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1. LIS1, a glyco-humanized swine polyclonal anti-lymphocyte globulin, as a novel induction treatment in solid organ transplantation

Author

Juliette Rousse, Pierre-Joseph Royer, Gwénaëlle Evanno, Elsa Lheriteau, Carine Ciron, Apolline Salama, Françoise Shneiker, Roberto Duchi, Andrea Perota, Cesare Galli, Emmanuele Cozzi, Gilles Blancho, Odile Duvaux, Sophie Brouard, Jean-Paul Soulillou, Jean-Marie Bach, and Bernard Vanhove

Subjects
solid organ transplantation, induction, anti-lymphocyte globulin, anti-lymphocyte antibody, polyclonal antibodies (PAbs), kidney trans plantation, Immunologic diseases. Allergy, RC581-607
Abstract

Anti-thymocyte or anti-lymphocyte globulins (ATGs/ALGs) are immunosuppressive drugs used in induction therapies to prevent acute rejection in solid organ transplantation. Because animal-derived, ATGs/ALGs contain highly immunogenic carbohydrate xenoantigens eliciting antibodies that are associated with subclinical inflammatory events, possibly impacting long-term graft survival. Their strong and long-lasting lymphodepleting activity also increases the risk for infections. We investigated here the in vitro and in vivo activity of LIS1, a glyco-humanized ALG (GH-ALG) produced in pigs knocked out for the two major xeno-antigens αGal and Neu5Gc. It differs from other ATGs/ALGs by its mechanism of action excluding antibody-dependent cell-mediated cytotoxicity and being restricted to complement-mediated cytotoxicity, phagocyte-mediated cytotoxicity, apoptosis and antigen masking, resulting in profound inhibition of T-cell alloreactivity in mixed leucocyte reactions. Preclinical evaluation in non-human primates showed that GH-ALG dramatically reduced CD4+ (p=0.0005,***), CD8+ effector T cells (p=0.0002,***) or myeloid cells (p=0.0007,***) but not T-reg (p=0.65, ns) or B cells (p=0.65, ns). Compared with rabbit ATG, GH-ALG induced transient depletion (less than one week) of target T cells in the peripheral blood (

Published
2023
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2. Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS

Author

Paola Crociara, Maria Novella Chieppa, Elena Vallino Costassa, Elena Berrone, Marina Gallo, Monica Lo Faro, Maria Domenica Pintore, Barbara Iulini, Antonio D'Angelo, Giovanni Perona, Alberto Botter, Donato Formicola, Alberto Rainoldi, Marianna Paulis, Paolo Vezzoni, Federica Meli, Fiorenzo Antonio Peverali, Caterina Bendotti, Maria Chiara Trolese, Laura Pasetto, Valentina Bonetto, Giovanna Lazzari, Roberto Duchi, Andrea Perota, Irina Lagutina, Corinne Quadalti, Maria Silvia Gennero, Daniela Dezzutto, Rosanna Desiato, Marina Boido, Matilde Ghibaudi, Maria Consuelo Valentini, Maria Caramelli, Cesare Galli, Cristina Casalone, and Cristiano Corona

Subjects
SOD1, Transgenic pig, Amyotrophic lateral sclerosis, ALS, TDP-43, Neurosciences. Biological psychiatry. Neuropsychiatry, RC321-571
Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Published
2019
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3. Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs.

Author

Olivier Reynard, Frédéric Jacquot, Gwénaëlle Evanno, Hoa Le Mai, Apolline Salama, Bernard Martinet, Odile Duvaux, Jean-Marie Bach, Sophie Conchon, Jean-Paul Judor, Andrea Perota, Irina Lagutina, Roberto Duchi, Giovanna Lazzari, Ludmilla Le Berre, Hélène Perreault, Elsa Lheriteau, Hervé Raoul, Viktor Volchkov, Cesare Galli, and Jean-Paul Soulillou

Subjects
Medicine, Science
Abstract

Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Published
2016
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4. High neutralizing potency of swine glyco-humanized polyclonal antibodies against SARS-CoV-2

Author

Dominique Blanchard, Melody Paulus, Gwénaëlle Evanno, Roberto Bruzzone, Matthieu Ledure, Sophie Brouard, Irina Lagutina, Sophie Conchon, Emanuele Cozzi, Roberto Duchi, Cesare Galli, Juliette Rousse, Bernard Vanhove, Elsa Lheriteau, Laurent Vacher, Apolline Salama, Romain Oger, Pierre-Joseph Royer, Andrea Perota, Nadine Gervois, Marc Bouillet, Yannick Jacques, Odile Duvaux, Ray T.Y. So, Jean-Marie Bach, Jean-Paul Soulillou, Chris Ka Pun Mok, Carine Ciron, Philippe Delahaut, Xenothera [Nantes, France], Anti-Tumor Immunosurveillance and Immunotherapy (CRCINA-ÉQUIPE 3), Centre de Recherche en Cancérologie et Immunologie Nantes-Angers (CRCINA), 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)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA)-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)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Centre National de la Recherche Scientifique (CNRS)-Université d'Angers (UA), Immunobiology of Human αβ and γδ T Cells and Immunotherapeutic Applications (CRCINA-ÉQUIPE 1), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Université de Nantes (UN)-Université de Nantes (UN), Avantea [Cremona, Italy], CER Groupe Marloie, The University of Hong Kong (HKU), University Hospital of Padua, Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Bpifrance, Xenothera, Société d'Accélération et Transfert de Technologie Ouest Valorisation, Région Pays de la Loire, Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes)-Université d'Angers (UA)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre hospitalier universitaire de Nantes (CHU Nantes), CER Groupe, Azienda Ospedale Università di Padova = Hospital-University of Padua (AOUP), Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and Bernardo, Elizabeth

Subjects
0301 basic medicine, pig, Swine, Spike, Antibodies, Viral, medicine.disease_cause, SARS‐CoV‐2, Epitope, Animals, Genetically Modified, chemistry.chemical_compound, 0302 clinical medicine, Neuraminic acid, Immunology and Allergy, polyclonal antibodies, Research Articles, Coronavirus, Covid-19, SARS-CoV-2, Galactosyltransferases, 3. Good health, Spike Glycoprotein, Coronavirus, Research Article|Basic, Antibody, medicine.drug_class, Immunology, Immunity to infection, Heterologous, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, Monoclonal antibody, Article, 03 medical and health sciences, Immune system, [SDV.CAN] Life Sciences [q-bio]/Cancer, Antigen, COVID‐19, medicine, Animals, Humans, Basic, COVID-19 Serotherapy, Immunization, Passive, Antibodies, Neutralizing, Virology, HEK293 Cells, 030104 developmental biology, chemistry, Polyclonal antibodies, Sialic Acids, biology.protein, 030215 immunology
Abstract

Heterologous polyclonal antibodies might represent an alternative to the use of convalescent plasma or monoclonal antibodies (mAbs) in coronavirus disease (COVID‐19) by targeting multiple antigen epitopes. However, heterologous antibodies trigger human natural xenogeneic antibody responses particularly directed against animal‐type carbohydrates, mainly the N‐glycolyl form of the neuraminic acid (Neu5Gc) and the α1,3‐galactose, potentially leading to serum sickness or allergy. Here, we immunized cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase and α1,3‐galactosyl‐transferase (GGTA1) double KO pigs with the Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike receptor binding domain to produce glyco‐humanized polyclonal neutralizing antibodies lacking Neu5Gc and α1,3‐galactose epitopes. Animals rapidly developed a hyperimmune response with anti‐SARS‐CoV‐2 end‐titers binding dilutions over one to a million and end‐titers neutralizing dilutions of 1:10 000. The IgG fraction purified and formulated following clinical Good Manufacturing Practices, named XAV‐19, neutralized spike/angiotensin converting enzyme‐2 interaction at a concentration, GGTA1/CMAH double KO pigs produced high titers of XAV‐19, an anti‐SARS‐Cov‐2 RBD glyco‐humanized polyclonal antibody. XAV‐19 is presented with a high complement activation and a high neutralization potency in vitro and in cell‐based neutralization assays. XAV‐19 does not interact with human Fc gamma receptors, preventing Fc‐dependent enhancement or immune cells skewing.

Published
2021

5. Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy

Author

Xi Chen, Roberto Duchi, Vered Padler-Karavani, Shirley Abramovitch, Eliran Moshe Reuven, Shani Leviatan Ben-Arye, Jean-Paul Soulillou, Andrea Perota, Cesare Galli, Sophie Conchon, Hai Yu, Department of Cell Research and Immunology, Tel Aviv University [Tel Aviv], Department of Chemistry [Davis, CA, USA], University of California [Davis] (UC Davis), University of California-University of California, Avantea Laboratory of Reproductive Technologies [Cremona, Italy], Immunoregulation And Immunointervention in Transplantation and Autoimmunity (Team 4 - U1064 Inserm - CRTI), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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)-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), Fondazione Avantea [Cremona, Italy], European Project: ERC-2016-STG-716220, Tel Aviv University (TAU), Department of Chemistry [Univ California Davis] (Chemistry - UC Davis), University of California (UC)-University of California (UC), Le Bihan, Sylvie, and European Union H2020 Program - ERC-2016-STG-716220 - INCOMING

Subjects
Swine, medicine.medical_treatment, General Physics and Astronomy, 02 engineering and technology, 01 natural sciences, Mice, Cancer immunotherapy, Biomimetic Materials, General Materials Science, Cancer, Mice, Knockout, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, General Engineering, biomimetic, 021001 nanoscience & nanotechnology, 3. Good health, 5.1 Pharmaceuticals, glycan microarray, sialic acid, Colonic Neoplasms, Immunotherapy, Development of treatments and therapeutic interventions, Antibody, 0210 nano-technology, Biotechnology, N-glycolylneuraminic acid, Knockout, Context (language use), Adenocarcinoma, 010402 general chemistry, Cancer Vaccines, Article, Vaccine Related, Immune system, Antigen, medicine, Animals, Particle Size, Nanoscience & Nanotechnology, glyconanoparticle, Nutrition, cancer immunotherapy, Prevention, N-Acetylneuraminic Acid, neoantigen, 0104 chemical sciences, Good Health and Well Being, Immunization, Cancer research, biology.protein, Nanoparticles, Neuraminic Acids, Cancer vaccine, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NG[pos]) or lack expression (NG[neg]) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah[−/−] mice with NG[pos] glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets.

Published
2019

6. Neu5Gc and α1-3 GAL Xenoantigen Knockout Does Not Affect Glycemia Homeostasis and Insulin Secretion in Pigs

Author

Andrea Perota, Pierre Weiss, Nicolas Gaide, Steffi Bosch, Anne Moure, Jean-Paul Concordet, Emanuele Cozzi, Mathilde Mosser, Cesare Galli, Jérôme Guicheux, Jeremy Hervouet, David Minault, Jean-Marie Bach, Jean-Paul Soulillou, Jérôme Abadie, Sylvie Pogu, Apolline Salama, Xavier Lévêque, Olivier Gauthier, Dominique Jégou, Roberto Duchi, Gilles Blancho, Jean-Paul Judor, Sophie Brouard, Stéphanie Le Bas-Bernardet, Giovanna Lazzari, Ludmilla Le Berre, Sophie Conchon, David Riochet, Corentin Danna, Irina Lagutina, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Immuno-Endocrinologie Cellulaire et Moléculaire [Nantes] (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Société d'Accélération du Transfert de Technologies (SATT OUEST VALORISATION), Avantea Laboratory of Reproductive Technologies, Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Animaux Modèles pour la Recherche en Oncologie [Nantes] (AMaROC), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Muséum national d'Histoire naturelle (MNHN), Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-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), Laboratoire d'ingénierie osteo-articulaire et dentaire (LIOAD), Université de Nantes (UN)-IFR26-Institut National de la Santé et de la Recherche Médicale (INSERM), PHU 4 OTONN, Centre hospitalier universitaire de Nantes (CHU Nantes), CIC - Nantes, Avantea Foundation, Transplantation Immunology Unit, Universita degli Studi di Padova, Consortium for Research in Organ Transplantation (CORIT), University of Bologna, Université de Nantes (UN), Avantea Laboratory of Reproductive Technologies [Cremona, Italy], Département de Chirurgie Expérimentale [Nantes] (Oniris - Centre de recherche et d'investigation préclinique), Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Centre de recherche et d'investigation préclinique [Nantes], Museum National d'Histoire Naturelle de Paris, Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Département de pédiatrie [CHU Nantes], Université de Nantes - UFR Odontologie, Ostéo-articulaire - Tête et cou - Odontologie - Neurochirurgie - Neurotraumatologie [CHU Nantes] (Pôle hospitalo-universitaire PHU4 - OTONN), CIC biothérapies CBT 0503 [Nantes], Hôtel-Dieu de Nantes-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre hospitalier universitaire de Nantes (CHU Nantes), Avantea Foundation [Cremona, Italy], Transplantation Immunology Unit [Padua, Italy] (Department of Transfusion Medicine), University of Padua–Ospedale Giustinianeo [Padua, Italy], Consortium for Research in Organ Transplantation [Padua, Italy] (CORIT), Department of Veterinary Medical Sciences [Ozzano dell'Emilia, Italy], Pays de la Loire Region (France) (Xenothera academic program and to A.S.), the Société d’Accélération du Transfert de Technologies Ouest Valorisation (to A.S.)., ANR: ANR-10IBHU-005, ANR: ANR-II-INSB-0014, European Project: LSHB-CT-2006-037377, European Project: 603049, European Project: ECTIS, Université de Nantes (UN)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS)-Institut National de la Recherche Agronomique (INRA), Animaux modèles pour la recherche en oncologie comparée (AMaROC), 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)-Muséum national d'Histoire naturelle (MNHN), Institut de Transplantation Urologie Néphrologie, AMaROC - Oniris Nantes, Centre d'Investigation Clinique (CIC) Biotherapy, This work was supported by Pays de la Loire Region (France) (Xenothera academic program and to A.S.), the Société d’Accélération du Transfert de Technologies Ouest Valorisation (to A.S.), the European Center for Transplantation and Immunotherapy Sciences (ECTIS IHU, Nantes, France), the National Research Agency 'Investment Into The Future' programs (ANR-10-IBHU-005, to X.L., and ANR-II-INSB-0014), the European Commission’s Xenome Sixth Framework Programme (LSHB-CT-2006-037377), and the European Seventh Framework Programme 'Translink' research program (grant agreement 603049 and to L.L.B.)., ANR-10-IBHU-0005,CESTI (TSI-IHU),Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU)(2010), ANR-11-INBS-0014,TEFOR,Transgenèse pour les Etudes Fonctionnelles sur les Organismes modèles(2011), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Università degli Studi di Padova = University of Padua (Unipd), Consorzio per la Ricerca sul Trapianto d'Organ = Consortium for Research in Organ Transplantation (CORIT), University of Bologna/Università di Bologna, European Project: 603049,EC:FP7:HEALTH,FP7-HEALTH-2013-INNOVATION-1,TRANSLINK(2013), Mosser, Mathilde, Lévêque, Xavier, Perota, Andrea, Galli, Cesare, Soulillou, Jean-Paul, Bach, Jean-Marie, Jehan, Frederic, Instituts Hospitalo-Universitaires B - Centre Européen des Sciences de la Transplantation et de l'Immunothérapie (TSI-IHU) - - CESTI (TSI-IHU)2010 - ANR-10-IBHU-0005 - IBHU - VALID, Infrastructures - Transgenèse pour les Etudes Fonctionnelles sur les Organismes modèles - - TEFOR2011 - ANR-11-INBS-0014 - INBS - VALID, and Defining the role of xeno-directed and autoimmune events in patients receiving animal-derived bioprosthetic heart valves - TRANSLINK - - EC:FP7:HEALTH2013-09-01 - 2017-08-31 - 603049 - VALID

Subjects
Blood Glucose, Male, 0301 basic medicine, Swine, Endocrinology, Diabetes and Metabolism, medicine.medical_treatment, Islets of Langerhans Transplantation, 030230 surgery, Gene Knockout Techniques, 0302 clinical medicine, Insulin Secretion, Homeostasis, Insulin, Glucose homeostasis, [SDV.MHEP.RSOA] Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Heterologous, geography.geographical_feature_category, [SDV.MHEP.GEG] Life Sciences [q-bio]/Human health and pathology/Geriatry and gerontology, C-Peptide, [SDV.MHEP.GEG]Life Sciences [q-bio]/Human health and pathology/Geriatry and gerontology, Islet, 3. Good health, Heterophile, medicine.anatomical_structure, [SDV.MHEP.RSOA]Life Sciences [q-bio]/Human health and pathology/Rhumatology and musculoskeletal system, Type 1, medicine.medical_specialty, endocrine system, Transplantation, Heterologous, Animals, Antigens, Heterophile, Diabetes Mellitus, Type 1, Galactose, Glucagon, Glucose, Islets of Langerhans, Neuraminic Acids, Pancreas, Purinergic P1 Receptor Antagonists, Theophylline, Carbohydrate metabolism, Biology, 03 medical and health sciences, Internal medicine, Diabetes mellitus, Diabetes Mellitus, Internal Medicine, medicine, Antigens, Transplantation, Type 1 diabetes, geography, Pancreatic islets, medicine.disease, 030104 developmental biology, Endocrinology, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited anti-bodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc 2/2 mice exhibit glycemic dys-regulations and pancreatic b-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities , and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Published
2017

7. 203 SINGLE-STEP GENE EDITING OF 3 XENOANTIGENS IN PORCINE FIBROBLASTS USING PROGRAMMABLE NUCLEASES

Author

J. P. Soulillou, Cesare Galli, G. Crotti, Giovanna Lazzari, Silvia Colleoni, Irina Lagutina, Roberto Duchi, Corinne Quadalti, Andrea Perota, Sophie Conchon, Jean-Paul Concordet, P. Turini, Perota, A., Lagutina, I., Quadalti, C., Duchi, R., Turini, P., Crotti, G., Colleoni, S., Conchon, S., Concordet, J.-P., Lazzari, G., Soulillou, J.-P., and Galli, C.

Subjects
Cloning, Genetics, Transcription activator-like effector nuclease, Expression vector, Reproductive technology, Biology, Programmable nucleases, swine fibroblast, xenoantigens, Endocrinology, Reproductive Medicine, Genome editing, Somatic cell nuclear transfer, CRISPR, Animal Science and Zoology, Molecular Biology, Gene, Developmental Biology, Biotechnology
Abstract

Programmable nucleases (ZFN, Tal Effector Nucleases, and CRISPR) opened a new era for mammal genome editing, in particular for the pigs used for xenotransplantation. Multiple gene editing events are required both for knockout (KO) of xenoantigens and for targeted integration of human protective genes (Perota et al. 2016 J. Genet. Genomics 43, 233–23). The objective of the present work was to edit selected pig lines to KO the enzymes coding for the most relevant xenoantigens (i.e. GGTA1, CMAH, and B4GalNT2), combining Talens and CRISPR/Cas9 technologies to magnetic beads selection (Li et al. 2013 Xenotransplantation 22, 20–31). Primary porcine adult fibroblasts were transfected using Nucleofector (V-024 program). In a single reaction 2 × 106 fibroblasts were co-transfected using 2 different sets of TALENS (4 μg/set) specific for CMAH (Conchon et al., 2013) and GGTA1 (Perota et al., 2015) genes together with B4GalNT2-specific CRISPR/Cas9 expression vector (2 μg; pX330-B4GalNT2; Estrada et al., 2015). Eight days post-transfection (DPT), Gal–/– cells were selected initially using biotin-conjugated IB4 lectin (Sigma, St. Louis, MO, USA) and magnetic beads (Dynabeads M-280, Thermo Fisher Scientific, Waltham, MA, USA). The selected cells were then plated on 150-mm Petri dishes (200 cells/dish) and cultured for 10 days. Selected colonies were expanded for PCR analysis and cryopreserved for somatic cell nuclear transfer (SCNT). All colonies were analysed by PCR for CMAH gene and their resulting products were digested with HindIII (HindIII-RFLP). Colonies that lost wild-type HindIII as a consequence of Talens effected deletion were PCR characterised for GGTA1, selecting those that had detectable Indels after gel electrophoresis and finally analysed by PCR for B4GalNT2. All PCR products were validated by sequencing for all the 3 genes of interest (TopoTA, Thermo Fisher Scientific). Selected colonies were used as nuclear donors for SCNT (Lagutina et al., 2006). Eight DPT we obtained 3.45 ×106 cells. About 6.0 × 103 Gal-negative cells (0.17%) were collected from the supernatant after magnetic beads separation. Eighteen DPT, 120 colonies were picked up and their HindIII-RFLP analyses on CMAH gene revealed that 22 colonies (18.3%) were KO for both CMAH alleles. Of these 22 colonies following electrophoretic analyses of GGTA1-PCR products, 13 colonies had detectable Indels. These 13 colonies were finally PCR analysed and sequenced for B4GalNT2 and sequenced. Final sequencing results confirmed that 2 colonies (1.6%) resulted in KO for the 3 genes. Three different zona-free SCNT experiments were done and 579 reconstructed embryos were obtained. On Day 7, 322 morulae or blastocysts (56%) were transferred in 3 synchronised sows and 2 (66%) became pregnant. In conclusion, after gene editing with programmable nucleases, combining beads-mediated selection with well-designed molecular analyses, we developed a multistep assay that can be used efficiently to detect desired gene edited events in cell colonies suitable for the SCNT. Embryos generated after SCNT were able to establish pregnancies at a high rate. This work is supported by European FP7 grants Translink (n° 603049) and Xenoislet (n° 601827).

Published
2017

8. Anti-EBOV GP IgGs Lacking α1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs

Author

Odile Duvaux, Andrea Perota, Hélène Perreault, Elsa Lheriteau, Bernard Martinet, Irina Lagutina, Gwénaëlle Evanno, Ludmilla Le Berre, Hervé Raoul, Olivier Reynard, Cesare Galli, Giovanna Lazzari, Apolline Salama, Frédéric Jacquot, Roberto Duchi, Sophie Conchon, Jean-Paul Judor, Viktor E. Volchkov, Jean-Marie Bach, Jean-Paul Soulillou, Hoa Le Mai, Bases moléculaires de la pathogénicité virale – Molecular Basis of Viral Pathogenicity (BMPV), 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), Laboratoire P4 - Jean Mérieux, Centre Européen de Virologie/Immunologie-Institut National de la Santé et de la Recherche Médicale (INSERM), Xenothera [Nantes, France], Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Centre hospitalier universitaire de Nantes (CHU Nantes), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Ecole Nationale Vétérinaire de Nantes-Université de Nantes (UN)-Institut National de la Recherche Agronomique (INRA), Avantea Laboratory of Reproductive Technologies [Cremona, Italy], University of Manitoba [Winnipeg], Anti-EBOV GP IgGs Lacking alpha 1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs, Reynard, Olivier, Jacquot, Frédéric, Evanno, Gwénaëlle, Mai, Hoa Le, Salama, Apolline, Martinet, Bernard, Duvaux, Odile, Bach, Jean-Marie, Conchon, Sophie, Judor, Jean-Paul, Perota, Andrea, Lagutina, Irina, Duchi, Roberto, Lazzari, Giovanna, Le Berre, Ludmilla, Perreault, Hélène, Lheriteau, Elsa, Raoul, Hervé, Volchkov, Viktor, Galli, Cesare, Soulillou, Jean-Paul, 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), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes-École nationale vétérinaire, agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Davoine, Laure-Hélène, 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), Bases moléculaires de la pathogénicité virale – Molecular Basis of Viral Pathogenicity, Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, University of Manitoba, and University of Bologna

Subjects
0301 basic medicine, Male, Physiology, Swine, lcsh:Medicine, medicine.disease_cause, Antibodies, Viral, Biochemistry, Epitope, Immunoglobulin G, 0302 clinical medicine, Viral Envelope Proteins, Pig Models, Intraperitoneal Injections, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Immune Physiology, Medicine and Health Sciences, 030212 general & internal medicine, Viral, Enzyme-Linked Immunoassays, lcsh:Science, Routes of Administration, Mammals, Multidisciplinary, Immune System Proteins, Immunogenicity, Vaccination, Agriculture, Animal Models, Hematology, Viral Load, Ebolavirus, 3. Good health, Antibodies, Anti-Idiotypic, Body Fluids, Titer, Blood, Anti-Idiotypic, Vertebrates, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Ebola, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.IMM]Life Sciences [q-bio]/Immunology, Antibody, Anatomy, [SDV.IMM.IMM] Life Sciences [q-bio]/Immunology/Immunotherapy, Viral load, Research Article, EBOV, Livestock, Immunology, Guinea Pigs, α1-3-Galactose, Biology, rabbit antithymocyte globulin, hemorrhagic-fever, virus-infection, nonhuman-primates, sialic-acid, molecular characterization, monoclonal-antibody, immunoglobulin-g, disease, immunotherapy, Research and Analysis Methods, Rodents, Virus, Antibodies, 03 medical and health sciences, Model Organisms, medicine, Animals, Antigens, Ebola Vaccines, Immunoassays, Pharmacology, Ebola virus, lcsh:R, Organisms, Biology and Life Sciences, Proteins, Galactose, Anti-EBOV, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Hemorrhagic Fever, Ebola, Guinea pig, Virology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, Amniotes, biology.protein, Immunologic Techniques, Hemorrhagic Fever, lcsh:Q, Neuraminic Acids
Abstract

International audience; Polyclonal xenogenic IgGs, although having been used in the prevention and cure of severe infectious diseases, are highly immunogenic, which may restrict their usage in new applications such as Ebola hemorrhagic fever. IgG glycans display powerful xenogeneic antigens in humans, for example α1-3 Galactose and the glycolyl form of neuraminic acid Neu5Gc, and IgGs deprived of these key sugar epitopes may represent an advantage for passive immunotherapy. In this paper, we explored whether low immunogenicity IgGs had a protective effect on a guinea pig model of Ebola virus (EBOV) infection. For this purpose, a double knock-out pig lacking α1-3 Galactose and Neu5Gc was immunized against virus-like particles displaying surface EBOV glycoprotein GP. Following purification from serum, hyper-immune polyclonal IgGs were obtained, exhibiting an anti-EBOV GP titer of 1:100,000 and a virus neutralizing titer of 1:100. Guinea pigs were injected intramuscularly with purified IgGs on day 0 and day 3 post-EBOV infection. Compared to control animals treated with IgGs from non-immunized double KO pigs, the anti-EBOV IgGs-treated animals exhibited a significantly prolonged survival and a decreased virus load in blood on day 3. The data obtained indicated that IgGs lacking α1-3 Galactose and Neu5Gc, two highly immunogenic epitopes in humans, have a protective effect upon EBOV infection.

Published
2016

9. Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus in vitro culture for different domestic species

Author

Dario Brunetti, Irene Tessaro, Cesare Galli, Irina Lagutina, P. Turini, Roberto Duchi, G. Crotti, Giovanna Lazzari, Silvia Colleoni, Lazzari G., Colleoni S., Lagutina I., Crotti G., Turini P., Tessaro I., Brunetti D., Duchi R., and Galli C

Subjects
animal structures, Swine, Embryonic Development, Fertilization in Vitro, Embryo Culture Techniques, CULTURE, Andrology, 03 medical and health sciences, 0302 clinical medicine, Food Animals, In vivo, Animals, Horses, Small Animals, Ovis, Fallopian Tubes, 030304 developmental biology, 0303 health sciences, Sheep, 030219 obstetrics & reproductive medicine, biology, Equine, EMBRYO, Embryogenesis, LIVESTOCK, Embryo, Embryo culture, biology.organism_classification, In vitro, Sexual reproduction, Immunology, Oviduct, Cattle, Female, Animal Science and Zoology
Abstract

The culture of early embryos in the surrogate xeno-oviduct was first developed in the early 1950s to allow transport of embryos at long distances. Later, it was applied to the study of culture requirements of the early embryo especially that of bovine origin. In this article, we review the data available on the culture of in vitro-matured and in vitro-fertilized embryos of Bos taurus, Sus scrofa, Equus caballus and Ovis aries in the surrogate sheep oviduct compared with data on in vitro culture in different media. Short-term and long-term cellular and molecular effects are described mainly for the bovine species where more extensive use of this technique has been made. A comparison with in vitro culture in various conditions and species indicate that embryos cultured in the sheep oviduct have close similarities to totally in vivo-derived embryos. The data provided demonstrate that the technique of in vivo culture in the surrogate sheep oviduct is versatile and allows a high rate of embryonic development in all species examined.

Published
2010
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10. Bortezomib, C1-inhibitor and plasma exchange do not prolong the survival of multi-transgenic GalT-KO pig kidney xenografts in baboons

Author

Andrea Perota, Simon C. Robson, David H. Sachs, Cesare Galli, Gilles Blancho, Mark B. Nottle, Emanuele Cozzi, Paolo Simioni, Béatrice Charreau, S. Le Bas-Bernardet, N. Klar, Giovanna Lazzari, Nahzli Dilek, Anthony J. F. D'apice, Evelyn J Salvaris, Irina Lagutina, Nicolas Poirier, Kazuhiko Yamada, Jeremy Hervouet, J. P. Soulillou, Julien Branchereau, Linda Scobie, Claire Crossan, Xavier Tillou, Karine Renaudin, Michael E. Breimer, Bernard Vanhove, M. Châtelais, Y Takeuchi, Peter J. Cowan, M. Diswall, Roberto Duchi, David Minault, Mohamed R. Daha, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), Transplant Immunology Unit [Padua, Italy], Padua General Hospital [Padua, Italy], Consorzio per la Ricerca sul Trapianto d'Organ = Consortium for Research in Organ Transplantation (CORIT), Effimune SAS [Nantes], Département de Pathologie [CHU Nantes], Centre hospitalier universitaire de Nantes (CHU Nantes), Department of Biological & Biomedical Sciences [Glasgow, UK], Glasgow Caledonian University (GCU), University College of London [London] (UCL), Department of Surgery [Gothenburg, Sweden] (Institute of Clinical Sciences), Sahlgrenska Academy at University of Gothenburg [Göteborg], Department of Nephrology [Leiden, The Netherlands], Leiden University Medical Center (LUMC), Department of Cardiologic, Thoracic and Vascular Sciences [Padua, Italy], Università degli Studi di Padova = University of Padua (Unipd), Department of Medicine [Boston, MA, USA], Beth Israel Deaconess Medical Center [Boston] (BIDMC), Harvard Medical School [Boston] (HMS)-Harvard Medical School [Boston] (HMS), School of Paediatrics and Reproductive Health [Adelaide, Australia], University of Adelaide-Robinson Research Institute, University of Adelaide, Immunology Research Centre [Melbourne, VIC, Australia], St Vincent's Hospital Melbourne [Australia], Transplantation Biology Research Center [Boston, MA, USA], Harvard Medical School [Boston] (HMS)-Massachusetts General Hospital [Boston], Avantea [Cremona, Italy], Department of Veterinary Medical Science [Ozzano Emilia, Italy], University of Bologna/Università di Bologna, This work was supported by the European Commission’s Sixth Framework Programme, under the priority thematic area Life Sciences, Genomics and Biotechnology for Health, contract N°. LSHB-CT- 2006- 037377, XENOME and by NIH Grant #5P01AI45897., Le Bihan, Sylvie, Consortium for Research in Organ Transplantation [Padua, Italy] (CORIT), University of Padua [Italy], University of Bologna, Le Bas-Bernardet, S., Tillou, X., Branchereau, J., Dilek, N., Poirier, N., Chatelais, M., Charreau, B., Minault, D., Hervouet, J., Renaudin, K., Crossan, C., Scobie, L., Takeuchi, Y., Diswall, M., Breimer, M.E., Klar, N., Daha, M.R., Simioni, P., Robson, S.C., Nottle, M.B., Salvaris, E.J., Cowan, P.J., D'Apice, A.J.F., Sachs, D.H., Yamada, K., Lagutina, I., Duchi, R., Perota, A., Lazzari, G., Galli, C., Cozzi, E., Soulillou, J.-P., Vanhove, B., and Blancho, G.

Subjects
basic (laboratory) research/science, translational research/science, medicine.medical_treatment, Sus scrofa, Cytomegalovirus, kidney transplantation/nephrology, immunosuppression/immune modulation, 030230 surgery, Pharmacology, Kidney, Virus Replication, xenoantibody, Animals, Genetically Modified, Bortezomib, Gene Knockout Techniques, 0302 clinical medicine, Models, xenotransplantation, Innate, Immunology and Allergy, genetics, Pharmacology (medical), 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, biology, Plasma Exchange, Medicine (all), Graft Survival, Immunosuppression, Galactosyltransferases, Boronic Acids, Papio anubis, 3. Good health, Pyrazines, Models, Animal, Heterografts, Complement C1 Inhibitor Protein, Immunosuppressive Agents, medicine.drug, plasmapheresis/plasma exchange, Xenotransplantation, Genetically Modified, Animal models: nonhuman primate, immunosuppressive regimens, Animals, Autoimmune Diseases, Immunity, Innate, Kidney Transplantation, Transplantation, Article, 03 medical and health sciences, Classical complement pathway, biology.animal, medicine, 030304 developmental biology, business.industry, Animal, Immunity, Complement system, immunosuppressive regimen, Immunology, Alternative complement pathway, genetic, business, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, Baboon
Abstract

Galactosyl-transferase KO (GalT-KO) pigs represent a potential solution to xenograft rejection, particularly in the context of additional genetic modifications. We have performed life supporting kidney xenotransplantation into baboons utilizing GalT-KO pigs transgenic for human CD55/CD59/CD39/HT. Baboons received tacrolimus, mycophenolate mofetil, corticosteroids and recombinant human C1 inhibitor combined with cyclophosphamide or bortezomib with or without 2-3 plasma exchanges. One baboon received a control GalT-KO xenograft with the latter immunosuppression. All immunosuppressed baboons rejected the xenografts between days 9 and 15 with signs of acute humoral rejection, in contrast to untreated controls (n = 2) that lost their grafts on days 3 and 4. Immunofluorescence analyses showed deposition of IgM, C3, C5b-9 in rejected grafts, without C4d staining, indicating classical complement pathway blockade but alternate pathway activation. Moreover, rejected organs exhibited predominantly monocyte/macrophage infiltration with minimal lymphocyte representation. None of the recipients showed any signs of porcine endogenous retrovirus transmission but some showed evidence of porcine cytomegalovirus (PCMV) replication within the xenografts. Our work indicates that the addition of bortezomib and plasma exchange to the immunosuppressive regimen did not significantly prolong the survival of multi-transgenic GalT-KO renal xenografts. Non-Gal antibodies, the alternative complement pathway, innate mechanisms with monocyte activation and PCMV replication may have contributed to rejection.

Published
2015

11. hCTLA4-Ig transgene expression in keratocytes modulates rejection of corneal xenografts in a pig to non-human primate anterior lamellar keratoplasty model

Author

Jean-Marie Bach, Jean-Paul Soulillou, Flora Coulon, Gilles Blancho, David Minault, Stéphanie Le Bas-Bernardet, Eric Venturi, Pascal Mermillod, Peter J. Cowan, Roberto Duchi, Cesare Galli, Mark B. Nottle, Yan Cherel, Philippe Brachet, Simon C. Robson, Jeremy Hervouet, Yvette Ducournau, Anne Moreau, Veronique Daguin, Irina Lagutina, David Riochet, Bernard Vanhove, Annaïck Pallier, Bertrand Vabres, Sophie Brouard, Centre hospitalier universitaire de Nantes (CHU Nantes), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), 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), Institut de Transplantation Urologie Néphrologie, Université de Nantes (UN), Développement et Pathologie du Tissu Musculaire (DPTM), Ecole Nationale Vétérinaire de Nantes-Institut National de la Recherche Agronomique (INRA), Department of Medicine, Transplant Center, Harvard Medical School [Boston] (HMS), Department of Obstetrics and Gynecology, University of Adelaide, Immunology Research Center, St Vincent's Hospital, Department of Medicine, University of Melbourne-Royal Melbourne Hospital, UE 1297 Unité Expérimentale de Physiologie Animale de l'Orfrasière, Institut National de la Recherche Agronomique (INRA)-Physiologie Animale et Systèmes d'Elevage (PHASE), Institut National de la Recherche Agronomique (INRA)-Unité Expérimentale de Physiologie Animale de l'Orfrasière (UE PAO), Physiologie de la reproduction et des comportements [Nouzilly] (PRC), Centre National de la Recherche Scientifique (CNRS)-Université de Tours-Institut Français du Cheval et de l'Equitation [Saumur]-Institut National de la Recherche Agronomique (INRA), Unité Mixte de Recherche en Santé Végétale (INRA/ENITA) (UMR SAVE), Institut National de la Recherche Agronomique (INRA)-École Nationale d'Ingénieurs des Travaux Agricoles - Bordeaux (ENITAB)-Institut des Sciences de la Vigne et du Vin (ISVV), Laboratorio di Tecnologie della Riproduzione, Dipartimento di Scienze Mediche Veterinarie, Alma Mater Studiorum University of Bologna (UNIBO), Immuno-Endocrinologie Cellulaire et Moléculaire (IECM), Institut National de la Recherche Agronomique (INRA)-Université de Nantes (UN)-Ecole Nationale Vétérinaire de Nantes, Pays de la Loire Regional Council, European Commission LSHB-CT-2006-037377, French government ANR-10-IBHU-005, Nantes Metropole, Pays de la Loire Region, Vabres B, Le Bas-Bernardet S, Riochet D, Chérel Y, Minault D, Hervouet J, Ducournau Y, Moreau A, Daguin V, Coulon F, Pallier A, Brouard S, Robson SC, Nottle MB, Cowan PJ, Venturi E, Mermillod P, Brachet P, Galli C, Lagutina I, Duchi R, Bach JM, Blancho G, Soulillou JP, Vanhove B, Physiopathologie Animale et bioThérapie du muscle et du système nerveux (PAnTher), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire, Agroalimentaire et de l'alimentation Nantes-Atlantique (ONIRIS), Unité Expérimentale de Physiologie Animale de l‘Orfrasiére (UE PAO), Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche en Santé Végétale (INRA/ENITA) (UMRSV), Institut National de la Recherche Agronomique (INRA)-Ecole Nationale Vétérinaire de Nantes, and Institut National de la Recherche Agronomique (INRA)-Institut Français du Cheval et de l'Equitation [Saumur]-Université de Tours-Centre National de la Recherche Scientifique (CNRS)

Subjects
Graft Rejection, Male, [SDV.OT]Life Sciences [q-bio]/Other [q-bio.OT], Immunoconjugates, Transgene, Xenotransplantation, medicine.medical_treatment, CD3, Sus scrofa, Transplantation, Heterologous, Immunology, Heterologous, Corneal Keratocytes, Abatacept, Animals, Genetically Modified, Corneal Transplantation, Andrology, Stroma, medicine, Animals, Transgenes, Corneal transplantation, hctla4-ig, corneal blindness, alpha1, Transplantation, biology, pig to non-human primate model, Graft Survival, Immunosuppression, transgenic pig line, 3. Good health, Macaca fascicularis, corneal blindne, Models, Animal, biology.protein, corneal xenograft, lamellar keratoplasty, 3-galactosyl transferase knockout, Biomarkers
Abstract

Background Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. Methods In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. Results Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. Conclusions Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.

Published
2014

12. Fertilization and development of oocytes after ICSI with semen of stallions with different in vivo fertility

Author

Irina Lagutina, Cesare Galli, C. Baca Castex, Giovanna Lazzari, Gaetano Mari, Roberto Duchi, Silvia Colleoni, S. Colleoni, G. Lazzari, R. Duchi, C. Baca Castex, G. Mari, I. Lagutina, and C. Galli

Subjects
Andrology, Human fertilization, Equine, In vivo, OOCYTES, media_common.quotation_subject, Semen, Fertility, STALLION, Biology, INTRACYTOPLASMIC SPERM INJECTION, media_common
Published
2012

13. Comparative aspects of somatic cell nuclear transfer with conventional and zona-free method in cattle, horse, pig and sheep

Author

Irina Lagutina, Irene Tessaro, Roberto Duchi, Dario Brunetti, Cesare Galli, G. Crotti, Giovanna Lazzari, Silvia Colleoni, P. Turini, 55. Lagutina I, Lazzari G, Duchi R, Turini P, Tessaro I, Brunetti D, Colleoni S, Crotti G, and Galli C.

Subjects
Nuclear Transfer Techniques, endocrine system, Swine, Cloning, Organism, Enucleation, Biology, Cryopreservation, CLONAZIONE, Cell Fusion, Andrology, Species Specificity, Food Animals, medicine, NUCLEAR TRANSFER, Animals, Horses, Blastocyst, Small Animals, Cells, Cultured, Zona Pellucida, reproductive and urinary physiology, Cloning, Genetics, Sheep, Cell fusion, Equine, urogenital system, Horse, Embryo, medicine.anatomical_structure, embryonic structures, Oocytes, Somatic cell nuclear transfer, Cattle, Animal Science and Zoology, ANIMALI DOMESTICI
Abstract

Nuclear transfer (NT) is a complex procedure that requires considerable technical skills. Over the years attempts have been made to simplify the micromanipulations involved and to make the procedure more user-friendly. A significant step forwards has been the development of the zona-free NT methods. We have used zona-free NT with mechanical aspiration of the metaphase plate as a mean of enucleation, in a comparative approach with the conventional nuclear transfer zona-enclosed method in cattle, horse, sheep and pig. The absence of the zona considerably facilitates the enucleation step and significantly increases cell fusion success. On the other hand, the culture of zona-free NT embryos requires the embryos to be cultured individually or anyway separated from each other to avoid aggregation and also requires to prolong the in vitro culture up to the blastocyst stage before transfer. Blastocyst rate is equal or higher with zona-free method as compared to zona-enclosed method while survival after cryopreservation and development to term is comparable. In conclusion, our findings, together with published data, demonstrate that the zona-free system described in this paper can significantly increase the output of NT blastocysts over the conventional zona-enclosed system.

Published
2007

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