Kazuhiro Sugi

Anti‐gp210 and anti‐centromere antibodies are different risk factors for the progression of primary biliary cirrhosis

The predictive role of antinuclear antibodies (ANAs) remains elusive in the long‐term outcome of primary biliary cirrhosis (PBC). The progression of PBC was evaluated in association with ANAs using stepwise Cox proportional hazard regression and an unconditional stepwise logistic regression model based on the data of 276 biopsy‐proven, definite PBC patients who have been registered to the National Hospital Organization Study Group for Liver Disease in Japan (NHOSLJ). When death of hepatic failure/liver transplantation (LT) was defined as an end‐point, positive anti‐gp210 antibodies (Hazard ratio (HR) = 6.742, 95% confidence interval (CI): 2.408, 18.877), the late stage (Scheuers stage 3, 4) (HR = 4.285, 95% CI:1.682,10.913) and male sex (HR = 3.266, 95% CI: 1.321,8.075) were significant risk factors at the time of initial liver biopsy. When clinical progression to death of hepatic failure/LT (i.e., hepatic failure type progression) or to the development of esophageal varices or hepatocellular carcinoma without developing jaundice (Total bilirubin < 1.5 mg/dL) (i.e., portal hypertension type progression) was defined as an end‐point in the early stage (Scheuers stage 1, 2) PBC patients, positive anti‐gp210 antibodies was a significant risk factor for hepatic failure type progression [odds ratio (OR) = 33.777, 95% CI: 5.930, 636.745], whereas positive anti‐centromere antibodies was a significant risk factor for portal hypertension type progression (OR = 4.202, 95% CI: 1.307, 14.763). Histologically, positive anti‐gp210 antibodies was most significantly associated with more severe interface hepatitis and lobular inflammation, whereas positive anticentromere antibodies was most significantly associated with more severe ductular reaction. Conclusion: These results indicate 2 different progression types in PBC, hepatic failure type and portal hypertension type progression, which may be represented by positive‐anti‐gp210 and positive‐anticentromere antibodies, respectively. (HEPATOLOGY 2007;45:118–127.)

The role of hepatitis B virus-specific memory CD8 T cells in the control of viral replication

BACKGROUND/AIMS The aim of this study is to clarify the differences of host immune responses between acute self-limited and chronic persistent hepatitis B virus (HBV) infections by quantitative and qualitative analysis of HLA-A*2402-restricted HBV-specific CD8+ T cells. METHODS HBV-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV were analyzed by flow cytometry using two HLA-A*2402-HBV peptide tetrameric complexes. RESULTS High numbers of HBV-specific CD8+ T cells were detected in acute phase PBMCs from most individuals with acute HBV infection while the number of these cells was greatly reduced in recovery phase PBMCs. HBV-specific CD8+ T cells were not detected in PBMCs from individuals with chronic HBV infection except for one patient during acute exacerbation. HBV-specific CD8+ T cells were induced by in vitro peptide stimulation in PBMCs from chronic HBV carriers with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. CD28CD45RA phenotype analysis showed that HBV-specific CD8+ T cells in acute phase PBMCs predominantly express a memory T cell phenotype. CONCLUSIONS HBV-specific memory CD8+ T cells may play a crucial role in complete clearance of HBV from patients with acute HBV hepatitis.

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia

BACKGROUND/AIMS The aim of this study was to identify and characterize hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) epitopes presented by human leukocyte antigen (HLA)-A*2402, most common HLA class I allele in East Asia. METHODS HLA-A*2402-restricted CTL epitopes were identified by reverse immunogenetics. Immunogenecity of these epitopes was investigated using peripheral blood mononuclear cell (PBMC) from HLA-A24+ patients with acute hepatitis B. RESULTS An HLA-A*2402 stabilization assay demonstrated that 36 of 63 HBV peptides carrying HLA-A*2402 anchor residues have high- and medium-HLA-A*2402 binding affinity. Two (C117-125 and P756-764) of the 36 peptides induced peptide-specific CTLs. CTL clones and lines specific for these peptides killed HBV recombinant vaccinia virus-infected target cells expressing HLA-A*2402, indicating that these two peptides are CTL epitopes presented by HLA-A*2402. These two peptides were able to induce specific CTLs in 7 and 11 of 12 HLA-A24+ patients with acute hepatitis B, respectively. CONCLUSIONS We identified two immunodominant CTL epitopes restricted by HLA-A*2402. Because HLA-A*2402 is the most common allele in East Asia, a region in which there are approximately 200 million HBV carriers, these epitopes will be useful for analysis of CTL responses in patients from East Asia.

Genetic polymorphisms in CTLA4 and SLC4A2 are differentially associated with the pathogenesis of primary biliary cirrhosis in Japanese patients

BackgroundAnti-gp210 and anti-centromere antibodies are different risk factors for the progression of primary biliary cirrhosis (PBC). In order to dissect the genetic basis for the production of these autoantibodies, as well as the development and progression of PBC in Japanese patients, we examined single nucleotide polymorphisms (SNPs) in cytotoxic T-lymphocyte antigen 4 (CTLA4) and solute carrier family 4 anion exchanger, member 2 (SLC4A2), which have been associated with the pathogenesis of PBC in Caucasian patients.MethodsFour SNPs for both CTLA4 and SLC4A2 were genotyped, using the polymerase chain reaction–restriction fragment length polymorphism method and TaqMan assay, in 450 Japanese PBC patients and 371 sex-matched healthy controls.ResultsThe CTLA4 rs231775, rs3087243, and rs231725 SNPs were significantly associated with PBC susceptibility. The CTLA4 rs231725 SNP was significantly associated with progression to late-stage disease. The CTLA-4 haplotype 1 (rs231775 G, rs231777 C, rs3087243 G, rs231725 A; GCGA) was a risk factor for PBC susceptibility but a protective factor for PBC progression. Conversely, the CTLA-4 haplotype 2 (ACAG) was a protective and risk factor, respectively, for PBC susceptibility and progression. In addition, the CTLA4 rs231777 SNP and haplotype 3 (ATGG) was significantly associated with anti-gp210 antibody production, while SLC4A2 haplotype 4 (rs2069443 A, rs2303933 G, rs2303937 A, rs2303941 T; AGAT) and haplotype 3 (AAGC) were significantly associated with PBC susceptibility and anti-centromere antibody production, respectively.ConclusionsCTLA4 and SLC4A2 genetic polymorphisms are differentially associated with PBC development and progression, as well as anti-gp210 or anti-centromere antibody production, in Japanese PBC patients.

Degradation of plasma bilirubin by a bilirubin oxidase derivative which has a relatively long half-life in the circulation

To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.

Circulating microRNA Profiles in Patients with Type-1 Autoimmune Hepatitis

Recent studies have demonstrated that micro (mi)RNA molecules can be detected in the circulation and can serve as potential biomarkers of various diseases. This study used microarray analysis to identify aberrantly expressed circulating miRNAs in patients with type 1 autoimmune hepatitis (AIH) compared with healthy controls. Patients with well-documented and untreated AIH were selected from the National Hospital Organization (NHO)-AIH-liver-network database. They underwent blood sampling and liver biopsy with inflammation grading and fibrosis staging before receiving treatment. To further confirm the microarray data, circulating expression levels of miR-21 and miR-122 were quantified by real-time quantitative polymerase chain reaction in 46 AIH patients, 40 patients with chronic hepatitis C (CHC), and 13 healthy controls. Consistent with the microarray data, serum levels of miR-21 were significantly elevated in AIH patients compared with CHC patients and healthy controls. miR-21 and miR-122 serum levels correlated with alanine aminotransferase levels. Circulating levels of miR-21 and miR-122 were significantly reduced in AIH patients with liver cirrhosis, and were inversely correlated with increased stages of fibrosis. By contrast, levels of circulating miR-21 showed a significant correlation with the histological grades of inflammation in AIH. We postulate that aberrantly expressed serum miRNAs are potential biomarkers of AIH and could be implicated in AIH pathogenesis. Alternations of miR-21 and miR-122 serum levels could reflect their putative roles in the mediation of inflammatory processes in AIH.

Association of STAT4 Polymorphisms with Susceptibility to Type-1 Autoimmune Hepatitis in the Japanese Population

Background/Aims Recent studies demonstrated an association of STAT4 polymorphisms with autoimmune diseases including systemic lupus erythematosus and rheumatoid arthritis, indicating multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 polymorphisms on the susceptibility and phenotype of type-1 autoimmune hepatitis in a Japanese National Hospital Organization (NHO) AIH multicenter cohort study. Methodology/Principal Findings Genomic DNA from 460 individuals of Japanese origin including 230 patients with type-1 autoimmune hepatitis and 230 healthy controls was analyzed for two single nucleotide polymorphisms in the STAT4 gene (rs7574865, rs7582694). The STAT4 rs7574865T allele conferred risk for type-1 autoimmune hepatitis (OR = 1.61, 95% CI = 1.23–2.11; P = 0.001), and patients without accompanying autoimmune diseases exhibited an association with the rs7574865T allele (OR = 1.50, 95%CI = 1.13–1.99; P = 0.005). Detailed genotype-phenotype analysis of type-1 autoimmune hepatitis patients with (n = 44) or without liver cirrhosis (n = 186) demonstrated that rs7574865 was not associated with the development of liver cirrhosis and phenotype (biochemical data and the presence of auto-antibodies). Conclusions/Significance This is the first study to show a positive association between a STAT4 polymorphism and type-1 autoimmune hepatitis, suggesting that autoimmune hepatitis shares a gene commonly associated with risk for other autoimmune diseases.

Genome-wide association studies identify PRKCB as a novel genetic susceptibility locus for primary biliary cholangitis in the Japanese population

&NA; A previous genome‐wide association study (GWAS) performed in 963 Japanese individuals (487 primary biliary cholangitis [PBC] cases and 476 healthy controls) identified TNFSF15 (rs4979462) and POU2AF1 (rs4938534) as strong susceptibility loci for PBC. In this study, we performed GWAS in additional 1,923 Japanese individuals (894 PBC cases and 1,029 healthy controls), and combined the results with the previous data. This GWAS, together with a subsequent replication study in an independent set of 7,024 Japanese individuals (512 PBC cases and 6,512 healthy controls), identified PRKCB (rs7404928) as a novel susceptibility locus for PBC (odds ratio [OR] = 1.26, P = 4.13 × 10‐9). Furthermore, a primary functional variant of PRKCB (rs35015313) was identified by genotype imputation using a phased panel of 1,070 Japanese individuals from a prospective, general population cohort study and subsequent in vitro functional analyses. These results may lead to improved understanding of the disease pathways involved in PBC, forming a basis for prevention of PBC and development of novel therapeutics.

Asialofetuin metabolism in animals with obstructive jaundice

Abstract In patients with chronic liver diseases, plasma levels of asialoglycoproteins are elevated. To elucidate the pathogenesis of the increase in plasma asialoglycoproteins, kinetic disposition of asialofetuin was studied in bile-duct-ligated rats. When 125 I-labeled asialofetuin was injected intravenously to normal rats, the radioactivity in the blood decreased rapidly with a half-life of 5.9 min. Most radioactivity accumulated in the liver and underwent biliary excretion. In bile-duct-ligated rats, the half-life of the radioactivity is prolonged significantly (10.2 min) compared with the control rats. Radioactivity accumulated in the liver remained high even after 120 min of administration when most of the radioactivity was excreted in the bile of intact rats. Recanalization of the ligated bile duct on days 3 and 7 failed to normalize the biliary excretion. These observations suggest that hepatic uptake, endosomal degradation and biliary excretion of asialoglycoproteins were significantly disturbed in rats whose bile ducts were transiently ligated and recanalized.

Effect of obstructive jaundice on the fate of a nephrophilic organic anion in the rat

Renal transtubular transport of phenolsulfophthalein (PSP), a nephrophilic organic anion that circulates bound to albumin, was studied in normal and bile-duct-ligated rats. Intravenously injected PSP disappeared from the circulation more rapidly in bile-duct-ligated jaundiced rats than in intact animals. However, urinary excretion of PSP was significantly lower in the former than in the latter. Kinetic analysis revealed that binding of PSP to plasma protein(s) was significantly lower with jaundiced rats than with intact animals. Addition of albumin to plasma samples from bile-duct-ligated rats markedly increased PSP binding. The decreased PSP binding returned to normal levels after treating the jaundiced plasma with bilirubin oxidase, an enzyme that degrades amphiphilic bilirubin to water soluble metabolites. These results suggest that bilirubin might be the major metabolite that occupied the PSP binding site(s) on albumin in jaundiced rats. When PSP was injected bound to equimolar amount of albumin, the rate of PSP disappearance from the circulation decreased and urinary excretion of the ligand increased markedly; urinary excretion of PSP was significantly larger in bile-duct-ligated rats than in intact animals. These results suggest that the renal transport capacity for amphiphilic organic anions, such as PSP, might be increased compensatively in bile-duct-ligated animals, and that the apparent decrease in renal secretory transport for PSP might result from, at least in part, random distribution of the ligand to extrarenal tissues due to decrease in the binding activity of albumin.