:10.1007/s10654-015-0092-8) contains supplementary material, which is available to authorized users. Despite decades of research, the relation between alcohol intake and mortality is not entirely clear. Most observational studies have shown a J-shaped association, with mortality lowest among light/moderate drinkers and higher risk among abstainers and heavy drinkers –4. UncerThe harmful effects of alcohol and drinking patterns are an important public health issue, and it is particularly pertinent for Central and Eastern Europe (CEE) and the former Soviet Union (FSU). The estimates of alcohol intake and alcohol-related mortality in the region are high , alcoholIn this report, we analysed data from a prospective cohort of a well -characterized general population sample in four countries of the region in order to investigate the association between volume, frequency and pattern of drinking with mortality from all causes, CVD, CHD and a priori defined category of alcohol-related causes of death.The Health, Alcohol and Psychosocial factors In Eastern Europe (HAPIEE) study is a multi-centre prospective cohort study conducted in Novosibirsk (Russia), Krakow (Poland), Kaunas (Lithuania) and six middle-sized Czech towns . Random The study was approved by Institutional Ethics Review Boards locally and at University College London and University College Hospital. All participants gave written informed consent.Follow-up of participants was available to 31 December 2010 in Novosibirsk and Krakow, 31 December 2011 in Czech towns and 31 December 2012 in Kaunas. Deaths were identified through record linkage with national (Czech) or local mortality registers, which recorded date of death and underlying cause of death (ICD-10 coded) . In the Participants who were lost to follow-up because they moved away were censored at their last date of contact. In addition to all-cause mortality, causes of death were divided into deaths from CVD (ICD-10 codes I00-99), CHD (ICD-10 codes I20-25), and pre-specified alcohol-related causes as previously defined by Zaridze et al. ; this grAt baseline, participants completed an extensive structured questionnaire, underwent a brief examination in a clinic, and provided a fasting blood sample. Alcohol consumption in the last 12 months was self-reported using the graduated frequency questionnaire (GFQ) , containSeveral alcohol indices were derived from the harmonised GFQ for the main analyses, with the following cut-offs in men/women: (1) average daily alcohol intake ; (2) drinking frequency ; (3) drinking pattern, combining information on drinking frequency with quantity per occasion: light drinker (≤2/0.5 drinks per occasion), moderate drinker (≤4/2 drinks per occasion), occasional heavy drinker (>4 drinks per occasion less than once a week), and regular heavy drinker (>4 drinks per occasion more than once a week); and (4) binge drinking (≥100/60 g of ethanol in one episode at least monthly).For all alcohol measures, participants who did not report any alcohol consumption in the past year were categorized as non-drinkers. Information on past drinking was also available in one of the centres (Novosibirsk). Additional measures on weekly alcohol intake and alcohol intake in the last 3 months from the food frequency questionnaire (FFQ) administered separately were strongly correlated with the GFQ data and therefore were not used in this report. Repeated measures of alcohol consumption, collected on average 3.5 years after the baseline examination, were available in all centres except Kaunas. Alcohol indices measured at re-examination, used in sensitivity analysis, were strongly correlated with alcohol measures at baseline, suggesting considerable stability of alcohol consumption over time. In addition, serum GGT was available for nearly all Russian participants and subsample of 4778 participants in the remaining centres, and showed expected associations with self-reported alcohol consumption in both genders.Covariates, measured at baseline, included study centre, age, education ; current economic activity ; marital status (married/cohabiting vs. single/separated/divorced/widowed); household asset index, as the number of items owned ; subjective hardship score ; smoking status ; leisure-time physical activity (none vs. some), high depressive symptoms (yes vs. no) measured by the CESD -10;  BMI from clinical examination, and self-reported history or presence of major chronic conditions .Means with standard deviations for continuous covariates and proportions, expressed as percentages, for categorical covariates were used to describe baseline characteristics of the cohort, separately for men and women.Cox regression was used to estimate hazard ratios (HR) with 95 % confidence intervals (CIs) for associations between alcohol measures and each mortality end-point using time-on-study as the time scale. Lowest-level drinkers were used as reference category. For binge drinking, non-binge drinkers were used as reference category. All analyses were conducted separately for men and women. We first conducted analyses separately for each centre; after checking for interactions between centres and alcohol, data from all cohorts were combined .For each combination of alcohol measures and mortality end-points, the first Cox model was adjusted only for age and centre, and the second model was additionally adjusted for all remaining covariates. We also repeated the analyses excluding participants with major chronic conditions at baseline but the results were similar. For each Cox model, proportional hazards assumption was checked by including interactions with log(time) and by examination of Schoenfeld residuals. A suggestion of violation of proportional hazards assumption with age was dealt with by including its interaction with time but this had no appreciable effect on associations between alcohol and mortality.Missing data on baseline covariates were filled in using multiple imputations by chained equations implemented in Stata 13. Imputation models included vital status, alcohol, covariates and a small number of auxiliary variables, predictive either of missingness or values of incomplete variables, and the Nelson-Aalen estimator of cumulative baseline hazard . Ten impOver the median follow up of 6.9 years , 2895 deaths from all causes occurred among 34,304 participants included in the analysis; about 43.0 % of all deaths were from CVD n = 1222), 672 deaths were from CHD and 489 from pre-specified alcohol-related conditions . Results for the Czech and Polish cohorts vs. the Russian and Lithuanian cohorts are shown in Supplementary Tables 8 and 9; the differences between these two sets of populations were not statistically significant. In the Russian cohort, where information on former drinking was available, the excess risk in non-drinkers was larger in female  former drinkers (Supplementary Table 10).Using the prevalence of heavy drinking and the associated adjusted hazard ratios, the population attributable risk fractions (PARF) for all-cause mortality were 2.8 % in men and 5.5 % in women; PARF calculated under different assumptions for all cohorts combined and for the Russian cohort are shown in Supplementary Table 11. Finally, we estimated how much of the hazard ratios for mortality in the Russian cohort compared to the Czech cohort can be explained by alcohol. In men, the age-adjusted HR was 1.92 [1.69–2.18] and 1.88 [1.65–2.14] after additionally controlling for alcohol consumption; in women, these two HRs were 1.21 [1.01–1.45] and 1.23 [1.02–1.48].This prospective study in well characterized middle-aged and older Central and Eastern European participants found associations between high alcohol intake and increased mortality from all causes and CVD in both men and women. Binge drinking was significantly associated with ARD in women but not with CVD and total mortality in either gender. Due to the low proportions of heavy drinkers in these cohorts, population risk fractions of mortality attributable to alcohol were modest, under 6 %. Alcohol did not seem to explain differences in all-cause mortality between cohorts.Several limitations of this study should be considered when interpreting the results. First, these urban population samples are not necessarily representative of whole countries. That said, their mortality rates and trends and risk factors are similar to national data –24 and tThird, measurement of alcohol consumption is notoriously difficult; this study, similarly to most other studies, relied on participants’ self-report. Although some underreporting is likely, particularly among women , 27, ourFourth, as the participants in these cohorts were 45 years old and older and, as drinking in most populations declines with age, there were fewer heavy drinkers than in the younger age groups. The prevalence of heavy drinking, however, was similar to that reported by the Zaridze et al. , as wellAs well as limitations, our study also has important strengths. First, the study participants were well characterized, with detailed assessment of alcohol intake and potential confounders, repeated assessment of alcohol consumption and with one  biomarker associated with alcohol. This provides confidence that, even if absolute alcohol intakes were underestimated, the rank of participants by intake is reasonably reliable. Second, this study included four population samples with different mortality rates and different drinking patterns. The consistency of the results across cohorts supports the reliability of the findings. Third, since we had access to death registers covering the whole population, the follow up is virtually complete. Due to differential access to register data, the length of follow up differs between cohorts but this should not affect the results.Our findings are consistent with the evidence that both non-drinkers and heavy drinkers have increased risk of all-cause and cardiovascular mortality –4. RegarIncreased mortality risk in non-drinkers relative to light/moderate drinkers observed in our study is consistent with the J-shaped dose–response association between daily alcohol consumption and mortality which has been widely reported by observational studies . This coIt is not clear how the findings from cohort studies relate to higher estimates of both absolute and relative effects of alcohol in Russia reported in case–control studies,  particulThe absence of an effect of drinking pattern is noteworthy. There is a growing literature suggesting that, in addition to regular heavy drinking, episodic heavy drinking is associated with increased total and CVD mortality , 6, 34. Most previous studies on alcohol in Eastern Europe focused on males. Our findings and the studies by Zaridze et al. , 14 suggIt has been suggested that some CVD deaths in Russia could be wrongly coded alcohol poisoning . For thiAn important issue is the population impact of alcohol on mortality. Alcohol has been widely seen as a major cause of premature deaths in Russia, particularly at younger ages , 37, 38.There is no doubt that alcohol is an important cause of mortality in Eastern Europe and globally, and the associations of alcohol with mortality in this study were broadly in line with those reported elsewhere. It remains uncertain, however, whether the high long-term mortality rates of middle aged and older persons in Russia are caused predominantly by alcohol and what is the contribution of other factors.Supplementary material 1 (docx 206 kb)


Vaccination against seasonal influenza (SI) is recommended for healthcare workers (HCW) in acute and long-term care facilities. Since vaccination cannot be made mandatory in Switzerland, an alternative was implemented since 2012 at HUG: HCW were obliged to be vaccinated or to wear a mask in ward corridors and patient rooms during SI epidemics. We describe the 20 years’ experience at HUG.We describe the 20 years’ experience at HUG.From 1994, data are collected annually about vaccinated HCWs at HUG. In addition, regular audits are performed to assess compliance with recommendations. Since 2012, during annual SI epidemics, audits recorded compliance of HCWs with a badge meaning “Vaccinated” and those with correct use of a mask over several 2 week-periods. Compliance was assessed as follows: (number of HCWs wearing a colored badge + number of HCWs wearing a mask correctly)/number of HCWs observed = number of compliant HCWs/number of HCWs observed, expressed as a percentage.In 1994, 14% of HCWs were vaccinated against SI. Following an investigation, information campaigns, lectures about influenza and the influenza vaccine, and on-site vaccination were offered from 1995 to 2008. During this period, the proportion of vaccinated HCWs’ varied between 21% and 27%. In 2009, HCWs had to choose between vaccination attested with a badge or correct mask wear. In 2009, the year of H1N1 pandemic, 49% of HCWs were vaccinated, in 2010 27%, in 2011 29%.Since winter 2012, HCWs vaccinated against SI wear a badge with the text "I am vaccinated to protect you" and those who were not vaccinated wear a badge with the text "I wear a mask to protect you". Of 1390 HCWs observed in winter 2012, 469 wore a badge or mask . In winter 2013, 2070/2937 observed HCWs were compliant (70.5%), in winter 2014, 3516 /4459 observed HCW (70.5%). Data collection is in progress for winter 2015. In parallel, we observed an increase of vaccinated HCW: 46.6% in winter 2015.Offering the choice between vaccination and mask wear, associated with mandatory badge wear and assessment of mask compliance respectively, can be an effective approach to increase HCW vaccination rate against SI.None declared.


Obstructive sleep apnea (OSA) is a chronic, multisystem disorder that has a bidirectional relationship with several major neurological disorders, including Alzheimer's dementia. Treatment with Continuous Positive Airway Pressure (CPAP) offers some protection from the effects of OSA, although it is still unclear which populations should be targeted, for how long, and what the effects of treatment are on different organ systems. We investigated whether cognitive improvements can be achieved as early as one month into CPAP treatment in patients with OSA.55 patients (mean (SD) age: 47.6 (11.1) years) with newly diagnosed moderate–severe OSA (Oxygen Desaturation Index: 36.6 (25.2) events/hour; Epworth sleepiness score (ESS): 12.8 (4.9)) and 35 matched healthy volunteers were studied. All participants underwent neurocognitive testing, neuroimaging and polysomnography. Patients were randomized into parallel groups: CPAP with best supportive care (BSC), or BSC alone for one month, after which they were re-tested.One month of CPAP with BSC resulted in a hypertrophic trend in the right thalamus , which was absent in the BSC group . Significant improvement was also recorded in ESS, in the CPAP plus BSC group, following treatment , correlated to neuroplastic changes in brainstem , and improvements in delayed logical memory scores .One month of CPAP treatment can lead to adaptive alterations in the neurocognitive architecture that underlies the reduced sleepiness, and improved verbal episodic memory in patients with OSA. We propose that partial neural recovery occurs during short periods of treatment with CPAP. •One month of CPAP treatment leads to adaptive alterations in the neuroanatomy and neurocognition in patients with OSA.•Improvements in sleepiness and verbal episodic memory were demonstrable following only one month of treatment with CPAP.•Our findings support the clinical rationale for the use of CPAP treatment to relieve sleepiness, even for a short duration.Obstructive sleep apnea (OSA) is a highly prevalent sleep disorder that has been linked with dementia, stroke and increased risks of cardiovascular disease. Continuous positive airway pressure (CPAP) treatment has been shown to be effective in reducing sleepiness in moderate to severe OSA. However, the evidence for its use to reverse or slow the rate of cognitive decline is unclear. Our findings show that one month of CPAP treatment in patients with moderate to severe OSA leads to adaptive alterations in the brain networks associated with reduced sleepiness and improved memory. It has Numerous clinical studies over the years have demonstrated changes in the central nervous system (CNS) of patients with OSA, including altered resting cerebral blood flow pattern  with hypThe additive impact of progressive changes in sleep quality and structure, changes in cerebral blood flow, neurovascular and neurotransmitters, plus the cellular redox status are all likely to contribute to the cognitive deficits reported in up to one out of four newly diagnosed OSA patients . DespiteIn a recent study of patients with OSA, augmentation of subjective experience, attention and vigilance has been demonstrated after only one night of CPAP . However22.1Patients with newly diagnosed OSA (18–65 years old) were recruited from Royal Brompton and Harefield Hospitals' sleep clinics. Inclusion criterion was an apnea/hypopnea index (AHI) > 10 events/h . Apneas The same exclusion criteria were used for healthy controls (age- and education-matched) recruited from a database of healthy volunteers. Those with a history of sleep problems on questionnaires, or evidence of OSA on pulse-oximetry (ODI > 5 events/h) were excluded.All enrolled patients were randomly allocated , by an independent study coordinator, to receive CPAP with best supportive care (BSC), or BSC alone, for one month and then re-assessed .The study was part of an ongoing research to investigate the impact of OSA on the brain and was approved by the UK central research ethics committee (10/H0706/51). All patients gave written informed consent.2.2CPAP treatment was initiated using standard clinical practice at each center .BSC comprised of advice on minimizing daytime sleepiness through sleep hygiene, naps, caffeine, exercise and weight loss as appropriate to each patient. Both groups were provided with BSC and asked to continue with their usual medical care during the trial.2.3Structured assessments were performed at baseline (patients and controls) and a follow-up after one month (patients only). In addition, all patients received a telephone call at 1 week to record symptoms, side-effects, and to optimize CPAP adherence. All OSA patients completed an inpatient polysomnography  prior to CPAP initiation. Domiciliary overnight pulse-oximetry  was performed at one month. Treatment compliance was measured objectively by download of the CPAP smart card during the one-month visit.2.4Cognitive function of all participants was assessed using a battery of tests comprising the Addenbrooke's Cognitive Examination-Revised (ACE-R) , Trail MACE-R is a brief battery that provides evaluation of six cognitive domains  . It is uThe TMT consists of two parts, A and B; it is one of the most widely used neuropsychological tests that provides information on visual conceptual and visuomotor tracking, motor speed, attention and executive functions . It is aWechsler Adult Intelligence Scale, third edition (WAIS-III)  is a com2.5All participants underwent MR imaging and T1-weighted MR-images were acquired using a 1.5 T scanner  and a 3D MP-RAGE sequence  with contiguous 2 mm coronal slices. The T1-weighted images were processed, and volumetry was performed using the automated method FreeSurfer, as previously described by our group .2.6t-test was applied (http://www.statsoft.com).The Kolmogorov–Smirnov test was used to test the normality of distributions. To analyze differences in a variety of demographic parameters between healthy controls and OSA patients, Student's  applied . All stat-test, P = .514) and Student's t-test was done on the ICV normalized data (i.e. volume/ICV) to assess between-group differences (Supplementary Table S2). Hierarchical cluster analysis (HCA) .Investigation of a priori hypotheses was focused on group differences for several neuroanatomical structures that had been previously highlighted by our data and that of other groups, to present network hubs of OSA-affected neurocircuitry: the hippocampus, amygdala, basal ganglia, thalamus, brainstem, corpus callosum (and its subdivisions) and cerebellum . In addiis (HCA)  was furtis (HCA) . The clupre − V/domainpost] / V/domainpre × 100). All results were Bonferroni corrected for multiple comparisons. Finally, partial correlations  and verbal episodic memory (delayed LM test scores) were explored with Pearson correlations; controlled for ICV and normalized for differential changes (diff ∆%) from the baseline (diff ∆ =  and NIHR Respiratory and Cardiac Biomedical Research Units, at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London. The funders had no role in study design, data collection, data analysis, interpretation or writing of the report.


Understanding the underlying pleiotropic relationships among quantitative traits is necessary in order to predict correlated responses to artificial selection. The availability of large-scale next-generation sequence data in cattle has provided an opportunity to examine whether pleiotropy is responsible for overlapping QTL in multiple economic traits. In the present study, we examined QTL affecting cattle stillbirth, calf size, and adult stature located in the same genomic region.A genome scan using imputed whole genome sequence variants revealed one QTL with large effects on the service sire calving index (SCI), and body conformation index (BCI) at the same location (~39 Mb) on chromosome 6 in Nordic Red cattle. The targeted region was analyzed for SCI and BCI component traits. The QTL peak included LCORL and NCAPG genes, which had been reported to influence fetal growth and adult stature in several species. The QTL exhibited large effects on calf size and stature in Nordic Red cattle. Two deviant haplotypes (HAP1 and HAP2) were resolved which increased calf size at birth, and affected adult body conformation. However, the haplotypes also resulted in increased calving difficulties and calf mortality due to increased calf size at birth. Haplotype locations overlapped, however linkage disequilibrium (LD) between the sites was low, suggesting that two independent mutations were responsible for similar effects. The difference in prevalence between the two haplotypes in Nordic Red subpopulations suggested independent origins in different populations.Results of our study identified QTL with large effects on body conformation and service sire calving traits on chromosome 6 in cattle. We present robust evidence that variation at the LCORL and NCAPG locus affects calf size at birth and adult stature. We suggest the two deviant haplotypes within the QTL were due to two independent mutations.The online version of this article (doi:10.1186/s12863-015-0202-3) contains supplementary material, which is available to authorized users. Genetic correlation between two complex (quantitative) traits may be due to pleiotropy, i.e. the same polymorphism affecting both traits, or linkage disequilibrium (LD) between loci affecting individual traits. Genome-wide association studies (GWAS) have identified many genetic loci that harbor variants associated with multiple traits, which highlights the traits sharing common genetic pathways, and emphasizes the relevance of pleiotropy in complex traits . TherefoIn a genome scan using whole genome sequence variants, we discovered QTL with a large effect on the service sire calving index (SCI), and body conformation index (BCI) at the same location (~39 Mb) on chromosome 6 (BTA6) in a Nordic Red cattle population  variants for Nordic Red cattle bulls was done in two steps. First, high density (HD) genotypes were imputed from 50 k genotypes using HD multi-breed reference genotype data. Then the imputed HD genotypes were further imputed to WGS variants using a multi-breed WGS reference. Genome-wide association analysis with the imputed WGS variants was done in two steps: 1) using a linear mixed model without considering the relationship among all individuals except half-sib family (sire-son) relationships and 2) followed by linear mixed model (LMM) analysis within the targeted region considering relationships among all of the bulls. A genome-wide scan with WGS variants using LMM considering the relationships among all individuals is computationally prohibitive. Haplotypes were constructed using top associated markers assuming they all had high linkage disequilibrium with the causal variant and haplotype based association was carried out using LMM analysis.http://www.nordicebv.info.No animal experiments were performed in this study, and, therefore, approval from the ethics committee was not required. The study was conducted in Nordic Red cattle (RDC) from Denmark (RDCDNK), Finland (RDCFIN), and Sweden (RDCSWE). The number of progeny tested bulls with breeding values for traits and genotypes is provided in Table BCI) is a compound index describing a sire’s genetic potential to produce desirable characteristics for various body measures in progeny. It is predicted as a linear combination of breeding values for stature, body depth, chest width, dairy form, top line, rump width, and rump angle. The definition of conformation traits used in this study was consistent with the International Committee for Animal Recording (ICAR) standards (http://www.icar.org/Documents/Rules%20and%20regulations/Guidelines/Guidelines_2012.pdf). The type traits were scored using a linear scale from 1 – 9 in Denmark, Sweden, and Finland. These records were corrected for calving age, lactation number, interval from calving, and optimum values fixed for each individual trait for each breed. In the joint Nordic evaluation for conformation traits, data from Finland, Sweden and Denmark were included.The indices for linear conformation traits describe different aspects of cow conformation. The linear traits were combined into trait groups describing the overall body form. The body conformation index (CI) is a compound index reflecting a sire’s additive genetic effects for calving ease and calf survival in progeny. SCI was predicted by combining service sire breeding values for the following: 1) stillbirth in first (SSBF) and later (SSBL) lactations; 2) calving ease in first (SCEF) and later (SCEL) lactations; and 3) calf size in first (SCSF) and later (SCSL) lactations. We followed Boelling et al. to estimate breeding values for calving traits in Denmark, Sweden, and Finland [Service sire calving index (S Finland . The her Finland .Accuracies (r) of de-regressed proofs used in this study were high. Average accuracies for DRPs ranged from 0.73 (chest width) to 0.89 (stature) for type traits. Average accuracies ranged from 0.57 (SCSL) to 0.75 (SSBF) for SCI component traits. For example, the DRP accuracy distribution for trait stature is presented in Additional file 2-test, 1 df, P < 0.00001) were excluded. The SNP number after quality control was 43,415 in the 50 k dataset. In addition, a multi-breed reference of 2,036 genotypes  using the Illumina BovineHD Genotyping BeadChip was available in-house, and from the EuroGenomics consortium [Genomic DNA was extracted from whole blood or semen. Bulls were genotyped using the Illumina Bovine SNP50 BeadChip  version 1 or 2. The SNP selection quality parameters were an 85% minimum call rate for individuals and 95% for loci. Marker loci without a known map position, with minor allele frequencies (MAFs) < 5%, and deviation from Hardy Weinberg proportions  < 0.90 were removed. The total marker number for chromosomes 1–29 was 8,938,927. The imputation accuracy for this data was reported earlier [The 242 dairy cattle whole genome sequences were from Aarhus University, and the 1000 bull genomes project. The reference included 132 Holstein, 42 Jersey, 52 Nordic Red, and 16 Brown-Swiss bulls . Markers10  = 8.25).A genome scan was conducted for BCI and SCI using a sire model without considering relationships among individuals other than between sire and son. The model is described in the Additional file j is the phenotype (de-regressed EBV) for individual j, μ is the overall mean, b is the allelic substitution effect, xj is the number of copies of an allele (with arbitrary labeling) of the SNP count in individual j , uj are random polygenic effects, рk is the effect of the k-th population , and ei is the random environmental deviate for individual j. The vector u = {uj} follows a multivariate normal distribution A is the additive relationship matrix, and e = {ej} follows a multivariate normal distribution W is the diagonal matrix containing weights of de-regressed estimated breeding values. The weight wj for individual j is estimated as rj2 is the reliability of the de-regressed EBV of individual j. Values of t-test against a null hypothesis of H0: b = 0, and the same Bonferroni multiple testing corrected significance threshold  = 8.25) was used as in the genome scan  was applied for association analyses of the BTA6 targeted region for the two indices (BCI and SCI), and the component traits Table . The asssoftware . DMU wasmentclass2pt{minimqh1j and qh2j are random effects of the two haplotypes carried by the j-th individual. Each haplotype effect is assumed independent and identically distributed (i.i.d), with a normal prior distribution 2 distribution with 1 df. The analysis was performed using the DMU software package [Haplotypes were constructed for markers selected from LMM analyses using BEAGLE software . We used package .Tests were conducted to examine if a significant candidate SNP or haplotype alone was sufficient to explain the variation resulting from a QTL region. Analyses were repeated with incorporation of the candidate SNP or haplotype as a fixed effect. If other SNPs or haplotypes remained significant in the presence of the new systematic effect, the original candidate alone was not sufficient to account for the entire effect of the QTL region.10  = 57.5, Figure The genome-wide associations scan using the sire model  was 89.4  = 57.9). This SNP was also the most significantly associated SNP for calf survival, i.e. stillbirth in first (SSBF) and later (SSBL) lactations. The SNP located at 38,956,499 bp showed the most significant association with calf size and calving ease. The allele substitution effect of this SNP on calf size and calving ease exhibited an inverse association, i.e. the allele increasing calf size at birth increased calving difficulty. The SNP most significantly associated with body conformation was at 38,914,033 bp  = 80.4). It was located 213,330 bp from the SNP most associated with SCI. The SNP most significantly associated with stature was at 38.13 Mb  = 81.9). However, a number of SNPs around 39.77 Mb showed a very strong association with stature  = 80.6).The Linear Mixed Model (LMM) analyses for conformation and calving indices and their component traits revealed overlapping peaks in the targeted region on BTA6 (~37.5-40.5 Mb). The most significant associated SNPs from LMM for calving, conformation and the component traits are presented in Table 10  at ~106  at ~80  for the targeted region among all traits analyzed was observed for SCSL  and later (SCEL) lactations, and calf size in first (SCSF) and later (SCSL) lactations. The second haplotype was constructed using the most significant markers for BCI  and in later (SSBL) lactations, and body-depth. These two haplotypes were fitted individually and jointly in the RHM to determine if one or both haplotypes were sufficient to explain the QTL variance observed in the targeted region for the traits analyzed.Two genomic regions were selected for haplotype based association analyses. The highest –logL Figure . The posL Figure , 3 and 4I Figure , 3 and 410 (P) = 106.9  between the two haplotype doses was 0.064, indicating very low LD between the two haplotype blocks.HAP1 and HAP2 were fitted as fixed effects in the mixed model to determine variance explained by these two haplotypes. A significant association for BCI Figure  and SCI In the present study, we reported QTL with a large effect on calf size and stature in Nordic Red cattle. We identified two deviant haplotypes that increased calf size at birth, and altered adult body conformation. However, the haplotypes also increased calving difficulties, and subsequently calf mortality due to the increase in calf size. Results indicated the haplotype locations overlapped. However, the LD between sites was low, suggesting two independent mutations responsible for similar effects. Several studies reported QTL in the same genomic region on chromosome 6 impacting birth weight, growth, and carcass traits in beef breeds and crossbreds -19. In dThe cattle population we used for association analysis had both population structure (three sub-populations) and family relationship . Therefore a linear model including the full relationship matrix is necessary to avoid false positive associations ,34. HoweWe observed reduced haplotype diversity at the QTL peak compared to the rest of the chromosome Figure . One of Soranzo et al.  studied The NCAPG and LCORL loci have been investigated in beef cattle populations for effects on carcass traits and body weight. A missense variant (rs109570900) at 38,777,311 bp within NCAPG, which induced a Ile-442-Met substitution in amino acid sequence was associated with carcass weight, the longissimus muscle area, and subcutaneous fat thickness in Japanese Black and Brown cattle . The samr = 0.62 in RDCDNK. This NCAPG gene missense variant was earlier reported from several cattle populations, e.g. the variant exhibited the strongest effect on birth weight in Holstein × Charolais [We observed two haplotypes within overlapping regions on BTA6, which affected SCI and BCI in Nordic Red cattle. Correlation between imputed haplotype doses for the two haplotype blocks was low, indicating low LDs. Haplotype distinctness was also supported by the model fit. The best model fit was obtained when HAP1 and HAP2 were included in the model. Marked frequency differences were observed in HAP1 and HAP2 from the three country RDC populations. HAP1 was common in RDCDNK (frequency = 0.42), but rare in RDCFIN 0.003) and RDCSWE (0.019). In contrast, HAP2 was rare in RDCDNK (frequency = 0.04), but common in RDCFIN (0.32) and RDCSWE (0.21). We checked the functional annotations for markers included in HAP1 and HAP2. All 15 markers from the LCORL gene, which form HAP1 were intronic variants  with similar effects on BCI and SCI. The two haplotype locations overlap, but LD between the sites was low. HAP1 frequency was common in RDCDNK, but rare in RDCFIN and RDCSWE, while HAP2 showed reverse frequencies, i.e. common in RDCFIN and RDCSWE, but rare in RDCDNK. Principal component analysis provided clear evidence RDCDNK was genetically divergent from RDCFIN and RDCSWE (results not shown). Therefore, the two haplotypes might have had independent origins in different populations. If the two haplotypes represent two distinct mutations on the same gene, then this would provide an explanation why they had the same effect on calf size at birth and adult stature.https://www.cdcb.us/eval/summary/trend.cfm?R_Menu=HO.sb#StartBody eval/summary/trend.cfm?R_Menu = HO.sb#StartBody, accessed 30 June 2014). In contrast, first lactation stature in Canadian Holstein has demonstrated steady genetic gain; average stature has increased from < 143 cm in 1991 to 147 cm in 2001 . The haplotypes identified in Nordic Red cattle increased adult body conformation. The present BCI breeding goal in Nordic Red cattle is for an optimum; however, the genetic trend for stature over time has been positive [Opposite trends for calving ease and stature had been reported during the last three decades. For example, the average breeding value (PTA) for daughter calving ease in US Holstein cattle have decreased from 9.3 in 1980 to 6.6 in 2009, while PTA for daughter stillbirths has decreased from 10.6 to 7.2 for the same period  responsible for the mutation(s) requires further study.ftp://ftp.ensembl.org/pub/release-73/fasta/bos_taurus/dna). All variations used in the mapping study have been submitted by the 1000 bull genomes project for inclusion in dbSNP (http://www.ncbi.nlm.nih.gov/SNP). Whole genome sequence data for the 234 individuals included in run2 of the 1000 bull genomes project are available at NCBI using SRA no. SRP039339 (http://www.ncbi.nlm.nih.gov/bioproject/PRJNA238491). All annotations were obtained from a publicly available source (http://www.ensembl.org) downloadable including through Variant Effect Predictor (http://www.ensembl.org/info/docs/tools/vep/script/index.html).All DNA sequences used were taken from a publicly available assembly. The assembly is available for download (


We investigated prognostic parameters of patients who underwent surgical intervention for acute mesenteric ischemia by evaluating demographic characteristics and laboratory data on admission.The hospital records of 30 patients who underwent surgical interventions due to acute mesenteric ischemia between January 2008 and December 2012, were reviewed retrospectively. The records were investigated with regard to demographic data, the presence of co-morbid diseases, presenting complaints, time elapsed between symptom onset and hospital admission, laboratory findings at admission, findings at surgical exploration, surgical methods used, and treatment outcomes. The patients were divided into two groups, according to death (Group 1) or survival (Group 2), and the two groups were compared in terms of the specified parameters.Of the patients, 15 were male (50%) and 15 female (50%); their mean age was 71.4 (29–94) years. Abdominal pain was the chief complaint in all patients (100%) and mean time from pain onset to hospital admission was 21 (1–72) h. In abdominal exploration, total small bowel (SB) ischemia and necrosis was found in 6 (20%) patients and other patients had subtotal SB, segmental SB, segmental SB with colon, or isolated colon ischemia. Treatment in 15 patients (50%) ended in mortality. Mean age (p = 0.038), urea (p = 0.002), AST (p < 0.001), ALT (p < 0.001), mean platelet volume , and amylase (p = 0.022) levels in Group 1 were significantly higher versus Group 2, whereas Ca (p = 0.024) and albumin (p = 0.002) levels were significantly lower.In this study, unlike other parameters that have been shown to be of prognostic significance in mesenteric ischemia, MPV values at presentation were higher among non-survivors than survivors. Acute mesenteric ischemia (AMI) can result from vascular occlusive or non-occlusive conditions. Non-occlusive mesenteric ischemia is caused by conditions such as hypovolemia, sepsis, and cardiogenic shock, whereas the underlying cause of ischemia in 70–80% of cases with AMI is the occlusion of the superior mesenteric artery, caused by embolism or thrombosis . IrreverDespite advances in diagnosis, treatment, and post-operative care in recent years, AMI still has a high mortality rate, ranging between 40 and 70% . The mosThe aim of the present study was to evaluate the outcomes of surgical treatment over a period of 5 years in patients who underwent surgery with a diagnosis of AMI and to investigate the parameters of prognostic significance.The hospital records of 30 patients who underwent surgical intervention due to acute mesenteric ischemia in the Department of General Surgery, Sakarya University Faculty of Medicine between January 2008 and December 2012 were reviewed retrospectively. The records were investigated regarding demographic data, presence of co-morbid diseases, presenting complaints, time elapsed between symptom onset and hospital admission, laboratory findings at admission, findings at surgical exploration, surgical methods used, and treatment outcomes. The patients were divided into two groups, according to death (Group 1) or survival (Group 2), and they were compared in terms of the specified parameters.Among the parameters in complete blood counts, leukocytes (WBC), hematocrit (Htc), hemoglobin (Hb), mean platelet volume (MPV), and total platelet count (PC) were evaluated. Among the biochemical parameters, urea, creatinine, sodium (Na), potassium (K), calcium (Ca), chlorine (Cl), aspartate amino transferase (AST), alanine amino transferase (ALT), gamma glutamyl transferase (GGT), alkaline phosphatase (ALP), total bilirubin, albumin, and amylase were evaluated.Survivors in whom more than 2 years had elapsed since their operation were contacted by phone to obtain their latest condition.t-test and Fischer’s exact test were used for comparison between subgroups. Statistical analyses were performed using the SPSS software . P values < 0.05 were considered to indicate statistical significance.In total, 21 variables were compared between the groups. Student’s n = 16; 72.7%). Abdominal pain was the chief complaint in all patients (100%) and mean time from pain onset to hospital admission was 21 (1–72) h. All patients underwent computed tomography (CT) of the abdomen and the use of intravenous contrast agent was avoided in 5 (16.6%) patients due to impaired renal function (creatinine > 2.5).Of the patients, 15 were male (50%) and 15 female (50%); their mean age was 71.4 (29–94) years. Of the patients, 22 (73.3%) had a history of comorbid disease and cardiovascular disorders were the most common  patients were found to have diffuse small bowel (SB) ischemia and necrosis, and 17 (56.6%) had subtotal (> 100 cm) SB ischemia; of the 17, 8 (47.0%) had right colonic ischemia. Five (16.6%) patients only had segmental SB ischemia and necrosis (<100 cm) and 1 (3.3%) patient had isolated right-sided colonic ischemia and necrosis. The operation was terminated without performing further intervention in patients suffering from diffuse SB ischemia and necrosis , whereas various resections were performed in the remaining 23 patients (76.6%): 9  patients underwent subtotal SB resection, 8  underwent subtotal SB resection plus right hemicolectomy, 5  underwent segmental SB resection, and 1  patients underwent a right hemicolectomy.One patient (3.3%) was admitted to the hospital 1 h after the onset of abdominal pain and CT scans showed occlusion of the superior mesenteric artery (SMA). This patient subsequently underwent an embolectomy due to the presence of subtotal ischemic changes  in the SB but without necrosis.Demographic features and exploration findings of the patients are presented in Table The treatment resulted in mortality in 15 patients (50%)  and there were 15 survivors (50%), discharged after a mean follow-up of 5 days -12. In an = 15) and survivors , mean age (p = 0.038), urea (p = 0.002), AST (p = 0.001), MPV (p = 0.002), and amylase (p = 0.022) levels in Group 1 were significantly higher than in Group 2, whereas Ca (p = 0.024) and albumin (p = 0.002) levels were significantly lower. No significant difference was found between the groups in terms of other parameters.In comparisons of the non-survivors , and high D-dimer levels can be found in the presence of AMI. Studies have shown that these findings are not useful in the early diagnosis of AMI and can even be elevated in acute abdominal conditions other than AMI due to their low sensitivity -9. BasedPredictive factors affecting mortality in patients with AMI upon admission to the hospital have been analyzed in various studies, which yielded different results for many parameters. Aliosmanoglu et al.  reportedMany studies have described advanced age and colonic ischemia accompanying small bowel ischemia as factors indicating poor prognosis -17. In tPlatelets play a critical role in the regulation of blood flow and thrombogenic cascades. MPV is a marker of the size and activation of platelets, and elevated levels of MPV reflect increased production and activation of platelets. Large platelets possess higher metabolic and enzymatic activity, and show higher thrombogenic potential . SeveralNo published study has examined the relationship between MPV and AMI. AMI is a cardiovascular disease in origin, although its consequences affect predominantly the gastrointestinal system. As a matter of course, a relationship between AMI and increased MPV is considered to indicate increased thrombogenic activity. In the current study, MPV in Group 1 was significantly higher than in Group 2.However, it would not be appropriate to consider that this result indicates that “increased MPV is a predictive factor for prognosis in AMI,” because a high MPV is found in other atherosclerosis-related conditions  . High meConsidering the significantly higher MPV in Group 1 in the current study: 1) MPV could be used to predict the potential for vascular damage in other organs, such as the liver and kidneys , and 2) because it reflects a tendency for thrombosis, MPV could be useful to justify re-operation when a second-look decision could not be made otherwise.In conclusion, AMI continues to have a high mortality rate despite advances in diagnosis, treatment, and post-operative care. MPV can be beneficial in predicting patients with poor prognosis and in the planning of re-operations.The authors declare that they have no competing interests.FA, YA and OVO contributed to study design. YA, OY and YU contributed to data collection. FA and YA contributed to data analysis and writing. All authors read and approved the final manuscript.


We report the case of a 38-year-old female patient with systemic lupus erythematosus (SLE) and Jaccoud arthritis (JA) that sequentially developed digital ischemic lesions of the hands. In spite of follow-up treatment with glucocorticoids, immunosuppressant, antiaggregant, and potent vasodilatator agents, a serious progression to digital gangrene over a one-month period was observed. Surprisingly, her nonhealing digital lesions improved after two cycles of rituximab (RTX) administration. Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease that is associated with considerable morbidity and mortality. Digital ischemia, digital ulcer, or gangrenous lesions have been described in SLE , 2. VascPresented here is an SLE-JA patient with subluxation of MCPs swan neck deformities who sequentially developed digital ischemic lesions after stopping immunosuppressive treatment at her own discretion due to exorbitant side effects. Digital ischemia of hands showed a serious progression to digital gangrene over a month in spite of follow-up treatment with glucocorticoids, immunosuppressant, antiaggregant, and potent vasodilatator agents.Surprisingly, her nonhealing ulcers improved dramatically after two cycles of rituximab RTX administration. To the best of our knowledge, this is the first SLE-JA case presenting with nonhealing digital ischemic lesions responding dramatically to RTX therapy in the medical literature.A 38-year-old Turkish woman with a history of SLE presented in 2003 with photosensitive rash on her face and chest, oral aphthous lesions, and arthritis with pain and motion restriction in hands, wrists, elbows, ankles, and knees. A diagnosis of SLE associated arthritis was made and treatment with 5 mg of prednisone once daily and 200 mg of hydroxychloroquine twice daily was started requiring the addition of methotrexate in escalation until 25 mg weekly was reached. Her joint manifestations were partially responsive; however, arthritis remained active in the small joints of her hands with pain and swelling. Six years later, the patient developed avascular necrosis during follow-up for which she was operated on at an outside setting and a hip prosthesis was implanted.Two years following surgical treatment for avascular necrosis, the patient presented to our rheumatologic polyclinic with complaints of pain, swelling, warmth, and cyanosis over the fingers of both hand and toe tips. Physical examination revealed swelling, warmth, and tenderness in the proximal interphalangeal (PIP) joints of both hands, Ulnar deviation, and bilateral swan neck deformities. Right is greater than left. “Z” deformities of thumbs  and cyanLaboratory findings were as follows: WBC: 5.6 × 109/L, Hb: 11.9 g/dL, platelet: 276 × 109/L, Hct: 35.4%, ESR: 120 mm/h, CRP: 80 mg/L, microscopic hematuria and trace proteinuria on urinalysis.Immunologic studies revealed an ANA titer of 1 : 3200, , U1RNP (+++), p-ANCA (+), antibeta 2 GP1-IgA isotype . There was hypocomplementemia . ds-DNA and Sm antibodies and rheumatoid factor were negative.Review of hand radiographs showed no erosive changes consistent with a diagnosis of JA . Electro2).Results from laboratory studies showed high disease activity of lupus. Initial treatment was started with glucocorticoid (1 mg/kg/day), aspirin (300 mg/day), IV prostaglandin (2 mcg/kg/min), and IV cyclophosphamide  with 2 infusions of 1 g at a 14-day interval in combination with hydroxychloroquine and aspirin was initiated during the second month. Clinical and laboratory response to treatment began to be observed during the third month of RTX therapy. Laboratory evaluation revealed the following: ESR: 22, CRP: 3 mg/dL, and normal levels of C3 and C4 complement levels. (Results from a complete blood count panel were unremarkable). A combining dosage of 100 mg/day azathioprine (AZA) was added to the treatment protocol after two infusions of RTX therapy. Also, corticosteroid dosage which was started at 1 mg/kg was tapered gradually by 10% per kilogram for every 10 days till it reached 5 mg/day over the three-month period.Eventually, digital gangrenous lesions started to regress after the first cycle (5 months) of RTX therapy . A seconVasculitis and digital gangrene have been described in SLE. Thromboembolism, premature atherosclerosis, overlap syndrome, and especially antiphospholipid antibodies (aPLs) may contribute to the development of gangrene , 4. In aThe prevalence of digital gangrene in APS patients has been reported to be between 3.3 and 7.5% and the presence of a positive anti-RNP has been described in some APS digital ulcer cases . The relRTX has been shown to be effective in the reduction of B-cell production and used in the treatment of refractory SLE or vasculitis. In a prospective study conducted by Smith et al., rituximab was shown to play a role in B-cell depletion offering the prospect of sustained disease remission and improved disease control with low toxicity in patients with active refractory SLE or ANCA associated vasculitis (AAV) .In the presence of Apl/LAC, patients were first anticoagulated with IV heparin or low molecular weight heparin (LMWH) before long-term treatment with warfarin . ClinicaIn our patient, digital ischemic lesions were seen two years after stopping treatment at her own will. We added a treatment regimen of Iloprost and acetyl salicylic acid to her traditional immunosuppressive agents which included steroids, hydroxychloroquine, and cyclophosphamides. Her nonhealing ulcers however prompted us to start an infusion dosage of 1000 mg rituximab (per 0–15 days) therapy considering its role in B-cell depletion and low toxicity in patients with ischemic lesions. Surprisingly, her digital ulcers responded dramatically to rituximab therapy after 2 cycles and a recession in acute phase markers was observed.To the best of our knowledge, our case is the first case in the medical literature reporting an SLE-JA association developing digital ischemic lesions as a late clinical complication and responding dramatically to rituximab therapy.As a conclusion in lupus patients with arthritis associated nonhealing digital ulcers under traditional immunosuppressive agents, RTX can be a treatment option.


That has broad biological and pathophysiological implications.Structural Equation Models (SEM) can explicitly distinguish “dementia-relevant” variance in cognitive task performance . In prior work, δ appears to uniquely account for dementia severity regardless of the cognitive measures used to construct it. In this study, we test δ as a mediator of age's prospective association with future cognitive performance and dementia severity in a large, ethnically diverse longitudinal cohort, the Texas Alzheimer's Research and Care Consortium (TARCC). Age had adverse effects on future cognition, and these were largely mediated through δ, independently of education, ethnicity, gender, depression ratings, serum homo-cysteine levels, hemoglobin A1c, and apolipoprotein e4 status. Age explained 4% of variance in δ, and through it, 11-18% of variance in future cognitive performance. Our findings suggest that normative aging is a dementing condition . While the majority of variance in dementia severity must be independent of age, age's specific effect is likely to accumulate over the lifespan. Our findings also constrain age's dementing effects on cognition to the age-related fraction of “general intelligence” (Spearman's “ Cognitive performance is associated with age, and seemingly healthy elderly persons may be impaired relative to young adult norms . Age is We do not yet know the phenotype of age-specific cognitive change. Many studies describe longitudinal age-related trends in cognition -5. Howevdementing processes. If Aging Proper is dementing, then even normative cognitive aging may be a matter of concern, and a potential target for therapeutic intervention.We have used a Growth Mixture Model (GMM) analysis, to better precise the ASCP . This alfor dementia itself, as distinct from either global cognitive performance or performance in any single cognitive domain . g's AUC for the same discrimination was at a near chance level [AUC = 0.630 (CI: 0.601-0.65.9)]. This is consistent with past findings, across batteries, in this and other cohorts.2 = 48.33 (22), p < 0.001; CFI = 0.996; RMSEA = 0.020]. Baseline Age was significantly associated with Wave 3 cognitive performance and with Wave 3 CDR. Age's effects on Wave 3 cognitive performance were significant and inverse (adverse), ranging from Boston  to Animal  , p < 0.001; CFI = 0.999; RMSEA = 0.013]. Independently of the covariates  baseline Age was significantly directly associated with Wave 3 Animal, Boston and CERAD. These direct associations were statistically weak  to moderate  and attenuated relative to the Base Model. Age's direct association with Wave 3 CDR was also attenuated and now statistically weak . Age was significantly associated with Wave 2 dEQ , but not with the Wave 2 g’ composite . Age's significant association with Wave 2 dEQ scores was moderately strong and in a positive direction . Given Age's unstandardized association of 0.02 with dEQ, each year of increasing age is associated with an 0.02 SD increase in dEQ.The final mediation model Figure  also hadBecause age was not significantly associated with g’, it cannot be a mediator of Wave 3 cognitive performance. g’ was therefore omitted from further consideration. Independently of both Wave 2 g’ ‘s adverse effects, and Wave 3 g's, Wave 2 dEQ was strongly inversely related to Wave 3 cognitive performance (and positively with CDR). Thus, the age-related effects mediated by dEQ were functionally adverse and likely to reinforce Age's attenuated and relatively weak direct effects.Age's significant direct effects in Model 1 were largely or fully mediated by dEQ Table . SimilarThe constrained model's fit was statistically indistinguishable from the unconstrained model. Therefore age's associations with all Wave 2 and Wave 3 measures remain stable across both subsamples (Table 2 difference = 0.09 (1), p = 0.75]. Nor was it affected by a diagnosis of dementia  .In post hoc analyses, Age's direct association with dEQ was statistically indiscriminable in the young old  vs. the elder old  .These models were all constructed in an SEM framework, using raw data. No statistical transformation was applied to biomarker data. Modern Missing Data Methods were automatically applied by the AMOS software (see below).Following the approach of MacKinnon et al. , we firsAccording to Kraemer et al. , the tim888 subjects had complete biomarker data. In contrast, psychometrics and adjudicated clinical diagnoses were available on 2017 subjects. Rather than using case-wise deletion, the missing biomarker and psychometric data were handled by Modern Missing Data Methods .Only the ROC analyses, which were performed in Statistical Package for the Social Sciences (SPSS)  were limModel fit was assessed using four common test statistics: chi-square, the comparative fit index (CFI), and the root mean square error of approximation (RMSEA). Where two nested models were compared, Akaike's Information Criterion (AIC) was added. A lower AIC statistic indicates better fit. A non-significant chi-square signifies that the data are consistent with the model . HoweverThe diagnostic performance or accuracy of a test to discriminate diseased from normal cases can be evaluated using ROC curve analysis -58. Brie


Background and Aim. The aim of this study was to determine the actions of caffeic acid phenethyl ester (CAPE) on the changes of endothelin-1 (ET-1) level, tumor necrosis factor- (TNF-) alpha, and oxidative stress parameters such as superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels in experimental sepsis model in rats. Materials and Methods. Twenty-four rats were randomly divided into three experimental groups: sham (group 1), sepsis (group 2), and sepsis + CAPE (group 3), n = 8 each. CAPE was administered (10 µmol/kg) intraperitoneally to group 3 before sepsis induction. Serum ET-1, serum TNF-alpha, tissue SOD activity, and tissue MDA levels were measured in all groups. Results. Pretreatment with CAPE decreased ET-1, TNF-alpha, and MDA levels in sepsis induced rats. Additionally SOD activities were higher in rats pretreated with CAPE after sepsis induction. Conclusion. Our results demonstrate that CAPE may have a beneficial effect on ET and TNF-alpha levels and oxidative stress parameters induced by sepsis in experimental rat models. Therefore treatment with CAPE can be used to avoid devastating effects of sepsis. Sepsis is a complex process characterized by uncontrolled systemic inflammation that may eventually lead to multiorgan dysfunction and even death .Endothelin-1 (ET-1) is the main isoform of the endothelin family, which have important roles in a wide range of diseases affecting the vascular system, kidney, heart, and lungs. Overexpression of ET-1 during sepsis, as a result of endothelial dysfunction, causes imbalance in local tissue perfusion , 3.α might be critical for treating septic organ dysfunction [Excessive release of proinflammatory mediators which are induced by endotoxin like tumor necrosis factor-alpha  and reactive oxygen species (ROS) causes systemic inflammatory response during sepsis . TNF-alpfunction , 6.ROS have been associated with the onset, progression, and outcome of sepsis, both in experimental and in clinical studies , 8. ExceμM [Caffeic acid phenethyl ester (CAPE) is one of the major components of honeybee propolis and has been used in traditional medicine. It was found to be a potent free radical scavenger and antioxidant . CAPE inμM . It has μM .The purpose of the current study was to evaluate the effects of CAPE on ET-1 levels, TNF-alpha, and oxidative stress in a rat sepsis model.Animal experiment was performed in accordance with the National Institute of Health guidelines for animal research and was approved by the Animal Research Ethics Committee of Fatih University School of Medicine, Istanbul. 24 adult male Wistar rats  were used for the experiment. All animals had access to commercial standard diet and water ad libitum throughout the study. The rats were divided randomly into three groups of eight animals each: sham group (group 1), sepsis group (group 2), and sepsis group treated with CAPE (group 3).10 CFU of Escherichia coli ATCC 25922. E. coli ATCC 25922 was grown in brain-heart infusion broth. In the logarithmic phase of the growth, the suspension was centrifuged at 1000 g for 15 minutes, the supernatant was discarded, and the bacteria were resuspended and diluted in sterile saline. The rats received an i.p. inoculum of 1 mL of saline containing 2 × 1010 CFU of E. coli ATCC 25922 [μmol/kg) by intraperitoneal injection. The CAPE was synthesized by standard method of Grunberger to prepare 25 micromol/mL of CAPE solution [Sham group (group 1) received only 1 mL intraperitoneal (i.p.) injection of 0.9% saline solution. Sepsis in group 2 and group 3 was induced by intraperitoneal (i.p.) injection of 2 × 10CC 25922 . Six housolution .24 hours after bacterial challenge, all animals were sacrificed using ketamine and cardiac puncture. Blood samples were drawn from vena cava inferior; liver tissue samples were immediately removed and stored at −80°C for the determination of tissue associated malondialdehyde (MDA) levels and superoxide dismutase (SOD) activities, as the parameters of oxidative stress. Blood samples were collected for the determination of endothelin and TNF-alpha levels, centrifuged at 3000 g for 10 minutes, and stored at −80°C.Rat big ET-1 levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits  following the manufacturer's instructions. The results are presented as pg/mL.Tumor necrosis factor-alpha rat ELISA kits produced by Biovendor Research and Diagnostic Products were utilised as described by the manufacturer. The results were expressed as pg/mL.Reaction with thiobarbituric acid (TBA) at 90–100°C was used as the basis for the determination of tissue thiobarbituric acid-reactive substance (TBARS) levels . In the −1 protein.SOD activity was determined according to the method defined by Sun et al. . This meFor statistical evaluation, we used the statistical software package SPSS 15.0 and probability value of less than 0.05 was accepted as statistically significant. As the data were independent and showed normal distribution, statistical analysis was performed using analysis of variance (ANOVA) followed by Tukey's test when comparing groups. The results are given as the mean ± standard deviation of the mean (SD).ET-1, TNF-alpha, and MDA levels and SOD activities for all groups are presented in Figures P = 0.028). Similarly, ET-1 levels in sham group (G1) were lower than in sepsis group (group 2) (P = 0.001).ET-1 levels were determined as it is one of the major peptides in the pathogenesis of sepsis . ET-1 leP = 0.035). When sepsis was induced in rat pretreated with CAPE (group 3), TNF-alpha levels were found to be similar to group 1 (P > 0.05) and significantly lower compared to group 2 (P = 0.039).TNF-alpha levels were determined since they have an important role in the pathogenesis of sepsis . TNF-alpP = 0.001). When rats were pretreated with CAPE before sepsis induction, MDA levels were significantly lower compared to those in group 2 (P = 0.002).Tissue MDA levels, used as a marker of oxidative stress, were determined . Tissue P = 0.001). When rats were pretreated with CAPE prior to sepsis induction, SOD activities were significantly higher compared to group 2 (P = 0.002). Tissue SOD activities in group 1 were higher than group 3 (P = 0.001).SOD activities were used as a marker of oxidative stress . Tissue Overall, results revealed that pretreatment with CAPE significantly reduced serum ET-1, TNF-alpha, and MDA levels while increasing SOD activities, when sepsis was induced in a rat sepsis model.Sepsis syndromes range from the systemic inflammatory response syndrome to severe sepsis and septic shock. These syndromes are the major causes of death in critical care units worldwide . AlthougET-1 levels increase with a septic process in correlation with the circulatory dysfunction and sepsis severity , 18. ItsIncreased oxidative stress, one of many factors involved in development of multiorgan dysfunction syndrome during sepsis, is the result of imbalance between antioxidant and oxidant status. ROS levels rise due to increased production as a part of the proinflammatory response during sepsis , 22. MorTNF-alpha is one of the proinflammatory cytokines which has been accepted as important mediators of sepsis –28. TherDeath from sepsis has decreased recently depending on various adjunctive therapies. However, it remains high when compared with other critical illnesses . In addiA growing number of reports demonstrate that a proinflammatory and oxidative condition is related to the pathogenesis and the progression of endotoxin-induced septic shock and that antioxidants may have therapeutic potential in lipopolysaccharide- (LPS-) induced sepsis .Because of this, in current study we investigate the roles of antioxidant therapy using CAPE in the treatment of sepsis. CAPE is a major component of honey bee propolis which is commonly used in Chinese traditional medicine. CAPE was also investigated in various organs such as intestine, lung, and spinal cord in a number of animal models and it has been reported that it has anti-inflammatory, radical scavenging and, therefore, antioxidant activities , 33–37. Our study demonstrated that effects of CAPE seemed to depend not only on the diminution of oxidative damage but also on its anti-inflammatory activity in rats in vivo. Therefore, it is reasonable to propose CAPE as a molecule with therapeutic potential for the treatment of systemic inflammation by interfering at the earliest steps of activation of the oxidative and proinflammatory cascade.These data demonstrate the protective effect of CAPE treatment in experimental sepsis by reducing the inflammatory process and oxidative stress.


PRCP and KLKB1 polymorphisms with cardiovascular disease (CVD). DNA was obtained from 2243 individuals from the Prevention of Events with Angiotensin Converting Enzyme trial. Two PRCP SNPs, rs7104980 and rs2298668, and two KLKB1 SNPs, rs3733402 and rs3087505, were genotyped. Logistic regression models were performed for history of diabetes, myocardial infarction, stroke, angina, angiographic coronary disease, CABG, intermittent claudication, percutaneous transluminal coronary angioplasty (PTCA), and transient ischemic attack. The PRCP SNP rs7104980 increased the odds of having a history of PTCA by 21% ; P = 0.041, but was non-significant after Bonferroni correction. Alternatively, having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% ; P = 0.007 that was statistically significant (P < 0.01) after Bonferroni correction for multiple hypothesis testing. When the best-fit model based on the Akaike information criterion controlled for age, weight, gender, hypertension, and history of angina, the G allele of KLKB1 rs3733402 that is associated with less plasma kallikrein activity correlated with reduced history of CVD.Plasma kallikrein formed from prekallikrein (PK) produces bradykinin from kininogens and activates factor XII. Plasma PK is activated by factors αXIIa, βXIIa, or prolylcarboxypeptidase (PRCP). A cross-sectional investigation determined if there is an association of  KLKB1 rs3087505 is associated with venous thrombosis and this SNP is in linkage disequilibrium (LD) with two F11 SNPs  also associated with thromboembolism  is a zymogen whose enzyme plasma kallikrein is known to initiate and amplify factor XII activation on collagen and polyphosphates in plasma in the intravascular compartment to activate intrinsic blood coagulation. Formed plasma kallikrein is also the major enzyme to generate bradykinin from high molecular weight kininogen (HK) and to convert plasminogen to plasmin and single-chain urokinase to two-chain urokinase for fibrinolysis, C3 and C5 to C3a and C5a, respectively, in the complement system and prorenin to renin in the renin angiotensin system. There are several SNPs that have been recognized to impact PK function. 1–13  indicates risk for chronic hypertension and preeclampsia in an African-American patient population (P = 0.3 × 10−10] (gt/gtprcp) mice that have 25% PRCP normal protein levels in their organs are constitutively hypertensive and have a heightened risk for arterial thrombosis (Prolylcarboxypeptidase (PRCP) is a membrane-associated serine protease that cleaves peptides where the penultimate position is a Pro-X bond , 11. As 1–13 –12. We a1–13 . Geneticsyndrome , 17. An pulation . In a Hapulation . In anot× 10−10] . PRCP-derombosis . Likewisrombosis . OppositPRCP intronic rs7014980 associates with CVD but does not meet statistical significance. Alternatively, the minor G allele of an exonic SNP rs3733402 (N124S) of KLKB1 is significantly associated with reduced CVD. These data suggest that a selected KLKB1 SNP (rs3733402) mirrors the phenotype observed in patient studies with diabetes and on murine deletions of klkb1 . In a single cross-sectional study, we show that the G allele for https://biolincc.nhlbi.nih.gov/home/)  Inhibition Trial that were in the NIH Biorepository BioLincc (v/home/) . In the v/home/) . The goav/home/) . The carv/home/) . The outN = 2243) plasma samples from the PEACE trial were used for DNA isolation. DNA was extracted by the NucleoSpin Plasma XS technique  yielding 0.03–0.1 μg/sample. The extracted DNA had whole-genome amplification using REPLI-g Mini Kit (Cat# 150023) (QIAGEN) to yield 10 mg/ml. DNA samples were quantified using Qubit DNA quantification assay and a Qubit 2.0 Fluorometer (Life Technologies).The subsample of subject plasmas from the repository were de-identified from the individual donors but were linked by code to clinical information in the data dictionary . The samPRCP that have been associated with hypertension, intronic rs7104980 (11:82864153)    and exon2864153) , 19 wereF11 SNPs rs2036914 and rs3756008, respectively. Unconditional logistic regression models assessed the association between candidate SNPs and various disease outcomes calculating ORs and 95% CI. For each candidate SNP, having the rarer allele was compared to not having the rarer allele. Other covariates included in the logistic regression models were age, weight, gender, hypertension status, history of diabetes, history of angina, and/or cigarette use. Hypertension in the PEACE trial was defined as having systolic blood pressure greater or equal to 140 mmHg and/or having diastolic blood pressure greater or equal to 90 mmHg  and pairwise LD were assessed using standard methods. A heatmap was used to determine if SNP rs3733402 is in LD with  90 mmHg . This va 90 mmHg . The resPatient characteristics are presented in Table PRCP and KLKB1 genes. Across all four SNPs, the frequency of undetermined genotypes ranged from 5.1 to 8.4%. For rs7104980 in the PRCP gene, the less common allele was G (44.7%) and the rarer genotype was the G homozygote (G/G) (29%). For rs2298668 in the PRCP gene, the less common allele was the G allele (8.3%) and the rarer genotype was the G homozygote (G/G) (5.8%). For rs3087505 in the KLKB1 gene, the less common allele was the A allele (10.29%) and the rarer genotype was the A homozygote (A/A) (3.3%). For rs3733402 in the KLKB1 gene, the less common allele was the G allele (48.1%) and the rarer genotype was G/G (31.4%).Allele and genotype frequencies are presented in Table P-values associated with the chi-square statistic for the HWE tests were observed that were the result of a large sample size, suggesting that the SNPs were in violation of HWE. This finding was due to having a statistical power large enough to detect small differences in this study that were not truly biologically meaningful. Hence, no SNPs were removed. In addition, both the allele and genotype frequencies for all tested SNPs were very similar to those from the dbSNP Hapmap CEU frequencies , as well as the LD correlation coefficients between each SNP per gene, were computed. Low es Table . There wPRCP rs2298668 and KLKB1 rs3087505 SNPs. Having the rare G allele compared to not having the G allele for rs7104980 (PRCP gene) increased the odds of having a history of PTCA by 21% . Alternatively, having the rarer G allele compared to not having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% . After correcting for testing multiple SNPs by the Bonferroni correction method, only SNP rs3733402 (KLKB1 gene) was significantly associated with an outcome of having a history of angiographic coronary disease (adjusted P < 0.0125)  increased the odds of having hypertension and a history of angina by 22% ; P = 0.034, but this analysis was not statistically significant (P > 0.01) after Bonferroni correction. Alternatively, having the G allele compared to not having the G allele for rs3733402 (KLKB1 gene) decreased the odds of the baseline covariates, hypertension, and history of angina by 24% , which was statistically significant (P ≤ 0.01) for multiple testing after Bonferroni correction.In order to better understand the potential association between having the G alleles for rs7104980 and rs3733402 and CVD, we calculated the −2 log-likelihood values in order to calculate the AIC to determine the best-fitting model . As showKLKB1 associated with reduced PK binding to HK leading to a less actable zymogen is associated with reduced CVD. However, we are cautious in over interpreting the results of this investigation because it has several limitations. The PEACE trial was performed on low-risk individuals with CVD who had stable coronary artery disease and normal or slightly reduced left ventricular function. The patient population was 82% male with the majority of patients between 51 and 70 years of age with only 14% frequent smokers. The majority of patients did not have TIA, stroke, claudication, diabetes, hypertension, CABG, or PTCA. The present subset analysis of samples from the PEACE trial was similar in most ways, but actually had subjects with significantly reduced incidence of hypertension, PTCA, and MI. Since these three indicators are important disease markers of CVD, their reduction may have influenced the outcomes of the present studies. Overall, the present study examined a healthier subset of individuals with lower risk CVD than in the original PEACE trial itself.The present cross-sectional study suggests that a polymorphism in KLKB1 exonic SNP, rs3733402 (N124S), was shown to be protective in the studied subjects for having a history of angiographic coronary artery disease  have reduced arterial thrombosis providing support in mouse models to enhance the observation made in human association studies , but the Bonferroni correction of the P-value for the total number of SNPs (P < 0.0125) shows non-significance  that was associated with chronic hypertension with or without increased risk of preeclampsia or resistance to an ACE-inhibitor therapy for hypertension (Furthermore, our studies like others did not show any significant association with the rs2298668 rtension , 19. Firrtension , 19. Thirtension –20. The When we performed this investigation, we were concerned whether we were able to get adequate DNA from stored plasma samples. We avoided serum samples for DNA preparation in this investigation since it has been associated with degraded DNA . We reasAnother caution in interpreting these findings is that this investigation is based only on a single cross-sectional study of samples. It needs to be confirmed by similar investigations on additional sample sets. Furthermore, the results of this cross-sectional study should be confirmed in a case-controlled study to obviate any unintended selection bias.KLKB1 SNP, rs3733402, is associated with reduced hypertension and coronary artery disease. These data suggest that reduction of PK activity may reduce CVD consistent with our previous studies in diabetic patients (In sum, we postulate that the G allele of a patients , 9. Our KLKB1 SNPs on a group of patients with mild CVD. When subject age, male gender, and weight are taken into account, KLKB1 SNP rs3733402 significantly associates with those with a reduced history of angiographic coronary artery disease. When subjects have covariates of age, weight, gender, hypertension, rs3733402 best associates with those individuals who both have a reduced history of angina. Rs3733402, an exonic SNP that is associated with reduced PK activation, also is associated with reduced CVD.This paper is the first cross-sectional investigation on two JB-S and AS designed the project. AM, OA, MV, and ES performed experimental work. HG, JB-S, MV, and AS performed data analysis. HG, JB-S, MV, ES, and AS performed manuscript drafting and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Calcium supplements are widely used among older adults for osteoporosis prevention and treatment. However, their effect on creatinine levels and kidney function has not been well studied.3 (1000 IU), both, or placebo for three or five years. Changes in blood creatinine and total calcium concentration were measured after one year of treatment and multiple linear regression was used to estimate effects on creatinine concentrations.We investigated the effect of calcium supplementation on blood creatinine concentration in a randomized controlled trial of colorectal adenoma chemoprevention conducted between 2004–2013 at 11 clinical centers in the United States. Healthy participants  aged 45–75 with a history of colorectal adenoma were assigned to daily supplementation with calcium , vitamin D2 at baseline. The effect of calcium treatment on creatinine was only partially mediated by a concomitant increase in blood total calcium concentration and was independent of randomized vitamin D treatment. There did not appear to be further increases in creatinine after the first year of calcium treatment.After one year of treatment, blood creatinine was 0.013±0.006 mg/dL higher on average among participants randomized to calcium compared to placebo after adjustment for other determinants of creatinine (P = 0.03). However, the effect of calcium treatment appeared to be larger among participants who consumed the most alcohol (2–6 drinks/day) or whose estimated glomerular filtration rate (eGFR) was less than 60 ml/min/1.73 mAmong healthy adults participating in a randomized clinical trial, daily supplementation with 1200 mg of elemental calcium caused a small increase in blood creatinine. If confirmed, this finding may have implications for clinical and public health recommendations for calcium supplementation.NCT00153816ClinicalTrials.gov  Calcium supplements are widely recommended and commonly used in the United States, especially by postmenopausal women for preventing and treating osteoporosis In a randomized controlled trial of the effects of calcium and/or vitamin D supplementation on colorectal adenoma recurrence, changes in blood creatinine concentration were measured as a pre-specified interim outcome for safety to assess potential renal effects. We observed that calcium treatment for one year was associated with a small increase in blood creatinine concentration, suggesting the possibility of an effect on renal function. In the current report, we analyze the association of calcium treatment with changes in blood creatinine among the predominantly male, 45–75 year old participants in the full factorial component of our trial.www.clinicaltrials.gov) on September 7, 2005, when the investigators first became aware of the opportunity for trial registration. The authors confirm that all ongoing and related trials for this drug/intervention are registered. The following institutional review boards (IRBs) approved the study prior to its initiation at that site : Dartmouth College Committee for the Protection of Human Subjects (8/25/03), University of Southern California IRB (3/17/04), Kaiser Permanente IRB (5/27/04), Colorado Multiple Institutional Review Board (7/23/04), Emory University IRB (4/12/04), Atlanta Veterans Affairs Medical Center (VAMC) IRB (4/12/04), University of Iowa IRB (6/9/04), University of Minnesota IRB (6/3/04), Minneapolis VAMC IRB (6/29/04), Portsmouth Regional Hospital IRB (3/24/04), Concord Hospital IRB (4/1/04), Maine Medical Center IRB (6/7/04), White River Junction VAMC IRB (3/4/04), University of North Carolina IRB (5/24/04), Cleveland Clinic IRB (5/21/04), University of Puerto Rico IRB (9/25/06), San Juan VAMC IRB (11/7/06), Palmetto Health IRB (4/13/04), University of Texas/MD Anderson Cancer Center IRB (4/7/04). All participants provided written informed consent at enrollment. An independent Safety and Data Monitoring Committee reviewed the study semiannually.This study was registered at ClinicalTrials.gov as NCT00153816 , both vitamin D3 and calcium, or placebo. In this report, which analyzes the effect of calcium treatment, we compare the two groups randomized to calcium  to the two “control” groups randomized to no calcium . In the “Two Arm” component of the study, women who chose to receive calcium supplementation were randomized with equal probability to either vitamin D3 plus calcium or to calcium alone, and are excluded from this report since they were not randomized to calcium treatment. The expected duration of the intervention was either 3 or 5 years, according to the colonoscopy follow-up interval recommended by the participants' physicians. Sample size was determined by the hypothesized treatment effect on the primary outcome of adenoma recurrence.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Participants were recruited from gastroenterology departments and practices associated with eleven clinical centers in the United States; the Project Coordination Center and trial database are located at The Geisel School of Medicine at Dartmouth.Eligible patients were 45–75 years of age, in good general health, and had at least one large bowel adenoma removed at a colonoscopy within four months before enrollment with a recommended follow-up interval of either 3 or 5 years. Other inclusion criteria included blood total calcium within the normal range, creatinine ≤20% above the upper limit of normal, and 25-hydroxyvitamin D between 12–90 ng/ml. Exclusion criteria included a history of a familial colorectal cancer syndromes, invasive large bowel cancer, malabsorption syndromes, and indications or contraindications to the study agents .3 (500 IU), both agents, or placebo with instructions to take one tablet twice daily with food. Participants, health care providers and investigators were blinded to treatment assignments.Participant recruitment began on 5/15/04 and enrollment occurred between July 16, 2004 and July 31, 2008. At enrollment, a detailed questionnaire was administered to collect demographic, medical and lifestyle data. Race and Hispanic ethnicity were self-reported. Current height and weight were documented, blood was collected, and participants completed a Block Brief 2000 Food Frequency Questionnaire . Participants were counseled to avoid additional supplementation with calcium or vitamin D during the study and to reduce their dietary consumption of calcium if it exceeded 1200 mg/day. Study multivitamins, formulated without any calcium or vitamin D, were provided to participants if desired. At enrollment, participants began a blinded placebo run-in period (56–84 days) to assess adherence to study procedures before randomization; participants who took <80% of their study pills were not randomized. A web-based program was used to randomize participants in blocks with computer-generated random numbers; stratification factors included full factorial participation, clinical center, sex, and colonoscopic follow-up interval (3 or 5 years). Identical appearing study pills contained calcium , vitamin DParticipants were interviewed every six months by telephone regarding adherence to study treatment; use of medications and nutritional supplements; symptoms, illnesses, medical procedures and hospitalizations; and dietary intake of calcium and vitamin D. As pre-specified in the protocol, interim visits were scheduled to measure blood total calcium, creatinine, and 25-hydroxyvitamin D one year after randomization, and three years after randomization for participants with a 5-year follow-up. Participants were removed from treatment if their calcium was above the normal range, if their creatinine was >20% above the upper limit of normal, or if their 25-hydroxyvitamin D was <10 or>100 ng/ml. In addition, 25-hydroxyvitamin D was measured at “end-of-treatment visits” scheduled shortly before the participants' follow-up colonoscopies. Creatinine was measured at this time as well for participants who elected to take part in a “kidney sub-study” of the renal effects of vitamin D, which was active between September 2010 and April 2012. Additionally, in October 2011 the Safety and Data Monitoring Committee unblinded the study investigators to the observed association of calcium treatment with elevated blood creatinine levels. As a result, the protocol was modified to include creatinine and calcium measurements annually, to remove participants from randomized calcium treatment if their creatinine was above the upper limit of normal or if there was an increase of ≥0.3 mg/dL from baseline, and to retrospectively measure creatinine and calcium on stored specimens from “end-of-treatment visits” that were completed previously without creatinine measurements . Fasting prior to study blood draws was recommended but not required. Participant follow-up in the treatment phase of the study ended on August 31, 2013 and the main results are currently being analyzed.Measurements of creatinine and calcium were performed on fresh serum or plasma samples according to standard procedures at laboratories associated with the eleven participating clinical centers. Local laboratory reference ranges were used to identify out-of-range values. Three clinical centers changed laboratories during the study. Additionally, measurements of creatinine and calcium on frozen serum samples collected at “end-of-treatment visits” completed prior to October 2011 were performed at Quest Diagnostics : creatinine was measured using a kinetic modified Jaffe method  and total calcium was measured using an Arsenazo assay (Beckman Coulter Inc.). All 25-hydroxyvitamin D measurements were performed on frozen serum samples at the University of California (Los Angeles) at the Nutrition Research Center Laboratory using a radioimmunoassay kit from Immunodiagnostic Systems .2. Analyses were conducted according to the intent-to-treat principle, except as indicated. All statistical tests were two-sided and considered significant at a value of P<0.05. Statistical analyses were performed using SAS .Study sample size was based on power calculations for the primary outcome of adenoma recurrence. T-tests were used to compare blood creatinine and total calcium concentrations between treatment groups. A chi-square test was used to compare the number of participants by treatment group with elevated (out-of-range) creatinine concentrations or with increases of ≥0.3 mg/dL between baseline and year one. Estimated glomerular filtration rate (eGFR) was calculated using the CKD-EPI equation 2, a level that, if persistent, commonly defines chronic kidney disease A total of 1,675 participants were randomized in the Full Factorial component of the study . BaselinNumbers of and reasons for unobtainable blood draws at years one, three and five were similar between the treatment arms . OverallAt year one, randomized calcium treatment was associated with a slightly larger increase in blood creatinine concentration: 0.038±0.004 versus 0.021±0.004 mg/dL (mean ± SE) (P = 0.01) . Among p2r = 0.596). After controlling for characteristics listed in Next, we examined the effect of calcium supplementation on year one creatinine concentration adjusted for baseline creatinine and other potential determinants of renal function . OverallTo determine whether the concomitant increase in blood calcium mediated the effect of calcium supplementation on creatinine concentration , year one blood calcium concentration was added to the regression model. For every 1 mg/dL increase in year one calcium concentration there was an increase of 0.039±0.008 mg/dL (mean ± SE) in year one creatinine (P<0.0001) and the coefficient for calcium treatment decreased by about 23% to 0.010±0.006 mg/dL (mean ± SE) (P = 0.11).interaction = 0.54). It also did not statistically significantly effect blood calcium concentration at year one, although it was associated with a net increase in 25-hydroxyvitamin D concentration of 6.7 ng/ml (P<0.001).Notably, randomized vitamin D treatment did not effect year one creatinine concentration  or modif2: at year one creatinine was on average 0.10±0.05 mg/dL higher (mean ± SE)  and the adjusted year one eGFR was on average 4.9±2.7 ml/min/1.73 m2 lower (mean ± SE) . Due to the high rate of missing data on baseline dietary calcium intake, it was excluded from this sub-group analysis; however, its inclusion did not change the results. Second, greater baseline alcohol consumption appeared to be associated with a larger effect of calcium treatment, although the interaction was not statistically significant. Among participants in the highest category of baseline alcohol consumption , year one creatinine was on average 0.034±0.016 mg/dL (mean ± SE) higher among participants randomized to calcium treatment .In exploratory analyses, there was suggestive evidence from subgroup analyses that two factors modified the effect of calcium supplementation. First, the effect of calcium appeared to be more pronounced among participants (N = 62) with baseline eGFR <60 ml/min/1.73 m2 or among those consuming the most alcohol (2–6 drinks/day). There was no evidence that randomized vitamin D treatment independently affected creatinine or modified the effect of calcium supplementation. In addition, in exploratory analyses, the effect appeared to be sustained but not progressive beyond the first year of calcium supplementation.Calcium supplementation  led to a very modest increase in blood creatinine concentration in a population of generally healthy 45–75 year old adults, predominantly white (85%) and male (85%), who were participating in a randomized clinical trial. After one year, creatinine concentration was 0.013 mg/dL higher on average among participants in the calcium treatment group compared to the control group after adjusting for other determinants of creatinine concentration. However, in exploratory analyses, the effect of calcium supplementation appeared to be more pronounced among participants with baseline eGFR <60 ml/min/1.73 mAlthough numerous studies have investigated randomized calcium supplementation for osteoporosis prevention or treatment i.e., without reducing glomerular filtration). For example, treatment with an active vitamin D analogue increased creatinine production and serum creatinine without modifying the glomerular filtration rate The mechanism by which calcium supplementation increases blood creatinine is unknown, but could be due to an effect on renal function. Some of the effect could be due to the very modest increase in blood calcium due to calcium supplementation, possibly through a hemodynamic mechanism. Our statistical analyses suggest that the increase in blood calcium concentration explained only about 23% of the increase in creatinine concentration due to calcium supplementation at year one. Other potential mechanisms include mild vasoconstriction due to increased calciuria or induction of natriuresis by calcium 2) have been associated with increased cardiovascular risk and mortality The clinical relevance of the increase in creatinine associated with one year of calcium supplementation in our study is uncertain, but our exploratory analyses suggest that it may be larger and therefore more important in individuals with pre-existing renal impairment or alcohol consumption. Notably, rapid declines in kidney function of the magnitude reported here for the participants with pre-existing renal impairment Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.