Biomedical subjects

Dina Kremsdorf

Publications and source records attributed to Dina Kremsdorf.

11 recordsLinked to original sources

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↗

PubMed2006

Plasticity of hepatic cell differentiation: bipotential adult mouse liver clonal cell lines competent to differentiate in vitro and in vivo.

In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes). The persistence of such progenitor cells in adult mouse liver is still debated. In damaged liver of adult murine animals, when hepatocyte proliferation is compromised, bipotential oval cells emerge, probably from bile ducts, proliferate, and differentiate to regenerate the liver. However, treatment to elicit oval cell proliferation is not necessary to obtain bipotential stem cells from adult mouse liver. Here, we have isolated bipotential clonal cell lines from healthy liver of 8-10-week-old C57BL/6 mice. Primary cultures established from hepatocyte-enriched suspensions were characterized by time-lapse image acquisition, immunocytology, and RNA transcript analysis. Although hepatocytes dedifferentiated with loss of apical polarity and other hepatocyte markers, they rapidly activated expression of bile duct/oval cell markers. Reversibility of these processes was achieved in part by culture under dilute Matrigel or by aging of confluent cultures. Cell lines were obtained at high frequency from mass cultures, from isolated colonies, and by primary cloning of the hepatocyte-enriched suspension. Cells of the clonal cell lines do not grow in soft agar and are nontumorigenic, and they express cytokeratin 19, A6 antigen, and alpha6 integrin, as well as a large panel of hepatocyte functions. Furthermore, they can participate in liver regeneration in albumin-urokinase-type plasminogen activator/severe combined immune-deficient mice, where they differentiate in clusters of hepatocytes and occasionally bile ducts. These results demonstrate the existence, in normal adult mouse liver, of a significant pool of clonogenic cells that are (or can become) bipotential.

Animals↗

PubMed2005

Human MxA protein participates to the interferon-related inhibition of hepatitis B virus replication in female transgenic mice.

BACKGROUND/AIMS: The interferon (IFN) inducible MxA protein is endowed with antiviral activity against a broad range of RNA viruses. In a previous in vitro study, we demonstrated that MxA inhibits hepatitis B virus (HBV) replication, arguing that the antiviral activity of MxA is not restricted to RNA viruses but also includes a DNA virus. The aim of the present study was to further demonstrate in vivo the antiviral action of MxA against HBV. METHODS: We generated HBV and HBV/MxA transgenic mice lacking a functional IFN-alpha/beta receptor and thus constituting a good model to evaluate MxA-induced virus resistance. HBV proteins expression, viral load and HBV replication were compared in HBV and HBV/MxA mice. RESULTS: An MxA-dependent moderate inhibitory effect on HBV expression was only observed in female HBV/MxA mice, in which MxA downregulates (i) viral HBeAg and capsid protein expression, (ii) viremia and (iii) HBV replication by decreasing the synthesis of HBV DNA replicative intermediates. Furthermore, these effects were not associated with changes to steady-state levels of HBV RNAs. CONCLUSIONS: Our results show that in vivo, MxA is able per se to reduce HBV expression by a post-transcriptional mechanism, and thus participates in the antiviral activity of IFN-alpha against HBV.

Animals↗

PubMed2004

Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes.

Cell lines have many advantages: they can be manipulated genetically, expanded, and stockpiled for organ transplantation. Freshly isolated hepatocytes, oval cells, pancreatic cells, and hematopoietic stem cells have been shown to repopulate the damaged liver. Here we show that bipotential mouse embryonic liver (BMEL) stem cell lines participate in liver regeneration in albumin-urokinase plasminogen activator/severe combined immunodeficiency disease (Alb-uPA/SCID) transgenic mice. In the liver, BMEL-GFP cells proliferate and differentiate into both hepatocytes and bile ducts, forming small to large clusters detected throughout the 3-8 weeks analyzed after transplantation. Moreover, they respond like host cells to signals for growth, differentiation, and even zonal expression of metabolic enzymes, showing regulated expression of cytokeratins and liver-enriched transcription factors. Immunostaining for MHC class I molecules revealed that cells do not coexpress donor and recipient H-2 haplotypes, as would be the case had cell fusion occurred. This report shows that immortalized stem cell lines not only are competent to participate in the repair of a damaged tissue but also can differentiate into the two major epithelial cell types of a complex organ, hepatocytes and bile ducts.

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

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

BACKGROUND/AIMS: The 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. METHODS: Using 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. RESULTS: Heterogeneous 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). CONCLUSIONS: We 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.

Adult↗

PubMed2003

A highly efficient, stable, and rapid approach for ex vivo human liver gene therapy via a FLAP lentiviral vector.

Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.

Animals↗

PubMed2002

Paracrine in vivo inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: an alternative mechanism of HBx-related pathogenesis.

The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose in vivo relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the in vivo impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act, at least in part, through paracrine biological pathways.

Animals↗

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↗

PubMed2002

Hepatitis C virus core protein expression in human B-cell lines does not significantly modify main proliferative and apoptosis pathways.

Hepatitis C virus (HCV) chronic infection has been associated with many lymphoproliferative disorders. Several studies performed on hepatoma and fibroblast cell lines suggest a role of the HCV core protein in activation of cellular transduction pathways that lead to cell proliferation and inhibition of apoptosis. However, no data are available concerning the effects of HCV core expression on B-lymphocyte proliferation and apoptosis. B-lymphocyte cell lines permanently expressing full-length HCV 1b core sequences isolated from chronically infected patients were established using B-cell lines at different degrees of differentiation. Clones and pools of clones permanently expressing the HCV core were selected and characterized for protein expression by Western blot and FACS. Expression of HCV core proteins did not significantly enhance cell proliferation rates under normal culture conditions or under mitogenic stimulation. Analysis of NF-kappa B, CRE, TRE and SRE pathways by luciferase reporter genes did not show a significant influence of HCV core expression on these signal transduction cascades in B-lymphocytes. The effects of HCV core on anti-IgM and anti-FAS-induced apoptosis in B-cell lines was also analysed. In this experimental model, HCV core expression did not significantly modify the apoptotic profile of the B-lymphocyte cell lines tested. These data underline a cell type-specific effect of HCV core expression. In fact, it was not possible to show a significant contribution of the HCV core protein in activation of the major B-cell signal transduction pathways involved in the regulation of proliferation and programmed cell death, which is in contrast with the results reported in hepatoma cell lines.

Antibodies, Anti-Idiotypic↗

PubMed2001

Vaccination of chronic hepatitis B virus carriers with preS2/S envelope protein is not associated with the emergence of envelope escape mutants.

PreS2/S vaccination of chronic hepatitis B virus (HBV) carriers led to a reduction in HBV replication or clearance of virus in 30% of treated patients. This study assessed whether vaccinotherapy of chronic HBV carriers induced the selection of escape mutants in the envelope 'a' determinant and whether envelope genetic variability might affect the response to vaccination. No amino acid differences were observed in the 'a' determinant between sequences obtained before and after treatment (five responders and seven non-responders). However, alignment with HBV prototype sequences revealed seven amino acid changes. Two mutations (T140S and P127L) diverged from subtype variations. In the complete envelope sequence (five non-responders and five responders), ten amino acid modifications were detected between sequences obtained before and after treatment. The absence of any common mutations did not enable the definition of a hot spot of mutations implicated in the response to vaccination. Moreover, vaccinotherapy does not induce the selection of escape mutants in the 'a' determinant.

Amino Acid Sequence↗