Ikuo Sakuma

Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection

BACKGROUND A region associated with sensitivity to interferon has been identified in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. The region spans amino acid residues 2209 to 2248 (NS5A2209-2248) of HCV-J, a strain of HCV-1b whose complete genomic sequence has been identified. We examined whether the NS5A2209-2248 sequence present before therapy could be used as a predictor of the response to interferon therapy in patients with chronic HCV-1b infection. METHODS We retrospectively analyzed 84 patients with chronic HCV-1b infection who had received interferon alfa (total dose, 516 million to 880 million units) for six months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209-2248 was determined by direct sequencing of the HCV genome amplified by the polymerase chain reaction (PCR) and was compared with the established sequence for HCV-J. RESULTS A complete response, as evidenced by the absence of HCV RNA in serum on nested reverse-transcription PCR for six months after therapy, did not occur in any of the 30 patients whose NS5A2209-2248 sequences were identical to that of HCV-J (wild type). Five of 38 patients (13 percent) with 1 to 3 changes in NS5A2209-2248 (intermediate type) had complete responses, as did all 16 patients with 4 to 11 amino acid substitutions (mutant type), indicating that the mutant type was significantly associated with a complete response (P < 0.001). Although baseline serum HCV RNA levels, as measured by a branched-chain DNA assay, were lower in patients with the mutant type of NS5A2209-2248 than in those with the other types (P < 0.001), multivariate analyses revealed that the number of amino acid substitutions in NS5A2209-2248 was the only variable associated with an independent effect on the outcome of interferon therapy (odds ratio, 5.3; 95 percent confidence interval, 1.6 to 18; P = 0.007). CONCLUSIONS In patients with chronic HCV-1b infection, there is a substantial correlation between responses to interferon and mutations in the NS5A gene.

Mutations in the core promoter region of hepatitis B virus in patients with chronic hepatitis B

The core promoter region of hepatitis B virus genomes regulates transcription of the precore and pregenomic mRNAs encoding hepatitis B e antigen (HBeAg) and core antigen that contain target epitopes for cytotoxic T lymphocytes. The prevalence and clinical significance of mutations in this region were investigated. DNA was extracted from six asymptomatic carriers positive for HBeAg, eight asymptomatic carriers positive for an anti‐HBe antibody, and 24 patients with chronic liver disease. The core promoter and precore regions of hepatitis B virus genomes were amplified by polymerase chain reaction, and predominant sequences were determined by direct sequencing. Mutations were found in none of the HBeAg‐positive asymptomatic carriers but in all of the anti‐HBe‐positive asymptomatic carriers and the patients with chronic liver disease. Especially, A to T mutations at nucleotide 1762 and G to A mutations at nucleotide 1764 were found in five anti‐HBe‐positive asymptomatic carriers, and 22 patients with chronic liver disease. These two mutation hot spots were located within binding sites of the nuclear factors, and nucleotide 1762 was also involved in the A, T rich sequence that is located 28 base pairs upstream of the precore mRNA initiation site. Serum HBeAg and DNA polymerase levels were significantly lower in patients with these mutations than those without these mutations, and five individuals with these mutations were positive for anti‐HBe despite the absence of the precore stop codon mutation. These mutants may be selected by host immune response to HBeAg and/or core antigen.

Efficacy of endoscopic clipping for bleeding gastroduodenal ulcer: comparison with topical ethanol injection.

Objective:Although endoscopic clipping is used widely for the treatment of bleeding gastroduodenal ulcers, clinical trials on its efficacy are scarce. The aim of this study is to assess the efficacy and safety of endoscopic clipping for hemostasis from bleeding gastroduodenal ulcers.Methods:The present study was designed as a retrospective study using historical controls. One hundred consecutive patients with bleeding gastroduodenal ulcers were treated by endoscopic clipping. The preceding 91 consecutive patients treated by endoscopic pure ethanol injection were regarded as controls. Forty-nine of the clipping group and 41 of the ethanol group had lesions at sites difficult to perform endoscopic manipulation. Hemostatic rates, rebleeding rates, amounts of blood transfusion, and durations of hospital stay were analyzed.Results:The hemostatic rate was 96% in both clipping and ethanol groups, whereas the rebleeding rate was lower (15%vs 29%, p= 0.023) in the former than the latter. In technically difficult cases, the hemostatic rate was comparable (96 vs 90%).Conclusion:In patients with bleeding gastroduodenal ulcers, endoscopic clipping may be a choice of therapy because of a low rebleeding rate compared with pure ethanol injection.

Sequential changes in full-length genomes of hepatitis B virus accompanying acute exacerbation of chronic hepatitis B

BACKGROUND/AIM During the course of persistent hepatitis B virus infection, viral replication markedly decreases after acute exacerbation of liver inflammation accompanied by emergence of antihepatitis B e antibody (anti-HBe) and/or anti-hepatitis B surface antibody (anti-HBs). In some cases, however, persistent viral replication continues even after such exacerbation with or without HBeAg/anti-HBe seroconversion. The aim of the present study was to investigate the extent of genetic variations of HBV in this phenomenon. METHODS Full-length HBV genomes were amplified by polymerase chain reaction from sera of three patients before and after acute exacerbation and were directly sequenced. RESULTS In the whole genomes of 3215 nucleotides, only six nucleotide mutations for six amino acid substitutions (2 in the surface gene, 2 in the X gene, 1 in the core gene and 1 in the polymerase gene) were observed in patient 1, 15 mutations for 14 amino acid substitutions (1 in the pre-core codon 28, 4 in the surface gene, 4 in the core gene and 5 in the polymerase gene) were observed in patient 2, and 5 mutations for 6 amino acid substitutions (2 in the surface gene, 2 in the X gene, pre-core stop codon mutation and 1 in the polymerase gene) were observed in patient 3. Substitution in the a determinant of the surface gene, which encodes target epitopes for neutralizing antibodies, as well as those in the pre-core/core gene, which encodes epitopes for cytotoxic T cells, were mainly found. CONCLUSION HBV that remained after the emergence of anti-HBe and anti-HBs are considered to possess mutations in epitopes for both humoral and cellular immunity. These mutant HBV may be involved in the pathogenesis of persistent hepatic injury after acute exacerbation.

Complete nucleotide sequences of hepatitis B virus genomes associated with epidemic fulminant hepatitis

Pre‐core/core mutants are frequently observed in patients with fulminant hepatitis. To investigate the extent of molecular characteristics of hepatitis B virus (HBV) genomes implicated in the development of fulminant hepatitis, full‐length HBV genomes were sequenced directly from sera of two patients with epidemic fatal fulminant hepatitis, after amplification by the polymerase chain reaction. These two genomes, of 3215 nucleotides, were 99.6% identical, indicating that a common source of HBV potentially caused fulminant hepatitis. Thirty unique nucleotide mutations were commonly found in the two entire HBV genomes. Three were located in the stem‐loop structure, changing this element to a more stable structure. Twenty‐five unique amino acid substitutions were found in each open reading frame, except for the X and pre‐surface 2 genes. One was located in the pre‐surface 1 gene; two were in the surface gene; three were in the pre‐core gene, including codons 28 (tryptophan to stop codon) and 29 (glycine to aspartic acid); eight were in the core gene; and 11 were in the polymerase gene. The pre‐core mutations at codons 28 and 29 were common to the two HBV strains reported previously in patients with epidemic fulminant hepatitis. Thus, HBV genomes associated with epidemic fatal fulminant hepatitis have numerous unique mutations, located mainly in the polymerase gene, as well as the pre‐core/core gene, including mutations in the stem‐loop structure of the pregenome encapsidation signal sequence. These mutations may be associated with the development of fulminant hepatitis.

Selection of hepatitis C virus quasispecies during interferon treatment

SummaryPreliminary studies have shown that hepatitis C virus (HCV) quasi-species populations after interferon therapy are different from those before interferon, suggesting that selection of HCV quasispecies occurs during IFN treatment. To confirm this, fluctuations of HCV quasispecies populations were investigated by single strand conformation polymorphism analysis in eight patients who remained viremic during interferon treatment. In all patients, HCV quasispecies populations changed in 1 to 4 months after the start of interferon therapy. In seven patients, a minor population of HCV quasispecies that was present before the interferon therapy was selected and became predominant during the therapy, whereas the abundance of the other quasispecies was reduced. In the other patient, new HCV quasispecies that had not been detected before interferon therapy appeared and replaced the pretreatment HCV. In contrast, no significant changes were observed during the pretreatment period in these patients. In two patients, deduced amino acid sequences of the hypervariable region were identical despite the difference in nucleotide sequences between interferon-sensitive and interferon-resistant HCV. Thus, selection of HCV quasispecies occurs during interferon treatment and immune responses to the hypervariable region may not be the determining factor of the selection.

Differential Effect of Interferon on Hepatitis C Virus 1b Quasispecies in the Nonstructural Protein 5A Gene

A close correlation was reported between amino acid mutations in the nonstructural protein 5A (interferon [IFN] sensitivity-determining region [ISDR]) of hepatitis C virus (HCV)-1b and the response to IFN therapy. The dynamic change of ISDR quasispecies during IFN treatment was investigated in 22 patients. In 18 nonresponders, the number of ISDR mutations in major quasispecies decreased after therapy (P=.039). In each nonresponder, the percentage of wild-type (no mutations in the ISDR) quasispecies increased after treatment (P=.008), whereas the percentages of intermediate- (1-3 mutations) and mutant-type (>/=4 mutations) quasispecies decreased (P=.037 and P=.043, respectively). No mutant-type quasispecies were detected after therapy. Four complete responders had only quasispecies with >/=3 mutations before therapy. Thus, HCVs have fewer mutations in the ISDR after IFN therapy than those before therapy. These IFN-resistant HCVs were already present before therapy as minor quasispecies and were selected by IFN in nonresponders.