利用丙酮酸脱氢酶复合物原核表达产物检测原发性胆汁性肝硬变患者血清自身抗体

目的 建立一种利用重组表达的丙酮酸脱氢酶复合物E2亚单位(PDC-E2)和E3结合蛋白(PDC-E3BP)检测原发性胆汁肝硬变(PBC)患者血清的方法。方法 以适当的条件诱导PDC-E2和PDC-E3BP表达质粒pExSecI/PDC-E2和pET28/E3BP,利用表达产物通过酶联免疫吸附试验检测PBC、正常人及其他原因肝硬变患者血清,并与欧盟的检测试剂盒比较。结果 显示PDC-E2和PDC-E3BP表达产物检测PBC患者血清中的自身抗体阳性率为93.3％,与欧盟试剂盒比较,总符合率为98.03％。结论 建立了利用重组表达丙酮酸脱氢酶复合物E2亚单位和E3结合蛋白检测PBC患者血清中自身抗体的方法,且获得PDC-E2和PDC-E3BP的高效表达产物。     

用重组丙酮酸脱氢酶复合物E2亚单位检测M2抗体及其临床意义

目的用重组表达的丙酮酸脱氢酶复合物E2亚单位（PDC-E2）检测M2抗体，以利于原发性胆汁性肝硬化（PBC）的早期诊断。方法采用重组表达的PDC—E2建立了免疫印迹法（IBT）和酶联免疫吸附试验（ELISA）。检测40例PBC患者血清的M2抗体，以其他肝病患者、自身免疫病患者、健康体检者作对照。结果40例PBC患者血清中检测出抗PDC—E2抗体阳性37例，阴性3例，阳性率为92．5％，而疾病对照组和健康体检者血清中该抗体检测均为阴性。结论用重组表达的人PDC-E2检测抗体有较好的敏感性和特异性，有助于PBC的临床诊断。     

用重组丙酮酸脱氢酶复合物E2亚单位检测M2抗体及其临床意义

目的用重组表达的丙酮酸脱氢酶复合物E2亚单位(PDC-E2)检测M2抗体,以利于原发性胆汁性肝硬化(PBC)的早期诊断。方法采用重组表达的PDC-E2建立了免疫印迹法(IBT)和酶联免疫吸附试验(ELISA)。检测40例PBC患者血清的M2抗体,以其他肝病患者、自身免疫病患者、健康体检者作对照。结果40例PBC患者血清中检测出抗PDC-E2抗体阳性37例,阴性3例,阳性率为92.5%,而疾病对照组和健康体检者血清中该抗体检测均为阴性。结论用重组表达的人PDC-E2检测抗体有较好的敏感性和特异性,有助于PBC的临床诊断。     

M2抗体重组人源靶抗原二联体在原发性胆汁性肝硬化患者中的检测及应用

目的 利用重组抗原BCOADA-E2和PDC-E2的二联体（BP），检测PBC患者血清中的M2抗体并探讨其临床意义。方法 经Ni—NTA亲和柱纯化重组表达的BP融合蛋白，分别建立免疫印迹法（IBT）和酶链免疫吸附（ELISA）法检测60份PBC患者血清，以60份其他肝病患者、60份自身免疫病患者、80例正常人血清为对照组。结果 经常规试剂盒检测为M2（＋）的60份患者血清，利用重组抗原检测阳性53例，阴性7例，阳性率为88．3％；经常规试剂盒检测为M2（-）的60份自身免疫病患者血清、60份其他肝病患者和80例正常人血清，利用重组抗原检测为M2（-）。结论 利用重组抗原BP检测M2抗体敏感性较高。对临床辅助诊断PBC提供一定的手段。     

原发性胆汁性肝硬化血清学抗体与戊型肝炎病毒抗体间的相关性分析

目的回顾性研究127例原发性胆汁性肝硬化(PBC)相关血清学自身抗体与戊型肝炎病毒(HEV)抗体之间的相关性。方法与PBC相关自身抗体:抗线粒体抗体M2亚型(AMA-M2)、重组M2融合蛋白抗体(M2-3E/BPO)、抗核点型靶抗原蛋白100(抗-sp100)、抗核孔复合物210跨膜糖蛋白抗体(抗-gp210)、抗着丝粒蛋白B抗体(CENP B),以上抗体中单一或多抗体阳性血清127例与93例系统性红斑狼疮(SLE)患者、92例类风湿性关节炎(RA)患者和122例健康人血清样本同期分别采用酶联免疫吸附实验(ELISA)检测HEV IgG/IgM抗体。结果 PBC相关自身抗体阳性组、健康人对照组、SLE和RA组的HEV-IgG抗体阳性率分别是55.9%、27.0%、23.7%、40.2%,经χ2检验PBC相关自身抗体阳性组与其他三组差异有统计学意义(P0.05)。PBC相关单一自身抗体阳性组和多抗体阳性组之间HEV-IgG抗体阳性率差异无统计学意义(χ2=0.319,P0.05)。性别差异无统计学意义(χ2=0.281,P0.05)。结论 PBC相关自身抗体阳性血清存在HEV抗体高阳性率。PBC相关自身抗体和HEV-IgG抗体的实验室检测可能存在相互干扰。     

MIT3、抗gp210和抗sp100联检在原发性胆汁性肝硬化诊断中的价值

目的比较新型研发的PBC筛查试剂盒与单个MIT3,以及2个抗核抗体(ANA)gp210和sp100 ELISA检测试剂盒在中国人群中PBC诊断的性能。方法使用PBCscreen ELISA筛查试剂盒和单独针对MIT3、gp210和sp100抗原的ELISA试剂盒,对95例确诊PBC患者、125例非PBC患者(自身免疫性肝炎/原发性硬化性胆管炎18例,病毒性肝炎32例,其他肝脏疾病21例),以及健康人54例的血清样本进行检测。结果 PBC Screen ELISA法检测敏感性为76.8%,特异性为95.2%。特异性与结合单独ELISA法检测MIT3、gp210和sp100抗体的特异性96.0%相当。在32例用间接免疫荧光(indirect immunofluorescence,ⅡF)检测为阴性的PBC患者中,有15(46.9%)例PBC Screen ELISA检测结果为阳性。结论 PBC Screen ELISA是理想的一线PBC筛查试剂,尤其对于ⅡF筛查为阴性的PBC患者展现出优良的评估性能。     

抗线粒体抗体M2亚型抗体三种检测方法的比较及其对原发性胆汁性肝硬化的诊断价值研究

目的 比较三种方法检测抗线粒体抗体M2亚型(AMA-M2)抗体在原发性胆汁性肝硬化诊断中的应用价值.方法 分别用以丙酮酸脱氢酶复合体(PDC)为靶抗原的ELISA法;以三联体(BPO)为靶抗原的ELISA法;以天然M2抗原和BPO融合蛋白M2-3E(BPO)为靶抗原的ELISA法检测原发性胆汁性肝硬化患者,自身免疫性疾病患者和健康体检者血清中的AMA-M2抗体.结果 以PDC为靶抗原的ELISA法测定AMA-M2抗体检出率(敏感性)达81.25%,特异性达97.15%;以BPO为靶抗原的ELISA法测定AMA-M2抗体检出率(敏感性)达88.54%,特异性达98.37%;以M2-3E(BPO)为靶抗原的ELISA法测定AMA-M2抗体检出率(敏感性)达96.88%,特异性达97.15%.结论 包被有天然M2抗原和BPO融合蛋白这两种抗原联合制备的三联体检测AMA-M2抗体的阳性率最高,特异性达到97.15%,为原发性胆汁性脉硬化的诊断提供了简便、快速而有效的手段.     

风疹病毒E1蛋白克隆表达及应用

目的 构建表达风疹病毒蛋白抗原的重组质粒及工程菌,获得纯化的E1蛋白抗原,用于检测风疹病毒特异性抗体IgM.方法 克隆表达风疹病毒E1重组蛋白,利用产物建立IgM捕获ELISA方法鉴定其抗原性及实用性.结果 表达纯化的E1蛋白经酶标记建立IgM捕获ELISA方法,检测53份抗风疹病毒IgM阳性血清和67份阴性血清,用酶标记E1蛋白建立的捕获ELISA法阳性检出率98.1%,阴性检出率100%,与意大利SORIN公司试剂盒检测结果比较,无统计学意义(P＞0.05);其中1份风疹病毒(IgM)阳性血清1∶16稀释后仍能与抗原反应,初步表明E1蛋白抗原表位有较好的抗原特异性.结论 高效表达纯化的E1蛋白抗原性强,利用其建立的IgM捕获ELISA方法,可用于检测风疹病毒抗体.     

用蛋白质抗原检测肝硬化病人的自身抗体

原发性胆汁性肝硬化(PBC)是一种病因未明、以肝内胆管损伤为特征的自身免疫病。已知这种病人血清中有抗线粒体抗体(AMA)。为检测这种抗体,作者用实验室分离的编码人线粒体74kd和52kd 蛋白质的两种cDNA 克隆,应用基因工程技术生产出分别与谷胱甘肽-S-转移酶融合的两种重组纯化蛋白质。经鉴定,74kd 蛋白质为丙酮酸脱氢酶复合体中的二氢硫辛酰胺乙酰基转移酶(E2),简写为PDH-E2;52kd 蛋白质为分支链α-酮酸脱氢     

利用重组人源靶抗原二联体OP检测M2抗体及临床意义

目的 利用重组抗原检测自身免疫病患者血清中抗OP自身抗体并探讨其临床意义。方法 经Ni-NTA柱纯化表达重组OP融合蛋白作为抗原，分别用免疫印迹法（IBT）和酶链免疫吸附试验（ELISA）检测70份PBC患者血清，80份其它肝病患者血清，80份自身免疫病患者血清和100份正常人血清。结果 70份PBC患者血清中检测出阳性患者62例，阴性8例，阳性率为87．6％。其它肝病患者血清、自身免疫病患者血清和正常人血清均为阴性。重组抗原检测M2抗体有一定的敏感性。结论 利用重组抗原OP检测M2抗体，有较好的敏感性及特异性，有助于PBC的临床诊断。     

The E1 alpha and beta subunits of the pyruvate dehydrogenase complex are M2'd' and M2'e' autoantigens in primary biliary cirrhosis

1. Sera from 76 patients with primary biliary cirrhosis (PBC) and 66 control subjects (53 with chronic liver disease and 13 healthy normal women) were immuno-blotted against purified E1 component of bovine pyruvate dehydrogenase complex (PDC) and bacterial PDC. 2. Thirty-one out of seventy-six (41%) sera from PBC patients showed a positive response to bovine E1 alpha, and five of these 31 (7% of total) reacted with bovine E1 beta. None of the control sera reacted with bovine E1 alpha or beta. 3. None of the PBC sera that recognized bovine E1 subunits reacted with bacterial PDC E1. 4. In the PBC patients there was no correlation between presence of antibodies to E1 alpha and beta subunits and histological stage of the disease. 5. Our data demonstrate that the E1 alpha and beta components of mammalian PDC are the M2'd' and 'e' mitochondrial autoantigens, respectively.     

Identification and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis.

The immunodominant antimitochondrial antibody response in patients with primary biliary cirrhosis (PBC) is directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Based on our earlier observations regarding peripheral blood mononuclear cell (PBMC) T cell epitopes, we reasoned that a comparative analysis of the precursor frequencies of PDC-E2 163-176-specific T cells isolated from PBMC, regional hepatic lymph nodes, and from the liver of PBC patients would provide insight regarding the role of T cells in PBC. Results showed a disease-specific 100-150-fold increase in the precursor frequency of PDC-E2 163-176-specific T cells in the hilar lymph nodes and liver when compared with PBMC from PBC patients. Interestingly, autoreactive T cells and autoantibodies from PBC patients both recognize the same dominant epitope. In addition, we demonstrated cross-reactivity of PDC-E2 peptide 163-176-specific T cell clones with PDC-E2 peptide 36-49 and OGDC-E2 peptide 100-113 thereby identifying a common T cell epitope "motif" ExETDK. The peptide 163-176-specific T cell clones also reacted with purified native PDC-E2, suggesting that this epitope is not a cryptic determinant. These data provide evidence for a major role for PDC-E2 peptide 163-176 and/or peptides bearing a similar motif in the pathogenesis of PBC.     

Molecular mimicry in primary biliary cirrhosis. Evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2.

Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC.     

Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.     

Bcl-2–dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis

The close association between autoantibodies against pyruvate dehydrogenase-E2 (PDC-E2), a ubiquitous mitochondrial protein, and primary biliary cirrhosis (PBC) is unexplained. Many autoantigens are selectively modified during apoptosis, which has focused attention on apoptotic cells as a potential source of "neo-antigens" responsible for activating autoreactive lymphocytes. Since increased apoptosis of bile duct epithelial cells (cholangiocytes) is evident in patients with PBC, we evaluated the effect of apoptosis on PDC-E2. Autoantibody recognition of PDC-E2 by immunofluorescence persisted in apoptotic cholangiocytes and appeared unchanged by immunoblot analysis. PDC-E2 was neither cleaved by caspases nor concentrated into surface blebs in apoptotic cells. In other cell types, autoantibody recognition of PDC-E2, as assessed by immunofluorescence, was abrogated after apoptosis, although expression levels of PDC-E2 appeared unchanged when examined by immunoblot analysis. Both overexpression of Bcl-2 and depletion of glutathione before inducing apoptosis prevented this loss of autoantibody recognition, suggesting that glutathiolation, rather than degradation or loss, of PDC-E2 was responsible for the loss of immunofluorescence signal. We postulate that apoptotic cholangiocytes, unlike other apoptotic cell types, are a potential source of immunogenic PDC-E2 in patients with PBC.     

M2抗体人源靶抗原三联体的克隆表达及其在原发性胆汁性肝硬化中的应用

目的 设计克隆表达抗原蛋白三联体BPO，利用纯化的重组BPO，建立原发性胆汁性肝硬化(primary biliary cirrhosis，PBC)特异性免疫学诊断方法。方法 利用基因工程方法克隆表达M2抗原及其三联体BP0，并加以鉴定。纯化BPO，建立ELISA法。应用ELISA法检测17例PBC患血清M2抗体，以167例非PBC患和1225例健康人作对照。结果 17例临床诊断为PBC的患，M2抗体均为阳性，而对照组M2抗体均为阴性。结论 本法检测M2抗体有较好的敏感性及特异性，为PBC的临床诊断提供了有效手段。     

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen- specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.