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.

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.

Interferon-gamma is produced by CD8 T cells in response to HLA-A24-restricted hepatitis C virus epitopes after sustained virus loss.

Differences in cytotoxic T lymphocyte activity in hepatitis C virus infection may account for the outcome of interferon monotherapy. To investigate this hypothesis, we analysed the response of peripheral CD8(+) T cells that recognized epitopes presented by HLA-A*2402. We synthesized HLA/beta2-microglobulin/peptide complexes using two epitopes. Production of interferon-gamma by CD8(+) T cells in response to plastic-bound monomeric HLA/peptide complex was observed frequently in sustained virus responders (SVR) (n = 13) against all the peptides, NS31296-1304 (the percentage of responding patients, 61.5%) and core 129-137 (53.8%), while no interferon-gamma production was observed in non-responders (NR) (n = 13) for any of the peptides. Tetramer-staining showed the presence of CD8(+) T cells specific for all the peptides except NS31296-1304 in two SVR at the end of interferon monotherapy, although hardly any such cells were found in four NR. Specific killing was observed against peptides NS31296-1304 (3/4) and core 129-137 (1/4) in sustained responders but none in non-responders. These results suggest that the responses of cytotoxic T lymphocytes (CTLs) were induced during interferon therapy in these patients and that interferon-gamma production by CD8(+) T lymphocytes against HCV NS31296-1304 and core 129-137 are well maintained in patients with SVR compared with those with NR. These findings emphasize the importance of the CD8(+) T cell response in controlling HCV infection.

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

BACKGROUND: Fulminant hepatitis is a clinical syndrome characterized by sudden and severe liver dysfunction. METHODS: We analyzed two patients with a superacute form of fulminant hepatitis B and compared findings with those of four patients with acute self-limited hepatitis B, two patients with acute exacerbation of chronic hepatitis B and four healthy individuals. RESULTS: In fulminant hepatitis, an increased population of human leukocyte antigen (HLA)-DR(+) CD8(+) lymphocytes was observed in peripheral blood by flow cytometry, which was accompanied by the presence of HLA-DR(hi) lymphocytes. The phenotype of CD8(+) T lymphocytes from patients with fulminant hepatitis was mostly that of the effector T lymphocytes in peripheral blood, whereas lymphocytes with CD45RA(-) CCR7(-) phenotype dominated in the liver of these patients. A larger population of hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) appeared in peripheral circulation of the fulminant hepatitis patients compared with that in a patient with acute hepatitis. HBV-specific CTLs were highly concentrated in the liver, although epitopes recognized by these CTLs in the peripheral blood and in the liver were similar. Peripheral CTLs were mostly functional as indicated by intracellular perforin and interferon-gamma. CONCLUSIONS: These results suggest the presence of vigorous activation of CD8(+) T cells in vivo in fulminant hepatitis and the necessity of extensive therapy in patients with this disease.

HBe seroconversion and HBs mutation among Japanese hepatitis B virus carriers.

It has been reported in Germany that seroconversion to anti-HBe or anti-HBs is frequently associated with genotype changes of hepatitis B virus (HBV) from genotype A to genotype D. We previously reported that the HBeAg-negative state in Japan was significantly more common in patients infected with genotype B HBV than those infected with genotype C HBV. To determine whether the high prevalence of genotype B in the HBeAg-negative state is due to a change from genotype C to genotype B, 72 pairs of serum samples before and after HBe seroconversion were examined for nucleotide sequences in the S gene (amino acids 42-164) among Japanese HBV carriers. No one was identified to have undergone genotype change during HBe seroconversion. A total of 71 codon mutations were observed. Sixty-two of these 71 codon mutations (87.3%) were non-synonymous. Genotype B HBV had no mutational hot spots. In genotype C, there was a mutational hot spot at amino acid 126 of the S protein, and it was suggested that Thr126 before HBe seroconversion was more susceptible to becoming an asymptomatic carrier after HBe seroconversion than Ile126. In conclusion, genotype changes during HBe seroconversion were not found to be common in Japan.

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 Sjøgren's 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.

Lambda-opt neural approaches to quadratic assignment problems.

In this article, we propose new analog neural approaches to combinatorial optimization problems, in particular, quadratic assignment problems (QAPs). Our proposed methods are based on an analog version of the lambda-opt heuristics, which simultaneously changes assignments for lambda elements in a permutation. Since we can take a relatively large lambda value, our new methods can achieve a middle-range search over possible solutions, and this helps the system neglect shallow local minima and escape from local minima. In experiments, we have applied our methods to relatively large-scale (N = 80 - 150) QAPs. Results have shown that our new methods are comparable to the present champion algorithms; for two benchmark problems, they are obtain better solutions than the previous champion algorithms.

[Genoepidemiology and pathogenicity of TT virus in Japanese men with history of intravenous drug abuse and tattoo].

Blood contamination has been proposed as TTV transmission. We studied the genoprevalence of TTV in Japanese men with history of intravenous drug abuse and/or tattoo. TTV was identified in serum by a polymerase chain reaction. TTV was detected in 89.7 percent of the men with history of intravenous drug abuse and/or tattoo, 74.4 percent of chronic hepatitis C patients, 78.0 percent of the chronic hepatitis B, and 65.8 percent of chronic hepatitis nonB nonC patients. Serum ALT levels of those infected with TTV alone were 27.2 +/- 17.5 IU/L. In the patients with chronic hepatitis C, serum ALT levels of those coinfected with TTV were similar to serum ALT levels of those without TTV infection. These results suggest that TTV causes no or mild hepatitis.

Low level hepatitis B viremia detected by polymerase chain reaction accompanies the absence of HBe antigenemia and hepatitis in hepatitis B virus carriers.

OBJECTIVES: Because outcome of antiviral treatment in patients with chronic hepatitis (CH) B is difficult to predict, we compared the severity of hepatitis with serum hepatitis B virus (HBV) DNA concentration. METHODS: We studied 40 HBV carriers with distinct stages of chronic infection, 32 HBe antigen (HBeAg)-negative or low-grade positive carriers whose HBV strains did not contain a point mutation at nucleotide 1896, 37 HBeAg-negative carriers with or without hepatitis, and 51 HBeAg-positive CH patients treated with interferon. Serum HBV DNA concentration was measured by the end-point dilution method using a polymerase chain reaction (PCR). The point mutation at nucleotide 1896 was detected by restriction fragment length polymorphism with PCR. RESULTS: Among the stages of chronic HBV infection, the serum HBV DNA concentration was lowest (10(0.67 +/- 0.71) copies/microliter) in HBeAg-negative asymptomatic carriers. A low-level viremia (10(2.10 +/- 1.45) copies/microliter) of HBV strains without the mutation at nucleotide 1896 was associated with an HBeAg-negative state. In HBeAg-negative carriers, the serum HBV DNA concentration in those without hepatitis was significantly lower than in those with hepatitis (10(1.00 +/- 0.89) vs 10(3.31 +/- 1.25) copies/microliter, p < 0.0001); 20 of 21 asymptomatic carriers had an HBV DNA concentration below 10(2) copies/microliter. Patients with serum HBV DNA concentrations below 10(1) copies/microliter. at the end of interferon treatment maintained normal serum alanine aminotransferase concentrations. CONCLUSIONS: A serum HBV DNA concentration below 10(1) copies/ microliter is an important goal for successful treatment of CH-B. PCR is necessary to assess such low-level viremias.

Genoepidemiology and pathogenicity of hepatitis G virus in Japan.

A recently discovered non-A non-B hepatitis virus has been designated hepatitis G virus (HGV). Blood contamination has been proposed as its mode of transmission. We studied the genoprevalence of HGV in Japanese people at high risk. HGV was identified in serum by a reverse-transcription polymerase chain reaction. HGV was detected in 16.0% of intravenous drug users (IDUs) (n = 25), 16.2% of those with tattoos (n = 37), 10.9% of IDUs with tattoos (n = 55), 5.7% of chronic hepatitis (CH)-C patients (n = 87), and in none of the CH-B (n = 50) or CH non-B non-C (n = 46) patients. Serum alanine aminotransferase (ALT) levels of those infected with HGV alone (n = 3) were all within normal range. In the patients with CH-C, serum ALT levels of those coinfected with HGV were similar to serum ALT levels of those without HGV infection. A phylogenetic tree of isolated HGV clones showed that the HGVs of these subjects bore only a distant-resemblance to clones reported from Africa and North America, and that variation in the phylogenetic index of HGV clones was small. These results suggest that HGV clones from different areas have genetic heterogeneity and that HGV causes no or mild hepatitis.

Prevalence of precore-defective mutant of hepatitis B virus in HBV carriers.

Two hundred and seventy-three serum specimens from hepatitis B virus (HBV) carriers were examined for the presence of a characteristic one point mutation at nucleotide (nt) 1896 from the EcoRI site of the HBV genome in the precore region (the preC mutant) using restriction fragment length polymorphism (RFLP) analysis. This assay approach could detect preC mutants or wild-type sequences when either form constituted more than 10% of the total sample. Overall, 65.5% (76/116) of HBeAg-positive carriers had only the preC wild-type. All HBeAg-positive asymptomatic carriers (n = 14) had only the preC wild-type. In patients with chronic hepatitis B and in anti-HBe-positive asymptomatic carriers, increased prevalence of the preC mutant was associated with the development of anti-HBe antibodies and normalization of the serum alanine aminotransferase concentration. Furthermore, 27 (29.0%) of 93 HBeAg-negative carriers had unexpectedly preC wild-type sequences only. Direct sequencing of the HBV precore region of HBV specimens from 24 patients revealed no mutation at nt 1896, supporting the specificity of the RFLP analysis. These results suggest that RFLP analysis was accurate for the detection of the preC mutation and that the absence of serum HBeAg cannot be explained solely by the dominance of the preC mutant.

Changes of serum hepatitis C virus levels in patients with chronic hepatitis C treated with two courses of interferon administration.

To assess the efficacy of repeated interferon (IFN) administration in patients with chronic hepatitis C unresponsive to initial therapy, serum hepatitis C virus (HCV)-RNA levels were measured in 12 patients who had failed prior IFN therapy. Serum HCV-RNA was assayed by measuring DNA complementary to HCV-RNA using a quantitative reverse transcription-polymerase chain reaction assay. The mean total dose of IFN was 227.8 mega-units for first treatment and 270.7 mega-units for second treatment. Five responders with a normal alanine aminotransferase (ALT) concentration (less than 40 IU/liter) at the end of the first treatment also had a normal ALT concentration at the end of the second treatment. By contrast, all nonresponders with an elevated ALT concentration during the first treatment likewise had an elevated ALT concentration at the end of the second treatment. HCV-RNA levels before the first treatment varied from 10(6) to 10(8) copies/microliters. The serum HCV-RNA levels fell in 9 out of 10 patients after the first treatment and in 11 out of 12 patients after the second treatment. One patient had unchanged normal serum ALT levels after two courses of IFN treatment. These results suggested that the outcome of a second course of IFN treatment was similar biochemically and virologically to a first course, and that patients who did not respond initially seldom respond to additional IFN therapy. Therefore, readministration of IFN should be restricted to patients who respond biochemically and virologically to initial treatment. The optimal second dose shall be determined with further studies.