Koju Kobayashi

PRIMARY CULTURE OF CHOLANGIOCYTES FROM NORMAL MOUSE LIVER

Dear Editor: Cholangiocytes have been thought to play a role as antigen presenting cells or target cells in immune-mediated cholangitis. The aberrant expression of MHC class II proteins on cholangiocytes in primary biliary cirrhosis raises the possibility that cholangiocytes can function as antigen presenting ceils and, in immune-mediated cholangitis, cholangiocytes appeared to be targeted by lymphocytes (8). To explore the immune mechanisms underlying cholangitis, a culture of mouse cholangiocytes is essential, because the mouse is indispensable for immunological study. Cholangiocytes are a minor component of liver ceils, comprising only 3-4% of the rodent liver cells. Most extensively studied cultured

Vigorous response of cytotoxic T lymphocytes associated with systemic activation of CD8 T lymphocytes in fulminant hepatitis B.

Abstract: Background: Fulminant hepatitis is a clinical syndrome characterized by sudden and severe liver dysfunction.

Hepatitis B Virus Replication Could Enhance Regulatory T Cell Activity by Producing Soluble Heat Shock Protein 60 From Hepatocytes

BACKGROUND HBcAg-specific regulatory T (T(reg)) cells play an important role in the pathogenesis of chronic hepatitis B. Soluble heat shock proteins, especially soluble heat shock protein 60 (sHSP60), could affect the function of T(reg) cells via Toll-like receptor. METHODS We analyzed the relationship between soluble heat shock protein production and hepatitis B virus (HBV) replication with both clinical samples from HBeAg-positive patients with chronic hepatitis B (n= 24) and HBeAb-positive patients with chronic hepatitis B (n= 24) and in vitro HBV-replicating hepatocytes. Thereafter, we examined the biological effects of sHSP60 with isolated T(reg) cells. RESULTS The serum levels of sHSP60 in patients with chronic hepatitis B were statistically significantly higher than those in patients with chronic hepatitis C (P<.01), and the levels of sHSP60 were correlated with the HBV DNA levels (r = 0.532; P<.001) but not with the alanine aminotransferase levels. Moreover, the levels of sHSP60 in HBV-replicating HepG2 cells were statistically significantly higher than those in control HepG2 cells. Preincubation of CD4(+) CD25(+) cells with recombinant HSP60 (1 ng/mL) statistically significantly increased the frequency of HBcAg-specific interleukin 10-secreting T(reg) cells. The frequency of IL7R(-)CD4(+)CD25(+) cells, the expression of Toll-like receptor 2, and the suppressive function of T(reg) cells had declined during entecavir treatment. CONCLUSION The function of HBcAg-specific T(reg) cells was enhanced by sHSP60 produced from HBV-infected hepatocytes. Entecavir treatment suppressed the frequency and function of T(reg) cells; this might contribute to the persistence of HBV infection.

Profiles of cytokines produced by CD4-positive T lymphocytes stimulated by anti-CD3 antibody in patients with chronic hepatitis C

Abstract: Helper T cells (Th) are classified as type 1 (Th1) and type 2 (Th2) according to the cytokines they produce; interferon-γ is produced by Th1, and interleukin-4 by Th2. We counted the circulating CD4-positive Th cells that produce interferon-γ or interleukin-4 with an enzyme-linked immunospot assay. CD4-positive T cells isolated from patients with chronic hepatitis B (n = 10), chronic hepatitis C (n = 16), and healthy subjects (n = 10) were stimulated with anti-CD3 antibody in vitro. The number of interferon-γ-producing Th cells was significantly lower in patients with chronic hepatitis C than in healthy subjects (P = 0.0024), whereas in patients with chronic hepatitis B, the number was similar to that in healthy subjects (P = 0.8530). The number of interleukin-4-producing Th cells was significantly higher in patients with chronic hepatitis C (P = 0.0010) and chronic hepatitis B (P = 0.0089) than in healthy subjects. In chronic hepatitis C, the number of interferon-γ-producing Th cells was increased after incubation of the cells with interferon-α (P = 0.008) or with recombinant interferon-γla (P = 0.024), but not with interferon-β (P = 0.051). The number of interleukin-4-producing Th cells was decreased after incubation with interferon-α (P = 0.0004), with interferon-β (P = 0.003), and with recombinant interferon-γla (P = 0.0004). Changes in the numbers of interferon-γ- or interleukin-4-producing Th cells in vitro were more evident in sustained responders to interferon therapy than in non-responders. These results suggest that Th2 cells are the predominant cell type in chronic hepatitis C, and that their activity may be suppressed by the administration of interferon.

Genoepidemiology and its relationship to clinical features in patients infected chronically with hepatitis B virus (HBV)

The route of hepatitis B virus (HBV) infection and subsequent clinical course vary widely. It is not clear if the prevalence of HBV genotypes differs in the different clinical features of HBV carriers. The genotype of HBV was determined by direct sequencing of HBV DNA amplified with a PCR method from 310 Japanese HBV carriers (189 male and 121 female, aged from 14 to 82 years). Genotype A was detected in eight (2.6%) of 310 HBV carriers, genotype B was detected in 92 (29.7%) and genotype C was detected in 210 (67.7%). None of them had genotype D, E or F. Among the eight patients infected with genotype A, one patient had been infected with HBV in his adulthood. Furthermore, both of one married couple also had genotype A. On the other hand, a HBeAg-negative state and HBeAg-negative healthy carrier state were significantly more common in patients infected with genotype B than those with genotype C by univariate analysis (P<0.0001 and 0.0058) and multiple logistic regression (P<0.0001 and 0.0029). Hepatocellular carcinoma was present in two of eight patients with genotype A, 11 of 210 patients with genotype C and none of the 92 patients with genotype B. These results suggest that the genotype of HBV may be a determinant of the outcome after acute HBV infection and of chronic HBV infection.

Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity.

We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7-N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype-antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a beta-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled alpha-type antiidiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246+/-184 (S.D.) microg (n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC (n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 microg/mg Ucre with the average of 5.41+/-4.53 (S.D.) microg/mg Ucre.

Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan.

We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000–2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69‐year‐old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278–646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0–0.3% and 0–0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942–AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non‐Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non‐fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non‐fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis. J. Med. Virol. 80:967–973, 2008.

HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases

Background Various kinds of autoimmune diseases have been reported to have a significant relationship with persistent hepatitis c virus (HCV) infection and Th17 cells. Previously, our group reported that the existence of HCV in T lymphocytes could affect the development of CD4+ helper T cells and their proliferation, in addition to the induction of immunoglobulin hyper-mutation. Methods Therefore, we analyzed the relationship between persistent infection of HCV and the mechanism of Th17 cell induction ex vivo and in vitro. Results The prevalence of autoimmune-related diseases in chronic hepatitis c patients (CH-C) was significantly higher than in other types of chronic hepatitis (hepatitis B and NASH). A significantly higher frequency of IL6 and TGF-β double-high patients was detected in CH-C than in other liver diseases. Moreover, these double-high patients had significantly higher positivity of anti-nuclear antibody, cryoglobulinemia, and lymphotropic HCV and higher amounts of IL1-β, IL21, IL23. In addition to the previously reported lymphotropic SB-HCV strain, we found a novel, genotype 1b lymphotropic HCV (Ly-HCV), by deep sequencing analysis. Lymphotropic-HCV replication could be detected in the lymphoid cells with various kinds of cytokine-conditions including IL1β, IL23, IL6 and TGF-β in vitro. Infection by HCV could significantly enhance the development of Th17 cells. The HCV protein responsible for inducing the Th17 cells was HCV-Core protein, which could enhance the STAT-3 signaling and up-regulate the expression of RORγt as a Th17 master gene. Conclusion Infection by lymphotropic HCV might enhance the Th17 development and contribute to understanding the pathogenesis of autoimmune-related diseases.

Primary biliary cirrhosis with antibody against carbonic anhydrase II associates with distinct immunological backgrounds

Objective: a part of patients with primary biliary cirrhosis (PBC) has anti-human carbonic anhydrase II (CA II) autoantibodies, although several contradictional reports followed. Since immunization of mice with CA II results in cholangitis in a susceptible strain, PBC with anti-CA II antibody may have distinct clinical features. Thus, we tested the sera of patients with PBC for anti-CA II antibodies and compared clinical characteristics of PBC patients with and without anti-CA II antibodies in Japanese patients. Methods: anti-CA II antibodies were detected in nine of 50 (18%) PBC patients by immunoblotting. The evaluation of these patients included various clinical parameters, autoantibodies, and immunological backgrounds. Results: the levels of serum liver tests and the prevalence of serum anti-mitochondrial antibody (77.8 vs. 92.7%) were not different between the patients with and without anti-CA II antibody. However, the prevalence of anti-nuclear antibody (ANA) was significantly higher in the patients with anti-CA II antibody than that in the patients without anti-CA II antibody (66.7 vs. 25.6%, P=0.044), although their mean titers were not statistically different. Association of Sjøgrens syndrome tended to be more frequent in the patients with anti-CA II antibody than those without it (33.3 vs. 14.6%, P=0.327). Studies of HLA class I allotype revealed that three of five (60.0%) patients with anti-CA II antibodies and one patients from 34 (3.0%) patients without anti-CA II antibodies had HLA B51 allotype; the difference in the prevalence of this allotype was significant (P=0.004, Pc=0.01), and the prevalence of other HLA class I and HLA DR allotypes was similar between the patients with and those without anti-CA II antibody. Administration of ursodeoxycholic acid (600 mg per day) was accompanied by change in liver tests in a similar way between the two patient groups. Conclusions: These results suggest that, although clinical features are not distinctive, PBC patients with anti-CA II antibody may have a genetic background, which may contribute to a susceptibility to immune-mediated cholangitis.

Primary culture of human gallbladder epithelial cells

SummaryGrowth requirements of epithelial cells isolated from the human gallbladder were examined. The growth of gallbladder epithelial cells (GBEC) was stimulated by medium conditioned with gallbladder fibroblasts (CM-GBF) in a dose-dependent manner. The conditioned medium derived from human embryo lung fibroblasts also showed similar growth stimulating activity for GBEC, suggesting that fibroblasts secrete a factor or factors which induce GBEC growthin vitro. CM-GBF in the presence of 10% fetal bovine serum increased GBEC growth up to 10 times the growth in the absence of CM-GBF supplement. GBEC cultured with CM-GBF showed hexagonal shape under a phase-contrast microscope, and also expressed cytokeratin and mucopolysaccharide in the cytoplasm, both of which are specific for GBEC. Electron microscopy revealed desmosomes and tight junctions between the cells and microvilli on the apical plasma membrane, suggesting that they regained morphological polarity in the medium containing CM-GBF. These results shows that CM-GBF is essential for the growth and the differentiation of GBEC in culture.