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。     

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。     

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。     

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。     

原发性胆汁性肝硬化特异性AMAM2抗体在5 011名体检者中的筛查分析

目的：在健康体检者中筛查原发性胆汁性肝硬化(PBC)特异性AMAM2抗体，并对AMAM2抗体阳性者进行分析。方法：设计克隆表达了一抗原蛋白三联体，命名为BPO，它包含了AMAM2抗体识别的人源BCOADC-E2、PDC-E2和OGDC-E2的抗原表位片断。利用纯化的重组BPO，建立了PBC特异性免疫学诊断方法。ELISA法筛查5011名体检者，进一步分析AMAM2抗体阳性者的生化和免疫指标。超声检查或ERCP检查排除非PBC异常。结果：5011名体检者中有8名AMAM2抗体阳性(阳性率0.16％、)，其中7名为女性，1名为男性，年龄均在40岁以上。4名AMAM2抗体阳性者有不明原因的碱性磷酸酶和γ-谷氨酸转肽酶的增高，虽无PBC的临床症状(如疲劳，皮肤瘙痒或黄疽)，但其中有3名符合美国肝脏病学会推荐的PBC诊断标准。有两名行肝穿刺检查，均符合PBC病理学特征。结论：无症状PBC患者在中国并不少见。     

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。     

汉族原发性胆汁性胆管炎患者抗线粒体抗体M2亚型抗原表位分布及其临床价值

目的探讨汉族原发性胆汁性胆管炎(PBC)患者抗线粒体抗体M2亚型(AMA-M2)抗原表位分布情况及其临床价值。方法采用Red/ET重组技术制备AMA-M2抗原表位蛋白PDC-E2、BCOADC-E2和OGDC-E2,建立相应的ELISA检测方法,对374例PBC患者抗原表位分布进行分析。比较AMA-M2主要抗原表位组合模式间清蛋白-胆红素评分(ALBI)结果的差异,熊去氧胆酸(UDCA)药物生化应答和不应答患者抗原表位分布的差异。结果 374例PBC患者血清与PDC-E2、BCOADC-E2和OGDC-E2抗原表位有反应率分别为86.6%、88.0%和35.0%。与PBC患者血清有反应性的常见抗原表位模式(PDC-E2+BCOADC-E2+OGDC-E2、PDC-E2+BCOADC-E2、PDC-E2和BCOADC-E2)间ALBI结果的差异有统计学意义(P0.05),UDCA生化不应答患者血清与BCOADC-E2的反应率(89.9%)高于应答患者(77.9%),差异有统计学意义(P0.05)。结论与AMA-M2抗原表位PDC-E2、 BCOADC-E2和OGDC-E2同时有反应性的PBC患者疾病预后不佳的风险较高,PDC-E2和BCOADC-E2抗原表位可能与UDCA治疗应答相关。     

用重组M2三联体抗原建立原发性胆汁性肝硬化免疫检测法

目的 建立原发性胆汁性肝硬化（PBC）特异性免疫学检测方法。方法 在重组质粒表达的基础上，用亲和层析进一步纯化重组蛋白后，用酶免疫吸附法检测M2抗体。结果 在PBC组11例患者哈部检出M2抗体，阳性率为1005，而非PBC组75例患者中无一检出M2抗体，本法与病理检查和临床诊断的相关性有非常显著意义（P＜0.01）。结论 本法检测M2抗体有较好的敏感性及特异性，为PBC的早期发现和临床诊断提供了有力的工具。     

抗M2-3E ELISA:一种新型、高效的PBC血清学诊断方法

原发性胆汁性肝硬化(PBC)是一种免疫介导的慢性炎症性胆汁性肝病,其病因不明。AMA-M2是PBC患者最为灵敏和最为特异的诊断标志,高达94%的PBC患者显示AMA-M2阳性。AMA-M2抗体的靶抗及OADC,由BCOADC-E2、PDC-E2、OGDC-E2、PDC-E1t和PDC-E3结合蛋白组成。德国欧蒙公司(EUROIMMUN)率先把融合重组蛋白BPO与天然的PDC纯化抗原相混合,开发了新型的抗M2-3EELISA检测法。该系统与仅采用天然M2抗原包被或仅包被BPO的ELISA检测系统相比,拥有敏感性较高的优点。     

原发性胆汁性肝硬化血清抗线粒体抗体亚型的检测及诊断价值

目的 探讨抗线粒体亚型（AMA亚型）对原发性胆汁性肝硬化（PBC）的诊断价值。方法 应用酶免疫斑点法检测血清中AMA—M2、M4、M9亚型。8例经临床诊断为PBC患者，47例非PBC肝胆病患者。结果 8例PBC患者M2均阳性、但M4及M9均阴性；47例非PBC肝胭病患者M2、M4、M9均阴性。4例PBC者肝活检Ⅰ期及Ⅳ期各1例、2例为Ⅱ期。结论 血清AMA-M2抗体检测可作为临床诊断PBC的重要血清免疫学诊断指标。     

原发性胆汁性肝硬化自身抗体谱的检测及临床应用

目的 探讨联合检测多种自身抗体在原发性胆汁性肝硬化(PBC)诊断中的价值及临床意义.方法 用免疫印迹法分别检测96例PBC患者,100例其他自身免疫性疾病(AID)患者和49名健康体检者血清中的抗线粒体(AMA)-M2亚型抗体、抗3E(BPO)抗体、抗Sp100抗体、抗旱幼粒细胞性白血病(PML)抗体、抗gp210抗体、抗肝肾微粒体-1(LKM-1)抗体、抗肝特异性胞质抗体-1(LC-1)、抗可溶性肝抗原/肝胰抗原抗体(SLA/LP).结果 96例PBC患者中AMA-M2、抗3E(BPO)抗体、抗Sp100抗体、抗PML抗体、抗gp210抗体阳性率分别为76.0%、84.4%、32.3%、28.1%、35.4%,而以上5种自身抗体在其他自身免疫性疾病中的阳性率分别为13.0%、9.0%、3.0%、2.0%、1.0%;AMA-M2对PBC诊断的敏感度达76.0%,特异度达87.0%;抗3E(BPO)抗体对PBC诊断的敏感度达84.4%,特异度达91.0%;抗Sp100抗体对PBC诊断的敏感度达32.3%,特异度达97.0%;抗PML抗体对PBC诊断的敏感度达28.1%,特异度达98.%;抗gp210抗体对PBC诊断的敏感度达35.4%,特异度达99.0%,未检测到抗LKM-1抗体、抗LC-1抗体及抗SLA/LP抗体;抗gp210抗体阳性患者肝功能衰竭的发生率明显高于抗体阴性患者(χ2 =11.17,P<0.01).结论 PBC患者中可检测到多种自身抗体,自身免疫性肝病自身抗体谱检测对PBC诊断、鉴别诊断、治疗及预后判断均有重要意义.     

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.     

血清抗线粒体抗体M_2亚型检测在原发性胆汁性肝硬化临床诊断中的价值

目的:探讨抗线粒体抗体(AMA)-M2亚型检测在原发性胆汁性肝硬化(primary billiary cirrhosis,PBC)诊断中的价值。方法:应用酶联免疫吸附试验,检测20名健康对照者、139例其他非PBC疾病患者和42例PBC患者血清中的AMA-M2水平,并采用矩阵图决策法分析血清AMA-M2检测对PBC的诊断效率。结果:健康对照者平均血清AMA-M2水平为(16.51±1.38)RU/mL,非PBC患者的平均血清AMA-M2水平为(24.33±7.29)RU/mL,两者比较差异无统计学意义(P>0.05)。PBC患者的平均AMA-M2水平为(211.51±126.68)RU/mL,与健康对照者比较,差异有统计学意义(P<0.01)。AMA-M2检测对PBC诊断的灵敏度为95.2%,特异度为97.4%,准确率达95.6%,阴性预测值为98.7%,阳性预测值为90.9%。结论:血清AMA-M2检测有较高的诊断效率,可作为PBC诊断的重要血清免疫学指标。     

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.     

用ELISA检测抗丙酮酸脱氢酶复合物E2和E3结合蛋白自身抗体

目的　用重组丙酮酸脱氢酶复合物E2亚单位和E3结合蛋白 (PDC E2 ,PDC E3BP)建立筛查原发性胆汁性肝硬化 (PBC)患者相应自身抗体的ELISA。方法　用本室制备的重组PDC E2和PDC E3BP蛋白包被酶联反应板 ,检测PBC患者、正常人及其他肝病患者血清。结果　组建成筛查PBC患者血清中自身抗体的试剂盒 ,能够准确地检测PBC患者血清中的自身抗体。结论　建立的试剂盒能用于PBC患者血清中丙酮酸脱氢酶复合物自身抗体的检测。     

Serial changes in enzyme inhibitory antibody to pyruvate dehydrogenase complex during the course of primary biliary cirrhosis

To assess the usefulness of enzyme inhibition assay for the diagnosis of primary biliary cirrhosis (PBC), we determined the serial changes in enzymatic inhibitory antibody to pyruvate dehydrogenase complex (PDC) in patients with PBC, and compared the results to those of immunofluorescence and immunoblotting. Forty-nine sera from 19 patients with PBC who were followed-up for at least 16 months were tested for antimitochondrial antibodies (AMA) by indirect immunofluorescence, immunoblotting on bovine heart mitochondria, and enzyme inhibition assay using commercially available TRACE Enzymatic Mitochondrial Antibody (M2) Assay (EMA) kit. Of the 49 sera, 39 (80%), 35 (71%), 38 (78%), 31 (63%), and 36 (73%) were positive for AMA by immunofluorescence, for immunoglobulin G (IgG), IgM, and IgA class antibody against E2 subunit of PDC (PDC-E2) by immunoblotting, and for enzymatic inhibitory antibody to PDC by EMA, respectively. AMA titers determined by immunofluorescence did not change in 9 patients (47%), increased in 4 (21%), decreased in 3 (16%), and fluctuated in 3 (16%) during follow-up. The number of anti-M2 bands by immunoblotting did not change in 9 (47%), increased in 6 (32%), decreased in 2 (11%), and fluctuated in 2 (11%). Units of PDC activity by EMA did not change markedly in 16 (84%), increased in 2 (11%), and fluctuated in 1 (5%). Positive EMA results were common in cases with high levels of serum alkaline phosphatase and IgM, and the units of PDC activity by EMA correlated significantly and inversely with AMA titers by immunofluorescence, and serum reactivity to PDC-E2 by immunoblotting, respectively. There was no correlation between serial changes in biochemical data and units of PDC activity by EMA. In three patients who showed a decrease in AMA titers, AMA titers correlated more with EMA results than immunoblotting. Moreover, in a patient with fluctuating AMA titers, the units of PDC activity by EMA paralleled AMA titers. Our results suggest that EMA is useful for the diagnosis of AMA-positive PBC, and also could be used for monitoring the disease course in PBC.     

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.