Preliminary results of interferon-alpha therapy on woodchuck hepatitis virus-induced hepatocarcinogenesis: possible benefit in female transgenic mice.

BACKGROUND: C-myc activation is a potent oncogenic event in hepatocarcinogenesis. The aim of this study was to test the preventive effect of interferon-alpha (IFN-alpha) on the development of dysplasia and subsequent hepatocellular carcinoma (HCC) in transgenic (Tg) mice overexpressing c-myc in the liver. METHODS: The WHV/c-myc Tg mice recapitulating woodchuck hepatitis virus-induced hepatocarcinogenesis were treated with IFN-alpha, starting early in life until sacrifice at pre-neoplastic or neoplastic stages. Transgene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), hepatocyte proliferation was assessed by bromodeoxyuridine incorporation and RT-PCR for proliferating cell nuclear antigen, and apoptosis was assessed by in situ nick-end-labeling of DNA. RESULTS: C-myc expression and hepatocyte proliferation were significantly reduced in treated female mice, without modification of apoptosis, correlating with a lower severity of dysplasia in 9/12 treated animals at pre-neoplastic stages. At the neoplastic stage, 2/3 treated females neither exhibited carcinoma nor dysplasia, while all 6/6 untreated mice and 3/3 treated males developed carcinomas. CONCLUSIONS: Inhibition of c-myc and hepatocyte proliferation by long-term administration of IFN-alpha was associated with a decrease, or a delay, of oncogenesis in the mouse Tg HCC model. Whether c-myc and hepatocyte proliferation down-regulation could be relevant parameters of IFN-alpha efficiency for hepatocarcinogenesis prevention in cirrhotic patients should be established.

Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity.

BACKGROUND/AIMS: We aimed to clarify the clinical relevance of hepatitis B virus pre-core mutant detection in patients with chronic hepatitis B using a newly developed assay. METHODS: Viral genotypes and pre-core mutations were studied in relation to viral persistence and liver disease severity using INNO-LiPA methodology. The study group included 151 patients with chronic hepatitis B, 85 positive for HBeAg (group I) and 66 positive for anti-HBe (group II). RESULTS: The prevalence of viral genotypes in group I was: 64% A, 1% B, 15% C, 19% D, 0% E, 0% F and in group II: 39% A, 0% B, 2% C, 56% D, 2% E, 2% F (p<0.001). The prevalence of mutations at pre-core codon 28 (M2) was lower in group I (5%) than in group II (64%) (p<0.001). The prevalence of pre-core promoter mutations was also lower in group I (21%) than in group II (61%) (p<0.001). M2 mutations were more frequently detected in genotype D than in genotype A (p<0.001), while the other mutations were not influenced by viral genotype. Serum HBV DNA levels were significantly lower in group II versus group I (p<0.001), and in patients with any of the pre-core mutations versus wild-type sequence (p<0.01). Although cirrhosis was more frequent in group II (37%) versus group I (22%) and in patients with either one of the pre-core mutation (31%) versus wild-type sequence (25%), there was no statistical difference in liver severity assessed by ALT levels and Knodell score. CONCLUSION: Pre-core mutants, whose molecular pattern is strongly dependent on viral genotypes, are associated with viral persistence in anti-HBe positive patients with ongoing chronic hepatitis B. The availability of this rapid assay should allow a precise monitoring of viral pre-core mutants during the course of chronic hepatitis B.

Immune responses against hepatitis C virus structural proteins following genetic immunisation.

We have used direct DNA inoculation to study the in vivo induction of both humoral and cellular immune responses to hepatitis C virus (HCV) encoded structural antigens. Following immunisation of mice, immune responses were compared using plasmids encoding full-length or partial HCV gene sequences for the nucleocapsid and envelope E2 proteins. Plasmids encoding secreted or non-secreted forms of the immunogens, including constructs expressing HCV sequences fused with the hepatitis B virus surface antigen (HCV-HBV chimeras), were evaluated. Results indicate that: (i) all constructs induced specific anti-HCV antibodies; (ii) antibody titres ranged from 1:100 to > 1:100,000; (iii) all HCV DNA immunogens induced a predominant Th1 response with the induction of IgG2a antibodies; (iv) the secretion level of the antigens and immune responses was not always correlated and (v) CTL could be detected against both HCV and HBV determinants.

Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid: comparative studies of antibody and T-helper responses following genetic immunization.

In a murine model, we have compared humoral and T-helper (Th) responses induced following genetic immunization with two hepatitis C virus (HCV) plasmid-derived immunogens: a plasmid expressing the full-length nucleocapsid (CAP) as a nonsecreted antigen (pCMVC2) and a plasmid expressing the amino-terminal part of CAP as a secreted antigen (pS2S.C2N). In BALB/c mice, intramuscular injection of either plasmid induced IgG2a antibodies associated with a Th1-like profile characterized by the in vitro splenic production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma). The pS2S.C2N plasmid induced antibody titers three- to five-fold higher than those obtained with the pCMVC2 plasmid (maximal titers 1:1,500 versus 1:500). In control experiments, immunization using purified CAP antigen induced a predominant, but not exclusive, Th2-like profile as determined by the splenic production of IL-4 and IL-10. Six putative Th determinants were identified using a panoply of overlapping synthetic peptides in in vitro stimulation assays: amino acids 20-44, 39-63, 79-113, 89-113, 118-142, and 138-152. For all CAP immunogens, MHC haplotype of immunized mice was found to influence seroconversion rates but not the type of cytokines produced in vitro. H-2d mice were faster responders and displayed recall T-cell activation by a larger number of peptides than H-2b mice, whereas H-2s mice were overall very poor responders. Splenic stimulation by at least one determinant, amino acids 79-103, appeared to be highly specific of the H-2b background and of DNA immunization only. These data indicate that DNA immunogens expressing different forms of HCV-CAP are not associated with different Th profiles but rather different seroconversion rates and antibody titers and that collaboration of distinct T-help epitopes can be restricted by the MHC background.