Naomi Kawaguchi

Prevalence of antimitochondrial antibody in Japanese corporate workers in Kanagawa prefecture

BackgroundThe prevalence of antimitochondrial antibody (AMA) in humans and its relationship to the development of primary biliary cirrhosis (PBC) are not well known. We have estimated the frequency of AMA in the general population, and studied its association with PBC.MethodsWe studies 1714 corporate workers (median age, 48 years; range, 30 to 59 years) who had an annual health check from 1998 to 1999 at Kawasaki Social Insurance Hospital in Japan. We used an indirect immunofluorescence method for screening serum AMA. We applied the prevalence of AMA-positive people in the study group to the general population in Japan. Then the inferred AMA-positive population was compared to the actual number of patients with PBC in statistics published by the Japanese Government.ResultsAMA was detected in 11 of 1714 people (0.64%; 95% confidence interval, 0.26% to 1.02%). All these 11 sera reacted with 2-oxoacid-dehydrogenase complex by immunoblotting. Of these 11 individuals, none had subjective symptoms, all had normal serum bilirubin levels, and 6 had abnormal liver function test results. Using published statistics for the Japanese population, we inferred that there were approximately 336 472 AMA-positive people in Japan from age 30 to 59 years. The number of patients with symptomatic PBC recorded by the nationwide epidemiological survey of the Japanese Government was 2459. Thus, we inferred the rate of symptomatic PBC among AMA-positive persons to be about 0.73% (2459/336 472).ConclusionsAMA is not a rare antibody in the general population, but few people develop recognizable PBC even if they have AMA.

Low frequency of anti-SLA/LP autoantibody in Japanese adult patients with autoimmune liver diseases: analysis with recombinant antigen assay

Anti-soluble liver antigen/liver pancreas (SLA/LP) autoantibody has been proposed to be one of the autoantibodies characterizing autoimmune hepatitis (AIH). Recently, one of the autoantigens to anti-SLA/LP was identified as a UGA suppressor tRNA-associated protein. Although the function of this protein remains unknown, the recombinant protein has been prokaryotically expressed. Using this protein as an antigen, a recombinant immunoassay for anti-SLA/LP autoantibody has been established and the frequency and significance of this autoantibody have been discussed in European countries. So, in the present study, we investigated anti-SLA/LP autoantibodies in Japanese patients with autoimmune liver diseases using the recombinant antigen ELISA and Western blot assay. Seventy-five patients with AIH type 1, 5 with AIH type 2, 46 with primary biliary cirrhosis, 10 with primary sclerosing cholangitis, 47 with chronic hepatitis C, 48 with systemic lupus erythematosus, 3 with cryptogenic hepatitis, and 40 normal controls were the subjects of the present study. Anti-SLA/LP autoantibodies were detected in only 5 of 75 (6.7%) patients with AIH type 1, but in none of the other 159 patients or 40 normal controls. The clinicopathologic features of anti-SLA/LP-positive AIH type 1, including carriers of HLA DR locus variations, were not significantly different from anti-SLA/LP-negative patients except for the mortality rate. Anti-SLA/LP autoantibody was detected at a low frequency in Japanese patients with AIH type 1 and did not significantly influence clinical features. However, since it has high disease-specificity to AIH type 1, further analysis of SLA/LP may contribute to help clarify the pathogenesis of AIH type 1.

Chronic hepatitis C associated with anti-liver/kidney microsome-1 antibody is not a subgroup of autoimmune hepatitis

To determine whether “autoimmune hepatitis type IIb” should be categorized as a subgroup of autoimmune hepatitis, we conducted a clinicopathological study of 25 adult Japanese patients who were positive for anti-liver/kidney microsome-1 (anti-LKM-1) antibody and infected with the hepatitis C virus (HCV). Anti-LKM-1 was determined by indirect immunofluo-rescence and by the double immunodiffusion assays we have developed. Twenty-two patients did not present any unusual symptoms or any associated diseases during the course of their chronic HCV infection. The spectrum of HCV genotypes of these patients did not significantly differ from that of anti-LKM-1-negative Japanese patients with chronic hepatitis C. Histological examination of liver biopsy specimens showed the usual characteristics of chronic hepatitis C and lack of characteristics of autoimmune hepatitis type I. No disease-specific HLA haplotypes were noted, and HLA-DR4, which is detectable in 88.7% of Japanese patients with autoimmune hepatitis type I, was detected in only 50.0% of our group, the same rate as the background frequency. Prednisolone was effective in none of the six patients treated, but interferon was effective in six of ten treated patients (60%). From these results, we conclude that “autoimmune hepatitis type IIb” should not be categorized as autoimmune hepatitis, and that this subgroup is essentially chronic hepatitis C in which an autoantibody has been produced during the course of chronic HCV infection.

Eta-1/osteopontin genetic polymorphism and primary biliary cirrhosis

Early T-lymphocyte activation 1 (Eta-1)/osteopontin is a soluble ligand with pleomorphic immunologic activities including activation of macrophage chemotaxis, promotion of Th1 responses, and activation of B1 B-cells. A recent study suggested that a single-nucleotide polymorphism (SNP) at position nt 9250 (C to T) in exon 7 was highly associated with systemic lupus erythematosus (SLE). Eta-1/osteopontin was reported to be highly expressed in the MRL/lpr mouse, which is recognized as one of the spontaneous autoimmune models of SLE. In the present study, we first investigated the association with this SNP and susceptibility to primary biliary cirrhosis (PBC). The allele frequencies of C/C, C/T, and T/T at position nt 9250 on the Eta-1/osteopontin gene in 50 PBC patients were 20, 32, and 48%, respectively, compared with 9, 47, and 44% in 34 healthy controls (P<0.16-0.72). The gene frequencies of C and T at this position in such PBC patients were 0.36 and 0.64, whereas those in the healthy controls were 0.32 and 0.68 (P<0.91), respectively. Moreover, clinical findings and pathologic stages were not correlated with the variation of SNP. Those findings suggest no associations with Eta-1/osteopontin genetic polymorphism and susceptibility to PBC.

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

Abstract: 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.

Serial changes of serum anti-M2 proteins in patients with primary biliary cirrhosis: a follow-up study by immunoblotting

Abstract To investigate the pathogenic role of M2 components in primary biliary cirrhosis (PBC), we studied the serial changes of serum anti-M2 protein profiles by immunoblotting in 21 patients with PBC who were observed for at least 3 years. Immunoblotting was done using bovine heart mitochondrial fraction as the antigen. First, we confirmed the antigen specificity by inhibition tests using recombinant proteins associated with two types of major M2 proteins. At the initial examination, 15 patients showed positivity for the 74-kDa protein corresponding to anti-PDC-E2, and 19 patients showed the 50-kDa protein corresponding to anti-BCOADC-E2. In subsequent examinations, four of the 21 patients continuously did not show the 74-kDa protein and only one patient showed the 50-kDa protein although it was not detected at the initial examination. The anti-M2s other than 74-kDa protein, especially 50-kDa protein, showed almost no changes from the initial examinations. These data indicate that part of the onset and development of PBC is highly associated with M2 proteins other than PDC-E2.

Detection of anti-LKM-1(anti-CYP2D6) by an enzyme-linked immunosorbent assay in adult patients with chronic liver diseases

Anti-liver kidney microsome-1 (LKM-1) autoantibody, which is a serological marker for autoimmune hepatitis type II, recognizes Cytochrome P450 IID6 (CYP2D6). This autoantibody is also detected in a portion of patients with chronic hepatitis C. Anti-LKM-1 has been measured by indirect immunofluorescence (IF) using rat liver and kidney sections. However, this method has some problems in specificity and is so laborious to handle with many samples. In this study, in order to determine anti-LKM-1, we established an enzyme-linked immunosorbent assay (ELISA) for anti-CYP2D6 using a recombinant CYP2D6 fusion protein. We studied sera from 29 patients positive for anti-LKM-1 by the new ELISA. We further studied sera from a total of 301 patients with various liver diseases and 100 sera from normal controls negative for anti-LKM-1 by the new ELISA. The specificity of the ELISA was ascertained by absorption tests using sera positive for anti-LKM-1. In 29 sera from patients positive for anti-LKM-1 by IF, we found a good correlation between the logarithms of the antibody titers determined by IF and ELISA indexes obtained by our new method. Anti-CYP2D6 was positive in 12 of 12 (100%) patient with autoimmune hepatitis type II and 16 of 17(94.1%) with chronic hepatitis C positive for anti-LKM-1 by IF. In other 401 sera negative for anti-LKM-1 by IF, anti-CYP2D6 was all negative except a few sera. We established a new ELISA for anti-LKM-1 (anti-CYP2D6). This ELISA system is sensitive, antigen-specific and easy to be done. Therefore, this assay allows a routine test of many serum samples, especially for diagnosing autoimmune hepatitis type II.

Detection of anti-branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 antibody in primary biliary cirrhosis by ELISA using recombinant fusion protein.

Anti-M2 of anti-mitochondrial antibody (AMA) is a serological marker of primary biliary cirrhosis (PBC). Anti-pyruvate dehydrogenase complex-E2 (anti-PDC-E2) is recognized as the most frequently occurring anti-M2, and a routine laboratory test for this antibody has already been established. However, it is also known that there are patients with PBC who are negative for anti-PDC-E2. For the serological diagnosis of these patients, immunoblotting for anti-M2s is indicated. However, the technique currently utilized is too laborious to allow testing of a large number of samples. In this study, we have developed an enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion protein in order to evaluate anti-branched chain 2-oxo-acid dehydrogenase complex-E2 (anti-BCOADC-E2), another frequently occurring anti-M2 in PBC patients. KB cell lines (CCL 17) were utilized as source material, and BCOADC-E2 cDNA (971 bp) including the lipoic acid binding domain was amplified by polymerase chain reaction. The amplified region was subcloned into pEX-3 vectors and expressed, and the resulting fusion protein (beta-galactosidase/BCOADC-E2) was utilized as antigen for an ELISA. We ascertained the specificity of this antigen by inhibition tests with ELISA and immunoblotting. We defined the cut-off optical density (OD) value as the mean + 3 SD (0.146) of sera from 60 normal controls. Anti-BCOADC-E2 could not be detected with this assay in sera from normal controls and from patients with autoimmune hepatitis and chronic viral hepatitis. Anti-BCOADC-E2 was detected in 119 of 210 sera (56.7%) from patients with PBC. In addition, anti-BCOADC-E2 was detected in 48 of 99 (48.5%) sera from PBC patients who were negative for anti-PDC-E2. Here, we have succeeded in developing a new ELISA for detecting anti-BCOADC-E2. This system is antigen-specific and easily performed. This assay should allow routine testing of a large number of serum samples, and should become especially useful for the serodiagnosis of anti-PDC-E2-negative PBC patients.

Combination assays for IgG class and IgM class anti-pyruvate dehydrogenase complex(PDC)-E2 by ELISA using recombinant autoantigen to diagnose primary biliary cirrhosis

Abstract IgG and IgM class anti-pyruvate dehydrogenase complex (PDC)-E2 were studied in sera from anti-M2-positive 84 patients with PBC by enzyme-linked immunosorbent assay (ELISA) to assess the usefulness of combination assays. We used recombinant antigen coding the autoantigenic epitopes of PDC-E2 comprising the lipoic acid binding sites. Antigen specificity of this ELISA was confirmed by inhibition test with pre-incubation of recombinant antigen with tested sera. Neither IgG class nor IgM class anti-PDC-E2 could be detected in sera from healthy controls and patients with chronic liver diseases other than PBC. In 84 sera from patients with PBC, the positive rate of IgG class anti-PDC-E2 (66.7%) was almost the same as that of IgM class anti-PDC-E2 (65.5%). Seven sera were positive for IgG class anti-PDC-E2 but negative for IgM class anti-PDC-E2. In contrast, six sera were positive for IgM class anti-PDC-E2 but negative for IgG class anti-PDC-E2. In total, 62 (73.8%) of 84 sera were positive for IgG class and/or IgM class anti-PDC-E2. This rate was higher than with assay of IgG class anti-PDC-E2 or IgM class anti-PDC-E2 alone. The positive rate of IgG class and IgM class anti-PDC-E2s in sera with high AMA titers was higher than that in sera with low AMA titers. The sensitivity of ELISA matched that of immunoblotting in 67 sera (79.7%). We conclude that the combination ELISAs for IgG class and IgM class anti-PDC-E2 are useful for diagnosing PBC.

Association of single nucleotide polymorphisms of the interleukin-10 promoter gene and susceptibility to primary biliary cirrhosis: immunogenetic differences in Italian and Japanese patients.

Several lines of data suggest that genetic factors play an important role in the onset and/or progression of primary biliary cirrhosis (PBC). Since PBC is an autoimmune disease, it is reasoned to assume that genes encoding cytokines may confer susceptibility to disease. Amongst these factors, interleukin-10 (IL-10) has received significant attention. The promoter region of IL-10 gene has three single nucleotide polymorphisms (SNPs) at positions m 1082, m 819 and m 592. To elucidate the association of the three SNPs of IL-10 promoter region with susceptibility of PBC in two different genetic populations, 159 unrelated patients with PBC (94 Italian and 65 Japanese) and 143 local controls (72 Italian and 71 Japanese) were enrolled. SNPs were determined using allele-specific PCR/RFLP. In Italian PBC patients, the frequency of homozygosity for G/G at position m 1082 was significantly higher than that of local controls (p <0.041, OR=2.44, 95% C.I.; 1.02-5.86). The frequencies of haplotype GCC in PBC patients, possibly linked to higher IL-10 production, were also significant higher than local controls (p <0.033). However, in Japanese population, there were no significant differences in the three SNPs and haplotypes between PBC patients and controls. Excessive production of IL-10 may play an important role in some populations in modulating the onset of PBC. Further, immunogenetic studies of PBC should take into account ethnic and geographic variations; this makes such studies in heterogeneous population, like the USA, more difficult.