Helmholtz Centre for Infection Research 11 articles published in JoVE Immunology and Infection Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test Julia Häring*1, Tanja Michel*1, Matthias Becker1, Daniel Junker1, Tatia Tchitchagua2, Olaf Leschnik2, Berit Lange3,4, Stefanie Castell3,4, Gérard Krause3,4, Monika Strengert3, Alex Dulovic1, Nicole Schneiderhan-Marra1 1NMI Natural and Medical Sciences Institute at the University of Tübingen, 2Department of Neurology, Sächsisches Krankenhaus Rodewisch, 3Department of Epidemiology, Helmholtz Centre for Infection Research, 4German Centre for Infection Research (DZIF) A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America. Bioengineering Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates Mahnaz Sabeti Azad*1, Angelo Cardoso Batista*1, Jean-Loup Faulon1, Chase L. Beisel2,3, Jerome Bonnet4, Manish Kushwaha1 1INRAe, AgroParisTech, Micalis Institute, Université Paris-Saclay, 2Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz-Centre for Infection Research (HZI), 3Medical Faculty, University of Würzburg, 4Centre de Biochimie Structurale, INSERM U1054, CNRS UMR 5048, University of Montpellier Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (ΔrecBCD and ΔrecB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates. Cancer Research Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo Julia Volckmar1,2, Laura Knop1,2,3, Tatjana Hirsch1, Sarah Frentzel1,2, Christian Erck4, Marco van Ham4, Sabine Stegemann-Koniszewski*1,2,5, Dunja Bruder*1,2 1Immune Regulation Group, Helmholtz Centre for Infection Research, 2Infection Immunology Group, Institute of Medical Microbiology, Infection Prevention and Control, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke University Magdeburg, 3Institute for Molecular and Clinical Immunology, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke University Magdeburg, 4Cellular Proteome Research, Helmholtz Centre for Infection Research, 5Experimental Pneumology, University Hospital of Pneumology, Health Campus Immunology, Infectiology and Inflammation, Otto-von-Guericke University Magdeburg We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation. Immunology and Infection P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives Carlos Victor Montefusco-Pereira*1,2, Justus C. Horstmann*1,2, Thomas Ebensen3, Christoph Beisswenger4, Robert Bals4, Carlos A. Guzmán3, Nicole Schneider-Daum1, Cristiane de Souza Carvalho-Wodarz1, Claus-Michael Lehr1,2 1Department of Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 2Department of Pharmacy, Saarland University, 3Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 4Department of Internal Medicine V - Pulmonology, Allergology, Respiratory Intensive Care Medicine, Saarland University Hospital We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers. Developmental Biology Development of a Larval Zebrafish Infection Model for Clostridioides difficile Junkai Li1, Can M. Ünal2, Kazuhiko Namikawa1, Michael Steinert3,4,5, Reinhard W. Köster1 1Division of Cellular and Molecular Neurobiology, Zoological Institute, Technische Universität Braunschweig, 2Department of Molecular Biotechnology, Turkish-German University, 3Institut für Mikrobiologie, Technische Universität Braunschweig, 4Braunschweig Integrated Centre of Systems Biology, 5Helmholtz Centre for Infection Research Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage. Environment Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector Michaela Hoenigsberger1, Alexey G. Kopchinskiy2, Alexandra Parich1, Karsten Hiller3,4, Alice Laciny5, Herbert Zettel5, Linda B.L. Lim6, Kamariah A. Salim7, Irina S. Druzhinina2, Rainer Schuhmacher1 1Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna (BOKU), Austria, 2Institute of Chemical, Environmental and Biological Engineering, TU Vienna, Austria, 3Department of Computational Biology of Infection Research, Helmholtz Centre for Infection Research, Germany, 4Institute for Biochemistry, Biotechnology and Bioinformatics, University of Technology (TU) Braunschweig, Germany, 52nd Zoological Department, Natural History Museum Vienna, Austria, 6Chemical Sciences, Universiti Brunei Darussalam, Brunei, 7Environmental and Life Sciences, Universiti Brunei Darussalam, Brunei Minor workers of the ant species Colobopsis explodens are dissected to isolate the wax-like content stored in their hypertrophied mandibular gland reservoirs for subsequent solvent-extraction and analysis by gas chromatography-mass spectrometry. The annotation and identification of volatile constituents using the open-source software MetaboliteDetector is also described. Medicine Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development Darío Lirussi1, Thomas Ebensen1, Kai Schulze1, Elena Reinhard1, Stephanie Trittel1, Peggy Riese1, Blair Prochnow1, Carlos A. Guzmán1 1Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines. Biology Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators Georgi Dimchev1,2, Klemens Rottner1,2 1Division of Molecular Cell Biology, Zoological Institute, Technische Universität Braunschweig, 2Department of Cell Biology, Helmholtz Centre for Infection Research We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton. Neuroscience Optimized Management of Endovascular Treatment for Acute Ischemic Stroke Katharina Schregel1,2, Daniel Behme1, Ioannis Tsogkas1, Michael Knauth1, Ilko Maier3, André Karch4, Rafael Mikolajczyk4,5, Mathias Bähr3, Jörn Schäper6, José Hinz6, Jan Liman3, Marios-Nikos Psychogios1 1Institute of Neuroradiology, University Medical Center Goettingen, 2 The outcome of patients with acute ischemic stroke depends on swift restoration of cerebral blood flow. This protocol aims at optimizing the management of such patients by minimizing peri-procedural timings and rendering the time from hospital admission to reperfusion as short as possible. Developmental Biology Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange Tim Pieters1,2,3,4, Lieven Haenebalcke1,2, Kenneth Bruneel1,2,4, Niels Vandamme1,2,4, Tino Hochepied1,2, Jolanda van Hengel5, Dagmar Wirth6, Geert Berx1,2,4, Jody J. Haigh7, Frans van Roy1,2,4, Steven Goossens1,2,3,4 1Department of Biomedical Molecular Biology, Ghent University, 2Inflammation Research Center, VIB, 3Center for Medical Genetics, Ghent University Hospital, 4Cancer Research Institute Ghent (CRIG), 5Department of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, 6Helmholtz Center for Infection Research, 7Mammalian Functional Genetics Laboratory, Division of Blood Cancers, Australian Centre for Blood Diseases, Department of Clinical Haematology, Monash University and Alfred Health Alfred Centre Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein. Immunology and Infection Study of Phagolysosome Biogenesis in Live Macrophages Marc Bronietzki1, Bahram Kasmapour1, Maximiliano Gabriel Gutierrez1,2 1Research Group Phagosome Biology, Helmholtz Centre for Infection Research, 2Division of Mycobacterial Research, National Institute for Medical Research