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Université Paris Descartes

16 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides
Nazzy Pakpour 1, Kong Wai Cheung 1, Lattha Souvannaseng 1, Jean-Paul Concordet 2, Shirley Luckhart 1
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.

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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.

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Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion
Magali Soyer 1, Guillaume Duménil 1
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.

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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.

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JoVE Core

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR
Riccardo Balzan 1, Laetitia Fernandes 1, Arnaud Comment 2, Laetitia Pidial 3, Bertrand Tavitian 3, Paul R. Vasos 1
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.

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Biology

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress
Luke A. White 1, Emily V. Stevenson 1, J. Winny Yun 1, Randa Eshaq 1, Norman R. Harris 1, David K. Mills 2, Alireza Minagar 3, Pierre-Olivier Couraud 4, J. Steven Alexander 1
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.

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Medicine

ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection
Guillaume Perrod 1,2,3, Laetitia Pidial 3, Sophie Camilleri 1,4, Alexandre Bellucci 1,3,5, Amaury Casanova 3, Thomas Viel 3, 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.

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Medicine

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload
Bérengère Koehl 1, Florence Missud 1, Laurent Holvoet 1, Ghislaine Ithier 1, Oliver Sakalian-Black 2, Zinedine Haouari 1, Emmanuelle Lesprit 3, André Baruchel 4, Malika Benkerrou 1
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.

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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 Alifano 4, Diane Damotte 2,3, Emmanuel Donnadieu 1
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.

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Cancer Research

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells
Clémence Granier 1, Emeline Vinatier 2,3,4, Elia Colin 2,3, Marion Mandavit 2,3, Charles Dariane 2,3,5, Virginie Verkarre 6, Lucie Biard 7, Rami El Zein 2, Corinne Lesaffre 2, Isabelle Galy-Fauroux 2, Hélène Roussel 2,3,6, Eléonore De Guillebon 8, Charlotte Blanc 2,3, Antonin Saldmann 4, 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.

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Neuroscience

Long-term Sensory Conflict in Freely Behaving Mice
Filipa França de Barros 1,2, Julie Carcaud 2, 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.

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Genetics

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis
Sophie Outh-Gauer 1, Jérémy Augustin 1, Marion Mandavit 2, Ophélie Grard 2, Thomas Denize 1, Marine Nervo 1, Charles Lépine 1, Marc Rassy 2, 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.

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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.

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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.

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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 Lantieri 4, 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.

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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. Andrews 2, 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.

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