Dina Kremsdorf

Hepatitis C virus genotype 4 is highly prevalent in central Africa (Gabon)

Following a survey of hepatitis C virus (HCV) infection recently carried in central Africa (Gabon), we cloned and sequenced PCR products of the 5 non-coding and capsid-encoding regions of HCV RNA from three randomly selected HCV RNA-positive Gabonese subjects. In the capsid-encoding region, the identity between the three Gabonese isolates was 91 to 98%. The three Gabonese sequences showed a divergence of 11 to 17% from published HCV genotypes I to IV (1a, 1b, 2a and 2b) isolates and of 6 to 11% from HCV genotype 4 isolates. Thus the Gabonese isolates, termed HC-G, belong to HCV genotype 4. Based on the sequences of the three isolates, a specific probe (cpsG) was designed to detect the HC-G genotype in 30 randomly selected anti-HCV-positive Gabonese subjects, 14 of whom were HCV RNA-positive. Analysis with cpsG showed that 10 of 14 of the HCV RNA-positive subjects were infected by the HC-G genotype. HC-G is therefore highly prevalent in the HCV RNA-positive Gabonese population. The availability of these Gabonese sequences should facilitate the design of specific serological tests for African HCV isolates.

Liver-Stage Development of Plasmodium falciparum in a Humanized Mouse Model

BACKGROUNDnThe liver stage of the human malaria parasite Plasmodium falciparum is the least known, yet it holds the greatest promise for the induction of sterile immunity and the development of novel drugs. Progress has been severely limited by the lack of adequate in vitro and in vivo models.nnnMETHODSnRecently, it was found that immunodeficient mice transgenic for the urokinase plasminogen activator allow survival of differentiated human hepatocytes. We confirm this finding but show that hepatocyte survival is short lived unless nonadaptive defenses are simultaneously depleted.nnnRESULTSnBy controlling macrophages and NK cells, we readily effected the long-term secretion of human serum albumin and human alpha-1 antitrypsin in mouse serum (at 3 months, the proportion of repopulated mice increased from 0% to 60% and from 22% to 80%, respectively; P<.0001). P. falciparum sporozoites delivered intravenously into mice readily infected transplanted human hepatocytes and developed into liver schizonts. Their size was twice as large as what was seen in vitro and was comparable to that found in humans and chimpanzees.nnnCONCLUSIONnThese results emphasize the importance of nonadaptive defenses against xenotransplantation and lead to development of small laboratory models that, because they can harbor human hepatocytes, provide novel opportunities to study intrahepatic pathogens, such as those causing malaria and hepatitis.

The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis

BACKGROUND/AIMSnWe have previously demonstrated the in vivo expression of a new spliced hepatitis B virus (HBV) protein (HBSP) encoded by a singly spliced pregenomic RNA. The present study was designed to evaluate the impact of HBSP expression on the clinical status and liver pathology of HBV infection.nnnMETHODSnSera from 125 chronic HBV carriers were tested for the presence of HBSP antibodies by an indirect enzyme-linked immunosorbent assay test. The severity of liver damage was evaluated using the Knodell score.nnnRESULTSnAnti-HBSP antibody prevalence in HBV chronic carriers was 46%. We highlighted the concomitant expression of HBSP protein and anti-HBSP antibody. An association between anti-HBSP antibody detection and serum markers of HBV replication was demonstrated. With respect to HBV-related liver disease, an association was only observed with the severity of fibrosis. Furthermore, an elevation of secreted tumor necrosis factor alpha (TNFalpha), but not of soluble TNFalpha receptor 75, was observed in anti-HBSP-antibody-positive patients. Multivariate analysis showed that anti-HBSP antibody detection was independently associated with viral replication, severity of fibrosis and elevated TNFalpha secretion.nnnCONCLUSIONSnOur data suggest the hypothesis that HBSP might play a role in the natural history of HBV infection and may be involved in the pathogenesis and/or persistence of HBV infection.

Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection

BACKGROUND/AIMSnThe analysis of hepatitis B virus (HBV) X protein genetic variability and is correlation with liver disease severity have only been addressed, so far, on whole liver extracts. We have studied, therefore, the HBV X protein (HBx) gene sequence in morphologically well-characterised tumour and non-tumour liver cells from patients with HBV-related hepatocellular carcinoma.nnnMETHODSnUsing laser capture microdissection (LCM), we picked up six to eight groups of tumour and non-tumour hepatocytes in serial frozen sections from six patients. After global DNA preamplification followed by HBx-specific polymerase chain reaction, the HBx gene was sequenced in each group of microdissected cells. We also validated the quantification of HBV-DNA in microdissected hepatocytes using HBV Amplicor.nnnRESULTSnHeterogeneous mutations in HBx gene were found in distinct cirrhotic nodules and tumour areas from the same patient. Mutations at aa 127, 130 and 131 were frequently detected but there was no distinct point mutation profile between tumour and non-tumour samples. In contrast, deletions in HBx gene, which were found in five/six patients, were more frequent in tumour-derived sequences (6/18) than in non-tumour-derived sequences (1/20).nnnCONCLUSIONSnWe have shown that LCM provides a direct insight of intrahepatic HBV infection. Using this technique, we demonstrated the persistence of distinct HBx encoding sequences in clonally expanding cells, thus supporting the hypothesis that HBx deletions may be implicated in liver carcinogenesis.

Antiviral Activity of Bay 41-4109 on Hepatitis B Virus in Humanized Alb-uPA/SCID Mice

Current treatments for HBV chronic carriers using interferon alpha or nucleoside analogues are not effective in all patients and may induce the emergence of HBV resistant strains. Bay 41-4109, a member of the heteroaryldihydropyrimidine family, inhibits HBV replication by destabilizing capsid assembly. The aim of this study was to determine the antiviral effect of Bay 41-4109 in a mouse model with humanized liver and the spread of active HBV. Antiviral assays of Bay 41-4109 on HepG2.2.15 cells constitutively expressing HBV, displayed an IC(50) of about 202 nM with no cell toxicity. Alb-uPA/SCID mice were transplanted with human hepatocytes and infected with HBV. Ten days post-infection, the mice were treated with Bay 41-4109 for five days. During the 30 days of follow-up, the HBV load was evaluated by quantitative PCR. At the end of treatment, decreased HBV viremia of about 1 log(10) copies/ml was observed. By contrast, increased HBV viremia of about 0.5 log(10) copies/ml was measured in the control group. Five days after the end of treatment, a rebound of HBV viremia occurred in the treated group. Furthermore, 15 days after treatment discontinuation, a similar expression of the viral capsid was evidenced in liver biopsies. Our findings demonstrate that Bay 41-4109 displayed antiviral properties against HBV in humanized Alb-uPA/SCID mice and confirm the usefulness of Alb-uPA/SCID mice for the evaluation of pharmaceutical compounds. The administration of Bay 41-4109 may constitute a new strategy for the treatment of patients in escape from standard antiviral therapy.

Partial nucleotide sequence analysis of a French hepatitis C virus : implications for HCV genetic variability in the E2/NS1 protein

To contribute to the study of the genetic variability of hepatitis C virus (HCV) we have determined the nucleotide sequence of the E2/NS1 and NS3/NS4 regions of a French isolate using the polymerase chain reaction. Comparison of these nucleotide sequences with those available for American and Japanese isolates showed a significant genetic variability: 5 to 33% and 2 to 30% at the nucleic acid and amino acid levels, respectively. The genetic variability is higher in the E2/NS1 (13 to 33% and 12 to 30% at the nucleic acid and amino acid levels, respectively) than in the NS3/NS4 (5 to 21% and 2 to 7%) regions. The sequence of the French isolate is more closely related to that of the American HCV prototype than to the Japanese HCV isolates. This study confirms the extent of HCV genetic variability.

Hepatitis B virus HBx protein impairs liver regeneration through enhanced expression of IL-6 in transgenic mice

BACKGROUND & AIMSnConflicting results have been reported regarding the impact of hepatitis B virus X protein (HBx) expression on liver regeneration triggered by partial hepatectomy (PH). In the present report we investigated the mechanisms by which HBx protein alters hepatocyte proliferation after PH.nnnMETHODSnPH was performed on a transgenic mouse model in which HBx expression is under the control of viral regulatory elements and liver regeneration was monitored. LPS, IL-6 neutralizing antibody, and SB203580 were injected after PH to evaluate IL-6 participation during liver regeneration.nnnRESULTSnCell cycle progression of hepatocytes was delayed in HBx transgenic mice compared to WT animals. Moreover, HBx induced higher secretion of IL-6 soon after PH. Upregulation of IL-6 was associated with an elevation of STAT3 phosphorylation, SOCS3 transcript accumulation and a decrease in ERK1/2 phosphorylation in the livers of HBx transgenic mice. The involvement of IL-6 overexpression in cell cycle deregulation was confirmed by the inhibition of liver regeneration in control mice after the upregulation of IL-6 expression using LPS. In addition, IL-6 neutralization with antibodies was able to restore liver regeneration in HBx mice. Finally, the direct role of p38 in IL-6 secretion after PH was demonstrated using SB203580, a pharmacological inhibitor.nnnCONCLUSIONSnHBx is able to induce delayed hepatocyte proliferation after PH, and HBx-induced IL-6 overexpression is involved in delayed liver regeneration. By modulating IL-6 expression during liver proliferation induced by stimulation of the cellular microenvironment, HBx may participate in cell cycle deregulation and progression of liver disease.

Morphogenetic competence of HNF4α-deficient mouse hepatic cells

BACKGROUND/AIMSnTo specify roles of HNF 4 alpha in mouse liver development, we have analyzed the ex vivo morphogenetic potential of HNF4 alpha-null embryonic hepatic cells.nnnMETHODSnUsing mice with floxed or deficiency alleles of HNF4 alpha, hepatic cells lacking this transcription factor were explanted into primary culture and derived into cell lines.nnnRESULTSnContrary to behavior in vivo where HNF4 alpha-null liver cells fail to show normal polarity and epithelialization, e18.5 hepatic cells in primary culture from mutant embryos show restoration of apical expression of tight junction protein-1 and of transcripts for E-cadherin. Clones of control and HNF4 alpha-null cell lines were indistinguishable, even when differentiation of bile canalicular formation was induced. HNF4 alpha-null and control cell lines showed similar potential to colonize livers of the murine ALB-uPA/SCID model of liver regeneration, but null cells formed only bile ducts and not clusters of hepatocytes. Finally, analysis of mutant embryonic livers revealed a transcriptional signature consistent with a stress response, which could underlie the morphogenetic defects observed in vivo.nnnCONCLUSIONSnWe conclude that the lack of epithelialization characteristic of the HNF4 alpha-null embryonic liver is due, at least in part, to non-cell autonomous defects, and that null cells do not suffer intrinsic defects in polarization.

Prospective comparison of Abbott RealTime HBV DNA and Versant HBV DNA 3.0 assays for hepatitis B DNA quantitation: Impact on HBV genotype monitoring

The quantitation of human hepatitis B virus (HBV) in the serum of infected patients is recommended to characterize the course of chronic HBV infection. The aim of this prospective study was to evaluate the performance of the Abbott RealTime PCR assay for HBV DNA quantitation by comparison with the standard Versant HBV DNA 3.0 assay. The better sensitivity and broader dynamic range of HBV DNA quantitation using the Abbott RealTime PCR assay was confirmed by the study of 362 serum samples from 311 patients. In addition, data analysis revealed the concordance of HBV DNA quantitations between the two assays. When this evaluation was assessed as a function of HBV genotype, there was discordance for HBV genotype C samples. Thus, we performed an in-house PCR to confirm the discrepancy observed regarding the HBV genotypes. The in-house PCR results agreed better with the Abbott RealTime PCR method when compared with the standard hybridization assay. In conclusion, the wide dynamic range of HBV DNA quantitation achieved with the Abbott RealTime PCR assay makes it appropriate for the clinical monitoring of HBV infected patients. However, a change of HBV DNA quantitation method could influence results on the follow-up of HBV genotype C infected patients.

Production of hepatitis B defective particles is dependent on liver status

Defective hepatitis B virus (dHBV) generated from spliced RNA is detected in the sera of HBV-chronic carriers. Our study was designed to determine whether the proportion of dHBV changed during the course of infection, and to investigate whether dHBV might interfere with HBV replication. To achieve this, HBV wild-type and dHBV levels were determined by Q-PCR in sera from 56 untreated chronic patients and 23 acute patients, in sequential samples from 4 treated-patients and from liver-humanized mice after HBV infection. The proportion of dHBV was higher in patients with severe compared to null/moderate liver disease or with acute infection. Follow-up showed that the proportion of dHBV increased during disease progression. By contrast, a low and stable proportion of dHBV was observed in the humanized-mouse model of HBV infection. Our results highlight a regulation of the proportion of dHBV during liver disease progression that is independent of interference with viral replication.