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Allelic variants of HLA-DRB1 have been associated with a variety of autoimmune and infectious diseases. Although the precise molecular mechanisms by which HLA-DRB1 alleles predispose to a particular disease are currently unclear, it has been shown that mRNA expression levels of HLA-DRB1 are dependent on the different alleles. We aimed to measure HLA-DR beta chain levels in peripheral blood mononuclear cells of individuals carrying HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*15:01 alleles by western blotting, using five commercially-available HLA-DRB antibodies. We observed highly heterogeneous binding of the tested antibodies to the different allelic forms of the HLA-DR beta chain. Overall, we show that current immunological research that employs available antibodies to detect HLA-DR beta chains is biased towards detection of specific variants of the protein; this may cause significant discrepancy in quantification of protein expression in a heterogeneous human population.

To gain further insight in the immunopathology underlying multifocal motor neuropathy (MMN) by exploring the association between MMN and the human leukocyte antigen (HLA) class II DRB1, DQB1, and DQA loci in depth and by correlating associated haplotypes to detailed clinical and anti-ganglioside antibody data.We performed high-resolution HLA-class II typing for the DRB1, DQB1, and DQA1 loci in 126 well-characterized MMN patients and assessed disease associations with haplotypes. We used a cohort of 1305 random individuals as a reference for haplotype distribution in the Dutch population.The DRB1*15:01-DQB1*06:02 haplotype (OR 1.6 [95% CI 1.1-2.2], p0.05) and the DRB1*12:01-DQB1*03:01 haplotype (OR 2.7 [95% CI 1.2-5.5], p0.05) were more frequent in patients with MMN than in controls. These haplotypes were not associated with disease course, response to treatment or anti-ganglioside antibodies.MMN is associated with the DRB1*15:01-DQB1*06:02 and DRB1*12:01-DQB1*03:01 haplotypes. These HLA molecules or gene variants in their immediate vicinity may promote the specific inflammatory processes underlying MMN.

Background Little is known about the genetic determinants of severe asthma exacerbations. Objectives We aimed to identify genetic variants associated with asthma hospitalizations. Methods We conducted a genome-wide association study of asthma hospitalizations in 34,167 white British adults with asthma, 1,658 of whom had at least 1 asthma-related hospitalization. This analysis was conducted by using logistic regression under an additive genetic model with adjustment for age, sex, body mass index, smoking status, and the first 5 principal components derived from genotypic data. We then analyzed data from 2 cohorts of Latino children and adolescents for replication and conducted quantitative trait locus and functional annotation analyses. Results At the chromosome 6p21.3 locus, the single-nucleotide polymorphism (SNP) rs56151658 (8 kb from the promoter of HLA-DQB1) was most significantly associated with asthma hospitalizations (for test allele A, odds ratio = 1.36 [95% CI = 1.22-1.52]; P = 3.11 × 10–8); 21 additional SNPs in this locus were associated with asthma hospitalizations at a P value less than 1 × 10–6. In the replication cohorts, multiple SNPs in strong linkage disequilibrium with rs56151658 were associated with severe asthma exacerbations at a P value of .01 or less in the same direction of association as in the discovery cohort. Three HLA genes (HLA-DQA2, HLA-DRB6, and HLA-DOB) were also shown to mediate the estimated effects of the SNPs associated with asthma hospitalizations through effects on gene expression in lung tissue. Conclusions We identified strong candidate genes for asthma hospitalizations in adults in the region for class II HLA genes through genomic, quantitative trait locus, and summary data–based mendelian randomization analyses.

Background Diversity at the Major Histocompatibility Complex (MHC) is critical to health and fitness, such that MHC genotype may predict an individual’s quality or compatibility as a competitor, ally, or mate. Moreover, because MHC products can influence the components of bodily secretions, an individual’s body odors may signal its MHC composition and influence partner identification or mate choice. Here, we investigated MHC-based signaling and recipient sensitivity by testing for odor-gene covariance and behavioral discrimination of MHC diversity and pairwise dissimilarity in a strepsirrhine primate, the ring-tailed lemur (Lemur catta). Methods First, we coupled genotyping of the MHC class II gene, DRB, with gas chromatography-mass spectrometry of genital gland secretions to investigate if functional genetic diversity is signaled by the chemical diversity of lemur scent secretions. We also assessed if the chemical similarity between individuals correlated with their MHC-DRB similarity. Next, we assessed if lemurs discriminated this chemically encoded, genetic information in opposite-sex conspecifics. Results We found that both sexes signaled overall MHC-DRB diversity and pairwise MHC-DRB similarity via genital secretions, but in a sex- and season-dependent manner. Additionally, the sexes discriminated absolute and relative MHC-DRB diversity in the genital odors of opposite-sex conspecifics, suggesting that lemur genital odors function to advertise genetic quality. Conclusions In summary, genital odors of ring-tailed lemurs provide honest information about an individual’s absolute and relative MHC quality. Complementing evidence in humans and Old World monkeys, we suggest that reliance on scent signals to communicate MHC quality may be important across the primate lineage. Electronic supplementary material The online version of this article (10.1186/s12862-019-1486-0) contains supplementary material, which is available to authorized users.

The phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) are the two major autoantigens in primary membranous nephropathy (MN), and define two molecular subclasses of this disease. Both proteins are large transmembrane glycoproteins expressed by the podocyte, and both induce IgG4-predominant humoral immune responses that produce circulating autoantibodies that can be used clinically for diagnostic and monitoring purposes. The biologic roles of these proteins remain speculative, although several features of THSD7A suggest a role in adhesion. PLA2R-associated MN was initially found to associate with risk alleles within HLA-DQA1, but subsequent studies have shifted the focus to the HLA-DRB locus. Three distinct humoral epitope-containing regions have been defined within the extracellular portion of PLA2R, and it appears that the number of targeted epitopes may determine disease severity. Although similar information is not yet available for THSD7A-associated MN, this form of MN may have a unique association with malignancy. Finally, it appears likely that other autoantigens in primary MN exist. Although protocols similar to those that identified PLA2R and THSD7A may be successful in the identification of novel antigenic targets in MN, newer techniques such as laser-capture mass spectrometry or protein arrays may be helpful as well.

The definition of biological donor organ age rather than chronological age seems obvious for the establishment of a valid pre-transplant risk assessment. Therefore, we studied gene expression for candidate markers in 60 zero-hour kidney biopsies. Compared with 29 younger donors under age 55, 31 elderly donors age 55 and older had significant mRNA expression for immunoproteasome subunits (PSMB8, PSMB9 and PSMB10), HLA-DRB, and transcripts of the activating cytotoxicity receptor NKG2D. Gene expression was validated in an independent donor cohort consisting of 37 kidneys from donors 30 years and under (Group I), 75 kidneys from donors age 31-54 years (Group II) and 75 kidneys from donors age 55 and older (Group III). Significant gene induction was confirmed in kidneys from Group III for PSMB9 and PSMB10. Strikingly, transcripts of NKG2D had the significantly highest gene induction in Group III versus Group II and Group I. Similar results were obtained for CDKN2A, but not for telomere length. Both NKG2D and CDKN2A mRNA expression were significantly correlated with creatinine levels at 24 months after transplantation. Univariate regression analysis showed significant predictive power regarding graft function at 6 and 12 months for NKG2D and CDKN2A. However, only NKG2D remained significantly predictive in the multivariate model at 12 months. Thus, our results reveal novel candidate markers in aged renal allografts, which could be helpful in the assessment of organ quality.

The aetiology of multiple sclerosis (MS) is as yet poorly understood. Multiple mechanisms in different disease stages are responsible for immunopathology in MS. HLA Class II DR2b (DRB1*1501 β, DRA1*0101 α) is the strongest genetic risk factor for MS. Remnants of ancient retroviruses in the human genome, termed human endogenous retroviruses (HERV), and Epstein-Barr virus (EBV) infection are also associated with MS. In silico analyses of human endogenous retroviral envelope (HERV env) proteins and three myelin proteins that are principal targets of an autoimmune response in MS showed sequence similarities between potential TH epitopes within pairs of viral and myelin peptides predicted to bind HLA DR2b. This led to the proposal that such molecular mimicry may potentially trigger MS. HLA DR2b binding characteristics of previously identified peptides from the three myelin proteins and HERV env proteins as well as additional in silico predicted peptides from other encephalitogenic brain proteins and EBV proteins were studied to further investigate molecular mimicry. Peptides containing potential TH epitopes from the myelin oligodendrocyte glycoprotein and HERV env previously predicted to bind HLA DR2b as well as other pertinent potential HLA DR2b-restricted TH epitopes were confirmed to bind HLA DR2b molecules. Molecular modelling of HLA DR2b in complex with high affinity peptides derived from MOG and HERV env proteins showed that their binding could occur in a similar manner to a HLA DR2b-binding peptide containing a known TH epitope. A structurally related pair of peptides predicted to bind HLA DR2b from the EBV protein EBNA1 and β synuclein, a brain protein implicated in MS, were also shown to similarly bind HLA DR2b. The findings justify investigating CD4+ T cell responses to the identified peptides.

Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis characterized by asthma, hypereosinophilia, and progressive multiorgan involvement. Although not fully elucidated, advancement in our understanding of the pathophysiology of EGPA has led to the development of multiple new treatment targets.Herein we review the epidemiology, clinical manifestations, pathophysiology, treatments, and ongoing research in the management of EGPA. The central role of Interleukin-5 (IL-5) in the development and maintenance of hypereosinophilia will be discussed. The value of mepolizumab, an anti-IL-5 monoclonal antibody, in the treatment of EGPA is reviewed in detail.The available literature supports the use of mepolizumab for the induction and maintenance of remission of refractory, relapsing, or glucocorticoid-dependent EGPA with potentially greater benefit in those who are ANCA-positive or those with greater eosinophilia (

Comprehensive knockout of HLA class II (HLA-II) β-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing–based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II–expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

The major histocompatibility complex (MHC) is a highly polymorphic and polygenic genomic region that plays a crucial role in immune-related diseases. Given the need for comparative studies on the variability of immunologically important genes among wild populations and species, we investigated the allelic variation of MHC class II DRB among three congeneric true lemur species: the red-fronted lemur (Eulemur rufifrons), red-bellied lemur (Eulemur rubriventer), and black lemur (Eulemur macaco). We noninvasively collected hair and faecal samples from these species across different regions in Madagascar. We assessed DRB exon 2 polymorphism with a newly developed primer set, amplifying nearly all non-synonymous codons of the antigen-binding sites. We defined 26 DRB alleles from 45 individuals (17 alleles from E. rufifrons (N = 18); 5 from E. rubriventer (N = 7); and 4 from E. macaco (N = 20). All detected alleles are novel and show high levels of nucleotide (26.8%) and non-synonymous codon polymorphism (39.4%). In these lemur species, we found neither evidence of a duplication of DRB genes nor a sharing of alleles among sympatric groups or allopatric populations of the same species. The non-sharing of alleles may be the result of a geographical separation over a long time span and/or different pathogen selection pressures. We found dN/dS rates > 1 in the functionally important antigen recognition sites, providing evidence for balancing selection. Especially for small and isolated populations, quantifying and monitoring DRB variation are recommended to establish successful conservation plans that mitigate the possible loss of immunogenetic diversity in lemurs. Electronic supplementary material The online version of this article (10.1007/s00251-018-1085-z) contains supplementary material, which is available to authorized users.

Background Maintaining variation in immune genes, such as those of the major histocompatibility complex (MHC), is important for individuals in small, isolated populations to resist pathogens and parasites. The golden snub-nosed monkey (Rhinopithecus roxellana), an endangered primate endemic to China, has experienced a rapid reduction in numbers and severe population fragmentation over recent years. For this study, we measured the DRB diversity among 122 monkeys from three populations in the Qinling Mountains, and estimated the relative importance of different agents of selection in maintaining variation of DRB genes. Results We identified a total of 19 DRB sequences, in which five alleles were novel. We found high DRB variation in R. roxellana and three branches of evidence suggesting that balancing selection has contributed to maintaining MHC polymorphism over the long term in this species: i) different patterns of both genetic diversity and population differentiation were detected at MHC and neutral markers; ii) an excess of non-synonymous substitutions compared to synonymous substitutions at antigen binding sites, and maximum-likelihood-based random-site models, showed significant positive selection; and iii) phylogenetic analyses revealed a pattern of trans-species evolution for DRB genes. Conclusions High levels of DRB diversity in these R. roxellana populations may reflect strong selection pressure in this species. Patterns of genetic diversity and population differentiation, positive selection, as well as trans-species evolution, suggest that pathogen-mediated balancing selection has contributed to maintaining MHC polymorphism in R. roxellana over the long term. This study furthers our understanding of the role pathogen-mediated balancing selection has in maintaining variation in MHC genes in small and fragmented populations of free-ranging vertebrates. Electronic supplementary material The online version of this article (10.1186/s12862-018-1148-7) contains supplementary material, which is available to authorized users.

BACKGROUND AND AIMS The genetic contribution to the prognosis of ulcerative colitis [UC] is poorly understood, and most currently known susceptibility loci are not associated with prognosis. To identify genetic variants influencing the prognosis of UC, we performed an Immunochip-based study using an extreme phenotype approach. METHODS Based on the finding that the only association, Pdiscovery-meta

Malaria is an infectious disease caused by parasites from the genus Plasmodium (P. falciparum and P. vivax are responsible for 90% of all clinical cases); it is widely distributed throughout the world’s tropical and subtropical regions. The P. vivax Pv12 protein is involved in invasion, is expressed on merozoite surface and has been recognised by antibodies from individuals exposed to the disease. In this study, B- and T-cell epitopes from Pv12 were predicted and characterised to advance in the design of a peptide-based vaccine against malaria. For evaluating the humoral response of individuals exposed to natural P. vivax infection from two endemic areas in Colombia, BepiPred-1.0 software was used for selecting B-cell epitopes. B-cell epitope 39038 displayed the greatest recognition by naturally-acquired antibodies and induced an IgG2/IgG4 response. NetMHCIIpan-3.1 prediction software was used for selecting peptides having high affinity binding for HLA-DRβ1* allele lineages and this was confirmed by in-vitro binding assays. T-epitopes 39113 and 39117 triggered a memory T-cell response (Stimulation Index2) and significant cytokine production. Combining in-silico, in-vitro and functional assays, two Pv12 protein regions (containing peptides 39038, 39040, 39113 and 39117) have thus been characterised as promising vaccine candidates against P. vivax malaria. © 2018 Yepes-Perez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Major histocompatibility complex (MHC) class II molecules have an important role in vertebrate adaptive immunity, being responsible for recognizing, binding, and presenting specific antigenic peptides to T lymphocytes. Here, we study the MHC class II DQB and DRB exon 2 genes of the Australian sea lion (Neophoca cinerea), an endangered pinniped species that experiences high pup mortality. Following characterization of N. cinerea DQB and DRB by molecular cloning, and evaluation of diversity in pups across 2 colonies using variant screening (n = 47), 3 DQB alleles and 10 DRB variants (including 1 pseudogene allele) were identified. The higher diversity at DRB relative to DQB is consistent with other studies in marine mammals. Despite overall lower MHC class II allelic diversity relative to some other pinniped species, we observed similar levels of nucleotide diversity and selection in N. cinerea. In addition, we provide support for recent divergence of MHC class II alleles. The characterization of MHC class II diversity in the Australian sea lion establishes a baseline for further investigation of associations with disease, including endemic hookworm infection, and contributes to the conservation management of this species.

Summary B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long-lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo-human leucocyte antigen (HLA) antibodies pre- and post-transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA-I alleles and the HLA reactivity of IVIg is lost after its HLA-E reactivity is adsorbed out. Therefore, we have generated an anti-HLA-E monoclonal antibody that mimics the HLA-reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well-defined culture system. The anti-HLA-E monoclonal antibody (mAb) significantly suppressed the allo-HLA class-II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA-I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti-HLA-E mAb for peptide sequences shared (i.e. shared epitopes) between HLA-E and other β2-microglobulin-free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti-HLA-E monoclonal antibody may also be useful to suppress allo-HLA IgG production in vivo.

Topological and stereo-electron characteristics are essential in major histocompability class II-peptide-T-cell receptor (MHC-p-TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche+ orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them. © 2014 Elsevier Ltd.

We established CD4 T-cell clones, Mz-1B7, and Ue-21, which recognized the NY-ESO-1 121-138 peptide from peripheral blood mononuclear cells (PBMCs) of an esophageal cancer patient, E-2, immunized with an NY-ESO-1 protein and determined the NY-ESO-1 minimal epitopes. Minimal peptides recognized by Mz-1B7 and Ue-21 were NY-ESO-1 125-134 and 124-134, respectively, both in restriction to DRB1*08:03. Using a longer peptide, 122-135, and five other related peptides, including either of the minimal epitopes recognized by the CD4 T-cell clones, we investigated the free peptide/DR recognition on autologous EBV-B cells as APC and peptide/DR tetramer binding. The results showed a discrepancy between them. The tetramers with several peptides recognized by either Mz-1B7 or the Ue-21 CD4 T-cell clone did not bind to the respective clone. On the other hand, unexpected binding of the tetramer with the peptide not recognized by CD4 T-cells was observed. The clone Mz-1B7 did not recognize the free peptide 122-135 on APC, but the peptide 122-135/DRB1*08:03 tetramer bound to the TCR on those cells. The failure of tetramer production and the unexpected tetramer binding could be due to a subtly modified structure of the peptide/DR tetramer from the structure of the free peptide/DR molecule. We also demonstrated that the NY-ESO-1 123-135/DRB1*08:03 tetramer detected ex vivo CD4 T-cell responses in PBMCs from patients after NY-ESO-1 vaccination in immunomonitoring.

Hydatidosis constitutes a major public health problem in kingdom of Saudi Arabia. The variability on susceptibility to hydatidosis has been related to the HLA system. So this study aimed to identifying the possible association between the class-II HLA-DRB1 alleles with the occurrence of hydatidosis and the clinical course in Saudi patients using Micro SSP HLA-DRB1 kits. Since HLA-disease associations might vary in relation to gender, so this study aims also to determining the HLA-DRB1 alleles- hydatidosis association in relation to gender. The results proved that HLA-DR16 and HLA-DR7 alleles were the suitable markers of susceptibility association while HLA-DR1 and HLA-DR10 alleles might confer protection against hydatidosis. All the previous susceptible and resistance associations were statistically significant. Also, the majority of the female patients (81.8%) had HLA-DR14 compared to 9.1% of female controls while 50% of the male patients had HLA-DR7 compared to 4.5% of male controls. These results were statistically associated and this is the first study that found an association between certain HLA-DRB 1 alleles and the occurrence of human hydatidosis in relation to gender. Also, significant relations were detected between HLA-DR16 and hepatic and single cyst, HLA-DR7 and lung cyst, HLA-DR14 and combined cysts. Also, no statistical significance was found between other cystic characteristics and any one of the susceptible HLA-DRBs. On conclusion beside the role HLA-DRB 1 on the susceptibility or the resistance to hydatidosis disease occurrence among Saudi population also it may have an important role in the prevalence of the disease in relation to gender.

The majority of clear-cell renal cell carcinomas (ccRCC) show high and homogeneous expression levels of the tumor associated antigen (TAA) carbonic anhydrase IX (CAIX), and treatment with interleukin-2 (IL-2) based immunotherapy can lead to cure in patients with metastatic renal cell carcinoma (mRCC). However, the involvement of CAIX specific CD8+ T cells and/or NK cells in the tumor eradication is unknown. We investigated T cell and antibody reactivity against overlapping 15-mer CAIX-peptides as well as HLA haplotype frequency and NK cell cytotoxicity in 11 patients with no evidence of disease (NED) following treatment with IL-2 based immunotherapy, and thus potentially cured. Immune reactivity in these patients was compared with samples from patients with dramatic tumor response obtained immediately at the cessation of therapy, samples from patients that experienced progressive disease during treatment and samples from healthy controls. We observed more focused but only weak and not consistent CAIX specific T-cells in the late observation and early observation response groups compared with the healthy control group. An increased frequency of the class II alleles HLA-DRB4 01:01, HLA-DPB 01:01 and HLA-DPB 03:01 was noted in the NED patients. In contrast, NK cytotoxicity was low even in the late observation response group as compared with controls. In particular, a HLA-B*40:01 restricted CD8+ T cell response recognizing the CAIX- derived peptide SEEEGSLKL was identified. This may have interest in future cancer vaccines, but more studies are needed to elucidate the immunological mechanisms of action in potentially cured patients treated with an immunotherapeutic agent.

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and β chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the ‘MHCII RNA operon’ in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5′- and 3′-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Mousavi jazi M, Solgi G, Asl roosta H, Noshad S, Moslemi N, Sadrimanesh R,Moradi B, Amirzargar A. HLA-DRB and HLA-DQA/HLA-DQB allele andhaplotype frequencies in Iranian patients with aggressive periodontitis. J PeriodontRes 2013; doi: 10.1111/jre.12043. © 2013 John Wiley & Sons A/SBackground and Objective: Genetic backgrounds play a key role in susceptibilityto and protection against a spectrum of periodontal diseases. Like other infec-tious diseases, the human leukocyte antigen (HLA) have been found to beassociated with periodontitis. This study aimed to investigate differences in alleleand haplotype frequencies of HLA class II antigens in a sample of Iranianpatients with aggressive periodontitis compared with a healthy control group.Material and Methods: Fifty unrelated patients with aggressive periodontitis and130 healthy volunteers were enrolled in this study. HLA genotyping for HLA-DRB, HLA-DQA1 and HLA-DQB1 was performed using the PCR withsequence-specific primers. Allele and haplotype frequencies were comparedacross groups.Results: The frequencies of HLA-DQA1*03:01, HLA-DQB1*03:02 and HLA-DQB1*03:05 alleles, as well as that of the HLA-DRB1*04:01 allele, were signifi-cantly higher in patients with aggressive periodontitis compared with controlsubjects (p = 0.01, p = 0.04, p = 0.05 and p = 0.04, respectively). In contrast, thefrequency of the HLA-DQB1*0603 allele was significantly lower in patients withaggressive periodontitis compared with control subjects (p = 0.006; odds ratio =0.20). With regard to haplotype association, a significantly higher frequency oftwo haplotypes – HLA-DRB1*04:01/HLA-DQA1*03:01/HLA-DQB1*03:02 andHLA-DRB1*16:01/HLA-DQA1*01:03/HLA-DQB1*05:01 – was observed inpatients with aggressive periodontitis compared with healthy controls (p = 0.01,odds ratio = 2.56 and p = 0.05, odds ratio = 5.38, respectively).Conclusion: These results provide additional evidence that class II HLA poly-morphisms, particularly in the DQ locus, are associated with protection againstand susceptibility to aggressive periodontitis.M. Mousavi jazi

A comprehensive structural portrait of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in rheumatoid arthritis., Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine–containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01+ individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4+ T cells in the peripheral blood of HLA-DRB1*04:01+ RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Leukocytospermia is a physiologic condition defined as human semen with a leukocyte count of >1 x 106 cells/ml that is often correlated with male infertility. Moreover, bacteriospermia has been associated with leukocytospermia ultimately leading to male infertility. We have found that semen samples with >1 x 106/ml leukocytes and/or bacteriospermia have oxidative predominance as evidenced by augmented protein carbonyl and lipid peroxidation status of the semen which is implicated in sperm dysfunction. It has been reported that Streptococcus agalactiae is present in bacteriospermic samples. Previous research has shown that human leukocyte antigen beta chain paralog (HLA-DRB) alleles interact best with the infected sperm cells rather than the non-infected cells. Little is known about the interaction of major histocompatibility complex (MHC) present on leukocytes with the sperm upon bacterial infection and how it induces an immunological response which we have addressed by epitope mapping. Therefore,...

Fully-protective, long-lasting, immunological (FPLLI) memory against Plasmodium falciparum malaria regarding immune protection-inducing protein structures (IMPIPS) vaccinated into monkeys previously challenged and re-challenged 60 days later with a lethal Aotus monkey-adapted P. falciparum strain was found to be associated with preferential high binding capacity to HLA-DR?1* allelic molecules of the major histocompatibility class II (MHC-II), rather than HLA-DR?3*, ?4*, ?5* alleles. Complete PPIIL 3D structure, a longer distance (26.5 Å ± 1.5 Å) between residues perfectly fitting into HLA-DR?1*PBR pockets 1 and 9, a gauche? rotamer orientation in p8 TCR-contacting polar residue and a larger volume of polar p2 residues was also found. This data, in association with previously-described p3 and p7 apolar residues having gauche+ orientation to form a perfect MHC-II-peptide-TCR complex, determines the stereo-electronic and topochemical characteristics associated with FPLLI immunological memory. © 2016 Elsevier Inc.

The importance of CSP- and STARP-derived ? and ? dihedral angles in mHABP structure was analysed by 1H NMR in the search for molecules which can be included as components of a first-line-of-defence Plasmodium falciparum sporozoite multi-epitope vaccine against the most lethal form of human malaria. Most of the aforementioned dihedral angles were left-hand-like polyproline type II (PPIIL) structures whilst others had right-hand-like ?-helix (?R), thus allowing mHABPS to fit better into MHCII molecules and thereby form an appropriate pMHCII complex and also establish the H-bonds which stabilise such complex and by this means induce an appropriate immune response. This information has great implications for vaccine development, malaria being one of them. © 2012 Elsevier Inc.

The HLA region shows diversity concerning the number and content of DRB genes present per haplotype. Similar observations are made for the equivalent regions in other primate species. To elucidate the evolutionary history of the various HLA-DRB genes, a large panel of intron sequences obtained from humans, chimpanzees, rhesus macaques, and common marmosets has been subjected to phylogenetic analyses. Special attention was paid to the presence and absence of particular transposable elements and/or to their segments. The sharing of different parts of the same long interspersed nuclear element-2 (LINE2, L2) and various Alu insertions by the species studied demonstrates that one precursor gene must have been duplicated several times before the Old World monkey (OWM) and hominid (HOM) divergence. At least four ancestral DRB gene families appear to have been present before the radiation of OWM and HOM, and one of these even predates the speciation of Old and New World primates. Two of these families represent the pseudogenes DRB6/DRB2 and DRB7, which have been locked in the genomes of various primate species over long evolutionary time spans. Furthermore, all phylogenies of different intron segments show consistently that, apart from the pseudogenes, only DRB5 genes are shared by OWM and HOM, and they demonstrate the common history of certain DRB genes/lineages of humans and chimpanzees. In contrast, the evolutionary history of some other DRB loci is difficult to decipher, thus illustrating the complex history of the evolution of DRB genes due to a combination of mutations and recombination-like events. The selected approach allowed us to shed light on the ancestral DRB gene pool in primates and on the evolutionary relationship of the various HLA-DRB genes.

Background: Ubiquitination is essential for regulation of MHC class II molecules. Results: Multiple elements in the transmembrane region of HLA-DR influence interaction with MARCH8. Conclusion: Sequences in the TM region of MHC class II molecules control recognition by different MARCH E3 ligases. Significance: MHC class II isotypes are subject to differential post-translational regulation by different MARCH E3 ligases., MARCH E3 ligases play a key role in controlling MHC class II surface expression by regulated ubiquitination of a lysine residue in the β-chain. Little is known concerning how these enzymes target their specific substrates. Here we show that recognition of HLA-DR by MARCH proteins is complex. Several features associated with the transmembrane domain and bordering regions influence the overall efficiency of receptor internalization. A cluster of residues at the interface of the lipid bilayer and the cytosol plays the most important role in MARCH8 recognition of HLA-DRβ. Variation in this sequence also determines specificity of MARCH9 for HLA-DQ. Residues located in helical face four of HLA-DRβ together with a charged residue at the boundary with the stalk region also contribute significantly to recognition. Truncation analysis suggested that a dileucine-like motif in the DRβ cytoplasmic tail influences the efficiency of co-localization of HLA-DR with MARCH8. The DRβ-encoded acceptor lysine functioned optimally when placed in its natural location relative to the bilayer. In the DRα/DRβ dimer most other amino acids in the cytoplasmic tail could be substituted for alanine with minimal influence on function. Our data support a model whereby multiple features of HLA-DR are involved in substrate recognition by MARCH8. The single most important region is located at the interface between the transmembrane domain and the cytosol. Variation in sequence in this location between different class II isotypes controls efficiency of recognition by different MARCH E3 ligases.

To the Editor: Anticitrullinated peptide/protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific antibodies that recognize various protein sequences containing citrulline, the deiminated form of arginine produced posttranslationally by peptidylarginine deiminase (PAD) enzymes. Recently, viral citrullinated peptides (VCP) derived from Epstein-Barr virus nuclear proteins EBNA-1 and EBNA-2 have been described as a target of ACPA in RA sera1,2. Genetic factors predisposing to RA, namely HLA-DRB1 shared epitope (SE) alleles and PTPN22 gene variants, as well as the TRAF1/C5 locus and PADI4 susceptible haplotype, have been found to be predominantly associated with ACPA-positive RA, even if the association with ACPA-negative RA cannot be excluded. We evaluated the influence of genes conferring susceptibility to RA on the production of anti-VCP antibodies and tested the interaction of VCP with HLA in vitro . We analyzed 172 patients with RA of French white origin3. All provided informed consent and the Ethics Committee of Hopital Bicetre (AP-HP, Paris, France) approved the study. HLA-DRB1 typing and subtyping were performed by a polymerase chain reaction (PCR)-based method, using a panel of sequence-specific oligonucleotide probes. PADI4 haplotypes were obtained by typing 3 single-nucleotide polymorphisms (SNP: rs1886303, PADI4\_92, PADI4\_96 – CGC haplotype) by PCR-restriction fragment-length polymorphism (RFLP)4. PTPN22 (R620W; rs24766601 – T variant) and C5/TRAF1 region polymorphisms (rs10818488 – A variant) were genotyped by PCR-RFLP5,6. … Address correspondence to Dr. P. Migliorini; E-mail: paola.migliorini{at}med.unipi.it

The major histocompatibility complex (MHC) on chromosome 6p is an established risk locus for ulcerative colitis (UC) and Crohn's disease (CD). We aimed to better define MHC association signals in UC and CD by combining data from dense single-nucleotide polymorphism (SNP) genotyping and from imputation of classical human leukocyte antigen (HLA) types, their constituent SNPs and corresponding amino acids in 562 UC, 611 CD and 1428 control subjects. Univariate and multivariate association analyses were performed, controlling for ancestry. In univariate analyses, absence of the rs9269955 C allele was strongly associated with risk for UC (P = 2.67 × 10(-13)). rs9269955 is a SNP in the codon for amino acid position 11 of HLA-DRβ1, located in the P6 pocket of the HLA-DR antigen binding cleft. This amino acid position was also the most significantly UC-associated amino acid in omnibus tests (P = 2.68 × 10(-13)). Multivariate modeling identified rs9269955-C and 13 other variants in best predicting UC vs control status. In contrast, there was only suggestive association evidence between the MHC and CD. Taken together, these data demonstrate that variation at HLA-DRβ1, amino acid 11 in the P6 pocket of the HLA-DR complex antigen binding cleft is a major determinant of chromosome 6p association with UC.

Absent in Melanoma 2 (AIM2) is a member of the HIN-200 family of hematopoietic, IFN-inducible, nuclear proteins, associated with both, infection defense and tumor pathology. Recently, AIM2 was found to act as a DNA sensor in innate immunity. In addition, we and others have previously demonstrated a high frequency of AIM2-alterations in microsatellite unstable (MSI-H) tumors. To further elucidate AIM2 function in colorectal tumors, we here addressed AIM2-responsive target genes by microarray based gene expression profiling of 22 244 human genes. A total of 111 transcripts were significantly upregulated, whereas 80 transcripts turned out to be significantly downregulated in HCT116 cells, constitutively expressing AIM2, compared with AIM2-negative cells. Among the upregulated genes that were validated by quantitative PCR and western blotting we recognized several interferon-stimulated genes (ISGs: IFIT1, IFIT2, IFIT3, IFI6, IRF7, ISG15, HLA-DRA, HLA-DRB, TLR3 and CIITA), as well as genes involved in intercellular adhesion and matrix remodeling. Expression of ISGs correlated with expression of AIM2 in 10 different IFN-γ treated colorectal cancer cell lines. Moreover, small interfering RNA-mediated knock-down of AIM2 resulted in reduced expression of HLA-DRA, HLA-DRB and CIITA in IFN-γ-treated cells. IFN-γ independent induction of HLA-DR genes and their encoded proteins was also demonstrated upon doxycyclin-regulated transient induction of AIM2. Luciferase reporter assays revealed induction of the HLA-DR promoter upon AIM2 transfection in different cell lines. STAT-signaling was not involved in IFN-γ independent induction of ISGs, arguing against participation of cytokines released in an autostimulating manner. Our data indicate that AIM2 mediates both IFN-γ dependent and independent induction of several ISGs, including genes encoding the major histocompatibility complex (MHC) class II antigens HLA-DR-α and -β. This suggests a novel role of the IFN/AIM2/ISG cascade likewise in cancer cells.

The polymorphism of immunogenes of the major histocompatibility complex (MHC) is thought to influence the functional plasticity of immune responses and, consequently, the fitness of populations facing heterogeneous pathogenic pressures. Here, we evaluated MHC variation (allelic richness and divergence) and patterns of selection acting on the two highly polymorphic MHC class II loci (DRB and DQB) in the endangered primate Madame Berthe's mouse lemur (Microcebus berthae). Using 454 pyrosequencing, we examined MHC variation in a total of 100 individuals sampled over 9 years in Kirindy Forest, Western Madagascar, and compared our findings with data obtained previously for its sympatric congener, the grey mouse lemur (Microcebus murinus). These species exhibit a contrasting ecology and demography that were expected to affect MHC variation and molecular signatures of selection. We found a lower allelic richness concordant with its low population density, but a similar level of allelic divergence and signals of historical selection in the rare feeding specialist M. berthae compared to the widespread generalist M. murinus. These findings suggest that demographic factors may exert a stronger influence than pathogen-driven selection on current levels of allelic richness in M. berthae. Despite a high sequence similarity between the two congeners, contrasting selection patterns detected at DQB suggest its potential functional divergence. This study represents a first step toward unravelling factors influencing the adaptive divergence of MHC genes between closely related but ecologically differentiated sympatric lemurs and opens new questions regarding potential functional discrepancy that would explain contrasting selection patterns detected at DQB., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 728

Background & objectives: Human papillomavirus (HPV) is the main causative agent for cervical cancer. Variability in host immunogenetic factors is important in determining the overall cellular immune response to the HPV infection. This study was carried out to confirm the association between human leukocyte antigen (HLA) class II alleles and cervical cancer in HPV infected women. Methods: Both low and high resolution methods were used to genotype HLA class II (DRB1 and DQB1) alleles in 75 women with cervical cancer (cases) and 75 HPV positive women and 100 HPV negative women with healthy cervix (controls). Odds ratio and 95% confidence interval were calculated. Co-occurring HLA alleles (haplotype) across cases and controls were also studied. Results: Significant association was found for HLA-DRB1*03(*13:01) and - DQB1*02(*02:01) with increased risk for cervical cancer. Also, HLA-DRB1*13(*13:01); -DQB1*06 and -DQB1*03:02 were significantly associated with decreased risk for cervical cancer. Haplotype analysis highlighted the significant association of HLA- DRB1*07:01-DQB1*02:02 and HLA DRB1*10:01-DQB1*05:01 with cervical cancer, while HLA-DRB1*14:04-DQB1*05:03 and DRB1*15:01-DQB1*06:01 conferred decreased risk for cervical cancer. Multivariate analysis highlighted the association of specific alleles with cervical cancer after adjusting for confounding factor age. Interpretation & conclusions: There were possible associations of specific HLA class II alleles either with risk of developing cervical cancer, or with its protection. Our results confirmed the assessment of DRB1*13 as a protective marker in HPV infection outcome. Our study also revealed protective association of homozygous haplotype DRB1*15- DQB1*06 with cervical cancer.

Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti-IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ-dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.

Specific human leucocyte antigen (HLA) alleles are considered a genetic risk factor for the progression of haemorrhagic fever with renal syndrome (HFRS) caused by hantaviruses. The aim of this study was to establish whether HLA-DRB alleles are associated with the severity of HFRS caused by different types of hantaviruses in a Chinese Han population from Hubei Province of central China. Twenty-two specific HLA-DRB alleles were analysed by sequence-specific primer-polymerase chain reaction (SSP-PCR) in 100 HFRS patients and 213 healthy volunteers. Associations of HLA-DRB alleles with the severity and clinical parameters of HFRS caused by Hantaan virus (HTNV) or Seoul virus (SEOV) infection were evaluated. Six alleles (HLA-DRB1*0401-0411, HLA-DRB1*1001, HLA-DRB1*1101-1105, HLA-DRB1*1201-1202, HLA-DRB1*1305 and DRB5*0101-0201) demonstrated strong associations with HFRS caused by HTNV and SEOV infections. Further comparison of these HLA-DRB1 allele frequencies between HFRS patients with differing severities and healthy controls demonstrated that the HLA-DRB1*0401-0411, HLA-DRB1*1001 and DRB1*1305 alleles were more frequent in the moderate course of HTNV-infected HFRS. Meanwhile, the DRB1*1101-1105 allele was more frequently observed in the severe course of HTNV-infected HFRS. We also found that the HLA-DRB1*1201-1202 allele frequency was higher in the moderate course of SEOV-infected HFRS, whereas the DRB5*0101-0201 allele may play a protective role in moderate HFRS caused by both HTNV and SEOV infections. These results provide evidence of the influence of HLA-DRB on the severity of HFRS and confirm the effect of HLA-DRB on HFRS during different types of hantavirus infection in a Chinese Han population in Hubei Province, China.

The defective major histocompatibility complex (MHC) DRB6 gene is transcribed into mRNA in human [peripheral blood lymphocytes, transfected and Epstein-Barr virus (EBV)] and chimpanzee EBV cell lines. MHC-DRB6 presents several anomalies, which include stop codons in exon 2, lack of the usual polyadenilation signal of other MHC-DRB genes, and a promoter region and exon 1 taken from a locally inserted retrovirus. The complete cDNA sequences from human DRB6*0201 and three common chimpanzee alleles (Patr-DRB6*0108, Patr-DRB6*0109, Patr-DRB6*0111) have been obtained; two exon 1-exon 2 cDNA sequences from bonobos (Papa-DRB6*0101 and Papa-DRB6*0102) are also shown. In contrast to chimpanzee DRB6 transcripts, the human ones: (1) present an exon 1-exon 2 splicing site that includes the transcription of the first 141 nucleotides of intron 1, rendering a longer exon 1, and (2) show a duplication of exon 6, which would render a longer cytoplasmic tail in a putative DRB6 protein. These two characteristics are found in all the human sequences obtained, regardless of the cellular type tested, and they are not present in any of the chimpanzee alleles reported; consequently, they are human-specific. All the alleles reported here bear stop codons in the three possible reading frames; however, a certain level of expression of DRB6 has been observed by cytofluorometry. This could be due to the presence of a selenocysteine insertion sequence (SECIS) stem-loop structure located at the 3 untranslated region of the DRB6 mRNA, which directs selenocysteine incorporation at UGA codons. DRB6 transcription and translation would be the first gene model of a readingthrough stop codon mechanism in primate MHC. It is also feasible that the DRB6 gene might generate a population of short polypeptides, bound to plasmatic membranes, having non-antigen-presenting functions or which are presented by other MHC molecules as HLA-E presents HLA-G and -B leader sequence-derived peptides.

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera.

HLA-DRB6 is one of the human major histocompatibility complex (MHC) genes present in DR1, DR2, and DR10 haplotypes (approximately 26% of individuals). It shows several anomalies in human and non-human primates, including exon 2 stop codons (non-randomly grouped between codons 74 and 94) and a promoter region, and an exon 1 coming from an inserted retrovirus. It has been shown that not only chimpanzee but also human Mhc-DRB6 lack the usual 3' untranslated (UT) polyadenylation signal, and in the present work it was found that the human DRB6 gene coming from an HLA-DR2 haplotype is effectively transcribed after transfection in mouse L cells, and that HLA-DRB6 molecules may be expressed on the cell surface. DRB6 transcription level is remarkably lower in human than in chimpanzee. Moreover, their exons 1 (both taken from the 3'LTR region of a mammary tumor retrovirus) are also different; this shows that these viral insertions may be an important mechanism for different evolutionary changes in orthologous genes of different species. The pathways by which DRB6 molecules may be expressed on the membrane are unclear but other examples of truncated protein expression have also been described, even within the human major histocompatibility complex (i. e., in HLA-G). Finally, the presence of mature HLA-DRB6 mRNA molecules supports the notion that splicing may take place even in the absence of a canonical 3'UT polyadenylation signal.

The region of the HLA complex occupied by the DRB genes has undergone many rearrangements in the course of primate evolution. The rearrangements have produced a number of haplotypes differing from one another in the number and composition of the DRB genes. Some of the rearrangements also affected the DRB genes themselves. Selective intron sequencing has revealed the DR10 haplotype to be composed of at least three segments, each of different origin. The haplotype carries three DRB genes (gene fragments): DRB1*10, DRB6, and DRB9. The 5' end of the DRB1*10 gene, from the promoter region to a site in intron 1 approximately 500 bp from the beginning of exon 2, is derived from a DRB1*03-like gene. The segment of the DR10 haplotype encompassing the rest of the DRB1*10 gene and extending to the region between the DRB1 and DRB6 genes is of independent origin; it diverged from other DRB genes (DRB1*01 and DRB1*03) approximately 30 million years ago. Finally, the third segment encompassing the remainder of the DR10 haplotype is derived from a DR1-like haplotype. Since the functional part of the DR10 haplotype is of independent origin, there is little justification for the currently common practice of placing the haplotype together with DR1 in the group of DR1 haplotypes. The rearrangements in the DR haplotypes may constitute one of several mechanisms for increasing diversity at the DRB loci. The region of high instability seems to be flanked by conservatively evolving regions.

A single mismatch in highly expressed HLA-A, -B, -C, and -DRB1 loci (HEL) is associated with worse outcomes in hematopoietic stem cell transplantation, while less is known about the cumulative impact of mismatches in the lesser expressed HLA loci DRB3/4/5, DQ, and DP (LEL). We studied whether accumulation of LEL mismatches is associated with deleterious effects in 3853 unrelated donor transplants stratified according to number of matches in the HEL. In the 8/8 matched HEL group, LEL mismatches were not associated with any adverse outcome. Mismatches at HLA-DRB1 were associated with occurrence of multiple LEL mismatches. In the 7/8 HEL group, patients with 3 or more LEL mismatches scored in the graft-versus-host vector had a significantly higher risk of mortality (1.45 and 1.43) and transplant-related mortality (1.68 and 1.54) than the subgroups with 0 or 1 LEL mismatches. No single LEL locus had a more pronounced effect on clinical outcome. Three or more LEL mismatches are associated with lower survival after 7/8 HEL matched transplantation. Prospective evaluation of matching for HLA-DRB3/4/5, -DQ, and -DP loci is warranted to reduce posttransplant risks in donor-recipient pairs matched for 7/8 HEL.

Persistent infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer. Environmental, behavioral, and ill-defined genetic factors have also been implicated in the pathogenesis of this disease. To determine whether human leukocyte antigen (HLA) DRB alleles are associated with cervical cancer and HPV infections in the Chinese population, HLA genotypes were examined in 69 cervical cancer patients and 201 controls.Polymorphisms in HLA-DRB genes were genotyped using oligoneucleotide arrays, and the magnitude of associations was determined by logistic regression analysis.HLA-DRB1*13 (OR = 4.01 95% CI, 1.703-9.442) and HLA-DRB1*3(17) (OR = 2.661 95% CI, 1.267-5.558) were associated with an increased risk of cervical cancer, and DRB1*09012 (OR = 0.182, 95% CI, 0.079-0.418 and DRB1*1201 (OR = 0.35 95% CI, 0.142-0.863 were associated with a decreased risk. The risk associations of HPV infection were increased in women carrying the HLA-DRB1*09012 (OR = 1.924; 95% CI, 1.08 -3.427) and DRB3(52)*0101 (OR = 7.527 95% CI, 0.909-62.347) alleles. Among cervical cancer patients, the risk associations differed between HPV positive and negative cases for several alleles; increased risk of cervical cancer was associated with DRB3 (52)*02/03 (OR, 12.794; 95% CI, 5.007-32.691) and DRB1*3(17) (OR = 3.48; 95% CI, 1.261-9.604), and decreased risk was associated with DRB1*09012 and DRB5(51)*01/02. Furthermore, HPV16-containing cervical cancer cases differed from non-HPV16 subjects in their positive association with DRB1*1501 (OR = 4.173; 95% CI, 1.065-16.356) and DRB5(51)*0101/0201, and their negative association with DRB4(53)*0101 (OR = 0.329; 95% CI,0.122-0.888).The present results provide further evidence that certain HLA class II allele polymorphisms are involved in the genetic susceptibility to cervical cancer and HPV infection in the Chinese population from an area with a high incidence of this neoplasia.

The major histocompatibility complex (MHC) plays an important role in the immune response and may thus crucially affect an individual's fitness, relevant also for studies on evolutionary ecology and wildlife conservation. Detailed knowledge on the genomic organization, polymorphism and diversity of the MHC has a narrow taxonomic focus though and among macaques is only available for rhesus and long-tailed macaques-the species most commonly kept for biomedical research. The lack of data on wild populations is largely due to the difficulty of obtaining blood or tissue samples necessary for genotyping approaches. Here, we aimed at analyzing MHC-DRB from non-invasively collected fecal samples in wild Assamese macaques (Macaca assamensis), utilizing the MHC-DRB-STR (D6S2878) microsatellite marker. Due to the fecal DNA source incomplete genotypes occurred, which may be improved in the future by method refinement. We detected 28 distinct DRB-STR lengths in 43 individuals with individual genotypes containing 1-9 MHC-DRB-STRs and defined four haplotypes segregating between families in Mendelian fashion. Our results indicate that variability and diversity of MHC-DRB in Assamese macaques is comparable to that of other macaque species and importantly, that fecal samples can be used for non-invasive analysis of MHC genes after refinement of the applied methods, opening a number of opportunities for MHC research on natural populations.

Open source tools are needed to facilitate the construction, analysis, and visualization of gene-gene interaction networks for sequencing data. To address this need, we present Encore, an open source network analysis pipeline for genome-wide association studies and rare variant data. Encore constructs Genetic Association Interaction Networks or epistasis networks using two optional approaches: our previous information-theory method or a generalized linear model approach. Additionally, Encore includes multiple data filtering options, including Random Forest/Random Jungle for main effect enrichment and Evaporative Cooling and Relief-F filters for enrichment of interaction effects. Encore implements SNPrank network centrality for identifying susceptibility hubs (nodes containing a large amount of disease susceptibility information through the combination of multivariate main effects and multiple gene-gene interactions in the network), and it provides appropriate files for interactive visualization of a network using tools from our online Galaxy instance. We implemented these algorithms in C++ using OpenMP for shared-memory parallel analysis on a server or desktop. To demonstrate Encore's utility in analysis of genetic sequencing data, we present an analysis of exome resequencing data from healthy individuals and those with Systemic Lupus Erythematous (SLE). Our results verify the importance of the previously associated SLE genes HLA-DRB and NCF2, and these two genes had the highest gene-gene interaction degrees among the susceptibility hubs. An additional 14 genes previously associated with SLE emerged in our epistasis network model of the exome data, and three novel candidate genes, ST8SIA4, CMTM4, and C2CD4B, were implicated in the model. In summary, we present a comprehensive tool for epistasis network analysis and the first such analysis of exome data from a genetic study of SLE.

An unexpected probe reaction pattern was observed in two samples during HLA-DR typing by PCR-Sequence Specific Oligonucleotide Probes. In order to confirm the unusual typings, samples were analyzed by PCR-Sequence Specific Primers, cloning, and nucleotide sequencing of the second exon of the HLA-DRB-genes. The confirmed DR, DQ phenotype for one sample was DRB1*0701, DRB4*01, DRB5*0101, DRB6*0201, DQB*0602, DQB1*0202. The phenotype of other sample was DRB1*1602, DRB1*1302, DRB3*0301, DRB6*0101, DQB1*0501, DQB1*0502. The first sample has the novel combination of DRB1*0701 with DRB5*0101 and DRB6*0201. The second sample has either DRB6*0101 together with DRB 1*1602 in absence of any DRB5 allele or DRB6*0101 together with DRB1*1302, DRB 3*0301. We postulate that the most likely haplotype in sample #1 is DRB1*0701, DRB5*0101, DRB1*0602 which could have arisen from gene conversion. The most likely haplotype in sample #2, DRB 1*1602, DRB6*0101, DQB1*0502 would have arisen from an homologous recombination event.

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.

DRB6 has been found to be transcribed in human and apes. Promoter region and exon 1 come from a 5' LTR from a mammary tumour retrovirus. However, the putative protein structure would be very different to other DR molecules and it is doubtful that it may function as an antigen presenting molecule. Primate DRB6 alleles previously published together with the two new macaque sequences reported here support the existence of a strong selective pressure working on exon 2 to generate stop codons at the end of the exon (between codons 74 and 94) during at least 23 million years. The topology of dendrograms constructed with different primate DRB6 alleles supports the "trans-species" evolution proposed for MHC class I, class II and possibly C4 genes. Finally, DRB6, which is one of the oldest DRB genes, has been lost in the HLA-DRB3 (or DR52) group of haplotypes (DR3, DR5, DR6 and DR8) and a small DRB6 sequence is present at the exon 2 first hypervariable region of DRB4 (or DR53) gene, which is present in DR4, DR7 and DR9 haplotypes.

Sexual selection theory suggests that choice for partners carrying dissimilar genes at the major histocompatibility complex (MHC) may play a role in maintaining genetic variation in animal populations by limiting inbreeding or improving the immunity of future offspring. However, it is often difficult to establish whether the observed MHC dissimilarity among mates drives mate choice or represents a by-product of inbreeding avoidance based on MHC-independent cues. Here, we used 454-sequencing and a 10-year study of wild grey mouse lemurs (Microcebus murinus), small, solitary primates from western Madagascar, to compare the relative importance on the mate choice of two MHC class II genes, DRB and DQB, that are equally variable but display contrasting patterns of selection at the molecular level, with DRB under stronger diversifying selection. We further assessed the effect of the genetic relatedness and of the spatial distance among candidate mates on the detection of MHC-dependent mate choice. Our results reveal inbreeding avoidance, along with disassortative mate choice at DRB, but not at DQB. DRB-disassortative mate choice remains detectable after excluding all related dyads (characterized by a relatedness coefficient r > 0), but varies slightly with the spatial distance among candidate mates. These findings suggest that the observed deviations from random mate choice at MHC are driven by functionally important MHC genes (like DRB) rather than passively resulting from inbreeding avoidance and further emphasize the need for taking into account the spatial and genetic structure of the population in correlative tests of MHC-dependent mate choice.

Modified HABP (mHABP) regions interacting with HLA-DRβ1 ∗ molecules have a more restricted conformation and/or sequence than other mHABPs which do not fit perfectly into their peptide binding regions (PBR) and do not induce an acceptable immune response due to the critical role of their Φ and Ψ torsion angles. These angle’s critical role was determined in such highly immunogenic, protection-inducing response against experimental malaria using the conformers (mHABPs) obtained by 1 H-NMR and superimposed into HLA-DRβ1 ∗ -like Aotus monkey molecules; their phi ( Φ ) and psi ( Ψ ) angles were measured and the H-bond formation between these molecules was evaluated. The aforementioned mHABP propensity to assume a regular conformation similar to a left-handed polyproline type II helix (PPII L ) led to suggesting that favouring these conformations according to their amino acid sequence would lead to high antibody titre production and sterile protective immunity induction against malaria, thereby adding new principles or rules for vaccine development, malaria being one of them.