Characterization of primary biliary cirrhosis (PBC) specific mitochondrial determinants by immunoblotting

Antimitochondrial antibodies (AMA) are detected in up to 100% of patients with primary biliary cirrhosis (PBC); subtypes of AMA are disease specific. Sera from 21 patients with PBC and from 50 patients with various hepatic and non-hepatic diseases were tested for AMA by indirect immunofluorescence, radioimmunoassay for PBC-specific subtype anti M2 and characterized by western blot analysis and agarose-IEF-immunoblotting. Sera from patients with PBC reacted with up to 7 different mitochondrial polypeptides on western blots, mol. wt. 24,000-62,000 dalton. Sera from 50 patients with various hepatic and non-hepatic diseases did not react with these polypeptides. Sera with other AMA subtypes were included in this study (anti M1, anti M3, and anti M5). These mitochondrial polypeptides were associated with inner mitochondrial membranes (mitoplasts). Sonification led to a solubilization of several mitochondrial polypeptides (p 62, p 48, p 40, p 24). On agarose-IEF-immunoblotting sera from patients with PBC and 3 sera from patients with AMA positive cholestatic CAH but no other sera reacted with a protein band at pI 4.0; seven PBC sera reacted in addition with a protein band at pI 4.4. Western blot and agarose-IEF-immunoblotting are sensitive and specific tools to identify and characterize mitochondrial target antigens in PBC. Furthermore these techniques allow to study the clinical relevance of the heterogeneity of AMA in cholestatic liver disease.     

人源M2抗原检测M2抗体诊断抗线粒体抗体阴性原发性胆汁性肝硬化

目的 分析抗线粒体抗体（AMA）阴性的原发性胆汁性肝硬化（PBC）患者与AMA阳性PBC患者在临床症状，实验室诊断指标等方面的差异；对比用克隆表达的人源M2抗原建立的酶联免疫吸附试验（ELISA）法测定M2抗体，与经典的间接免疫荧光法测定AMA的敏感性高低。方法 测定63例PBC患者外周血中的AMA与M2抗体。结果 AMA在63例PBC患者中的阳性率为81%。而M2抗体为100%；AMA阴性的PBC患者抗核抗体（ANA），抗平滑肌抗体（ASMA）的阳性率高于AMA阳性的PBC患者，但在临床症状和其他实验室诊断标准上差异并无显著性。结论 酶联免疫吸附试验（ELISA）法检测M2抗体的敏感性高于间接免疫荧光法测定AMA，PBC患者外周血中AMA的阳性与否和疾病并无明显关联。     

Autoantibody IgG subclasses to recombinant antigens and the role of bacterial stimuli in primary biliary cirrhosis

Aim:  Serum antimitochondrial antibody (AMA) of the IgG2 and IgG3 subclasses has been reported to be predominant in patients with primary biliary cirrhosis from developed countries. No data are available as to the significance of AMA subtypes in Japanese primary biliary cirrhosis (PBC) patients who have previously manifested unique serological features, nor it is known whether AMA subclasses are influenced by bacterial stimuli, as suggested by the molecular theory of PBC. We undertook a three-step study to address these questions.
Methods:  First, Japanese PBC sera were tested using the established triple recombinant antigen (pML-MIT3) to find AMA subclass distribution. Second, we used the three recombinant mitochondrial antigens in PBC sera of Japanese and USA patients to explore the ethnic difference. Third, we used CpG oligodeoxynucleotides and a B cell mitogen to challenge ex vivo peripheral leukocytes from indirect immunofluorescence (IIF)-AMA-positive patients with Japanese PBC.
Results:  We detected most frequently IgG2-AMA followed by IgG3-AMA, with the latter being more common in IIF-AMA-positive cases, and demonstrated that the IgG3 reactivity against the dominant antigen was significantly higher in PBC sera from the USA. We determined that the bacterial stimulus was superior to the mitogen at inducing a predominant production of IgG2-AMA and CD20+ B cell activation.
Conclusion:  Our data cumulatively supported the hypothesis that IgG2 AMA subtypes are predominant in PBC and suggest that this might be favored by an innate immune reaction against bacterial particles, such as CpG DNA.     

原发性胆汁性肝硬化患者的免疫学特点分析

目的 分析原发性胆汁性肝硬化(PBC)出现的自身抗体等免疫学指标及其临床意义。 方法对3000例肝功能异常患者采用间接免疫荧光法检测抗核抗体(ANA)、抗线粒体抗体(AMA)、抗平滑肌抗体(SMA)和抗肝肾微粒体抗体(抗-LKM)等,并对ANA和AMA亚型及抗可溶性肝抗原/肝胰抗原(抗-SLA/LP)、LKM-1和抗肝特异性胞浆抗原型1抗体(抗-LC-1)等肝脏疾病相关的自身抗体进行了检测。结果 3000例肝病患者中,PBC 52例占1.7％。PBC患者的AMA和AMA-M2抗体均为阳性,52例PBC中,94.0％呈AMA高滴度(≥1:320)阳性,79.0％M2>200 RU/L,78.0％ANA阳性。ANA的主要荧光模式为细胞核膜型、细胞核点型和着丝点型。少见的荧光模式有抗干燥综合征A/B(SS-A/SS-B)、细胞核均质型、核仁型及颗粒型等。PBC患者免疫球蛋白M、碱性磷酸酶和γ-谷氨酰转肽酶高于乙型肝炎肝硬化患者;其白细胞介素(IL)-6、IL-10、肿瘤坏死因子α和干扰素γ水平高于正常人。5例表现为自身免疫性肝病重叠综合征,其中2例抗-SLA/LP阳性,提示PBC与自身免疫性肝炎(AIH)3型的重叠;1例抗-LKM-1阳性,提示PBC与AIH 2型的重叠;2例ANA阳性,且肝活体组织检查证实存在AIH和PBC的病理改变,提示为PBC与AIH 1型的重叠综合征。 结论 PBC在我国肝病患者中约占1％~2％。临床已出现典型症状者一     

The antimitochondrial antibody anti-M9: A marker for the diagnosis of early primary biliary cirrhosis

The clinical relevance of a new antimitochondrial antibody, anti-M9, reacting with an outer membrane-associated antigen on liver mitochondria is described. Sera from 22 anti-M2-negative patients with histologically proven primary biliary cirrhosis (PBC) who had been followed for 5-15 years were tested for anti-M9 in the ELISA using a purified M9-fraction. 18 (82%) were anti-M9-positive, and 17 of them (94%) were in stage I/II. None of the 17 anti-M9-positive/anti-M2-negative patients with early PBC progressed to stage III/IV during the observation period of 5-15 years, and in all instances anti-M9 remained of the IgM-type. In one anti-M9-positive patient anti-M2 of the IgM type appeared 2 years after the first demonstration of anti-M9. Among 156 patients with anti-M2-positive PBC, 58 (37%) had anti-M9, and 39 of them (67%) were in stage I/II. 19 of these 39 stage I/II patients (49%) had anti-M9 exclusively of the IgM-type in contrast to none of the 19 stage III/IV patients. Using the purified M9-fraction in ELISA and Western blotting, anti-M9 antibodies were confined only to patients with PBC or overlap syndromes between PBC and autoimmune chronic active hepatitis (10% of 133 patients) and were not found in patients with other hepatic and non-hepatic disorders. We conclude that the determination of anti-M9 may be helpful for the diagnosis of early and asymptomatic PBC. From follow-up studies of anti-M9-positive but anti-M2-negative patients it emerges that this antibody type may be associated with a benign course of PBC.     

Antimitochondrial autoantibodies in saliva and sera from patients with primary biliary cirrhosis

Background and Aims: Primary biliary cirrhosis (PBC) is a cholestatic autoimmune liver disease characterized by antimitochondrial autoantibodies (AMA) in serum, for which the reactants are E2 subunits of the three 2‐oxoacid dehydrogenase (2‐OAD) enzymes, particularly pyruvate dehydrogenase complex (PDC‐E2). Some 70% of patients with PBC have a coexisting autoimmune disease including Sjögren's syndrome. We aimed to ascertain the frequency and isotype of AMA in saliva in PBC. Methods: Serum and saliva from 12 patients with PBC were tested for AMA by immunoblotting on bovine heart mitochondria, and by an automated microassay based on inhibition of the enzymatic activity of PDC. Results: Autoantibodies of the immunoglobulin (Ig)G, IgM, and IgA immunoglobulin isotypes against the E2 subunits of 2‐OAD enzymes were demonstrable in PBC in serum (12 of 12 cases) and saliva (nine of 12 cases). Salivary autoantibodies, like serum autoantibodies, were predominantly reactive with PDC and of the IgG isotype. Results for serum and saliva corresponded closely with regard to reactivity with individual enzymes of the 2‐OAD enzyme family, and to the autoantibody isotype that was predominantly expressed, and also in the capacity to inhibit the enzymatic activity of PDC. Conclusions: The presence of AMA in saliva to 2‐OAD enzymes indicates that salivary glands could participate in the pathogenetic process of PBC. The detection of salivary AMA by a semi‐automated enzyme inhibition assay offers possibilities for rapid population screening for detection of preclinical PBC among at‐risk individuals, middle‐aged to older women.     

Clinicopathological study of primary biliary cirrhosis negative for antimitochondrial antibodies

Abstract: Primary biliary cirrhosis (PBC) is characterized by the occurrence of antimitochondrial antibodies (AMA) and the progressive destruction of intrahepatic bile ducts, followed by biliary cirrhosis. However, there are about 5% of PBC patients who show clinicopathological features of PBC but are negative for AMA. In this study, clinicopathological features, as well as antibody reactivity against recombinant (r)-mitochondrial polypeptides, were examined in 30 AMA negative PBC patients and 38 AMA positive PBC patients, in whom the presence of AMA had been determined by indirect immunofluorescence (IF). There were few differences in the clinical and serological features between both groups. Histopathologic features, including staging, bile duct lesions and granuloma, were also similar in both groups. Among the 30 IF-tested AMA negative patients, 29 were also negative against beef heart mitochondrial proteins, but 24 reacted to one or more of the following r-polypeptides, as determined by immunoblotting: E1 alpha of pyruvate dehydrogenase complex, the E2 subunit of pyruvate dehydrogenase complex, and the branched-chain 2-oxo-acid dehydrogenase complex. The remaining six AMA-negative patients were asymptomatic, and histologically resembled having stage 1 of the disease, with relatively mild lymphocytic piecemeal necrosis. One case was positive for anti-smooth muscle antibody. The other clinicopathological features of these patients were similar to those of other AMA negative patients. The present study found that a majority of the AMA-negative patients fulfilling other clinicopathological criteria of PBC, had features similar to the AMA-positive PBC patients, and that a majority of IF AMA-negative patients were positive for r-polypeptides of the 2-oxo-acid dehydrogenase complex. It seems that nearly all the AMA negative patients possess a broad spectrum of antibody profile of AMA, in addition to clinicopathological and serological features.     

Immunoreactivity to M2 proteins in antimitochondrial antibody-negative patients with primary biliary cirrhosis

Abstract Although antimitochondrial auto-antibodies are characteristically present in the serum of patients with primary biliary cirrhosis (PBC), there is a discrepancy between the positivity for antimitochondrial antibody (AMA) and that for anti-M2 auto-antibody. In an attempt to explain the discrepancy, this study investigates the relationship between the AMA titre, determined by indirect immunofluorescence, and immunoreactivity to four inner mitochondrial membrance proteins (M2 proteins) with molecular weights of 70, 50, 47, and 40 kDa in 129 patients with PBC. Antimitochondrial antibody positivity was identified in 114 (88%) of 129 patients with clinically and histologically confirmed PBC. There were no significant differences between the AMA-negative and AMA-positive groups in clinical characteristics or histologically determined disease stage. Immunoblot analysis showed that all patients had anti-M2 auto-antibodies to one or more of the four M2 proteins. Nine (60%) of the 15 AMA-negative patients had antibodies to only one M2 protein (either 70 or 47 kDa). In contrast, 34 (53%) of the 64 patients with high AMA titres ( 1: 320) had antibodies to all four M2 proteins. There was a significant rank correlation between the AMA titre and the number of antibodies to M2 proteins ( P < 0.01). These findings indicate that the AMA titre is not influenced by the immunogenicity of M2 protein but by the number of M2 proteins that elicit an antibody response and that decreased immunoreactivity to M2 proteins may induce AMA negativity in PBC serum samples.     

High prevalence of primary biliary cirrhosis and disease-associated autoantibodies in Japanese patients with systemic sclerosis

Objective

To investigate the prevalence of primary biliary cirrhosis (PBC) and PBC-associated autoantibodies in Japanese systemic sclerosis (SSc) patients.

Methods

Clinical data from 225 Japanese SSc patients were retrospectively obtained. Serum samples from these patients were examined for PBC-associated autoantibodies, anti-mitochondrial M2 antibodies (AMA), anti-sp100 antibodies (anti-sp100), and anti-gp210 antibodies (anti-gp210) by enzyme-linked immunosorbent assay.

Results

Of 225 patients, 37 (16.4%) had AMA, 13 (5.8%) had anti-sp100, and 3 (1.3%) had anti-gp210. Three patients were positive for both AMA and anti-sp100, and 2 were positive for both AMA and anti-gp210. PBC was found in 22 (9.8%) patients positive for AMA with or without anti-sp100 or anti-gp210, but not in those with anti-sp100 or anti-gp210 without AMA. Furthermore, 13 patients lacking these three antibodies were diagnosed with or suspected of PBC by liver biopsy and/or their clinical manifestation. Multivariable analysis revealed that AMA and anti-centromere antibodies were independently associated with PBC in SSc patients, while anti-sp100 and anti-gp210 were not.

Conclusions

This study has demonstrated even higher prevalence of both PBC-associated autoantibodies and PBC in the Japanese SSc population than in the Caucasian SSc population. AMA and anti-centromere antibodies are likely to indicate increasing risk of PBC in SSc patients.     

原发性胆汁性肝硬化患者自身抗体测定的意义

目的:探讨自身抗体测定对诊断原发性胆汁性肝硬化(PBC)的临床意义.方法:PBC患者52例和非PBC患者202例,其中包括自身免疫性肝炎(AIH)41例,原发性硬化型胆管炎(PSC)18例,乙型肝炎(HBV)89例,丙型肝炎(HCV)54例以及健康体检者40例,采用间接免疫荧光法(IIF)检测抗核抗体(ANA)、抗平滑肌抗体(SMA)、抗线粒体抗体(AMA)、抗心肌抗体(HRA)、抗骨骼肌抗体(ASA)、抗胃壁细胞抗体(PCA)、抗双链DNA(ds-DNA)抗体、抗肝肾微粒体抗体(LKM)、抗可溶性肝抗原(SLA)抗体和抗中性粒细胞胞质抗体(ANCA)等自身抗体,ELISA法检测抗髓过氧化酶抗体(MPO),并对其结果进行回顾性分析.结果:PBC患者中AMA阳性例数最高为46例(88.5%),ANA阳性率为71.2%(37/52);非PBC患者中阳性例数则分别为20例(9.9%)和51例(25.2%),两组比较,有非常显著性意义(P<0.01).AMA、SMA抗体检测在PBC与AIH患者中,均有非常显著性意义(P<0.01).但两组之间的ANA阳性率无显著性意义(P>0.05).PSC患者18例ANA阳性6例,AMA阳性7例均低于PBC患者.HBV,HCV感染患者检测ANA阳性率分别只有9.0%和22.2%;AMA阳性率也只有7.9%和3.7%,与PBC患者比较均有显著性差异(P<0.01).PBC患者及对照组检测ds-DNA,ANCA,LKM,SLA和MPO抗体结果显示PBC患者检测最高的ANCA阳性率为26.9%(14/52),其次是MPO阳性率为25.0%(13/52),与AIH比较,均有非常显著性意义(P<0.01).结论:血清自身抗体的检测对诊断、治疗和阻止原发性胆汁性肝硬化的发展有着十分重要作用.对提高PBC同其他疾病鉴别诊断和治疗有着非常重要的意义.     

Antimitochondrial antibodies in acute liver failure: implications for primary biliary cirrhosis

In our previous work, including analysis of more than 10,000 sera from control patients and patients with a variety of liver diseases, we have demonstrated that with the use of recombinant autoantigens, antimitochondrial autoantibodies (AMAs) are only found in primary biliary cirrhosis (PBC) and that a positive AMA is virtually pathognomonic of either PBC or future development of PBC. Although the mechanisms leading to the generation of AMA are enigmatic, we have postulated that xenobiotic-induced and/or oxidative modification of mitochondrial autoantigens is a critical step leading to loss of tolerance. This thesis suggests that a severe liver oxidant injury would lead to AMA production. We analyzed 217 serum samples from 69 patients with acute liver failure (ALF) collected up to 24 months post-ALF, compared with controls, for titer and reactivity with the E2 subunits of pyruvate dehydrogenase, branched chain 2-oxo-acid dehydrogenase, and 2-oxo-glutarate dehydrogenase. AMAs were detected in 28/69 (40.6%) ALF patients with reactivity found against all of the major mitochondrial autoantigens. In addition, and as further controls, sera were analyzed for autoantibodies to gp210, Sp100, centromere, chromatin, soluble liver antigen, tissue transglutaminase, and deaminated gliadin peptides; the most frequently detected nonmitochondrial autoantibody was against tissue transglutaminase (57.1% of ALF patients). CONCLUSION: The strikingly high frequency of AMAs in ALF supports the thesis that oxidative stress-induced liver damage may lead to AMA induction. The rapid disappearance of AMAs in these patients provides further support for the contention that PBC pathogenesis requires additional factors, including genetic susceptibility.     

Antimitochondrial antibodies and reactivity to N. aromaticivorans proteins in Icelandic patients with primary biliary cirrhosis and their relatives

OBJECTIVES: Primary biliary cirrhosis (PBC) is a chronic, progressive cholestatic disease of unknown etiology characterized by serum antimitochondrial antibodies (AMA) directed against a functionally related family of mitochondrial enzymes. We recently suggested that N. aromaticivorans might be the trigger of autoimmunity in PBC. No data are available on the specificity and crossreactivity of AMA in a genetically homogenous group of patients, such as the Icelandic population. METHODS: To address these issues and to confirm previous findings in a unique population, we obtained sera from 14 PBC patients and 85 first-degree relatives, all of Icelandic descent. We analyzed such sera for AMA specificity using recombinant mitochondrial antigens and for reactivity against N. aromaticivorans proteins. RESULTS: Thirteen of the 14 Icelandic patients with PBC (93%) were found AMA positive. We found that 5/13 AMA positive sera (38%) reacted against PDC-E2 only; 5/13 (or 38%) reacted against BCOADC-E2; and 2/13 (15%) reacted against all three antigens. There was no reactivity against OGDC-E2. Reactivities of patients' sera against N. aromaticivorans were consistent with the AMA status. One serum among the 85 first-degree relatives (1.2%) was found to be AMA-positive, as well as reactive against N. aromaticivorans. CONCLUSIONS: Interestingly, despite the homogenous genetic background, the group of Icelandic patients with PBC was heterogeneous in their AMA reactive patterns and also reacted with N. aromaticivorans proteins.     

Demonstration of PDC-E1 subunits as major antigens in the complement-fixing fraction M4 and re-evaluation of PDC-E1-specific antibodies in PBC patients.

BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. PATIENTS AND METHODS: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). RESULTS: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CONCLUSIONS: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.     

原发性胆汁性肝硬化免疫学指标检测的临床意义

为研究原发性胆汁性肝硬化(PBC)免疫学指标测定的临床意义,选取32例确诊PBC的住院患者,采用ELISA法检测患者血清中的抗线粒体抗体M2亚型(AMA-M2);间接免疫荧光法检测抗线粒体抗体(AMA),抗核抗体(ANA),抗平滑肌抗体(SMA);斑点法检测抗肝肾微粒体抗体(LKM-1),抗肝溶质Ⅰ型抗原抗体(LC-1),抗可溶性肝抗原/肝胰抗原抗体(SLA/LP);免疫比浊法检测免疫球蛋白(IgG,IgA,IgM)和类风湿因子(RF)。同时观察AMA-M2与生化肝功指标的关系,比较PBC患者治疗前后AMA-M2的变化。结果显示诊断PBC意义最大的是AMA-M2,其阳性率最高达94%,其次是AMA和ANA,阳性率分别为88%和86%;AMA-M2值的高低与血清转氨酶、碱性磷酸酶和γ-谷氨酰转肽酶无关,熊去氧胆酸治疗前后AMA-M2变化不大。     

Mucosal immunity and primary biliary cirrhosis: presence of antimitochondrial antibodies in urine

We have shown that IgA-class antimitochondrial autoantibodies (AMA) can be detected in the bile and saliva of patients with PBC, suggesting that AMA are secreted into the luminal fluid across bile ducts and salivary glands. These data prompted us to determine whether AMA of the IgA isotype may be transported across other epithelial mucosa. Therefore, we tested for the presence of AMA in the urine specimens of 83 patients with PBC and 58 non-PBC controls including healthy individuals and patients with other liver diseases. Patients enrolled in this study had no history of renal disease, and we confirmed there was less than 50 microgram/mL of protein in each of the urine specimens. Interestingly, we found that AMA were present in the urine of 71/83 (86%) of all patients with PBC and in 71/78 (91%) of patients with PBC that were serum AMA positive. In contrast, AMA were not detected in any of the 58 control urine specimens. Of particular interest, AMA of the IgA isotype was present in 57/83 (69%) of patients with PBC, and in 52 of these 57, we found secretory-type IgA. In a nested random subgroup of urine samples, the prevalence of the IgA2 AMA was 6/18 (33%), significantly lower than in matched serum samples, 13/16 (81%, P =.007). These data show that AMA of the IgA isotype is secreted into urine from the uroepithelium of patients with PBC, and support the thesis that PBC originated from either a mucosal challenge or a loss of mucosal tolerance.     

Identification and analysis of the major M2 autoantigens in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.     

Immunology of primary biliary cirrhosis

(1) The serological diagnosis of PBC is possible in almost 100% of cases when appropriate methods and specific antigen preparations are used such as the purified ATPase fraction by ELISA for the detection of anti-M2, sonicated mitochondria by immunodiffusion for the demonstration of precipitating antibodies against M-A or M-B, and cell cultures by immunofluorescence for the detection of antibodies against nuclear dots. (2) The establishment of AMA profiles obtained by ELISA and CFT seems to be a sensitive approach to a better definition of the natural course of PBC. A distinction between a rather benign and a more progressive course seems especially possible in the presence of the AMA profiles A and B (anti-M9 and/or anti-M2-positive only by ELISA) versus D (anti-M2-, anti-M4-, anti-M8-positive in the CFT). (3) The analysis of cellular immune reactions in vitro and in vivo suggests an activation of cytotoxic T cells as well as a defect in the function of T suppressor cells. (4) Although the aetiology of PBC is unknown, the detection of MHC Class II antigens on bile duct epithelial cells in liver biopsies of patients with PBC but not of normal individuals may imply that an infectious agent being exposed in association with these MHC structures may trigger the disease. The inability of the immune system in controlling this infectious process would then lead to an ongoing inflammatory reaction which is responsible for the continuous destruction of bile ducts within portal triads.     

Evidence for a locally driven mucosal response and the presence of mitochondrial antigens in saliva in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is often considered to be a dry gland disease caused by frequent involvement of salivary and lacrimal glands. Although high titers of antimitochondrial autoantibodies (AMA) have long been recognized in PBC, little is known about the presence of mitochondrial autoantigens in mucosal compartments such as saliva. We investigated saliva and sera in PBC patients and controls for the presence of AMA and mitochondrial antigens. In PBC saliva, AMA were detected in 45 of 49 (92%), with specificity directed against pyruvate dehydrogenase complex (PDC-E2) alone in 22 of 49 (45%), against PDC-E2 and branched-chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) in 4 of 49 (8%), to PDC-E2 and 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) in 9 of 49 (18%), and to the 3 antigens together in 10 of 49 (20%). Isotyping of the saliva AMA showed that 80% of the patients had immunoglobulin A (IgA) against PDC-E2, 18% had IgM-specific PDC-E2, and 35% had IgG specific PDC-E2. Similar to serum and bile anti-PDC-E2 IgA antibodies, the saliva autoantibodies localized their reactivity to the inner lipoyl domain of PDC-E2. Furthermore, saliva from patients with PBC but not controls inhibited pyruvate dehydrogenase enzyme activity in vitro. In addition, and of particular interest, we detected a molecule with a molecular weight corresponding to PDC-E2 (74 kd) in PBC but not control saliva. These findings make several important points: first, there appears to be localized mucosal immunity in the secretory system of PBC; second, AMA are readily detected in PBC saliva; and third, PDC-E2 may be present in the saliva of PBC.     

High sensitivity of a novel ELISA for anti-M2 in primary biliary cirrhosis

The use of an ELISA for the detection of anti-M2, a specific autoantibody in primary biliary cirrhosis (PBC), has been common in Japan. However, there are some problems in the sensitivity of this ELISA, especially in PBC patients showing antimitochondrial antibody (AMA)-negative sera or low AMA titers by immunofluorescence. Recently, a new ELISA for anti-M2 was developed, using porcine heart mitochondrial protein as the antigen. We report here comparative studies of the new and the former anti-M2 ELISAs. Porcine heart mitochondrial protein was prepared and used as the antigen for the new ELISA for anti-M2. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this protein showed three major M2 antigen proteins. As the second antibody, peroxidase-conjugated anti-human mouse monoclonal IgM, in addition to monoclonal IgG, was included. The sera of 171 PBC patients were examined. As controls, we examined the sera of 167 non-PBC patients and the sera of 115 normal controls. The cut-off index was set at 10 U/ml, based on the results for the normal controls. No sera from the non-PBC patients or the normal controls were positive for anti-M2 by either the new or the former ELISA. However, the positivity rate for anti-M2 in PBC patients with the new ELISA was 78%; in contrast, that with the former ELISA was only 54%; this difference was significant (P = 0.00001). In particular, in 65 patients showing AMA titers of 1 : 20 or less, the positivity rate with the new ELISA was 51%; in contrast, that with the former ELISA was only 17%. As the sensitivity of the new ELISA is significantly higher than that of the former ELISA, especially for sera from patients showing AMA-negativity or low titers of AMA, the new ELISA is considered to be more effective than the former ELISA for use in anti-M2 screening assays in patients with PBC. Received: May 17, 2000 / Accepted: September 1, 2000     

No prognostic significance of antimitochondrial antibody profile testing in primary biliary cirrhosis

BACKGROUND/AIMS: The rate of disease progression varies considerably between individuals with primary biliary cirrhosis (PBC). On the basis of serological subtyping 4 antimitochondrial antibody (AMA) profiles (A, B, C and D) can be defined. The finding of previous studies that profile C/D is associated with a progressive course, in contrast to profile A/B, is a question of debate. The aim of the study was to investigate whether AMA profiles predicted the course for a cohort of Dutch PBC patients. METHODOLOGY: Patients with an established diagnosis of AMA-positive PBC, AMA-negative PBC patients, non-PBC decompensated cirrhotics and healthy volunteers. Serum samples from 38 AMA-positive progressive patients, 31 AMA-positive patients without evidence of progression for at least 6 years, 5 AMA-negative PBC patients, 5 non-PBC decompensated cirrhotics and 5 healthy volunteers were assessed. AMA profiles were determined without knowledge of the clinical data. RESULTS: In the progressive AMA-positive group, 13% had profile A/B and 84% had profile C/D. In the non-progressive group, 13% had profile A/B, 77% profile C/D; 10% had no profile. During follow-up, a change from profile A/B to profile C/D or vice versa was not observed. CONCLUSIONS: This study found that not only PBC patients with AMA profile C/D but also patients with profile A/B may run a progressive course and therefore does not support the suggestion that AMA profiles can be used as independent prognostic indicator. The divergent results of this and previous studies may be explained by the selection of different patient populations.