Long-term entecavir therapy results in falls in serum hepatitis B surface antigen levels and seroclearance in nucleos(t)ide-naïve chronic hepatitis B patients.

Entecavir (ETV) is reported to result in suppression of hepatitis B virus DNA (HBV DNA) replication with minimal drug resistance. However, information on the long-term effect of such therapy on serum hepatitis B surface antigen (HBsAg) level and elimination of HBsAg is not available. ETV therapy was started in 553 nucleos(t)ide-naïve patients with chronic hepatitis B infection (HBeAg positive: 45%) in our hospital. Serum HBsAg levels were measured serially by the Architect assay. The median baseline HBsAg was 2180 IU/mL (0.12-243 000 IU/mL), and median follow-up period was 3.0 years, with 529, 475, 355, 247 and 163 patients followed-up for 1, 2, 3, 4 and 5 years, respectively. At year 5, the mean log HBsAg decline from baseline was -0.48 log IU/mL, and the cumulative HBsAg clearance rate was 3.5%. Multivariate analysis identified HBV DNA level at baseline (<3.0 log copies IU/mL, odd ratio = 10.2; 95% confidence interval = 1.87-55.5, P = 0.007) and HBsAg level (<500 IU/mL, odd ratio = 29.4; 95% confidence interval = 2.80-333, P = 0.005) as independent predictors of HBsAg seroclearance. These results indicate that although serum HBsAg level declines gradually during ETV therapy, HBsAg seroclearance remains a rare event.

Occult hepatitis B virus infection increases hepatocellular carcinogenesis by eight times in patients with non-B, non-C liver cirrhosis: a cohort study.

An impact of serum hepatitis B virus (HBV) DNA on hepatocarcinogenesis has not been investigated in a cohort of patients with non-B, non-C cirrhosis. Eighty-two consecutive Japanese patients with cirrhosis, who showed negative hepatitis B surface antigen and negative anti-hepatitis C virus, were observed for a median of 5.8 years. Hepatitis B virus core (HBc) region and HBx region were assayed with nested polymerase chain reaction. Both of HBc and HBx DNA were positive in 9 patients (11.0%) and both were negative in 73. Carcinogenesis rates in the whole patients were 13.5% at the end of the 5th year and 24.6% at the 10th year. The carcinogenesis rates in the patients with positive DNA group and negative DNA group were 27.0% and 11.8% at the end of the 5th year, and 100% and 17.6% at the 10th year, respectively (P = 0.0078). Multivariate analysis showed that men (P = 0.04), presence of HBc and HBx DNA (hazard ratio: 8.25, P = 0.003), less total alcohol intake (P = 0.010), older age (P = 0.010), and association of diabetes (P = 0.005) were independently associated with hepatocellular carcinogenesis. Existence of serum HBV DNA predicted a high hepatocellular carcinogenesis rate in a cohort of patients with non-B, non-C cirrhosis.

Single nucleotide polymorphism of the MxA gene promoter influences the response to interferon monotherapy in patients with hepatitis C viral infection.

The biological activity of interferon (IFN) is mediated by the induction of intracellular antiviral proteins, such as 2'-5' oligoadenylate synthetase, dsRNA-activated protein kinase and MxA protein. Among these, MxA protein is assumed to be the most specific surrogate parameter for IFN action. This study was performed to elucidate whether a single nucleotide polymorphism (SNP) (G/T at nt-88) in the promoter region of the MxA gene influences the response to IFN therapy in patients with chronic hepatitis C virus (HCV) infection. Polymorphisms of the MxA gene in 235 HCV patients were determined by polymerase chain reaction-restriction fragment length polymorphism. The frequency of SNP was compared between sustained-responders (n = 78) and nonresponders (n = 157), as determined by biochemical and virological responses to IFN. Multivariate analysis showed that among all patients, HCV genotype, HCV RNA level and the SNP of the MxA gene were independent and significant determinants of the outcome of IFN therapy [odds ratio 3.8 (95% confidence interval 2.0-7.0), P < 0.0001; 0.27 (0.15-0.50), P < 0.0001; 1.8 (1.0-3.4), P = 0.0464, respectively]. Furthermore, among patients with a low viral load (< or =2.0 Meq/mL), MxA-T-positive patients were more likely to show a sustained response compared with MxA-T-negative patients [2.87 (1.3-6.3); 62%vs 36%; P = 0.0075]. Our findings suggested that the SNP of the MxA gene is one of the important host factors that independently influences the response to IFN in patients with chronic HCV infection, especially those with a low viral load.

Wild-type precore and core promoter sequences in patients with acute self-limited or chronic hepatitis B.

BACKGROUND: Mutations in the precore region and core promoter were compared between patients with acute and chronic hepatitis B. METHODS: There were 69 patients with acute self-limited hepatitis B and 210 with chronic hepatitis B who had been followed for > 15 years. The hepatitis B virus (HBV) of genotypes A, B and C was detected in 14 (23%), 8 (13%) and 28 (45%) of the patients with acute self-limited hepatitis, respectively, in contrast to 11 (5%), 25 (12%) and 167 (80%) of those with chronic hepatitis. RESULTS: At presentation, hepatitis B e antigen (HBeAg) in serum was the more common (82% versus 65%, P < 0.05), and the wild-type sequences of the precore region (100% versus 74%, P < 0.001) and core promoter (88% versus 36%, P < 0.00001) were more frequent in the 50 patients with acute self-limited hepatitis than the 203 patients with chronic hepatitis B who were infected with HBV of genotype A, B or C. Wild-types of both the precore region and core promoter persisted in acute self-limited hepatitis, while they decreased from 28% to 10% in chronic hepatitis over the course of > 15 years. CONCLUSION: HBV with the wild-type sequences of the precore region and core promoter prevails in patients with acute self-limited hepatitis, unlike in patients with chronic hepatitis.

Pathogenic significance and organic virus levels in patients infected with TT virus.

Previous reports documented the recovery of a DNA virus from a patient with posttransfusion non-A-G hepatitis and named TT virus (TTV). Although the virus was initially detected as a causative agent of hepatitis, there is doubt about its pathogenicity. The aim of this study was to clarify the relationship between TTV and liver diseases. Histopathological examination of liver biopsies from 14 patients with TTV genotype 1 positive non-B, non-C and non-G chronic hepatitis showed mild fibrosis and periportal/piecemeal necrosis. Using the real-time detection (RTD)-PCR method, we found that TTV DNA levels of genotype 1 in liver samples from 3 such patients were 100- to 1,000-fold higher than those in the paired serum samples. Further investigation using various tissues from 2 autopsies of patients with hepatitis C with hepatocellular carcinoma revealed that the concentrations of TTV DNA in the liver were also higher than in serum samples. However, the highest TTV DNA concentrations in these 2 autopsies were found in the lung and bone marrow, respectively. Our results suggest that TTV may replicate in various tissues including the liver and may cause only mild liver damage.

Randomized controlled clinical trial of lymphoblastoid interferon-alpha for chronic hepatitis C.

Objective: Most of chronic hepatitis C patients with HCV-genotype 1 and a high virus load fail to eradicate the HCV-RNA by the interferon (IFN) or IFN/ribavirin therapy. But in these patients, IFN is often effective with regard to normalization of alanine aminotransferase (ALT). We had therefore the following two randomized controlled clinical trials to evaluate the effect of IFN which reduce ALT and maintain normalization of ALT. One approach (study 1) was to compare the efficacy of a 6 month course of three different dosages of recombinant IFN-alpha-2a in patients with chronic hepatitis C associated with HCV-genotype 1b and a high serum HCV-RNA level of more than 1 Meq/ml. Another approach (study 2) was to make clear the significance of an additional 6 month course of IFN in patients who had biochemical response during the first 6 month course of IFN (study 1). Methods: (1) Study 1; 45 patients with HCV-genotype 1b and a high serum HCV-RNA level of more than 1 Meq/ml were randomly assigned into three equal groups; group 1 was treated with 3 million units (MU), group 2 with 6 MU and group 3 with 9 MU. They were treated with IFN 3 times weekly for 6 months. Biochemical response was defined as normalization of ALT at the 6 month after initiation of IFN; (2) Study 2; Subsequently, of 23 patients with biochemical response by the first study, 22 were randomly assigned to two groups; patients in group A were continued to receive 3 MU of IFN-alpha-2a three times a week for an additional 6 months and patients in group B were discontinued IFN therapy. Results: (1) Study 1; One patient in group 1, three in group 2 and five in group 3 withdrew from IFN therapy because of IFN-related side-effects. Biochemical response was 10 (66.7%) patients of group 1,8 (53.3%) of group 2 and 5 (33%) of group 3 by the intention-to-treat (ITT) analysis. The biochemical response rate in group 1 was slightly higher than that in other two groups by the Cochran Armitage two-tailed test (P=0.066). With respect to serum HCV-RNA level, one patient in group 1, six patients in group 2 and four patients in group 3 became negative for HCV-RNA by reversed transcription nested-polymerase chain reaction (RT nested-PCR) at the end point of first 6 month course of IFN; (2) Study 2; The maintenance rate of ALT normalization was 88.9% (9/11) in group A and 11.1% (2/11) in group B. The maintenance rate of ALT normalization in group A was significantly higher than that in group B by the Fisher exact's test (P=0.0089). With respect to serum HCV-RNA level by RT nested-PCR, four patients in group A had negative HCV-RNA at the end of an additional IFN therapy. On the other hand, all the patients in group B had positive HCV-RNA at the same time. Conclusion: Our data suggested that a prolonged IFN therapy using a dose of 3 MU of IFN-alpha-2a is safe strategy to reduce ALT and to maintain ALT normalization in patients with HCV-genotype 1b and a high serum HCV-RNA level of more than 1 Meq/ml.

Evaluation of quantitative measurements of hepatitis C virus RNA to predict sustained response to interferon by genotype.

Hepatitis C virus (HCV) virus load is one of the most important predictive factors for the outcome of interferon (IFN) therapy. Recent technological advances have allowed a more precise measurement of HCV load. However, the exact cutoff values that could be used to predict the outcome of IFN have not been established for each assay. Five recent quantitative assays were evaluated for the measurement of HCV (Amplicor monitor ver 1.0, Amplicor monitor ver 2.0 (GT), Amplicor monitor ver 2.0 (Cobas), Quantiplex branched DNA amplification (bDNA) ver 2.0 and HCV core protein level by enzyme immunosorbent assay) in 209 consecutive patients with chronic hepatitis C, who received IFN therapy. The results of the two second generation Amplicor monitor tests (GT and Cobas) showed the best correlation (r = 0.930), but the other tests also showed relatively good correlations (r = 0.646-0.925). Each method predicted the effect of IFN with comparable predictive efficacy, ranging from 77.0 to 80.8%. Receiver operating characteristic (ROC) curve analysis showed that Amplicor monitor ver 2.0 and bDNA ver 2.0 are superior in predicting the response in genotype 2a. The best cutoff value for predicting the response to IFN was different by genotype, which should be considered in selecting candidates for IFN treatment.

Risk factors of hepatitis C virus-related liver cirrhosis in young adults: positive family history of liver disease and transporter associated with antigen processing 2(TAP2)*0201 Allele.

The aim of this study was to clinically characterize young patients with hepatitis-C-related cirrhosis. We compared 27 patients with liver cirrhosis (Group LC) who were anti-HCV positive, aged 40 years or less at the time of diagnosis, with 323 consecutive patients with HCV-related chronic hepatitis (Group CH) matched for age and gender. Furthermore, Group LC was divided into two arbitrary groups (29-35 years, n = 8 /36-40 years, n = 19), based on the age of patients at the time of diagnosis of liver cirrhosis. Patients' characteristics and family history were investigated, and the frequency of transporter associated with antigen processing 2 (TAP2) was determined. A family history of liver disease was present in 40.7% of Group LC but in 18.0% of Group CH (P < 0.05). The younger the age of diagnosis of cirrhosis in Group LC, the higher the frequency of a positive family history (29-35 years, 87.5%; 36-40 years, 21.1%, P < 0.05). The frequency of TAP2*0201 was significantly higher in young adult patients with HCV-related liver cirrhosis than in HCV carriers with normal ALT (P < 0.05), and tended to be higher than in uninfected normal subjects (P = 0.05). The cumulative survival rate of cirrhosis patients with family history of liver diseases was significantly lower than that of cirrhosis patients without such history (P < 0.05). Our findings suggest that a positive family history of liver disease and TAP2*0201 polymorphism may be risk factors for HCV-related liver cirrhosis in young adults.

Twice-daily administration of interferon-beta for chronic hepatitis C is not superior to a once-daily regimen.

PURPOSE: Although interferon (IFN) is commonly used for the treatment of chronic hepatitis C virus (HCV) infection, eradication of the virus occurs in only a small proportion of patients with genotype 1b and a high virus titer. Modified IFN therapies have been tried, with only limited benefit. Recently, the administration of IFN-beta twice per day has been reported to be more effective than the usual once-daily administration regimen. The aim of this study was to evaluate whether twice-daily IFN results in a sustained response in patients with chronic HCV infection with genotype 1b, and a high virus titer. METHODS: Twenty patients with genotype 1b and high HCV RNA level (more than 1 MEq/ml by branched DNA probe assay) were randomly assigned to receive either twice-daily 3 MU of IFN beta (group A) or once-daily 6 MU of IFN-beta (group B) for 4 weeks. All patients received a further daily dose of 6 MU IFN-beta for 12 weeks, followed by IFN-alfa three times a week for 16 weeks. RESULTS: Although a rapid fall in HCV RNA levels was noted in group A, a sustained response was observed in only one of nine patients in this group, and none of group B. Adverse effects of IFN were more frequent and pronounced in group A than in group B. CONCLUSIONS: We conclude that further modification, which combines the early strong anti-viral effects of the twice-daily regimen with long-term sustained response, is necessary for effective therapy of HCV patients with genotype 1b and high HCV RNA levels.

Relationship between five-year histological outcome and serial changes in serum alanine aminotransferase in patients with biochemical and virological relapse after interferon treatment for chronic hepatitis C.

OBJECTIVE: The aim of this study was to assess the correlation between serial changes in serum alanine aminotransferase (ALT) levels and histological outcome 5 years after treatment of patients with chronic hepatitis C with interferon (IFN). METHODS: We retrospectively evaluated 61 consecutive patients who underwent two liver biopsies, just before and 5 years after a 6-month course of IFN therapy, and who showed a relapse after therapy. The extent of liver fibrosis was estimated using a scale with seven grades. RESULTS: At the end of 6-month IFN therapy, 40 (65.6%) patients had normal serum ALT concentrations. However, 13 of the 40 patients relapsed within 6 months after IFN therapy. The average ALT during 5 years in 40 patients was less than or equal to 75 IU/l, while in the other 21 patients it was more than 75 IU/l. Among the 40 patients with a mean ALT less than 75 IU/l, 13 (33%) patients showed histological improvement, 26 (65%) showed no changes and only 1 patient (2%) showed worsening of liver fibrosis. On the other hand, among the 21 patients with a mean ALT of more than 75 IU/l, only 1 (5%) patient showed improvement, 8 (38%) showed no changes and 12 patients (57%) showed worsening of liver fibrosis. CONCLUSION: There is a significant relationship between 5-year histological outcome and serial changes in serum ALT in patients with biochemical and virological relapse after IFN treatment for chronic hepatitis C.

Mutations of CLCN5 in Japanese children with idiopathic low molecular weight proteinuria, hypercalciuria and nephrocalcinosis.

The annual urinary screening of Japanese children above three years of age has identified a progressive renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrocalcinosis. The disorder has been observed in over 60 patients and has a familial predisposition. Mutations of a renal chloride channel gene, CLCN5, have been reported in four such families, and we have undertaken studies in additional patients from 10 unrelated, non-consanguineous Japanese families to further characterize such CLCN5 mutations and to ascertain their prevalence. CLCN5 abnormalities we identified in 7 of the 10 unrelated patients and consisted of 5 mutations (2 nonsense, 1 frameshift and 2 missense), 1 deletion and 1 silent polymorphism. A clustering of these mutations in CLCN5 exons 8 and 10 was observed. Over 80% of the CLCN5 mutations could be readily detected by single stranded conformational polymorphism (SSCP) analysis, thereby providing a useful mutation screening method. Our results, which indicate that over 70% of Japanese patients with this renal tubulopathy have CLCN5 mutations, will help in the genetic and clinical evaluation of children at risk from this disorder.

[Bone marrow transplantation for girls with aplastic anemia utilizing modified field of total lymphoid irradiation and cyclophosphamide: with emphasis on the field of pelvic cavity].

A preparative regimen for allogeneic bone marrow transplantation, consisting of total lymphoid irradiation (TLI) with 750 cGy and cyclophosphamide (CY), was used in five girls with aplastic anemia. All patients received bone marrow from HLA matched/mixed lymphocyte culture negative siblings. In our regimen the "inverted Y" field to irradiate the pelvic nodes was modified, which did not include the whole pelvic cavity in an attempt to protect the ovaries from irradiation. Although some of the pelvic nodes was supported not to be irradiated in order to protect the ovaries, engraftment occurred in all five patients including four who had been transfused prior to transplantation. All five are alive from 47 days to 1378 days (median 285 days) after transplantation without transplantation-associated complications. The calculated dose to the ovaries was sixteen percent of the entire dose of the regimen. Both of the two evaluable patients that had received transplantation just before or during the puberty are developing normal sex maturity including menstruation. This study suggests that our preparative regimen is effective not only for engraftment of the donor marrow but also for protecting the ovaries from irradiation.