PubMed Health⌕ Search

Biomedical subjects

Christian Trépo

Publications and source records attributed to Christian Trépo.

At least 19 recordsLinked to original sources
PubMed2006

euHCVdb: the European hepatitis C virus database.

The hepatitis C virus (HCV) genome shows remarkable sequence variability, leading to the classification of at least six major genotypes, numerous subtypes and a myriad of quasispecies within a given host. A database allowing researchers to investigate the genetic and structural variability of all available HCV sequences is an essential tool for studies on the molecular virology and pathogenesis of hepatitis C as well as drug design and vaccine development. We describe here the European Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr), a collection of computer-annotated sequences based on reference genomes. The annotations include genome mapping of sequences, use of recommended nomenclature, subtyping as well as three-dimensional (3D) molecular models of proteins. A WWW interface has been developed to facilitate database searches and the export of data for sequence and structure analyses. As part of an international collaborative effort with the US and Japanese databases, the European HCV Database (euHCVdb) is mainly dedicated to HCV protein sequences, 3D structures and functional analyses.

Databases, Protein↗
PubMed2006

Novel alpha interferon (IFN-alpha) variant with improved inhibitory activity against hepatitis C virus genotype 1 replication compared to IFN-alpha2b therapy in a subgenomic replicon system.

Hepatitis C virus (HCV) treatment is based on the association of pegylated alpha interferon (IFN-alpha) and ribavirin. To improve the level of sustained virological response to treatment, especially in patients infected with HCV genotype 1, new IFNs with improved efficacy and toxicity profiles may be developed. In this report, we show that, in the BM4-5 cell line harboring an HCV subgenomic replicon, a novel and naturally occurring human IFN-alpha17 variant, GEA007.1, which was discovered by using an original population genetics-based drug discovery approach, inhibits HCV genotype 1 RNA replication more efficiently than does IFN-alpha2b. Moreover, we show that complete viral clearance is obtained in BM4-5 cells after long-term treatment with GEA007.1, while HCV subgenomic RNA is still detected in cells treated with other IFN-alpha variants or with standard IFN-alpha2b. Eventually, we demonstrate that the better inhibitory activity of GEA007.1 compared to that of standard IFN-alpha is likely to be due to stronger and faster activation of the JAK-STAT signaling pathway and to broader expression of IFN-alpha-responsive genes in cells. Our results demonstrate a superior inhibitory activity of GEA007.1 over that of IFN-alpha2b in the HCV replicon system. Clinical trials are required to determine whether GEA007.1 could be a potent "next generation" IFN for the treatment of HCV infection, especially in nonresponders or relapsing patients infected with HCV genotype 1 who currently represent a clinical unmet need.

Antiviral Agents↗
PubMed2006

Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient.

BACKGROUND & AIMS: Sequential anti-hepatitis B virus (HBV) therapy may lead to the selection of complex mutants. We analyzed the genetic and phenotypic evolution of the viral quasispecies of a patient who received successively lamivudine, add-on adefovir+lamivudine, followed by lamivudine+adefovir+hepatitis B immunoglobulins (HBIg) after orthotopic liver transplantation. METHODS: For genotypic analysis, a 1310-bp region of the polymerase gene was amplified, cloned, and sequenced. Huh-7 cells were transfected to compare the replication fitness of HBV mutants and their susceptibility to drugs. RESULTS: At baseline, all HBV genomes carried a wild-type (wt) RT gene but 22% harbored the sP120S and 55% the sC107stop mutations within the surface (S) gene associated with vaccine escape. Following viral breakthrough to lamivudine monotherapy, a complex mixture of lamivudine-resistant HBV strains prevailed. Interestingly, among these mutants emerged a population harboring only the rtL180M+A181V mutations, conferring lamivudine-resistance in vitro. After addition of adefovir to the ongoing treatment, viral load dropped, and the patient underwent an orthotopic liver transplantation and received HBIg. As viral load rose again, a single viral population was progressively selected, harboring the rtV173L+L180M+A181V+N236T and sP120S mutations. In vitro, this last mutant showed a level of replication reduced by only 30% compared to wt HBV and a strong resistance to both lamivudine (>1000-fold) and adefovir (>10-fold). It remained sensitive to tenofovir both in vitro and in vivo. CONCLUSIONS: We report the selection of a complex HBV mutant that escaped the antiviral pressure of lamivudine, adefovir, and HBIg, and provide insight on the process of selection via genotypic and phenotypic analysis.

Adenine↗
PubMed2006

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein.

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.

Alleles↗
PubMed2006

Antiviral effect of alpha-glucosidase inhibitors on viral morphogenesis and binding properties of hepatitis C virus-like particles.

Hepatitis C virus (HCV) infections are a major public-health concern. New antiviral drugs are needed urgently to complement and improve the efficacy of current chemotherapies. The morphogenesis of HCV represents an interesting, and still unexploited, novel molecular target. alpha-Glucosidase inhibitors derived from the glucose analogue deoxynojirimycin (DNJ) inhibit viral morphogenesis in cellulo via perturbation of the N-glycosylation pathway and hence the misfolding of viral glycoproteins that depend on certain N-glycans for correct folding. Due to the heavy N-glycosylation of HCV glycoproteins, it was hypothesized that such inhibitors would also affect HCV morphogenesis. To study the effect of alpha-glucosidase inhibitors on viral morphogenesis and binding properties, HCV virus-like particles (VLPs) were produced by using baculovirus loaded with HCV structural-protein genes. Here, it is demonstrated that, in the presence of these alpha-glucosidase inhibitors, viral glycoproteins synthesized and retained in the endoplasmic reticulum (i) contain unprocessed, triglucosylated N-glycans, (ii) are impaired in their interaction with calnexin and (iii) are at least partially misfolded. Moreover, it is shown that, although the production of VLPs is not affected by alpha-glucosidase inhibitors, these VLPs contain unprocessed, triglucosylated N-glycans and potentially misfolded glycoproteins. Finally, it is demonstrated that VLPs produced in the presence of alpha-glucosidase inhibitors have impaired binding properties to hepatoma cells. The inhibitors of morphogenesis studied here target steps of the HCV viral cycle that may prevent or delay viral resistance. These alpha-glucosidase inhibitors may prove to be useful molecules to fight HCV infection in combination protocols.

Animals↗
PubMed2006

Preferential association of Hepatitis C virus with apolipoprotein B48-containing lipoproteins.

Hepatitis C virus (HCV) in cell culture has a density comparable to that of other members of the family Flaviviridae, whereas in vivo infectious particles are found partially in low-density fractions, associated with triacylglycerol (TG)-rich lipoproteins (TRLs). In the blood of infected patients, HCV circulates as heterogeneous particles, among which are lipo-viroparticles (LVPs), globular particles rich in TG and containing viral capsid and RNA. The dual viral and lipoprotein nature of LVPs was addressed further with respect to apolipoprotein composition and post-prandial dynamic lipid changes. The TRLs exchangeable apoE, -CII and -CIII, but not the high-density lipoprotein apoA-II, were present on LVPs, as well as the viral envelope proteins. apoB100 and -B48, the two isoforms of the non-exchangeable apoB, were represented equally on LVPs, despite the fact that apoB48 was barely detectable in the plasma of these fasting patients. This indicates that a significant fraction of plasma HCV was associated with apoB48-containing LVPs. Furthermore, LVPs were enriched dramatically and rapidly in triglycerides after a fat meal. As apoB48 is synthesized exclusively by the intestine, these data highlight the preferential association of HCV with chylomicrons, the intestine-derived TRLs. These data raise the question of the contribution of the intestine to the viral load and suggest that the virus could take advantage of TRL assembly and secretion for its own production and of TRL fate to be delivered to the liver.

Apolipoprotein B-48↗
PubMed2006

Hepatitis viruses, alcohol, and tobacco in the etiology of hepatocellular carcinoma in Italy.

Mortality rates of hepatocellular carcinoma (HCC) are high in Italy compared with other Western countries. To elucidate further the role of hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol drinking, and tobacco smoking in the etiology of HCC, we carried out a hospital-based case-control study in two areas of Italy: the province of Pordenone in the Northeast and the town of Naples in the South. A total of 229 HCC cases (median age, 66 years) and 431 controls (median age, 65 years) answered a questionnaire and provided blood samples between 1999 and 2002. Odds ratios (OR), percent attributable risks, and corresponding 95% confidence intervals were computed using unconditional multiple logistic regression. ORs for hepatitis B surface antigen (HBsAg) positive versus HBsAg negative and for anti-HCV antibody positive versus anti-HCV antibody negative were 20.2 and 15.6, respectively. Positivity for both markers was associated with an OR of 51.6. Sensitive molecular techniques applied to sera in a subset of HCC cases disclosed a very small number of occult hepatites. Maximal lifetime alcohol intake of > or =35 versus <7 drinks/wk was associated with an HBV/HCV adjusted OR of 5.9. Tobacco smoking was unrelated to HCC risk overall but seemed to enhance HCC risk among virus carriers. Overall, 61% of HCC were attributable to HCV, 13% to HBV, and 18% to heavy alcohol drinking. In conclusion, our study confirms the importance of HCV in HCC etiology in Italy where the widespread dissemination of the virus dates back four or five decades.

Adult↗
PubMed2006

HDV genotypes in the Western Brazilian Amazon region: A preliminary report.

In Brazil, hepatitis delta virus (HDV) is only reported in Western Amazonia, where severe cases of acute and chronic HDV hepatitis have been described. The study area was chosen in the States of Acre and Rondonia where most cases of hepatitis B virus (HBV)/HDV are reported. From December 2003 to October 2004, 40 HBsAg carriers with anti-HDV IgM were selected. An epidemiologic questionnaire, including demographic and clinical/epidemiologic variables was filled out. HDV amplification and genotyping were performed. Genotype I was detected in 22 patients (55.0%), whereas genotype III was identified in 18 (45.0%). Patients who were infected with genotype I were older (45.1 +/- 17.8 years) than patients infected with genotype III (32.8 +/- 10.9 years; P = 0.01). No symptoms were reported by 21 (52.5%) patients. Otherwise, 19 (47.5%) had symptoms (fatigue, abdominal pain, weight loss, and decompensated liver disease) that motivated them to seek medical care. Genotype III carriers were more symptomatic, but no statistical significance was achieved. Our preliminary results show that HDV genotypes I and III are present in Brazilian Amazonia and that HDV genotype III is not limited to the Amerindian population.

Adolescent↗
PubMed2005

Amantadine triple therapy for non-responder hepatitis C patients. Clues for controversies (ANRS HC 03 BITRI).

BACKGROUND/AIMS: To determine whether addition of amantadine to pegylated interferon/ribavirin improved response rates among chronic hepatitis C patients, non-responders to interferon/ribavirin and study the dynamic of response. METHODS: In a double blind, multicenter, randomized trial, 200 non-responder patients received pegylated interferon 1.5 microg/kg per week and ribavirin 800-1200 mg/day, plus either amantadine 200 mg/day or placebo for 48 weeks. Endpoints were virological responses, ALT normalization, and histological benefit overtime. RESULTS: Twenty percent of all patients achieved a sustained virological response (SVR). This rate was 8% higher in the triple therapy group (24%) compared with the double therapy group (16%) (P = 0.22). A better virological response rate at week 24 was observed in the triple regimen group (43 vs 29%; P = 0.06), which was lost at week 48 suggesting viral escape. The biochemical response rate was also significantly higher with triple therapy at week 12 (63 vs 49%; P = 0.05) and week 24 (64 vs 49%; P = 0.03). Fibrosis stabilized or improved in 77% of all patients. CONCLUSIONS: Re-treatment of interferon/ribavirin non-responder patients should be encouraged since a substantial proportion benefits from re-treatment with pegylated interferon/ribavirin +/- amantadine. In triple therapy involving amantadine, a time wise response and an increased SVR rate in subgroups less prone to viral breakthrough suggest clues for existing controversies.

Adult↗
PubMed2005

Oncogenic role of the frizzled-7/beta-catenin pathway in hepatocellular carcinoma.

BACKGROUND/AIMS: The molecular mechanisms of hepatocarcinogenesis remain largely unknown. Previous studies suggest that activation of the Wnt/beta-catenin pathway is important during hepatocyte transformation but the role of Frizzled receptor (FZD) in this process has not been defined. Here we investigate activation of this pathway by FZD using transgenic hepatocellular carcinoma (HCC) murine models. METHODS: We employed single (c-myc, SV40-Tag) and established double [insulin receptor substrate-1 (IRS-1/c-myc) and hepatitis Bx protein (X/c-myc)] transgenic lines and all developed HCC. Expression of 9 FZD was measured by real time RT-PCR and Western blot analysis. Phosphorylation and cellular accumulation of beta-catenin were assessed in both dysplastic tissue and tumors. We investigated the effect of a dominant negative (DN) FZD7 on TCF transcriptional activity in a SV40 derived HCC cell line. RESULTS: FZD7 was highly overexpressed at the mRNA and protein level(s) in HCC and occurred in dysplasia. Upregulation of FZD7 was associated with reduced phosphorylation of beta-catenin and led to nuclear accumulation in HCC tumors. Ectopic expression of a DN FZD7 construct decreased TCF transcriptional activity in tumor cells. CONCLUSIONS: These observations suggest that upregulation of FZD7 receptors in association with activation of the canonical Wnt/beta-catenin pathway is a common molecular event in HCC.

Animals↗
PubMed2005

Enveloped particles in the serum of chronic hepatitis C patients.

HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06-1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.

Centrifugation, Density Gradient↗
PubMed2005

Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir.

Mutations within the hepatitis B virus (HBV) polymerase gene conferring drug-resistance are selected during prolonged lamivudine (3TC) or adefovir dipivoxil (ADV) treatment. Because there is no other approved drug against HBV, treatments with 3TC or ADV are used either sequentially or in addition, depending on treatment response or failure. Considering the use of de novo or add-on 3TC+ADV bitherapy, we investigated the possibility of the emergence of an HBV strain harboring polymerase mutations conferring resistance to both 3TC (rtL180M+M204V) and ADV (rtN236T). We constructed the L180M+M204V+N236T mutant and determined its replication capacity and its susceptibility to different nucleos(t)ide analogs in transiently transfected hepatoma cell lines. The triple mutant replicates its genome in vitro, but less efficiently than either the wild-type (wt) HBV or L180M+M204V and N236T mutants. Phenotypic assays indicated that the L180M+M204V+N236T mutant is resistant to pyrimidine analogs (3TC, -FTC, beta-L-FD4C, L-FMAU). Compared with wt HBV, this mutant displays a 6-fold decreased susceptibility to ADV and entecavir and a 4-fold decreased susceptibility to tenofovir. Interferon alfa inhibited equally the replication of wt and L180M+M204V+N236T HBV. In conclusion, the combination of rtL180M+M204V and rtN236T mutations impairs HBV replication and confers resistance to both 3TC and ADV in vitro. These results suggest that the emergence of the triple mutant may be delayed and associated with viral resistance in patients treated with 3TC+ADV. However, other nucleos(t)ide analogs in development showed an antiviral activity against this multiresistant strain in vitro. This provides a rationale for the clinical evaluation of de novo combination therapies.

Adenine↗
PubMed2005

Increase of doxorubicin sensitivity by doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is known to be chemoresistant to anticancer drugs due to the multidrug resistant (MDR) transporters expression. Here, we compared in vitro and in vivo the anti-tumor efficacy of doxorubicin-loaded polyisohexylcyanoacrylate nanoparticles (PIHCA-Dox) versus free doxorubicin (Dox). These nanoparticles are known to overcome the MDR phenotype. METHODS: We first determined in vitro the 50% inhibition concentration (IC(50)) of these drugs on different human hepatoma cell lines. Secondly, the efficacy of the drugs in vivo was determined on the X/myc transgenic murine model of HCC by histological counting of apoptotic tumorous hepatocytes and by TUNEL labeling. We characterized by semi-quantitative RT-PCR the MDR-related gene (mdr1, mdr3, mrp1) expression pattern in this model. RESULTS: In vitro, IC(50) was reduced with PIHCA-Dox versus Dox for Huh7 (1.7-fold reduction; P<0.001), HepaRG (4.5-fold reduction; P<0.01), HepG2 (1.5-fold reduction; P<0.001), and HepG2.2.15 (1.5-fold reduction; P=0.059). In vivo, HCC in transgenic mice overexpressed the mdr1 and mdr3 genes and the antitumor drugs efficacy was greatly enhanced after injection of PIHCA-Dox (9.0+/-5.0%; n=15) versus Dox (4.6+/-3.3%; n=13; P=0.01) for apoptotic bodies count. CONCLUSIONS: These promising data showing a higher anti-tumor efficacy on HCC of PIHCA-Dox versus Dox, warrant further studies in both animals and humans.

Animals↗
PubMed2004

A new strategy for studying in vitro the drug susceptibility of clinical isolates of human hepatitis B virus.

Resistance of hepatitis B virus (HBV) to antivirals has become a major clinical problem. Our objective was to develop a new method for the cloning of naturally occurring HBV genomes and a phenotypic assay capable of assessing HBV drug susceptibility and DNA synthesis capacity in vitro. Viral DNA was extracted from sera and was amplified by polymerase chain reaction, and amplicons were cloned into vectors that enable, after cell transfection, the initiation of the intracellular HBV replication cycle. Single or multiple clones were used to transfect Huh7 cells. The viral DNA synthesis capacity and drug susceptibility were determined by measuring the level of intracellular DNA intermediate, synthesized in absence or presence of antiviral, using Southern blot analysis. We have developed, calibrated, then used this phenotypic assay to determine the drug susceptibility of HBV quasispecies isolated throughout the course of therapy from patients selected according to their mutation profile. A multiclonal and longitudinal analysis enabled us to measure the variation of drug susceptibility of different viral quasispecies by comparison of IC(50)/IC(90)s with standards. The presence of famciclovir- or lamivudine-induced mutations in the viral population caused a change in viral DNA synthesis capacity and drug susceptibility in vitro, demonstrating the clinical relevance of the assay. In conclusion, our phenotypic assay enables the in vitro characterization of DNA synthesis capacity and drug susceptibility of HBV quasispecies isolated from patients. This assay should allow a better monitoring of patients undergoing antiviral therapy, as well as the screening of novel drugs on emerging resistant strains.

Calibration↗
PubMed2004

Analysis of B-lymphocyte differentiation in patients infected with hepatitis C virus.

To clarify whether some of the functions of B lymphocytes could be affected during hepatitis C virus (HCV) infection, phenotypic characteristics of B lymphocytes from HCV-infected patients and their capacity to differentiate into immunoglobulins (Ig)-secreting cells were studied. B lymphocytes differentiation was investigated for patients untreated and non-responders (n=9), treated and non-responders (n=6), responders (n=6), long-term responders (n=9) to therapy and seronegative controls (n=14) following in vitro stimulation with S. aureus strain Cowan I mitogen. HCV sequences in purified B lymphocytes were detected by RT-PCR. It was found that HCV-patients harbor a similar mean percentage of B cells and a normal level of naïve B cells (% IgM+/IgD+ cells=79.7 +/- 15.4 for untreated non-responders, 57.1 +/- 22.9 for treated non-responders, 44.3 +/- 29.1 for responders, 75.7 +/- 16 for long-term responders) as compared with controls. It was also found that peripheral blood mononuclear cells (PBMCs) of patients or controls produced similar amounts of IgG, A, and M in vitro. A total of 57% of untreated non-responders versus 17% of treated non-responders were able to produce HCV-specific antibodies. Interestingly, B lymphocytes from PBMCs able to secrete anti-HCV antibodies contained HCV positive strand RNA, although no systematic detection of the negative strand was found. These data suggest that signaling through the B cell receptor (BCR) in B lymphocytes of HCV-infected patients appears normal whatever their response to therapy. The capacity to secrete HCV-specific IgG seemed to be linked to the presence of positive strand RNA rather than virus replication.

Adult↗
PubMed2004

[Autoimmunization induced by interferon alpha therapy in chronic hepatitis C].

We report the case of a 56 year-old woman with post-transfusion chronic hepatitis C who presented with a severe ALT flare up associated with a rapid progression of liver fibrosis during interferon alpha 2b therapy. Several hypotheses were considered to explain the etiology of this ALT flare: there was no viral super infection by other hepatotropic viruses, no toxic hepatitis, no metabolic disease, and no other specific liver diseases could be identified. HLA typing showed a specific profile A1 B8 DR3 (risk factor of auto-immunization during interferon alpha therapy) with antinuclear antibodies and anti smooth muscle antibodies. This case suggests that auto-immunization induced by interferon alpha should be investigated in case of ALT flare that is not followed by an HCV breakthrough.

Antiviral Agents↗