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433 results on '"Immunologie Moléculaire"'

1. Rgg proteins associated with internalized small hydrophobic peptides: a new quorum-sensing mechanism in streptococci

Author: INRA Jouy-en-Josas - UMR1319 MICALIS, INRA Jouy-en-Josas - UR892 Virologie Immunologie Moléculaire, INRA Jouy-en-Josas - UR1077 Mathématique, Informatique et Génome, UCL - SST/ISV - Institut des sciences de la vie, Nancy-Université - UMR1128, IFR110, Laboratoire de Génétique et Microbiologie, Fleuchot, Betty, Gitton, Christophe, Guillot, Alain, Vidic, J., Nicolas, P., Besset, Colette, Fontaine, Laetitia, Hols, Pascal, Leblond-Bourget, Nathalie, Monnet, Véronique, Gardan, Rozenn, INRA Jouy-en-Josas - UMR1319 MICALIS, INRA Jouy-en-Josas - UR892 Virologie Immunologie Moléculaire, INRA Jouy-en-Josas - UR1077 Mathématique, Informatique et Génome, UCL - SST/ISV - Institut des sciences de la vie, Nancy-Université - UMR1128, IFR110, Laboratoire de Génétique et Microbiologie, Fleuchot, Betty, Gitton, Christophe, Guillot, Alain, Vidic, J., Nicolas, P., Besset, Colette, Fontaine, Laetitia, Hols, Pascal, Leblond-Bourget, Nathalie, Monnet, Véronique, and Gardan, Rozenn
Abstract: We identified a genetic context encoding a transcriptional regulator of the Rgg family and a small hydrophobic peptide (SHP) in nearly all streptococci and suggested that it may be involved in a new quorumsensing mechanism, with SHP playing the role of a pheromone. Here, we provide further support for this hypothesis by constructing a phylogenetic tree of the Rgg and Rgg-like proteins from Gram-positive bacteria and by studying the shp/rgg1358 locus of Streptococcus thermophilus LMD-9. We identified the shp1358 gene as a target of Rgg1358, and used it to confirm the existence of the steps of a quorumsensing mechanism including secretion, maturation and reimportation of the pheromone into the cell. We used surface plasmon resonance to demonstrate interaction between the pheromone and the regulatory protein and performed electrophoretic mobility shift assays to assess binding of the transcriptional regulator to the promoter regions of its target genes. The active form of the pheromone was identified by mass spectrometry. Our findings demonstrate that the shp/rgg1358 locus encodes two components of a novel quorum-sensing mechanism involving a transcriptional regulator of the Rgg family and a SHP pheromone that is detected and reimported into the cell by the Ami oligopeptide transporter.
Published: 2011

2. Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency

Author: David Fenard, Ornellie Bernadin, Anais Girard-Gagnepain, Camille Lévy, Chantal Lagresle-Peyrou, Kuiying Ma, Rana Mhaidly, Hanem Sadek, Marina Cavazzana, Fouzia Amirache, Caroline Costa, Ranjita Devi Moirangthem, Isabelle André, François-Loïc Cosset, Els Verhoeyen, Christian Reimann, Didier Nègre, Agata Cieslak, Vahid Asnafi, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Human Lymphohematopoiesis Laboratory (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Lucerne Cantonal Hospital [Luzern, Switzerland], Généthon, CHU Necker - Enfants Malades [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut Necker Enfants-Malades (INEM - UM 111 (UMR 8253 / U1151)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Clinical Investigation Center in Biotherapy, Institut National de la Santé et de la Recherche Médicale (INSERM), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015 - 2019) (COMUE UCA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), Human Virology Department, Virologie humaine, École normale supérieure - Lyon (ENS Lyon)-IFR128-Institut National de la Santé et de la Recherche Médicale (INSERM), Developpement Normal et Pathologique du Système Immunitaire, Université Nice Sophia Antipolis (UNS), Département de Biothérapie [CHU Necker], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Université Paris Descartes - Paris 5 (UPD5)-CHU Necker - Enfants Malades [AP-HP], Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)
Subjects: 0301 basic medicine, medicine.medical_treatment, Genetic enhancement, [SDV]Life Sciences [q-bio], CD34, chemical and pharmacologic phenomena, Mice, SCID, Hematopoietic stem cell transplantation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, complex mixtures, Mice, 03 medical and health sciences, 0302 clinical medicine, Mice, Inbred NOD, medicine, Animals, Progenitor cell, ComputingMilieux_MISCELLANEOUS, Severe combined immunodeficiency, integumentary system, Stem Cells, Lentivirus, hemic and immune systems, Gene Therapy, Hematology, bacterial infections and mycoses, medicine.disease, T cell deficiency, 3. Good health, Cell biology, Haematopoiesis, 030104 developmental biology, 030220 oncology & carcinogenesis, Stem cell, Papio
Abstract: T cells represent a valuable tool for treating cancers and infectious and inherited diseases; however, they are mainly short-lived in vivo. T-cell therapies would strongly benefit from gene transfer into long-lived persisting naive T cells or T-cell progenitors. Here we demonstrate that baboon envelope glycoprotein pseudotyped lentiviral vectors (BaEV-LVs) far outperformed other LV pseudotypes for transduction of naive adult and fetal interleukin-7–stimulated T cells. Remarkably, BaEV-LVs efficiently transduced thymocytes and T-cell progenitors generated by culture of CD34+ cells on Delta-like ligand 4 (Dll4). Upon NOD/SCIDγC−/− engraftment, high transduction levels (80%-90%) were maintained in all T-cell subpopulations. Moreover, T-cell lineage reconstitution was accelerated in NOD/SCIDγC−/− recipients after T-cell progenitor injection compared with hematopoietic stem cell transplantation. Furthermore, γC-encoding BaEV-LVs very efficiently transduced Dll4-generated T-cell precursors from a patient with X-linked severe combined immunodeficiency (SCID-X1), which fully rescued T-cell development in vitro. These results indicate that BaEV-LVs are valuable tools for the genetic modification of naive T cells, which are important targets for gene therapy. Moreover, they allowed for the generation of gene-corrected T-cell progenitors that rescued SCID-X1 T-cell development in vitro. Ultimately, the coinjection of LV-corrected T-cell progenitors and hematopoietic stem cells might accelerate T-cell reconstitution in immunodeficient patients.
Published: 2019
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3. In Vivo Monitoring of Calpain Activity by Forster Resonance Energy Transfer

Author: Israeli, David, Cosette, Jérémie, Corre, Guillaume, Amor, Fatima, Poupiot, Jérôme, Montus, Marie, Gjata, Bernard, Hoch, Lucile, Henriques, Sara, Bruge, Celine, Marsolier, Justine, Benabides, Manon, Bourg, Nathalie, Tournois, Johana, Mahé, Gurvan, Morizur, Lise, Jarrige, Margot, Bigot, Anne, Nissan, Xavier, Lostal, William, Stockholm, Daniel, Richard, Isabelle, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Evry, GENETHON, Genethon, Généthon, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris-Saclay, Université d'Évry-Val-d'Essonne (UEVE), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, and École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon
Subjects: 0301 basic medicine, Proteases, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, γ-sarcoglycanopathy, MESH: Calpain, MESH: Fluorescence Resonance Energy Transfer, MESH: Proteolysis, 030204 cardiovascular system & hematology, Imaging, 03 medical and health sciences, 0302 clinical medicine, translational medicine, Downregulation and upregulation, In vivo, MESH: Animals, limb girdle muscular dystrophy, ComputingMilieux_MISCELLANEOUS, MESH: Humans, biology, Chemistry, MESH: Molecular Biology, mechanical stress, Calpain, AAV, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, gene therapy, Cell biology, Muscle fibers, 030104 developmental biology, Förster resonance energy transfer, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, biology.protein, FRET, LGMD2C, miRNA biomarker, Calpain activity
Abstract: International audience; Calpains are a 15-member class of calcium-activated nonlysosomal neutral proteases. They are involved in many cellular processes and are highly upregulated in pathological conditions. Some are ubiquitously expressed (CAPN1, CAPN2, CAPN4, CAPN5, CAPN7, and CAPN10), but others are thought to be localized in specific tissues. The monitoring of in vivo calpain activity is required for physiological, pathological, and therapeutic evaluations. This past decade, a tool for monitoring calpain activity in such conditions was developed using Forster resonance energy transfer (FRET). Studies showed that the level of calpain activity correlates with a decrease in FRET between the two fluorescent proteins. This chapter describes the methodologies from the design of the construct to the imaging procedure and analysis to evaluate ubiquitous calpain activity in vivo.
Published: 2019
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4. Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia

Author: Laurence Ma, Roger Frutos, Dysoley Lek, Saorin Kim, Odile Mercereau-Puijalon, Sovannaroth Siv, Didier Menard, Emmanuel Cornillot, Chanra Khean, Christelle Reynes, Ankit Dwivedi, Pheaktra Chim, Nimol Khim, Christiane Bourchier, Frédéric Ariey, Magali Tichit, Dany Dourng, Patrice Ravel, Institut de Biologie Computationnelle (IBC), Institut National de la Recherche Agronomique (INRA)-Institut National de Recherche en Informatique et en Automatique (Inria)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Institut du Cancer de Montpellier (ICM), Laboratoire d'épidémiologie moléculaire, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Institut de Génomique Fonctionnelle (IGF), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Génopole, Institut Pasteur [Paris] (IP), National Center for Parasitology, Entomology and Malaria Control [Phnom Penh, Cambodia] (CNM), Interactions hôtes-vecteurs-parasites-environnement dans les maladies tropicales négligées dues aux trypanosomatides (UMR INTERTRYP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut de Recherche pour le Développement (IRD)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université de Bordeaux (UB), Institut d’Electronique et des Systèmes (IES), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Immunologie Moléculaire des Parasites, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], National Centre for Parasitology, Entomology and Malaria Control, Immunologie Moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Herrada, Anthony, Université de Bordeaux (UB)-Institut de Recherche pour le Développement (IRD)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)
Subjects: 0301 basic medicine, Drug Resistance, Drug resistance, Flux de gènes, L73 - Maladies des animaux, Gene flow, MESH: Genotype, 0302 clinical medicine, MESH: Genetic Variation, Malaria, Falciparum, Artemisinin, MESH: Plasmodium falciparum, education.field_of_study, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Mefloquine, MESH: Malaria, Falciparum, 3. Good health, Parasite, Infectious Diseases, S50 - Santé humaine, MESH: Drug Resistance, Cambodia, medicine.drug, Gene Flow, Genotype, Résistance aux médicaments, 030231 tropical medicine, 030106 microbiology, Population, Plasmodium falciparum, Biology, Antimalarials, 03 medical and health sciences, parasitic diseases, medicine, DNA Barcoding, Taxonomic, Humans, Allele, education, MESH: DNA Barcoding, Taxonomic, MESH: Gene Flow, MESH: Humans, business.industry, Research, MESH: Cambodia, Genetic Variation, biology.organism_classification, medicine.disease, Virology, MESH: Antimalarials, Biotechnology, Artémisinine, Malaria, Parasitology, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: Background Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008–2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. Methods To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010–2011 from 16 health centres in malaria endemics areas in Cambodia. Results Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. Conclusions The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1370-y) contains supplementary material, which is available to authorized users.
Published: 2016
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5. Functional analysis of monoclonal antibodies against the Plasmodium falciparum PfEMP1-VarO adhesin

Author: Françoise Marchand, Farida Nato, Inès Vigan-Womas, Micheline Guillotte, Audrey Hessel, Odile Mercereau-Puijalon, Anita Lewit-Bentley, Graham A. Bentley, Alexandre Juillerat, Immunologie Moléculaire des Parasites, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Parasitologie moléculaire et Signalisation, Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris] (IP), Biochimie Structurale et Cellulaire, Immunologie structurale, Immunologie moléculaire des parasites, Unité d'immunologie des maladies infectieuses [Antananarivo, Madagascar] (IPM), Institut Pasteur de Madagascar, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), This work was supported by the French National Research Agency (Agence Nationale de la Recherche, Programme Microbiologie, Immunologie et Maladies Emergentes, grant ANR-07-MIME-021-0). The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under grant agreement No 242095., ANR-07-MIME-0021,ROSETTE,Analyses sérologiques, fonctionnelles et structurales des facteurs de virulence, PfEMP1, impliqués dans le rosetting et l'auto-agglutinantion(2007), European Project: 242095,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,EVIMALAR(2009), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]
Subjects: 0301 basic medicine, Antigenicity, medicine.drug_class, 030231 tropical medicine, Plasmodium falciparum, Protozoan Proteins, Enzyme-Linked Immunosorbent Assay, Rosetting, Monoclonal antibody, Epitope, 03 medical and health sciences, Mice, Epitopes, 0302 clinical medicine, parasitic diseases, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, biology, Research, Antibodies, Monoclonal, Rosette-disrupting antibodies, biology.organism_classification, Monoclonal antibodies (mAbs), Virology, Molecular biology, 3. Good health, Malaria, Bacterial adhesin, 030104 developmental biology, Infectious Diseases, Biotinylation, biology.protein, Parasitology, Antibody, Cell Adhesion Molecules, PfEMP1 adhesin, Protein Binding
Abstract: Background Rosetting, namely the capacity of the Plasmodium falciparum-infected red blood cells to bind uninfected RBCs, is commonly observed in African children with severe malaria. Rosetting results from specific interactions between a subset of variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesins encoded by var genes, serum components and RBC receptors. Rosette formation is a redundant phenotype, as there exists more than one var gene encoding a rosette-mediating PfEMP1 in each genome and hence a diverse array of underlying interactions. Moreover, field diversity creates a large panel of rosetting-associated serotypes and studies with human immune sera indicate that surface-reacting antibodies are essentially variant-specific. To gain better insight into the interactions involved in rosetting and map surface epitopes, a panel of monoclonal antibodies (mAbs) was investigated. Methods Monoclonal antibodies were isolated from mice immunized with PfEMP1-VarO recombinant domains. They were characterized using ELISA and reactivity with the native PfEMP1-VarO adhesin on immunoblots of reduced and unreduced extracts, as well as SDS-extracts of Palo Alto 89F5 VarO schizonts. Functionality was assessed using inhibition of Palo Alto 89F5 VarO rosette formation and disruption of Palo Alto 89F5 VarO rosettes. Competition ELISAs were performed with biotinylated antibodies against DBL1 to identify reactivity groups. Specificity of mAbs reacting with the DBL1 adhesion domain was explored using recombinant proteins carrying mutations abolishing RBC binding or binding to heparin, a potent inhibitor of rosette formation. Results Domain-specific, surface-reacting mAbs were obtained for four individual domains (DBL1, CIDR1, DBL2, DBL4). Monoclonal antibodies reacting with DBL1 potently inhibited the formation of rosettes and disrupted Palo Alto 89F5 VarO rosettes. Most surface-reactive mAbs and all mAbs interfering with rosetting reacted on parasite immunoblots with disulfide bond-dependent PfEMP1 epitopes. Based on competition ELISA and binding to mutant DBL1 domains, two distinct binding sites for rosette-disrupting mAbs were identified in close proximity to the RBC-binding site. Conclusions Rosette-inhibitory antibodies bind to conformation-dependent epitopes located close to the RBC-binding site and distant from the heparin-binding site. These results provide novel clues for a rational intervention strategy that targets rosetting. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-1016-5) contains supplementary material, which is available to authorized users.
Published: 2016
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6. Immunogenicity of the Plasmodium falciparum PfEMP1-VarO Adhesin: Induction of Surface-Reactive and Rosette-Disrupting Antibodies to VarO Infected Erythrocytes

Author: Inès Vigan-Womas, Micheline Guillotte, Cécile Le Scanf, Alexandre Juillerat, Odile Mercereau-Puijalon, Audrey Hessel, Stéphane Petres, Anita Lewit-Bentley, Elodie Crublet, Graham A. Bentley, Sebastien Igonet, Immunologie moléculaire des parasites, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunologie structurale, Production de Protéines Recombinantes et d'Anticorps (Plate-Forme), Institut Pasteur [Paris], CHU Bordeaux [Bordeaux], This work was supported by the French National Research Agency (Agence Nationale de la Recherche, Programme Microbiologie, Immunologie et Maladies Emergentes, grant ANR-07-MIME-021-0, Project Rosettes, PIs OMP and GAB), which supported a fellowship to A. J. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement N° Evimalar 242095. OMP is a member of the Evimalar Network of Excellence. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., We are indebted to the Cytometry Platform, Centre for Human Immunology, Institut Pasteur, for access to FACS analyser. We thank the colleagues of the Unité d'Immunologie Moléculaire des Parasites for helpful discussions. We are indebted to Thierry Blisnick for help in preparing some figures., ANR-07-MIME-0021,ROSETTE,Analyses sérologiques, fonctionnelles et structurales des facteurs de virulence, PfEMP1, impliqués dans le rosetting et l'auto-agglutinantion(2007), European Project: 242095,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,EVIMALAR(2009), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: Antigenicity, Erythrocytes, Rosette Formation, Plasmodium falciparum, Protozoan Proteins, Antibodies, Protozoan, lcsh:Medicine, Antigens, Protozoan, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cross Reactions, Epitope, Epitopes, Mice, Antigen, Malaria Vaccines, parasitic diseases, Animals, Humans, Malaria, Falciparum, Adhesins, Bacterial, lcsh:Science, Mice, Inbred BALB C, Multidisciplinary, biology, Immunogenicity, lcsh:R, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Virology, Recombinant Proteins, Protein Structure, Tertiary, 3. Good health, Bacterial adhesin, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, lcsh:Q, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Antibody, Research Article, Binding domain
Abstract: International audience; Adhesion of Plasmodium falciparum-infected red blood cells (iRBC) to human erythrocytes (i.e. rosetting) is associated with severe malaria. Rosetting results from interactions between a subset of variant PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) adhesins and specific erythrocyte receptors. Interfering with such interactions is considered a promising intervention against severe malaria. To evaluate the feasibility of a vaccine strategy targetting rosetting, we have used here the Palo Alto 89F5 VarO rosetting model. PfEMP1-VarO consists of five Duffy-Binding Like domains (DBL1-5) and one Cysteine-rich Interdomain Region (CIDR1). The binding domain has been mapped to DBL1 and the ABO blood group was identified as the erythrocyte receptor. Here, we study the immunogenicity of all six recombinant PfEMP1-VarO domains and the DBL1- CIDR1 Head domain in BALB/c and outbred OF1 mice. Five readouts of antibody responses are explored: ELISA titres on the recombinant antigen, VarO-iRBC immunoblot reactivity, VarO-iRBC surface-reactivity, capacity to disrupt VarO rosettes and the capacity to prevent VarO rosette formation. For three domains, we explore influence of the expression system on antigenicity and immunogenicity. We show that correctly folded PfEMP1 domains elicit high antibody titres and induce a homogeneous response in outbred and BALB/c mice after three injections. High levels of rosette-disrupting and rosette-preventing antibodies are induced by DBL1 and the Head domain. Reduced-alkylated or denatured proteins fail to induce surface-reacting and rosette-disrupting antibodies, indicating that surface epitopes are conformational. We also report limited cross-reactivity between some PfEMP1 VarO domains. These results highlight the high immunogenicity of the individual domains in outbred animals and provide a strong basis for a rational vaccination strategy targeting rosetting.
Published: 2015
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7. Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory

Author: Yoshiaki Yamagata, Ophélie Arnaud, José Viñuelas, Thi My Anh Neildez-Nguyen, Guillaume Corre, Guillaume Beslon, Gaël Kaneko, Jean-Jacques Kupiec, Daniel Stockholm, Andras Paldi, Olivier Gandrillon, École pratique des hautes études (EPHE), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Artificial Evolution and Computational Biology (BEAGLE), Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire d'InfoRmatique en Image et Systèmes d'information (LIRIS), Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon-Université de Lyon-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA), Laboratoire d'InfoRmatique en Image et Systèmes d'information (LIRIS), Department of Obstetrics and Gynaecology, Yamaguchi University Graduate School of Medicine, La République des savoirs : Lettres, Sciences, Philosophie, Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS)-Département de Philosophie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris), Centre Cavaillès, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS)-Département de Philosophie - ENS Paris, Généthon, Genethon, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris sciences et lettres (PSL), Immunologie moléculaire et biothérapies innovantes (IMBI), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-École Centrale de Lyon (ECL), Université de Lyon-Université Lumière - Lyon 2 (UL2)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Lyon (ECL), Université de Lyon-Université Lumière - Lyon 2 (UL2), Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)-Département de Philosophie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)-Département de Philosophie - ENS Paris, École Pratique des Hautes Études (EPHE), Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Université Lumière - Lyon 2 (UL2)-École Centrale de Lyon (ECL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), and Kupiec, Jean-Jacques
Subjects: Epigenomics, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Heredity, Cell division, Transcription, Genetic, Expression stochastique des genes, lcsh:Medicine, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Genes, Reporter, Gene expression, Biochemical Simulations, Tumor Cells, Cultured, lcsh:Science, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, Genetics, Regulation of gene expression, Gene knockdown, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, Retinoblastoma, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Cell biology, Gene Expression Regulation, Neoplastic, Phenotype, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.IMM]Life Sciences [q-bio]/Immunology, Epigenetics, Stochastic Gene Expression, Research Article, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Gene product, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BDD] Life Sciences [q-bio]/Development Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell Lineage, Computer Simulation, Physiologie cellulaire, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Gene, Reporter gene, [SDV.GEN]Life Sciences [q-bio]/Genetics, Stochastic Processes, lcsh:R, Biology and Life Sciences, Computational Biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Embryo, Mammalian, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, Cellular physiology, lcsh:Q
Abstract: International audience; Despite the stochastic noise that characterizes all cellular processes the cells are able to maintain and transmit to their daughter cells the stable level of gene expression. In order to better understand this phenomenon, we investigated the temporal dynamics of gene expression variation using a double reporter gene model. We compared cell clones with transgenes coding for highly stable mRNA and fluorescent proteins with clones expressing destabilized mRNA-s and proteins. Both types of clones displayed strong heterogeneity of reporter gene expression levels. However, cells expressing stable gene products produced daughter cells with similar level of reporter proteins, while in cell clones with short mRNA and protein half-lives the epigenetic memory of the gene expression level was completely suppressed. Computer simulations also confirmed the role of mRNA and protein stability in the conservation of constant gene expression levels over several cell generations. These data indicate that the conservation of a stable phenotype in a cellular lineage may largely depend on the slow turnover of mRNA-s and proteins.
Published: 2014
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8. Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle

Author: Adrien Morin, Amalia Stantzou, Olga N. Petrova, John Hildyard, Thomas Tensorer, Meriem Matouk, Mina V. Petkova, Isabelle Richard, Tudor Manoliu, Aurélie Goyenvalle, Sestina Falcone, Markus Schuelke, Corinne Laplace-Builhé, Richard J. Piercy, Luis Garcia, Helge Amthor, Handicap neuromusculaire : Physiopathologie, Biothérapie et Pharmacologies appliquées (END-ICAP), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Institut National de la Santé et de la Recherche Médicale (INSERM), Royal Veterinary College [London], University of London [London], Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), Charité - UniversitätsMedizin = Charité - University Hospital [Berlin], Berlin Institute of Health (BIH), Généthon, Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Analyse moléculaire, modélisation et imagerie de la maladie cancéreuse (AMMICa), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut Gustave Roussy (IGR), Centre Scientifique de Monaco (CSM), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Hôpital Raymond Poincaré [Garches], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université Paris-Saclay, Association Française contre les Myopathies, AFM, Agence Nationale de la Recherche, ANR, Deutsch-Französische Hochschule, DFH: CDFA-06-11, We thank Thomas Bestetti, Chloé Dambrune, Karima Relizani, and Cécile Gastaldi for technical assistance. We thank Dr. Feng Zhang for providing the plasmid pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA, and U6::BsaIsgRNA. We also thank Frederic De Leeuw for assistance with multiphoton imaging and Matthieu Dos Santos for preliminary RNA scope experiments. This work was supported by the Association Monegasque contre les Myopathies (AMM, Monaco), the Université Franco-Allemande (CDFA-06-11, UFA), the Association Française contre les Myopathies (AFM, France), and the Agence National de la Recherche (ANR, France).
Subjects: Gene Editing, Multidisciplinary, [SDV]Life Sciences [q-bio], Muscles, dystrophin-EGFP, nuclear domain, Dystrophin, Muscular Dystrophy, Duchenne, Mice, Disease Models, Animal, Treatment Outcome, Mice, Inbred mdx, Animals, Female, mdx mouse, myotendinous junction, Muscle, Skeletal
Abstract: Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin ( Dmd EGFP ), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called “basal sarcolemmal dystrophin units (BSDUs).” These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber—with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle–tendon junctions.
Published: 2023
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9. Cardiac characterization of sgca-null mice using high resolution echocardiography

Author: Fayssoil, Abdallah, Renault, Gilles, Guerchet, Nicolas, Marchiol-Fournigault, Carmen, Fougerousse, Françoise, Richard, Isabelle, Fougerousse, Françoise, Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire d'Imagerie Fonctionnelle (LIF), Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-IFR49-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Généthon R&D, Genethon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), and École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Null mice, medicine.medical_specialty, Pathology, alpha-sarcoglycanopathy, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Diastole, High resolution, Neurosciences. Biological psychiatry. Neuropsychiatry, Doppler echocardiography, Article, 03 medical and health sciences, 0302 clinical medicine, Internal medicine, medicine, echocardiography, Muscular dystrophy, Prospective cohort study, 030304 developmental biology, SGCA, 0303 health sciences, LV function, Ejection fraction, medicine.diagnostic_test, business.industry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, Sgca-null mice, RC31-1245, Sgca-null mice, alpha-sarcogly-canopathy, LV function, echocardiography, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Cardiology, Medicine, Neurology (clinical), alpha-sarcogly-canopathy, business, 030217 neurology & neurosurgery, RC321-571
Abstract: Limb-girdle muscular dystrophy 2D (LGMD2D) is an inherited myogenic disorder belonging to the group of muscular dystrophies. Sgca-null mouse is a knock-out model of LGMD2D. Little is known about cardiac phenotype characterization in this model at different ages. We conducted a prospective study to characterize cardiac sgca-null mice phenotype using high resolution Doppler echocardiography at different ages. Conventional echocardiography was performed on anesthetised mice using a Vevo 770 (Visualsonics) with 30 MHz cardiac probe. Wild Type (WT) and sgca-null mice were scanned at 13, 15 and 17 months. From M-mode, we measured interventricular septal (IVS) wall thickness, posterior wall (PW) thickness, and end-left ventricular diameter in systolic and diastolic. From the above parameters, we calculated left ventricular (LV) shortening fraction (SF), LV ejection fraction (EF) and LV mass. At age 13 months, PW diastolic thickness was increased in sgca-null mice (0.89±0.14 mm vs 0.73±0.2 mm; P=0.020) and LV mass was higher in sgca-null mice (LV mass 205.2 mg vs 143 mg; P=0.001). We found also dilation of the LV (LVEDD: 4.84 mm vs 4.29 mm; P=0.019) in sgca-null mice. At age 15 months, dilation of the LV (LVEDD: 4.86 mm vs 4 mm; P=0.05) with an increase of the LV mass (165.7 mg vs 127.12; P=0.03) are found in sgca-null mice. At age 17 months, we found a decrease of the PW thickening (17% vs 30%; P=0.036). This work provides echocardiographic insights for the assessment of pharmaceutical therapies in sgca-null mice.
Published: 2013
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10. Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4-L1

Author: Daniel Scherman, Sandrine Genries, David Fenard, Anne Galy, Samia Martin, Antoine Kichler, Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Unité de pharmacologie chimique et génétique et d'imagerie (UPCGI - UMR 8151 / UMR_S 1022 ), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC), Généthon, This work was supported by the Association Française contre les Myopathies., Fenard, David, École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), École Pratique des Hautes Études (EPHE), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon-École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon-Généthon, Association française contre les myopathies ( AFM-Téléthon ) -Association française contre les myopathies ( AFM-Téléthon ), Association française contre les myopathies ( AFM-Téléthon ), École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, Unité de pharmacologie chimique et génétique et d'imagerie ( UPCGI - UMR 8151 / UMR_S 1022 ), and Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL ( ENSCP ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS )
Subjects: MESH : Hematopoietic stem cell, MESH : retrovirus, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, Virus Attachment, Biology, Hematopoietic stem cell, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Gene therapy, MESH: Gene therapy, Transduction, Genetic, MESH : Gene therapy, Virology, medicine, Humans, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, MESH : cationic peptide, Cells, Cultured, 030304 developmental biology, Infectivity, 0303 health sciences, MESH: cationic peptide, Lentivirus, lentiviral vector, MESH: Hematopoietic stem cell, [ SDV.BIO ] Life Sciences [q-bio]/Biotechnology, Transfection, cationic amphipathic peptide, Recombinant Proteins, Fibronectins, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 3. Good health, [INFO.INFO-BT] Computer Science [cs]/Biotechnology, medicine.anatomical_structure, 030220 oncology & carcinogenesis, MESH: retrovirus, Stem cell, Peptides, [ INFO.INFO-BT ] Computer Science [cs]/Biotechnology, Ex vivo, Antimicrobial Cationic Peptides
Abstract: International audience; Lentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols.
Published: 2013
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11. Cardiac Characterization of mdx Mice Using High-Resolution Doppler Echocardiography

Author: Fayssoil, Abdallah, Renault, Gilles, Guerchet, Nicolas, Marchiol-Fournigault, Carmen, Fougerousse, Françoise, Richard, Isabelle, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Imagerie Fonctionnelle (LIF), Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR14-IFR49-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Généthon R&D, Genethon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), and École pratique des hautes études (EPHE)
Subjects: musculoskeletal diseases, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, MESH: Echocardiography, Doppler, MESH: Algorithms, Sensitivity and Specificity, Mice, Ventricular Dysfunction, Left, MESH: Mice, Inbred C57BL, Image Interpretation, Computer-Assisted, MESH: Ventricular Dysfunction, Left, MESH: Muscular Dystrophy, Duchenne, Animals, MESH: Animals, MESH: Mice, MESH: Mice, Inbred mdx, Reproducibility of Results, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Image Enhancement, Echocardiography, Doppler, MESH: Sensitivity and Specificity, Mice, Inbred C57BL, Muscular Dystrophy, Duchenne, MESH: Reproducibility of Results, MESH: Cardiomyopathies, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Mice, Inbred mdx, MESH: Image Enhancement, Cardiomyopathies, MESH: Image Interpretation, Computer-Assisted, Algorithms
Abstract: Duchenne muscular dystrophy is an X-linked neuromuscular disorder. The heart is traditionally involved, leading to heart failure. The mdx mouse is a natural animal model of the disease. We conducted a prospective study to analyze left ventricular (LV) function in mdx mice at different ages using high-resolution Doppler echocardiography.Echocardiography was performed with a 30-MHz cardiac probe. Wild-type and mdx mice were scanned at 10 and 12 months. We measured the interventricular septal wall thickness, posterior wall thickness, and LV diameter in systole and diastole. The LV shortening fraction, LV ejection fraction, and LV mass were then calculated.At 10 months, the shortening fractions in mdx and wild-type mice were relatively close (29% ± 5% versus 25% ± 4%). We found a significant difference in the posterior wall thickness change (40% ± 12% in mdx versus 28% ± 10% in wild-type; P = .048). The LV mass/body weight ratio was higher in mdx than wild-type mice (3.67 ± 0.25 versus 3.39 ± 0.26; P = .05). At 12 months, the LV mass was elevated in mdx mice compared to wild-type (152 ±16 versus 135 ± 3.7 mg; P = .04). The diastolic posterior wall thickness change was decreased in mdx mice at 12 months compared to wild-type (21% ± 7% versus 33% ± 4%; P = .01). The LV ejection fraction was not statistically different between mdx and wild-type mice (50% ± 6% versus 54% ± 2%).Ten-month-old mdx mice had a significantly higher posterior wall thickness than wild-type mice, but it was not significant at 12 months. In 12-month-old mdx mice, the posterior wall thickness change was significantly lower, and the LV mass was significantly higher. These findings indicate the role of LV function in the early stages of Duchenne muscular dystrophy.
Published: 2013
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12. Lymphoma B-cell responsiveness to CpG-DNA depends on the tumor microenvironment

Author: Hanane Ouakrim, Sabrina Donnou, Catherine Sautès-Fridman, Aouni Mahjoub, Lucile Crozet, Rym Ben Abdelwahed, Sylvain Fisson, Wolf H. Fridman, Jérémie Cosette, Faculté de Pharmacie [Monastir] (FPHM), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Matière et Systèmes Complexes (MSC), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, This work was supported by the Institut National du Cancer (Grants RC013- C06N631-2005 and C06N748-2006), the Association pour la Recherche contre le Cancer (ARC), the Institut National de la Santé et de la Recherche Médicale (INSERM), the University Pierre and Marie Curie (UPMC, Convergence project), the University Paris-Descartes, the pole de compétitivité Ile de France (ImmuCan project), the Tunisian Direction Générale de la Recherche Scientifique, and the French-Tunisian DGRS-INSERM and CMCU (Egide-PHC Utique) projects. RBA received grants from the DGRS-INSERM and the CMCU. S.D. received a grant from the Institut National du Cancer (INCa)., Faculté de Pharmacie, Faculté des Sciences de Monastir-Université de Monastir ( UM ), Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire Matière et Systèmes Complexes ( Laboratoire MSC ), Université Paris Diderot - Paris 7 ( UPD7 ) -UFR de Physique, Genethon, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Assistance publique - Hôpitaux de Paris (AP-HP)-Association française contre les myopathies ( AFM-Téléthon ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Assistance publique - Hôpitaux de Paris (AP-HP)-Association française contre les myopathies ( AFM-Téléthon ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Institut des Biothérapies, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, BMC, Ed., Matière et Systèmes Complexes (MSC (UMR_7057)), École pratique des hautes études (EPHE), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)
Subjects: Primary cerebral lymphoma, Cancer Research, Lymphoma, B-Cell, Primary intraocular lymphoma, medicine.medical_treatment, Apoptosis, [SDV.CAN]Life Sciences [q-bio]/Cancer, Cell Growth Processes, Biology, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, 03 medical and health sciences, Mice, 0302 clinical medicine, [SDV.CAN] Life Sciences [q-bio]/Cancer, Non-Hodgkin B-cell lymphoma, In vivo, TLR, medicine, Animals, Humans, B cell, Tumor microenvironment, Mice, Inbred BALB C, Brain Neoplasms, Research, Eye Neoplasms, TLR9, Immunotherapy, DNA, Neoplasm, CpG-DNA, medicine.disease, 3. Good health, Lymphoma, Disease Models, Animal, medicine.anatomical_structure, Subcutaneous lymphoma, Oncology, CpG site, Oligodeoxyribonucleotides, 030220 oncology & carcinogenesis, Toll-Like Receptor 9, Cancer research, CpG Islands, Female, Intraocular lymphoma, 030215 immunology
Abstract: Background Toll-like receptor (TLR) agonists have important properties that can be exploited for immunotherapy against tumors. Locally injected immunostimulatory oligodeoxynucleotides containing CpG motifs (CpG-ODNs), which are TLR9 agonists, have shown promise in cancer models. Several studies have demonstrated that these motifs have immunologic effects similar to those of bacterial DNA and can stimulate monocytes, macrophages, dendritic, and B cells, which then produce several proinflammatory cytokines. However, these CpG-ODNs appear to produce opposite effects on tumor B cells. Methods In this study, we investigated the direct effects of a murine class B CpG (1826) ODNs on lymphoma B cells in vitro and in vivo, using mouse models of non-Hodgkin B lymphomas developing in immunoprivileged sites, specifically the brain and the eye, and in subcutaneous sites. Results In vitr o, CpG-ODNs produced antiproliferative and proapoptotic effects on lymphoma B cells. In vivo, it had an antitumor effect when injected into tumors in murine models of subcutaneous lymphoma (SCL) and primary cerebral lymphoma (PCL). However, its intravitreal administration into a primary intraocular lymphoma (PIOL) mouse model did not produce an antitumor effect. In vitro experiments using supernatant from mouse PIOL samples demonstrated that the PIOL molecular microenvironment inhibits the antiproliferative effect of CpG-ODNs on lymphoma B-cells. Conclusions Responsiveness to CpG stimulation differs in subcutaneous, cerebral, and ocular tumors, according to the tumoral and molecular microenvironment, and this should be considered for further therapeutic approaches.
Published: 2013
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13. Hyperosmotic stress contributes to mouse colonic inflammation through the methylation of protein phosphatase 2A

Author: Xavier Wertz, Laurent Schwartz, Philippe Chaumet-Riffaud, Jean-Marc Steyaert, Mohammad Pooya, Maurice Israël, Adeline Guais, Mohammad Abolhassani, Laboratoire d'informatique de l'École polytechnique [Palaiseau] (LIX), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), BCG department, Institut Pasteur d'Iran, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Algorithms and Models for Integrative Biology (AMIB ), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Laboratoire de Recherche en Informatique (LRI), Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), Chaire Immunologie moléculaire, Collège de France (CdF (institution)), Laboratoire de neurobiologie cellulaire et moléculaire (NBCM), Centre National de la Recherche Scientifique (CNRS), Biorébus, Service de Biophysique et de Médecine Nucléaire, AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Recherche en Informatique (LRI), and Collège de France - Chaire Immunologie moléculaire
Subjects: Male, medicine.medical_specialty, Physiology, Inflammation, Biology, Methylation, Inflammatory bowel disease, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Stress, Physiological, Physiology (medical), Internal medicine, medicine, Animals, Gene Silencing, Protein Phosphatase 2, RNA, Small Interfering, Colitis, 030304 developmental biology, Mice, Inbred BALB C, 0303 health sciences, Hepatology, Osmotic concentration, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Dextran Sulfate, Osmolar Concentration, Gastroenterology, Protein phosphatase 2, Okadaic acid, medicine.disease, digestive system diseases, 3. Good health, Enzyme Activation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Endocrinology, chemistry, 030220 oncology & carcinogenesis, Immunology, Cytokines, Cytokine secretion, Mannitol, medicine.symptom, medicine.drug
Abstract: International audience; There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation attributable to methylation of the catalytic subunit of protein phosphatase 2A (PP2A) and subsequent NF-kappaB activation resulting in cytokine secretion. In this study, we demonstrate that dextran sulfate sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm, and 627 mOsm) for a duration of 3 wk or with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm) for a duration of 12 wk. Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-kappaB activation. and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.
Published: 2008
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14. Many human peripheral VH5-expressing IgM+ B cells display a unique heavy-chain rearrangement

Author: Philippe Kourilsky, Xavier Wertz, François Huetz, Brigitte Lemercier, Annick Lim, Sarah Lesjean Pottier, Développement des Lymphocytes, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Chaire Immunologie moléculaire, Collège de France (CdF (institution)), Cytokines et Développement Lymphoïde, Immunité Cellulaire Antivirale, Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Collège de France - Chaire Immunologie moléculaire, and Institut Pasteur [Paris] (IP)
Subjects: Male, Gene Rearrangement, B-Lymphocyte, Heavy Chain, Immunoglobulin Variable Region, MESH: Immunoglobulin Variable Region, MESH: Flow Cytometry, Polymerase Chain Reaction, law.invention, 0302 clinical medicine, law, Immunology and Allergy, MESH: Gene Rearrangement, B-Lymphocyte, Heavy Chain, Polymerase chain reaction, 0303 health sciences, Heavy chain, B-Lymphocytes, MESH: Middle Aged, biology, medicine.diagnostic_test, General Medicine, Middle Aged, Flow Cytometry, MESH: Immunoglobulin M, Peripheral, Immunoglobulin Isotypes, [SDV.IMM]Life Sciences [q-bio]/Immunology, Female, Immunoglobulin Heavy Chains, MESH: Immunoglobulin Heavy Chains, Adult, Immunology, Immunoglobulin light chain, Flow cytometry, 03 medical and health sciences, MESH: B-Lymphocytes, medicine, Humans, 030304 developmental biology, MESH: Humans, MESH: Adult, MESH: Polymerase Chain Reaction, T lymphocyte, Molecular biology, MESH: Male, Immunoglobulin M, MESH: Immunoglobulin Isotypes, biology.protein, Immunoglobulin heavy chain, MESH: Female, 030215 immunology
Abstract: International audience; The immunoscope methodology has proven useful to analyze T-cell repertoires in mice and humans. We adapted it to the analysis of VH chains of human peripheral B cells by setting up a quantification of various VH and JH segments and the profiling of IgM-, IgG-, IgA- and IgE-expressing B cells. We then tested the hypothesis that the human B-cell and T-cell repertoires have a similar diversity of VH and V-beta rearrangements. We studied in more detail the VH5 family because it is not abundantly used, which facilitated the analysis. The data showed that the number of distinct VH5 rearrangements in all samples studied is close to the number of cells in the sample. This contrasts with T cells in which we previously showed that distinct V-beta rearrangements amount to a few percent of the number of T cells because each V-beta chain is on the average paired with approximately 25 alpha chains. Thus, in the VH5 family, the light chains add little quantitative diversity to that produced by the heavy chain alone. Whether this feature can be generalized to other VH chains is discussed.
Published: 2007
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15. Delayed and separate costimulation in vitro supports the evidence of a transient 'excited' state of CD8+ T cells during activation

Author: Christophe Cambouris, Nadège Bercovici, Fabrice Lemaître, Roland S. Liblau, Nathalie Pardigon, Philippe Kourilsky, Biologie Moléculaire du Gène, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Immunologie Cellulaire et Clinique, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Institut National de la Santé et de la Recherche Médicale (INSERM), Collège de France - Chaire Immunologie moléculaire, Collège de France (CdF (institution)), We thank S. Darche for SC-Kd preparation, Drs. O. Acuto, E. G. Strauss, J. Bradley, and D. Guy-Grand for critical reading of the manuscript, and Ph. Bousso and Drs. J. Kanellopoulos and D. Laouini for helpful discussions., Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), and Chaire Immunologie moléculaire
Subjects: MESH: Signal Transduction, Time Factors, Cell Culture Techniques, Stimulation, Cell Communication, CD8-Positive T-Lymphocytes, Lymphocyte Activation, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, Major Histocompatibility Complex, Mice, 0302 clinical medicine, Immunology and Allergy, Cytotoxic T cell, MESH: Animals, Cells, Cultured, 0303 health sciences, biology, Long-term potentiation, MESH: Receptors, Antigen, T-Cell, MESH: CD8-Positive T-Lymphocytes, Cell biology, medicine.anatomical_structure, MESH: Oligopeptides, MESH: Cell Division, Oligopeptides, Cell Division, MESH: Dose-Response Relationship, Immunologic, Signal Transduction, MESH: Cells, Cultured, MESH: Mice, Transgenic, T cell, Immunology, Dose-Response Relationship, Immunologic, Receptors, Antigen, T-Cell, Mice, Transgenic, Major histocompatibility complex, MESH: Interphase, 03 medical and health sciences, MESH: Major Histocompatibility Complex, MESH: Cell Communication, medicine, Animals, MESH: Lymphocyte Activation, Interphase, MESH: Mice, 030304 developmental biology, MESH: Cell Culture Techniques, T-cell receptor, MESH: Time Factors, In vitro, biology.protein, CD8, 030215 immunology
Abstract: Although the two-signal model for T cell activation states that a signal-1 through the TCR and a costimulatory signal-2 are required for optimal stimulation, it is now clear that the requirement for costimulation can be bypassed under certain conditions. We previously reported that this is the case for naive CD8+ T cells in vitro. In the present study we tested the effect of signal-2 when delivered after signal-1 has been disrupted. Naive CD8+ T cells from TCR transgenic mice were stimulated in vitro by using immobilized recombinant single-chain MHC molecules alone as signal-1. This signal was then stopped after different lengths of time, and anti-CD28 mAb as signal-2 was given either immediately or after a time lag. We found that signal-2 can potentiate a short signal-1 when added sequentially. Moreover, a time lag between the two signals does not abolish this potentiation. If the strength of signal-1, but not its duration, is increased, then the time lag between the delivery of signals 1 and 2 can be lengthen without loss of potentiation. Together, our results indicate that the two signals do not need to be delivered concomitantly to get optimal T cell activation. We suggest that the CD8+ T cells can reach a transient “excited” state after being stimulated with signal-1 alone, characterized by the cell’s ability to respond to separate and delayed signal-2.
Published: 2000
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16. The complete nucleotide sequence of the mitochondrial DNA of the agnathan Lampetra fluviatilis : bearings on the phylogeny of cyclostomes

Author: Christiane Delarbre, Philippe Janvier, Vincent Laudet, Cyril Gallut, Philippe Kourilsky, Gabriel Gachelin, Hector Escriva, Véronique Barriel, Collège de France - Chaire Immunologie moléculaire, Collège de France (CdF (institution)), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chaire Immunologie moléculaire, École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon
Subjects: 0106 biological sciences, Mitochondrial DNA, [SDV]Life Sciences [q-bio], mitochondrial DNA, phylogeny, DNA, Mitochondrial, Polymerase Chain Reaction, 010603 evolutionary biology, 01 natural sciences, cyclostomes, 03 medical and health sciences, Lampetra fluviatilis, Lampetra, Phylogenetics, biology.animal, Genetics, Animals, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Synapomorphy, 0303 health sciences, biology, Lamprey, Lampreys, Vertebrate, Sequence Analysis, DNA, biology.organism_classification, Sister group, Evolutionary biology, Hagfish
Abstract: International audience; There are two competing theories about the interrelationships of craniates: the cyclostome theory assumes that lampreys and hagﬁshes are a clade, the cyclostomes, whose sister group is the jawed vertebrates (gnathostomes); the vertebrate theory assumes that lampreys and gnathostomes are a clade, the vertebrates, whose sister group is hagﬁshes. The vertebrate theory is best supported by a number of unique anatomical and physiological characters. Molecular sequence data from 18S and 28S rRNA genes rather support the cyclostome theory, but mtDNA sequence of Myxine glutinosa rather supports the vertebrate theory. Additional molecular data are thus needed to elucidate this three-taxon problem. We determined the complete nucleotide sequence of the mtDNA of the lamprey Lampetra ﬂuviatilis. The mtDNA of L. ﬂuviatilis possesses the same genomic organization as Petromyzon marinus, which validates this gene order as a synapomorphy of lampreys. The mtDNA sequence of L. ﬂuviatilis was used in combination with relevant mtDNA sequences for an approach to the hagﬁsh/lamprey relationships using the max- imum-parsimony, neighbor-joining, and maximum-likelihood methods. Although trees compatible with our present knowledge of the phylogeny of craniates can be reconstructed by using the three methods, the data collected do not support the vertebrate or the cyclostome hypothesis. The present data set does not allow the resolution of this three-taxon problem, and new kinds of data, such as nuclear DNA sequences, need to be collected.
Published: 2000
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17. Dynamics of centriole amplification in centrosome-depleted brain multiciliated progenitors

Author: Marion Faucourt, Olivier Mercey, Alexia Mahuzier, Alice Meunier, Aurélien Fortoul, Amelie-Rose Boudjema, Nathalie Spassky, Adel Al Jord, Philippe Rostaing, Virologie et Immunologie Moléculaire, Faculté des Sciences Pharmaceutiques, Institut National de la Recherche Agronomique (INRA)-Université Francois Rabelais [Tours], Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des sciences et techniques de l'information, de la communication et de la connaissance (Lab-STICC), École Nationale d'Ingénieurs de Brest (ENIB)-Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Télécom Bretagne-Institut Brestois du Numérique et des Mathématiques (IBNM), Université de Brest (UBO)-Université européenne de Bretagne - European University of Brittany (UEB)-École Nationale Supérieure de Techniques Avancées Bretagne (ENSTA Bretagne)-Institut Mines-Télécom [Paris] (IMT)-Centre National de la Recherche Scientifique (CNRS), Regionalisation du cerveau des vertébrés - Organogenèse précoce chez la souris et maladies génétiques associées, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: Centriole, sports, [SDV]Life Sciences [q-bio], Science, Fluorescent Antibody Technique, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Cellular imaging, Article, 03 medical and health sciences, Procentriole, 0302 clinical medicine, Microtubule, Humans, Cilia, 030304 developmental biology, Pericentriolar material, Centrioles, Centrosome, Organelles, 0303 health sciences, Cilium, Cell Cycle, Brain, Epithelial Cells, Cell biology, Molecular Imaging, sports.league, Deuterosome, Motile cilium, Medicine, 030217 neurology & neurosurgery, Biomarkers
Abstract: International audience; Reproductive and respiratory organs, along with brain ventricles, are lined by multiciliated epithelial cells (MCC) that generate cilia-powered fluid flows. MCC hijack the centrosome duplication pathway to form hundreds of centrioles and nucleate motile cilia. In these cells, the large majority of procentrioles are formed associated with partially characterized organelles called deuterosomes. We recently challenged the paradigm that deuterosomes and procentrioles are formed de novo by providing data, in brain MCC, suggesting that they are nucleated from the pre-existing centrosomal younger centriole. However, the origin of deuterosomes and procentrioles is still under debate. Here, we further question centrosome importance for deuterosome and centriole amplification. First, we provide additional data confirming that centriole amplification occurs sequentially from the centrosomal region, and that the first procentriole-loaded deuterosomes are associated with the daughter centriole or in the centrosomal centriole vicinity. Then, to further test the requirement of the centrosome in deuterosome and centriole formation, we depleted centrosomal centrioles using a Plk4 inhibitor. We reveal unexpected limited consequences in deuterosome/centriole number in absence of centrosomal centrioles. Notably, in absence of the daughter centriole only, deuterosomes are not seen associated with the mother centriole. In absence of both centrosomal centrioles, procentrioles are still amplified sequentially and with no apparent structural defects. They seem to arise from a focal region, characterized by microtubule convergence and pericentriolar material (PCM) assembly. The relevance of deuterosome association with the daughter centriole as well as the role of the PCM in the focal and sequential genesis of centrioles in absence of centrosomal centrioles are discussed.
Published: 2019
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18. Bistable Cell Fate Specification as a Result of Stochastic Fluctuations and Collective Spatial Cell Behaviour

Author: Peggy Sanatine, Yoshiaki Yamagata, Daniel Stockholm, Sophie Coutant, Laurent Le Guillou, Guillaume Corre, Andras Paldi, Thi My Anh Neildez-Nguyen, Frédérique Edom-Vovard, Généthon, Institut de Myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institute for Research and Innovation in Biomedicine (IRIB), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Département de Physique des Particules (ex SPP) (DPhP), Institut de Recherches sur les lois Fondamentales de l'Univers (IRFU), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Genethon, Université Pierre et Marie Curie - Paris 6 (UPMC)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), GENETHON, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Département de Physique des Particules (ex SPP) (DPP), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Normandie Université (NU)-Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)
Subjects: Cell signaling, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Bistability, Cellular differentiation, Population, Gene regulatory network, lcsh:Medicine, Cell Communication, Cell Separation, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Cell fate determination, Biology, Models, Biological, Myoblasts, 03 medical and health sciences, 0302 clinical medicine, Superoxides, Humans, Cell Lineage, Computer Simulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Epigenetics, lcsh:Science, education, Cell Biology/Gene Expression, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Genetics, Stochastic Processes, [SDV.GEN]Life Sciences [q-bio]/Genetics, 0303 health sciences, education.field_of_study, Multidisciplinary, lcsh:R, Cell Differentiation, Cell Biology, DNA Methylation, Flow Cytometry, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Phenotype, CD56 Antigen, [SDV.IMM]Life Sciences [q-bio]/Immunology, lcsh:Q, Computational Biology/Ecosystem Modeling, Neuroscience, 030217 neurology & neurosurgery, Research Article
Abstract: Cell differentiation is traditionally seen as a sequence of genetically predetermined gene expression changes. This view has been challenged recently by the discovery of important stochastic fluctuations in gene expression and by the identification of the role these fluctuations may play in the cell fate decision. In particularly, it has been proposed that fully multipotent cells fluctuate slowly between states with varying likelihoods of differentiation and this spontaneous heterogeneity or “noise” is the central driving force behind multipotency. It has been proposed that the different phenotypic states correspond to attractor states in the “epigenetic landscape” defined by the network of genes. In our study we used myoblasts that express the differentiation marker CD56 considered as a sign of definitive fate commitment to the muscle pathway. We show experimentally that these cells fluctuate between two phenotypes. Using spatial distribution analysis of the cells in growing population, computer simulations of these observations and experimental test of the new predictions made by the model we show that the fluctuations follow a bistable dynamics driven by a microenvironment and phenotype dependent noise. We show that fluctuation between phenotypes is not a distinguishing feature of multipotent cells. Committed tissue cells (myoblasts, used in our study) also may fluctuate between different phenotypes. We suggest that phenotypic fluctuations are a general feature of any non-terminally differentiated cell, thus, reversion to a previous differentiation state is a normal event. The cellular microenvironment contributes actively to the fluctuations by increasing the noise level in cells with a given phenotype and decreasing it in others. Importantly, the microenvironment is created by the cells themselves as a consequence of their motility that creates random cell interactions. In this way each cell contributes to put together its own microenvironment that in turn stimulates it to fluctuate between the phenotypes until the most appropriate phenotypic state with low noise is found. This means that the attractor state is not cell intrinsic; rather it is specified by the joint action of the cell-intrinsic gene network and the network of cell-cell interactions. Both networks are subject to stochastic fluctuations.
Published: 2010
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19. Human syncytins can pseudotype lentiviral vectors, enabling gene transfer into B cells

Author: Ferrand, M., Coquin, Y., Seye, A., Frin, S., Renaud, E., Danger, Y., Cogné, M, Galy, A., Généthon, Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Microenvironment and B-cells: Immunopathology,Cell Differentiation, and Cancer (MOBIDIC), Université de Rennes (UR)-Etablissement français du sang [Rennes] (EFS Bretagne)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Etablissement français du sang [Rennes] (EFS Bretagne)
Subjects: [SDV.CAN]Life Sciences [q-bio]/Cancer
Abstract: International audience; Meeting Abstract P194
Published: 2021

20. In vivo engineering of lymphocytes after systemic exosome-associated AAV delivery

Author: Casey A. Maguire, Federico Mingozzi, Benjamin P. Kleinstiver, James J. Moon, Killian S. Hanlon, Amine Meliani, Jeya-shree Natasan, Cort B. Breuer, Adrienn Volak, Massachusetts General Hospital [Charlestown, MA, États-Unis], Harvard Medical School [Boston] (HMS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Thérapie des maladies du muscle strié, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Massachusetts General Hospital [Boston], This work was supported by the National Institutes of Health (R01-AI107020, R21-AI124143 to J.J.M., and R01-DC017117 to C.A.M.) and the European Research Council (Consolidator Grant 617432 to F.M.)., European Project: 617432,EC:FP7:ERC,ERC-2013-CoG,MOMAAV(2014), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Bodescot, Myriam, and Molecular signatures and Modulation of immunity to Adeno-Associated Virus vectors - MOMAAV - - EC:FP7:ERC2014-07-01 - 2019-06-30 - 617432 - VALID
Subjects: Male, Viral vectors, Genetic enhancement, T cell, T-Lymphocytes, Genetic Vectors, Green Fluorescent Proteins, lcsh:Medicine, [SDV.GEN] Life Sciences [q-bio]/Genetics, Gene delivery, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Exosomes, Exosome, Article, 03 medical and health sciences, Mice, 0302 clinical medicine, Immune system, In vivo, Transduction, Genetic, medicine, Animals, Transgenes, lcsh:Science, 030304 developmental biology, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Multidisciplinary, lcsh:R, Gene Transfer Techniques, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Dependovirus, 3. Good health, Mice, Inbred C57BL, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Cancer research, lcsh:Q, Stem cell, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, Genetic Engineering, CD8
Abstract: Ex-vivo gene therapy using stem cells or T cells transduced by retroviral or lentiviral vectors has shown remarkable efficacy in the treatment of immunodeficiencies and cancer. However, the process is expensive, technically challenging, and not readily scalable to large patient populations, particularly in underdeveloped parts of the world. Direct in vivo gene therapy would avoid these issues, and such approaches with adeno-associated virus (AAV) vectors have been shown to be safe and efficacious in clinical trials for diseases affecting differentiated tissues such as the liver and CNS. However, the ability to transduce lymphocytes with AAV in vivo after systemic delivery has not been carefully explored. Here, we show that both standard and exosome-associated preparations of AAV8 vectors can effectively transduce a variety of immune cell populations including CD4+ T cells, CD8+ T cells, B cells, macrophages, and dendritic cells after systemic delivery in mice. We provide direct evidence of T cell transduction through the detection of AAV genomes and transgene mRNA, and show that intracellular and transmembrane proteins can be expressed. These findings establish the feasibility of AAV-mediated in vivo gene delivery to immune cells which will facilitate both basic and applied research towards the goal of direct in vivo gene immunotherapies.
Published: 2020
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21. Correction of the Exon 2 Duplication in DMD Myoblasts by a Single CRISPR/Cas9 System

Author: Stephanie Duguez, Annalisa Lattanzi, Kamel Mamchaoui, Araksya Izmiryan, Fulvio Mavilio, Samia Martin, Matteo Bovolenta, Francesco Bernardi, Arianna Moiani, Elena Barbon, Vincent Mouly, Généthon, Thérapie des maladies du muscle strié, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Sorbonne Université (SU), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Evry, GENETHON, Genethon, Association française contre les myopathies (AFM-Téléthon), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Université Pierre et Marie Curie - Paris 6 (UPMC), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)
Subjects: 0301 basic medicine, [SDV]Life Sciences [q-bio], CRISPR/Cas9, duplication, dystrophin, gene editing, lentivirus, Socio-culturale, Biology, 03 medical and health sciences, Exon, 0302 clinical medicine, Economica, Genome editing, 030225 pediatrics, Drug Discovery, Gene duplication, dystrophin, CRISPR/Cas9, duplication, lentivirus, gene editing, medicine, Myocyte, CRISPR, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Guide RNA, Muscular dystrophy, Genetics (clinical), Genetics, [SDV.GEN]Life Sciences [q-bio]/Genetics, Cas9, lcsh:RM1-950, Ambientale, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, lcsh:Therapeutics. Pharmacology, 030104 developmental biology, Neurology, Pediatrics, Perinatology and Child Health, biology.protein, Molecular Medicine, Original Article, Neurology (clinical), Dystrophin, 030217 neurology & neurosurgery
Abstract: Exonic duplications account for 10%–15% of all mutations in Duchenne muscular dystrophy (DMD), a severe hereditary neuromuscular disorder. We report a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-based strategy to correct the most frequent (exon 2) duplication in the DMD gene by targeted deletion, and tested the efficacy of such an approach in patient-derived myogenic cells. We demonstrate restoration of wild-type dystrophin expression at transcriptional and protein level in myotubes derived from genome-edited myoblasts in the absence of selection. Removal of the duplicated exon was achieved by the use of only one guide RNA (gRNA) directed against an intronic duplicated region, thereby increasing editing efficiency and reducing the risk of off-target effects. This study opens a novel therapeutic perspective for patients carrying disease-causing duplications., Graphical Abstract
Published: 2017
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22. Massive centriole production can occur in the absence of deuterosomes in multiciliated cells

Author: Olivier Mercey, Alice Meunier, Eva J. Brotslaw, Michelle S. Levine, Andrew J. Holland, Brian J. Mitchell, Nathalie Spassky, Philippe Rostaing, Abhijay Kumar, Gina M. LoMastro, Valerie Gomez, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des sciences et techniques de l'information, de la communication et de la connaissance (Lab-STICC), École Nationale d'Ingénieurs de Brest (ENIB)-Université de Bretagne Sud (UBS)-Université de Brest (UBO)-Télécom Bretagne-Institut Brestois du Numérique et des Mathématiques (IBNM), Université de Brest (UBO)-Université européenne de Bretagne - European University of Brittany (UEB)-École Nationale Supérieure de Techniques Avancées Bretagne (ENSTA Bretagne)-Institut Mines-Télécom [Paris] (IMT)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), The Open University [Milton Keynes] (OU), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Virologie et Immunologie Moléculaire, Faculté des Sciences Pharmaceutiques, Institut National de la Recherche Agronomique (INRA)-Université Francois Rabelais [Tours], and École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Centriole, [SDV]Life Sciences [q-bio], [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Biology, Article, 03 medical and health sciences, Mice, Xenopus laevis, 0302 clinical medicine, Organelle, Basal body, Animals, Humans, Cilia, Multiciliogenesis, Process (anatomy), Cells, Cultured, 030304 developmental biology, Centrioles, 0303 health sciences, Extramural, Cell Biology, Cell biology, Mice, Inbred C57BL, HEK293 Cells, 030220 oncology & carcinogenesis, Motile cilium
Abstract: International audience; Multiciliated cells (MCCs) amplify large numbers of centrioles that convert into basal bodies, which are required for producing multiple motile cilia. Most centrioles amplified by MCCs grow on the surface of organelles called deuterosomes, whereas a smaller number grow through the centriolar pathway in association with the two parent centrioles. Here, we show that MCCs lacking deuterosomes amplify the correct number of centrioles with normal step-wise kinetics. This is achieved through a massive production of centrioles on the surface and in the vicinity of parent centrioles. Therefore, deuterosomes may have evolved to relieve, rather than supplement, the centriolar pathway during multiciliogenesis. Remarkably, MCCs lacking parent centrioles and deuterosomes also amplify the appropriate number of centrioles inside a cloud of pericentriolar and fibrogranular material. These data show that the centriole number is set independently of their nucleation platforms and suggest that massive centriole production in MCCs is a robust process that can self-organize.
Published: 2019
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23. Alpha-1-Antitrypsin Promoter Improves the Efficacy of an Adeno-Associated Virus Vector for the Treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy

Author: Ramon Martí, Ferran Vila-Julià, Raquel Cabrera-Pérez, Federico Mingozzi, Javier Torres-Torronteras, Michio Hirano, Universitat Autònoma de Barcelona (UAB), Instituto de Salud Carlos III [Madrid] (ISC), Columbia University Medical Center (CUMC), Columbia University [New York], Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, Bitoun, Marc, and École Pratique des Hautes Études (EPHE)
Subjects: [SDV]Life Sciences [q-bio], Genetic Vectors, Gene Expression, Alpha (ethology), Mitochondria, Liver, Mitochondrion, Biology, medicine.disease_cause, thymidine, Mice, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Mitochondrial Encephalomyopathies, Transduction, Genetic, Gene Order, Genetics, medicine, Animals, Homeostasis, Vector (molecular biology), Thymidine phosphorylase, Promoter Regions, Genetic, Molecular Biology, Adeno-associated virus, Research Articles, 030304 developmental biology, Mice, Knockout, Thymidine Phosphorylase, 0303 health sciences, promoter, AAV, Genetic Therapy, Dependovirus, Molecular biology, mitochondria, [SDV] Life Sciences [q-bio], Disease Models, Animal, Liver, chemistry, alpha 1-Antitrypsin, 030220 oncology & carcinogenesis, MNGIE, Molecular Medicine, Thymidine
Abstract: International audience; Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a devastating disease caused by mutations in TYMP, which encodes thymidine phosphorylase (TP). In MNGIE patients, TP dysfunction results in systemic thymidine and deoxyuridine overload, which interferes with mitochondrial DNA replication. Preclinical studies have shown that gene therapy using a lentiviral vector targeted to hematopoietic stem cells or an adeno-associated virus (AAV) vector transcriptionally targeted to liver are feasible approaches to treat MNGIE. Here, we studied the effect of various promoters (thyroxine-binding globulin [TBG], phosphoglycerate kinase [PGK], hybrid liver-specific promoter [HLP], and alpha-1-antitrypsin [AAT]) and DNA configuration (single stranded or self complementary) on expression of the TYMP transgene in the AAV8 serotype in a murine model of MNGIE. All vectors restored liver TP activity and normalized nucleoside homeostasis in mice. However, the liver-specific promoters TBG, HLP, and AAT were more effective than the constitutive PGK promoter, and the self-complementary DNA configuration did not provide any therapeutic advantage over the single-stranded configuration. Among all constructs, only AAV-AAT was effective in all mice treated at the lowest dose (5 × 1010 vector genomes/kg). As use of the AAT promoter will likely minimize the dose needed to achieve clinical efficacy as compared to the other promoters tested, we propose using the AAT promoter in the vector eventually designed for clinical use.
Published: 2019
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24. Constraints on Human CD34+ Cell Fate due to Lentiviral Vectors Can Be Relieved by Valproic Acid

Author: Guillaume Corre, Rudiyanto Gunawan, Alice Moussy, Saliha Majdoul, Andras Paldi, Nan Papili Gao, David Fenard, Valentina Poletti, Daniel Stockholm, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Università degli Studi di Modena e Reggio Emilia (UNIMORE), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Genethon - Inserm UMR_S951, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Généthon, Genethon, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-Université d'Évry-Val-d'Essonne (UEVE), École Pratique des Hautes Études (EPHE), Università degli Studi di Modena e Reggio Emilia = University of Modena and Reggio Emilia (UNIMORE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and ANR-17-CE12-0031,SinCity,Analyses transcriptomiques sur cellules uniques dont la généalogie est identifiée au cours d'un processus de différentiation(2017)
Subjects: Myeloid, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, medicine.drug_class, Virus Integration, Genetic Vectors, CD34, Gene Expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Genetics, medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transgenes, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Progenitor, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Chemistry, Valproic Acid, Histone deacetylase inhibitor, Lentivirus, Gene Transfer Techniques, Hematopoietic stem cell, Cell Differentiation, Fetal Blood, Hematopoietic Stem Cells, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Cell biology, Crosstalk (biology), medicine.anatomical_structure, Phenotype, 030220 oncology & carcinogenesis, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Biomarkers
Abstract: The initial stages following the in vitro cytokine stimulation of human cord blood CD34+ cells overlap with the period when lentiviral gene transfer is typically performed. Single-cell transcriptional profiling and time-lapse microscopy were used to investigate how the vector-cell crosstalk impacts on the fate decision process. The single-cell transcription profiles were analyzed using a new algorithm, and it is shown that lentiviral transduction during the early stages of stimulation modifies the dynamics of the fate choice process of the CD34+ cells. The cells transduced with a lentiviral vector are biased toward the common myeloid progenitor lineage. Valproic acid, a histone deacetylase inhibitor known to increase the grafting potential of the CD34+ cells, improves the transduction efficiency to almost 100%. The cells transduced in the presence of valproic acid can subsequently undergo normal fate commitment. The higher gene transfer efficiency did not alter the genomic integration profile of the vector. These observations open the way to substantially improving lentiviral gene transfer protocols.
Published: 2019
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25. Histamine releasing factor and elongation factor 1 alpha secreted via malaria parasites extracellular vesicles promote immune evasion by inhibiting specific T cell responses

Author: Shuai Ding, Thibault Lagache, Jacques Bellalou, Vincenzo Di Bartolo, Salaheddine Mécheri, Neta Regev-Rudzki, François Guillonneau, Sébastien Brûlé, Chris J. Janse, Claudia Demarta-Gatsi, Roger Peronet, Anna Rivkin, Paula Abou Karam, Sidney R. Cohen, Pierre-Henri Commere, Biologie des Interactions Hôte-Parasite - Biology of Host-Parasite Interactions, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], Immunologie Moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Cytométrie (Plate-forme), Institut Pasteur [Paris], Plateforme protéomique 3P5 [Institut Cochin] (3P5), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Production de Protéines Recombinantes (Plate-Forme) (PRPF), Biophysique Moléculaire (Plate-forme), Analyse d'Images quantitatives (AIQ), Parasitology, Center of Infectious Diseases, Leiden University Medical Center (LUMC), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Weizmann Institute of Science [Rehovot, Israël], Biologie Cellulaire des Lymphocytes - Lymphocyte Cell Biology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagopole (CITECH), Institut Pasteur [Paris] (IP), Centre de Ressources et de Recherche Technologique - Center for Technological Resources and Research (C2RT), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Columbia University [New York], MECHERI, Salah, Universiteit Leiden, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Analyse d'images biologiques - Biological Image Analysis (BIA)
Subjects: MAPK/ERK pathway, CD4-Positive T-Lymphocytes, Plasmodium berghei, T cell, T-Lymphocytes, Immunology, Antigen presentation, T cells, Biology, Microbiology, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, 03 medical and health sciences, Extracellular Vesicles, Immune system, [SDV.IMM.VAC] Life Sciences [q-bio]/Immunology/Vaccinology, Peptide Elongation Factor 1, Virology, medicine, Biomarkers, Tumor, Animals, Humans, Secretion, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.IMM.ALL]Life Sciences [q-bio]/Immunology/Allergology, Antigens, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, 030304 developmental biology, Cell Proliferation, Immune Evasion, 0303 health sciences, Antigen Presentation, immunosuppression, 030306 microbiology, T-cell receptor, Tumor Protein, Translationally-Controlled 1, In vitro, Eukaryotic translation elongation factor 1 alpha 1, 3. Good health, Cell biology, Malaria, medicine.anatomical_structure, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, [SDV.IMM.IA] Life Sciences [q-bio]/Immunology/Adaptive immunology, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology, [SDV.IMM.ALL] Life Sciences [q-bio]/Immunology/Allergology, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology
Abstract: Protozoan pathogens secrete nanosized particles called extracellular vesicles (EVs) to facilitate their survival and chronic infection. Here, we show the inhibition by Plasmodium berghei NK65 blood stage-derived EVs of the proliferative response of CD4(+) T cells in response to antigen presentation. Importantly, these results were confirmed in vivo by the capacity of EVs to diminish the ovalbumin-specific delayed type hypersensitivity response. We identified two proteins associated with EVs, the histamine releasing factor (HRF) and the elongation factor 1 alpha (EF-1 alpha) that were found to have immunosuppressive activities. Interestingly, in contrast to WT parasites, EVs from genetically HRF- and EF-1 alpha-deficient parasites failed to inhibit T cell responses in vitro and in vivo. At the level of T cells, we demonstrated that EVs from WT parasites dephosphorylate key molecules (PLC gamma 1, Akt, and ERK) of the T cell receptor signalling cascade. Remarkably, immunisation with EF-1 alpha alone or in combination with HRF conferred a long-lasting antiparasite protection and immune memory. In conclusion, we identified a new mechanism by which P. berghei-derived EVs exert their immunosuppressive functions by altering T cell responses. The identification of two highly conserved immune suppressive factors offers new conceptual strategies to overcome EV-mediated immune suppression in malaria-infected individuals.
Published: 2019
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26. Twelfth Annual Meeting of the French Society of Cell and Gene Therapy

Author: Sophie Gomez, Els Verhoeyen, Pierre Cordelier, Anne Galy, Patrick Midoux, Eduard Ayuso, Michel Pucéat, Georges Vassaux, Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (Equipe EVIR), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Equipe 'Contrôle Métabolique des Morts Cellulaires' (INSERM U1065 - C3M), Centre méditerranéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Alliance Nationale pour les Sciences de la vie et de la Santé [Paris] (AVIESAN), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Laboratoire de Thérapie Génique Translationnelle des Maladies Génétiques (Inserm UMR 1089), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génétique Médicale et Génomique Fonctionnelle (GMGF), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Département de génétique médicale [Hôpital de la Timone - APHM], Institut National de la Santé et de la Recherche Médicale (INSERM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Assistance Publique - Hôpitaux de Marseille (APHM)-Aix Marseille Université (AMU), UMR E4320 (TIRO-MATOs), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Centre de Recherches en Cancérologie de Toulouse (CRCT), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Virus enveloppés, vecteurs et immunothérapie – Enveloped viruses, Vectors and Immuno-therapy (EVIR), Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Côte d'Azur (UCA), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), École pratique des hautes études (EPHE), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)
Subjects: 0303 health sciences, medicine.medical_specialty, business.industry, Genetic enhancement, 03 medical and health sciences, 0302 clinical medicine, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], 030220 oncology & carcinogenesis, Family medicine, Genetics, Molecular Medicine, Medicine, business, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract: International audience
Published: 2016
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27. Extract from Aphloia theiformis, an edible indigenous plant from Reunion Island, impairs Zika virus attachment to the host cell surface

Author: Clain, Elodie, Sinigaglia, Laura, Koishi, Andrea Cristine, Gorgette, Olivier, Gadea, Gilles, Viranaicken, Wildriss, Krejbich-Trotot, Pascale, Mavingui, Patrick, Desprès, Philippe, Nunes Duarte dos Santos, Claudia, Guiraud, Pascale, Jouvenet, Nolwenn, El Kalamouni, Chaker, Processus Infectieux en Milieu Insulaire Tropical (PIMIT), Université de La Réunion (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-IRD-Centre National de la Recherche Scientifique (CNRS), Génomique virale et vaccination, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire des parasites, Laboratoire de Biométrie et Biologie Evolutive - UMR 5558 (LBBE), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de Recherche en Informatique et en Automatique (Inria)-VetAgro Sup (VAS)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-IRD-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de La Réunion (UR), Instituto de Biofísica Carlos Chagas Filho [Rio de Janeiro] (IBCCF / UFRJ), Universidade Federal do Rio de Janeiro (UFRJ), Plateforme BioImagerie Ultrastructurale – Ultrastructural BioImaging Platform (UTechS UBI), Institut Pasteur [Paris], Univ, Réunion, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
Subjects: Carcinoma, Hepatocellular, viruses, lcsh:Medicine, Virus Attachment, Article, Magnoliopsida, Chlorocebus aethiops, Animals, Humans, [SDV.BV]Life Sciences [q-bio]/Vegetal Biology, [SDV.BV] Life Sciences [q-bio]/Vegetal Biology, lcsh:Science, Vero Cells, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Cell Proliferation, Plant Extracts, Zika Virus Infection, lcsh:R, Cell Cycle, Liver Neoplasms, Zika Virus, Virus Internalization, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, lcsh:Q, Plants, Edible, Reunion
Abstract: International audience; The mosquito-borne Zika virus (ZIKV) belongs to the flavivirus genus of the Flaviviridae family. Contemporary epidemic strains of ZIKV are associated with congenital malformations in infants, including microcephaly, as well as Guillain-Barré syndrome in adults. A risk of human-to-human transmission of ZIKV is also well documented. A worldwide research effort has been undertaken to identify safe and effective strategies to prevent or treat ZIKV infection. We show here that extract from Aphloia theiformis, an edible endemic plant from Indian Ocean islands, exerts a potent antiviral effect against ZIKV strains of African and Asian lineages, including epidemic strains. The antiviral effect of A. theiformis extract was extended to clinical isolates of dengue virus (DENV) of the four serotypes in human hepatocytes. A. theiformis inhibited virus entry in host cells by acting directly on viral particles, thus impairing their attachment to the cell surface. Electron microscopic observations revealed that organization of ZIKV particles was severely affected by A. theiformis. We propose a model of antiviral action for A. theiformis against flaviviruses that highlights the potential of medicinal plants as promising sources of naturally-derived antiviral compounds to prevent ZIKV and DENV infections. Zoonotic Zika virus (ZIKV) is a mosquito-borne virus that emerged in 2007 in Micronesia and since has caused important outbreaks in the South Pacific, Americas and SouthEast Asia. These recent ZIKV epidemics were associated with severe fetal brain injuries and neurological defects in adults, such as Guillain-Barre syndrome 1,2. ZIKV infection is now identified as a sexually-transmitted illness as well 3-5. In 2016, Zika infection was declared an emerging epidemic threat worldwide by the World Health Organization. ZIKV is a member of the flavivirus genus, a group of small, enveloped viruses, which also includes Dengue virus (DENV), West Nile virus (WNV) and Yellow fever virus 6. The genome consists of a single-stranded, positive-sense RNA molecule of around 10,7 kb, encoding a polyprotein precursor that is processed by the viral protease NS3 to give rise to 7 non-structural (NS) proteins and 3 structural proteins (Capsid C, pre-membrane prM and Envelope E). The NS proteins are mainly involved in viral RNA replication, while the structural proteins constitute the virion 7,8. The early stages of ZIKV infection require the attachment of the virion to the cell surface. This first step is mainly mediated by the interaction between phosphatidylserine exposed at the surface of the virus and the cellular receptor Axl 9 and probably also mobilizes close contacts between the E protein and the cell membrane. Following Axl mediated-binding, the virus enters target cells through clathrin-mediated endocytosis 9. The low-pH environment of endosomes triggers fusion between the viral envelope and the endosomal membrane. This fusion event leads to the release of the viral nucleocapsid into the cytosol.
Published: 2018
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28. Dosage Thresholds and Influence of Transgene Cassette in Adeno-Associated Virus–Related Toxicity

Author: Hanen Khabou, Deniz Dalkara, Laure Pacot, Sylvain Fisson, Chloé Cordeau, Institut de la Vision, Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes (IMBI), GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Généthon, and Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon)
Subjects: 0301 basic medicine, Retinal Disorder, MESH: gene therapy, retina, AAV, GFP, toxicity, promoter, Transgene, Genetic enhancement, viruses, Genetic Vectors, Green Fluorescent Proteins, Gene Dosage, medicine.disease_cause, Virus, Green fluorescent protein, 03 medical and health sciences, Capsid, 0302 clinical medicine, Genetics, medicine, Animals, Transgenes, Molecular Biology, Adeno-associated virus, Retina, [SDV.GEN]Life Sciences [q-bio]/Genetics, business.industry, Immunity, Genetic Therapy, Dependovirus, Virology, 3. Good health, Mice, Inbred C57BL, 030104 developmental biology, medicine.anatomical_structure, [SDV.TOX]Life Sciences [q-bio]/Toxicology, Toxicity, Molecular Medicine, business, 030217 neurology & neurosurgery
Abstract: International audience; Today, there are >500 published studies and 40 clinical trials to treat retinal disorders using gene therapy. The great majority of them rely on the use of adeno-associated virus vectors (AAV) for therapeutic gene delivery. Thus far, AAVs have an excellent safety profile in the clinic. Nevertheless, it is known that AAV-mediated gene delivery leads to toxicity at higher input doses in experimental gene therapy. This study reveals the factors that contribute to retinal toxicity after subretinal administration of AAV vectors in wild-type mice. The study shows that alongside the input dose, the nature of the transgene and the cells mediating the expression determine the extent of toxicity. Importantly, the study shows that AAV vectors encoding green fluorescent protein (GFP) used as controls in experimental gene therapy are toxic at doses as low as 5 × 109 vg, confounding the observed therapeutic effect in gene therapy paradigms. Altogether, the data show the importance of reducing input doses while increasing transgene expression levels via the use of more efficient capsids and promoters in order to avoid side effects in AAV-mediated gene therapy. Furthermore, the toxicity observed with AAV-GFP vectors imply a reinterpretation of previous gene therapy studies where the therapeutic effect was measured in relation to this control.
Published: 2018
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29. Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions

Author: Erwann Philippe, Athanassia Sotiropoulos, Hervé Le Stunff, Agnieszka Blachnio-Zabielska, Julien Véret, Paola Giussani, Fabienne Foufelle, Cécile L Bandet, Maxime Poirier, Xavier Le Liepvre, Olivier Bourron, Pascal Ferré, Eric Hajduch, Raphaëlle Ballaire, Jan Górski, Dušan Berkeš, Rana Mahfouz, Mélanie Campana, Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Institute of cardiometabolism and nutrition (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [APHP]-Sorbonne Université (SU), Centre de Recherche des Cordeliers (CRC), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Diderot - Paris 7 (UPD7), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Pathogenèse cellulaire et clinique du diabète (CRC - Inserm U1138), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Università degli Studi di Milano-Bicocca [Milano] (UNIMIB), Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Nutrition, diabète et cerveau, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Bialystok Med Univ, Virologie et Immunologie Moléculaire, Faculté des Sciences Pharmaceutiques, Institut National de la Recherche Agronomique (INRA)-Université Francois Rabelais [Tours], Centre de Recherche des Cordeliers (CRC (UMR_S_1138 / U1138)), École pratique des hautes études (EPHE)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Service de diabétologie [CHU Pitié-Salpétrière], Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-CHU Pitié-Salpêtrière [APHP], Slovak University of Technology in Bratislava, Department of Physiology, Medical University of Bialystok, Pathologies nutritionnelles et métaboliques : obésité et diabète, Université Pierre et Marie Curie - Paris 6 (UPMC)-IFR58-Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Biologie Fonctionnelle et Adaptative (BFA (UMR_8251 / U1133)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Recherche sur les Maladies Cardiovasculaires, du Métabolisme et de la Nutrition = Research Unit on Cardiovascular and Metabolic Diseases (ICAN), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut de Cardiométabolisme et Nutrition = Institute of Cardiometabolism and Nutrition [CHU Pitié Salpêtrière] (IHU ICAN), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Diderot - Paris 7 (UPD7)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Milano-Bicocca = University of Milano-Bicocca (UNIMIB), Medical University of Białystok (MUB), Service de Diabétologie [CHU Pitié-Salpétrière], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Hajduch, Eric, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Université de Paris (UP), Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)
Subjects: 0301 basic medicine, Adult, Male, Ceramide, Endocrinology, Diabetes and Metabolism, medicine.medical_treatment, Golgi Apparatus, Mice, Transgenic, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Protein Serine-Threonine Kinases, Ceramides, Endoplasmic Reticulum, 03 medical and health sciences, chemistry.chemical_compound, Mice, Insulin resistance, Internal Medicine, medicine, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Myocyte, Animals, Humans, Insulin, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, biology, Muscles, Fatty Acids, medicine.disease, Sphingolipid, Cell biology, Mice, Inbred C57BL, Insulin receptor, 030104 developmental biology, chemistry, Lipotoxicity, biology.protein, Signal transduction, Insulin Resistance, Signal Transduction
Abstract: International audience; One main mechanism of insulin resistance (IR), a key feature of type 2 diabetes, is the accumulation of saturated fatty acids (FAs) in the muscles of obese patients with type 2 diabetes. Understanding the mechanism that underlies lipid-induced IR is an important challenge. Saturated FAs are metabolized into lipid derivatives called ceramides, and their accumulation plays a central role in the development of muscle IR. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi apparatus through a transporter called CERT, where they are converted into various sphingolipid species. We show that CERT protein expression is reduced in all IR models studied because of a caspase-dependent cleavage. Inhibiting CERT activity in vitro potentiates the deleterious action of lipotoxicity on insulin signaling, whereas overexpression of CERT in vitro or in vivo decreases muscle ceramide content and improves insulin signaling. In addition, inhibition of caspase activity prevents ceramide-induced insulin signaling defects in C2C12 muscle cells. Altogether, these results demonstrate the importance of physiological ER-to-Golgi ceramide traffic to preserve muscle cell insulin signaling and identify CERT as a major actor in this process.
Published: 2018
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30. Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients

Author: Olivier Boyer, Fabienne Jouen, Virginie Latournerie, Carole Masurier, Marcelo Simon Sola, Federico Mingozzi, Diana Desgue, Severine Charles, Philippe Veron, Aliénor Quéré, Elisa Masat, Christian Leborgne, Elodie Pignot, Sylvie Boutin, Fanny Collaud, Généthon, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes, Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie, Autoimmunité, maladies Neuromusculaires et THErapies Régénératrices (PANTHER), Généthon, Evry, Centre de recherche et d'applications sur les thérapies géniques (CRATG), Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Laboratoire des Sciences de l'Information et des Systèmes (LSIS), and Aix Marseille Université (AMU)-Université de Toulon (UTLN)-Arts et Métiers Paristech ENSAM Aix-en-Provence-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Adult, AAV humoral immunity, Adolescent, Duchenne muscular dystrophy, Genetic enhancement, [SDV]Life Sciences [q-bio], viruses, Immunology, Genetic Vectors, Duchenne Muscular Dystrophy, Biology, medicine.disease_cause, Antibodies, Viral, Virus, Cohort Studies, 03 medical and health sciences, Young Adult, 0302 clinical medicine, Gene therapy, Seroepidemiologic Studies, medicine, Humans, Longitudinal Studies, Seroconversion, Adeno-associated virus, ComputingMilieux_MISCELLANEOUS, Immunosuppression Therapy, Antibody titer, Age Factors, Dependovirus, medicine.disease, Antibodies, Neutralizing, 3. Good health, Immunity, Humoral, Muscular Dystrophy, Duchenne, 030104 developmental biology, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Immunoglobulin G, Humoral immunity, biology.protein, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, 030215 immunology
Abstract: International audience; Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.
Published: 2018
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31. Influence of Pre-existing Anti-capsid Neutralizing and Binding Antibodies on AAV Vector Transduction

Author: Severine Charles, Elisa Masat, Giuseppe Ronzitti, Christian Leborgne, Alban Vignaud, Marcelo Simon Sola, Fanny Collaud, Fabienne Jouen, Laetitia van Wittenberghe, Olivier Boyer, Zachary Fitzpatrick, Elena Barbon, Federico Mingozzi, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Physiopathologie, Autoimmunité, maladies Neuromusculaires et THErapies Régénératrices (PANTHER), Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), HAL UPMC, Gestionnaire, Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Généthon, Evry, GENETHON, Genethon, Université Paris-Saclay, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Coeur, Muscle et Vaisseaux, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, and École Pratique des Hautes Études (EPHE)
Subjects: 0301 basic medicine, NAb, binding antibody, liver gene transfer, lcsh:QH426-470, [SDV.IMM] Life Sciences [q-bio]/Immunology, [SDV]Life Sciences [q-bio], Transgene, media_common.quotation_subject, viruses, Article, Virus, 03 medical and health sciences, Transduction (genetics), Immunity, Genetics, lcsh:QH573-671, Neutralizing antibody, Internalization, liver transduction, Molecular Biology, ComputingMilieux_MISCELLANEOUS, media_common, biology, lcsh:Cytology, AAV immune-response, neutralizing antibody, Virology, 3. Good health, AAV vector, lcsh:Genetics, 030104 developmental biology, pre-existing antibodies, vector transduction, Capsid, biology.protein, Molecular Medicine, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, BAb
Abstract: Pre-existing immunity to adeno-associated virus (AAV) is highly prevalent in humans and can profoundly impact transduction efficiency. Despite the relevance to AAV-mediated gene transfer, relatively little is known about the fate of AAV vectors in the presence of neutralizing antibodies (NAbs). Similarly, the effect of binding antibodies (BAbs), with no detectable neutralizing activity, on AAV transduction is ill defined. Here, we delivered AAV8 vectors to mice carrying NAbs and demonstrated that AAV particles are taken up by both liver parenchymal and non-parenchymal cells; viral particles are then rapidly cleared, without resulting in transgene expression. In vitro, imaging of hepatocytes exposed to AAV vectors pre-incubated with either NAbs or BAbs revealed that virus is taken up by cells in both cases. Whereas no successful transduction was observed when AAV was pre-incubated with NAbs, an increased capsid internalization and transgene expression was observed in the presence of BAbs. Accordingly, AAV8 vectors administered to mice passively immunized with anti-AAV8 BAbs showed a more efficient liver transduction and a unique vector biodistribution profile compared to mice immunized with NAbs. These results highlight a virtually opposite effect of neutralizing and binding antibodies on AAV vectors transduction., Graphical Abstract
Published: 2018
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32. Emerging Issues in AAV-Mediated In Vivo Gene Therapy

Author: Giuseppe Ronzitti, Pasqualina Colella, Federico Mingozzi, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Association française contre les myopathies ( AFM-Téléthon ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, lcsh:QH426-470, Genetic enhancement, viruses, AAV capsid, Gene transfer, Computational biology, Article, 03 medical and health sciences, Transduction (genetics), In vivo, [ SDV.MHEP ] Life Sciences [q-bio]/Human health and pathology, Genetics, Medicine, lcsh:QH573-671, liver immunogenicity, Molecular Biology, AAV genome, lcsh:Cytology, business.industry, genotoxicity, AAV, persistence, gene therapy, Bench to bedside, 3. Good health, Clinical trial, lcsh:Genetics, Safety profile, 030104 developmental biology, Drug development, Molecular Medicine, business, inherited diseases, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract: International audience; In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective “therapeutic products” is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies.
Published: 2018
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33. Diagnostic anoctamin-5 protein defect in patients with ANO5-mutated muscular dystrophy

Author: Mikaela Lindfors, Sini Penttilä, H. Luque, Talita C. Conte, Bernard Brais, Giorgio Tasca, Bruno Eymard, Karine Charton, Isabelle Richard, Anna Vihola, Marco Savarese, F. Leturcq, Bjarne Udd, Lääketieteen ja biotieteiden tiedekunta - Faculty of Medicine and Life Sciences, University of Tampere, University of Helsinki, Tampere University Hospital, Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Università cattolica del Sacro Cuore [Roma] (Unicatt), McGill University = Université McGill [Montréal, Canada], Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Vaasa Central Hospital, CHU Cochin [AP-HP], Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Università Cattolica del Sacro Cuore [Roma] (Unicatt), McGill University, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Richard, Isabelle, Helsingin yliopisto = Helsingfors universitet = University of Helsinki, Università cattolica del Sacro Cuore = Catholic University of the Sacred Heart [Roma] (Unicatt), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and École Pratique des Hautes Études (EPHE)
Subjects: 0301 basic medicine, Male, Histology, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Blotting, Western, Anoctamins, Biology, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, Compound heterozygosity, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, ANO5, Limb-Girdle Muscular Dystrophy, Pathology and Forensic Medicine, 03 medical and health sciences, 0302 clinical medicine, Antibody Specificity, Physiology (medical), Lääketieteen bioteknologia - Medical biotechnology, medicine, Humans, Muscular dystrophy, LGMD2L, limb-girdle muscular dystrophy, Point mutation, Skeletal muscle, Antibodies, Monoclonal, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DMM3, medicine.disease, Molecular biology, 3. Good health, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 030104 developmental biology, medicine.anatomical_structure, Neurology, Membrane protein, Muscular Dystrophies, Limb-Girdle, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Mutation, Immunohistochemistry, Female, Neurology (clinical), ITGA7, 030217 neurology & neurosurgery, Limb-girdle muscular dystrophy
Abstract: Aims Previously, detection of ANO5 protein has been complicated by unspecific antibodies, most of which have not identified the correct protein. The aims of the study were to specify ANO5 protein expression in human skeletal muscle, and to investigate if the ANO5 protein levels are affected by different ANO5 mutations in anoctaminopathy patients. Methods Four different antibodies were tested for ANO5 specificity. A sample preparation method compatible with membrane proteins, combined with tissue fractionation was used to determine ANO5 expression in cell cultures expressing ANO5, in normal muscles and eight patient biopsies with six different ANO5 mutations in homozygous or compound heterozygous states, and in other dystrophies. Results Only one specific monoclonal N-terminal ANO5 antibody was efficient in detecting the protein, showing that ANO5 is expressed as a single 107 kD polypeptide in human skeletal muscle. The truncating mutations c.191dupA and c.1261C>T were found to abolish ANO5 expression, whereas the studied point mutations had variable effects, however, all the ANO5 mutations resulted in clearly reduced ANO5 expression in the patient muscle membrane fraction. Attempts to detect ANO5 using immunohistochemistry were not yet successful. Conclusions The data presented here indicate that the ANO5 protein expression is decreased in ANO5-mutated muscular dystrophy and that most of the non-truncating pathogenic ANO5 mutations likely destabilize the protein and cause its degradation. The method described here allows direct analysis of human ANO5 protein, which can be used in diagnostics, for evaluating the pathogenicity of the potentially harmful ANO5 variants of uncertain significance. This article is protected by copyright. All rights reserved.
Published: 2018
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34. A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity

Author: Caroline Denevault-Sabourin, Philippe Jacquiet, Espérance Moine, Olivier Gorgette, Jean-Christophe Barale, Laurence Silpa, Françoise Debierre-Grockiego, Fabien Brossier, Alain Gueiffier, Isabelle Dimier-Poisson, Cécile Enguehard-Gueiffier, Infectiologie et Santé Publique (UMR ISP), Institut National de la Recherche Agronomique (INRA)-Université de Tours (UT), Université Francois Rabelais [Tours], Immunologie moléculaire des parasites, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Interactions hôtes-agents pathogènes [Toulouse] (IHAP), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Nutrition, croissance et cancer (U 1069) (N2C), Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT), Infectiologie Animale et Santé Publique - IASP (Nouzilly, France), Institut National de la Recherche Agronomique (INRA), UR Infectiologie animale et Santé publique (UR IASP), Université de Tours-Institut National de la Santé et de la Recherche Médicale (INSERM), Infectiologie Santé Publique (ISP-311), and Institut National de la Recherche Agronomique (INRA)-Université de Tours
Subjects: Cell Survival, Pyridines, Plasmodium falciparum, Antiprotozoal Agents, Toxoplasma gondii, Neospora caninum, Anti-apicomplexan, Chemical synthesis, Cell Line, Chemical library, Small Molecule Libraries, Structure-Activity Relationship, chemistry.chemical_compound, parasitic diseases, Drug Discovery, Humans, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, ComputingMilieux_MISCELLANEOUS, Pharmacology, Molecular Structure, biology, Chemistry, Biphenyl Compounds, Organic Chemistry, Imidazoles, Imidazo[1,2-b]pyridazine, Besnoitia besnoiti, General Medicine, biology.organism_classification, Small molecule, Virology, 3. Good health, Pyridazines, Biochemistry, Imidazo[1,2-a]pyridine, Apicomplexa, Toxoplasma, Eimeria tenella, Intracellular
Abstract: International audience; An in vitro screening of the anti-apicomplexan activity of 51 compounds, stemming from our chemical library and from chemical synthesis, was performed. As a study model, we used Toxoplasma gondii (T. gondii), expressing β-galactosidase for the colorimetric assessment of drug activity on parasites cultivated in vitro. This approach allowed the validation of a new series of molecules with a biphenylimidazoazine scaffold as inhibitors of T. gondii growth in vitro. Hence, 8 molecules significantly inhibited intracellular replication of T. gondii in vitro, with EC50 < 1 μM, while being non-toxic for human fibroblasts at these concentrations. Most attractive candidates were then selected for further biological investigations on other apicomplexan parasites (Neospora caninum, Besnoitia besnoiti, Eimeria tenella and Plasmodium falciparum). Finally, two compounds were able to inhibit growth of four different apicomplexans with EC50 in the submicromolar to nanomolar range, for each parasite. These data, including the broad anti-parasite spectrum of these inhibitors, define a new generation of potential anti-parasite compounds of wide interest, including for veterinary application. Studies realized on E. tenella suggest that these molecules act during the intracellular development steps of the parasite. Further experiments should be done to identify the molecular target(s) of these compounds.
Published: 2015
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35. Peptides derived from evolutionarily conserved domains in Beclin-1 and Beclin-2 enhance the entry of lentiviral vectors into human cells

Author: Sophie Frin, Nathalie Chazal, Saliha Majdoul, Lucile Espert, Laurence Briant, David Fenard, Nathalie Holic, Jérémie Cosette, Anne Galy, Ababacar K. Seye, Eric Bernard, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), École pratique des hautes études (EPHE), Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Infections rétrovirales et signalisation cellulaire (IRSC), Université Montpellier 1 (UM1)-Centre National de la Recherche Scientifique (CNRS), Groupe Immunologie et Thérapie Génique, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Evry, Genethon, Centre d’études d’Agents Pathogènes et Biotechologies pour la Santé (CPBS), Genethon - Inserm UMR_S951, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Association française contre les myopathies (AFM-Téléthon), École pratique des hautes études ( EPHE ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Genethon, Centre de Recherche des Cordeliers ( CRC (UMR_S 872) ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Recherche en Infectiologie de Montpellier ( IRIM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Infections rétrovirales et signalisation cellulaire ( IRSC ), Université Montpellier 1 ( UM1 ) -Centre National de la Recherche Scientifique ( CNRS ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), and Chazal, Nathalie
Subjects: 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, [SDV]Life Sciences [q-bio], Biochemistry, Transduction (genetics), 0302 clinical medicine, Cells, Cultured, Conserved Sequence, ComputingMilieux_MISCELLANEOUS, Cell Line, Transformed, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Hematopoietic Stem Cell Transplantation, Intracellular Signaling Peptides and Proteins, Specific Pathogen-Free Organisms, Up-Regulation, 3. Good health, Cell biology, 030220 oncology & carcinogenesis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Beclin-1, tat Gene Products, Human Immunodeficiency Virus, Recombinant Fusion Proteins, Protein domain, Mice, Transgenic, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viral vector, 03 medical and health sciences, Viral entry, Cell Line, Tumor, Autophagy, Animals, Humans, Amino Acid Sequence, Progenitor cell, Enhancer, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Lentivirus, Genetic Therapy, Cell Biology, Virus Internalization, Hematopoietic Stem Cells, Virology, Peptide Fragments, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, 030104 developmental biology, HIV-1
Abstract: International audience; Autophagy-related proteins such as Beclin-1 are involved in an array of complex processes, including antiviral responses, and may also modulate the efficiency of gene therapy viral vectors. The Tat-Beclin-1 (TB1) peptide has been reported as an autophagy-inducing factor inhibiting the replication of pathogens such as HIV, type 1 (HIV-1). However, autophagy-related proteins are also essential for the early steps of HIV-1 infection. Therefore, we examined the effects of the Beclin-1 evolution-arily conserved domain in TB1 on viral transduction and autophagy in single-round HIV infection or with nonreplicative HIV-1-derived lentiviral vectors. TB1 enhanced transduction with various pseudotypes but without inducing the autophagy process. TB1 augmented the transduction of human CD34؉ hematopoietic stem/progenitor cells while maintaining their capacity to engraft in vivo into humanized mice. TB1 was as effective as other transduction additives and functioned by enhancing the adhesion and fusion of viral particles with target cells but not their aggregation. We also found that the N-terminal L1 loop was critical for TB1 transduction-enhancing activity. Interestingly, the Tat-Beclin-2 (TB2) peptide, derived from the human Beclin-2 protein, was even more potent than TB1 in promoting viral transduction and infection. Taken together, our findings suggest that the TB1 and TB2 peptides enhance the viral entry step. Tat-Beclin peptides therefore represent a new family of viral transduction enhancers for potential use in gene therapy.
Published: 2017
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36. CDC20B is required for deuterosome-mediated centriole production in multiciliated cells

Author: Diego R. Revinski, Laure-Emmanuelle Zaragosi, Camille Boutin, Sandra Ruiz-Garcia, Marie Deprez, Virginie Thomé, Olivier Rosnet, Anne-Sophie Gay, Olivier Mercey, Agnès Paquet, Nicolas Pons, Gilles Ponzio, Brice Marcet, Laurent Kodjabachian, Pascal Barbry, Institut de Biologie du Développement de Marseille (IBDM), Aix Marseille Université (AMU)-Collège de France (CdF (institution))-Centre National de la Recherche Scientifique (CNRS), Institut de signalisation, biologie du développement et cancer (ISBDC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), MetaGenoPolis, Institut National de la Recherche Agronomique (INRA), Biologie et physiopathologie cutanées : expression génique, signalisation et thérapie, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Collège de France (CdF)-Centre National de la Recherche Scientifique (CNRS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Virologie et Immunologie Moléculaire, Faculté des Sciences Pharmaceutiques, Institut National de la Recherche Agronomique (INRA)-Université Francois Rabelais [Tours], US 1367 MetaGénoPolis, Institut National de la Recherche Agronomique (INRA)-Département Microbiologie et Chaîne Alimentaire (MICA), Institut National de la Recherche Agronomique (INRA)-MetaGénoPolis (MGP), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-IFR50-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Centriole, Cdc20 Proteins, [SDV]Life Sciences [q-bio], Science, Cell, Xenopus, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Article, 03 medical and health sciences, Mice, Xenopus laevis, 0302 clinical medicine, Ependyma, medicine, Animals, Humans, Cilia, lcsh:Science, Mitosis, ComputingMilieux_MISCELLANEOUS, Separase, 030304 developmental biology, Centrioles, 0303 health sciences, Cilium, biology.organism_classification, 3. Good health, Cell biology, medicine.anatomical_structure, Deuterosome, Vertebrates, Motile cilium, lcsh:Q, Female, Epidermis, Single-Cell Analysis, Transcriptome, 030217 neurology & neurosurgery, Protein Binding
Abstract: Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules., In vertebrate multiciliated cells, most centrioles are synthesized and released by poorly characterized structures called deuterosomes. Here, the authors report that the previously-uncharacterized Cdc20b in mouse and Xenopus associates with deuterosomes and contributes to centriole release.
Published: 2017
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37. Enhanced liver gene transfer and evasion of preexisting humoral immunity with exosome-enveloped AAV vectors

Author: Christian Leborgne, Béatrice Marolleau, Alain Brisson, Casey A. Maguire, Federico Mingozzi, Fabienne Jouen, Laetitia van Wittenberghe, Alban Vignaud, Florence Boisgerault, Olivier D. Christophe, Fanny Collaud, Solenne Marmier, Giuseppe Ronzitti, Olivier Boyer, Zachary Fitzpatrick, Sisareuth Tan, Amine Meliani, Marcelo Simon Sola, Severine Charles, Centre de recherche en myologie, Université Pierre et Marie Curie - Paris 6 (UPMC)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Généthon, Université Paris-Saclay, Coeur, Muscle et Vaisseaux, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Physiopathologie et biothérapies des maladies inflammatoires et autoimmunes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU), Chimie et Biologie des Membranes et des Nanoobjets (CBMN), Université de Bordeaux (UB)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hémostase et biologie vasculaire, Université Paris-Sud - Paris 11 (UP11)-IFR93-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagerie Moléculaire et Nanobiotechnologies - Institut Européen de Chimie et Biologie (IECB), Université Sciences et Technologies - Bordeaux 1-Centre National de la Recherche Scientifique (CNRS), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Centre de Recherche en Myologie, Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Normandie Université (NU)-Institut National de la Santé et de la Recherche Médicale (INSERM), École Pratique des Hautes Études (EPHE), Université Sciences et Technologies - Bordeaux 1 (UB)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Université Pierre et Marie Curie - Paris 6 (UPMC), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Généthon, Evry, Université Pierre et Marie Curie - Paris 6 (UPMC)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Recherche et développement [Généthon, Evry] (R & D Généthon), Généthon [Evry], Université Sciences et Technologies - Bordeaux 1-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire et biothérapies innovantes, and École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon
Subjects: 0301 basic medicine, Transgene, viruses, [SDV]Life Sciences [q-bio], Hematology, Gene Therapy, Gene delivery, Biology, Exosome, Virology, Virus, 3. Good health, 03 medical and health sciences, Transduction (genetics), 030104 developmental biology, 0302 clinical medicine, 030220 oncology & carcinogenesis, hemic and lymphatic diseases, Humoral immunity, biology.protein, Cytotoxic T cell, [SDV.IMM]Life Sciences [q-bio]/Immunology, Neutralizing antibody, ComputingMilieux_MISCELLANEOUS
Abstract: Results from clinical trials of liver gene transfer for hemophilia demonstrate the potential of the adeno-associated virus (AAV) vector platform. However, to achieve therapeutic transgene expression, in some cases high vector doses are required, which are associated with a higher risk of triggering anti-capsid cytotoxic T-cell responses. Additionally, anti-AAV preexisting immunity can prevent liver transduction even at low neutralizing antibody (NAb) titers. Here, we describe the use of exosome-associated AAV (exo-AAV) vectors as a robust liver gene delivery system that allows the therapeutic vector dose to be decreased while protecting from preexisting humoral immunity to the capsid. The in vivo efficiency of liver targeting of standard AAV8 or AAV5 and exo-AAV8 or exo-AAV5 vectors expressing human coagulation factor IX (hF.IX) was evaluated. A significant enhancement of transduction efficiency was observed, and in hemophilia B mice treated with 4 × 1010 vector genomes per kilogram of exo-AAV8 vectors, a staggering ∼1 log increase in hF.IX transgene expression was observed, leading to superior correction of clotting time. Enhanced liver expression was also associated with an increase in the frequency of regulatory T cells in lymph nodes. The efficiency of exo- and standard AAV8 vectors in evading preexisting NAbs to the capsid was then evaluated in a passive immunization mouse model and in human sera. Exo-AAV8 gene delivery allowed for efficient transduction even in the presence of moderate NAb titers, thus potentially extending the proportion of subjects eligible for liver gene transfer. Exo-AAV vectors therefore represent a platform to improve the safety and efficacy of liver-directed gene transfer.
Published: 2017
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38. Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

Author: Moussy, Alice, Cosette, Jérémie, Parmentier, Romuald, da Silva, Cindy, Corre, Guillaume, Richard, Angélique, Gandrillon, Olivier, Stockholm, Daniel, Paldi, Andras, Huang, Sui, Centre de Recherche des Cordeliers (CRC (UMR_S 872)), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), GENETHON 3, Université d'Évry-Val-d'Essonne (UEVE), Genethon - Inserm UMR_S951, Immunologie moléculaire et biothérapies innovantes, École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Genethon-Généthon, Association française contre les myopathies (AFM-Téléthon)-Association française contre les myopathies (AFM-Téléthon), Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE - UMR 8587), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Généthon, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Saclay (COmUE), and Université Paris Descartes - Paris 5 (UPD5)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], ComputingMilieux_MISCELLANEOUS
Abstract: International audience
Published: 2017
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39. Gait characteristics in a canine model of X-linked myotubular myopathy

Author: Alan H. Beggs, Emily Burlingame, Melissa A. Goddard, Anthony P. Marsh, Martin K. Childers, Anna Buj-Bello, Valerie E. Kelly, Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Wake Forest University, Genomics Program and Division of Genetics, Boston Children's Hospital-The Manton Center for Orphan Disease Research-Harvard Medical School [Boston] (HMS), Institut de génétique et biologie moléculaire et cellulaire (IGBMC), Université Louis Pasteur - Strasbourg I-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), University of Washington [Seattle], Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Harvard Medical School [Boston] (HMS)-Boston Children's Hospital-The Manton Center for Orphan Disease Research, Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Louis Pasteur - Strasbourg I, École Pratique des Hautes Études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon
Subjects: medicine.medical_specialty, Kinematics, Time Factors, Neuromuscular disease, [SDV]Life Sciences [q-bio], STRIDE, Article, Motion, 03 medical and health sciences, Dogs, 0302 clinical medicine, Physical medicine and rehabilitation, Animals, Medicine, Biomechanics, Gait, 030304 developmental biology, 0303 health sciences, business.industry, Outcome measures, Extremities, medicine.disease, X-linked myotubular myopathy, Animal models, Myotubular myopathy, Disease Models, Animal, Neurology, Gait analysis, Physical therapy, Neurology (clinical), business, Canine model, Locomotion, 030217 neurology & neurosurgery, Myopathies, Structural, Congenital
Abstract: International audience; X-linked myotubular myopathy (XLMTM) is a fatal pediatric disease where affected boys display profound weakness of the skeletal muscles. Possible therapies are under development but robust outcome measures in animal models are required for effective translation to human patients. We established a naturally-occurring canine model, where XLMTM dogs display clinical symptoms similar to those observed in humans. The aim of this study was to determine potential endpoints for the assessment of future treatments in this model. Video-based gait analysis was selected, as it is a well-established method of assessing limb function in neuromuscular disease and measures have been correlated to the patient's quality of life. XLMTM dogs (N = 3) and their true littermate wild type controls (N = 3) were assessed at 4-5 time points, beginning at 10 weeks and continuing through 17 weeks. Motion capture and an instrumented carpet were used separately to evaluate spatiotemporal and kinematic changes over time. XLMTM dogs walk more slowly and with shorter stride lengths than wild type dogs, and these differences became greater over time. However, there was no clear difference in angular measures between affected and unaffected dogs. These data demonstrate that spatiotemporal parameters capture functional changes in gait in an XLMTM canine model and support their utility in future therapeutic trials.
Published: 2014
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40. Acute effect of androgens on maximal force-generating capacity and electrically evoked calcium transient in mouse skeletal muscles

Author: Gillian Butler-Browne, Daniel Metzger, Arnaud Ferry, Alban Vignaud, Bourama Fane, Mélanie Schuh, Bodvael Fraysse, Service d'Oto-Rhino-Laryngologie et d'otoneurochirurgie, Hôpital Purpan [Toulouse], CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, UMRS974, Université Pierre et Marie Curie - Paris 6 (UPMC), U974, Institut National de la Santé et de la Recherche Médicale (INSERM), UMR7215, Centre National de la Recherche Scientifique (CNRS), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), École Pratique des Hautes Études (EPHE), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), and Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Male, medicine.medical_specialty, Electric Stimulation, medicine.drug_class, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Muscle Contraction/*drug effects, chemistry.chemical_element, Stimulation, Hindlimb, Calcium, Biochemistry, Contractility, Mice, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, Internal medicine, Receptors, Calcium Signaling/*drug effects, medicine, Animals, Myocyte, Calcium Signaling, Muscle, Skeletal, Molecular Biology, Skeletal/*cytology/drug effects/metabolism/*physiology, 030304 developmental biology, Pharmacology, 0303 health sciences, Organic Chemistry, Skeletal muscle, Androgen, Androgen receptor, Androgen/deficiency, medicine.anatomical_structure, chemistry, Receptors, Androgen, Androgens, Muscle, Androgens/*pharmacology, 030217 neurology & neurosurgery, Muscle Contraction
Abstract: International audience; As androgens might have rapid androgen-receptor (AR) independent action on muscle cells, we analysed the in vivo acute effect of androgens on maximal force generation capacity and electrically evoked calcium transient responsible for the excitation-contraction coupling in skeletal muscle from wild-type male mice and muscle fibre androgen receptor (AR) deficient (AR(skm-/y)) male mice. We tested the hypothesis that acute in vivo androgen treatment improves contractility and modifies calcium transient in mouse hindlimb muscles. In addition, we determined whether the reduced maximal force generation capacity of AR(skm-/y) mice is caused by an alteration in calcium transient. We found that acute dehydrotestosterone (DHT) and testosterone treatment of mice does not change in situ maximal force, power or fatigue resistance of tibialis anterior muscles. In agreement with this observation, maximal force and twitch kinetics also remained unchanged when both whole extensor digitorum longus (EDL) muscle or fibre bundles were incubated in vitro with DHT. Electrically evoked calcium transient, i.e. calcium amplitude, time to peak and decay, was also not modified by DHT treatment of EDL muscle fibre bundles. Finally, we found no difference in calcium transient between AR(skm-/y) and wild-type mice despite the reduced maximal force in EDL fibre bundles of AR(skm-/y) mice. In conclusion, acute androgen treatment has no ergogenic effect on muscle contractility and does not affect calcium transient in response to stimulation. In addition, the reduced maximal force of AR(skm-/y) mice is not related to calcium transient dysfunction.
Published: 2014
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41. A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer

Author: Anne Galy, Florence Boisgerault, Maxime Ferrand, Institut de l’Élevage, Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut de l'élevage (IDELE), École Pratique des Hautes Études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon
Subjects: CD4-Positive T-Lymphocytes, CD8-Positive T-Lymphocytes/metabolism, Antigen-Presenting Cells/metabolism, [SDV]Life Sciences [q-bio], Priming (immunology), CD8-Positive T-Lymphocytes, Inbred C57BL, dosage, Mice, 0302 clinical medicine, Muscular Dystrophy, 0303 health sciences, CD11b Antigen, Muscles, Gene Transfer Techniques, Sarcoglycans/*genetics/metabolism, Dependovirus, medicine.anatomical_structure, Molecular Medicine, Original Article, Transgene, Genetic Vectors, Antigen-Presenting Cells, Genetic Vectors/administration &amp, Biology, Viral vector, Dependovirus/*genetics/immunology, 03 medical and health sciences, Immune system, Antigen, Sarcoglycans, Macrophages/metabolism, Genetics, medicine, Animals, Muscles/*immunology/pathology, Antigens, Antigen-presenting cell, Molecular Biology, 030304 developmental biology, CD4-Positive T-Lymphocytes/metabolism, CD11b/metabolism, Macrophages, Skeletal muscle, Muscular Dystrophy, Animal, Molecular biology, Mice, Inbred C57BL, MicroRNAs, MicroRNAs/*genetics, Animal/*immunology/therapy, 030217 neurology & neurosurgery, CD8
Abstract: International audience; Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of alpha-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic alpha-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.
Published: 2014
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42. Cis -splicing and Translation of the Pre- Trans -splicing Molecule Combine With Efficiency in Spliceosome-mediated RNA Trans -splicing

Author: Florence Le Roy, Isabelle Richard, François Monjaret, Nathalie Bourg, Carinne Roudaut, Laurence Suel, Karine Charton, Baculovirus et Thérapie, Centre National de la Recherche Scientifique (CNRS), Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE - UMR 8587), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Cergy Pontoise (UCP), Université Paris-Seine-Université Paris-Seine-Université d'Évry-Val-d'Essonne (UEVE)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), CHU Pontchaillou [Rennes], Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire Analyse, Modélisation et Matériaux pour la Biologie et l'Environnement (LAMBE - UMR 8587), Université d'Évry-Val-d'Essonne (UEVE)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-CY Cergy Paris Université (CY), Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), École Pratique des Hautes Études (EPHE), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon
Subjects: [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Trans-splicing, Muscular Dystrophies, Trans-Splicing, Mice, 0302 clinical medicine, MESH: Molecular Targeted Therapy, Drug Discovery, RNA Precursors, MESH: Animals, Molecular Targeted Therapy, Dysferlin, Genetics, 0303 health sciences, MESH: Trans-Splicing, MESH: Alternative Splicing, Non-coding RNA, 3. Good health, RNA silencing, RNA editing, RNA splicing, Molecular Medicine, Original Article, MESH: Membrane Proteins, MESH: RNA Precursors, Computational biology, Biology, Cell Line, Open Reading Frames, 03 medical and health sciences, MESH: Mice, Inbred C57BL, Animals, Humans, RNA, Messenger, MESH: Protein Kinases, MESH: Mice, Molecular Biology, MESH: RNA, Messenger, 030304 developmental biology, Pharmacology, MESH: Dysferlin, MESH: Humans, Alternative splicing, Intron, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Open Reading Frames, MESH: Muscular Dystrophies, MESH: Cell Line, Mice, Inbred C57BL, Alternative Splicing, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Protein Kinases, 030217 neurology & neurosurgery, Minigene
Abstract: International audience; Muscular dystrophies are a group of genetically distinct diseases for which no treatment exists. While gene transfer approach is being tested for several of these diseases, such strategies can be hampered when the size of the corresponding complementary DNA (cDNA) exceeds the packaging capacity of adeno-associated virus vectors. This issue concerns, in particular, dysferlinopathies and titinopathies that are due to mutations in the dysferlin (DYSF) and titin (TTN) genes. We investigated the efficacy of RNA trans-splicing as a mode of RNA therapy for these two types of diseases. Results obtained with RNA trans-splicing molecules designed to target the 3' end of mouse titin and human dysferlin pre-mRNA transcripts indicated that trans-splicing of pre-mRNA generated from minigene constructs or from the endogenous genes was achieved. Collectively, these results provide the first demonstration of DYSF and TTN trans-splicing reprogramming in vitro and in vivo. However, in addition to these positive results, we uncovered a possible issue of the technique in the form of undesirable translation of RNA pre-trans-splicing molecules, directly from open reading frames present on the molecule or associated with internal alternative cis-splicing. These events may hamper the efficiency of the trans-splicing process and/or lead to toxicity.
Published: 2014
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43. Concurrent Measures of Fusion and Transduction Efficiency of Primary CD34+ Cells with Human Immunodeficiency Virus 1-Based Lentiviral Vectors Reveal Different Effects of Transduction Enhancers

Author: Anne Galy, Saliha Majdoul, Ababacar K. Seye, Dina Ingrao, David Fenard, Immunologie moléculaire et biothérapies innovantes (IMBI), École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, Association française contre les Myopathies (AFM), Fondation pour la recherche médicale (FRM), and École Pratique des Hautes Études (EPHE)
Subjects: Cell type, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Genetic enhancement, Genetic Vectors, Antigens, CD34, Biology, Receptor, Nerve Growth Factor, Applied Microbiology and Biotechnology, beta-Lactamases, Article, Flow cytometry, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Genetics, medicine, Humans, Progenitor cell, Enhancer, Cells, Cultured, Genetics (clinical), 030304 developmental biology, Pharmacology, 0303 health sciences, medicine.diagnostic_test, Lentivirus, HCT116 Cells, Hematopoietic Stem Cells, Molecular biology, Fusion protein, 3. Good health, Haematopoiesis, 030220 oncology & carcinogenesis, HIV-1, Cytokines, Molecular Medicine
Abstract: International audience; Lentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.
Published: 2014
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44. Global analysis of Plasmodium falciparum Na+/H+ exchanger (pfnhe-1) allele polymorphism and its usefulness as a marker of in vitro resistance to quinine

Author: Nimol Khim, Lydie Canier, Valérie Andriantsoanirina, Arsène Ratsimbasoa, Benoit Witkowski, Didier Menard, Odile Mercereau-Puijalon, Christophe Benedet, Rémy Durand, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Laboratoire de Parasitologie-Mycologie [CHU Avicenne], Hôpital Avicenne [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Programme National de Lutte contre le Paludisme [Antananarivo, Madagascar] (PNLP), Ministère de la Santé Publique - Ministry of Public Health [Antananarivo, Madagascar], Immunologie moléculaire des parasites, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by Grants from Natixis Banques and the Genomics Platform, Pasteur Génopôle, Pasteur Institute, France. Sample collection was funded in Comoros Islands by the French Foreign Ministry (FSP-RAI project) and in Madagascar by the Global Fund project round 3 (Grant MDG-304-G05-M). Didier Ménard is supported by the French Ministry of Foreign Affairs, Benoit Witkowski by a post-doctoral fellowship from the Division International – Institut Pasteur (2011–2013) and Christophe Benedet by a grant from the Fondation Pierre Ledoux – Jeunesse Internationale (2012)., We thank the patients and healthcare workers involved in the studies performed in Madagascar and Comoros Islands. We are grateful to Christiane Bouchier and Magali Tichit for performing sequencing reactions (Genomics Platform, Pasteur Génopôle, Pasteur Institute, France) and Carol H. Sibley for her advices., Ministère de la Santé Publique [Antananarivo, Madagascar], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: Quinine resistance, Plasmodium falciparum, 030231 tropical medicine, Article, 03 medical and health sciences, chemistry.chemical_compound, Na+/H+ exchanger, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Polymorphism (computer science), Molecular marker, medicine, Pharmacology (medical), Allele, Gene, 030304 developmental biology, Pharmacology, Genetics, 0303 health sciences, Quinine, Genetic polymorphism, biology, biology.organism_classification, medicine.disease, Malaria, 3. Good health, Infectious Diseases, chemistry, GenBank, Parasitology, medicine.drug
Abstract: International audience; The aim of this study was to provide a comprehensive analysis of the worldwide genetic polymorphism of ms4760 alleles of the pfnhe-1 gene and to discuss their usefulness as molecular marker of quinine resistance (QNR). A new numbering of ms4760 allele, classification grouping ms4760 alleles according to the number of DNNND and DDNHNDNHNND repeat motifs in blocks II and V was also proposed. A total of 1508 ms4760 sequences from isolates, culture-adapted parasites or reference strains from various geographical regions were retrieved from GenBank (last update on 15th June 2012) or from publications and were used for genetic analyses. The association of different alleles of pfnhe-1 with resistance to quinoline antimalarial drugs showed marked geographic disparities. The validity and reliability of candidate polymorphisms in pfnhe-1 gene as molecular markers of QNR appeared restricted to endemic areas from South Asia or possibly East African countries and needs to be confirmed.
Published: 2013
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45. Gene therapy on the move

Author: Kerstin B. Kaufmann, Anne Galy, Hildegard Büning, Axel Schambach, Manuel Grez, Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, and École pratique des hautes études (EPHE)
Subjects: Pathology, medicine.medical_specialty, viral vectors, [SDV]Life Sciences [q-bio], medicine.medical_treatment, Genetic enhancement, Reviews, Gene transfer, monogenic disorders, Bioinformatics, stem cell therapy, Viral vector, 03 medical and health sciences, 0302 clinical medicine, Genome editing, medicine, Humans, ddc:610, 030304 developmental biology, Clinical Trials as Topic, clinical trials, 0303 health sciences, business.industry, iPS, Gene Transfer Techniques, Gene targeting, Treatment options, Genetic Therapy, Stem-cell therapy, 3. Good health, Clinical trial, 030220 oncology & carcinogenesis, Molecular Medicine, business
Abstract: International audience; The first gene therapy clinical trials were initiated more than two decades ago. In the early days, gene therapy shared the fate of many experimental medicine approaches and was impeded by the occurrence of severe side effects in a few treated patients. The understanding of the molecular and cellular mechanisms leading to treatment- and/or vector-associated setbacks has resulted in the development of highly sophisticated gene transfer tools with improved safety and therapeutic efficacy. Employing these advanced tools, a series of Phase I/II trials were started in the past few years with excellent clinical results and no side effects reported so far. Moreover, highly efficient gene targeting strategies and site-directed gene editing technologies have been developed and applied clinically. With more than 1900 clinical trials to date, gene therapy has moved from a vision to clinical reality. This review focuses on the application of gene therapy for the correction of inherited diseases, the limitations and drawbacks encountered in some of the early clinical trials and the revival of gene therapy as a powerful treatment option for the correction of monogenic disorders.
Published: 2013
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46. Self-inactivating MLV vectors have a reduced genotoxic profile in human epidermal keratinocytes

Author: Alessia Cavazza, Carlo Pincelli, Fabienne Cocchiarella, Fulvio Mavilio, Manfred Schmidt, Fernando Larcher, Cynthia C. Bartholomae, European Commission - Joint Research Centre [Ispra] (JRC), Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, European Commission, Comunidad de Madrid, Consiglio Nazionale delle Ricerche, and Instituto de Salud Carlos III
Subjects: Keratinocytes, Virus Integration, [SDV]Life Sciences [q-bio], viruses, Transgene, Genetic enhancement, Genetic Vectors, Retroviral integration, Biology, Mice, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Genetics, medicine, Animals, Humans, Transgenes, Vector (molecular biology), Epidermolysis bullosa, Insertional mutagenesis, Promoter Regions, Genetic, Molecular Biology, epidermolysis bullosa, insertional mutagenesis, retroviral integration, 030304 developmental biology, Swiss 3T3 Cells, Regulation of gene expression, 0303 health sciences, fungi, Genetic Therapy, biochemical phenomena, metabolism, and nutrition, Molecular biology, 3. Good health, Transplantation, medicine.anatomical_structure, Gene Expression Regulation, 030220 oncology & carcinogenesis, HIV-1, Virus Inactivation, Molecular Medicine, Moloney murine leukemia virus, Stem cell, Epidermolysis Bullosa, Keratinocyte, Cell Adhesion Molecules, HeLa Cells
Abstract: et al., Transplantation of epithelia derived from keratinocyte stem cells transduced by retroviral vectors is a potential therapy for epidermolysis bullosa (EB), a family of inherited skin adhesion defects. The biosafety characteristics of retroviral vectors in keratinocytes are, however, poorly defined. We developed self-inactivating (SIN) vectors derived from the Moloney murine leukemia (MLV) and the human immunodeficiency (HIV) viruses expressing therapeutic levels of LAMB3, a transgene defective in junctional EB, and tested their integration profile in human primary keratinocytes. The SIN–HIV vector showed the expected preference for transcribed genes while the SIN–MLV vector integrated preferentially in regulatory elements, but showed a significantly lower tendency to target cell growth-related genes, transcription start sites and epigenetically defined promoters compared with a wild-type MLV vector in an epithelial cell context. A quantitative gene expression assay in individual keratinocyte clones showed that MLV-derived vectors deregulate expression of targeted genes at a lower frequency than in hematopoietic cells, and that the SIN–MLV design has the lowest activity compared to both MLV and SIN–HIV vectors. This study indicates that SIN–MLV vectors may have a better safety profile in keratinocyte than in hematopoietic cells, and be a reasonable alternative to lentiviral vectors for gene therapy of inherited skin disorders., This work was supported by grants from the European Research Council (GT-SKIN) and the Italian National Research Council (EPIGEN). FL was supported by grants PI11/01225 from ISCIII and S2010/BMD-2359 from CM.
Published: 2013
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47. Reduced Artemisinin Susceptibility of Plasmodium falciparum Ring Stages in Western Cambodia

Author: Rithea Leang, Sophie Chy, Antoine Berry, Pascal Ringwald, Saorin Kim, Odile Mercereau-Puijalon, Rupam Tripura, Pheaktra Chim, Socheat Duong, Françoise Benoit-Vical, Jean-Christophe Barale, Didier Menard, Nimol Kloeung, Nimol Khim, Olivier Gorgette, Sopheakvatey Ke, Benoit Witkowski, Frédéric Ariey, Arjen M. Dondorp, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), National Center for Parasitology, Entomology and Malaria Control [Phnom Penh, Cambodia] (CNM), World Health Organization [Geneva], Mahidol University [Bangkok], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Immunologie moléculaire des parasites, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), CHU Toulouse [Toulouse], and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)
Subjects: medicine.medical_treatment, Plasmodium falciparum, 030231 tropical medicine, Drug Resistance, Dihydroartemisinin, Drug resistance, Antimalarials, 03 medical and health sciences, 0302 clinical medicine, Parasitic Sensitivity Tests, Gentamicin protection assay, Pulse exposure, parasitic diseases, medicine, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Pharmacology (medical), Malaria, Falciparum, Artemisinin, Survival rate, ComputingMilieux_MISCELLANEOUS, Pharmacology, 0303 health sciences, biology, 030306 microbiology, Cell Cycle Checkpoints, medicine.disease, biology.organism_classification, Virology, Artemisinins, 3. Good health, Infectious Diseases, Susceptibility, Cambodia, Malaria, medicine.drug
Abstract: The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA.
Published: 2013
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48. Prolonged Gene Expression in Muscle Is Achieved Without Active Immune Tolerance Using MicrorRNA 142.3p-Regulated rAAV Gene Transfer

Author: Razzak, A, al Samarrai, A, Al Samarrai, Razzak, Boisgérault, Florence, Gross, David-Alexandre, Ferrand, Maxime, Poupiot, Jérôme, Darocha, Sylvie, Richard, Isabelle, Galy, Anne, Immunologie moléculaire et biothérapies innovantes (IMBI), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Généthon, Tolérance immunitaire et présentation antigénique: impact en auto-immunité et en transplantation, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Approches génétiques intégrées et nouvelles thérapies pour les maladies rares (INTEGRARE), École pratique des hautes études (EPHE)-Université d'Évry-Val-d'Essonne (UEVE)-GENETHON 3-Institut National de la Santé et de la Recherche Médicale (INSERM), Généthon, Evry, Association française contre les myopathies (AFM-Téléthon), and École pratique des hautes études (EPHE)
Subjects: CD4-Positive T-Lymphocytes, Male, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, MESH: Dependovirus, CD8-Positive T-Lymphocytes, MESH: Muscular Dystrophy, Animal, Immune tolerance, Transduction (genetics), Mice, 0302 clinical medicine, MESH: Genetic Vectors, Gene expression, MESH: Animals, Transgenes, 0303 health sciences, MESH: Muscle, Skeletal, Gene Transfer Techniques, MESH: CD4-Positive T-Lymphocytes, Dependovirus, MESH: CD8-Positive T-Lymphocytes, Cell biology, Sarcoglycan, 030220 oncology & carcinogenesis, Molecular Medicine, Female, Expression cassette, MESH: Gene Expression, MESH: Immune Tolerance, Transgene, Genetic Vectors, MESH: Transgenes, MESH: Gene Transfer Techniques, Gene delivery, Biology, Viral vector, 03 medical and health sciences, MESH: Mice, Inbred C57BL, Sarcoglycans, Genetics, Immune Tolerance, Animals, MESH: Sarcoglycans, Muscle, Skeletal, Molecular Biology, MESH: Mice, 030304 developmental biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Muscular Dystrophy, Animal, Molecular biology, MESH: Male, Mice, Inbred C57BL, MicroRNAs, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, MESH: MicroRNAs, MESH: Female
Abstract: International audience; Gene transfer efficacy is limited by unwanted immunization against transgene products. In some models, immunization may be avoided by regulating transgene expression with mir142.3p target sequences. Yet, it is unclear if such a strategy controls T-cell responses following recombinant adeno-associated viral vector (rAAV)-mediated gene transfer, particularly in muscle. In mice, intramuscular rAAV1 gene delivery of a tagged human sarcoglycan muscle protein is robustly immunogenic and leads to muscle destruction. In this model, the simple insertion of mir142.3p-target sequences in the transgene expression cassette modifies the outcome of gene transfer, providing high and persistent levels of muscle transduction in C57Bl/6 mice. Such regulated vector fails to prime specific CD4 and CD8 T cells; although, transgene tolerance seems to result from ignorance and could be broken by a robust antigenic challenge. While effective in normal mice, the mir142.3p-regulated transgene remains immunogenic in sarcoglycan-deficient dystrophic mice. In these mice, transgene expression is only prolonged but does not persist as effector CD4 and CD8 T-cell responses develop. Thus, using a mir142.3p-regulated transgene can improve rAAV muscle gene transfer results, but the level of efficacy depends on the context of application. In normal muscle, this strategy is sufficient to prevent immunization and functions even more effectively than tissue-specific promoters. In dystrophic models, additional strategies are required to fully control T-cell responses.
Published: 2013
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49. Plasmodium falciparum: multifaceted resistance to artemisinins

Author: Arba Pramundita Ramadani, Jean-Michel Augereau, Françoise Benoit-Vical, Odile Mercereau-Puijalon, Lucie Paloque, Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées, Laboratoire de chimie de coordination (LCC), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie de Toulouse (ICT-FR 2599), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Immunologie moléculaire des parasites, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), ANR-13-BSV3-0018,MALARTRES,Résistance de Plasmodium aux antipaludiques de la famille des artémisinines(2013), ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), BMC, BMC, Blanc 2013 - Résistance de Plasmodium aux antipaludiques de la famille des artémisinines - - MALARTRES2013 - ANR-13-BSV3-0018 - Blanc 2013 - VALID, Integrative Biology of Emerging Infectious Diseases - - IBEID2010 - ANR-10-LABX-0062 - LABX - VALID, Institut de Chimie de Toulouse (ICT-FR 2599), Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Institut de Chimie de Toulouse (ICT), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut de Recherche pour le Développement (IRD)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Artemisinins, [SDV]Life Sciences [q-bio], Plasmodium falciparum, Resistance, Drug Resistance, Review, Drug resistance, Biology, Quiescence, Models, Biological, Antimalarials, 03 medical and health sciences, parasitic diseases, medicine, Humans, Malaria, Falciparum, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, Transcription factor, PI3K/AKT/mTOR pathway, Apicoplast, PfK13, biology.organism_classification, medicine.disease, Virology, Artemisinin-based combination therapy, 3. Good health, Cell biology, Malaria, [SDV] Life Sciences [q-bio], 030104 developmental biology, Infectious Diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Unfolded protein response, Parasitology
Abstract: Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1206-9) contains supplementary material, which is available to authorized users.
Published: 2016
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50. Large-scale screening for genes involved in T-cell signaling: do we know all the players now?

Author: Vincenzo Di Bartolo, Oreste Acuto, Immunologie Moléculaire, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)
Subjects: MESH: Signal Transduction, MESH: Gene Expression, Transcription, Genetic, T cell, Lymphocyte, Immunology, Cell, Gene Expression, Biology, Transcriptome, 03 medical and health sciences, MESH: Gene Expression Profiling, 0302 clinical medicine, medicine, Immunology and Allergy, Humans, Mass Screening, Serial analysis of gene expression, MESH: Mass Screening, Gene, 030304 developmental biology, 0303 health sciences, MESH: Humans, Gene Expression Profiling, MESH: Transcription, Genetic, Cell biology, medicine.anatomical_structure, Membrane protein, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, MESH: T-Lymphocytes, Cytotoxic, Intracellular, 030215 immunology, Signal Transduction, T-Lymphocytes, Cytotoxic
Abstract: Approaches that allow global analysis of cell transcriptomes are greatly accelerating the dissection of lymphocyte signaling. Using serial analysis of gene expression, a recent study has provided evidence that most plasma membrane proteins of T cells have now been identified. Is this also true for the proteins governing intracellular signaling circuitries? This article comments on the recent study and describes other high-throughput screenings aimed at identifying and functionally characterizing signaling proteins.
Published: 2016
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