Masanao Matsushita

Relationship between gene polymorphisms of mannose-binding lectin (MBL) and two molecular forms of MBL

Mannose‐binding lectin (MBL) activates complement through MBL‐associated serine proteases (MASP). A deficiency in MBL due to mutations at exon 1 of the human MBL gene is reported to cause vulnerability to infection. We examined sera of known MBL genotype by gel filtration and assessed their elution patterns using an ELISA for MBL and identified two MBL forms, a high‐molecular‐mass form and a lower‐molecular‐mass form. By the identification of either or both forms in individual sera, three types of patterns emerged: type 1 consisted of a high‐molecular form; type 2, of a low‐molecular form; and type 3, of both forms. Types 1, 2 and 3 corresponded, respectively, to a wild type (A/A), a homozygous mutation at codon 54 (B/B) and their heterozygote (A/B). One exception was a heterozygous LXPA/LYPB phenotype that exhibited the type‐2 pattern. Binding to mannan and MASP‐1/3 occurred exclusively with the high‐molecular form. An apparent MBL deficiency does not in fact representa deficiency in MBL molecules but rather the presence of circulating oligomeric mutant MBL with impaired function.

Genetic Polymorphisms of Transforming Growth Factor β-1 Promoter and Primary Biliary Cirrhosis in Japanese Patients

Abstract:  As suggested by concordance rates in twins, genetic factors are critical to the susceptibility and progression of primary biliary cirrhosis (PBC). Among cytokines, transforming growth factor beta‐1 (TGF‐β1) plays an important role in autoimmunity and liver fibrosis and a TGF‐β1 receptor knockout mouse has been recently proposed as a model for PBC. The promoter region of the TGF‐β1 gene has two single nucleotide polymorphisms (SNPs) at positions −800 and −509, which influence serum concentrations of latent and active TGF‐β1. We studied genomic DNA from 65 Japanese patients with PBC and 71 matched healthy controls for the association of TGF‐β1 SNPs analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) with susceptibility and disease progression of PBC. The −800 G to A SNP was not found in the Japanese population and no significant difference in the distribution of TGF‐β1 promoter gene −509 SNP was found between PBC cases and controls. Further, TGF‐β1 genotypes failed to correlate with clinical parameters, including histological stage and prognostic score. In conclusion, the TGF‐β1 promoter gene SNPs are not associated with disease susceptibility or progression in Japanese patients with PBC.

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.

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.

Hepatic sarcoidosis found as a small liver SOL in a patient with type C cirrhosis

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