Hirofumi Niitsuma

Two Subtypes of Genotype B (Ba and Bj) of Hepatitis B Virus in Japan

We have previously reported 2 subtypes of hepatitis B virus (HBV) genotype B, one of which has the recombination with genotype C over the precore region plus core gene (Ba) and the other of which does not (Bj). A restriction fragment-length polymorphism method with 2 endonucleases was newly developed for distinguishing between subtypes Ba and Bj and was applied to 313 carriers of HBV genotype B in Japan. Subtype Ba was detected in 38 (12%) and subtype Bj in 275 (88%) of the carriers of HBV genotype B. Hepatitis B e antigen in serum was found more frequently in patients with chronic infection with subtype Ba than in those with chronic infection with subtype Bj (8 [32%] of 25 vs. 25 [9%] of 273; P<.01). The new method for distinguishing between Ba and Bj by restriction fragment-length polymorphism would be useful in examining the distribution of these 2 subtypes in situations in which HBV genotype B is prevalent.

Four‐year study of lamivudine and adefovir combination therapy in lamivudine‐resistant hepatitis B patients: influence of hepatitis B virus genotype and resistance mutation pattern

Summary.  To investigate the efficacy of long‐term lamivudine (3TC) and adefovir dipivoxil (ADV) combination therapy in 3TC‐resistant chronic hepatitis B virus (HBV) infected patients, we analysed 28 3TC‐resistant patients treated with the combination therapy during 47 months (range, 9–75). At 12, 24, 36, and 48 months, the rates of virological response with undetectable HBV DNA (≤2.6 log copies/mL) were 56, 80, 86, and 92%, respectively. Among 17 hepatitis B e antigen (HBeAg)‐positive patients, HBeAg disappeared in 24% at 12 months, 25% at 24 months, 62% at 36 months, and 88% at 48 months. When HBV genotypes were compared, patients with genotype B achieved virological response significantly more rapidly than those with genotype C (P = 0.0496). One patient developed virological breakthrough after 54 months, and sequence analysis of HBV obtained from the patient was performed. An rtA200V mutation was present in the majority of HBV clones, in addition to the 3TC‐resistant mutations of rtL180M+M204V. The rtN236T ADV‐resistant mutation was observed in only 25% clones. In vitro analysis showed that the rtA200V mutation recovered the impaired replication capacity of the clone with the rtL180M+M204V mutations and induced resistance to ADV. Moreover, rtT184S and rtS202C, which are known entecavir‐resistant mutations, emerged in some rtL180M+M204V clones without rtA200V or rtN236T. In conclusion, 3TC+ADV combination therapy was effective for most 3TC‐resistant patients, especially with genotype B HBV, but the risk of emergence of multiple drug‐resistant strains with long‐term therapy should be considered. The mutation rtA200V with rtL180M+M204V may be sufficient for failure of 3TC+ADV therapy.

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.

Ribavirin upregulates interleukin-12 receptor and induces T cell differentiation towards type 1 in chronic hepatitis C

Background and Aim:  The mechanisms of ribavirin as an immune modulator have not been fully revealed, contrary to its clinical benefit in chronic hepatitis C. Recently, host immune defense, especially cytotoxic T lymphocytes and T helper cells, have been considered to be closely related to the pathophysiology of chronic viral hepatitis. The aim of the present study was to evaluate the function of ribavirin in cellular immunity.

Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

Enhanced Replication of Hepatitis B Virus With Frameshift in the Precore Region Found in Fulminant Hepatitis Patients

BACKGROUND The genotype B of hepatitis B virus (HBV) was reported to associate with fulminant hepatitis (FH). We aimed to clarify the characteristics of HBV obtained from FH patients in an area of Japan where genotype B HBV is prevalent. METHODS Using serum samples of 16 HBV-associated FH patients, partial HBV sequences were determined. The effects of HBV mutation/insertion/deletion were evaluated using an in vitro HBV replication system. RESULTS Of the 16 HBV isolates, 31% belonged to subgenotype B1/Bj, 38% were subgenotype B2/Ba, and 31% were subgenotype C2/Ce. Notably, the single nucleotide insertion/deletion that resulted in a frameshift of the precore protein was found exclusively in 60% of B1/Bj strains. An in vitro study showed that all of the frameshift mutants had significantly higher amounts of HBV DNA than did the wild type. One of the isolates had a novel insertion of A between nucleotides 1900 and 1901, which resulted in a 3-nucleotide change within the Kozak sequence of the core protein and enhanced the core protein expression in vitro. CONCLUSIONS The frameshift insertion/deletion in the precore region enhanced HBV replication and might be associated with the development of FH by the subgenotype B1/Bj HBV.

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.

Association between S21 substitution in the core protein of hepatitis B virus and fulminant hepatitis

BACKGROUND The viral factors of hepatitis B virus (HBV), such as genotypes and mutations, were reported to affect the development of fulminant hepatitis B (FHB), but the mechanism is still unclear. OBJECTIVES To investigate HBV mutations associated with FHB, especially in the subgenotype B1/Bj HBV (HBV/B1), which are known to cause FHB frequently in Japan. STUDY DESIGN A total of 96 serum samples from acute self-limited hepatitis B (AHB) patients and 13 samples from FHB patients were used for full-genome/partial sequencing. A total of 107 chronic infection patients with HBV were also examined for the distribution of mutants. RESULTS In the analysis of full-genome sequences of HBV/B1 (FHB, n=11; non-FHB, n=35) including those from the databases, mutations at nt 1961 [T1961V (not T)] and nt 1962 [C1962D (not C)], which change S21 in the core protein, were found more frequently in FHB than in non-FHB (100% vs. 20%, 55% vs. 3%, respectively). When our FHB and AHB samples were compared, T1961V and C1962D were significantly more frequent in FHB than in AHB, both in the overall analysis (46% vs. 6%, 39% vs. 3%, respectively) and in HBV/B1 (100% vs. 29%, 100% vs. 14%, respectively). A newly developed PCR system detecting T1961V showed that HBV/B1 and low viral load were independent factors for the mutation among chronic infection patients. CONCLUSIONS T1961V/C1962D mutations were found frequently in FHB, especially in HBV/B1. The resulting S21 substitution in the core protein may play important roles in the development of FHB.