Universite-Paris-Descartes

20 articles published in JoVE

• Bioengineering

  
  A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies Victor Pozzo^1,2,4, Golda Romano^1,2,4, Marion Goutard^1,2,4, Elise Lupon^1,2,4, Pierre Tawa^1,2,4, Aylin Acun^3,4,5, Alec R. Andrews², Corentin B. Taveau^1,2,4,6, Basak E. Uygun^1,2,3,4, Mark A. Randolph^1,2,4, Curtis L. Cetrulo^1,2,4, Alexandre G. Lellouch^{1,2,4,6 1}Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, ²Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, ³Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, ⁴Shriners Hospital for Children, ⁵Department of Biomedical Engineering, Widener University, ⁶Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université Paris Descartes The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

• Immunology and Infection

  
  Partial Heterotopic Hindlimb Transplantation Model in Rats Marion Goutard^1,2,3, Mark A. Randolph^1,2,3, Corentin B. Taveau^1,2,3,4, Elise Lupon^1,2,3, Laurent Lantieri⁴, Korkut Uygun^1,2,3, Curtis L. Cetrulo Jr.*^1,2,3, Alexandre G. Lellouch*^{1,2,3,4 1}Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, ²Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, ³Shriners Hospital for Children, ⁴Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université de Paris This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

• Immunology and Infection

  
  Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue Nikaïa Smith^1,2,3,4, Nassima Bekaddour^2,3,4, Nicolas Leboulanger^4,5, Yolande Richard*⁶, Jean-Philippe Herbeuval*^{2,3,4 1}Institute of Molecular Virology, Ulm University Medical Center, ²CNRS UMR-8601, Centre Interdisciplinaire Chimie Biologie, ³Team Chemistry & Biology, Modeling & Immunology for Therapy, Université Paris Descartes, ⁴Université Paris Descartes, Sorbonne Paris Cité, ⁵Pediatric Otolaryngology Department, Hôpital Necker-Enfants Malades, Assistance Publique Hôpitaux de Paris, ⁶Université de Paris, Institut Cochin In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

• Genetics

  
  Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis Sophie Outh-Gauer¹, Jérémy Augustin¹, Marion Mandavit², Ophélie Grard², Thomas Denize¹, Marine Nervo¹, Charles Lépine¹, Marc Rassy², Eric Tartour^2,3, Cécile Badoual^{1,2 1}Department of Pathology, Georges Pompidou European Hospital, Assistance publique - Hôpitaux de Paris (APHP), Paris Descartes University, ²Paris Cardiovascular Research Center (PARCC), Institut national de la santé et de la recherche médicale (Inserm) U970, ³Laboratory of Immunology, Georges Pompidou European Hospital, APHP, Paris Descartes University Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

• Neuroscience

  
  Long-term Sensory Conflict in Freely Behaving Mice Filipa França de Barros^1,2, Julie Carcaud², Mathieu Beraneck^{1,2 1}Integrative Neuroscience and Cognition Center, UMR8002, CNRS, ²Sorbonne Paris Cité, Université Paris Descartes The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

• Genetics

  
  Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions Charles Decraene^1,2, Amanda Bortolini Silveira¹, Marc Michel¹, François-Clément Bidard^1,3,4, Jean-Yves Pierga^1,3,5, Marc-Henri Stern^1,6, Charlotte Proudhon^{1 1}Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department, Institut Curie, PSL Research University, ²CNRS UMR144, Institut Curie, PSL Research University, ³Department of Medical Oncology, Institut Curie, PSL Research University, ⁴University Versailles Saint-Quentin-en-Yvelines, ⁵University Paris Descartes, ⁶Inserm U830, Institut Curie, PSL Research University Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

• Cancer Research

  
  Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1⁺ Tim-3⁺ CD8⁺ T Cells Clémence Granier¹, Emeline Vinatier^2,3,4, Elia Colin^2,3, Marion Mandavit^2,3, Charles Dariane^2,3,5, Virginie Verkarre⁶, Lucie Biard⁷, Rami El Zein², Corinne Lesaffre², Isabelle Galy-Fauroux², Hélène Roussel^2,3,6, Eléonore De Guillebon⁸, Charlotte Blanc^2,3, Antonin Saldmann⁴, Cécile Badoual^2,6, Alain Gey*⁴, Éric Tartour*^{2,4 1}PARCC-INSERM U970, Université Paris Descartes, ²INSERM U970, Université Paris Descartes, ³Equipe Labellisée Ligue Contre le Cancer, ⁴Department of Immunology, Hopital Européen Georges Pompidou, ⁵Department of Medical Urology, Hopital Européen Georges Pompidou, ⁶Department of Pathology, Hopital Européen Georges Pompidou, ⁷Université Paris Diderot Paris 7, ⁸Department of medical oncology, Hopital Européen Georges Pompidou Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8⁺ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

• Cancer Research

  
  Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy Elisa Peranzoni*¹, Houcine Bougherara*¹, Sarah Barrin*¹, Audrey Mansuet-Lupo^2,3, Marco Alifano⁴, Diane Damotte^2,3, Emmanuel Donnadieu^{1 1}Institut Cochin, Inserm U1016-CNRS UMR8104, Université Paris Descartes, ²Department of Pathology, Paris Centre University Hospitals, Université Paris Descartes, ³INSERM U1138, Cancer and Immune Escape, Cordeliers Research Center, University Pierre and Marie Curie, ⁴Department of Thoracic Surgery, Paris Centre University Hospitals, University Paris Descartes This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

• Immunology and Infection

  
  Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells Pascale Vonaesch¹, Philippe J. Sansonetti^1,2, Pamela Schnupf^{3 1}Unité de Pathogénie Microbienne Moléculaire, INSERM U786, Institut Pasteur, ²Microbiologie et Maladies Infectieuses, Collège de France, ³Laboratory of Intestinal Immunity, Institut Imagine-INSERM UMR 1163, Université Paris Descartes-Sorbonne Paris Cité We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

• Genetics

  
  Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions Oriana Romano^1,2, Ingrid Cifola³, Valentina Poletti⁴, Marco Severgnini³, Clelia Peano³, Gianluca De Bellis³, Fulvio Mavilio^1,4, Annarita Miccio^{1,2,4,5 1}Center for Genome Research, Department of Life Sciences, University of Modena and Reggio Emilia, ²Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, ³Institute for Biomedical Technologies, CNR, ⁴Généthon, ⁵Sorbonne Paris Cité - Université Paris Descartes Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

• Medicine

  
  Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload Bérengère Koehl¹, Florence Missud¹, Laurent Holvoet¹, Ghislaine Ithier¹, Oliver Sakalian-Black², Zinedine Haouari¹, Emmanuelle Lesprit³, André Baruchel⁴, Malika Benkerrou^{1 1}Reference Centre of Sickle Cell Disease, Hematology Unit, Robert Debré Hospital, AP-HP, ²School of Pharmacy, Université Paris Descartes, ³Etablissement Français du Sang, Robert Debré Hospital, AP-HP, ⁴Hematology Unit,, Robert Debré Hospital, AP-HP, Univsité Paris Diderot We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

• Medicine

  
  ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection Guillaume Perrod^1,2,3, Laetitia Pidial³, Sophie Camilleri^1,4, Alexandre Bellucci^1,3,5, Amaury Casanova³, Thomas Viel³, Bertrand Tavitian^1,3,5, Chirstophe Cellier^1,2, Olivier Clément^1,3,5, Gabriel Rahmi^{1,2,3 1}Assistance Publique-Hôpitaux de Paris, Université Paris Descartes Sorbonne Paris Cité, ²Department of Gastroenterology, Hôpital Européen Georges Pompidou, ³UMR-S970, Université Paris Descartes Sorbonne Paris Cité, ⁴Department of Pathology, Hôpital Européen Georges Pompidou, ⁵Department of Radiology, Hôpital Européen Georges Pompidou This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

• Biology

  
  The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress Luke A. White¹, Emily V. Stevenson¹, J. Winny Yun¹, Randa Eshaq¹, Norman R. Harris¹, David K. Mills², Alireza Minagar³, Pierre-Olivier Couraud⁴, J. Steven Alexander^{1 1}Molecular and Cellular Physiology, Louisiana State University Health Sciences Center in Shreveport, ²Biological Sciences, Louisiana Tech University, ³Neurology, Louisiana State University Health Sciences Center in Shreveport, ⁴Institut Cochin, Inserm U1016, Cnrs Umr8104, Université Paris Descartes Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

• Neuroscience

  
  The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation Félix Leroy¹, Boris Lamotte d'Incamps^{1 1}Centre National de la Recherche Scientifique (UMR 8119), Centre de Neurophysique, Physiologie et Pathologie, Université Paris Descartes We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

• Immunology and Infection

  
  "Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM) Florence Marie-Anaïs¹, Julie Mazzolini¹, Pierre Bourdoncle¹, Florence Niedergang^{1 1}Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

• Chemistry

  
  Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR Riccardo Balzan¹, Laetitia Fernandes¹, Arnaud Comment², Laetitia Pidial³, Bertrand Tavitian³, Paul R. Vasos^{1 1}LCBPT - UMR8601, Institut de Chimie, Université Paris Descartes, ²Institute of Physics of Biological Systems, EPFL, ³PARCC - INSERM U970, Université Paris Descartes The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

• Medicine

  
  Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue Elena Dossi¹, Thomas Blauwblomme^2,3, Rima Nabbout^2,4, Gilles Huberfeld^2,5, Nathalie Rouach^{1 1}Neuroglial Interactions in Cerebral Physiopathology, Center for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Collège de France, ²Infantile Epilepsies & Brain Plasticity, INSERM U1129, PRES, Paris Descartes University, Sorbonne Paris Cité, CEA, ³Neurosurgery Department, Necker Hospital, AP-HP, Paris Descartes University, ⁴Rare Epilepsies Reference Center, Necker Hospital, AP-HP, Paris Descartes University, ⁵Neurophysiology Department, La Pitié-Salpêtrière Hospital, AP-HP, Sorbonne and Pierre and Marie Curie University We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

• Immunology and Infection

  
  Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature Keira Melican^1,2, Flore Aubey^1,2, Guillaume Duménil^{1,2 1}INSERM U970, Paris Cardiovascular Research Centre, ²Faculté de Médecine Paris Descartes, Université Paris Descartes Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

• Immunology and Infection

  
  Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes Hélène Salmon^1,2, Ana Rivas-Caicedo^1,2, François Asperti-Boursin^1,2, Camille Lebugle^1,2, Pierre Bourdoncle^1,2, Emmanuel Donnadieu^{1,2 1}Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), ²Inserm, U1016, Paris, France This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

• Immunology and Infection

  
  Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides Nazzy Pakpour¹, Kong Wai Cheung¹, Lattha Souvannaseng¹, Jean-Paul Concordet², Shirley Luckhart^{1 1}Department of Medical Microbiology and Immunology, University of California, Davis, ²Département Génétique et Développement, Institut Cochin, Université Paris Descartes Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.