Hepatitis B Virus Chronic Hepatitis Research Articles

Association of an HLA-G 14-bp Insertion/Deletion polymorphism with high HBV replication in chronic hepatitis.

Identification of an HLA-G 14-bp Insertion/Deletion (Ins/Del) polymorphism at the 3' untranslated region of HLA-G revealed its importance in HLA-G mRNA stability and HLA-G protein level variation. We evaluated the association between the HLA-G 14-bp Ins/Del polymorphism in patients with chronic Hepatitis B virus (HBV) infection in a case-control study. Genomic DNA was extracted from 263 patients with chronic HBV hepatitis and 246 control subjects and was examined for the HLA-G 14-bp Ins/Del polymorphism by PCR. The polymorphic variants were genotyped in chronic HBV seropositive cases stratified according to HBV DNA levels, fibrosis stages and in a control population. There was no statistical significant association between the 14-bp Ins/Del polymorphism and increased susceptibility to HBV infection neither for alleles (P = 0.09) nor for genotypes (P = 0.18). The stratification of HBV patients based on HBV DNA levels revealed an association between the 14-bp Ins/Del polymorphism and an enhanced HBV activity with high HBV DNA levels. In particular, the Ins allele was significantly associated with high HBV DNA levels (P = 0.0024, OR = 1.71, 95% CI 1.2-2.4). The genotype Ins/Ins was associated with a 2.5-fold (95% CI, 1.29-4.88) increased risk of susceptibility to high HBV replication compared with the Del/Del and Ins/Del genotypes. This susceptibility is linked to the presence of two Ins alleles. No association was observed between the 14-bp Ins/Del polymorphism and fibrosis stage of HBV infection. We observed an association between the 14-bp Ins/Del polymorphism and high HBV replication characterized by high HBV DNA levels in chronic HBV patients. These results suggest a potential prognostic value for disease outcome evaluation.

Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA

To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA. Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis. HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice). The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.

RNA-Seq Based Transcriptome Analysis of Hepatitis E Virus (HEV) and Hepatitis B Virus (HBV) Replicon Transfected Huh-7 Cells

Pathogenesis of hepatitis B virus (HBV) and hepatitis E virus (HEV) infection is as varied as they appear similar; while HBV causes an acute and/or chronic liver disease and hepatocellular carcinoma, HEV mostly causes an acute self-limiting disease. In both infections, host responses are crucial in disease establishment and/or virus clearance. In the wake of worsening prognosis described during HEV super-infection over chronic HBV hepatitis, we investigated the host responses by studying alterations in gene expression in liver cells (Huh-7 cell line) by transfection with HEV replicon only (HEV-only), HBV replicon only (HBV-only) and both HBV and HEV replicons (HBV+HEV). Virus replication was validated by strand-specific real-time RT-PCR for HEV and HBsAg ELISA of the culture supernatants for HBV. Indirect immunofluorescence for the respective viral proteins confirmed infection. Transcription profiling was carried out by RNA Sequencing (RNA-Seq) analysis of the poly-A enriched RNA from the transfected cells. Averages of 600 million bases within 5.6 million reads were sequenced in each sample and ∼15,800 genes were mapped with at least one or more reads. A total of 461 genes in HBV+HEV, 408 in HBV-only and 306 in HEV-only groups were differentially expressed as compared to mock transfection control by two folds (p<0.05) or more. Majority of the significant genes with altered expression clustered into immune-associated, signal transduction, and metabolic process categories. Differential gene expression of functionally important genes in these categories was also validated by real-time RT-PCR based relative gene-expression analysis. To our knowledge, this is the first report of in vitro replicon transfected RNA-Seq based transcriptome analysis to understand the host responses against HEV and HBV.

Hepatitis B virus-mediated effects on host expression of the proprotein convertase Furin

To study the effects of hepatitis B virus (HBV) infection on the expression of Furin, an important proprotein convertase, in liver cells to provide insights towards its potential as a therapeutic target for improved antiviral efficacy. Furin expression was measured in human liver specimens (infected tissues from patients with chronic HBV hepatitis vs. normal tissues from healthy donors) and in hepatoma cell lines (HBV-infected HepG2.2.15 cells vs. uninfected parental cell lines HepG2) using quantitative real-time RT-PCR (for mRNA), western blotting and immunohistochemistry (for protein). Compared to the uninfected tissues and cells, the HBV-infected tissue and cells showed down-regulated expression of furin at both the mRNA and protein levels. In particular, the HepG2.2.15 cells showed -50% less furin mRNA expression than the HepG2 cells and the difference was statistically significant (P less than 0.05). HBV may suppress the host cell's expression of furin, possibly to benefit its survival and replication in the host cell.

The pharmacology and activity of non-steroidal anti-inflammatory drugs (NSAIDs): a review of their use as an adjuvant treatment in patients with HBV and HCV chronic hepatitis

Introduction: Different DNA and RNA viruses exploit common strategies to support their persistence and replication in infected individuals. In particular, the hepatitis B virus (HBV) and the hepatitis C virus (HCV) cause major health problems worldwide. These pathogens exert an immunosuppressive role by inducing the persistent activation of cyclooxygenase-2 (COX-2) and an increased synthesis of prostaglandin E2 (PGE2). The suppression of this proinflammatory network by non-steroidal anti-inflammatory drugs (NSAIDs) has been proposed as a therapeutic approach to decrease viral replication. Materials and methods: In this review, the role of inflammation in the support of viral replication and NSAIDs and ketoprofen pharmacology are briefly discussed. In addition, studies that have investigated the use of NSAIDs for the treatment of HBV and HCV chronic hepatitis, which were identified by a systematic literature search of PubMed and MEDLINE, are reported. Results: To date, pegylated-interferon (PEG-IFN) and/or nucleot(s)ide analogues and PEG-IFN and ribavirin remain the standard therapy for HBV and HCV chronic hepatitis, respectively. Discussion: The use of NSAIDs in patients with chronic viral hepatitis has only a ‘‘historical’’ interest. Nevertheless, the possible usefulness of ketoprofen with PEG-IFN and ribavirin for HCVinfected patients, non-responders to standard therapy or with genotype 1, should be evaluated in future clinical studies.

HLA class II associated with outcomes of hepatitis B and C infections

Several factors influence the clinical course of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. The human leukocyte antigen (HLA) system, the major histocompatibility complex (MHC) in humans, has been considered one of the most important host factors with respect to outcomes. To date, conventional genotyping studies have shown that HLA class II loci are mainly associated with spontaneous clearance of HBV and HCV. However, the specific HLA locus associated with the outcomes of hepatitis virus infection remains unclear. A recent genome-wide association study (GWAS) using a comprehensive approach for human genotyping demonstrated single nucleotide polymorphisms (SNPs) associated with the outcomes of hepatitis virus infection. Examination of large numbers of cohorts revealed that several SNPs in both HLA-DPA1 and HLA-DPB1 loci are associated with persistent HBV infection in Asian populations. To date, however, few studies have focused on HLA-DP because polymorphisms of HLA-DP haplotype do not vary greatly as compared with other loci of HLA. There are not enough studies to reveal the function of HLA-DP. GWAS additionally detected candidate SNPs within HLA loci associated with chronic HBV or HCV hepatitis, hepatic fibrosis, and the development of hepatocellular carcinoma. The results of one cohort were not always consistent with those of other cohorts. To solve several controversial issues, it is necessary to validate reported SNPs on HLA loci in global populations and to elucidate the HLA-allele-regulated molecular response to hepatitis virus infection.

Predictive value of serum gelsolin in hepatitis B virus (HBV)-related chronic liver disease

Gelsolin, an actin-binding protein, which serves as a substrate of caspase in tissue injury has been proposed as a prognostic marker in acute liver injury, but its relationship with human hepatitis B virus (HBV)-related hepatitis at various stages is still unclear. This study was conducted in order to investigate the predictive value of serum gelsolin in HBV-related chronic liver disease. The observation group included 101 patients with HBV-related chronic liver disease and 96 healthy adults were selected as the control group. The enzyme linked immunosorbent assay (ELISA) method was used to detect the gelsolin level in the serum. The concentration of serum gelsolin level of the observation group (54.099 ± 23.688 ig/ml) was much lower (P = 0.000) than that of the control group (186.372 ± 37.549 ig/ml). The concentration of serum gelsolin in chronic HBV hepatitis patients (64.158 ± 21.389 ig/ml), liver cirrhosis patients (50.067 ± 21.658 ig/ml) and acute-on-chronic liver failure patients (37.012 ± 21.231 ig/ml) was considerably different (P =0.013) and decreased as the severity of the pathogenetic condition increased. Serum gelsolin levels of patients with HBV-related chronic liver disease had negative correlations with model for end-stage liver disease (MELD) scores (r = -0.348, P = 0.001) and Child-Pugh scores (r = -0.487, P = 0.001). The serum gelsolin level may be a potential marker of the severity of hepatic injury. Key words : Gelsolin, hepatitis B virus (HBV)-related chronic liver disease, predictive value.

Reactivation of Hepatitis B Virus (HBV) Associated with Rituximab Use in Lymphoma: Under Reporting and Lack of Data Quality Reported to the FDA.

Abstract 1370Poster Board I-392 Background:HBV is a major public health problem with infection that can lead to cirrhosis, liver failure, and death. Immunosuppressive therapy, such as steroids and/or chemotherapy, is known to cause a flare or “reactivation” of HBV (HBV-react). Rituximab was approved in 1997 for the treatment of B-cell lymphoma. In 2004, based on 3 case reports, the FDA warned healthcare professionals of rituximab-associated HBV-react. Since that time, multiple cases and retrospective series of rituximab-induced HBV-react have been reported in the literature. However, the characteristics and scope of this association still remains largely unknown. We evaluated the characteristics of patients with lymphoma who developed HBV-react after exposure to rituximab, and the quality of case reports available in the medical literature and at the FDA. Methods:Data sources included 2 observations at Northwestern Memorial Hospital, 83 reports from the medical literature, and 10 reports obtained from the FDA MedWatch database (n=97). Two reports of rituximab-related HBV-react not associated with lymphoma (vasculitis and gloumerulonephritis) were excluded. HBV-react was defined as ≥ 2-fold increase in serum HBV DNA with an increase in serum ALT compared with baseline. A completeness analysis was performed comparing cases submitted to the FDA MedWatch database vs. the medical literature and active surveillance. Results:Of 83 unique cases of HBV-react associated with rituximab reported in the literature, 28 were published as case reports, while 55 were included in case series. Of these 83 cases, 46 occurred in patients (pts) with anti-HBV core antibody (HBcAb+) in the absence of HBV surface antigen (HBsAg-), while 37 cases involved pts with chronic HBV hepatitis (i.e, HBsAg+). Among the 28 case reports and 2 Northwestern cases (n=30; HBcAb+ n=16, HBsAg+ n=14), the median age at HBV-react was 55 years (23M/9F). Histology of these cases were diffuse large B-cell lymphoma (n=19), indolent lymphoma (n=8), CLL (n=2), and mantle-cell lymphoma (n=1). In terms of concomitant treatment at time of HBV-react, 25 pts were receiving concurrent immunosuppressive therapy (chemotherapy +/- steroids n=22 and steroids n=3), while only 5 cases involved single-agent rituximab treatment. Each of these 5 latter pts had received chemotherapy prior to rituximab treatment (2, 3, 12, 24, and 34 months). The median number of rituximab doses received prior to HBV-react was 6 (range 3-10). The median time from last rituximab dose to HBV-react was 5 months (0-21 months); of note, 30% of cases occurred >6 months from last rituximab dose. In terms of outcome, 60% of pts experienced fulminant liver failure, while the remaining had HBV-related hepatitis. Furthermore, 40% (12/30) of pts died due to HBV-react. Quality comparison of source data of the literature and surveillance reports vs. the FDA is contained in Table 1. Overall completeness ratio for literature and observed reports vs. the FDA was 2.18. Of rituximab-related HBV-react occurrences (n=55) reported in 8 case series, five studies included a control group; there was a suggestion of increased risk of HBV-react with rituximab-based therapy, especially with concurrent steroids, although the absolute risk was not consistent (e.g., among HBcAb+ cases, reported rate of HBV-react: 2.7% to 23.8%). Conclusions:We have found 95 total cases of rituximab-associated HBV-react. A paucity of safety reports regarding rituximab-associated HBV-react have been reported to the FDA MedWatch. Furthermore, published cases in the medical literature and through active surveillance were superior in data quality compared with FDA reports. However, the absolute risks of rituximab-related HBV-react in HBsAg+ or HBcAb+ pts are still not known. Further examination of the relationship of HBV-react with single-agent rituximab and rituximab combined with immunosuppressive therapy, with and without steroids, using an active surveillance strategy is warranted.Table 1Completeness/quality of data.Type of InformationLiterature and Observed Cases (n=30) % ReportingFDA Cases (n=10) % ReportingCompleteness RatioHBV status*100205.00NHL subtype94801.18Age and gender94901.07Prior/concurrent immunosuppressive therapy100601.67Number of rituximab doses94601.57Time from last dose of rituximab100701.43Liver outcome97501.93Treatment of reactivation91204.55Survival97801.21*HBcAb+ and/or HBsAg+ Disclosures:No relevant conflicts of interest to declare.

Hepatitis D outbreak among children in a hepatitis B hyper-endemic settlement in Greenland

Hepatitis B virus (HBV) infection is endemic in Greenland with 5-10% of the population being HBsAg-positive (chronic carriers). Surprisingly, despite of the high prevalence of HBV infection, acute and chronic hepatitis B, liver cirrhosis and primary hepatocellular carcinoma appear much less frequently than expected. The reasons for the low frequencies are unknown, but as a consequence implementation of a childhood HBV vaccination programme, though debated for years, has never been instituted. We describe an outbreak of hepatitis D (HDV) infection among children in a hepatitis B hyper-endemic settlement of 133 inhabitants on the west coast of Greenland. In 2006 a total of 27% of the inhabitants were HBsAg-positive (chronic carriers) and 83% were HBcAb-positive (previously exposed). Forty-six percent of the HBsAg-positive persons were below 20 years of age. On follow-up 1 year later a total of 68% of the HBsAg-positive persons were HDV-IgG positive. Five children, who were HBsAg-positive in 2006, had HDV-seroconverted from 2006 to 2007, indicating a HDV-super-infection. Most of the HDV-IgG positive children had markedly elevated liver enzymes. In the multivariate analysis, among the HBV and HDV markers, presence of HDV-IgG was most strongly associated with elevation of liver enzymes. In conclusion, the HBV-HDV super-infection and presumed HDV outbreak in this settlement challenges the notion that HBV infection may not be as harmless in Greenland as previously anticipated. The findings strongly suggest that HBV vaccination should be included in the child-immunization program in Greenland.

Use of Tumor Necrosis Factor-α-blocking Agents in Hepatitis B Virus-positive Patients: Reports of 3 Cases and Review of the Literature

To evaluate the development of hepatitis B virus (HBV) infection in patients receiving tumor necrosis factor-alpha-blocking agents (TNFBA), and to evaluate whether lamivudine (LAM) prophylaxis can reduce the risk of viral reactivation in inactive HBsAg carriers. Local experience and published reports were reviewed. Patients with HBV infection were classified as having chronic HBV hepatitis, or being inactive HBsAg carriers or occult carriers. Three patients in our series and 24 patients in the literature were identified: 2 had active HBV-associated disease, 23 were inactive HBsAg carriers, and 2 occult carriers. When exposed to TNFBA, HBsAg-inactive carriers pretreated with LAM had lower risk of having detectable HBV-DNA (p=0.02) or viral reactivation (p=0.046) than those without LAM prophylaxis. In 3 patients who discontinued TNFBA, LAM prophylaxis was also discontinued 10-12 months thereafter without hepatitis flares. Two cases of reactivation in occult carriers (HBsAg-negative, anti-HBs+, anti-HBc+) were described in the literature. TNFBA should be avoided in patients with active HBV replication and should be used with caution in inactive HBsAg carriers. In these patients, the risk of viral reactivation seems to be high, but it might be reduced by prophylactic LAM, which should probably be given for a long time when TNFBA are discontinued (e.g., 12 mo). Potential occult carriers might carry a low, but not negligible, risk of viral reactivation. They should therefore be monitored with particular care.

Radiation Dosimetry and Biodistribution in Monkey and Man of 11C-PBR28: A PET Radioligand to Image Inflammation

(11)C-PBR28 ([methyl-(11)C]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine) is a recently developed radioligand to image peripheral benzodiazepine receptors (PBRs) in brain. The aim of this study was to estimate the human radiation doses of (11)C-PBR28 based on biodistribution data in monkeys and humans. In addition, we scanned 1 human subject who fortuitously behaved as if he lacked the PBR binding protein. Whole-body PBR images were acquired after intravenous bolus administration of (11)C-PBR28 in 7 healthy humans (651 +/- 111 MBq) and 2 rhesus monkeys (370 +/- 59.9 MBq). One monkey was scanned after receptor blockade with PK 11195 (10.7 mg/kg intravenously). For typical subjects (subjects 1-6), the 3 organs with highest exposure were those with the high PBR densities (kidneys, spleen, and lungs), and the effective dose was 6.6 microSv/MBq. The unusual subject (subject 7) had 60%-90% less uptake in these 3 organs, resulting in 28% lower effective dose. The activity in the baseline monkey scans was greater than that in humans for organs with high PBR densities. For this reason, the human effective dose was overestimated by 60% with monkey biodistribution data. The monkey with receptor blockade had an overall distribution qualitatively similar to that of the unusual human subject (subject 7), with decreased exposure to lungs, kidney, and spleen. The effective dose of (11)C-PBR28 was modest and was similar to that of several other (11)C-radioligands. Lack of receptor binding in the unusual human subject and in the monkey with receptor blockade decreased exposure to organs with high PBR densities and enhanced uptake in excretory and metabolic pathways.

Analysis of differential gene expression between chronic hepatitis B patients and asymptomatic hepatitis B carriers

Chronic hepatitis B virus (HBV) infection remains a serious global health problem, inducing a spectrum of diseases, including asymptomatic HBV carriage (ASC) and chronic hepatitis B (CHB). ASC and CHB represent different immunological states and their prognoses are diverse. To clarify molecular mechanisms underlying the two infection states, the differentially expressed genes between the two states were screened and identified. Subtracted complementary DNA libraries by suppression subtractive hybridization, dot blot hybridization and quantitative real-time PCR were used to identify the differentially expressed genes between subjects with CHB and those with ASC. RNA from peripheral blood mononuclear cells from CHB and ASC subjects was subjected to suppression subtractive hybridization and resulted in isolation of subtracted complementary DNA clones. Eighty-eight randomly sampled clones were rescreened by dot blot hybridization, from which 29 clones were identified as differentially expressed genes. The differential expression of three genes was confirmed by real-time PCR in 23 subjects with CHB and 21 with ASC. Differentially expressed genes in peripheral blood mononuclear cells between CHB and ASC have been isolated by suppression subtractive hybridization, including some new genes. Of the up-regulated genes in CHB, most are known to be responsive to inflammatory conditions. These genes might provide clues in elucidating the mechanisms of the two different HBV infection states and designing therapeutic targets for HBV infection.

Analysis of HLA‐DQB1 gene polymorphisms in asymptomatic HBV carriers and chronic hepatitis B patients in the Chinese Han population

Host genetic factors and environment factors including hepatitis B virus (HBV) genotype are widely viewed as common basis of the different outcomes of HBV infection. Human leucocyte antigen (HLA) plays an important role in immunological reaction to HBV infection. The study aimed to explore whether the HLA-DQB1 allele polymorphisms are associated with the outcomes of HBV infection in the Chinese Han population. One hundred and thirty-four asymptomatic HBV carriers and 139 chronic hepatitis B patients were recruited in this case-control study in Beijing, China. Sequence-specific primers-polymerase chain reaction was used to detect 13 alleles of HLA-DQB1 gene. The frequency distributions of alleles in two groups were analysed using sas 9.1.2 software. After adjustment of confounders the frequencies of HLA-DQB1*0503 allele and *0303 allele in chronic hepatitis B group were statistically significant lower than those in asymptomatic HBV carrier group (P = 0.04; P = 0.05), and the frequency of exposure to alcohol consumption in patients with chronic hepatitis B was clearly higher than that in asymptomatic HBV carrier group (P = 0.004). HLA-DQB1*0503 allele and *0303 allele are independently resistant genetic factors to chronic hepatitis B, and alcohol consumption is the risk factor of chronic hepatitis B.

Significance of hepatitis B virus genotypes A to E in a cohort of patients with chronic hepatitis B in the Seine Saint Denis District of Paris (France)

The aim of this study was to examine the genetic variability of the hepatitis B virus (HBV) and its significance. HBV genotypes, core promoter and precore mutants were characterized in 109 consecutive patients with biopsy-proven HBV chronic hepatitis. Genotypes A (26.6%), B (12.8%), C (18.3%), D (18.3%), and E (14.7%) indicate a wide genotypic distribution. Patients were from Asia (30.3%), Europe (28.4%), Sub Saharan Africa (23.9%), the Caribbean (10.1%), North Africa (5.5%), and Madagascar (1.8%). HBV genotypes A and D (HBV/A and /D) infected all subgroups except Asian patients. HBV/B or /C were found in 97% of Asian patients, whereas HBV/E only infected sub-Saharan African and Caribbean patients. Differences according to genotypes were: an increased prevalence of anti-HBe antibodies in patients infected with HBV/D (P = 0.003), higher serum transaminases in patients infected with HBV/A and/D (P = 0.043), more severe liver fibrosis in patients infected with HBV/A, /C and/D (P = 0.02). Precore and core promoter mutants were found in 87% of anti-HBe positive patients, and were associated with HBV/D (P = 0.04) and severe liver fibrosis (P = 0.002). It is concluded that HBV genotypes A, B, C, D, and E circulate in the Seine Saint Denis District, reflecting the geographical origin of patients. HBV/A, /C and/D seem to be associated with more severe hepatic disease.

Natural leukocyte interferon alfa for the treatment of chronic viral hepatitis in heart transplant recipients.

A more rapid and aggressive course of hepatitis B virus (HBV)-related and hepatitis C virus (HCV)-related infection in organ transplant recipients has been described. Interferon alfa is the most accepted drug for treating HBV and HCV chronic infections. However, the use of interferon alfa-N3 has been contraindicated in heart transplant (HTx) recipients because of the hypothesized greater risk of triggering acute cellular rejection. The aim of this clinical pilot study was to evaluate tolerability, safety, and efficacy of natural leukocyte interferon alfa in the treatment of chronic HBV and HCV in HTx recipients. Seven HTx recipients were enrolled in the study: two with HBV, four with HCV, and one with combined HBV-HCV chronic infection. The patients had a mean follow-up after heart transplantation of 8.5+/-3 years, before starting interferon alfa-N3 treatment at a dose of 6 MU three times per week, intramuscularly for 12 months. All patients completed the treatment with no major side effects. No unexpected episodes of acute cellular rejection were observed during the treatment. Mean aminotransferase serum levels were significantly lower than before transplantation at 3 (P<0.03), 6 (P<0.02), and 12 (P<0.02) months of treatment and at the 12-month follow-up (P<0.02). A complete and sustained response was achieved in all subjects with HBV-related chronic hepatitis, whereas sustained virologic response was observed in one of four HCV patients. The preliminary data emerging from our study indicate that natural leukocyte interferon alfa-N3 can be safely administered in HTx recipients with chronic HBV or HCV viral hepatitis. Further studies with larger numbers of patients are needed to assess the efficacy of interferon alfa-N3 on HCV virologic response.

Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China

We assessed the separate and combined effects of hepatitis B virus (HBV), hepatitis C virus (HCV), and aflatoxin in causing hepatocellular carcinoma (HCC) in Qidong, China. A consecutive series of 181 pathologic-diagnosed HCC cases were studied for hepatitis B surface antigen (HBsAg), anti-HBc, HBV X gene sequence, anti-HCV, the 249ser-p53 mutation, and chronic hepatitis pathology. Each of the 181 incident HCC cases had markers for HBV infection and hepatitis pathology; only 6 of 119 cases were coinfected with HCV. The 249ser-p53 mutation was found in 54% (97/181) of HCC cases and in all 7 cases with tissue for analysis from the hepatitis cohort but in none of 42 matched cases from Beijing. The estimated cumulative dose of aflatoxin B1 in these 7 cases ranged from 0.13 to 0.49 mg/kg. Follow-up data through 13.25 years on a cohort of 145 men with chronic HBV hepatitis showed that the relative risk from aflatoxin exposure was 3.5 (1.5-8.1). A similar relative risk was found using 249ser-p53 mutation as a marker for aflatoxin exposure. In conclusion, HBV hepatitis is ubiquitous in Qidong HCC cases, whereas HCV contributes little to its risk. The 249ser-p53 mutation appears to result from coexposure to aflatoxin and HBV infection. Even modest levels of aflatoxin exposure tripled the risk of HCC in HBV-infected men. (H EPATOLOGY 2002;36:1214-1220.)

Induction of oral tolerance towards hepatitis B envelope antigens in a murine model

Background: Hepatitis B virus (HBV) is a non-cytopathic virus, and the hepatocellular injury that occurs as a consequence of HBV infection is mediated by the host antiviral immune response. Subjects with natural tolerance to HBV have minimal or no liver injury despite chronic viremia. We have shown that immune tolerance towards viruses can be induced by oral administration of viral proteins. Aims: To test whether oral induction of tolerance can be induced towards HBV antigens, and whether oral tolerance induction downregulates preexisting anti-HBV immune response. Methods: Oral tolerance was induced via feeding of five low oral doses of HBV proteins (HBsAg+preS1+preS2, BioHepB). This was followed by two inoculations with the BioHepB vaccine. Humoral immune tolerance was evaluated by measuring serum levels of anti-HBs antibody titers at monthly intervals. To determine if oral tolerance induction downregulates pre-existing anti-HBs immunity, mice were inoculated twice with the BioHepB vaccine, followed by feeding of BioHepB-HBV proteins. Results: Feeding of HBV proteins markedly inhibited production of anti-HBs antibodies in naive mice. Anti-HBs titers were 45 versus 135 mIU/ml, in tolerized versus non-tolerized controls ( P<0.005). Moreover, oral tolerance induction effectively down-regulated pre-existing immunity and reduced the anti-HBs titers in previously immunized mice to 112 versus 223 mIU/ml, in tolerized compared with non-tolerized controls ( P<0.01). Conclusions: Induction of oral tolerance towards HBV proteins downregulates the antiviral humoral immune response in naive mice, and in the presence of preexisting anti-HBV immunity. This approach should be further investigated as a method for alleviation of antiviral-mediated liver injury in chronic HBV hepatitis.

Increased risk of hepatocellular carcinoma in male hepatitis B surface antigen carriers with chronic hepatitis who have detectable urinary aflatoxin metabolite M1.

We followed 145 men with chronic hepatitis B virus (HBV) hepatitis for 10 years to determine whether exposure to aflatoxin, or concomitant exposure to hepatitis C virus (HCV), or family history of hepatocellular carcinoma (HCC) increased the risk of developing HCC. We collected 8 monthly urine samples before beginning follow-up and pooled them to detect aflatoxin metabolite M1 (AFM1). AFM1 was detected in 78 (54%) of the subjects. The risk of HCC was increased 3.3-fold (with a 95% confidence interval of 1.2-8.7) in those with detectable AFM1 (above 3.6 ng/L). This relative risk was adjusted for age and for HCV status. The attributable risk from exposure to detectable AFM1 was 0.553 (0.087, 0.94). The relative risk of fatal cirrhosis for those with elevated AFM1 was 2.8 (0.6, 14.3), and the odds of having a persistently elevated alanine transaminase (ALT) were 2.5-fold greater in those with detectable AFM1 (P =.007). Concomitant infection with HCV increased the risk of HCC 5.8-fold (2. 0-17), adjusted for age and AFM1 status. A family history of HCC increased the risk of HCC 5.6-fold, adjusted for age and AFM1. Four men with detectable AFM1 and HCC all had missense mutation in codon 249 of the p53 gene in cancer tissues. This study shows that exposure to AFM1 can account for a substantial part of the risk of HCC in men with chronic HBV hepatitis and adds importantly to the evidence that HCV and family history of HCC increase the risk of HCC in men with chronic HBV hepatitis.

Replication of Hepatitis B Virus Which Carries Foreign DNA in Vitro

Targeting a specific DNA sequence to the desired tissues is an important step in gene therapy. The hepatitis B virus (HBV) is the only DNA virus that has hepatocyte specificity. We attempted to construct an HBV-based vector for targeting the liver. We observed the replication and secretion of virus particles in an HBV construct that lacks X gene and carries an extra 63 bp DNA fragment in vitro. Replication was observed in the cell line HuH-7 but not HepG2. From this construct, we designed an HBV-based vector that could carry foreign DNA. HBV based vectors provide for the possibilities of generating therapeutic agents for individual patients. Our host vector system may be used to clear out the HBV from the HBV carrier or chronic hepatitis B patients by introducing a genetically engineered HBV into these patients.

HEPATITIS B: Clinical Perspectives in Pediatrics

Hepatitis B virus (HBV) can cause acute and chronic liver disease in children. Age at the time of acquisition of primary HBV infection is the major determinant of chronicity, with the risk of chronic infection with HBV related inversely to the age at the time infection occurs. Perinatal transmission usually results in chronic HBV carrier status, although children are most often asymptomatic or have slow disease progression. The average time to onset of clinical liver disease following infection may range from 10 to 40 years. Many HBV carrier children, therefore, may not be brought to medical attention for appropriate counselling and anticipatory guidance in a timely fashion. Adolescence may present risk behaviors of sexual and illicit drug experimentation and alcohol use, which can additionally stress a compromised liver. Sequelae of chronic HBV infection include fulminant hepatic failure, chronic hepatitis, cirrhosis, and liver cancer. Hepatitis B is one of the major vaccine-preventable diseases of mankind. Significant progress has been seen in countries that have integrated the hepatitis B vaccination program into their Expanded Program of Immunization for children. The goal is universal vaccination for children in every country of the world. The pediatric perspective needs to emphasize an evolving continuity of care of chronic HBV hepatitis in the patient through infancy, childhood, adolescence, and adulthood. This article addresses these issues.