Evaluation of the Role of MHC Class II Alleles,Haplotypes and Selected Amino Acid Sequences in Primary Sclerosing Cholangitis

Background and Aims: Genetic susceptibility to primary sclerosing cholangitis is associated with several different HLA haplotypes, though a single "shared" susceptibility allele has yet to be identified. Most recently, attention has focussed on the MICA alleles in close proximity to the HLA class I, B locus. However, although there are strong associations with MICA*008, implicating this or a closely linked allele as major risk factors, this explanation alone does not account for all of the MHC-encoded susceptibility and resistance to PSC. The present study re-examines HLA class II associations in a large single centre series of well-characterised PSC patients. The specific aims of the study were to test existing associations and to develop hypotheses which together may account for all, or the majority, of the MHC-encoded susceptibility in PSC. Methods: A total of 148 adult white northern European patients and 134 control subjects were studied. HLA DRB1, DQA1, DQB1 alleles and DRB1*04, DRB1*13 and DRB3 subtypes were determined by standard PCR-genotyping. Results: The primary associations with the DRB3*0101-DRB1*0301-DQA1*0501-DQB1*0201 and DRB1*1301-DQA1*0103-DQB1*0603 haplotypes were confirmed (O.R.=2.69, p <0.0000025 and O.R.=3.8, p <0.0005). In addition the strong protective influence of the DRB1*04-DQB1*0302 haplotype was reaffirmed (O.R.=0.26, p <0.000025) and a previously unreported negative (i.e. protective) association with the DRB1*0701-DQB1*0303 haplotype was also demonstrated (O.R.=0.15, p <0.005). Further analysis suggested that susceptibility/resistance encoded by the second and third susceptibility haplotypes and by the two resistance haplotypes may be determined by specific amino acids at DQ&#103 -87 and DQ&#103 -55, respectively.     

A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: A study of human autoreactive CD4+T cells specific for GAD65

T cells specific for pancreatic islet proteins can be detected in type 1 diabetes patients and at-risk individuals, suggesting a failure of the central tolerance and negative selection. We addressed the question, how antigen dose shapes the diversity of CD4+ autoreactive T cells specific for glutamate decarboxylase 65 (GAD65) in a healthy HLA-DR*0404+ individual, with a persistent GAD65-specific T-cell response. CD4+T cells from this subject were stimulated with decreasing concentrations of the GAD65 555-567 (557I) peptide, and T-cell clones were derived from the tetramer-binding cell population. Functional and structural avidity, TcR-Vβ usage, and cytokine profiles were investigated at a clonal level. T-cell clones established with a low antigen dose (0.1 and 1 μg/ml) displayed higher avidity in contrast to the clones established with the highest antigen dose (10 μg/ml; Mann–Whitney U test, p = 0.003 and 0.006, respectively). The T-cell clones stimulated with the lowest peptide dose also had a higher tetramer-binding affinity than clones stimulated with the highest dose (p = 0.026). The majority (60.0%) of the high-avidity clones expressed TcR-Vβ5.1 chain whereas only one (12.5%) low-avidity clone did. All clones displayed Th0/Th2 cytokine profiles, but intermediate and high-avidity clones produced more IL-10 than low-avidity clones (p = 0.032). The results demonstrate an important role of the antigen dose in the determination of characteristics of the responding T-cell repertoire. High IL-13 and IL-10 production by GAD65-reactive T cells suggests a more anti-inflammatory profile of this healthy individual underlying protection from T1D.     

Autoantigens: Novel forms and presentation to the immune system

Abstract

It is clear that lupus autoimmunity is marked by a variety of abnormalities, including those found at a macroscopic scale, cells and tissues, as well as more microenvironmental influences, originating at the individual cell surface through to the nucleus. The convergence of genetic, epigenetic, and perhaps environmental influences all lead to the overt clinical expression of disease, reflected by the presences of autoantibodies and tissue pathology. This review will address several specific areas that fall among the non-genetic factors that contribute to lupus autoimmunity and related syndromes. In particular, we will discuss the importance of understanding various protein post-translational modifications (PTMs), mechanisms that mediate the ability of “modified self” to trigger autoimmunity, and how these PTMs influence lupus diagnosis. Finally, we will discuss altered pathways of autoantigen presentation that may contribute to the perpetuation of chronic autoimmune disease.     

Bacterial Polysaccharides with Zwitterionic Charge Motifs: Toll-Like Receptor 2 Agonists,T Cell Antigens,or Both?

Bacterial capsular polysaccharides (PS) which naturally contain zwitterionic charge motifs (ZPS) possess specific immunostimulatory activity, leading to direct activation of antigen-presenting cells (APCs) through Toll-like receptor 2 (TLR2) and of T cells in co-culture systems. When administered intraperitoneally, ZPS and bacteria expressing them are involved in the induction or regulation of T-cell dependent inflammatory processes such as intra-abdominal abscess formation. To generate vaccine candidates with antigen and adjuvant properties in one molecule we have chemically introduced zwitterionic motifs into naturally anionic PS and find that the resulting ZPS are TLR2 agonists, able to activate human and mouse APCs. Since T-regulatory cells and other T-cell subsets express TLR2, and TLR2 engagement modifies functionality and activation state of these cells, we speculate that most effects induced by natural and chemically derived ZPS may be explained by their TLR2 agonist properties, presumably through the combined action on TLR2-expressing APCs and T cells.     

Carbon monoxide decreases endosome–lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells.

Heme oxygenase‐1 (HO‐1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe²⁺, and biliverdin. We have previously shown that either induction of HO‐1 or treatment with exogenous CO inhibits LPS‐induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen‐specific inflammation. Here, we evaluated the capacity of HO‐1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS‐treated DCs. We observed that HO‐1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA‐derived peptides, bead‐bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO‐1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO‐1 and CO can inhibit the ability of LPS‐treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome–lysosome fusion.     

Complement opsonization of HIV‐1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells

Induction of optimal HIV‐1‐specific T‐cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV‐1 in human monocyte‐derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV‐1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7‐integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV‐1 exposure; complement‐opsonized HIV‐1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7‐integrin can promote or disfavor antigen presentation probably by routing HIV‐1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV‐1 affects the antigen‐processing machinery.     

Peritoneal cavity B‐1a cells promote peripheral CD4+ T‐cell activation

Innate‐like murine B‐1a cells are well known for their ability to secrete natural IgM. Their non‐Ab mediated functions, including Ag presentation to CD4⁺ T cells, are less well explored. Using combined adoptive transfer experiments with peptide‐pulsed peritoneal cavity (PerC)‐derived B‐1a cells and CFSE‐labeled T cells, we show that B‐1a cells present Ag to CD4⁺ T cells from the periphery in vivo. In vitro characterization, using co‐cultures in which B‐1a or splenic B cells presented whole OVA protein to OVA‐specific Tg T cells, shows that B‐1a cells differentially promote intracellular cytokine‐expressing T cells. PerC‐derived B‐1a cells increase the percentage of IL‐10‐producing T cells along with IL‐4‐ and IFN‐γ‐producing CD4⁺ T cells. These data suggest that B cells in the PerC have the potential to influence peripheral immune responses without the necessity to migrate out of this location. This, to our knowledge previously undescribed, immuno‐logical pathway potentially plays a role in the presentation of gut microbiota‐derived Ags to peripheral T cells.     

水痘-带状疱疹病毒抗原的ELISA定量检测方法的建立

目的:建立水痘-带状疱疹病毒(VZV)抗原的双抗体夹心ELISA定量检测方法,用于质控VZV灭活疫苗研发和生产中抗原含量.方法:以VZV中和单抗5F6C8为包被抗体、8H5D1为酶标抗体,构建定量检测VZV抗原的双抗体夹心ELISA方法,并对本方法的特异性、灵敏度、准确性、线性和稳定性等性能进行分析.结果:建立的双抗体夹心定量检测VZV抗原的ELISA方法,线性范围为0.4 μg～13 μg/ml,相关系数为R2=0.994,定量限度为0.4.μg/ml;变异系数CV＜ 15％、准确性回收率介于87.5％ ～ 111.6％之间,稳定性37℃6天的回收率＞80％.与VZV以外的相关病毒样本没有交叉反应.结论:构建的VZV抗原ELISA定量检测方法的各项性能符合定量检测需要,可用于VZV灭活疫苗的研发和生产过程的抗原含量检测.     

Diagnostic Role of Prostate Resection in the Elderly Patients Who Experience Significant Co-Morbidity with a High Clinical Suspicion of Prostate Cancer

The necessity of routine prostate biopsy prior to transurethral resection of the prostate (TURP) in elderly comorbid patients with a high prostate specific antigen (PSA) level remains controversial. We assessed the role of TURP in prostate cancer diagnosis in these individuals. A total of 197 patients underwent TURP in conjunction with prostatic needle biopsy. Pathologic reviews of specimens of TUR chips and biopsy cores were analyzed. Overall, prostate cancer (CaP) was detected in 114 patients (57.6%). Ninety-eight cancers (86%) were detected with TURP and biopsy, and seven cancers (6.1%) with only TURP. The Gleason score of a TUR-specimen was identical to that of the biopsy-core in 43.9% of cases. Variables associated with diagnostic accuracy in the TUR-specimens included the prebiopsy PSA level, prostate specific antigen density (PSAD), and the Gleason score in biopsy cores. In patients with a PSA level and a PSAD that was greater than 15.4 ng/mL and 0.69 ng/mL/g, respectively, 100% of the cancers were detected in the TUR-specimens. Our results suggest that a prostatic biopsy might be omitted prior to TURP in elderly patients with significant co-morbidity and levels for PSA of >15.4 ng/mL.     

CMIA法在输血前感染性指标检测中的应用研究

目的对化学发光微粒子免疫测定(CMIA)技术定量检测输血前感染性指标的临床应用进行评价。方法用CMIA技术对22235例患者进行输血前乙肝表面抗原(HBsAg)、抗丙型肝炎病毒抗体(Anti-HCV)、抗梅毒螺旋体特异性抗体(Anti-TP)及抗人类免疫缺陷病毒抗体(Anti-HIV)定量检测,所有阳性标本用相应的酶联免疫吸附法(ELISA)进行定性检测,Anti-TP阳性标本用梅毒螺旋体抗体明胶颗粒凝集试验(TPPA)进行确诊,Anti-HIV阳性标本送市疾控中心进行抗体确认,并对几种方法的检测结果进行分析。结果 HBsAg、Anti-HCV、Anti-TP、Anti-HIV定量检测阳性例数分别为3028、336、684、63;定量阳性标本经ELISA检测,定性阳性例数分别为2472,269,668,58;684例Anti-TP定量阳性样本中,用TPPA确认,阳性603例;63例Anti-HIV定量阳性样本送南充市疾控中心进行确认,48例确定为阳性,5例不确定,10例为阴性。结论 CMIA法定量检测输血前感染性指标可及早发现感染"窗口期"患者,降低漏检率,有利于患者的早期诊断和治疗,有利于医护人员自我防护及减少医疗纠纷。