Nuclear membrane-staining antinuclear antibody in patients with primary biliary cirrhosis

An antinuclear antibody specific for nuclear membrane (ANMA) was observed by the immunofluorescence method in sera from patients with primary biliary cirrhosis (PBC). ANMA was present in 18 of 63 PBC sera (28.5) and in 1 of 431 control sera (0.2%). Its reaction appeared as a thin fluorescent ring confined to the nuclear envelope and was more evident when the sera were highly diluted and the fluorescence, due to frequently associated antimitochondrial antibody, faded. The ANMA fluorescent pattern was confirmed by indirect immunoperoxidase staining. ANMA was seen on both tissue cryostat sections and HEp-2 cells. It was a poorly or non-complement-fixing IgG, specific for an antigen resistant to DNase I, RNase, and trypsin. The significance of its presence in PBC in unknown at present. Identification of its antigen with one of the centromeric antigens is suggested.     

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase.

Anti-M9 antibodies in sera from patients with primary biliary cirrhosis (PBC) were previously found to recognize two antigenic determinants at 98 and 59 kD, using a purified antigen fraction derived from rat liver mitochondria in the Western blot. Here we show that these antibodies are directed against an epitope of the enzyme glycogen phosphorylase. By Western blotting, a determinant at 98 kD was obtained testing anti-M9 positive sera against phosphorylase from skeletal muscle, and after plasmin treatment a degradation product appeared at 59 kD. Both determinants were identical to the M9-specific determinants 98 and 59 kD as shown by absorption studies. When these antibodies were eluted from the 98 and 59 kD determinants of the M9 antigen after immunoblotting, they again recognized the same epitopes on plasmin-treated phosphorylase. Furthermore, phosphorylase enzyme activity could be also demonstrated in the purified M9 fraction, and anti-M9-positive/anti-M2-negative but not anti-M9-negative/anti-M2-positive sera could be shown to stimulate phosphorylase activity. Testing sera from 1189 patients with different hepatic and non-hepatic disorders against M9 and phosphorylase from skeletal muscle by ELISA, 20% were positive with phosphorylase and only 2% with the M9 fraction. These data indicate that the commercially available phosphorylase from skeletal muscle cannot be recommended as M9 source. It may still contain non-PBC-specific epitopes which are probably recognized by naturally occurring antibodies directed against this highly conserved protein.     

Antibody to carbonic anhydrase II is present in primary biliary cirrhosis (PBC) irrespective of antimitochondrial antibody status

Antibody to carbonic anhydrase II, an enzyme abundantly present in biliary epithelium, has been proposed as a diagnostic marker for antimitochondrial antibody-negative PBC. In this study we determine its prevalence and clinical significance in a large series of patients with antimitochondrial antibody-positive and -negative PBC. Reactivity to carbonic anhydrase II was sought by Western immunoblotting in sera from 215 consecutive patients with PBC (26 antimitochondrial antibody-negative), 13 with autoimmune hepatitis, 25 with primary Sjögren's syndrome (pSS), 12 with systemic sclerosis, 19 with systemic lupus erythematosus and 73 healthy subjects. The prevalence of antibody to carbonic anhydrase II (titre 1:100) in PBC was 8%. No specific reactivity to carbonic anhydrase II was found in antimitochondrial antibody-negative PBC (7% versus 8% in antimitochondrial antibody-positive PBC). Ascites (P = 0.006) and Sjögren's syndrome (SS) (P = 0.022) in PBC were significantly associated with presence of the antibody. In patients with SS associated with PBC, the prevalence (19%) was similar to that observed in pSS (16%). At a serum dilution of 1:40, the prevalence of positive sera in PBC rose to 27% but disease specificity was reduced. Our findings in a large population of PBC patients rule out a relation between presence of antibody to carbonic anhydrase II and lack of antimitochondrial antibody. The higher prevalence of ascites found in positive patients warrants further evaluation.     

Diagnosis and management of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized histologically by destruction of intrahepatic bile ducts and serologically by the presence of antimitochondrial antibodies. The incidence and prevalence of PBC are increasing. Fatigue and pruritus are common symptoms in PBC, although the proportion of asymptomatic PBC is increasing due to the widespread use of screening biochemical tests and antimitochondrial antibody assays. PBC may eventually lead to cirrhosis and its consequent complications. In the 1980s, PBC was the leading indication for liver transplantation. Ursodeoxycholic acid is the only US FDA-approved therapeutic agent for PBC. Clinical trials have shown that the use of ursodeoxycholic acid in PBC results in reduction of liver biochemistries, a delay in histological progression, a delay in the development of varices and improvement in survival without liver transplantation.     

原发性胆汁肝硬化患者血清中抗线粒体抗体及其亚型和抗核抗体的联合检测的临床价值

为了分析原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者的抗线粒体抗体(anti-mitochondria antibody,AMA)及其M2、M4、M9亚型、抗核抗体(antinuclear antibody,ANA)的阳性率。应用间接免疫荧光法检测ANA、AMA,免疫斑点法检测AMA M2、M4、M9。结果显示91例PBC患者中AMA有89例为阳性。其中96.7%(88/91)的患者M2型阳性,45.1%(41/91)M4型阳性,2.2%(2/91)M9型阳性。39/91例患者ANA阳性,其中21例为核膜型。ANA、AMA及其M2、M4、M9亚型联合检测对于PBC患者的诊断有重要价值。     

Analysis of mitochondrial antigens reveals inner membrane succinate dehydrogenase flavoprotein subunit as autoantigen to antibodies in anti-M7 sera

The role of mitochondrial proteins as antigens to antibodies of anti-M7 sera was analysed by flavin fluorescence, one- and two-dimensional Western blots and blue native gel electrophoresis. Flavin fluorescence of succinate dehydrogenase (SucDH, complex II of the respiratory chain) of rat liver inner mitochondrial membranes correlated with the immunoreactivity of a representative anti-M7 myocarditis serum. Antigens of isolated bovine heart mitochondria reacting with antibodies of myocarditis serum on two-dimensional Western blots were identified by MALDI-TOF and NanoESI mass spectrometry as myosin heavy chain beta and as dihydrolipoamide dehydrogenase of the mitochondrial 2-oxoacid dehydrogenase complexes. The SucDH-flavoprotein was not resolved as a discrete protein spot on two-dimensional polyacrylamide gels. However, separation of the rat liver inner mitochondrial membrane complexes by blue native gel electrophoresis followed by Western blotting, and Western blots of purified Escherichia coli SucDH complex revealed that anti-M7 sera contained antibodies directed against the SucDH-flavoprotein subunit.     

Reduction in serum levels of antimitochondrial (M2) antibodies following immunoglobulin therapy in severe combined immunodeficient (SCID) mice reconstituted with lymphocytes from patients with primary biliary cirrhosis (PBC)

The effect of gammaglobulin treatment on autoantibody production was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells (PBMC) obtained from patients with PBC. All reconstituted mice displayed the presence of human antimitochondrial antibodies (αM2Ab) of both IgG and IgM types before treatment with human immunoglobulin. Two weeks after i.p. injection of 20 ×10⁶ PBMC into SCID mice, i.p. treatment with various preparations of human immunoglobulin was initiated. In control animals treated with saline, serum levels of human αM2Ab of the IgG type increased with time, peaking around 4 weeks after reconstitution. In contrast, human IgG autoantibodies rapidly decreased in all animals treated with human IgG. Treatment with a human IgM preparation had no effect on serum levels of αM2Ab of the IgG type. The results may suggest that the pronounced reduction of specific IgG autoantibodies was due to an increased catabolism of human IgG, including the autoantibodies, in the gammaglobulin-treated mice. Although the production of human αM2Ab in reconstituted mice could be easily shown, PBC-specific liver lesions or bile duct destruction were not observed, irrespective of treatment protocol.     

IgA class antibodies to 2-oxo-acid dehydrogenase complex are not predictive markers of histopathological progression in primary biliary cirrhosis

Although antimitochondrial antibody (AMA) is the characteristic serological feature of primary biliary cirrhosis (PBC), its pathogenic role remains unclear. In our previous study, we reported a positive correlation between immunoglobulin (Ig) A class anti-2-oxo-acid dehydrogenase complex (2-OADC) and histopathological stage. To determine whether the appearance of IgA class anti-2-OADC by immunoblotting represents an early marker of more aggressive disease or whether it is late finding during the disease course of PBC, we tested not only the entire IgA class but also IgA1, IgA2 and secretory IgA class anti-2-OADC in serial serum samples from 15 patients with PBC. During the median observation period of 51 months, four cases showed histopathological progression (from stage 1 to 2, stage 1 to 3, stage 1 to 4 and stage 2 to 4). There was no statistically significant correlation between the above IgA class anti-2-OADCs and histopathological progression. There was no significant correlation between histopathological stages and IgA2 class anti-2-OADC or secretory IgA class anti-2-OADC by immunoblotting. IgA class anti-2-OADC was more frequent in stages 3–4 than in stages 1–2 (p = 0.0049), but IgA1 class anti-2-OADC was more frequent in stages 1–2 than in stages 3–4 (p = 0.0232). Our present study demonstrated that serum IgA class 2-OADC was not a predictive marker of histopathological progression but was associated with the histopathological stage of PBC. Although the IgA class AMA may have a specific pathogenic role for PBC, the discrepant results between IgA and IgA1 class anti-2-OADC should be further assessed to investigate different functional activities depending on their molecular form.     

Further purification of the mitochondrial inner membrane autoantigen reacting with primary biliary cirrhosis sera

The mitochondrial inner membrane autoantigen reacting with sera from patients with primary biliary cirrhosis depends on lipid and protein moieties for complement-fixing activity, but its chemical analysis requires some degree of solubilization. Attemps to achieve this by citraconylation led to anticomplementary effects and inactivation, but treatment with 8 M urea fragmented the membranes sufficiently to allow gel filtration and estimation of its mol. wt at 180,000–200,000. The antigen was further purified by affinity chromatography using Igs from patients with anti-mitochondrial antibodies (AMA) coupled to Sepharose 4B, as immunosorbent. The 8 M urea eluate was about 100 times more active than crude inner membranes and showed a single band on polyacrylamide electrophoresis. Liver and brown fat gave the same band, brown fat having four times the potency of liver. Electron microscopy of the purified antigen from the two organs showed that it reaggregated into membranous vesicles when urea was removed. The purified antigen may be of use if an automated radioimmunoassay were to prove sensitive and specific for the detection of AMA as this antibody is an important marker for `autoimmune'' chronic liver disorders.     

Isotypic distribution of anti-pyruvate dehydrogenase antibodies in patients with primary biliary cirrhosis and their family members

IgG subclasses of anti-pyruvate dehydrogenase (PDH) antibodies were determined in 72 patients with primary biliary cirrhosis. All isotypes were detected, but IgG3, IgG1, and IgG2 predominated independently or in association. An average of 33.3±19.1% of the anti-PDH IgG was IgG1 (mean optical density, 0.863±0.783, vs 0.053±0.038 in the normal controls), 25.0±17.8 IgG2 (0.652±0.656 vs 0.062±0.030), 39.5±23.4% IgG3 (1.140±0.917 vs 0.010±0.023), and 2.4±7.4% IgG4 (0.060±0.182 vs 0.012±0.007). Anti-PDH IgG were restricted to IgG1 in the family members of patients (0.180±0.403).     

Spontaneous occurrence of chronic non-suppurative destructive cholangitis and antimitochondrial autoantibodies in MRL/lpr mice: Possible animal model for primary biliary cirrhosis

MRL/MP mice bearing the lymphoproliferative gene lpr (known as MRL/MP‐lpr/lpr or MRL/Ipr mice) are known to spontaneously develop severe autoimmune diseases such as glomerulonephritis, arteritis and arthritis at an early stage of their life. They have also been reported to develop severe sialadenitis, suggesting that this mouse could be a model for Sjögren’s syndrome. Primary biliary cirrhosis, an autoimmune disease characterized by chronic non‐suppurative destructive cholangitis and the occurrence of antimitochondrial antibodies, is frequently associated with Sjögren’s syndrome. In this study, we examined whether cholangitis and/or antimitochondrial antibodies occur in this mouse model, using more than 100 young and old MRL/Ipr mice. We frequently found portal inflammation associated with cholangitis of small intrahepatic bile ducts, especially in older mice. There was also infiltration of inflammatory cells (monocytes) as well as CD4‐positive T cells. Epithelioid granuloma and bile‐duct loss were also occasionally found. These histological features resemble primary biliary cirrhosis. In addition, antimitochondrial antibodies were shown by immunocytochemistry to be present in the sera of MRL/Ipr mice. There is currently no established animal model for primary biliary cirrhosis. Therefore, further studies on MRL/Ipr mice, with respect to pathogenesis of primary biliary cirrhosis, are warranted.     

四聚体技术检测原发性胆汁性肝硬化患者血循环中抗原特异性T细胞

目的定量检测原发性胆汁性肝硬化(PBC)患者体内抗原特异性T淋巴细胞含量,探讨其在PBC发病机制中的作用。方法采用四聚体技术检测15例HLA-A0201阳性(A2 )PBC患者外周血单个核细胞(PBMC)经抗原肽诱导生长的细胞毒性T淋巴细胞(CTL)中PDC-E2159~167aa与PDC-E2165~174aa特异性CD8 T细胞频率,以A0201阴性(A2-)PBC患者与A2 的其他慢性肝病和健康自愿者为对照组。结果在A2 PBC患者PDC-E2159~167aa与PDC-E2165~174aa诱导的CTL中可检测到其相应的四聚体/CD8 细胞,平均频率分别为0.42%±0.24%(0.17%~1.08%)、0.27%±0.17%(0.05%~0.56%),各对照组的四聚体阳性细胞频率均低于0.1%,差异非常显著(P<0.001);A2 PBC组中PDC-E2159~167aa特异性的CTL频率与PDC-E2165~174aa特异性的CTL无显著性差异(P>0.05)。处于临床Ⅰ、Ⅱ期的A2 PBC患者中CD8 特异性CTL频率均较Ⅲ期的要高(P<0.05)。PDC-E2159~167aa特异性CTL与PDC-E2165~174aa特异性CTL频率在抗-PDC阳性PBC组和抗-PDC阴性组之间均无显著性差异(P>0.05)。结论HLA-A0201限制性的PDC-E2159~167aa和PDC-E2165~174aa特异性CD8 CTL在PBC疾病进展中起重要作用,抗线粒体抗体阴性或抗-PDC阴性PBC患者与阳性患者可能有着相似的T细胞介导的免疫发病机制。     

新型的抗线粒体抗体M2亚型IgG和IgA抗体的检测方法在原发性胆汁性肝硬化诊断中的价值

目的: 评价以新型天然M2抗原和BPO融合蛋白M2-3E(BPO)为靶抗原的 ELISA法(抗-M2-3E ELISA)检测抗线粒体抗体M2亚型(AMA-M2)IgG和IgA抗体在原发性胆汁化肝硬化(PBC)诊断中的敏感性、特异性。 方法: 分别用间接免疫荧光法(IFL)、以丙酮酸脱氢酶复合体(PDC)为靶抗原的ELISA法(抗- PDC ELISA)、抗-M2-3E ELISA法检测107例PBC、31例自身免疫肝炎(AIH)、10例原发性硬化性胆管炎(PSC)、17例丙型病毒性肝炎(HCV)、29例乙型病毒性肝炎(HBV)患者和26例健康体检者。 结果: (1)107例PBC患者用IFL、抗-PDC ELISA和抗-M2-3E ELISA 3种方法检测AMA-M2 IgG的检出率分别为:87/107(81.3%)、78/107(72.9%)、97/107(90.6%)。特异性分别为98.1%、97.2%、98.3%。抗-M2-3E ELISA法AMA-M2 IgG的检出率(90.6%)显著高于IFL法(81.3%)和抗-PDC ELISA法(72.9%)(均P<0.01)。用抗-M2-3E ELISA法检测AMA-M2 IgA的检出率为59/107(55.1%),特异性为97.2%。而总体AMA-M2 IgG和/或IgA的检出率99/107(92.5%),特异性为95.2%。(2)IFL 和 抗-M2-3E ELISA的重叠度为85%,抗-M2-3E ELISA可以在超过一半的IFL阴性的患者中检出AMA-M2 IgG和/或IgA。 结论: 抗-M2-3E ELISA法具有比IFL和抗-PDC ELISA法更高的敏感性、特异性。但是,AMA-M2 IgG和IgA都不可以单独用于诊断PBC的标记。     

Immunoglobulin-containing cells in liver biopsies of patients with chronic hepatitis and primary biliary cirrhosis

The numbers of IgA, IgM and IgG-containing cells were studied by means of an indirect immunoperoxidase technique and morphometry in liver biopsies of patients with primary biliary cirrhosis and chronic hepatitis, in whom serum immunoglobulin concentrations were also determined. In patients with primary biliary cirrhosis the absolute and relative number of IgM-containing cells in the liver was significantly higher, whereas the absolute and relative number of IgG-containing cells in the liver was significantly lower compared to patients with chronic hepatitis. IgM-containing cells in liver biopsies of patients with primary biliary cirrhosis correlated strongly with their serum IgM levels. It is concluded that determination of the pattern of immunoglobulin containing cells in liver biopsies may help in the differentiation of primary biliary cirrhosis from chronic hepatitis and that local production of IgM in the liver may contribute significantly to the high serum IgM levels in patients with primary biliary cirrhosis.     

Preciitating antibodies to mitochondrial antigens in patients with primary biliary cirrhosis.

Sera of patients with primary biliary cirrhosis (PBC) were examined for the presence of precipitating antibodies to sonicated rat liver mitochondrial (M) fraction. Three distinct precipitating systems observed in double immunodiffusion were identified and called M-A, M-B and M-C. Unsonicated mitochondria did not form precipitin lines. Precipitating system M-A was found in nineteen of twenty (95 percent) sera from PBC. The mitochondrial antigen of M-A system had the unusual property of being resistant to enzymatic digestion with deoxyribonuclease (DNase), ribonuclease (RNase) and trypsin under standard conditions. The titres of antibody to M-A antigen correlated (P less than 0.05) with titres of mitochondrial immunofluorescence staining on unfixed mouse kidney sections. Precipitating systems M-B and M-C were present in seven of twenty ribonuclease and trypsin but resistant to ribonuclease indicating that it could be DNA-protein complex. The M-C antigen was destroyed by trypsin suggesting its protein character, but it was difficult to determine if nucleic acids might also be associated with antigenicity. The antibodies to mitochondrial antigens were not present in normals (fifteen health adults), systemic lupus erythematosus (forty patients), rheumatoid arthritis (fifteen patients) and chronic liver diseases (fifteen patients). The antibodies did not show identity with antibodies to ribosomal ribonucleo-protein and other known nuclear antigens previously reported. The data confirm previous reports concerning the heterogeneity of mitochondrial antibodies present in sera of patients with PBC. The antibody to M-A antigen appeared to be a diagnostically useful immunological marker since it was present in the majority of patients with PBC.     

Human leukocyte antigen class II alleles in Caucasian women with primary biliary cirrhosis

In the current study, we investigated human leukocyte antigen (HLA) class II alleles in Caucasian women with primary biliary cirrhosis (PBC), a disease that preferentially affects women. Alleles of DRB1, DQA1, and DQB1 were determined by DNA-based HLA typing for women with PBC (n = 72) and healthy women (n = 381). All study subjects were Caucasian. HLA DRB1*08 was significantly increased in women with PBC compared to healthy women. The increase was primarily due to the DRB1*0801 allele, also the most common DRB1*08 allele among controls. DQB1*04 and DQA1*0401 were significantly increased. DRB1*1501, DQA1*0102, and DQB1*0602 were associated with decreased risk. Analyses conducted comparing parous women with PBC to parous healthy women (n = 68 and n = 282, respectively) yielded similar significant results. Although the DRB1*08-DQA1*0401-DQB1*04 haplotype was significantly associated with PBC, consistent with other studies, this haplotype nevertheless represented only 19% (14/72) of all PBC patients and can account for only a minority of the risk of PBC.     

The value of antinuclear antibodies in primary biliary cirrhosis

Objective

Although autoantibodies have been used for the diagnosis of primary biliary cirrhosis (PBC), their role has not been clarified. In this study, we try to explore the value of gp210 antibody and anti-centromere antibodies (ACA) in PBC.

Methods

Anti-gp210 and ACA were tested in 140 PBC patients by ELISA and indirect immunofluorescence respectively. Their association with clinical, pathological data and prognosis was analysed.

Results

30.5% of PBC patients had positive anti-gp210 antibody and 29.2% had ACA. The anti-gp210 antibody positive group had higher Mayo risk scores and lower serum albumin levels compared to the negative one. Patients with positive anti-gp210 antibody were more likely to develop hepatic failure (p < 0.05, OR = 9.8460, 95% CI: 1.067–90.901) than patients with negative anti-gp210 antibody. More patients with positive ACA developed portal hypertension than patients with negative ACA (p < 0.05, OR = 9.259; 95% CI: 1.027–88.410). Furthermore, concurrent Sjögren’s syndrome (SjS) and PBC was significantly more likely in the ACA positive group than in the negative ones (68.4% in ACA positive group, 20.7% in ACA negative group p < 0.001).

Conclusions

Both anti-gp210 antibody and ACA are related to severe disease course and poor prognosis. For PBC patients with positive ACA, further examinations should be made to detect underlying SjS.

     

Mitochondrial antigens as targets of cellular and humoral auto-immunity in primary biliary cirrhosis

Several factors point toward an auto-immune pathogenesis for primary biliary cirrhosis (PBC), mostly based on the presence of serum auto-antibodies to mitochondrial antigens (AMAs) and autoreactive T cells (both helper and cytotoxic). Interestingly, epitopes recognized by AMA and T-cell clones are located within overlapping areas of the antigens. Moreover, a role for an imbalance in cytokine pattern and for natural-killer lymphocytes has also been proposed. Despite several experimental reports, no clear evidence is available regarding the interaction of these factors leading to bile duct destruction. This article reviews the current reports regarding the auto-immune reaction against mitochondrial auto-antigens in PBC.     

Endothelial cell damage in primary biliary cirrhosis: influence of cholestasis and immunological mechanisms.

In a study looking for evidence of endothelial cell damage in primary biliary cirrhosis, serum from patients was found to be significantly more cytotoxic to cultured endothelial cells in vitro than normal control serum (P less than 0.001). Serum also contained higher levels of von Willebrand factor antigen (vWFAg, a specific product of the endothelium) than control serum (P less than 0.001). Cytotoxicity correlated with serum levels of vWFAg (P less than 0.05) and with serum bilirubin (P less than 0.05). No correlations were found between cytotoxicity and circulating immune complexes, C-reactive protein or CH50. The study was controlled by serum from patients with other liver diseases. In these samples vWFAg (P less than 0.003), and bilirubin levels (P less than 0.001) were also raised relative to normal controls. vWFAg levels again correlated with levels of bilirubin (P less than 0.05). Tissue culture experiments showed that purified bilirubin was cytotoxic to human umbilical vein endothelial cells and induced the release of vWFAg. The case report of a patient with obstructive jaundice again indicated that levels of bilirubin and vWFAg were related. These results suggest that endothelial cell damage occurs in both primary biliary cirrhosis and to a lesser extent in other liver diseases, and may be mediated by cholestasis, not by humoral immunological mechanisms.