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Université Paris Descartes

16 ARTICLES PUBLISHED IN JoVE

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 Luckhart¹

¹Department 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.

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

¹Institut 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

Introducing Shear Stress in the Study of Bacterial Adhesion

Magali Soyer¹, Guillaume Duménil¹

¹Blood vessels as a target for infection, Paris center for cardiovascular research, INSERM U970

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

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

¹INSERM 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.

JoVE Core

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. Vasos¹

¹LCBPT - 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.

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 Alexander¹

¹Molecular 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.

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

¹Assistance 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.

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 Benkerrou¹

¹Reference 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.

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 Donnadieu¹

¹Institut 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.

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

¹PARCC-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.

Neuroscience

Long-term Sensory Conflict in Freely Behaving Mice

Filipa França de Barros^1,2, Julie Carcaud², Mathieu Beraneck^1,2

¹Integrative 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

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

¹Department 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

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Louis Richevaux^1,2, Louise Schenberg^1,2, Mathieu Beraneck^1,2, Desdemona Fricker^1,2

¹CNRS (Integrative Neuroscience and Cognition Center, UMR 8002), ²Université Paris Descartes, Sorbonne Paris Cité

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

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

¹Institute 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.

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

¹Division 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.

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

¹Division 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.