Maternal ethnicity is a recognized risk factor for stillbirth, such that South Asian women have higher rates than their Caucasian counterparts. However, whether maternal ethnicity is a risk factor for intrapartum outcomes is less clear. The aim of this study is to explore associations between maternal country of birth, operative vaginal delivery and emergency cesarean section, and to identify possible mechanisms underlying any such associations.We performed a retrospective cohort study of singleton term births among South Asian, South East/East Asian and Australian/New Zealand born women at an Australian tertiary hospital in 2009–2013. The association between maternal country of birth, operative vaginal birth and emergency cesarean was assessed using multivariate logistic regression.p < 0.001 for both) and emergency cesarean section . While prolonged labor was the predominant reason for cesarean section among Australian/New Zealand and South East/East Asian women, fetal compromise accounted for the majority of operative births in South Asian women.Of the 31,932 births, 54%  were to Australian/New Zealand-born women, 25% (7874) to South Asian, and 22% (6879) to South East/East Asian born women. Compared to Australian/New Zealand women, South Asian and South East/East Asian women had an increased rate of both operative vaginal birth (OR 1.43 [1.30–1.57] and 1.22 [1.11–1.35] respectively, South Asian and South East/East Asian women experience higher rates of both operative vaginal birth and cesarean section in comparison to Australian/New Zealand women, independent of other risk factors for intrapartum interventions. Maternal ethnicity is known to be an independent risk factor for a variety of adverse pregnancy and perinatal outcomes. In particular, women of Asian ethnicity giving birth in high-income countries exhibit higher rates of adverse pregnancy outcomes than other women accessing the same health services –7. For eWhile the increased rates of adverse antenatal outcomes for SA women compared to other women are consistent observations, studies exploring the relative rates of various intrapartum interventions, such as cesarean section and operative vaginal birth rates among women of different ethnicities are less so –11. SomeWe studied all singleton births ≥37 weeks gestation and free from congenital anomalies at Monash Women’s Services, Monash Health, a metropolitan university teaching hospital in Melbourne, Australia, from 2009 to 2013 inclusive. Data were extracted from the Birthing Outcomes System (BOS), an electronic database recording all births ≥20 weeks’ gestation. For each birth the attending midwife, supported by routine data validation, enters 46 data items into BOS . For thiWomen were classified into regional groups, as a surrogate measure of ethnicity, according to their self-reported country of birth as defined by the United Nations . Briefly2 or Kruskal-Wallis test. The rates of spontaneous vaginal birth, operative vaginal birth, planned cesarean and emergency cesarean were also determined for each risk factor. Logistic regression was used to examine the association between maternal country of birth and spontaneous vaginal birth, operative vaginal birth and planned and intrapartum cesarean delivery. Known and potential risk factors that were assessed for their inclusion in the final model were maternal age, BMI, parity, need for interpreter, labor type , epidural, birth weight over 4000 g, private patient, length of first and second stage of labor, past cesarean, first trimester ultrasound, pre-existing hypertension, pre-existing diabetes, gestational hypertension/pre-eclampsia/HELLP, gestational age, baby gender, and birthweight <10th centile (birthweight centiles were not customized for ethnicity). The association between maternal country of birth, spontaneous vaginal birth, operative vaginal birth, and planned and emergency cesarean, adjusted for confounders, was then assessed using stepwise multivariable logistic regression, including only confounders significant at the univariate level. The likelihood ratio was used to determine the final model. Not unexpectedly, the rates of operative vaginal birth and cesarean delivery differed by parity. Therefore, we also performed the regression stratified by parity. Doing so did not change the associations between maternal country of birth and the outcomes and so we have presented data for all women regardless of parity. The indications for intrapartum cesarean section and operative vaginal birth by maternal country of birth group were also tabulated. A p-value <0.05 (two-tailed) was regarded as significant. All analyses were performed using the SPSS statistical package .Maternal demographics, antenatal and intrapartum factors were tabulated by maternal country of birth. Differences across groups were determined by a chith centile) in comparison to 16.6% of those born to SA and 13% of those born to SE/EA.Between 2009 and 2013 there were 41,040 singleton births at the maternity service. Of these, 31,932 (78%) births were at term to women from one of the three regions of origin. Of these, 17,149 (54%) births were to AUS/NZ born women, 7874 (25%) to SA born women, and 6879 (22%) to SE/EA born women. The characteristics of the women are presented in Table p < 0.001). However, labor augmentation was more common in SE/EA (24.9%) and SA (23%) women than in AUS/NZ born mothers (19.1%) (p < 0.001). Use of epidural anesthesia was greatest in the AUS/NZ born population (21.1%). Overall, Asian women experienced a longer 1st and 2nd stage of labor compared to AUS/NZ women (p < 0.001 for both). APGAR scores at delivery were similar across ethnic groups. However admission rates to NICU were similar between the SA and AUS/NZ populations and lower in the SE/EA born women (p < 0.001).Regarding labor, women born in SE/EA were less likely to be induced (15.5%) than either AUS/NZ (23.9%) or SA (24.9%) women (p < 0.001) and a 24% (95% CI 0.69–0.84) decreased odds for SE/EA women (p < 0.001). The rate of planned cesarean section was comparable between SA and AUS/NZ women . However SE/EA born women had a significantly lower rate of planned cesarean section  (p = 0.02).The rate of spontaneous vaginal delivery differed amongst ethnic groups Table  and was p < 0.001) and an 14%  increased odds for SE/EA women (p < 0.001). After adjustment for confounders, compared to AUS/NZ born women, SA women had a 43%  and SE/EA women a 22%  increased odds of operative vaginal birth (Table p = 0.003). A prolonged labor was the most frequent indication for an operative vaginal birth among SE/EA women (42.4%) followed by SA (38%) and AUS/NZ (36.1%) women (p = 0.006).The rate of operative vaginal birth differed significantly by maternal country of birth. The rate of operative vaginal birth was highest in SA women (16.9%), followed by SE/EA women (14.6%) and then AUS/NZ women (13%), representing a 36%  increased odds of operative vaginal birth for SA women compared to AUS/NZ women (p < 0.001). After adjusting for confounders, SA women had a 67% increased odds  and SE-EA women a 16% increased odds  of intrapartum cesarean section, compared to AUS/NZ women (p = 0.007). The primary indication for intrapartum cesarean section also varied significantly between maternal country of birth. Fetal compromise was most common in SA women (42%) followed by AUS/NZ (36%) and then SE/EA (26.3%) women (p < 0.001). In contrast, prolonged labor was the most common indication in SE/EA (65%), followed by AUS/NZ (57%) and then SA (55%) women (p < 0.001).The rate of intrapartum cesarean also differed significantly by maternal country of birth. The rate of emergency cesarean was significantly higher in SA women (18.5%) than in either AUS/NZ(13.5) or SE/EA(13.3) women  may explain ethnic differences in Asian women. We were able to adjust for these and, consistent with other populations , 21–23, We were also able to show that the indications for operative births varied between ethnic groups. Specifically, the increased rate of cesarean section among SA women was largely due to an increased rate of intrapartum fetal compromise. This confirms previous studies , 23 and Whatever the underlying reasons for increased intervention rates, both operative vaginal birth and cesarean section are associated with significant maternal and perinatal morbidity. Operative vaginal births are associated with higher rates of both maternal and fetal trauma as well as postpartum hemorrhage –34. CesaOur study has a number of limitations. As a retrospective analysis drawn from a single center, the demographic of the population and the predispositions of physician practice may all have influenced mode of delivery and so limit generalizability. We don’t believe this to be likely because our broad findings are in accord with those of others. However, it would be useful for other centers to explore whether indications for cesarean section differed between maternal ethnic groups. Of course, the indication recorded for any operative delivery is dependent on the attending accoucher and therefore inter-practitioner variability is possible. Nonetheless, we believe that the large number of practitioners that would be represented in our study makes our findings more, and not, less generalizable. In this study, we were only able to define ethnicity by maternal country of birth and as such it is possible that some women within the AUS/NZ born group are of Asian ethnicity. However, we believe that this is more likely to have underestimated rather than overestimated the associations. Nonetheless, we would call for all jurisdictions to record both maternal ethnicity and maternal country of birth rather than one or the other. Lastly, non-independence is a recognized issue when using population based perinatal datasets. We assessed our associations in both nulliparous and parous women. Doing so did not substantially change our findings.Maternal country of birth is an independent risk factor for operative vaginal birth and emergency cesarean delivery. In regards to emergency cesarean section, the principal indications for surgery also differ by maternal country of birth with fetal compromise being more common in SA women and lack of progress of labor being more common in SE/EA women. Understanding these differences further is likely to improve maternity care for all women.


Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent. Culex tritaeniorhynchus. Birds are reservoirs for JEV. Pigs and ardeidae birds are the principal amplifying hosts and they play important roles in the maintenance and transmission of the disease  is vector-borne virus that causes Japanese encephalitis (JE), a severe zoonotic neurological disease. The virus is an enveloped single-stranded positive sense RNA virus that is endemic in Asian countries. JE is fatal to almost one third of affected human population. It is estimated that approximately three billion people worldwide are at risk of acquiring JE with 50,000–175,000 cases  Perak—Tapah (3) , Parit Buntar (4) , Tanjong Piandang (5) , and Gopeng (6) ; (II) Selangor—Serdang (9) , Putrajaya (10) , Sabak Bernam (11) , Jenderam Hulu (12) , and Kepong (13) ; (III) Perlis—Kangar (1)  and Arau (2) ; (IV) Kelantan—Kuala Krai (7)  and Machang (8) ; and (V) Pahang—Temerloh (14) . Figure The study was carried out in Peninsular Malaysia. Peninsular Malaysia is located between 1° N and 7° N of the equator covering a distance of 124,450 kmMacaca fascicularis) were collected from three different states: Pahang, Kelantan, and Perlis. Pig samples were collected from Tapah (Perak) 45 and Sepang (Selangor) 45, and all cattle samples (n = 90) were collected from Sabak Bernam and Serdang areas under Selangor state.A cross-sectional survey was carried out to determine the seroprevalence and risk factors of JEV in animals and avian species in Peninsular Malaysia. Sample collection was conducted between December (2015) to February in 2016 and March to August in 2016. Overall, 416 samples were collected from dogs, cats, water birds, village chickens, jungle fowl, long-tailed macaque, domestic pigs, and cattle. Blood samples were collected for serum separation in a total of 45 birds from Perak, Selangor, and Putrajaya. Cat samples were collected from shelters and domestic cats from Ipoh, Perak, Putrajaya, Selangor, and University Veterinary Hospital—Universiti Putra Malaysia , respectively. Ninety samples were collected from cat shelters and domestic cats from Perak and Selangor. However, only 45 dog samples were collected from shelters in Ipoh, Perak, and Selangor. A total of 56 serum samples from long-tailed macaque (g for 10 min. The serum was collected and stored at − 20 °C until further use. All samples used in this study were collected by the attending veterinary physician.Plain blood tube (3 ml) was used to collect blood from the jugular vein of each animal and birds. The serum was collected and centrifuged at 4000 ×NC) are less or equal to 0.15 and the mean value of positive control O.D (O.DPC) is more or equal to 1.00. For result interpretation, the cut off value was calculated using the following formula:Serological detection of antibodies to JEV was performed using a commercially available qualitative ELISA kit  (JE-IgG)  for chicken and water birds; Qualitative Canine Japanese Encephalitis IgG Antibody (JE-IgG)  for dogs; Cat (JE-IgG) Elisa kit  for cats; Bovine (JE-IgG) Elisa kit  for cattle; Porcine (JE-IgG) Elisa kit  and quantitative ELISA namely the Monkey Japanese Encephalitis Antibody IgG (JEAb-IgG),  for monkeys. All procedures were carried out according to the manufacturer’s protocol. Samples were duplicated and microplates were read at the wavelength of 450 nm within 15 min of adding the stop solution. The optical density (OD) was read using a TECAN infinite microplate reader  with Magellan v.6.5 software (Austria). The test is validated if the mean values of negative control O.D  and y = 1 / (a + bxc). The positive samples were range between 0.5 and 10 μg/ml according to the manufacturer’s protocol.To determine the antibody concentration of the samples, the standard curve was constructed by plotting mean absorbance for each standard  on the A 13-questionaire was distributed to the farmers in order to collect and identify the risk factors related with JEV seropositive. The survey questions were collected from farm owner the following: (a) Individual animal or bird: age, health status , source of animals or birds  and breed types , ownership of animals or birds , and presence of mosquitoes in sample collected areas (present or absent). (b) Environment factor: stagnant water (types of stagnant water are ditches or ponds within 100 to 500 m), paddy cultivation (within 200 to 500 m), presence of ardeid birds (within 1 to 2-km location of bird landing areas), locality , state , and nature of state located .χ2 test is not appropriate one of expected cell value was less than 5, Fisher’s exact test was used at α = 0.05) was used to determine the univariate association between each of the putative risk factors with JEV. Similarly, the odd ratio (OR) and its 95% confidence interval (CI) were calculated at a significance level of α = 0.05.All analyses were performed using the IBM SPSS Statistics V22.0 . Prevalence was expressed as a percentage and calculated by dividing the number of positive samples by the total number of animals tested. Chi-square analysis  followed by pig (44.4%), cattle (32.2%), and bird (28.9%). Cat and monkey both had the lowest prevalence among all animals sampled Table .Table 1SP > 0.05) of age, locality, breed, and source of cats to JEV exposure in these species. Other factors such as gender, health status, stagnant water, place, nature, and owner all did not show any significant influence on JEV (Table P < 0.05) seropositivity as cats from the same areas showed two times more seropositive rate than those from the same district.The individual risk factor assessment of JEV in cats showed significant influence (EV Table . Breed oP < 0.05) influenced JEV exposure, while all other factors evaluated did not have an influence on JEV exposure  to JEV exposure. All other factors evaluated were not statistically different  influence on JEV exposure in this species. Other risk factors assessed did not significantly affect the exposure of this animal to JEV  contributed to JEV exposure . This is considered a lower prevalence rate in comparison to dogs. There are several factors that can contribute to the prevalence rate of JEV among pigs in Malaysia. Pig farms in Malaysia are normally far away from residential areas and situated in rural areas which are not close to paddy cultivation areas. The chances of JEV transmission through Culex spp. is very common in certain areas. Bovine are dead-end hosts for JEV . Due to the semi-intensive nature of cattle husbandry in Malaysia, cattle exposure to Seroprevalence rate in birds was only 28.9%. This is a rather unexpected finding as the sampling area in the state is an area with high development of rice paddies. There is also a migratory bird landing areas there . There is no pig farming around the area, the residents normally engaged in fishing activities. The presence of numerous water bodies serves as suitable breeding sites for mosquitoes. A low-incidence case of JEV was previously reported in rice growing area in Thanjavur district in Tamil Nadu, India. This was attributed to the ratio of cattle to pigs in the district  revealed a prevalence rate of 44%  colonies in northern Thailand were reported to be seropositive for JE . However, all blood samples were found negative for JEV antigen detection. This rapid test did not detect antigen in both JEV seropositive and seronegative animals. This can be explained by the presence of low levels of viremia in the hosts which may have contributed to false negative at molecular assay (Cloherty et al. Since most JEV cases were detected during natural infection, the acute phase of virus replication is often unknown. Virus antigen was not detected in blood samples taken during non-acute phase or during neutralizing activities (Casenghi et al. This study has provided with the baseline information for the understanding of epidemiology and risk factors in JEV infection, transmission, and spread. This study demonstrated that most animals in Malaysia are susceptible to JEV infections. However, the pigs, dogs, and cattle are most susceptible to become JEV seropositive with exposure to the virus. JEV seropositivity is significantly influenced by several risk factors, where young aged animals, presence of mosquito vectors, and migrating birds had major influence on the development of infections in these animals. Evidently, the infection is highly prevalent in Peninsular Malaysia and control measures are to be in place to ensure that the disease is contained.


We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10–100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease ; Canavan disease ; and Tay-Sachs disease . We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples . Taken together, the results suggest coordinated selection in AJ population for higher CD risk alleles in general. The results and approach illustrate the value of exome sequencing data in case-control studies along with reference data sets like ExAC (sites VCF available via FTP at ftp.broadinstitute.org/pub/ExAC_release/release0.3/) to pinpoint genetic variation that contributes to variable disease predisposition across populations.As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at  The Ashkenazim are a people with ancestry in northern-European Jewish groups. A founder effect caused a bottleneck in this population approximately one thousand years ago, resulting in a group of enriched alleles in their genetic makeup. A higher documented prevalence of Crohn’s Disease in the Ashkenazim indicates that some enriched alleles may confer risk of having this disease. By studying which genes are enriched, and which of these contribute to Crohn’s Disease risk, we are better able to understand the genetic architecture of the affected population, and of the disease itself. Further, we are able to develop a resource containing tables of significantly enriched alleles that are known or suspected to contribute to other disease. Genetic population isolates like the Ashkenazim, Jews who trace their ancestry to eleventh century central European Jewish groups, have prWe generated a jointly called whole-exome sequence dataset consisting of 18,745 individuals from international Inflammatory Bowel Disease (IBD) and non-IBD cohorts,10 S1 F. Given tTo identify AJ individuals and estimate admixture fractions we used a set  of LD-pruned common variants  filtered for genotype quality (GQ>20). The 18,745 individuals were assigned to four groups (K = 4) using ADMIXTURE , AFR , and AMR ]. We focused on protein-coding alleles with estimated allele frequency of at least 0.002 and less than .1 in AJ . Since much rarer alleles have a higher probability of being damaging , the advantage to gene mapping arises from the fact that enriched alleles of a certain frequency are more damaging/deleterious on average than non-enriched alleles of the same frequency.To explore AJ exome population genetics, including proportion of enriched alleles and degree of enrichment, we used the observed alternate allele counts and total number of alleles available from ExAC release 0.3 dataset  version 83 with Gencode v19 on GRCh37. Loss-of-function (LoF) variants were annotated using LOFTEE  and —Credible sets of SNPs around index associations were defined as in  for fine-mapped loci, and for others credible sets were defined as all SNPs with rhttp://pngu.mgh.harvard.edu/~purcell/plink/summary.shtml#prune.As the present study aimed to focus on variation observed in Ashkenazi Jewish (AJ) population in comparison to reference populations in ExAC including (non-Finnish Europeans (NFE), Latino (AMR), and African/African-American (AFR)) we chose a model-based approach to estimate the ancestry of the study population using ADMIXTURE. To idenFor the parameters, we selected a window size of 50 SNPs, a window shift of 5 SNPs at each step, and the variance inflation factor (VIF) threshold equal to 2.The 18,745 samples were assigned to four groups (K = 4), as ancestry was defined as having a single estimated ancestry fraction ≥ 0.4, and remaining three fractions < 0.4 . IndividPrior to enrichment and association analysis, 81 samples  were also filtered due to possible contamination (heterozygous/homozygous ratio < 1), excess of singletons (n>2000), deletion/insertion ratio (>1.5) and mean genotype quality (<40). 275 samples were excluded for relatedness (>0.35 cut-off). Genotypes with low genotype quality (<20) were filtered, in addition to variants with low call rate (<80%) and allele balance deviating from 70:30 ratio for greater than 40% of heterozygous samples if at least 7 heterozygous samples were identified.https://github.com/broadinstitute/hail). Allele balance was calculated using PLINK/SEQ (https://atgu.mgh.harvard.edu/plinkseq/).As we were interested in computing an enrichment statistic that would not be affected by possible admixture, we obtained alternate allele frequency estimates by restricting the enrichment analysis to the 2,178 non-IBD Ashkenazi Jewish samples that passed QC and relatedness filtering and had AJ focused ancestry fraction > 0.9 . PrincipTo estimate which alleles are enriched in AJ compared to alleles in reference population groups in ExAC we applied Fisher’s exact test one-sided alternative (“greater”).http://www.itl.nist.gov/div898/software/dataplot/refman2/auxillar/logoddra.htm, and http://www.itl.nist.gov/div898/software/dataplot/refman2/auxillar/logodrse.htm)Using the number of alternate and reference alleles observed in AJ non-IBD samples and in the population  with the highest frequency from ExAC we compute a bias corrected log odds ratio estimate, Precisely, We classified a variant as ‘enriched’ if p-value was less than .05/73,228, where 73,228 is the number of variants analyzed with minor allele frequency between .002 and .1.To estimate allele enrichment in AJ compared to reference populations we used 2,178 non-IBD Ashkenazi Jewish samples, after sample and relatedness QC.total = 60,706) and focused on alleles where allele frequency information was available for AJ and the reference populations. For the enrichment plot we focused on alleles with estimated frequency of at least 0.002 in AJ  and with alleles observed with an estimated frequency of at least .0001 in the reference populations with depth of coverage of at least 20X in at least 80% of the samples in ExAC.We calculated alternate allele frequencies for the Ashkenazi Jewish population and used allele frequency information for NFE , AFR , and AMR  available from ExAC release 0.3 dataset (nhttps://github.com/macarthur-lab/clinvar). Briefly, we normalized variants using a Python implementation of vt normalize[We harmonized the XML and TXT releases of the ClinVar database  into a snormalize and de-dWe focused Crohn’s disease association analysis of protein-coding variant to alleles that may account for difference in disease prevalence in AJ population to reference populations. To do so we focused on alleles with high probability of belonging to the enriched group. We included all samples with ADMIXTURE estimated AJ ancestry fraction of at least 0.4 . Samples with Ulcerative Colitis (n = 700), unspecified and Indeterminate Colitis (n = 86) were excluded from subsequent analysis. This resulted in a dataset of 4,899 AJ samples .Study-specific association analysis was performed with Firth bias-corrected logistic regression test,26 and f10BF > 2). For each model we can obtain a Bayes factor (BF) for association by comparing it with the null model where all the prior weight is on an effect size of zero. We report p-value approximation using the Bayes factor as a statistic for model 2 where the effects are correlated between studies.For meta-analysis we combined association statistics using the Bayesian models and frequentist properties proposed in Band et al, which iAssociation statistics were combined based on association analysis across three study groups: 1) AJ ; 2) NFE ; and 3) Finnish (FINN)  for a total of 4,361 CD samples and 15,744 non-IBD samples.https://atgu.mgh.harvard.edu/plinkseq/), an open-source C/C++ library for working with human genetic variation data, and the Python bindings implemented in pyPLINKSEQ to perform Bayesian Model Averaging (BMA). We applied BMA[https://cran.r-project.org/web/packages/BMA/BMA.pdf).In the conditional haplotype-based testing (—chap) analysis we used PLINK v1.08p and set plied BMA using thIL23R from a recent fine-mapping study[NOD2 and LRRK2. We used 454 AJ controls; 1,938 AJ CD; 35,007 non-Jewish controls and 20,652 non-Jewish CD samples. Polygenic risk scores were calculated using array genotype data as the sum of the log odds ratio of the variants associated with CD. Scores for missing genotypes were replaced by the imputed expected value using PLINK[NOD2 and LRRK2 were excluded from the analysis to assess whether polygenic signal was independent of those genes.The polygenic risk scores were calculated for the international inflammatory bowel diseases consortium European samples. Details of these samples including the QC procedures were described in previous publications. We useding study, and excPRSi be the polygenic risk score of individual i, assuming additive effects on the log-odds scale, i.e.m and Gi,m denotes the genotype dosage of individual i for variant m. More specifically, m on a logit scale in conferring risk of CD in an individual.Let In the setting where effects are non-additive, i.e. a genotype-specific effect model,NOD2 alleles to genetic risk in two populations.For now, we consider the additive scenario, and later we return to the setting where non-additive effects exist, which is relevant for quantifying the differences in contribution of j isNj is the number of individuals sampled in population j. Substituting equation for PRSi and rearranging terms simplifies the equation as a function of variant frequency:m in population j. Thus, the estimated expected PRS value of an individual in population j is The estimated expected PRS value for an individual in population m is shared across both populations. Then, using the estimated expected PRS value we define estimated expected difference in contribution of the studied variants to the PRS as the difference in estimated expected PRS value in two populations:m to the polygenic risk score in two populations,Assume that we are interested in the expected difference in contribution of the studied variants to the PRS between two individuals, say from population 1 being AJ and population 2 being NFE. Also, assume that the effect size of variant NOD2 and LRRK2 alleles, used the list of estimated effect size from the published studies[To rank variants according to their relative differences in contribution to genetic risk we included the d studies,37, and PRS* for PRS,If we substitute PRS* when non-additive effects exist isThen, the estimated expected difference in p = (1 + exp(−η))−1, where η tends towards a normal distribution with parameters p0 refers to a baseline disease probability.Assuming log-additive effects in the logit risk model the disease probability for an individual is given as 1−fm)βm2 . Here p0We can see that μ may be expressed in terms of the expected polygenic risk score, i.e. μ we can express the shift as p for new values of μ to obtain a fold-increase in prevalence for a population that has undergone such a shift.To evaluate the impact of a shift in the expected value of polygenic risk score to the expected value of p0 and that log(p0/(1 − p0)) can be represented as a baseline risk score value β0.To get an estimate of the absolute prevalence of CD in the AJ population, we must choose a baseline β0, where p0 represents the expected prevalence with zero non-baseline alleles in the population[We see that this requires a value to be chosen for opulation, to whic0, we can calculate the ratio of expected prevalence as follows. First, use the means (μAJ and μNAJ) and variances  = (1 + eη−)−1, and ϕ is the standard normal density function.Once we have chosen a value for βNext, we integrate to get 0 = -20.5 was chosen in order to obtain a prevalence in the non-AJ population of ~0.5%. At this value of β0, the ratio of prevalence in the AJ population to that in the non-AJ population was estimated to be 1.5 Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S4 Fig(PNG)Click here for additional data file.S5 Fig(PNG)Click here for additional data file.S6 Fig(PNG)Click here for additional data file.S7 Fig(PNG)Click here for additional data file.S8 Fig(PNG)Click here for additional data file.S9 Fig(PNG)Click here for additional data file.S10 Fig(PNG)Click here for additional data file.S11 Fig(PNG)Click here for additional data file.S1 Data File(XLSX)Click here for additional data file.S2 Data File(TXT)Click here for additional data file.S1 Table(PNG)Click here for additional data file.S2 Table(PNG)Click here for additional data file.S3 Table(PNG)Click here for additional data file.S4 Table(PNG)Click here for additional data file.S5 Table(JPG)Click here for additional data file.


There are few studies on the regulation of RGC functions, but our previous investigations demonstrated that dopamine stimulates cyp19a1b expression in goldfish RGCs, while secretoneurin A (SNa) inhibits the expression of this enzyme. Here, we determine the range of proteins and cellular processes responsive to SNa treatments in these steroidogenic cells. The focus here is on SNa, because this peptide is derived from selective processing of secretogranin II in magnocellular cells embedded within the RGC-rich preoptic nucleus. Primary cultures of RGCs were treated (24 h) with 10, 100, or 1,000 nM SNa. By using isobaric tagging for relative and absolute quantitation and a Hybrid Quadrupole Obritrap Mass Spectrometry system, a total of 1,363 unique proteins were identified in RGCs, and 609 proteins were significantly regulated by SNa at one or more concentrations. Proteins that showed differential expression with all three concentrations of SNa included H1 histone, glutamyl-prolyl-tRNA synthetase, Rho GDP dissociation inhibitor γ, vimentin A2, and small nuclear ribonucleoprotein-associated protein. At 10, 100, and 1,000 nM SNa, there were 5, 195, and 489 proteins that were downregulated, respectively, whereas the number of upregulated proteins were 72, 44, and 51, respectively. Subnetwork enrichment analysis of differentially regulated proteins revealed that processes such as actin organization, cytoskeleton organization and biogenesis, apoptosis, mRNA processing, RNA splicing, translation, cell growth, and proliferation are regulated by SNa based on the proteomic response. Moreover, we observed that, at the low concentration of SNa, there was an increase in the abundance of proteins involved in cell growth, proliferation, and migration, whereas higher concentration of SNa appeared to downregulate proteins involved in these processes, indicating a dose-dependent proteome response. At the highest concentration of SNa, proteins linked to the etiology of diseases of the central nervous system  were also differentially regulated. These data implicate SNa in the control of cell proliferation and neurogenesis.Radial glial cells (RGCs) are the main macroglia in the teleost brain and have established roles in neurogenesis and neurosteroidogenesis. They are the only brain cell type expressing aromatase B ( Therefoin vivo and in vitro, SNa reduces cyp19a1b mRNA levels in goldfish RGCs, implicating SNa in the control of neuroestrogen production -immunoreactive magnocellular preoptic neurons and RGCs along the third ventricle in goldfish . The neuoduction . Furtheroduction . AltogetCarassius auratus) were purchased from a commercial supplier  and allowed to acclimate for at least 3 weeks prior to experimentation. Goldfish were maintained at 18°C under a stimulated natural photoperiod and fed standard flaked goldfish food. Sexually mature female goldfish (18–35 g) were anesthetized using 3-aminobenzoic acid ethyl ester (MS222) for all handling and dissection procedures.All procedures were approved by the University of Ottawa Protocol Review Committee and followed standard Canadian Council on Animal Care guidelines on the use of animals in research. Common adult female goldfish  with Antibiotic-Antimycotic solution (Gibco) and minced into small explants. RGCs were dissociated with trypsin  and cultured in Leibovitz’s L-15 medium (Gibco) with 15% fetal bovine serum (Gibco) and Antibiotic-Antimycotic solution. Cell culture medium was changed 4–7 days after isolation and then once a week thereafter. RGCs were subcultured by trypsinization (0.125%) for three passages and then used for experimentation. Goldfish SNa was synthesized as reported previously  acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid, pH 2.8]. The peptides were fractionated using an Agilent HPLC 1260 with a polysulfoethyl A column . Peptides were eluted with a linear gradient of 0–20% solvent B  over 50 min., followed by ramping up to 100% solvent B in 5 min. The absorbance at 280 nm was monitored, and a total of 14 fractions were collected. The fractions were lyophilized and resuspended in LC solvent A . A hybrid quadrupole Orbitrap (Q Exactive) MS system (Thermo Fisher Scientific) was used with high-energy collision dissociation in each MS and MS/MS cycle. The MS system was interfaced with an automated Easy-nLC 1000 system (Thermo Fisher Scientific). Each sample fraction was loaded onto an Acclaim Pepmap 100 pre-column  and separated using a PepMap RSLC analytical column  at a flow rate at 350 nL/min during a linear gradient from solvent A [0.1% formic acid (v/v)] to 25% solvent B [0.1% formic acid (v/v) and 99.9% acetonitrile (v/v)] for 80 min and to 100% solvent B for an additional 15 min.N-terminus, lysine, and variable iTRAQ modifications of tyrosine. The MS/MS spectra that (1) were unique to a specific protein and (2) showed a sum of the signal-to-noise ratios for all the peak pairs greater than 9 were used for quantification. A protein was quantified if it was represented with at least three unique spectra in at least two of the biological replicates, along with a Fisher’s combined probability of <0.05 and a fold change of <0.5 or >1.5.The raw MS/MS data files were processed by a thorough database searching approach considering biological modification and amino acid substitution against the National Center for Biotechnology Information Cyprinidae database  using the ProteinPilot v4.5 with the Fraglet and Taglet searches under ParagonTM algorithm (http://www.genecards.org) to map proteins into Pathway Studio. The number of proteins that successfully mapped to Pathway Studios using Name + Alias was as follows: 10 nM SNa = 99, 100 nM SNa = 231, and 1,000 nM SNa = 499. Interaction networks were based on expression, binding, and regulatory interactions in the database and were constructed using direct connections with one neighbor. Subnetwork enrichment analysis (SNEA) for cell processes was performed in Pathway Studio to determine if differentially expressed proteins were related to specific biological functions. P value for gene seeds was set at < 0.05 and the criterion of >5 members per cell process or group was required for inclusion as a significantly regulated gene network.Gene ontology terms were assigned using protein annotation through evolutionary relationships to classify all proteins identified in RGC cultures . GO caten = 1,363) that were identified in this study is provided in Table S1 in Supplementary Material. The annotated proteins were assigned GO terms and classified in three main GO categories: biological function, molecular function, and cellular component , diseases of the cardiovascular system , and neoplasms . An example of a network associated with Alzheimer, Parkinson, and neurodegenerative diseases is shown in Figure in vitro cell culture system. This effort to characterize the goldfish RGC proteome identified 1,363 unique proteins and adds to those previously reported  in RGCs in vivo and in vitro in a dose-dependent manner  is a calcium-dependent adhesion molecule (N-cadherin controls mouse RGC function in neuronal migration and directing axon formation (N-cadherin through its interactions with its effector β-catenin regulates RGC proliferation and differentiation (N-cadherin causes RGCs to migrate from the stem cell niche and stimulate neuronal differentiation (N-cadherin levels were increased at 10 nM and decreased at 1,000 nM SNa. Therefore, our data on N-cadherin expression indicate a role for SNa in the regulation of RGC-mediated neuronal migration, as well as RGC proliferation and differentiation.Following treatment with 1,000 nM SNa, subnetworks related to tight junction and gap junction assembly were enriched based on the proteins regulated. These cell processes were also identified by gene set enrichment analysis of transcriptomic data and were increased by SNa at the gene network level . Unlike molecule , and it molecule –42. N-caormation . Furtherntiation –46. ReduProteins involved in cellular processes such as neurite outgrowth and nerve fiber regeneration were also altered in abundance in RGCs following treatment with 1,000 nM SNa. The high dose of SNa significantly decreased the abundance of proteins known to be involved in neurite outgrowth. Although this cellular process was not enriched at lower doses, these data indicate that SNa has inhibitory effects on proteins that regulate neurite outgrowth. In contrast, SN stimulates neurite outgrowth in mouse cerebellar granule cells with a maximal concentration of 100 nM . ProteinMany proteome subnetworks were identified that correspond to diseases of the CNS and cardiovascular system along with several neoplasms. Protein networks in RGCs related to Alzheimer disease, Parkinson disease, brain ischemia, and brain injury were all regulated by 1,000 nM SNa. Changes in these networks are consistent with previous studies that have associated SN with these neuropathological conditions. SN is differentially regulated in brain ischemia , 54, 55,The teleost brain has become a popular model for neurogenesis and neuroendocrinology because of the persistent abundance of progenitor RGCs in the adult brain and the high aromatase activity present in RGCs , 66, 67.The study was carried out in accordance with the Canadian Council on Animal Care and approved by the University of Ottawa animal care and veterinary services.DD performed experiments, analyzed the data, and wrote the paper. CM performed experiments, analyzed the data, and contributed to writing the paper. LX performed experiments, analyzed the data, and contributed to writing the paper. VT helped design experiments and co-wrote the paper.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.


SP110 and PMP22 are candidate genes identified in this study as associated with human susceptibility to TB. Here we performed an association analysis in a case-control study of a Tibetan population (217 cases and 383 controls). Using bioinformatics methods, we identified two SNPs in SP110 that may decrease susceptibility to TB , whereas one SNP in PMP22 appeared to increase TB risk . SNPs rs4327230 and rs2114591 remained significant after Bonferroni correction (p<0.00178). We found that the “GC” haplotype in SP110 was protective against TB, with a 64% reduction in disease risk. “CA” and “CG” in PMP22 were also associated with a protective effect. Our study indicates there is an association between specific gene polymorphisms and TB risk in a Tibetan population, and may help to identify those TB-affected individuals most susceptible to disease.Susceptibility to tuberculosis (TB) is partially dependent on host genetic variability.  Mycobacterium tuberculosis, the causative agent of TB, only about 10% will eventually develop clinical disease [Tuberculosis (TB) is a highly infectious respiratory disease that remains one of the world's deadliest communicable diseases. According to the World Health Organization (WHO), an estimated 9 million people developed TB and 1.5 million died of the disease in 2013. Although one third of the world's population is infected with the bacillus SP110 gene on chromosome 2 encodes a leukocyte-specific nuclear body component that can function as an activator of gene transcription and may serve as a nuclear hormone receptor coactivator. SP110 is a promising candidate gene in the regulation of individual susceptibility to M. tuberculosis. First reported by Pan H, et al. [SP110 is the closest human homolog of the mouse gene Ipr1 (Intracellular Pathogen Resistance 1), which protects against tuberculosis by encoding Speckled proteins (SP) that regulate cell activation, division and apoptosis [SP110 polymorphisms and susceptibility to TB in north Indian [PMP22  gene on chromosome 17, previously reported to be associated with Charcot Marie Tooth (CMT) disease or hereditary neuropathy with pressure palsies (HNPP), may also play an important role in susceptibility to TB [SP110 nor PMP22 has yet been investigated with respect to TB in the Tibetan population.The , et al. , SP110 ipoptosis . Previouh Indian , Vietnamh Indian  and Chinh Indian . In addity to TB -15. HoweSP110 and PMP22 and TB risk in Tibetans, we performed an association analysis in a case-control study. Our findings will help to improve patient-specific TB treatment and diagnosis options in the Tibetan population.To investigate the association between We recruited 217 cases  and 383 controls  for this study (Table SP110 and three in PMP22) were identified in cases and controls. All SNP call rates exceeded 98.2%, which was considered high enough to perform association analyses. SNPs in controls were all in Hardy-Weinberg equilibrium (HWE)  and one in PMP22  that exhibited differential allele frequency distributions in cases vs. controls. Rs4327230 and rs2114591 in SP110 remained significant after Bonferroni correction (p<0.00178).To design the multiplexed SNPs MassEXTEND assay, we used Sequenom MassARRAY Assay Design 3.0 Software  and “AA” in rs13422  were associated with TB risk. A protective effect was also associated with genotypes “GG” in rs4327230  and “CC” in rs2114591 . The “GG” genotype of rs4327230 remained significant after Bonferroni correction.We then analyzed associations between ty Table . Genotypp=0.019, OR: 1.59, 95%CI: 1.06–2.39), while rs4327230 was significant in a dominant model . Two other SNPs were associated with TB in both dominant  and recessive models . Rs4327230 and rs2114591 remained significant in the dominant model after Bonferroni correction (p<0.00178).We assumed that the minor allele of each SNP was a TB risk factor compared to the wild type allele and analyzed associations between SNPs and TB in various inheritance models Table . Rs64369SP110 and PMP22 via haplotype analysis  was: total case = 434, total control = 766, global χ2 = 26.416 while df = 2, Pearson's p value<0.001. The result for Block 2 (rs13422 and rs230915) was: total case = 434, total control = 766, global χ2=10.567 while df = 3, Pearson's p value=0.014. We did not perform Fisher's exact probabilities because the total sample size exceeded 40 and all theoretical frequencies were >1.We detected two blocks in SP110 and PMP22 haplotypes and TB risk showed that haplotype “GC” in Block 1 was protective against TB , which remained significant after Bonferroni correction (p<0.00178)  and “CG”  were also protective against TB. These results suggested that rs13422 allele A might be a risk factor for TB susceptibility.Further analyses of associations between SP110 and TB susceptibility in a variety of populations, but these study results are generally considered controversial [SP110  and one in PMP22 (rs13422) were associated with TB risk. To the best of our knowledge, this is the first study to demonstrate an association between PMP22 and TB, and the first study of SP110 in Tibetans. We found that the “GC” haplotype in SP110 was protective against TB, with a 64% reduction in disease risk. “CA” and “CG” in PMP22 were also associated with a protective effect.Multiple groups have investigated associations between oversial , 16-18. SP110 SNPs, rs2114591 and rs6436917, were particularly notable in this study. The rs2114591 C allele in intron 11 of SP110 greatly reduced TB risk in the study population. However, a previous study by Abhimanyu, et al. [et al. [et al. [et al. [p=0.0057). Our study and others indicate that gene expression may differ among races, and this should be taken into consideration when determining an individual patient's susceptibility to TB.Two , et al.  showed n [et al.  nor Png, [et al.  did not PMP22 encodes an integral membrane protein that is a major component of myelin in the peripheral nervous system. It is expressed in Schwann cells and also in non-neural cells such as the lung epithelium, and influences cell proliferation [PMP22 3′UTR as a TB risk factor in a Tibetan population. This is the first study to associate PMP22 and TB risk.feration . In the M. tuberculosis strain virulence. Mouse models have been employed to explore the impacts of Ipr1, encoded within the sst1 locus, on TB susceptibility. Kramnik [sst1 gene expression may be regulated by macrophages in a cell-autonomous manner. Chackerian, et al. [+ T cells in the lungs of sst1-resisitant C57BL/6 inbred mice, and conjectured that TB susceptibility was related to the ability to recruit mycobacteria-specific IFN-γ-producing Th1 cells to the lungs. Mechanisms of TB resistance via Ipr1 and sst1 have been extensively explored, but studies of SP110 with respect to TB in humans are rare, and no work had yet addressed PMP22, which is primarily associated with neural diseases, in TB. Though we found that PMP22 is a risk factor for TB, the mechanisms by which PMP22 affects immunity are unknown and require further study.TB elicits a kind of type IV allergic reaction . The main pathological processes involved include exudation, alteration and proliferation, which depend on both the host immune system and  Kramnik  showed t, et al.  also fouThe Bonferroni correction is one of the most important methods used to address false discovery rates resulting from multiple testing. We found that SNPs rs4327230 and rs2114591 remained significant after Bonferroni correction, while rs6436917 and rs13422 were not associated with TB. This may due to our strict SNP filtering criteria and small sample size. Additionally, the Bonferroni correction adjusts the value of alpha based on the number of tests performed, and is thus conservative; in some cases, truly significant differences may be deemed non-significant as a result of type II errors [Our study faced some intrinsic limitations. Our patient sample size (217 cases and 383 controls) was not large enough for the association analysis. Other studies included more than 1000 samples , and theSP110 genotypes that appear protective against TB, and identified PMP22 genotypes as risk factors in TB susceptibility. Our study may help to identify those TB-affected individuals most susceptible to disease, and to improve patient-specific clinical TB diagnosis.Our study provides new evidence supporting the importance of host genetic variability in TB susceptibility. We identified We strictly obeyed the World Medical Association Declaration of Helsinki when using human tissue and signing the study protocol with subjects, which was approved by the Ethical Committee of Xi'an Jiaotong University. Each participant provided written, informed consent.M. tuberculosis sputum culture. All patients were HIV negative, which was confirmed by the HIV serology test. There were no age, sex or TB classification restrictions when enrolling the case group. Controls were healthy people receiving physical examinations in other clinical departments of Tangdu Hospital. Healthy controls did not have respiratory disease, immune system disease or any other infectious disease, such as acquired immunodeficiency syndrome (AIDS), that could have affected our study results. Peripheral blood was collected from both cases and controls for DNA extraction.All participants in our study were Tibetan Chinese. A total of 143 TB patients were consecutively recruited between March 2013 and June 2015 in the Tangdu Hospital, affiliated with The Fourth Military Medical University in Xi'an city, China. Patients were diagnosed with TB using symptoms and signs, chest X-ray, sputum smear test for acid fast bacilli (AFB) and A standardized epidemiological questionnaire was provided to all subjects to collect basic demographic information, including sex, age, residence, educational status, history of family cancer, history of smoking, alcohol consumption and TB contact history. Plasma carcinoembryonic antigen and alpha-fetoprotein levels were determined to ensure that no controls suffered any cancer.SP110 and PMP22 genes were selected from previous publications that associated polymorphisms with TB [SP110 and three in PMP22. The GoldMag-Mini Purification Kit  was used to extract genomic DNA from whole blood samples. DNA concentration was measured using a DU530 UV/VIS spectrophotometer . Using MassARRAY Assay Design 3.0 software , we designed a multiplexed SNP MassEXTENDED assay. SNPs were genotyped using the standard protocol recommended by the MassARRAY RS1000 (Sequenom) manufacturer and data were analyzed using Typer 4.0 Software (Sequenom).Candidate SNPs in the  with TB , 24. SNPp-values were two-sided and p<0.05 was considered statistically significant. A t test and Chi-square test were performed to compare sex and age differences between cases and controls. Fisher's exact test was applied to each SNP in the controls to test for departure from Hardy–Weinberg Equilibrium (HWE). Odds ratios (ORs) and 95% confidence intervals (CIs) for the allele and genotype frequencies were calculated using Pearson Chi-square test adjusted by age and gender. Cochran-Armitage trend test was performed to determine associations between SNPs and TB. Sex and age subgroups were not separated due to sample size limitations.We used Microsoft Excel and SPSS 16.0  statistical packages to perform statistical analyses. All http://pngu.mgh.harvard.edu/purcell/plink/) was used to assess SNP associations with TB risk in different genetic models (dominant and recessive). We used unconditional logistic regression analysis to calculate ORs and 95% CI adjusted for age and gender. Pairwise linkage disequilibrium and haplotype constructions were performed using HAPLOVIEW 4.1 (http://broad.mit.edu/mpg/haploview) [p values were Bonferroni corrected, and statistical significance was set at p<0.00178 (0.05/28).PLINK software (ploview) . All p v


Lactoferrin (Lf) is one of the antigens of antineutrophil cytoplasmic antibodies (ANCA) and functions as an endogenous suppressor of neutrophil extracellular trap (NET) formation. However, the prevalence and pathogenicity of anti-lactoferrin antibodies (aLf) in ANCA-associated vasculitis (AAV) remain unrevealed. This study aimed to examine the significance of aLf in AAV, initially. Sixty-five sera from AAV patients, including 41 microscopic polyangiitis, 5 granulomatosis with polyangiitis, and 19 eosinophilic granulomatosis with polyangiitis (EGPA) patients, were subjected to aLf detection using enzyme-linked immunosorbent assay. Clinical characteristics were compared between aLf-positive and aLf-negative patients. Neutrophils from healthy donors were exposed to suboptimal dose (10 nM) of phorbol myristate acetate (PMA) with aLf followed by evaluation of NET formation. Results demonstrated that 4 out of 65 AAV sera (6.2%) were positive for aLf. All of them were EGPA sera . In EGPA, the frequency of renal involvement, serum CRP levels, and Birmingham Vasculitis Activity Score (BVAS) in the aLf-positive patients was significantly higher than those in the aLf-negative patients, and the aLf titer correlated positively with the serum CRP level and BVAS. The NET formation was particularly enhanced by combined stimulation of 10 nM PMA and 1 µg/mL aLf. IgG isolated from sera of the aLf-positive EGPA patients (250 µg/mL) enhanced NET formation induced by 10 nM of PMA, and the effect was abolished completely by absorption of the aLf. This pilot study suggests that aLf enhance NET formation induced by PMA and are associated with disease activity of EGPA. Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is characterized by pauci-immune necrotizing small vessel vasculitis with the presence of ANCA in the serum. AAV includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA), and eosinophilic granulomatosis with polyangiitis (EGPA) . The majLactoferrin (Lf) is present in specific granules of neutrophils . It may Lactoferrin is immediately secreted by degranulation upon activation of neutrophils . RecentlIn the present study, sera from AAV patients and healthy controls were subjected to aLf detection in order to examine the prevalence of aLf in AAV. We investigated correlations between the titers of aLf and clinical parameters. Furthermore, we determined the contribution of aLf to NET formation.In order to examine the prevalence and pathogenicity of aLf in AAV initially, 65 AAV patients including 41 MPA, 5 GPA, and 19 EGPA patients, who were diagnosed and treated at the Department of Internal Medicine II, Hokkaido University Hospital or Department of Dermatology, St. Marianna University Hospital from January 2005 to April 2014, were enrolled in this study. For controls, 10 healthy volunteers were included. Because this is a pilot exploratory study, a random study population size was chosen. After acquirement of written informed consent, peripheral blood was obtained without anticoagulants, and the sera were stored at −80°C until use. Clinical information, including age and gender, laboratory data, such as serum levels of MPO–ANCA and CRP, and Birmingham Vasculitis Activity Score (BVAS) of the AAV patients at the point of blood sampling were collected from medical records retrospectively. The history of asthma and eosinophil count in the peripheral blood were also collected concerning EGPA patients. This study was approved for practice by our Institutional Ethical Committee, the Ethical Committee of the Faculty of Health Sciences, Hokkaido University (Permission No. 15-90).Titer of aLf was determined using the enzyme-linked immunosorbent assay (ELISA) kit .Human neutrophils were obtained from 20 mL of peripheral blood of healthy volunteers by density centrifugation using Polymorph Prep . After washing with PBS, the obtained cells were resuspended in RPMI 1640 medium supplemented with 5% fetal bovine serum.in vitro assay, rabbit polyclonal anti-human Lf antibodies (CSB-PA00870EORb)  and rabbit control IgG  were employed. Prior to use, the contaminating endotoxin was removed using ProteoSpin Endotoxin Removal Micro Kit . Residual endotoxin was ruled out using Limulus Color KY Test Kit . Phorbol myristate acetate (PMA) was purchased from Sigma-Aldrich .For 5/mL), incubated for 15 min at 37°C, and then exposed to 0 or 10 nM PMA combined with 1 µg/mL aLf (CSB-PA00870EORb) or control rabbit IgG. After incubation for 3 h at 37°C, the samples were fixed with 4% paraformaldehyde (PFA) followed by mounting with the solution containing 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). NET area was represented by DAPI-positive area, which was calculated using Image J software, as described previously  . Contamination by endotoxin in the IgG samples was ruled out using the endotoxin detection kit.Recombinant human Lf (10 µg)  was added to 500 µL patient sera. After incubation for 15 min at room temperature, IgG was purified using the immunoadsorbent column. The absorption of aLf was confirmed using WIESLAB ANCA Panel Kit . Residual Lf was confirmed as below the detection limit in Lf ELISA Kit .5/mL), incubated for 15 min at 37°C, and then exposed to 0 or 10 nM PMA combined with 250 µg/mL patient IgG (before and after absorption of aLf) or healthy control IgG. After incubation for 3 h at 37°C, the samples were fixed with 4% PFA followed by mounting with a solution containing DAPI. NET area was represented by DAPI-positive area, which was calculated using Image J software.Peripheral blood neutrophils obtained from healthy volunteers were seeded on chamber slides  and healthy volunteers (n = 10)  were significantly higher than those in the aLf-negative EGPA patients (n = 15)  and BVAS   Table . Corresp) Figure . These fin vitro. Although aLf alone (1 µg/mL) did not induce NET formation in neutrophils (1 × 105/mL), the aLf enhanced NET formation when combined with a suboptimal dose (10 nM) of PMA  and Group 2, aLf-positive/MPO–ANCA-positive (n = 3). Sera from a healthy volunteer and from MPA patients  were used as controls. As shown in Figure in vivo.Next, we examined if aLf in EGPA sera could enhance NET formation induced by PMA as well. For this purpose, EGPA sera with aLf were divided into the following two groups; Group 1, aLf-positive/MPO–ANCA-negative  can enhance NET formation induced by a suboptimal dose of PMA 5/mL). This finding is consistent with the presence of Lf in the specific granules of neutrophils under unstimulated conditions  did not induce NET formation in neutrophils   , histone−) , and mat−) , have be−) , 24, 25.−) . These rin vitro. Earlier studies have demonstrated that aLf could activate neutrophils primed by bacteria-derived f-Met–Leu–Phe (fMLP)  , but note (fMLP) . In addie (fMLP) . It has Among the three AAV types, including MPA, GPA, and EGPA, aLf were detected in the EGPA sera exclusively in this study. A characteristic of EGPA is the presence of asthma or paranasal sinusitis history preceding the development of the disease. A recent study has demonstrated that a specific allergen for asthma induced Lf secretion from neutrophils derived from asthmatic patients . In asthHS and YT carried out the experiments. HS, YK, AM, FH, and TK contributed to the collection of serum samples. HS, DN, SM, UT, TA, and AI analyzed the data. HS, UT, and AI designed the research and wrote the manuscript.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.


We hypothesized that the use of actual body weight might lead to more frequent misdiagnosis of acute kidney injury (AKI) than when ideal body weight is used in underweight and/or obese patients. We examined which definition of body weight is most effective in establishing a urinary diagnosis of AKI in septic patients.Consecutive patients aged ≥ 20 years admitted to the intensive care unit of a university hospital between June 2011 and December 2016 were analyzed. Sepsis was defined in accordance with the Sepsis-3 criteria. AKI was defined as a urinary output of < 0.5 mL/kg/6h during intensive care unit stay. Patients were divided into one of four body mass index-based classes. The severity of illness and 90-day mortality were compared across the body mass index subgroups in patients diagnosed using the actual body weight or ideal body weight.P = 0.033).Of 5764 patients, 569 septic patients were analyzed. One hundred and fifty-three (26.9%) and 140 (24.6%) patients were diagnosed as having AKI using actual body weight and ideal body weight, respectively. There were no significant differences in the severity of illness among these groups. Also, 90-day mortality did not differ significantly among these groups. According to body mass index, 90-day mortality significantly differed in patients diagnosed using their actual body weights (underweight vs. normal vs. overweight vs. obese: 76.7% vs. 39.5% vs. 26.0% vs. 35.7%, Generally, using actual body weight to calculate the weight-adjusted hourly urine output for diagnosing AKI increased the sensitivity compared to ideal body weight, irrespective of the severity of illness in septic patients. Delayed diagnosis, however, was more common among underweight patients in this situation, and clinicians should be cautious when diagnosing urinary AKI using actual body weight. Acute kidney injury (AKI) is a major complication in sepsis, and is associated with a high mortality rate. Nearly half of the patients in the intensive care unit (ICU) develop AKI, and the mortality rate among these patients ranges from 30% to 50% , 2. EarlThe definition of AKI is based on absolute or relative changes in serum creatinine levels and the weight-adjusted hourly urine output . The weiOnly one study  has evalTherefore, in this study, we investigated the influence of body weight definitions on the urinary diagnosis of AKI in septic patients who were at risk of AKI. In addition, we evaluated the severity of illness and 90-day mortality for each body weight type determined according to each patients’ body mass index (BMI).This was a single-center, retrospective, observational study that was conducted in a 14-bed general ICU of a university hospital . Clinical decisions were made at the discretion of the attending ICU physicians. The Institutional Research Ethics Committee of Jichi Medical University Hospital approved this study and waived the need for informed consent because of the retrospective nature of the study (No. 16–116).Data for consecutive patients admitted to the ICU between June 2011 and December 2016 were screened. Patients were included in the study if they were ≥ 20 years of age, admitted to the ICU for a minimum of 6 continuous hours, underwent hourly urine output measurements, and were diagnosed with sepsis according to the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) . PatientThe urinary diagnosis and staging of AKI were based on the Kidney Disease: Improving Global Outcomes (KDIGO) criteria . A urinaMales: IBW (kg) = 50 kg + 0.91 * (Height [cm] – 152.4).Females: IBW (kg) = 45.5 kg + 0.91 * (Height [cm] – 152.4).2  group, 122 (87.1%) patients were diagnosed with AKI and 18 (12.9%) without AKI based on the level of serum creatinine.n = 526). When using ABW to diagnose AKI, 31 patients (5.4%) were classified into higher AKI stages, while 12 patients (2.2%) were classified into lower AKI stages than those diagnosed using IBW  patients had stage 1 disease, 38 (6.7%) stage 2, and 64 (11.3%) stage 3. In the AKI [IBW] group, 45 (7.9%) patients had stage 1 disease, 32 (5.6%) stage 2, and 63 (11.1%) stage 3. The concordance rate between the groups was 92.4% (Table P = 0.033), although the severity of illness  was not significantly different. However, in the AKI [IBW] group, the mortality rate was not significantly different among BMI subgroups  when compared with the normal subgroup, 4.04  when compared with the overweight subgroup, and 1.98  when compared with the obese subgroup . However, in the AKI [ABW] group, creatinine levels did not significantly differ across BMI subgroups (P = 0.366).Table P = 0.036). In particular, the underweight and normal subgroups showed significantly higher cystatin C levels than the overweight group . However, in the AKI [IBW] group, serum cystatin C levels did not differ significantly among the BMI subgroups, and they also showed a homogeneous distribution (P = 0.596).On the other hand, in the AKI [ABW] group, serum cystatin C levels differed significantly across the BMI subgroups  have been used  mg/dL ha–6, 22. HMeasurement of urine output is not only more sensitive and quicker than measuring serum creatinine once a day in detecting AKI, but it is also important to realize that patients only fulfilling AKI definitions based on urine output criteria differ from patients fulfilling AKI definitions based on serum creatinine with or without urine output measurements. In this study, 15.7% of AKI [ABW] patients and 12.9% of AKI [IBW] patients were not diagnosed with AKI based on the serum creatinine definition. In this regard, serum creatinine and urine output for diagnosing AKI do not always act complementarily, but may show independent pathophysiologies. Further studies are needed to evaluate this concern in these populations.To evaluate renal function, serum creatinine was commonly used. However, its value was highly affected by body types. Recently, cystatin C has alternatively been recommended for the diagnosis of AKI instead of creatinine . CystatiSeveral limitations associated with the present study should be mentioned. First, this was a single-center, retrospective study. The sample size was relatively small, particularly when divided into BMI subgroups. Further studies are needed to confirm our findings, especially for the underweight patients. Second, we did not evaluate fluid balance before admission to the ICU; therefore, the baseline body weight might differ from that in the pre-morbid state. In addition, the timing of body weight measurement was not always just after ICU admission. However, we tried to select the most reliable body weight value after ICU admission in an effort to minimize any errors. Third, we did not measure other AKI biomarkers, such as urine neutrophil gelatinase-associated lipocalin (NGAL), to evaluate the accuracy of urinary AKI. Finally, whether or not the patients’ underweight statuses were due to malignant disease was unclear. The presence of such disease, for example, cachexia, might be associated with the relatively higher mortality rate observed in this subgroup than that in the other BMI subgroups.Despite these limitations, our study has several strengths. All of our patients were evaluated using the Sepsis-3 criteria as being at risk for AKI. In addition, to the best of our knowledge, our study is the first to focus on various body weight types in order to evaluate the homogeneity of the timing of establishing a urinary diagnosis of AKI.Using ABW to calculate the weight-adjusted hourly urine output for diagnosing AKI generally increased the sensitivity compared with IBW irrespective of the severity of disease among both groups. This result could be expected due to the increasing number of overweight patients who earlier meet the definition of urinary AKI. Delayed diagnosis, however, was more common among underweight patients in this situation, and clinicians should be cautious when diagnosing urinary AKI using actual body weight.