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Biomedical subjects

Christian Brechot

Publications and source records attributed to Christian Brechot.

14 recordsLinked to original sources
PubMed2006

Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis.

AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations. METHODS: The percentages of mononuclear diploid (2n), 4n, and 8n hepatocytes and those of binuclear 2 x 2n, 2 x 4n, and 2 x 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62 patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 x 4n and 2 x 8n hepatocytes in the binuclear hepatocyte population. RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 x 4n and 2 x 8n hepatocytes in binuclear hepatocytes were similar, regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus. CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.

Adult↗
PubMed2006

Liver-stage development of Plasmodium falciparum, in a humanized mouse model.

BACKGROUND: The 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. METHODS: Recently, 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. RESULTS: By 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. CONCLUSION: These 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.

Animals↗
PubMed2005

Hepatitis C virus core variants isolated from liver tumor but not from adjacent non-tumor tissue interact with Smad3 and inhibit the TGF-beta pathway.

Hepatitis C virus (HCV) is a major risk factor for human hepatocellular carcinoma (HCC) but the mechanisms underlying HCV-induced carcinogenesis are still poorly understood. We have hypothesized that viral variants, selected during long-term infection, might contribute to cellular transformation. To address this issue, we have investigated the effect of natural HCV core variants isolated from liver tumors (T), or their non-tumor (NT) counterparts, on the tumor growth factor-beta (TGF-beta) pathway, a major regulator of cellular proliferation, differentiation and apoptosis. We have found a significant reduction in TGF-beta reporter gene activity with the expression of core sequences isolated from liver tumors. In contrast, moderate or no effects were observed with non-tumor mutants or a core reference sequence. The molecular mechanisms have been characterized and involved the inhibition, by tumor-derived cores, of the DNA-binding activity of the Smad3/4 transcription factors complex. This inhibition occurs through a direct interaction between the central domain (amino acids 59-126) of tumor-derived core and the MH1 DNA-binding domain of Smad3, thus preventing its binding to DNA. We have therefore identified a new cell-signaling pathway targeted by HCV core and inhibited by tumor-derived core sequences. These results suggest that during chronic infection, there is selection of viral variants that may promote cell transformation by providing, to clonally expanding cells, resistance to TGF-beta antiproliferative effects.

Amino Acid Sequence↗
PubMed2004

Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors.

BACKGROUND/AIMS: Gene therapy is a promising approach for treatment of hepatocellular carcinoma (HCC). However, transduction of non-tumoral hepatocytes may lead to severe hepatitis when using suicide gene therapy approaches. The aim of our study was to evaluate the gene transfer efficiency into HCC cells and normal hepatocytes using human immunodeficiency virus (HIV)-derived lentiviral vectors in vitro and in vivo. METHODS: Lentiviral vectors encoding for the LacZ gene or the fusion gene HSV-Tk/GFP were tested in vitro in human HCC cells and human hepatocytes in primary culture and in vivo in a chemically induced rat model of HCC. RESULTS: We show that HIV-1-derived lentiviral vectors are efficient in transducing HCC cells in vitro and in vivo. No significant transduction of non-tumorous hepatocytes was observed in vivo whatever the route of administration used. Measurement of tumor growth following direct intratumoral injection of a lentiviral vector containing the HSV-Tk gene and GCV treatment showed a strong antitumoral efficacy in the absence of normal liver toxicity. CONCLUSIONS: These observations suggest that lentiviral vectors allow an antitumoral effect with low liver toxicity when using suicide gene therapy approach and could be efficient tools for HCC gene therapy.

Animals↗
PubMed2004

Intrahepatic hepatitis C virus RNA quantification in microdissected hepatocytes.

BACKGROUND/AIMS: Debate continues on whether serum and intrahepatic HCV viral loads are correlated and if HCV viral load correlates with the severity of liver disease. These difficulties may at least in part be linked to liver cell heterogeneity, when total liver extracts from HCV-infected individuals are tested for HCV RNA quantification. We have therefore investigated the feasibility of quantifying HCV replication using a laser-based microdissection technique. METHODS: We compared the results with those obtained for serum HCV RNA quantification and immunochemistry in the case of HCV antigen detection in the liver. Twenty-one HCV-positive patients with chronic active hepatitis (n=10) or cirrhosis (n=11) were analyzed. RESULTS: A positive correlation (P=0.0019) was observed between HCV RNA quantifications in sera and microdissected cells. Immunohistochemistry demonstrated that HCV antigen hepatocytes were randomly distributed within liver lobules. Their percentage varied in different patients (0-40%), but did not correlate with the HCV viral load. CONCLUSIONS: We have designed a sensitive methodology to evaluate the intrahepatic HCV viral load by combining a standardized RNA quantification method with microdissected hepatocytes from frozen liver needle biopsies. Our results directly demonstrate a positive correlation between serum and intrahepatic viral loads, which therefore provides a reliable reflection of intrahepatic HCV replication.

Adult↗
PubMed2004

HIP/PAP, a C-type lectin overexpressed in hepatocellular carcinoma, binds the RII alpha regulatory subunit of cAMP-dependent protein kinase and alters the cAMP-dependent protein kinase signalling.

HIP/PAP is a C-type lectin overexpressed in hepatocellular carcinoma (HCC). Pleiotropic biological activities have been ascribed to this protein, but little is known about the function of HIP/PAP in the liver. In this study, therefore, we searched for proteins interacting with HIP/PAP by screening a HCC cDNA expression library. We have identified the RII alpha regulatory subunit of cAMP-dependent protein kinase (PKA) as a partner of HIP/PAP. HIP/PAP and RII alpha were coimmunoprecipitated in HIP/PAP expressing cells. The biological relevance of the interaction between these proteins was established by demonstrating, using fractionation methods, that they are located in a same subcellular compartment. Indeed, though HIP/PAP is a protein secreted via the Golgi apparatus we showed that a fraction of HIP/PAP escaped the secretory apparatus and was recovered in the cytosol. Basal PKA activity was increased in HIP/PAP expressing cells, suggesting that HIP/PAP may alter PKA signalling. Indeed, we showed, using a thymidine kinase-luciferase reporter plasmid in which a cAMP responsive element was inserted upstream of the thymidine kinase promoter, that luciferase activity was enhanced in HIP/PAP expressing cells. Thus our findings suggest a novel mechanism for the biological activity of the HIP/PAP lectin.

Antigens, Neoplasm↗
PubMed2003

Liver cell polyploidization: a pivotal role for binuclear hepatocytes.

Polyploidy is a general physiological process indicative of terminal differentiation. During liver growth, this process generates the appearance of tetraploid (4n) and octoploid (8n) hepatocytes with one or two nuclei. The onset of polyploidy in the liver has been recognized for quite some time; however, the cellular mechanisms that govern it remain unknown. In this report, we observed the sequential appearance during liver growth of binuclear diploid (2 x 2n) and mononuclear 4n hepatocytes from a diploid hepatocyte population. To identify the cell cycle modifications involved in hepatocyte polyploidization, mitosis was then monitored in primary cultures of rat hepatocytes. Twenty percent of mononuclear 2n hepatocytes failed to undergo cytokinesis with no observable contractile movement of the ring. This process led to the formation of binuclear 2 x 2n hepatocytes. This tetraploid condition following cleavage failure did not activate the p53-dependent checkpoint in G1. In fact, binuclear hepatocytes were able to proceed through S phase, and the formation of a bipolar spindle during mitosis constituted the key step leading to the genesis of two mononuclear 4n hepatocytes. Finally, we studied the duplication and clustering of centrosomes in the binuclear hepatocyte. These cells exhibited two centrosomes in G1 that were duplicated during S phase and then clustered by pairs at opposite poles of the cell during metaphase. This event led only to mononuclear 4n progeny and maintained the tetraploidy status of hepatocytes.

Animals↗
PubMed2003

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/AIMS: We 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. METHODS: Sera 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. RESULTS: Anti-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. CONCLUSIONS: Our 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.

Adult↗
PubMed2003

HIP/PAP stimulates liver regeneration after partial hepatectomy and combines mitogenic and anti-apoptotic functions through the PKA signaling pathway.

The HIP/PAP (=human Reg-2) C-type lectin encoding gene is activated in primary liver cancers. In normal liver, the protein is undetectable in normal mature hepatocytes and found only in some ductular cells, representing potential hepatic progenitor cells. The aim of this study was to examine the consequences of human HIP/PAP expression in the liver of transgenic mice. We demonstrated that HIP/PAP stimulated liver regeneration after partial hepatectomy. To further investigate the enhanced liver regeneration observed in vivo, primary cultures of hepatocytes were used to evaluate the mitogenic and anti-apoptotic properties of HIP/PAP. HIP/PAP increased hepatocyte DNA synthesis and protected hepatocytes against TNF-alpha plus actinomycin-D-induced apoptosis. HIP/PAP anti-apoptotic effects against TNF-alpha were clearly demonstrated when protein kinase A activity was specifically inhibited by KT5720, and HIP/PAP stimulated PKA-dependent phosphorylation of the proapoptotic Bad protein at Ser-112, suggesting that HIP/PAP may compete with cAMP to stimulate PKA activity. Overall, our results led us to propose a new role for a C-type lectin, HIP/PAP, as a hepatic cytokine that combines mitogenic and anti-apoptotic functions regarding hepatocytes, and consequently acts as a growth factor in vivo to enhance liver regeneration.

Animals↗
PubMed2003

Quantitative fluorescence imaging approach for the study of polyploidization in hepatocytes.

We applied automatic quantitative fluorescence imaging of nuclear DNA to rat liver cells obtained from animals at various times after birth up to 3 months of age. We show that, in conditions best preserving the native cellular structures, DNA content measurements, performed on whole single cells in situ after Hoechst staining, were precise and accurate. Cells in the various ploidy and nuclearity classes could thus be identified correctly and their percentages were estimated on a total of 300 cells or more. DNA synthesis was shown to occur asynchronously in all ploidy and nuclearity classes around weaning time. Observation of the labeling patterns, after in vivo BrdU pulse and short-term culture (chase), showed that the cell cycle was shorter in diploid cells compared with cells undergoing polyploidization. These results show that the approach of fluorescence imaging is well suited to investigations on polyploidization mechanisms.

Age Factors↗
PubMed2002

Molecular pathogenesis of human hepatocellular carcinoma.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, the viral-chemical etiology as well as molecular mechanisms of HCC pathogenesis remains largely unknown. Recent studies in our laboratory have identified several potential factors that may contribute to the pathogenesis of HCC. Oxidative stress and chronic inflammation have been linked to an increased risk of liver cancer. For example, oxyradical overload diseases such as Wilson disease and hemochromatosis result in the generation of oxygen/nitrogen species that can cause mutations in the p53 tumor suppressor gene. The Hepatitis B virus X gene (HBx), a viral transactivator with oncogenic potentials, has been shown to bind to and inactivate p53-mediated apoptosis. HBx mutants derived from HCC have a diminished ability to act as a transactivator. However, they still retain the ability to bind to and abrogate p53-mediated apoptosis. The comparison of gene expression profiles between HBx-expressing primary human hepatocytes and HBV-infected liver samples by cDNA microarrays indicate a unique alteration of a subset of oncogenes and tumor suppressor genes including p53. Our studies implicate both viral and endogenous chemical processes in the etiology of HCC, and p53 may be a common target for the inactivation during liver carcinogenesis.

Carcinoma, Hepatocellular↗
PubMed2002

Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection.

BACKGROUND: Some epidemiologic studies suggest a link between hepatitis C virus (HCV) infection and some B-cell non-Hodgkin's lymphomas. We undertook this study after a patient with splenic lymphoma with villous lymphocytes had a hematologic response after antiviral treatment of HCV infection. METHODS: Nine patients who had splenic lymphoma with villous lymphocytes and HCV infection were treated with interferon alfa-2b (3 million IU three times per week) alone or in combination with ribavirin (1000 to 1200 mg per day). The outcomes were compared with those of six similarly treated patients with splenic lymphoma with villous lymphocytes who tested negative for HCV infection. RESULTS: Of the nine patients with HCV infection who received interferon alfa, seven had a complete remission after the loss of detectable HCV RNA. The other two patients had a partial and a complete remission after the addition of ribavirin and the loss of detectable HCV RNA. One patient had a relapse when the HCV RNA load again became detectable in blood. In contrast, none of the six HCV-negative patients had a response to interferon therapy. CONCLUSIONS: In patients with splenic lymphoma with villous lymphocytes who are infected with HCV, treatment with interferon can lead to regression of the lymphoma.

Adult↗
PubMed2002

Identification, using cDNA macroarray analysis, of distinct gene expression profiles associated with pathological and virological features of hepatocellular carcinoma.

It is still unclear as to whether the gene expression profile in HCV- or HBV-related HCC exhibits a degree of specificity and whether the development of HCC in a context of cirrhosis influences this gene profile. To address these issues, the expression profiles of 15 cases of HCC were analysed using cDNA macroarray. A global analysis and hierarchical clustering, demonstrated the heterogeneity of HCC patterns, with a majority of down-regulated genes. Statistical analysis clearly showed a distinction between the gene expression profiles of HCV- and HBV-related HCC. HBV-associated HCC exhibited involvement of different cellular pathways, those controlling apoptosis, p53 signalling and G1/S transition. In HCV-related HCC we identified a more heterogenous pattern with an over-expression of the TGF-beta induced gene. In HCC developing on non-cirrhotic tissues, beta-catenin encoding gene and genes implicated in the PKC pathway were specifically up-regulated. In addition, our investigation highlighted a distinct profiles of TGF-beta superfamily encoding genes in well, moderately or poorly differentiated HCC. Overall, our study supports the hypothesis that despite the heterogeneity of the HCC pattern, the large-scale screening of gene expression may provide data significant to our understanding of the mechanism of liver carcinogenesis.

Carcinoma, Hepatocellular↗