Hirotoshi Fujikawa

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.

CASE REPORT: Vanishing Bile Duct Syndrome Associated with Chronic EBV Infection

We reported here an adult patient with vanishing bile duct syndrome due to chronic EBV infection. A 22-year-old male was admitted to a nearby hospital complaining of a sore throat and jaundice. He received a high dose of prednisolone for bile stasis of acute viral hepatitis. However, the hepatitis did not improve, and he was transferred to our hospital. He had exhibited jaundice for one year as well as hemophagocytic syndrome and intestinal perforation. Subtotal intestinal resection was successfully performed. Three follow-up biopsied liver specimens indicated vanishing bile duct syndrome. Positive results of EBV-DNA in his serum and mRNA of EBV by in situ hybridization of his liver indicated that massive doses of prednisolone caused chronic EBV infection and vanishing bile duct syndrome.

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.

Differences in antigenic sites, recognized by anti-liver-kidney microsome-1 (LKM-1) autoantibody, between HCV-positive and HCV-negative sera in Japanese patients

Abstract: Anti-liver-kidney microsome-1(LKM-1), which reacts with cytochrome P450 IID6 (CYP2D6), is an autoantibody present in autoimmune hepatitis type II, which affects primarily young patients. Recently, it has been shown some adult patients with chronic hepatitis C are also positive for anti-LKM-1. Thus, anti-LKM-1-positive patients can be classified into two subgroups: (1) those with autoimmune hepatitis type II and (2) those with chronic hepatitis C. We investigated the antigenic epitopes of CYP2D6 with which each of these two anti-LKM-1-positive subgroups reacted. Multiple deletion mutants of CYP2D6 were constructed from a human liver cDNA library and five recombinant fusion proteins expressed. Antigenic epitopes were determined by immunoblot analysis using these proteins. Anti-LKM-1 present in HCV-negative sera recognized at least two peptide regions of aa213-280 and aa341-477 of human CYP2D6. In contrast, anti-LKM-1 present in HCV-positive sera recognized only a single region of aa341-477. Thus, the sera of patients with autoimmune hepatitis type II and patients with chronic hepatitis C recognize different antigenic epitopes of the CYP2D6 molecule. To our knowledge, this is the first time LKM-1 autoantigens have been analyzed at the molecular level in Japanese patients.

Intestinal cryptosporidiosis as an initial manifestation in a previously healthy Japanese patient with AIDS

Background:Cryptosporidium parvum infection has been recognized as one of the pathogens causing severe and persistent diarrhea in immunodeficient patients, such as those with AIDS, worldwide. However, in Japan, the frequency of this infection has been rare, except for environmental contamination through the water supply. In this communication, we describe a Japanese patient with AIDS presenting with intestinal Cryptosporidiosis as an initial manifestation. Methods: The oocysts of Cryptosporidium parvum in his stool were detected by the Ziehl-Neelsen method and electron microscopy. The antigen-specificity was proved by immunostaining, using a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody and enzyme-linked immunosorbent assay (ELISA), using Cryptosporidium-specific antibody. Results: A 28-year-old Japanese homosexual man was admitted to our hospital because of severe watery diarrhea of 1-week duration. Numerous oocysts of Cryptosporidium parvum were observed in his stool. Cryptosporidium parvum antigen was detected in stool samples. Serological examinations revealed that anti-HIV-1 antibody was positive, and HIV RNA was positive at a high level. He was diagnosed as having AIDS associated with intestinal Cryptosporidiosis. The circulating CD4+ T-cell count was 152/μl. His diarrhea was not alleviated by administration of loperamide and an ordinary antibiotic agent, but ultimately resolved by the administration of the macrolide antibiotic agent, clarithromycin. Conclusions: We emphasize that the presence of Cryptosporidium parvum infection should be kept in mind in searching for pathogens causative of severe diarrhea in AIDS patients.

Analysis of two major anti-M2 antibodies (anti-PDC-E2/anti-BCOADC-E2) in primary biliary cirrhosis: relationship to titers of immunofluorescent anti-mitochondrial antibody

To analyze anti-M2 components in primary biliary cirrhosis (PBC) we measured two major anti-M2 antibodies (anti-PDC-E2 and anti-BCOADC-E2) by immunoblotting and ELISA, and compared the results between 38 immunofluorescent anti-mitochondrial antibody (AMA)-negative PBC patients (group A) and 39 strongly AMA-positive PBC patients (group B) with titers of 1:640. Using bovine heart mitochondrial fraction as antigen, the immunoblot positivity rate of anti-PDC-E2 in group B was significantly higher than that in group A, whereas the positivity rate of anti-BCOADC-E2 was not significantly different between the two groups. This result was similar to that obtained by ELISA using recombinant fusion proteins. In group A there was a significant inverse correlation between ELISA optical density values of anti-PDC-E2 and of anti-BCOADC-E2, but in group B there was no correlation between the two values. Only three patients from group A and 21 from group B were positive for both antibodies. Taken together these results appear to indicate that the detection of anti-BCOADC-E2 is critical for the accurate serological diagnosis of AMA-negative PBC patients. The detection of anti-BCOADC-E2 may also help to distinguish between AMA-negative PBC and autoimmune cholangitis patients.