Etude du contrôle de la prolifération cellulaire par les interférons :Caractérisation et clonage de protéines induites à la membrane plasmique

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Etude du contrôle de la prolifération cellulaire par les interférons :Caractérisation et clonage de protéines induites à la membrane plasmique

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Stem and progenitor cells for liver regenerative medicine

Orthotopic liver transplantation is the gold standard treatment for end stage liver disease or inborn errors of liver metabolism. However organ shortage, severity of the surgical procedure and risks related to graft loss have stimulated the exploration of less invasive strategies. Liver cell transplantation has demonstrated short to medium-term efficacy in correcting inborn errors of metabolism and could offer a valuable bridging treatment for patients with acute liver failure awaiting liver transplant. However, hepatocyte transplantation in the clinic has shown important limitations including transient effect of donor cells, low proportion of engraftment and repopulation, poor resistance of hepatocytes to cryopreservation procedures and also shortage of procurement. Thus while hepatocyte transplantation remains a promising alternative to liver transplantation, replacement of hepatocytes by proliferating stem cells seems likely to circumvent many of the aforementioned obstacles. Building on the demonstration that various stem and progenitor cells can differentiate in vitro and in vivo into hepatocyte-like cells, these cells are now being investigated as an alternative to hepatocytes in liver regenerative medicine. In this study, we concentrate on cells sourced from human adult tissues only as the use of embryonic or fetal material still faces major ethical, safety and/or feasibility concerns. We summarize here the major progresses that have been achieved as regards the isolation and characterization of potential stem and progenitor cell candidates for liver cell therapies both from intra-and extra-hepatic sources. We also discuss animal transplantation data as well as considerations about the carcinogenic risk of such cell therapies. © 2012 The authors and IOS Press. All rights reserve

Xenopus Neuralized Is a Ubiquitin Ligase that Interacts with XDelta1 and Regulates Notch Signaling

AbstractNotch signaling in Drosophila requires a RING finger (RF) protein encoded by neuralized. Here we show that the Xenopus homolog of neuralized (Xneur) is expressed where Notch signaling controls cell fate choices in early embryos. Overexpressing XNeur or putative dominant-negative forms in embryos inhibits Notch signaling. As expected for a RF protein, we show that XNeur fulfills the biochemical requirements of ubiquitin ligases. We also show that wild-type XNeur decreases the cell surface level of the Notch ligand, XDelta1, while putative inhibitory forms of XNeur increase it. Finally, we provide evidence that XNeur acts as a ubiquitin ligase for XDelta1 in vitro. We propose that XNeur plays a conserved role in Notch activation by regulating the cell surface levels of the Delta ligands, perhaps directly, via ubiquitination

CD69 is expressed on Daudi cells in response to interferon-alpha.

An approach to obtain monoclonal antibodies directed against cell surface proteins induced by interferon has been developed in order to characterize such proteins and determine their role. Hybridomas obtained by fusion of murine myeloma cells and spleen cells of mice immunized with interferon-alpha-treated Daudi cells were screened for the production of antibodies reacting differentially with interferon-alpha-treated and untreated Daudi cells. One such hybridoma, 2D5, produced an antibody reacting with a 28/32 kDa homodimeric protein (p28/32) expressed at the surface of Daudi cells in response to IFN-alpha treatment. IFN-alpha treatment also increased the basal level of p28/32 detected on peripheral blood leukocytes (PBL). 2D5 Antibody was used to probe the expression of p28/32 on different cells and in response to various inducers. It appears that 2D5 reacted in fact with CD69, a marker of leukocyte activation and that, following IFN-alpha treatment, CD69 was not induced on all cultured cell lines tested. Interestingly, IFN-gamma was also able to induce CD69 expression on a restricted number of cell lines but the induction pattern only partially overlapped that of IFN-alpha. As expected, activation of cells with phorbol myristate acetate (PMA) resulted in a notable increase in the level of CD69 on all cell lines considered except for the epithelial and fibroblastic types.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Human progenitor cell quantification after xenotransplantation in rat and mouse models by a sensitive qPCR assay

Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation. Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50 to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intra-splenic transplantation and in rat liver four hours after intra-hepatic transplantation

Nrarp is a novel intracellular component of the Notch signaling pathway

The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Here we describe Nrarp as a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta–Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. We show that Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and that in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription

Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth.

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.Journal Articleinfo:eu-repo/semantics/publishe

Adult-derived human liver stem/progenitor cells infused 3 days post-surgery improve liver regeneration in a mouse model of extended hepatectomy.

There is growing evidence that cell therapy constitutes a promising strategy for liver regenerative medicine. In the setting of hepatic cancer treatments, cell therapy could prove a useful therapeutic approach for managing the acute liver failure that occurs following extended hepatectomy. In this study, we examined the influence of delivering adult human liver mesenchymal stem/progenitor cells (ADHLSCs) at two different early time points in an immunodeficient mouse model (Rag(2-/-)IL2Rγ(-/-) ) that had undergone a 70%-hepatectomy procedure. The hepato-mesenchymal cells were intrasplenically infused either immediately after surgery (n= 26) or following a critical 3-day period (n=26). We evaluated the cells' capacity to engraft at Day 1 and Day 7 following transplantation by means of human Alu qPCR quantification, along with histological assessment of human albumin and alpha-smooth muscle actin. In addition, cell proliferation (anti-mouse and human Ki67 staining) and murine liver weight were measured in order to evaluate liver regeneration. At Day 1 post-transplantation, the ratio of human to mouse cells was similar in both groups, whereas 1 week post-transplantation, this ratio was significantly improved (p <0.016) in mice receiving ADHLSC injection at Day 3 post-hepatectomy (1.7%), compared to those injected at the time of surgery (1%). Based on liver weight, mouse liver regeneration was more extensive 1 week post-transplantation in mice transplanted with ADHLSCs (+65.3%) compared to that of mice from the sham vehicle group (+42.7%). In conclusion, infusing ADHLSCs 3 days after extensive hepatectomy improves the cell engraftment and murine hepatic tissue regeneration, thereby confirming that ADHLSCs could be a promising cell source for liver cell therapy and hepatic tissue repair

Long-Term in Vivo Monitoring of Adult-Derived Human Liver Stem/Progenitor Cells by Bioluminescence Imaging, Positron Emission Tomography, and Contrast-Enhanced Computed Tomography

Adult-derived human liver stem/progenitor cells (ADHLSCs) have the potential to alleviate liver injury. However, the optimal delivery route and long-term biodistribution of ADHLSCs remain unclear. In this article, we used a triple fusion reporter system to determine the kinetic differences in the biodistribution of ADHLSCs following intrasplenic (IS) and intrahepatic (IH) administration in severe combined immunodeficiency/beige mice. ADHLSCs were transduced with a lentiviral vector expressing a triple fusion reporter comprising renilla luciferase, monomeric red fluorescent protein, and truncated HSV-1 thymidine kinase. The stability and duration of the transgenes, and the effects of transduction on the cell properties were evaluated in vitro. The acute retention and long-term engraftment in vivo were revealed by positron emission tomography and bioluminescence imaging (BLI), respectively, followed by histochemical analysis. We showed that ADHLSCs can be safely transduced with the triple fusion reporter. Radiolabeled ADHLSCs showed acute cell retention at the sites of injection. The IH group showed a confined BLI signal at the injection site, while the IS group displayed a dispersed distribution at the upper abdominal liver area, and a more intense signal. In conclusion, ADHLSCs could be monitored by BLI for up to 4 weeks with a spread out biodistribution following IS injection.SCOPUS: ar.jinfo:eu-repo/semantics/publishe