BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE
BERNHARD-NOCHT-INSTITUT F??R TROPENMEDIZIN
Scienti??c Report 2000 / 2001 T??tigkeitsbericht 2000 / 2001
An Institute of the Leibniz Association Ein Institut der Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz
Table of Contents Table of Contents/Inhaltsverzeichnis
Beitr??ge in deutscher Sprache sind in Blau gesetzt
Page/Seite General Information / Allgemeines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Management / Leitung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Board of Directors / Kuratorium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Scienti??c Advisory Board / Wissenschaftlicher Beirat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Einleitung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Parasitology Section / Sektion Parasitologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Chairman’s Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Zusammenfassung des Sprechers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Staff Parasitology Section / Arbeitsgruppen und Mitarbeiter der Sektion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Selected Scienti??c Projects / Ausgew??hlte wissenschaftliche Projekte: Characterization of Cysteine Proteinases in Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Can German Mosquitoes Transmit Plasmodium falciparum Malaria? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Glutathione-dependent Detoxi??cation Processes of Onchocerca volvulus and Caenorhabditis elegans . . . . . . . . . . . . . . . . 28 The Polyamine Biosynthesis in Plasmodium falciparum is Well Balanced by a Unique Bifunctional Ornithine Decarboxylase, S-adenosylmethionine Decarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . 30 The Redox Systems of the Human Malaria Parasite Plasmodium falciparum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Epidemiology and Detection of Lymphatic Filariasis on Alor Island, Indonesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Microsporidia: Investigation of the Chitinous Spore and Use of a Fluorescence-based Stain . . . . . . . . . . . . . . . . . . . . . . . . 36 Control of the Leishmania Life Cycle via HSP90 Homeostasis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Signal Transduction in Leishmania: Protein Kinases as Potential Drug Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 Medical Microbiology Section / Sektion Medizinische Mikrobiologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Chairman’s Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Zusammenfassung des Sprechers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Staff Medical Microbiology Section / Arbeitsgruppen und Mitarbeiter der Sektion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Selected Scienti??c Projects / Ausgew??hlte wissenschaftliche Projekte: Heat Shock Protein 60 (Hsp60) as a ??Dangerous“ Link in Innate and Adaptive Immune Responses . . . . . . . . . . . . . . . . . . 50 Murine Susceptibility to Chagas‘ Disease Maps to Chromosomes 5 and 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 CTLA-4 Expression During Blood-stage Malaria Could Prevent Immune Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Wolbachia Endobacteria in Filarial Nematodes as Targets for a Novel Chemotherapeutic Approach – From the Lab to the Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 Inhibition of Flaviviridae NTPase/helicase by Ring-Expanded Nucleosides (REN’s). New Class of Antiviral Agents . . . . . . 58 Imported Cases of Lassa fever: Molecular Epidemiology and Clinical Virology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 Serological Differentiation of Dengue Virus 1 - 4 and West Nile Virus Infections using Recombinant Antigens . . . . . . . . . . 62 Role of N-glycosylation of the HIV-1 Envelope gp120 for Interaction with the Viral Coreceptors CXCR4 und CCR5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Diagnostic PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Tropical Medicine Section / Sektion Tropenmedizinische Forschung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 Chairman’s Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Zusammenfassung des Sprechers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Staff Tropical Medicine Section / Arbeitsgruppen und Mitarbeiter der Sektion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Selected Scienti??c Projects / Ausgew??hlte wissenscha??iche Projekte: Human Genetic Variants In??uencing Resistance to Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Genetic Variants In??uencing Allergy-type Reactions and Immunosuppression in Human Onchocerciasis . . . . . . . . . . . . . . 76
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Table of Contents
Page/Seite Skin Changes Associated with GJB2-R143W Mutations in Ghana: Evidence for Balancing Selection of Hearing Impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Eosinophil Regulator Interleukin 5 (IL-5), Peripheral Eosinophil Granulocytes and Serum Eosinophil Cationic Proteins (ECP, EPX) in Case of Hypereosinophilia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Wolbachia Surface Protein (WSP) of Filarial Endobacteria Generates In??ammatory Monocyte and Interferon-(IFN)- γ Responses in Onchocerca volvulus-infected Persons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Expression of Prostaglandin E2 (PGE2) by Onchocerca volvulus and Human Immune Cells Exposed to O. volvulus Antigen and Endobacterial Wolbachia Surface Protein (WSP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 SIV/HIV Vaccines: Detecting Ef??cacy and Explaining Inef??cacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 Report on KCCR Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Bericht über die Aktivit??ten des KCCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Clinical Department / Klinische Abteilung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Department Head’s Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Zusammenfassung des Abteilungsleiters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Staff Clinicical Department / Mitarbeiter der Klinik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Selected Scienti??c Projects / Ausgew??hlte wissenschaftliche Projekte: The Role of Procalcitonin (PCT) in Human Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 Monocyte Activation in Malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Lymphoid Aggregates in Bone Marrow of Infected Patients Contain HIV Producing Cells . . . . . . . . . . . . . . . . . . . . . . . . . 100 Reisemedizinisches Zentrum (RMZ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Administration / Verwaltung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Bericht des Verwaltungsleiters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Administration Staff / Mitarbeiter der Verwaltung. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Public Relations Report / Bericht ??ffentlichkeitsarbeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Education and Teaching / Ausbildung und Lehre . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 Theses / Doktorarbeiten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Masters Theses / Diplomarbeiten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 Course on Tropical Medicine 2001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Kursus für Tropenmedizin 2001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 Lectures and Seminars University of Hamburg / Lehrveranstaltungen Universit??t Hamburg . . . . . . . . . . . . . . . . . . . . . . . . 120 Training for Physisians / Fortbildungsveranstaltungen für Mediziner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Seminar Programme / Seminare. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Meetings / Symposien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 International Scienti??c Meetings / Internationale Symposien . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Meetings of Cooperative Scienti??c Projects / Arbeitstreffen im Rahmen von Verbundprojekten. . . . . . . . . . . . . . . . . . . . . 130 Staff Activities / Aktivit??ten der Mitarbeiter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134 Publications / Publikationen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Publications 2000 / Publikationen 2000. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 Publications 2001 / Publikationen 2001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Appendix/Anhang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 Chronicle / Chronik . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 Centenary of the BNI in 2000 / 100-Jahrfeier des BNI 2000 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Index of Scienti??c Staff / Verzeichnis der wissenschaftlichen Mitarbeiter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Organization Chart / Organigramm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inside reverse cover/Innenseite Cover
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Blick von Norden auf Bernhard-Nocht-Institut und Hamburger Hafen Bernhard Nocht Institute and Hamburg harbour from the north
Foto: Bilderberg / Elleringmann
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Das Bernhard-Nocht-Institut für Tropenmedizin (BNI) ist ein Mitglied der Leibniz-Gemeinschaft und eine Einrichtung der Freien und Hansestadt Hamburg Beh??rde für Umwelt und Gesundheit (BUG) Pr??ses Senator Peter Rehaag, Beh??rde für Umwelt und Gesundheit seit 10/2001 Senatorin Karin Roth, Beh??rde für Arbeit, Gesundheit und Soziales bis 10/2001
Amt für Gesundheit Senatsdirektor Norbert Lettau Director / Direktor Professor Dr. med. Bernhard Fleischer Deputy Directors / Stellvertreter Professor Dr. med. Rolf Horstmann Professor Dr. med. Egbert Tannich Physician in Chief / Leitender Krankenhausarzt Professor Dr. med. Manfred Dietrich Administration / Verwaltungsleiter Oberregierungsrat Gerd Schlütemann
Staatsrat Gregor Kempkens seit 10/2001 Dr. med. Peter Lippert bis 10/2001
Scienti??c Coordinator / Wissenschaftsreferentin Dr. rer. nat. Barbara Ebert
Funding The Bernhard Nocht Institute for Tropical Medicine is ??nanced jointly by the Federal Government and the States of the Federal Republic of Germany. Additional ??nancial support for speci??c research projects was granted by the following organizations: Das Bernhard Nocht-Institut für Tropenmedizin wird gemeinsam von Bund und L??ndern ??nanziert. Darüber hinaus wurde von folgenden Organisationen weitere ??nanzielle Unterstützung gew??hrt:
?? Alexander von Humboldt-Stiftung ?? Beh??rde für Wissenschaft und Forschung, Hamburg ?? Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) ?? Bundesministerium für Gesundheit (BMG) ?? Bundesministerium für Wirtschaftliche Zusammenarbeit und Entwicklung (BWZE) ?? Caritas ?? Carl-Zeiss-Stiftung ?? Deutsche Forschungsgemeinschaft (DFG) ?? Deutsche Krebshilfe, Mildred-Scheel-Stiftung ?? Deutscher Akademischer Austauschdienst (DAAD) ?? Edna McConnell Clark Foundation (EMCF) ?? European Union ?? EUROSIDA, Copenhagen, Denmark ?? Fraunhofer-Gesellschaft (FhG) ?? Fritz Bender Stiftung ?? Hamburger Wissenschaftsstiftung ?? Hermann Friedrich Bruhn sen.-Stiftung zur F??rderung der Wissenschaften und Kultur
?? Hertha- und Eitel-Fritz-Haver-Stiftung ?? Indo-German Biotechnology Programme ?? Japan Health Sciences Foundation ?? K??rber-Stiftung ?? Lion’s Club, Hamburg ?? MSD Sharp and Dohme GmbH, Haar ?? National Instituts of Health, USA ?? Netherlands Organization for Scienti??c Research in the Tropics (NWO/WOTRO) ?? Robert-Koch-Institut, Berlin ?? Stifterverband für die Deutsche Wissenschaft ?? Strathmann Biotech AG ?? Studienstiftung des Deutschen Volkes ?? UNDP / World Bank ?? Vereinigung der Freunde des Tropeninstituts Hamburg e.V. ?? Visible Genetics Europe, Paris, France ?? Volkswagen-Stiftung (VW-Foundation) ?? Wellcome Trust ?? World Health Organization (WHO)
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Board of Directors / Kuratorium
Staatsrat Gregor Kempkens Chairman since 11/2001 Beh??rde für Umwelt und Gesundheit Freie und Hansestadt Hamburg Staatsrat Dr. Peter Lippert Chairman until 10/2001 Beh??rde für Arbeit, Gesundheit und Soziales Freie und Hansestadt Hamburg Thomas Delissen Finanzbeh??rde Freie und Hansestadt Hamburg Hanna Fangohr Beh??rde für Wissenschaft und Forschung Freie und Hansestadt Hamburg Dr. Peter Lange Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie Senatsdirektor Norbert Lettau Beh??rde für Umwelt und Gesundheit Freie und Hansestadt Hamburg Dr. Johannes Nie??en Beh??rde für Umwelt und Gesundheit Freie und Hansestadt Hamburg Dr. Norbert Schnitzler Bundesministerium für Gesundheit Ministerialrat Arnold Schreiber Bundesministerium für Gesundheit
Scienti??c Advisory Board / Wissenschaftlicher Beirat
Prof. Dr. Jürgen Heesemann Chairman since 2001 Max-von-Pettenkofer-Institut für Hygiene und Mikrobiologie Universit??t München Prof. Dr. Ernst Rietschel Chairman until 2001 Forschungszentrum Borstel Prof. Dr. Philippe Sansonetti Vice Chairman Unité de Pathogénie Microbienne Moléculaire Institut Nationale de la Santé et de la Recherche Médicale (INSERM) Institut Pasteur, Paris, Frankreich Prof. Dr. Roy Anderson Department of Infectious Diseases and Epidemiology Imperial College School of Medicine London, UK Prof. Dr. Bruno Gryseels Prins Leopold Instituut voor tropische Geneeskunde Antwerpen, Belgien Prof. Dr. Thomas Hünig since 2001 Institut für Virologie und Immunbiologie der Universit??t Würzburg Prof. Dr. Karl-Hermann Meyer zum Büschenfelde until 2001 I. Med. Universit??t, Klinik und Poliklinik Universit??t Mainz Prof. Dr. med Rainer Laufs Institut für Medizinische Mikrobiologie und Immunologie Universit??tsklinikum Hamburg-Eppendorf Prof. Eric Ottesen, MD Robert W. Wodruff Health Sciences Center Emory University, Atlanta, USA Prof. Dr. Dietmar Richter Institut für Zellbiochemie und Klinische Neurobiologie Universit??tsklinikum Hamburg-Eppendorf Prof. Dr. Angelika Vallbracht since 2001 Institut für Virologie Universit??t Bremen
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Introduction
Introduction
Since its foundation as Institute for Maritime and Tropical Diseases on 1st October 1900, the BNI is still Germany’s largest research institute for tropical medicine and still combines laboratory research with clinical studies and patient care under one roof. The BNI is a government institution af??liated to the Federal Ministry of Health and the Ministry of Health of the State of Hamburg and is ??nanced jointly by the Federal Government and the States of the Federal Republic of Germany. It is a member of the Leibniz Association, that comprises institutes of national scienti??c impact. The BNI has approx. 380 members (including the staff of the Clinical Department, the diploma students and guests working for short time periods), approx. 37 of them are full time institutional staff scientists for research.
research activities of the BNI concentrate on three areas: (1) cellular and molecular biology of infectious agents that cause tropical diseases, (2) the host response to those agents and its role in protection and pathology, and (3) a disease oriented approach to pathogenesis and pathology. Accordingly, the studies focus on infectious diseases caused by parasites and tropical viruses. Main topics of work are pathogenicity factors and cell biology of parasites, the analysis of the host-parasite-interaction including immunological defence mechanisms, and the de??nition of genes causing susceptibility to certain tropical infections. In all these ventures, special emphasis is put on two issues: relevance for disease, prevention and control, and use of tropical infections as models for general issues in medicine and biology.
Organization
To ful??l these aims, the BNI has a speci??c organizational structure. Three scienti??c sections (Parasitology, Medical Microbiology, Tropical Medicine) contain departments established for longer periods of time, temporary research groups and the closely associated Clinical Department. Research groups are usually installed for a limited period of time only and are replaced by new groups according to scienti??c necessity. The Parasitology Section consists of the Departments of Molecular Parasitology and of Biochemistry and several research groups working on pathogenicity factors, biology of pathogens including biochemical pathways and mechanisms of adaptation and evasion. The Medical Microbiology Section contains the Departments of Immunology and of Helminthology working on host-responses to parasites, the Department of Virology, concentrating mostly on tropical viruses, a Central Diagnostic Unit developing new diagnostic tools and the animal experimentation facilities. The Tropical Medicine Section contains the Departments of Molecular Medicine and Pathology and a research group on Bioinformatics. The Department of Molecular Medicine runs the Kumasi Centre for Collaborative Research in Tropical Medicine in Kumasi, Ghana. The presence of a Clinical Department performing diagnosis and treatment of patients provides a distinctive advantage to the BNI. It is a valuable addition to clinical research performed in the tropics because patients seen in Hamburg usually have primary infections with only one infecting agent, and intensive and longterm studies applying high technology are possible in Hamburg.
Mission of the BNI
As the German centre for research in tropical medicine the Bernhard Nocht Institute is dedicated to research, training and medical care in the area of human infectious diseases which are of particular relevance in the tropics. It is the primary mission of the BNI to develop means for the control of these diseases. Secondary missions are to provide expertise for regional and national authorities and to (directly and indirectly) improve health care for national and regional citizens in regard to diseases of the tropics. A clinical department with 68 beds and an out-patient clinic where patients with tropical infections are treated is an integral part of the BNI. The hospital and its staff are entirely ??nanced by health insurances. There is a travel medicine centre associated with the hospital to advise travellers on prophylaxis. The Central Diagnostic Unit of the BNI performs specialized diagnostic tests for the detection of tropical viruses and pathogens causing tropical infections diseases. The unit serves as the National Reference Centre for Tropical Infections for Germany. There is a biosafety level 4 laboratory available at the BNI for work with haemorrhagic fever viruses. The BNI has multiple educational activities. It is engaged in postgraduate training in the area of tropical medicine. Seventeen members of the BNI are teaching at the University of Hamburg at the Faculties of Medicine, Biology and Chemistry. Three members of the institute hold full professorships (for Molecular Parasitology, Immunology and Molecular Medicine) at the University of Hamburg. A three-month’s course on tropical medicine is held each year that is approved as an officially accredited diploma course by the German Medical Board and the American Society of Tropical Medicine and Hygiene.
Institute Programmes
Institute-wide research programmes are a prominent feature of the scienti??c work at the BNI. Most scienti??c work directly or indirectly leads to projects participating in one of the programmes. During the reported time period, three programmes have been maintained by the BNI on three important and exemplary diseases (??lariasis, malaria and amoebiasis) to foster the interdisciplinary communication and exchange of expertise leading to problem-ori-
Research
The Institute conducts disease-oriented basic research and applies contemporary techniques of cell biology, molecular genetics and immunology to characterize hostpathogen-interactions in tropical infectious diseases. The
7
Introduction
ented research. Other major topics of research are viral haemorrhagic fever, AIDS and leishmaniasis. Filariasis research has become the largest programme (coordinated by Achim Hoerauf) of the BNI. This research has a long history at the BNI having started already in the 1960s. The main topic is onchocerciasis (river blindness) caused by the ??laria Onchocerca volvulus. Work on lymphatic filariasis is also performed, especially on biochemical pathways exploitable for the design of new anti??larial drugs and on ??eld tests for diagnosis and epidemiology. Adult O. volvulus worms develop from infective larval stages and live in subcutaneous nodules from where they release micro??lariae (MF) that migrate within the skin. The Departments of Helminthology, of Tropical Medicine and Immunology cooperate in de??ning O. volvulus proteins and in the analysis of immunological effector mechanisms involved in anti-parasitic defence. The vectors transmitting infective larvae and the differential susceptibility of exposed individuals are studied. Individuals can be divided into i) patients with generalized disease, showing a high load of worms and MF accompanied by immunological tolerance against onchocercal antigens, ii) patients with low MF load and immune reactivity leading to severe dermatitis and iii) putatively immune individuals that remain free of worms despite exposure to infective larvae. The genetic differences underlying the various manifestations of the infection are identi??ed in a genomewide linkage analysis to identify host genes relevant to protection. T cells from putatively immune individuals proliferate to onchocercal antigens and produce IL-5 and IFN-γ in contrast to patients with generalized onchocerciasis. The defective response of the latter is due to production of the deactivating cytokines IL-10 and TGF-β. CD25+CTLA-4+ T cells resembling the Th3 or Tr1 regulatory T cells have been found in these patients. These T cells are speci??c for antigens of O. volvulus and their task may be a speci??c inhibition of T cell response by production of IL-10 and TGF and by scavenging IL-2. The recent ??nding by Achim Hoerauf and colleagues that endosymbiotic Rickettsia-like bacteria of the genus Wolbachia present in most ??larial species can serve as a target for chemotherapy has given the research programme an additional momentum. His group has previously described that in murine ??lariasis the depletion of the bacteria from the nematodes blocks worm development and abolishes fertility. Treatment studies of onchocerciasis patients were conducted in Ghana and have now shown that depletion of endobacteria can be achieved by doxycycline treatment. This leads to a complete and prolonged sterility of female worms and, in addition, has macro??laricidal effects. The ef??cacy thus surpasses that of ivermectin and shows that anti-Wolbachia therapy is a new option for the treatment of ??lariasis. In fact, a combination therapy of doxycycline and ivermectin resulted in a signi??cantly prolonged and enhanced amicro??laridermia compared to sole ivermectin treatment. This might provide the long expected tool to eradicate onchocerciasis and also lymphatic filariasis. The pathogenetic role that reactivity to Wolbachia antigens or to Wolbachia LPS and not to O. volvulus itself plays in onchocerciasis is also studied and has a bearing for therapeutical interventions. Malaria research is a second programme (coordinated by Rolf D. Walter) with institute-wide cooperations. Ongoing projects characterize the glutathione metabolism and polyamine synthesis of Plasmodium falciparum. These studies demonstrate that the plasmodicidal effect of an inhibitor of glutathione synthesis does not depend on its speci??city towards its target enzyme in the parasite, but on the changed physiological needs for the metabolite glutathione in the P. falciparum-infected red blood cells. Therefore, the depletion of glutathione is proposed as a chemotherapeutic strategy for malaria, and gammaglutamylcysteine synthetase is proposed as a potential drug target. P. falciparum, in contrast to other organisms, presents a unique bifunctional ornithine decarboxylase plus S-adenosylmethionine decarboxylase, an organization which is possibly exploitable for the design of new antimalarial drugs. Work in the Department of Parasitology characterized the repertoire of sporozoite gene expression in the mosquito and the proteomics of rhoptry proteins. Clinical studies on patients with severe malaria have supported the notion that the immune response of T cells may not be bene??cial but rather contribute to pathology. This is also supported by studies in mice showing that blockade of the negative T cell regulator molecule CTLA4 leads to exacerbation of P. berghei malaria. A molecular genetic approach is presently used by Prof. Horstmann and colleagues to identify genes involved in susceptibility for or resistance to severe malaria by a genome-wide linkage analysis within the German National Genome Network. Amoebiasis research was established as the ??rst institute-wide program in 1988, covering a variety of aspects concerning the biology and pathogenicity of Entamoeba histolytica. In 1989, the group of Egbert Tannich ??rst described that pathogenic and apathogenic amoeba can be distinguished by molecular genetic methods, a ??nding that had enormous impact on diagnosis and therapy. Topics of the present work are the identi??cation and characterization of molecules involved in pathogenicity, e.g. amoebapores, highly ef??cient and conserved pore-forming proteins, and cysteine proteinases. The comparison of the expression of such proteins in E. histolytica and E. dispar allows an estimate on their impact for pathogenicity. Moreover, transfection and stable expression of heterologous genes is now possible in E. histolytica and E. dispar allowing to elucidate the elucidation of the role of various gene products in pathogenesis. Mechanisms of metronidazole resistance in amoeba and the organization of the genome of E. histolytica are also investigated. In vitro diagnostic tests have been developed to differentiate infections by pathogenic amoeba from infections by apathogenic amoeba. A possible candidate antigen for a vaccine has been identi??ed, a 25mer peptide of the large 170 kD galactose speci??c surface lectin
8
Introduction
leads to protection in experimental infection of animals. In a collaborative research project with the Faculty of Medicine of the University of Hué, Vietnam, the epidemiology of amoebiasis is being investigated. In the Phu Cat district of Hué, the world-wide highest incidence of amoebiasis is found with approx. 6,5 % of the population being infected with E. histolytica. This is an appropriate place to investigate factors in??uencing the development of the disease, of immunity or latency. Research on haemorrhagic fever viruses has become another major topic at the BNI. The endemicity of Lassa virus infections was assessed in Guinea and shows a wider distribution and a higher variability as previously appreciated. The cellular immune response to Lassa virus antigens is characterized in seropositive individuals. Three imported cases of Lassa fever in Europe that were diagnosed in the BNI gave interesting virologic and pathogenetic results. Novel diagnostic methods for Dengue virus infection were established and used in epidemiological studies in Hué, Vietnam. New PCR methods for the detection of haemorrhagic fever viruses have been established and are used in the Central Diagnostic Unit. This has made the Department of Virology a European Reference Centre, and samples were received from several European countries. The Department is a member of the European Network for Imported Viral Diseases (ENIVD). Due to the acquisition of an additional scientist position, research on the molecular biology of yellow fever virus will be added. AIDS research is also performed in a collaborative effort of several departments. The distribution of HIV is studied in the lymphoid tissue of patients and the antibody response to major neutralizing epitopes of HIV and the cellular immune response are characterized. Data from the Department of Pathology together with the German Primate Research Center published in Science and the Journal of Virology show that the oral mucosal route is a most ef??cient route of infection due to rapid infection of oral mucosal-associated lymphoid tissue in the simian immunode??ciency virus system. The Department has especially close collaborations with colleagues at Harvard Medical School and at Rockefeller University. This work concentrates on mechanisms of persistence of HIV/SIV and on novel vaccine approaches. The quanti??cation of HIV in the lymphoid tissue and the enumeration of productively infected cells showed that the unenlarged lymph nodes of HIV-1-infected, asymptomatic patients with high CD4 T cell counts are sites for virus replication and CD4 T cell proliferation. This is also found in late stages of the disease and has important implications for therapeutical strategies. A European Union consortium lead by Prof. Racz on novel techniques to vaccinate against HIV was funded within the 5th Framework Program and an international meeting co-??nanced by the NIH on “Monitoring of HIV Vaccine Trials” was hosted by Prof. Racz. tutions in developing countries have led to longstanding partnerships. Most prominent is the Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) that exists has existed since 1997. With this center the BNI has established partnership with the School of Medical Sciences, Kwame Nkruma University of Science and Technology in Kumasi, Ghana, as a basis for research in the tropics. The cooperative centre was set up according to a state agreement between the Republic of Ghana and Free and Hanseatic City of Hamburg, to foster longstanding contacts to scientists of the host country. Its hallmark is that each research project is carried out jointly by scientists from Hamburg and from Kumasi. The KCCR has grown to more than 60 employees, most of them paid by grant money. It has 400 sqm of laboratory space equipped with modern research equipment and computer facilities. Peripheral laboratories are maintained in Agogo and Dunkwa. An own building for the KCCR is in the process of construction. For the construction and equipment of laboratories, approx. 1,000,000 Euro are available, half of which was provided through a grant from the Volkswagen Foundation and a donation from the Vereinigung der Freunde des Tropeninstituts Hamburg, and the other half was granted by the Senate of Hamburg and the Ministry of Health of the Federal Republic of Germany. A construction compound on the campus was provided by UST and the sod cutting ceremony took place in March 2001 in the presence of Professors Horstmann and Tannich. At the end of 2001 the construction plans of the laboratory building of the KCCR in Kumasi were ready. After the tender was completed construction has now started. The buildings will be ??nished in March 2003. Longterm cooperations under of??cial agreements are also going on with the Central Institute of Drug Research, Lucknow (India), the University of Hué Medical School (Vietnam), and the International Livestock Research Institute (formerly ILRAD), Nairobi (Kenya). Recently an agreement for scienti??c cooperations has been signed with the Ministry of Health of Uganda for work at the Ugandan Virus Research Institute in Entebbe.
Events in 2000 and 2001
A chronicle of important events during the period of this report is given in the appendix. The centenary celebrations of the Institute in 2000 have led to many activities. A special stamp issued by the German Mail on the occasion of the anniversary was presented to the Institute on September 7th by Secretary of State, Karl Diller, of the Federal Ministry of Finances. At the ??rst day of issue, September 14th, philatelists were standing in queue in the special postof??ce in the Institute’s library to obtain the ??rst-day cover with its special postmark. On October 4th, a ceremonial act took place in the large ceremonial hall of the city hall with more than 800 guests attending. The Mayor of the Free and Hanseatic City of Hamburg, Mr. Ortwin Runde, and the Federal Minister of Health, Mrs. Andrea Fischer delivered addresses. On this occasion, an exhibition showing history and work of the BNI on 48 posters to the public was presented in the city hall of
Collaborative research in the tropics
Several of the many collaborations of the BNI with insti-
9
Introduction
Hamburg. The BNI was recognized in the media even more than before, several TV features were broadcasted and a special article in GEO assigned the designation “nation’s body guards” (Leibw??chter der Nation) to the BNI members. In the year 2000, several international symposia that took place at the BNI on the occasion of its centenary, conferences on amoebiasis, malaria, polyamines, AIDS, ??lariasis and leishmaniasis (see chapter ??Meetings“). Also in the framework of the anniversary, from 19th to 22nd September 2001 an international conference on ??lariasis, cosponsored by the World Health Organization, was held in the Institute with participants from 22 countries. In spite of the terrible events of the 11th September, nearly all international guests managed to come to Hamburg. The meeting was made possible by grants from the Federal Ministry of Health and the Ministry of Science and Research of the Free and Hanseatic City of Hamburg. On several occasions, the BNI proved its role as a diagnostic centre for highly contagious diseases in Europe. The Department of Virology made diagnosis of several imported cases of Lassa fever. The Institute received great public attention by two suspected cases of Lassa virus haemorrhagic fever that had been repatriated to Hamburg from Sierra Leone in 2001. Scientists of the Department of Virology and the Clinical Department participated in the efforts to control an Ebola virus outbreak in Gulu, Uganda, in collaboration with the WHO. The Department of Virology received more than 45 samples of suspected viral hemorrhagic fever (VHF), among them samples from the Netherlands, Austria, Switzerland, France, Portugal and Saudi-Arabia. In recognition of the Institute’s competence the BNI was appointed as National Reference Center for Tropical Infections by the Federal Ministry of Health. During the time of the anthrax assaults in the US and the public fear in Germany, the BNI was one of the few institutions able to perform appropriate diagnostic tests for anthrax and received samples from various places in Germany for analysis. The Institute was again honoured by the visits of a number of VIP’s. Mrs. Karin Roth, Minister of Health, Occupation and Social Affairs of the City State of Hamburg, took part in several ceremonies at the BNI, e.g. to present the Medal for Arts and Science to Dr. TennerRacz and Prof. Racz. The State Secretaries of the Federal Ministry of Health, Erwin Jordan and Dr. Klaus-Theo Schr??der, came to Hamburg in 2000 and 2001, respectively, to visit the BNI and become acquainted with its accomplishments. The Ministers of Health of the Republic of Ghana, of the Sultanate of Oman, of the Republic of China Taiwan and the Minister of Economic Affairs of the Republic of Guinea visited the institute during their stays in Hamburg. The new president of the Leibniz-Association, Hans-Olaf Henkel, came to be introduced to the Institute. The medal was originally endowed in 1925 by the Association of Friends of the Tropical Institute (Vereinigung der Freunde des Tropeninstitutes Hamburg) as an award for special merits in “research and control of tropical diseases” (für Erforschung und Bek??mpfung von Tropenkrankheiten). Among the scientists the medal was awarded to are Albert Schweitzer 1927 (who received the medal for his humanitarian work), Gustav Giemsa 1930, and Manson Bahr in 1937. The medal had not been awarded since 1987. The BNI has asked the German Society for Tropical Medicine and International Health to biannually select the awardee jointly with the BNI, thus an of??cal Scienti??c Society participates in the selection. As from the inception the award is ??nanced by the Vereinigung der Freunde des Tropeninstituts Hamburg. With Eric Ottesen the medal was presented to a person that combines scienti??c excellence and a longstanding service to the international community in the control of a tropical disease, namely lymphatic ??lariasis. After his successful scienti??c career at the National Institutes of Health of the USA Dr. Ottesen was essential in putting the global alliance for eradication of lymphatic ??lariasis together, obtaining drugs for mass treatment, and establishing methods for detection.
Constructions
The entrance of the hospital was converted into the main entry of the BNI. Within 12 months, during normal operation of the hospital, the hospital entrance was transformed into a spatious lobby that allows better entry into the hospital while restricting access to the research tracts of the institute. Most important for the future of the Institute is the acquisition of more space for research work. The BNI had secured in 2000 a sum of 20 million DM for the construction of an extension building on the site of the animal house to provide the urgently required additional laboratory space. In fact, the shortness of space, predominantly of laboratories of higher biosafety levels, is now becoming an obstacle to use more grant money and to accomodate more personal. An architect bureau was appointed to perform a thorough calculation and rough planning of the new building. The calculations showed that the required space of laboratories, offices and animal facilities could be accomodated on the small triangular compound in 8 stories, 3 containing animal facilities and technical equipment under the ground and 5 above ground. However, the calculations showed that the budget for the construction had to be raised, and the ??nanciers of the BNI agreed in 2001 to increase the funds for construction to 38.5 Mio DM (19.68 Mio Euro). Due to the extremely exposed location of the new building on the height of the north bank of the river Elbe visible from the river, an architectural competition was advertised and 8 architect’s teams were invited to submit blueprints of the building. On 25 March 2002 a large jury chose the design of Kister, Scheithauer & Gross, Cologne. The detailed planning, especially of the new biosafety level 4 laboratories is underway. The construction work will start before the end of 2002 and should be completed in 2004.
Bernhard Nocht Medal
With the award of the Bernhard Nocht Medal presented to Dr. Eric A. Ottesen during the international Filariasis Conference in 2001 the BNI has revived an old tradition.
10
Introduction
Concomitantly the planning phase has started for the conversion of the quarantaine unit of the Clinical Department into a containment unit for patients with highly contagious infections such as Ebola or Marburg disease. This is necessary to secure the role of the BNI as a treatment center for these infections in Germany. viously at the Max-Planck-Institute for Biology in Tübingen) started in January 2001 with work on kinases in Leishmania, Dr. Volker Heussler (previously at University of Bern, Switzerland) started in January 2002 with research on the liverstage of Plasmodia.
Recognition
The work of members of the BNI again received appreciation. In 2000, Dr. Klara Tenner-Racz and Prof. Paul Racz were awarded the prestigious Medal for Art and Science of the Free and Hanseatic City of Hamburg and Dr. Tim Gilberger the Gerhard-Piekarski-Prize of the German Society for Parasitology. In 2001 Dr. Achim Hoerauf was awarded the Dr. Martini-Prize of the Dr. MartiniStiftung (founded in 1880 it is the oldest promotional award for young scientists). The offer of professorships to four BNI scientists (Barbara Br??ker, Matthias Leippe, Peter Zipfel and Achim Hoerauf) in the last 2 years was again a tribute to the work done at the BNI.
Financial matters
Due to the general economic situation in Germany, the BNI had to suffer from ??nancial cuts. It had to reduce in both 1999 and 2000 its personnel by 1.5% with a corresponding shortening of funds. However, the ??nanciers made funds for two scientist positions in 2000 available for additional virus and malaria research. As every year, 2.5% of its total budget was transferred to the Deutsche Forschungsgemeinschaft (German Research Society, DFG), i.e. approx. 230,000 Euro. As this amount has to be taken from ??exible accounts, the percentage reduction of accounts for scienti??c personnel and consumables is in fact much higher. In return to this deduction, the Institute was granted permission to apply for grants from the DFG also in its main ??elds of work which had not been possible before. This procedure is an instrument of competition for research funds which has been successfully faced by the BNI. Altogether the members of the BNI received grants worth more than 5.18 million DM (2.65 Mio Euro) in the year 2000, setting up a new record in its history. This was maintained in 2001 with 5.20 Mio DM (2.66 Mio Euro). Noteworthy is the success of Prof. Racz to establish a European consortium of 8 partners with 3.6 Mio DM funds in 2000 and of Prof. Horstmann to set up a network project in the Human Genome Network in 2001 together with the two other Northern Leibniz Institutes (Research Center Borstel and Heinrich Pette Institute). A total of 7.5 Mio DM was granted for the research on the genetics of malaria, tuberculosis and hepatitis. Since 1999, the budgets of the research groups are distributed according to their activities in publication (impact factors) and in obtaining grant money.
Acknowledgements
This is the place to thank several persons that have earned great merits for the BNI. Staatsrat Dr. Peter Lippert left his of??ce as Chairman of the Board of Directors of the BNI that he had held since 1988. Dr. Lippert has accompanied and escorted the BNI since more than 17 years. He had a decisive in??uence on the development of the institute during the phases of reorganisation and consolidation, both as a protector and as a challenger. The BNI recognizes with gratitude Dr. Lippert’s support without which the Institute would not have developed to the present stage. Two members of the Scienti??c Advisory Board left the Board in 2000 because of the end of their term of of??ce. Prof. Ernst Rietschel stepped down from the post as chairman of the SAB that he had held since 1996. He has used this of??ce to help the BNI whenever possible and has been a true friend in times of dif??culties and successes. Prof. Karl-Hermann zum Büschenfelde, whose advice has been highly valuable, especially in matters concerning clinical research, has also left the Board. We are grateful for the time and effort they have devoted to the BNI. The BNI would again like to thank its ??nanciers, the Federal Ministry of Health and the Department of Health of the Free and Hanseatic City of Hamburg, for their continuous support of the work of the Bernhard Nocht Institute. And all members of the BNI are commended upon their jointly performed work.
Organizational changes
As with every scienti??c institute the BNI has to be dynamic and ??exible in its scienti??c organisation to meet new research developments. Dr. Frank W. Tischendorf, Head of the Department of Clinical Chemistry retired in 2001. His department had performed research on effector mechanisms against helminths. Since there is no need for clinical chemistry as a research discipline any more, the department (that was created at the inception of the BNI and originally occupied by Gustav Giemsa from 1900 to 1933) was closed. Instead, a research group on bioinformatics was opened and Dr. Bertram Müller-Myhsok appointed as head, who had worked at Lion Biosciences after some years work at the BNI. The Department of Helminthology that had been closed after Prof. Dietrich Büttner’s retirement in 1998 was re-opend and assigned to Dr. Achim Hoerauf as head to prevent his leave to Texas. Due to the award of professorships to Prof. Leippe and Prof. Zipfel their groups were closed and two new research groups were established: Dr. Martin Wiese (pre-
Hamburg, May 2002 Bernhard Fleischer
11
Awards 2000/2001
Awards for Members of the BNI in 2000/2001
Awards in 2000
Dr. Klara Tenner-Racz und Prof. Paul Racz Medal for Art and Science of the Free and Hanseatic City of Hamburg Dr. Tim Gilberger Gerhard-Piekarski-Prize of the German Society for Parasitology
Offered Professorships
PD Dr. Barbara Br??ker (2000) Associate-Professor for Immunology, University of Greifswald Prof. Dr. Peter Zipfel (2000) Associate-Professor for Microbiology/ Infection Biology, University of Jena PD Dr. Matthias Leippe (2001) Associate-Professor for Parasitology, University of Würzburg PD Dr. Achim Hoerauf (2001) Associate Professor for Parasitology, University of Texas, Galveston, USA
Award in 2001
PD Dr. Achim Hoerauf Dr. Martini-Preis of the Martini-Foundation Hamburg
Fellowships for work abroad
PD Dr. Sylke Müller Wellcome Senior Research Fellowship in Basic Biomedical Science (Dundee, UK) Dr. Tim Gilberger Emmi-Noether-Fellowship, Deutsche Forschungsgemeinschaft (Melbourne, Australia) Dr. Christoph Hemmer Habilitation Fellowship, Deutsche Forschungsgemeinschaft (Rostock) Dr. Simone Korten Research Fellowship, Deutsche Forschungsgemeinschaft (Oxford, UK)
Award of the Venia legendi at the University of Hamburg
Dr. Iris Bruchhaus (2000) Dr. Eva Liebau (2000)
Award of the Vereinigung der Freunde des Tropeninstituts Hamburg e.V.
Dr. med. Jan Castan Best thesis 2000 Dr. rer. nat. Tim Gilberger Best thesis 2001
Wettbewerbs-Modell des geplanten Laboratoriumsgeb??udes auf dem Tierhausgel??nde. Rechts im Bild ist der Turm des SchumacherBaues von 1914 zu sehen. Model of the new laboratory building at the site of the animal facilities building. In the right corner, the tower of the old building constructed by Fritz Schumacher in 1914.
12
Introduction
Einleitung
Im Zeitraum dieses Berichtes liegt das 100. Jahr der Existenz des Bernhard-Nocht-Institutes (BNI) von Oktober 1900 bis Oktober 2000. Seit seiner Gründung als Institut für Schiffs- und Tropenkrankheiten ist das BNI Deutschlands gr????tes Institut für Forschung auf dem Gebiet der Tropenmedizin und vereint seither Forschung, Lehre und Patientenversorgung unter einem Dach. Als Institut der Wissenschaftsgemeinschaft Gottfried Wilhelm Leibniz, in der Institute mit überregionaler wissenschaftspolitischer Bedeutung vereint sind, wird es gemeinsam nach Artikel 91b des Grundgesetzes von Bund und L??ndern ??nanziert. Tr??ger des Institutes sind das Bundesministerium für Gesundheit und die Beh??rde für Umwelt und Gesundheit der Freien und Hansestadt Hamburg, deren Dienststelle es ist. Das Institut hat zur Zeit etwa 380 Mitarbeiter, inklusive der etwa 70 Mitarbeiter der Klinik, der Studenten und Praktikanten. Es verfügt in seiner Grundausstattung über rund 37 Wissenschaftlerstellen für die Forschung.
und Doktoranden aus diesen Fachbereichen arbeiten im Institut. Drei Mitglieder des Instituts haben C4-Professuren (für Molekulare Parasitologie, für Immunologie und für Tropenmedizinische Grundlagenforschung) im Fachbereich Medizin inne. Das BNI ist mit einem Kooperationsvertrag mit dem Fachbereich Medizin der Universit??t Hamburg assoziiert.
Wissenschaftliches Programm des BNI
Die Forschung des BNI konzentriert sich auf die Charakterisierung der Erreger-Wirt-Interaktion bei tropischen Infektionserregern mit folgenden Schwerpunkten: 1. die zellul??re und molekulare Charakterisierung der Erreger, 2. die Wirtsreaktion auf diese Erreger und ihre schützende oder pathologische Rolle, 3. die Mechanismen der Pathogenese und der Erkrankung. Bei all diesen Untersuchungen wird auf die Relevanz für die Bek??mpfung tropischer Infektionen Wert gelegt sowie auf die M??glichkeit, tropische Infektionen als Paradigmen grundlegender Prinzipien in Biologie und Medizin zu behandeln. Wegen seiner begrenzten ??nanziellen M??glichkeiten konzentriert sich das BNI in seiner Forschungsarbeit auf tropische Erreger, die durch die Zahl der von ihnen In??zierten bedeutend oder beispielhaft für grundlegende Prinzipien in Biologie und Medizin sind. Um diese Arbeiten durchführen zu k??nnen, verfügt das BNI über Abteilungen und Arbeitsgruppen mit unterschiedlicher Spezialisierung und Schwerpunktsetzung, die in drei wissenschaftlichen Sektionen (Parasitologie, Medizinische Mikrobiologie und Tropenmedizinische Grundlagenforschung) zusammengefasst sind. Abteilungen werden für l??ngere Zeitr??ume bestehen, Arbeitsgruppen sollen je nach wissenschaftlicher Notwendigkeit ersetzt werden und dienen der Flexibilit??t der wissenschaftlichen Arbeit. Die Verbindung mit der Klinischen Abteilung, die aktiv an den Forschungsarbeiten beteiligt ist, ist von besonderem Vorteil, da die in Hamburg behandelten Patienten anders als Patienten in den Tropen meist nur mit einem Erreger in??ziert sind und hier mit hochtechnologischen Methoden auch über l??ngere Zeitr??ume hinweg untersucht werden k??nnen.
Aufgaben des BNI
Als ein Zentrum für Tropenmedizin in Deutschland ist das BNI der Forschung, der Ausbildung und der Versorgung von Patienten auf dem Gebiet der tropischen Infektionskrankheiten des Menschen gewidmet. Hauptaufgabe des BNI ist die Erforschung dieser Erkrankungen, um Wege zu ??nden, sie wirkungsvoll zu bek??mpfen. Zus??tzliche Aufgaben liegen in der Ausbildung von ??rzten und Studenten und in der direkten und mittelbaren Versorgung von Patienten mit tropischen Infektionen. Integraler Teil des Institutes ist eine klinische Abteilung mit 68 Betten und einer Ambulanz, die der Versorgung von Kranken mit tropischen und anderen Infektionskrankheiten dienen. Der Betrieb der Klinik und die in der Krankenversorgung t??tigen Mitarbeiter werden von den Krankenkassen durch Pfleges??tze finanziert. Eine reisemedizinische Beratungsstelle gibt Auskünfte über Prophylaxe von tropischen Infektionen. Das BNI führt eine Diagnostik zum Nachweis von speziellen tropischen Krankheitserregern durch, die überregional in Anspruch genommen wird. Es ist Nationales Referenzzentrum für tropische Infektionserreger und Konsiliarlabor für Plasmodien, Leishmanien, Am??ben, Trypanosomen, Filarien und tropische Viren. Es unterh??lt zum Nachweis und zur Erforschung von hochinfekti??sen Erregern, wie z. B. h??morrhagischen Fieberviren, ein Hochsicherheitslabor der Stufe 4. Das BNI zeigt ein gro??es Engagement in der Lehre: Es veranstaltet j??hrlich verschiedene Kurse mit Bezug auf die Tropenmedizin, u. a. einen dreimonatigen Diplomkursus in Tropenmedizin und Parasitologie für die Zusatzbezeichnung ??Tropenmedizin“ für ??rzte, der bei der American Society of Tropical Medicine and Hygiene akkreditiert ist. Siebzehn Hochschullehrer des BNI führen Lehrveranstaltungen in den Fachbereichen Medizin, Biologie und Chemie der Universit??t Hamburg durch. Diplomanden
Institutsprogramme
Ein wesentliches Instrument der wissenschaftlichen Arbeiten sind Institutsprogramme, in denen Wissenschaftler aus verschiedenen Abteilungen oder Arbeitsgruppen an beispielhaften Erkrankungen (zur Zeit Am??biasis, Filariasis und Malaria) zusammenarbeiten. Das Filariasis-Programm (Koordinator PD Dr. Achim H??rauf) führt Arbeiten fort, die bereits in den 60er Jahren im BNI zur Flussblindheit oder Onchocerciasis begonnen wurden. Die Onchocerciasis wird durch die Filarie Onchocerca volvulus verursacht, die von Kriebelmücken (Simulien) als infekti??se Larve übertragen wird. Nach Ent-
13
Introduction
wicklung zum reifen Wurm setzt O. volvulus Mikro??larien frei, die in der Haut wandern und für die Pathologie verantwortlich sind. Im Filariasis-Programm werden immunologische, parasitologische, entomologische, molekularbiologische und genetische Ans??tze verfolgt. Proteine von O. volvulus, die Zielantigene für T- oder B-Zellen darstellen, werden kloniert und charakterisiert, ebenso Enzyme, die dem Parasiten Schutz vor oxidativem Stress verleihen und Angriffspunkte für chemotherapeutische Interventionen sein k??nnen, oder Proteine, die an der Wanderung der Mikro??larien beteiligt sind. Wichtiger Untersuchungsgegenstand ist die unterschiedliche Empf??nglichkeit betroffener Individuen. Die vorherrschende Form der Onchocerciasis, die generalisierte Onchocerciasis mit hoher Wurmlast und zahlreichen Mikro??larien, geht mit einer spezi??schen immunologischen Toleranz gegen O. volvulus einher, w??hrend die hyperreaktive Form durch eine lebhafte Immunreaktion und chronische, schwere Hautver??nderungen gekennzeichnet ist. Die dritte Reaktionsform auf die Infektion ??ndet sich in Personen, die in den endemischen Gebieten der Infektion mit dem Parasiten ausgesetzt sind, aber nicht erkranken und deshalb wahrscheinlich geschützt sind, die sogenannten ??putativ immunen“ Individuen. Eine genomweite Kopplungsanalyse dieser Individuen wird mit Patienten aus D??rfern in Ghana durchgeführt, um die Wirtsgene zu identi??zieren, die für die unterschiedliche Auspr??gung der Infektion verantwortlich sind. T-Zellen von putativ immunen Individuen proliferieren auf O. volvulus-Antigene und produzieren IL-5 und IFN-γ w??hrend Zellen von Patienten mit generalisierter Onchocerciasis deaktivierende Zytokine wie IL-10 und TGF-?? sezernieren. Ein den Th3Zellen ??hnlicher T-Zell-Typ konnte bei diesen Patienten gefunden werden, der m??glicherweise durch spezi??sche Produktion dieser Zytokine und Verbrauch von IL-2 für die spezi??sche Immunsuppression verantwortlich ist. In fast allen Filarien sind intrazellul??re Bakterien des Genus Wolbachia zu ??nden, die seit ca. 50 Mio Jahren als Symbionten leben und maternal übertragen werden. Produkte der Endobakterien wie LPS oder Hitzeschockproteine sind an der Pathogenese der Onchocerciasis und den Nebenwirkungen mancher Therapieformen beteiligt. Versuche von Achim H??rauf und Kollegen haben gezeigt, dass die Eliminierung dieser Bakterien durch Antibiotika die Wurmentwicklung und Fertili??t dramatisch st??rt. Klinische Studien in Ghana haben nun bewiesen, dass diese neue Strategie der Therapie der Filariasis auch beim Menschen angewendet werden kann. Die Wirkung der antibakteriellen Therapie ist nachhaltiger als die des derzeit verwendeten Ivermectins, insbesondere in einer Kombinationstherapie. Dies er??ffnet nun die M??glichkeit, die ??bertragung der Onchocerciasis und der lymphatischen Filariasis dauerhaft zu unterbrechen und diese Infektionen zu eliminieren. Für diese Untersuchungen wurde Dr. H??rauf 2001 mit dem Martini-Preis der Hamburger Martini-Stiftung ausgezeichnet.
Forschungen zur Malaria stellen das zweite Programm (Koordinator Prof. R.D. Walter) mit institutsweiter Zusammenarbeit dar. Laufende Arbeiten der Abteilung Biochemische Parasitologie behandeln den GlutathionStoffwechsel und die Polyaminsynthese der Plasmodien. Sie zeigen, dass die Depletion von Glutathion eine interessante Strategie der Chemotherapie der Malaria darstellt, und dass Plasmodium falciparum eine einzigartige bifunktionelle Ornithindecarboxylase besitzt, die eine günstige Zielstruktur zur Hemmung der Polyaminsynthese der Plasmodien ist. Die Etablierung des Transfektion von P. falciparum erlaubt nun die funktionelle Untersuchung dieser Moleküle in vivo. Weitere Arbeiten charakterisieren die Expression der Sporozoitengene in der Mücke und das Repertoire der Rhoptrienproteine. Untersuchungen der Abteilung Immunologie in einer experimentellen Infektion zeigen, dass die zellul??re Immunantwort gegen die Plasmodien an der Pathogenese der schweren Malaria beteiligt ist. Eine genomweite Kopplungsanalyse bei Bewohnern einer hochendemischen Region in der N??he von Kumasi, Ghana, wird unternommen, um Gene zu identi??zieren, die Empf??nglichkeit oder Resistenz gegenüber schwerer Malaria tropica vermitteln. Das Am??biasis-Programm wurde bereits 1988 begonnen. Als weltweit gr????tes Forschungsvorhaben auf diesem Gebiet deckt es Aspekte der Biologie und Pathogenit??t von Entamoeba histolytica, dem Erreger der Am??benruhr ab. Im Rahmen dieses Programmes hatte Egbert Tannich 1989 gezeigt, dass Entamoeba in 2 Spezies, pathogene E. histolytica und apathogene E. dispar, unterschieden werden kann, ein Befund mit weitreichenden Konsequenzen für Diagnostik und Therapie. Die aktuellen Arbeiten besch??ftigen sich mit der Identifizierung und Charakterisierung von Molekülen der Am??ben, die für die Auspr??gung des Krankheitsgeschehens von Bedeutung sind, so den Amoebaporen und den Cysteinproteasen. Der Vergleich ihrer Expression in E. histolytica und E. dispar erlaubt Rückschlüsse auf die Bedeutung dieser Moleküle. Die Transfektion und stabile Expression von heterologen Genen in die Am??ben ist inzwischen gelungen, so dass die Bedeutung einzelner Genprodukte für die Pathogenit??t nun untersucht werden kann. Ebenfalls bearbeitet werden Mechanismen der Metronidazol-Resistenz und der Stresstoleranz der Am??ben, sowie die chromosomale Organisation des Am??bengenoms. Die Prüfung verschiedener Bereiche des Lektins von E. histolytica auf ihre Eignung als Impfstoff führte zur Identifizierung eines Peptides, das im Tierversuch Schutz vor der Entwicklung von Leberabszessen verleiht. In einer von der Volkswagen-Stiftung gef??rderten Zusammenarbeit mit der Medizinischen Fakult??t von Hué, Vietnam, wird ein Gebiet mit der weltweit h??chsten Inzidenz von Am??biasis, der Stadtteil Phu Cat von Hué, epidemiologisch untersucht. Der hohe Anteil von In??zierten (etwa 6,5% der Bev??lkerung) wird es erm??glichen, Faktoren zu de??nieren, die an der Auspr??gung der Latenz oder der Immunit??t beteiligt sind.
14
Introduction
Die Erforschung der h??morrhagischen Fieberviren hat sich zu einem weiteren Schwerpunkt des Instituts entwickelt. Die Inzidenz der Lassa-Virusinfektion wird in Guinea, Westafrika, in einem von der Volkswagen-Stiftung unterstützten Projekt untersucht und zeigt eine weitere Verbreitung und eine h??here Variabilit??t des Virus als vorher angenommen. Die zellul??re Immunantwort gegen Antigene des Virus wird charakterisiert. Neue Methoden des molekularbiologischen Nachweises h??morrhagischer Fieberviren wurden etabliert und stehen der Diagnostik und epidemiologischen Projekten zur Verfügung. Ein Projekt zur Epidemiologie der Dengue-Viren wurde in Hué begonnen. Die Abteilung für Virologie des BNI, die in das europ??isches Netzwerk für importierte Virusinfektionen eingebunden ist, ist Anlaufpunkt für die Diagnostik der Fieberviren für Deutschland und mehrere europ??ische Staaten. Dieser Schwerpunkt wird durch eine Arbeitsgruppe für Gelb??eber-Virus verst??rkt werden. AIDS-Forschung ist ebenfalls ein Schwerpunkt des BNI, besonders von den Abteilungen für Pathologie und Virologie und der Klinischen Abteilung getragen. Themen sind die Verteilung des HIV im lymphatischen Gewebe, die Verbreitung des Virus nach Infektion über die Schleimh??ute, die zellul??re Immunantwort und die Bildung neutralisierender Antik??rper gegen das Glykoprotein gp120. Die Quanti??zierung der Viren und der virusproduzierenden Zellen im lymphatischen Gewebe zeigen, dass auch in asymptomatischen Patienten im Lymphknoten eine Virusreplikation statt??ndet und dass selbst unter hochaktiver antiretroviraler Therapie das Virus jahrelang persistiert und sogar repliziert. Diese Ergebnisse haben direkte Bedeutung für die Festlegung der notwendigen Intensit??t und Dauer der Therapie. Ein von der Europ??ischen Union finanziertes Consortium von Wissenschaftlern aus mehreren europ??ischen L??ndern wird von Prof. Racz geführt, das über neue Wege der Impfung gegen HIV arbeitet. Ein von den National Institutes of Health der USA veranstaltetes Meeting über das Monitoring von Impfstudien gegen HIV wurde von Prof. Racz am BNI organisiert.
nen sind eingeladen, dort zusammen mit ghanaischen Partnern Forschungsprojekte durchführen. Das KCCR ist bemüht, den wissenschaftlichen Nachwuchs zu f??rdern, ??rzten und Wissenschaftlern die Weiterbildung und internationalen Austausch zu erm??glichen. Gleichzeitig werden im Rahmen der Forschungsprojekte Stellen für ghanaische Wissenschaftler geschaffen. Das KCCR hat sich in den letzten 2 Jahren stark vergr????ert, es besch??ftigt inzwischen über 60 Angestellte, von denen die meisten aus Projektmitteln ??nanziert werden. Es hat an der School of Medical Sciences der Kwame Nkruma University of Science and Technology (KNUST) in Kumasi 400 qm Labor zur Verfügung, dazu periphere Laboratorien in Agogo und Dunkwa. Ein eigenes Laborgeb??ude für das KCCR wird derzeit mit Mitteln der Tr??ger in Bonn und Hamburg, der Volkswagen-Stiftung und der Vereinigung der Freunde des Tropeninstituts Hamburg e.V. gebaut. Der zeremonielle erste Spatenstich erfolgte im M??rz 2001, das Geb??ude wird im M??rz 2003 bezugsfertig sein. Andere langfristige Kooperationsprojekte mit of??ziellen Vereinbarungen werden mit dem Central Drug Research Institute in Lucknow (Indien), der Universit??t von Hué (Vietnam), dem International Livestock Research Institute (vormals ILRAD) in Nairobi, Kenia und seit kurzem mit dem Uganda Virus Research Institute in Entebbe durchgeführt.
Ereignisse 2000 und 2001
Eine Chronik der Ereignisse der Zeit dieses Berichtes ist im Anhang zu finden. Herausragend waren die Veranstaltungen zum 100. Jahrestag des BNI im Jahr 2000. Eine Sonderbriefmarke der Deutschen Post erschien am 14. September und wurde am 7. September von Staatssekret??r Karl Diller vom Bundesministerium der Finanzen dem BNI feierlich überreicht. Für den Ersttagsstempel wurde ein Sonderpostamt in der Bibliothek des BNI eingerichtet, unter gro??em Andrang der Philatelisten. Am 4. Oktober fand die Jubil??umsfeier im Festsaal des Hamburger Rathauses statt. Der Erste Bürgermeister, Herr Ortwin Runde, und die Bundesministerin für Gesundheit, Frau Andrea Fischer, hielten Ansprachen und versicherten dem BNI die andauernde Unterstützung der Hansestadt und des Bundes. Eine Ausstellung über die Geschichte und die Arbeiten des BNI war 3 Wochen in der Halle des Rathauses für die ??ffentlichkeit zu sehen, zahlreiche Artikel der Presse sowie Fernseh- und Rundfunkbeitr??ge besch??ftigten sich mit dem BNI. In einer Reportage der Zeitschrift GEO wurden die Mitarbeiter des BNI als ??Leibw??chter der Nation“ bezeichnet. Im Jubil??umsjahr fanden mehrere internationale Tagungen im BNI statt, die im Kapitel ??Symposien“ aufgezeichnet sind. Themen waren Am??biasis, Filariasis, Malaria, Leishmaniasis und Polyamine. Vom 19. bis 22. September 2001 veranstaltete das BNI zusammen mit der WHO eine internationale Konferenz über Filariasis, die von Teilnehmern aus 22 L??ndern besucht wurde. Die Tagung wurde von den schrecklichen Ereignissen des 11.
Kollaborative Forschung in den Tropen
Mehrere der Forschungsprojekte des BNI in den Tropen haben zu dauerhaften Partnerschaften geführt. Das Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR) ist das prominenteste, ein gemeinsames Projekt des BNI und der Medizinischen Fakult??t der Universit??t Kumasi, Ghana. Durch einen Staatsvertrag zwischen der Freien und Hansestadt Hamburg und der Republik Ghana wurde 1997 die Errichtung des KCCR vereinbart. Die Grundidee bei der Durchführung von Forschungsprojekten ist die Kooperation. Alle wissenschaftlichen Projekte werden gleichberechtigt von einem Mitarbeiter des Tropeninstituts und einem ghanaischen Wissenschaftler geleitet. Das KCCR soll sich langfristig zu einem Zentrum der internationalen Zusammenarbeit entwickeln, denn auch Wissenschaftler anderer Institutio-
15
Introduction
September überschattet, dennoch konnten fast alle eingeladenen Sprecher an der Tagung teilnehmen. Wiederholt musste das BNI seine Kapazit??t als diagnostisches Zentrum für hochkontagi??se Erreger zur Verfügung stellen. Gro??es ??ffentliches Interesse erregten Verdachtsf??lle von Lassa??eber, die aus Westafrika nach Hamburg einge??ogen wurden, um im BNI abgekl??rt zu werden. Auf Anforderung der WHO nahmen Wissenschaftler des BNI an der Bek??mpfung des Ebola-Ausbruchs in Gulu, Uganda, teil. Die Abteilung Virologie erhielt Proben zur diagnostischen Abkl??rung von tropischen Virusinfektionen aus verschiedenen L??ndern, wie Holland, ??sterreich, Frankreich, Portugal, der Schweiz und Saudi-Arabien. Zur Zeit der Milzbrand-Anschl??ge in den USA war das BNI das diagnostische Zentrum für Norddeutschland, da es eines der wenigen Institute in Deutschland war, die eine schnelle Abkl??rung verd??chtiger Proben gew??hrleisten konnten. Durch das hohe Probenaufkommen mussten die Mitarbeiter der Diagnostik manche Nachtschicht einlegen. Das Institut emp??ng wiederum eine Reihe von hochrangigen Besuchern. Frau Senatorin Karin Roth besuchte mehrfach zu besonderen Anl??ssen das BNI, z.B. zur Verleihung der Medaille für Wissenschaft und Kunst an Frau Dr. Tenner-Racz und Herrn Prof. Racz. Die Staatssekret??re des Bundesgesundheitsministeriums, Herr Erwin Jordan und Herr Klaus-Theo Schr??der, kamen eigens nach Hamburg, um das BNI kennenzulernen. Die Gesundheitsminister der Republik Ghana, des Sultanates Oman, der Republik China Taiwan und die Wirtschaftsministerin von Guinea besuchten das BNI w??hrend ihrer Hamburg-Aufenthalte. Auch der neue Pr??sident der Leibniz-Gemeinschaft, Hans-Olaf Henkel, kam, um sich über die Arbeiten des BNI berichten zu lassen.
Bethesda, mit hervorragenden Forschungsarbeiten zur Filariasis hat Eric Ottesen als Mitarbeiter der WHO eine globale Allianz zur Eradikation der lymphatischen Filariasis aufgestellt.
Baut??tigkeiten
Der Eingang der Klinik wurde zum Haupteingang des BNI umgebaut. In einer Bauzeit von 12 Monaten wurde (bei laufendem Betrieb der Klinik) ein ger??umiger Empfangsbereich geschaffen, der einen einfachen Zugang zur Klinik gew??hrleistet und dabei den Zutritt zum Institut sichert. Eine für die Zukunft des BNI entscheidende Entwicklung war die Bereitstellung von Mitteln für einen Erweiterungsbau auf dem Gel??nde des jetzigen Tierhauses. Die Knappheit an Labor????chen, insbesondere an solchen h??herer Sicherheitsstufen, hatte die Forschungsarbeiten schon wesentlich behindert und erlaubte kaum eine weitere Einwerbung von Drittmitteln. Nach der Entscheidung der Tr??ger, einen Anbau zu ??nanzieren, lie?? das BNI eine Machbarkeitsstudie durch das Architektenbüro APB durchführen, die zeigte, dass die ben??tigten 3000 qm Nutzfl??che an Labors und Tierhaltungsr??umen einschlie??lich eines neuen Labors der Sicherheitsstufe 4 auf dem kleinen dreieckigen Grundstück untergebracht werden kann. Obwohl die Kostensch??tzung von 38,5 Mio DM über der ursprünglich vorgesehenen Summe von 20 Mio DM lag, waren die Tr??ger des BNI bereit, diesen vorgesehenen Bau mit 8 Stockwerken (davon 3 unter der Erde) zu ??nanzieren und 19,68 Mio Euro in die mittelfristige Planung einzustellen. Wegen der exponierten Lage des Grundstücks am Elbhang über den Landungsbrücken wurde ein Architektenwettbewerb ausgeschrieben. Acht Büros wurden zur Einsendung von Entwürfen eingeladen. Am 25. M??rz 2002 tagte ein Preisgericht bestehend aus 13 Fach- und Sachpreisrichtern, darunter der Oberbaudirektor, Architekten, Ingenieure und Mitglieder des BNI, und pr??mierten den Entwurf von Frau Susanne Gross des K??lner Büros Kister, Scheithauer und Gross mit dem ersten Preis. Derzeit laufen die detaillierten Planungen, Baubeginn soll noch in 2002 sein, die Fertigstellung wird für 2004 vorgesehen. Parallel beginnen die Planungen für den Ausbau der Infektionsstation zu einer Quarant??ne-Einheit für Patienten mit hochkontagi??sen Infektionen wie Ebola- oder Lassa-Fieber. Mit dieser Einheit wird das BNI seiner Rolle als ein nationales Behandlungszentrum für diese tropischen Infektionen gerecht.
Bernhard-Nocht-Medaille
Mit der Verleihung der Bernhard-Nocht-Medaille an Dr. Eric Ottesen im September 2001 hat das BNI eine alte Tradition wieder au??eben lassen. Die Medaille war 1925 von der Vereinigung der Freunde des Tropeninstitutes Hamburg e.V. gestiftet worden, um besondere Verdienste um ??Erforschung und Bek??mpfung von Tropenkrankheiten“ zu ehren. Unter den mit der Medaille Ausgezeichneten sind Albert Schweitzer (1927), Gustav Giemsa (1930), und Manson Bahr (1937). Nachdem seit 1987 die Medaille nicht mehr vergeben worden war, hatte das BNI die Deutsche Gesellschaft für Tropenmedizin und Internationale Gesundheit gebeten, an der Verleihung mitzuwirken und hatte ein entsprechendes Statut aufgestellt. Somit wird die Medaille nun alle zwei Jahre vom BNI zusammen mit einer offiziellen wissenschaftlichen Gesellschaft verliehen werden. Wie von Beginn an ??nanziert die Vereinigung der Freunde die Verleihung. Mit Eric Ottesen in 2001 wurde die Medaille an einen herausragenden Wissenschaftler verliehen, der zugleich bedeutende Verdienste um die Bek??mpfung von Tropenkrankheiten erworben hat. Nach seiner erfolgreichen Karriere an den National Institutes of Health in
Finanzen
Durch die schwierige ??nanzielle Lage der ??ffentlichen Haushalte blieb das BNI von Sparma??nahmen nicht verschont. Es musste 2000 und 2001 jeweils 1,5% seiner Stellen abbauen mit entsprechender Kürzung der Mittel, erhielt allerdings ab 2000 zwei zus??tzliche Wissenschaftlerstellen für Malaria und Virusforschung. Personal- und Betriebsmittel wurden ansonsten überrollt. Au??erdem führte das BNI wie in jedem Jahr 2,5% seines
16
Introduction
Gesamtetats an die Deutsche Forschungsgemeinschaft (DFG) ab, also etwa 230.000 Euro. Da dieser Betrag aber nur aus ??exiblen Titeln entnommen werden kann, betrifft dies überproportional die Etats für wissenschaftliches Personal und für Sachmittel. Im Gegenzug hat das Institut die M??glichkeit, bei der DFG auch auf seinen Hauptarbeitsgebieten Antr??ge zu stellen. Diesem Instrument des Wettbewerbs um Forschungsgelder hat sich das BNI wiederum mit Erfolg gestellt. Seit 1999 gibt es Wettbewerb auch innerhalb des BNI: die Mittel für die Arbeitsgruppen werden nach Leistungen, gemessen an Publikationsaktivit??t und Drittmitteleinwerbung, verteilt. Insgesamt wurden im Jahr 2000 mehr als 2,65 Mio. Euro an Drittmitteln eingeworben, und dieser Rekord in der Geschichte des BNI im Jahr 2001 mit 2,66 Mio. Euro gehalten. Bemerkenswert sind die Erfolge von Prof. Racz, ein europ??isches Konsortium mit 3,6 Mio. DM für neun Partner-Institutionen zu etablieren, und von Prof. Horstmann zusammen mit dem Forschungszentrum Borstel und dem Heinrich-Pette-Institut ein Projekt von 7,5 Mio. DM im Rahmen des Nationalen Genomforschungsnetzes einzuwerben.
Gilberger den Gerhard-Piekarski-Preis für Parasitologie. Dr. Achim H??rauf erhielt den Dr. Martini-Preis der Dr. Martini-Stiftung (gestiftet 1880 ist er der ??lteste Nachwuchspreis für junge Wissenschaftler). Für ihre erfolgreichen Arbeiten wurden mehreren Mitarbeitern des BNI Positionen an anderen Orten angeboten oder Auslandsstipendien zugesprochen, um andernorts Forschungen durchzuführen. Die Rufe an Dres. Barbara Br??ker, Matthias Leippe, Peter Zipfel und Achim H??rauf sind auch eine Anerkennung für das BNI.
Danksagung
Das BNI hat vielen Personen für Hilfe und Unterstützung zu danken. An dieser Stelle soll drei Personen besonders gedacht werden, die nach langer T??tigkeit für das BNI aus ihren ??mtern ausgeschieden sind. Staatsrat Dr. Peter Lippert legte sein Amt als Vorsitzender des Kuratoriums im Oktober 2001 nieder, das er seit 1988 innehatte. Dr. Lippert hat das BNI seit mehr als 17 Jahren begleitet, zun??chst als Leiter des Amtes für Gesundheit. Er hat entscheidend an der Reorganisation mitgewirkt und war auch in der folgenden Phase der Konsolidierung sowohl Beschützer als auch Herausforderer des BNI. Ohne seine tatkr??ftige Unterstützung h??tte sich das BNI nicht zu dem heutigen Institut entwickeln k??nnen. Zwei Mitglieder des wissenschaftlichen Beirates verlie??en turnusgem???? den Beirat im Jahr 2000. Prof. Ernst Th. Rietschel legte zugleich den Vorsitz des Beirates nieder, den er seit 1996 hatte. Er hatte dieses Amt genutzt, um für das BNI zu streiten, und bew??hrte sich als wahrer Freund des Instituts, besonders in schwierigen Phasen. Prof. Karl-Hermann Meyer zum Büschenfelde verlie?? ebenfalls den Beirat, er hatte dem BNI insbesondere als Berater in Fragen der klinischen Forschung geholfen. Das Institut ist beiden Kollegen für die Zeit und Mühe dankbar, die sie ihm gewidmet haben. Den Tr??gern des BNI, der Beh??rde für Umwelt und Gesundheit der Freien und Hansestadt Hamburg und dem Bundesministerium für Gesundheit sei an dieser Stelle für ihr Engagement und ihre gro??zügige Unterstützung des BNI gedankt. Allen Mitarbeiterinnen und Mitarbeitern des Institutes soll hier zu der gemeinsam erbrachten Leistung gratuliert werden.
Strukturelle ??nderungen
Wie jedes wissenschaftliche Institut muss das BNI in seiner wissenschaftlichen Struktur ??exibel sein, um sich neuen Entwicklungen anpassen zu k??nnen. PD Dr. Frank Tischendorf, Leiter der Abteilung Klinische Chemie, ging Ende 2001 in den Ruhestand nach langj??hriger Arbeit über Effektormoleküle aus eosinophilen Granulozyten. Da die Klinische Chemie als Forschungsdisziplin nicht mehr ben??tigt wird, wurde die Abteilung (die bei der Gründung des BNI entstanden war und ursprünglich von Giemsa geleitet worden war) nicht fortgeführt. Stattdessen wurde eine Arbeitsgruppe Bioinformatik eingerichtet, mit PD Dr. Bertram Müller-Myhsok als Leiter, der nach einigen Jahren am BNI bei Lion Biosciences (Heidelberg) gearbeitet hatte. Die Abteilung Helminthologie, die nach Prof. Dietrich Büttners Pensionierung geschlossen worden war, wurde wieder eingerichtet und Dr. Achim H??rauf als Leiter zugeordnet, um seinen Ruf an die Universit??t Texas abzuwenden. Nach der Wegberufung der Drs. Matthias Leippe und Peter Zipfel wurden zwei neue Arbeitsgruppen eingerichtet. Dr. Martin Wiese (vorher am Max-Planck-Institut für Biologie in Tübingen) begann im Januar 2001 mit Arbeiten zu Kinasen von Leishmanien. Dr. Volker Heussler (vorher an der Universit??t Bern, Schweiz) begann im Januar 2002 mit seinen Arbeiten zur Leberphase der Plasmodieninfektion.
Hamburg, im Mai 2002 Bernhard Fleischer
Anerkennung
Die Arbeiten der Mitglieder des BNI wurden 2000 und 2001 mit Anerkennungen und Auszeichnungen gewürdigt. Dr. Klara Tenner-Racz und Prof. Paul Racz erhielten für ihre Arbeiten zur Verbreitung des HIV im lymphatischen Gewebe die Medaille für Wissenschaft und Kunst der Freien und Hansestadt Hamburg und Dr. Tim
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Introduction
Auszeichnungen für Mitglieder des BNI 2000 und 2001
Preise 2000
Dr. Klara Tenner-Racz und Prof. Paul Racz Medaille für Kunst und Wissenschaft der Freien und Hansestadt Hamburg Dr. Tim Gilberger Gerhard-Piekarski-Preis der Deutschen Gesellschaft für Parasitologie
Rufe auf Professuren
PD Dr. Barbara Br??ker (2000) Professur (C3) für Immunologie, Universit??t Greifswald Prof. Dr. Peter Zipfel (2000) Professur (C3) für Mikrobiologie/Infektionsbiologie, Universit??t Jena PD Dr. Matthias Leippe (2001) Professur (C3) für Parasitologie, Universit??t Würzburg PD Dr. Achim H??rauf (2001) Associate Professorship for Parasitology, University of Texas, Galveston, USA
Preis 2001
PD Dr. Achim H??rauf Dr.-Martini-Preis der Martini-Stiftung Hamburg
Verleihung von Stipendien
PD Dr. Sylke Müller Wellcome Senior Research Fellowship in Basic Biomedical Science (Dundee, GB) Dr. Tim Gilberger Emmi-Noether-Stipendium, Deutsche Forschungsgemeinschaft (Melbourne, Australien) Dr. Christoph Hemmer Habilitationsstipendium, Deutsche Forschungsgemeinschaf (Rostock) Dr. Simone Korten Forschungsstipendium, Deutsche Forschungsgemeinschaft (Oxford, GB)
Verleihung der Venia legendi an der Universit??t Hamburg
Dr. Iris Bruchhaus (2000) Dr. Eva Liebau (2000)
Preise der Vereinigung der Freunde des Hamburger Tropeninstitutes Hamburg e.V.
Dr. med. Jan Castan Doktorandenpreis 2000 Dr. rer. nat. Tim Gilberger Doktorandenpreis 2001
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Parasitology Section
Selected Scienti??c Projects Ausgew??hlte wissenschaftliche Projekte
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Parasitology Section
Parasitology Section
Chairman’s Summary
The Parasitology Section comprises a number of different research groups, working on a broad variety of medically important parasites. Emphasis was given to amoebiasis, malaria, ??lariasis and leishmaniasis, four of the most important parasitic diseases with high impact on morbidity and mortality in most tropical and subtropical countries. Work on amoebiasis concentrated on various aspects concerning the biology and pathogenicity of Entamoeba histolytica. This included: (i) Characterization of proteins speci??cally regulated during amoeba encystation (Frank Ebert); (ii) analysis of effector molecules such as cysteine proteinases and pore-forming peptides, responsible for the extraordinary high capacity of E. histolytica to destroy human tissues (Iris Bruchhaus, Matthias Leippe), and (iii) genetic comparisons between the two closely related amoeba species E. histolytica and E. dispar in aim to identify differences which might help to explain why only E. histolytica is able to cause disease (Ute Willh??ft). In addition, work has been continued to development an oral amoebiasis vaccine, which recently resulted in generation of recombinant bacteria with a high potential to protect against invasive amoebiasis (Hannelore Lotter). In order to obtain more accurate data on the epidemiology and treatment of amoebiasis, comprehensive studies were performed in Hué, Vietnam, (J??rg Blessmann) in close collaboration with the Medical College of the University of Hué. One of the outcomes of these studies was the identi??cation of relatively long asymptomatic intestinal persistance of the parasite with a half-life of infection of more than 15 months. Moreover, comparative treatment trials of asymptomatic E. histolytica carriers indicated signi??cant higher cure rates for paromomycin compared to diloxanide fuorate. During the last few years malaria research has expanded within the Institute and is currently being performed in all three sections. Within the Parasitology Section work was continued on antimalarial drug discovery (Rolf D. Walter). Recent achievements included identi??cation of a unique bifunctional enzyme complex of P. falciparum consisting of ornithine decarboxylase and Sadenosylmethionine decarboxylase and the discovery that thioredoxin reductase is absolutely required for survival of the parasite within red blood cells. The project on the P. falciparum rhoptry proteome was continued in order to characterize the molecules forming the organelle of the malaria parasite which is instrumentel for invading red blood cells (Matthias Leippe). Moreover, to study malaria immunology and the development of the malaria parasite within mosquitoes or liver cells, the complete mouse-malaria-cycle is run in the Institute, which requires the time-consuming breeding of the vector mosquito Anopheles stephensii (Andreas Krüger). Research groups of the Parasitology Section working on ??lariasis have focussed on the identi??cation and characterization of vaccine candidates against onchocerciasis, the development of new methods to better evaluate the success of intervention programmes for lymphatic ??lariasis and the characterization of Onchocerca volvolus glutathione-dependent detoxi??cation enzymes. Based on observations concernig the speci??c immune responses of onchocerciasis patients to various O. volvolus antigens, a DNA-based vaccine is currently assessed using O. ochengi infections in cattle as a model (Klaus Erttmann). To evaluate the prevalence of Brugia malayi and Wuchereria bancrofti, the causative agents of lymphatic ??lariasis, improved PCR-based test systems were developed, which allow highly sensitive detection of the two parasites in clinical samples as well as in transmitting mosquitoes (Peter Fischer). Studies on O. volvolus glutathione-dependent enzymes make use of the model organism Caenorhabditis elegans which provides a powerful system for investigating the molecular aspects of detoxi??cation processes in nematodes. Here the developmental and cellular biology is thoroughly understood and stable lines of transgenic worms can easily be generated (Eva Liebau). Research on Leishmania has been strengthened by the introduction of a new group headed by Martin Wiese. His major interest is the understanding of signal transduction in kinetoplastidae and in particular the role of protein kinases in aim to identify new anti-Leishmania drug targets. A second group headed by Joachim Clos has established genetic screening techniques for the identi??cation of Leishmania genes which contribute to virulence, tissue tropism or drug resistance. Moreover, this group has identi??ed the 90 kD heat shock protein of L. donovani as a key molecule in the conversion of the parasite from the vector-borne promastigote stage towards the mammalian amastigote stage. Leishmania stage conversion was further investigated by a proteom approach leading to the identification of 70 proteins which are speci??cally expressed in the promastigote or the amastigote stage of the parasite (Iris Bruchhaus). The two service units located within the Parasitology Section, the Electron Microscopy Unit and the DNA Sequencing Unit have proven to be of great help for many of the research groups, which is documented by the large number of investigations that have been performed in the two units within the last two years. Egbert Tannich
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Zusammenfassung des Sprechers
Die Sektion Parasitologie umfasst verschiedene Arbeitsgruppen, die sich mit einer Vielzahl medizinisch bedeutsamer Parasiten besch??ftigten. Schwerpunkte bildeten dabei Arbeiten zur Am??biasis, Malaria, Filariasis und Leishmaniasis, vier der wichtigsten Humanparasitosen mit erheblichem Ein??uss auf Morbidit??t und Mortalit??t in vielen tropischen und subtropischen L??ndern. Die Arbeiten zur Am??biasis konzentrierten sich auf unterschiedliche Aspekte bezüglich der Biologie und Pathogenit??t von Entamoeba histolytica. Diese umfassten: 1. Charakterisierung von Proteinen, die spezi??sch w??hrend der Enzystierung der Am??ben reguliert werden (Frank Ebert); 2. Analyse von Effektormolekülen, wie etwa Cysteinproteinasen oder porenbildende Peptide, die für die extreme, Am??ben-bedingte Zerst??rung von Wirtsgewebe verantwortlich sind (Iris Bruchhaus, Matthias Leippe), und 3. vergleichende genetische Analysen zwischen den beiden eng verwandten Am??benarten E. histolytica und E. dispar, mit dem Ziel, Anhaltspunkte darüber zu bekommen, warum nach Besiedlung des menschlichen Darms nur E. histolytica in der Lage ist Erkrankungen auszul??sen (Ute Willh??ft). Darüber hinaus wurde die Entwicklung einer Am??biasisvakzine weiter vorangetrieben. Dabei gelang es kürzlich, rekombinante Bakterien herzustellen, die ein hohes Ma?? an Protektion gegen eine invasive Am??biasis vermitteln (Hannelore Lotter). Zus??tzlich wurden umfangreiche Arbeiten zur Epidemiologie und Behandlung der Am??biais in Hué, Vietnam, durchgeführt (J??rg Blessmann). Dies geschah in enger Zusammenarbeit mit dem Medical College der Universit??t von Hué. Eines der Ergebnisse dieser Untersuchungen war der Befund, dass E. histolytica für relativ lange Zeit als asymptomatische Infektion den Darm des Menschen besiedeln kann und dass Paromomycin weitaus besser zur Behandlung asymptomatischer E. histolytica-Infektionen geeignet ist als Diloxanid Fuorat. W??hrend der vergangenen Jahre wurde die Malariaforschung am BNI weiter ausgebaut und wird gegenw??rtig in allen drei Sektionen des Instituts durchgeführt. Innerhalb der Sektion Parasitologie wurden frühere Arbeiten zur Identifizierung von Zielstrukturen für neue Malariamedikamente weitergeführt (Rolf D. Walter). Wichtige Ergebnisse waren dabei die Identifizierung eines einzigartigen Enzymkomplexes in P. falciparum, der sowohl Ornithin- als auch S-AdenosylmethioninDekarboxylaseaktivit??t besitzt, sowie der Befund, dass für das ??berleben des Parasiten in den Erythrozyten die Plasmodien-Thioredoxinreduktase von essentieller Bedeutung ist. Zus??tzlich wurde das Rhoptrien-Proteomprojekt weitergeführt, um die Moleküle der besonderen Organelle des Malariaparasiten zu charakterisieren, die für die Invasion roter Blutk??rperchen von Bedeutung ist (Matthias Leippe). Zum Studium der Malariaimmunologie
und der Entwicklung des Parasiten innerhalb der ??bertr??germücke sowie innerhalb von Leberzellen wird der gesamte Maus-Malaria-Zyklus vorgehalten, was mit der zeitaufwendigen Zucht von Anophelesmücken verbunden ist (Andreas Krüger). Forschergruppen der Sektion Parasitologie, die sich am Filariasis-Programm des Instituts beteiligten, haben sich konzentriert auf die Identi??zierung und Charakterisierung von m??glichen Vakzinekandidaten gegen Onchozerkiasis, die Entwicklung neuer Methoden zur Evaluierung von Bek??mpfungsma??nahmen gegen lymphatische Filariasis und die Charakterisierung von Glutathion-abh??ngigen Entgiftungsenzymen aus O. volvulus. Basierend auf Untersuchungen zur Immunreaktion von Onchzerkiasispatienten gegenüber verschiedenen O. volvolus Antigenen, wird zur Zeit eine DNA-Vakzine im O. ochengi Model beim Rind evaluiert. (Klaus Erttmann). Zur Bestimmung der Pr??valenz von Brugia malayi und Wuchereria bancrofti, den Erregern der lymphatischen Filariasis, konnten verbesserte PCR-Tests entwickelt werden, die mit hoher Sensitivit??t den Nachweis der beiden Parasiten in klinischem Material so wie in den ??bertr??germücken erm??glichen (Peter Fischer). Untersuchungen über Glutathion-abh??ngige Enzyme von O. volvolus wurden unter Verwendung des Modellorganismus Caenorhabditis elegans durchgeführt, da dieser Nematode genetischen Manipulationen zug??nglich ist und sich leicht stabile Linien transgener C. elegans herstellen lassen (Eva Liebau). Arbeiten über Leishmanien wurden verst??rkt durch die Etablierung einer neuen Gruppe, die sich mit der Signaltransduktion in Kinetoplastiden besch??ftigt und dabei insbesondere die Rolle von Proteinkinasen untersucht (Martin Wiese). In einer bereits l??nger bestehenden zweiten Arbeitsgruppe wurde ein genetisches ??Screening-System“ etabliert zur Identi??zierung von Genen, die für die Pathogenit??t, den Organtropismus und die Antibiotikaresistenz von Leishmanien eine Rolle spielen. Au??erdem wurde von dieser Gruppe gezeigt, dass das Hitzeschockprotein HSP90 von L. donovani ma??geblich an der Stadienkonversion des Parasiten von der promastigoten zur amastigoten Form beteiligt ist. Darüber hinaus wurde die Gesamtheit aller Proteine bestimmt, die w??hrend der Stadienkonversion von Leishmanien einer Regulation unterliegen. Dabei wurden bisher 70 Proteine identifiziert, die spezifisch in amastigoten bzw. promastigoten Parasiten exprimiert werden. Die beiden in der Sektion Parasitologie angesiedelten Serviceeinheiten, die Elektronenmikroskopie und die DNA-Sequenziereinheit haben sich als besonders hilfreich für die Umsetzung der verschidenen Forschungsaktivit??ten erwiesen, was unter anderem durch die gro??e Zahl an durchgeführten Untersuchungen deutlich wurde. Egbert Tannich
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Parasitology Section
Parasitology Section
Please note that not all persons listed were employed during the entre reporting period
Department of Molecular Parasitology
Scienti??c Staff Prof. Dr. Egbert Tannich, Head PD Dr. Iris Bruchhaus Dr. Hannelore Lotter Dr. Andreas Krüger Dr. Ute Willh??ft Associated Scienti??c Staff Dr. Frank Ebert Technical Staff Heidrun Bu?? Ina Hennings Sebastian Horstmann Susann Ofori Britta Weseloh Doctoral / Graduate Students Felix Asche J??rg Blessmann Eduardo Campos Góngora Sassia Touzni
Department of Biochemical Parasitology
Scienti??c Staff Prof. Dr. Rolf D. Walter, Head PD. Dr. Sylke Müller PD Dr. Eva Liebau Dr. Kai Lüersen Prof. Dr. Justus Schottelius Visiting Scientists Dr. Yenni Djuardi, Jakarta, Indonesia Prof. Dr. Rentala Madhubala, New Delhi, India Dr. Shailandra Saxena, New Delhi, India Dr. Arvind Srivastava, Lucknow, India M.Sc. Lyn-Marie Birkholtz, Pretoria, South Africa Dr. Ilia Bankov, So??a, Bulgaria Technical Staff B??rbel Bergmann Marie-Luise Eschbach Marzena Domagalski Doctoral / Graduate Students Benjamin C. Abo-Dalo Robin DasGupta Nashya Haider Tanja Krause Ingrid Müller Dieudonné Ndjonka Carsten Wrenger
Laboratory Bruchhaus
PD Dr. Iris Bruchhaus Technical Staff Maike Schwerdtfeger Doctoral / Graduate Students Verena Diehm Simone Harder Andrea Hellberg Kerstin Isermann Nicolas Nowak Meike Thiel Claudia Wa??mann
Laboratory Liebau
PD Dr. Eva Liebau Doctoral / Graduate Students Cora Burmeister Kristina Krause Stephanie Krause Alexandra Sommer Inga B. Wilde
Laboratory Müller
PD Dr. Sylke Müller Doctoral / Graduate Students Zita Krnajski Svenja Meierjohann
Laboratory Schottelius
Prof. Dr. Justus Schottelius
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Parasitology Section
Research Group Clos
PD Dr. Joachim Clos Technical Staff Manfred Kr??mer Andrea Macdonald Dorothea Zander Doctoral / Graduate Students Kohelia Choudhury Sandra Fleischer Cornelia Hoyer Linda Klaholz Katja Mellenthin Martina Wiesgigl
Reseach Group Wiese
since 03/2001 Dr. Martin Wiese Technical Staff Eva Kampen Doctoral / Graduate Students Daniela Kuhn Florian Bengs
Research Group Zipfel
until 05/2000 Scienti??c Staff Prof. Dr. Peter F. Zipfel PD Dr. Christine Skerka Technical Staff Alexandra Bialonski Eva Kampen Doctoral / Graduate Students Sylvain Djoha Christian K??hler Tamara Manuelian Nina Nehmann Helge Penski
Research Group Erttmann
PD Dr. Klaus Erttmann Technical Staff Silke van Hoorn Doctoral / Graduate Students André Kleensang
Research Group Fischer
Dr. Peter Fischer Technical Staff Insa Bonow Doctoral Student Simone Klüver
Electron Microscopy Unit
Christa Schmetz Ellen Torlach
Research Group Leippe
until 05/2001 Scienti??c Staff PD Dr. Matthias Leippe Dr. Heike Bruhn Technical Staff Ulrike Fr??hlke Claudia Ott Doctoral / Graduate Students Conny Brandt Christoph Gelhaus Marco Klapper Rosa Nickel Beate Riekens Maren Rudolph Julia Winkelmann
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Parasitology Section
Characterization of Cysteine Proteinases in Entamoeba histolytica
Zusammenfassung
Cystein-Proteinasen (CPs) von Entamoeba histolytica (EhCPs) gelten als wichtige Pathogenit??tsfaktoren. Insgesamt acht Gene für diese Klasse von Enzymen wurden bisher im Genom des Parasiten identi??ziert, wobei EhCP1, EhCP2 und EhCP5 am st??rksten exprimiert sind. Interessanterweise konnten wir zeigen, dass funktionsf??hige Gene für CP1 und CP5 in der nah verwandten, apathogenen Spezies Entamoeba dispar fehlen. Die bisher identi??zierten CPs der Am??ben haben Molekulargewichte von ca. 28 kDa. Weitere, in der Literatur als ??higher molecular mass proteinases“ beschriebene CPs mit Molekulargewichten von 35 und 48 kDa, wurden von uns gereinigt und als die schon bekannten Enzyme EhCP1 und EhCP2 identifiziert. Durch Gentransferexperimente gelang es, EhCP2 in E. histolytica und E. dispar verst??rkt zu exprimieren. Diese ??berexpression führte zu einer verst??rkten Zerst??rung von Zellverb??nden in Kultur, hatte aber keinen Ein??uss auf die Leberabszess-Bildung in experimentell in??zierten Versuchstieren. E64 or the addition of laminin, which blocks the substrate-binding pocket of CPs, greatly reduces the ability of the parasite to produce liver abscesses in laboratory animals. Likewise, amoebae genetically engineered to express low levels of CP activity were unable to induce liver abscess formation and produced signi??cantly less gut in??ammation and damage to the intestinal permeability barrier. Entamoeba histolytica contains a multitude of at least 8 different CPs. It remains to be determined, whether all of them or whether a particular CP is involved in amoeba pathogenicity. Thus, a detailed characterization of each of the various enzymes is required.
Project Description and Results Cystein proteinase genes of E. histolytica and E. dispar
Until now, a total of eight genes coding for CPs in E. histolytica (ehcp1-ehcp7, ehcp112) have been identified. Four of the corresponding proteins, namely EhCP1, EhCP2, EhCP5 and EhCP112, have been puri??ed and/or further characterised. In cultured E. histolytica trophozoites approximately 90% of total CP activity could be attributed to three enzymes only, namely EhCP1, EhCP2, and EhCP5, whereas the remaining CPs were found to be expressed at very low levels. Interestingly, in contrast to all other CPs, proteins corresponding to EhCP1 and EhCP5 were found to be absent in Entamoeba dispar. This amoeba species is closely related to and morphologically indistinguishable from E. histolytica. It also colonizes the human gut but is non-pathogenic. Interestingly, the comparison between the cp5-containing genomic region of E. histolytica and the respective orthologous sequence in E. dispar revealed that the position of cp5 within the genomic context is conserved between the two organisms. However, the gene is highly degenerated in E. dispar, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the cp5 reading frame. With respect to E. histolytica pathogenicity, EhCP5 appears to be of particular importance, as it is the only CP, which is present on the amoeba surface.
Summary
Cysteine proteinases (CPs) of Entamoeba histolytica are considered to be important virulence factors. A total of eight genes coding for CPs in E. histolytica has been identi??ed, of which EhCP1, EhCP2 and EhCP5 are most abundantly expressed. Interestingly, functional genes corresponding to EhCP1 and EhCP5 were found to be absent in the closely related but non-pathogenic species Entamoeba dispar. All CPs of E. histolytica have molecular masses of approximately 28 kDa. Puri??cation of the repeatedly described but as yet uncharacterised ??higher molecular mass proteinases“ of 35 and 38 kDa indicated that they in fact represent EhCP1 and EhCP2, respectively. By genetic manipulation we were able to overexpress EhCP2 in E. histolytica as well as in E. dispar. Compared with respective controls, these amoeba mutants had a signi??cant higher capacity to destroy cell monolayers in vitro, but revealed no difference in liver abscess formation of experimentally infected animals.
Introduction
Cysteine proteinases (CPs) have been identi??ed as important virulence factors of various infectious agents and are the main proteolytic enzymes in many parasites. The protozoon parasite Entamoeba histolytica, the causative agent of human amoebiasis, is characterized by its high capacity to destroy host tissues, leading to potentially life-threatening diseases such as ulcerative colitis or liver abscess. Convincing evidence exists that CPs are involved in E. histolytica-induced pathology. Treatment of amoebae with sublethal doses of the speci??c CP inhibitor
Characterization of higher molecular weight cysteine proteinases
With the exception of ehcp112, all E. histolytica CP genes identi??ed so far encode for mature proteins with calculated molecular masses of approximately 28kDa. However, substrate gel analysis of E. histolytica lysates indicated three CP activities, which correspond to proteins of 28, 35 and 48 kDa, respectively. To further characterize the higher molecular weight enzymes, the molecules were puri??ed by af??nity chromatography on bacitracinsepharose. Western blot analysis using speci??c antibod-
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Parasitology Section
ies and protein sequencing revealed that the 48-kDa molecule represents EhCP1, whereas the 35-kDa protein represents EhCP2. Thus, the repeatedly described but so far uncharacterized “higher molecular mass proteinases” are in fact already known amoeba enzymes. The reason for the disparate migration behaviour of the two CPs is not yet clear. Whether oligomerisation or aggregation of mature CPs or the presence of active proforms are responsible, remains to be determined. However, from the results of N-terminal sequencing of the puri??ed 35 and 48 kDa enzymes, the possibility of active proforms is rather unlikely.
Selected Publications
?? Hellberg A, Nickel R, Lotter H, Tannich E, Bruchhaus I (2001). Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils. Cell Microbiol 3:13-20 ?? Hellberg A, Leippe M, Bruchhaus I (2000). Two major ??higher molecular mass proteinases“ of Entamoeba histolytica are identi??ed as cysteine proteinases 1 and 2. Mol Biochem Parasitol 105:305-309 ?? Willh??ft U, Hamann L, Tannich E (1999). A DNA sequence corresponding to the gene encoding cysteine proteinase 5 in Entamoeba histolytica is present and positionally conserved but highly degenerated in Entamoeba dispar. Infect Immun 67:5925-5929 ?? Jacobs T, Bruchhaus I, Dandekar T, Tannich E, Leippe M (1998). Isolation and molecular characterization of a surface-bound proteinase of Entamoeba histolytica. Mol Microbiol 27:269-276 ?? Bruchhaus I, Jacobs T, Leippe M, Tannich E (1996). Entamoeba histolytica and Entamoeba dispar: differences in numbers and expression of cysteine proteinase genes. Mol Microbiol 22:255-263
Overexpression of EhCP2 in E. histolytica and E. dispar
To study the role of CPs in the pathogenicity of E. histolytica, we have attempted to overexpress the three main CPs (EhCP1, EhCP2, and EhCP5) of this parasite in trophozoites of E. histolytica as well as in non-pathogenic E. dispar by episomal transfection. Although each of the corresponding coding sequences were cloned in identical expression plasmids, we were unable to overexpress EhCP1 or EhCP5, but could substantially induce expression of EhCP2 in both amoeba species by 7-fold leading to a 3-fold increase in total cysteine proteinase activity. Overexpression of EhCP2 did not in??uence expression of other CPs and could be attributed to an increase of a single 35-kDa-activity band in substrate gel electrophoresis. In contrast to previous findings, which indicated that amoeba CPs are involved in erythrophagocytosis or liver abscess formation cells overexpressing EhCP2 revealed no differences in erythrophagocytosis or liver abscess formation compared to respective controls. However, overexpression of EhCP2 in both amoeba species resulted in a marked increase of in vitro monolayer destruction.
Funding
Deutsche Forschungsgemeinschaft (DFG)
Cooperating Partners
?? Prof. Dr. Matthias Leippe, University of Würzburg
Investigators
Iris Bruchhaus Nicolas Nowak Andrea Hellberg Ute Willh??ft Hannelore Lotter Egbert Tannich
Fig.1: Various cysteine proteinases (CPs) have been identi??ed in E. histolytica and E. dispar. However, CP1 and CP5 are absent in E. dispar.
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Parasitology Section
Can German Mosquitoes Transmit Plasmodium falciparum Malaria?
unusual cases occurred during periods of very hot weather in Europe, which in principle would allow completion of the sporogonic cycle of P. falciparum in indigenous Anopheles species. Recently, possible transmission of PFM in Europe by An. plumbeus has been suggested. This rare indigenous mosquito species which preferentially breeds in water-??lled holes of old beech trees has been shown to be susceptible to P. falciparum in experimental infections. So far, natural malaria transmission by An. plumbeus has not been demonstrated.
Zusammenfassung
F??lle von autochthoner ‘Malaria tropica’ sind in Mitteleuropa sehr selten, wurden aber wiederholt beschrieben. Dabei werden für die ??bertragung des Parasiten (Plasmodium falciparum) Bluttransfusionen oder importierte infekti??se Anopheles-Mücken verantwortlich gemacht. Wir berichten über zwei zeitnah aufgetretene F??lle von Malaria tropica bei deutschen Kindern ohne vorausgegangenen Tropenaufenthalt. Beide F??lle ereigneten sich in einem westdeutschen Krankenhaus, in dem zur gleichen Zeit ein angolanisches Kind mit chronischer P. falciparum-Infektion behandelt wurde. M??gliche ??bertragungswege, wie importierte Mücken oder Bluttransfusionen, konnten weitgehend ausgeschlossen werden. Stattdessen k??nnte eine ??bertragung durch die einheimische Mückenart Anopheles plumbeus stattgefunden haben.
Project Description and Results
We retrospectively analysed two cases of autochthonous PFM, which were suspected to be transmitted by local mosquitoes. The cases occurred within one week on the paediatric ward of a hospital in Duisburg, Germany, in the summer of 1997. Both patients had no travel history to malaria-endemic areas and had never received any blood transfusions. A six-year-old girl from Angola with chronic P. falciparum-infection was present at the same time for treatment of an extensive mandibular abscess (Fig. 1). As no clinical signs of malaria were evident, priority was given to abscess treatment. A speci??c antimalarial therapy was not performed until August 22, so that the Angolan child remained parasite-positive for about 6 weeks. The ??rst autochthonous case (‘A’), a four-year-old girl, was originally hospitalised from August 13 to August 28 for treatment of pyelonephritis. A few days after discharge she developed fever and was re-hospitalised on September 3 with suspected urosepsis. The diagnosis of PFM as revealed by microscopic examination of blood smears was made 6 days after re-admission.
Summary
Autochthonous Plasmodium falciparum malaria (PFM) in Central Europe has repeatedly been reported. Transmission of the parasite has been attributed to blood transfusion or imported P. falciparum-infected vectors. We report two cases of PFM in German children without travel history to malaria-endemic areas. Both infections occurred during a stay in a hospital where, at the same time, a child from Angola with chronic P. falciparum infection was hospitalised. Known routes of transmission, such as imported mosquitoes or blood transfusion, were very unlikely or could be excluded, whereas evidence was obtained for transmission by the indigenous mosquito species Anopheles plumbeus.
Introduction
Natural transmission of Plasmodium falciparum malaria (PFM) depends on the presence of infected individuals, a tropical climate, and the occurrence of susceptible Anopheles vectors. In Central Europe PFM has never been endemic, in particular due to the fact that the most common and widely distributed Anopheles species (members of the An. maculipennis complex) are refractory to P. falciparum. Therefore, autochthonous infections with P. falciparum are considered to be transmitted either by blood transfusions or by infected Anopheles imported from endemic areas. The latter is known as ‘airport’ or ‘baggage malaria’. Although transmission by blood transfusion or by imported mosquitoes is well documented or were most likely in most cases of autochthonous PFM, the route of transmission in a few cases remained dif??cult to explain. Interestingly, all these
Fig.1: Courses of hospitalisation of the two German patients (A,B) and the Angolan child (I) and corresponding temperature data. Shown are minimum, maximum (in grey) and mean (black line) daily temperatures in Duisburg during July to September 1997 and duration of stay in the hospital of the three patients. S, beginning of malaria symptoms; T, introduction of antimalarial treatment.
The other autochthonous case (‘B’) was a 22-year-old female with cystic ??brosis suffering from pneumonia. She was hospitalised from August 15 to September 24 and developed malaria symptoms on September 10. The im-
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Parasitology Section
mediate diagnosis of malaria was con??rmed by demonstration of P. falciparum in blood smears. All patients responded well to adequate antimalarial treatment and became free of parasite within a few days. Questions remained open about the way by which the two German patients had become infected with P. falciparum as, for the following reasons, transmission by infected mosquitoes imported from the tropics or from patient to patient during hospitalisation were very unlikely. (i) Visitors from tropical countries that might have introduced infected mosquitoes into the hospital were not identi??ed, and the Angolan child was treated for three weeks in another German hospital prior to transfer to Duisburg, which excludes survival of imported Anopheles up to the admission dates of the two German patients. (ii) The closest international airport is that of Düsseldorf about 13 km apart from the hospital, which is beyond the known migration distances of Anophelines. In addition, only a very limited number of ??ights from P. falciparum endemic countries are arriving at Düsseldorf airport. (iii) Beside the fact that the three malaria patients were located in different rooms of the paediatric ward, PCRbased microsatellite fingerprint analysis with parasite DNAs extracted from blood smears of the Angolan child and of patient A revealed different genotypes (Fig. 2), which likely rules out direct patient to patient transmission. However, assuming that the Angolan child had a mixed parasite infection, which is common in chronically infected individuals from malaria-endemic areas, new plasmodia genotypes may occur due to recombinational events taking place during parasite development in the mosquito but not during development in the human host. Thus, the microsatellite data and the time-courses of infections were in agreement with transmission from the Angolan child to the German patients via an indigenous Anopheles species. However, vector-borne P. falciparum transmission requires an extended and continuous period of elevated temperatures, which is necessary for development of the parasite within the mosquito. Interestingly, in August 1997, over a period of more than 3 weeks, relatively high temperatures were recorded for the area around Duisburg with mean daily values between 21°C and 27°C (Fig. 1). These conditions with daily maxima of up to 34° C are suf??cient for parasite development in less than 14 days. To investigate whether indeed a local Anopheles species might have been responsible for the two autochthonous malaria cases, a series of entomological surveys in the surrounding of the Duisburg hospital were performed in search for putative indigenous P. falciparum vectors. A number of potential mosquito breeding sites such as ponds, water-??lled drains as well as puddles on the ??at roof of the hospital were identi??ed, but all of them were found to be free of Anopheles larvae. However, only 700 meters apart from the hospital, within a small forest, a ??ooded hole in an old beech tree was discovered containing larvae and pupae with characteristics typical for those of An. plumbeus. The species An. plumbeus was further confirmed by the morphological characters of adult mosquitoes, which were reared in the laboratory from collected pupae, leaving no doubt that the potential P. falciparum vector An. plumbeus was breeding in the vicinity of the Duisburg hospital. This ??nding in conjunction with the presence of an individual chronically infected with P. falciparum, the climatic conditions in summer 1997 and the microsatellite data suggest that the two autochthonous malaria cases were likely the result of local parasite transmission by indigenous An. plumbeus. On the other hand, it has to be emphasised that the results provide accumulated evidence but no direct proof for this way of transmission and other ways such as baggage malaria can not be completely ruled out. Nevertheless, the results suggest that transmission by indigenous Anopheles should be considered in otherwise unexplained cases of autochthonous malaria. In addition, although coincidence of conditions as presented here are extremely rare in Central Europe, the possibility of malaria transmission by indigenous mosquitoes argues for continuation of malaria surveillance and rapid identi??cation and treatment of P. falciparum-carriers in Europe.
Selected Publication
?? Krüger A, Rech A, Su X-Z, Tannich E (2001). Two cases of autochthonous Plasmodium falciparum malaria in Germany with evidence for local transmission by indigenous Anopheles plumbeus. Trop Med Int Hlth 6 (12): 983-985
Cooperating Partners
?? Andreas Rech, Klinikum Duisburg, Germany ?? Xin-Zhuan Su, NAIAID, Maryland, USA ?? Birgit Muntau, Rolf D. Horstmann, Department of Molecular Medicine, BNI
Investigators
Andreas Krüger
Fig. 2: Electropherograms of multicopy microsatellite PCR ??ngerprints from parasite DNAs extracted from blood smears of the Angolan child (I) and of patient A. The ordinates measure ??uorescence intensity (arbitrary units) and the numbers indicate the lengths of ampli??ed fragments in base pairs.
Egbert Tannich
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Parasitology Section
Glutathione-dependent Detoxi??cation Processes of Onchocerca volvulus and Caenorhabditis elegans
Zusammenfassung
Die Bildung von Glutathion-Konjugaten durch Glutathion S-Transferasen ist für die Detoxi??kation der Helminthen von essentieller Bedeutung und wird mit der Ausbildung von Resistenzen in Zusammenhang gebracht. Der Schwerpunkt des Projektes liegt auf der Charakterisierung verschiedener Onchocerca volvulus Glutathion S-Transferasen auf Gen-, Transkriptund Proteinebene. Hierbei konnten erstmals membranst??ndige, glykosylierte Glutathion S-Transferasen nachgewiesen und deren Funktion in der Parasiten/Wirts-Interaktion untersucht werden. Bei einer weiteren O. volvulus Glutathion S-Transferase konnte mit Hilfe computergestützter 3-D-Analysen gezeigt werden, dass das Tyrosin-115 einen idealen Andockpunkt für β-Carbonyl-substituierte Glutathion-Konjugate darstellt. Desweiteren konnte der genomische Aufbau einer Glutathion S-Transferase der OmegaKlasse n??her untersucht werden. Funktionelle und Gen-regulatorische Analysen der O. volvulus Glutathion S-Transferase-Promotoren wurden in transgenen Caenorhabditis elegans durchgeführt. Mit Hilfe transgener Wurmlinien konnte die Stadien- und Gewebespezi??t??t der Parasitengene bzw. deren homologen C. elegans Gene verfolgt werden. Das Ausschalten des Glyoxalase-Systems im Parasiten stellt einen weiteren vielversprechenden chemotherapeutischen Ansatz dar. Daher wurde die Glyoxalase I aus O. volvulus isoliert und charakterisiert. Eine Strukturanalyse zeigte parasiten-spezifische Abweichungen zum humanen Enzym, die sich für die Entwicklung spezi??scher Hemmstoffe anbieten. They are detoxi??ed by the glutathione-dependent glyoxalase system. Due to critical differences observed in the hydrophobic binding pocket of the glyoxalase I of O. volvulus compared to the host enzyme, the glyoxalase system has been targeted for the development of lead substances.
Introduction
Glutathione S-transferases (GSTs) are multifunctional proteins that catalyze the conjugation of reduced glutathione (GSH) to a large variety of electrophilic compounds. GSTs are primarily involved in the detoxi??cation of cytotoxic products of the xenobiotic metabolism, the protection of tissues against oxidative damage and, as ligandins, in the intracellular transport of hydrophobic compounds. In a parasitic context it is especially important to consider their function in the regulation of oxidative stress response, in drug resistance and possibly in the modulation of host immune defense mechanisms. The glyoxalase system, consisting of two consecutive enzymatic reactions, represents another glutathione-dependent detoxification system. The physiological substrates are glyoxal, methylglyoxal and other α-oxoaldehydes formed by the lipid peroxidation, glycation and degradation of glycolytic intermediates.
Project Description and Results
The extracellular Onchocerca volvulus GST1a and 1b (Ov-GST1a/1b) possess a cleavable signal peptide and an uncommon N-terminal extension. Both enzymes are extensively glycosylated, bearing truncated Man2-9GlcNAc2 at their N-glycosylation sites as shown by HPLC-MS and ESI-MS/MS analyses (Fig.1). By ELISA we show that Ov-GST1a/1b are immunodominant antigens, with less than 7% nonresponder patients. The direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the gene structure and sequence revealed that the Ov-GST1a/1b belong to the sigma-class. LC-MS analysis identified prostaglandin H2 as the physiological substrate of the enzyme, the isomerization leading to prostaglandin D2 (Fig.2). Ov-GST2 is topologically related to the mammalian π-class GSTs but possesses a more open hydrophobic binding cleft site. The tyrosine 115 residue, located in the GSH-binding site, is a potential target for covalent binding of inhibitors bearing a carbonyl moiety β to the point of GSH conjugation. Derivatisation of β-keto substituted GSH S-conjugates increased selectivity and speci??city over human π-class GSTs. To examine the activity and specificity of the Ov-GST gene promoters, Caenorhabditis elegans was exploited as a heterologous transformation system. To analyse whether transgenic C. elegans are able to carry out processing and posttranscriptional modi??cations of the Ov-GST1a correctly, the protein was ectopically overexpressed in C. elegans.
Summary
The glutathione S-transferases are major cellular detoxification enzymes that catalyse the conjugation of reduced glutathione to a large variety of electrophiles and therefore detoxify both exogenously and endogenously derived toxic compounds. The aim of the project lies in the characterisation of Onchocerca volvulus glutathione S-transferases on genomic, transcript- and protein-level. Apart from several intracellular enzymes, we have identi??ed glycosylated glutathione S-transferases, that occur directly at the parasite-host interface. The design, optimisation and synthesis of β-carbonyl substituted glutathione conjugates directed against the major cytosolic glutathione S-transferase provided a new class of inhibitors. A highly stress-responsive omega-class related glutathione S-transferase was further investigated. Three cDNA sequences, which are alternatively spliced products of the same gene, were obtained. To determine the mechanisms and factors that are involved in the expression of these diverse genes, we used Caenorhabditis elegans for functional analysis of the promoter regions. α-Oxoaldehydes are formed during lipid peroxidation, glycation and degradation of glycolytic intermediates.
28
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The parasite-derived Ov-GST1a gene product was correctly processed in transgenic C. elegans and posttranslational modi??cations, such as signal cleavage and N-glycosylation, were performed successfully. This demonstrates the potential of C. elegans as a host for expression of candidate vaccine antigens from O. volvulus. The OvGST3, identified by differential display RT-PCR, is a highly stress responsive enzyme. The Ov-GST3 gene comprises 8 exons. It is alternatively spliced to 3 different transcripts. Northern blot analysis of the homologous genes in C. elegans revealed strong responses to various oxidative stress conditions. Owing to the high degree of sequence identity (60%) with human glyoxalase I, it has been possible to build a three-dimensional model of Ov-Glo I. The protein was recombinantly expressed and kinetics were determined. Whereas the modelled core of Ov-GloI is conserved compared with the human glyoxalase I, there are critical differences in the hydrophobic binding pocket. By semiquantitative PCR ELISA it was demonstrated that the OvGloI is expressed at elevated levels under oxidative stress conditions. To obtain a clearer picture of the role of the glyoxalases in nematodes we are currently analysing the structural basis of transcriptional regulation of the C. elegans glyoxalase I gene.
Fig. 1: MALDI/TOF spectrum of an glycopeptide fraction isolated by reverse phase HPLC from Ov-GST1a/1b following tryptic digestion. The peptide containing N-glycosylation site 1 (N50) is bearing mannose type glycans with truncated mannose residues (M2, M3, M4) (left side). The peptide containing glycosylation site 4 (N144) (right side) is bearing high-mannose glycans with 5 - 8 mannose residues. The dominant glycan structure of all four N-glycosylation sites is GlcNAc2Man5.
Selected Publications
?? ?? ?? ?? ?? Sommer A et al. (2001). Infect Immun 69: 7718-7728 Krause S et al. (2001). Mol Biochem Parasitol 117: 145-154 Sommer A et al. (2001). Biochem J 353: 445-452 Liebau E et al. (2000). Mol Biochem Parasitol 109: 101-110 Brophy PM et al. (2000). Bioorg Med Chem Lett 10: 979-981
Cooperating Partners
?? N. Nimtz, Gesellschaft für Biotechnologische Forschung mBH, Braunschweig ?? P.M. Brophy, University of Wales, Aberystwyth, UK
Fig. 2: Topology of Ov-GST1a and position of the N-glycan sites. Ribbon presentation of a 3D-model of the Ov-GST 1a as monomer. The structure is based on the squid sigma-class GST (PDB: 1GSQ). Domain I is built up in a βαβαββα structural motive and contains the principal determinants for GSH-binding, while domain II is composed of 5 α-helices, providing the structural elements respon-sible for second substrate binding.
Funding
Deutsche Forschungsgemeinschaft
Investigators
Eva Liebau Stefanie Krause Rolf D. Walter Inga Wilde Alexandra Sommer
Fig. 3: Expression of Ov-GST2::GFP (green ??uorescent protein)-constructs in adult transgenic C. elegans. Strongest staining is observed in the excretory glands and the posterior end of the intestine.
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Parasitology Section
The Polyamine Biosynthesis in Plasmodium falciparum is Well Balanced by a Unique Bifunctional Ornithine Decarboxylase, S-Adenosylmethionine Decarboxylase
Zusammenfassung
In Plasmodium falciparum, dem Erreger der Malaria, wird das Polyamin Spermidin durch die SpermidinSynthase aus den beiden Substraten Putrescin und decarboxyliertem S-Adenosylmethionin synthetisiert. Die Substrate für diese Reaktion werden von einem Protein gebildet, das aus Ornithin- und S-Adenosylmethionin-Decarboxylase (ODC, AdoMetDC) besteht. Dieses bifunktionelle Protein, das bisher ausschlie??lich bei Plasmodien gefunden wurde, bildet nach posttranslationeller Prozessierung aus zwei Pro-Proteinen einen heterotetrameren Enzymkomplex. Der Vorteil dieser Anordnung liegt nicht in einer Kanalisierung der Spermidinsynthese oder einer gegenseitigen Steuerung der ODC- und AdoMetDC-Aktivit??ten, sondern ausschlie??lich in einer geregelten stadienspezi??schen Proteinsynthese. Parasitenspezi??sch ist auch die Regulation beider Enzymaktivit??ten und damit die der Synthese von Spermidin. Putrescin stimuliert nicht wie bei anderen Eukaryonten die AdoMetDC-Aktivit??t, sondern inhibiert die ODC-Aktivit??t, was bei Berücksichtung von Substrat- und Produktkonzentrationen für eine Feed-Back-Regulation durch Putrescin spricht. Parasitenspezi??sche Struktur und Regulation des bifunktionellen ODC/AdoMetDC-Proteins bieten uns Ansatzpunkte, über die Synthese spezi??scher Enzyminhibitoren neue Malariamedikamente zu entwickeln.
Fig. 1: Polyamine metabolism in Plasmodium falciparum
The two rate limiting enzymes of polyamine synthesis are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Both are usually derived from separate genes and act individually as highly regulated monofunctional enzymes. In P. falciparum, however, ODC and AdoMetDC activities are located on a single polypeptide with the AdoMetDC domain in the N-terminal part connected to the C-terminal ODC domain by a hinge region (Fig. 2).
Introduction
Malaria is undergoing resurgence and the control of Plasmodium falciparum malaria has become a severe problem. Due to rapidly spreading drug resistance there is an urgent need for the identi??cation of novel targets in the metabolism of the parasite exploitable for the synthesis of enzyme inhibitors and their development to new antimalarial drugs. Polyamines are ubiquitous and essential cellular components involved in various metabolic processes including cell proliferation and differentiation. Interference with polyamine biosynthesis is considered as antitumour and antiparasitic strategy. We suggest that due to a higher polyamine requirement of rapidly proliferating cells the interference with their synthesis has more severe consequences for the parasite compared to the host organism.
Fig. 2: Schematic organisation of the PfODC/AdoMetDC and the positions of the changed amino acid residues
Project Description and Results
As shown in Fig.1 spermidine biosynthesis in Plasmodium depends on the decarboxylation of ornithine to putrescine and the subsequent attachment of an aminopropyl group to form spermidine.
The mature heterotetrameric enzyme complex consists of two bifunctional polypeptides posttranslationally cleaved in the AdoMetDC domain. Here we address possible functional consequences and the potential signi??cance of the bifunctional nature of this parasite protein, which may help to validate this protein as a target for a chemotherapeutic intervention of malaria. To analyse domain-domain interactions and to determine the steady state kinetic parameters of ODC and AdoMetDC activities, the PfODC/AdoMetDC was recombinantly expressed in E. coli. Attributable to the bifunctional nature of the Plasmodium protein, the similar speci??c activities of PfODC and PfAdoMetDC might lead to a balanced production of putrescine and decarboxylated AdoMet, depending on the availability of their respective substrates. The analyses revealed a sixfold higher concentration for ornithine compared to AdoMet. It is dif??cult to calculate precisely the molarity of both in
30
Parasitology Section
the Plasmodium cell, but based on the relative volume of parasite to host erythrocyte, non-saturated substrate levels of 28.5 ??M and 4.5 ??M for ornithine and AdoMet were estimated. Considering enzyme kinetics and substrate levels we expect that the reaction leading to putrescine is favoured, which should lead to higher putrescine levels in Plasmodium so that the availability of dcAdoMet might be the limiting factor for spermidine synthesis as it has been discussed for mammalian cells. Of some interest is that the PfODC activity of P. falciparum is strongly inhibited by putrescine which allows feed back control of putrescine synthesis. Another important feature in the regulation of polyamine synthesis in Plasmodium is that PfAdoMetDC is not stimulated by putrescine, which shows that P. falciparum lacks the regulatory mechanism proposed for mammalian cells to relate putrescine abundance to the synthesis of spermidine. The comparison of the deduced amino acid sequence from the bifunctional PfODC/AdoMetDC with those of the respective mammalian proteins revealed that the amino acids involved in catalytic activities, protein processing and cofactor binding are conserved in the Plasmodium sequence. To con??rm that these amino acids are essential for processing of the AdoMetDC domain and for ODC domain activity and polymerisation, mutagenic analyses of various residues were performed, their positions are shown in Fig. 2. The PfAdoMetDC domain contains the putative cleavage site 70LeuSerGluSerSer74. Mutation of Ser-73 into alanine prevented the posttranslational cleavage into the β and α subunit. The mutant protein was inactive because the cleavage of the proenzyme is essential to form the pyruvoyl prosthetic group derived from Ser-73. Interestingly, the activity of the PfODC domain was not affected by this mutation despite the fact that part of the recombinant protein did not fold properly anymore. Vice versa, the AdoMetDC activity was not affected by mutations of amino acid residues in the ODC domain, which are thought to be involved in catalysis and conformation of the active site and which destroyed ODC activity (Fig. 3).
The organization of bifunctional compared to monofunctional proteins might have a biological advantage. Data obtained from the mutagenesis experiments suggest however, that PfODC and PfAdoMetDC domains do not interact directly and operate independently of each other. Inactivation of the PfODC and PfAdoMetDC domains by speci??c enzyme inhibitors and by exchange of essential residues did not deplete or enhance the activity of the other domain in the mutant compared to the wild type protein. Furthermore, results with the separately expressed and enzymatically active PfODC and PfAdoMetDC domains support the data obtained with the bifunctional mutant proteins. A biological advantage of the plasmodial bifunctional ODC/AdoMetDC might be that the control of polyamine synthesis is achieved by only having to regulate the abundance and activity of one protein.
Selected Publications
?? Wrenger C et al (2001). J Biol Chem 276: 29651-29656 ?? Müller S et al (2001). Trends Parasitol. 17: 242-249 ?? Müller S et al (2000). J Biol Chem 275: 8097-8102 ?? Krause T et al. (2000). Biochem J 352: 287-292
Cooperating Partners
?? R. Madhubala, New-Delhi, India ?? A.I. Louw, Pretoria, South Africa ?? Novartis Pharma, Basel, Switzerland
Funding
Deutsche Forschungsgemeinschft Indo-German Biotechnology Programme Vereinigung der Freunde des Tropeninstituts Hamburg e.V.
Investigators
Rolf D. Walter Tanja Krause Sylke Müller Robin DasGupta Kai Lüersen Carsten Wrenger Nashya Haider Ingrid Müller
Fig. 3: Speci??c activities of the rPfODC/AdoMetDC wild type and mutant proteins
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Parasitology Section
The Redox Systems of the Human Malaria Parasite Plasmodium falciparum
Project Description and Results Zusammenfassung
Malaria tropica, verursacht durch den Einzeller Plasmodium falciparum, ist eine der wichtigsten Tropenerkrankungen. In den letzten Jahrzehnten haben die Parasiten Resistenzen gegen die g??ngigen Malariamedikamente entwickelt, d.h. die Identi??zierung und Validierung neuer Angriffspunkte für die Chemotherapie ist dringend erforderlich. In der Vergangenheit ist gezeigt worden, dass mit Plasmodium in??zierte Erythrozyten eine erh??hte Empfindlichkeit gegenüber reaktiven Sauerstoffspezies zeigen; Substanzen, wie Artemisinin-Derivate, die über die Erh??hung von oxidativem Stress auf den Erreger wirken, unterstützen die Theorie, dass die antioxidativen Systeme der Parasiten eine wichtige Funktion für deren ??berleben haben. In den vergangen Jahren haben wir uns mit der Rolle des Thioredoxin-Systems in P. falciparum besch??ftigt und konnten zeigen, dass dieses System eine wichtige Komponente für die Abwehr reaktiver Sauerstoffspezies in P. falciparum darstellt. Ferner sind wir daran interessiert, diesen Stoffwechselweg im Hinblick auf seine Eignung als Angriffspunkt für die Chemotherapie zu evaluieren.
Structural analysis of thioredoxin reductase
Plasmodium possesses a thioredoxin reductase (TrxR) that belongs to the family of high Mr TrxR. The protein has a high degree of similarity to other members of the ??avo oxido reductase family such as glutathione reductase, trypanothione reductase, mercuric ion reductase and dihydrolipoamide dehydrogenase in its primary structure and also in its catalytic mechanism. The mammalian TrxR (also a member of the high Mr TrxR) has a selenocysteine in its active site, which enables the enzyme to reduce a wide range of substrates, whereas the parasite TrxR possesses a cysteine residue at the equivalent position which makes it rather speci??c for its endogenous substrate thioredoxin. This difference gives hope for the development of inhibitors which act speci??cally on the parasite enzyme without affecting the mammalian enzyme and a high throughput screening programme is in process to identify such compounds. In order to learn more about the structure of the active site of the homodimeric Plasmodium TrxR, differentially tagged subunits of the enzyme were co-expressed in Escherichia coli. To elucidate whether the protein forms intersubunit active sites or intrasubunit active sites, the constructs co-expressed were wild-type and double mutants (altered in the N-terminal and C-terminal redox-active centres - C88 and C535 were changed to alanine, respectively) (Fig. 1). The differential tagging allowed the puri??cation of three different enzyme forms, which were synthesised by the bacteria, and the subsequent analysis of their activities revealed that the enzyme possesses intersubunit active sites (Fig. 2 and 3).
Introduction
During recent years the antioxidative and redox systems of the malaria parasite Plasmodium falciparum have attracted a lot of interest. Apart from an Fe dependent superoxide dismutase, a functional glutathione redox cycle and synthesis were determined and it was shown that the parasite possesses thioredoxin reductase (TrxR), which is distinct from the bacterial enzyme and shows parasite speci??c features such as the unique occurence of the Cterminal active site motif CXXXXC, where the human counterpart possesses a highly reactive selenocysteine residue. The involvement of these residues in the catalytic activity of the enzyme has been previously shown and the summary presented here focusses on our studies as to how the active sites of the parasite protein are organized. In addition, the identi??cation and isolation of the natural substrate of P. falciparum TrxR is essential to conduct inhibitor studies with TrxR because E. coli thioredoxin is only a moderate substrate for the parasite TrxR. Further, two peroxiredoxins were identi??ed and shown to be involved in the defence against reactive oxygen species.
Fig. 1: Two possible organisations of the active site of the homodimeric P. falciparum TrxR
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Parasitology Section
Fig. 2: Three protein species synthesised by E. coli co-transformed with plasmids containing Strep-tagged wild-type TrxR and His-tagged mutant TrxR.
state, which is reduced by TrxR. Subsequently electrons are transferred to an acceptor molecule. The recombinantly expressed Plasmodium Trx was reduced by TrxR with high speci??city. The Km values determined in coupled and non-coupled enzyme assays were found to be in the low micromolar range. The kcat (about 2000 min-1) is in agreement with pre-steady state kinetic analyses that were performed on TrxR and support that the reduction of the substrate is achieved via an intersubunit dithioldisulphide exchange reaction. Trx-Px are a family of proteins characterized by either the occurrence of one active site VCT/P or two VCP/T motifs and accordingly are categorised as 1-Cys or 2-Cys peroxiredoxins. It is established that 2-Cys peroxiredoxins are reduced by Trx whereas as yet only two members of the 1-Cys family of Trx-Px have been shown to accept reducing equivalents from the Trx system including the Plasmodium protein. Our results clearly show that both, 1-Cys and 2-Cys, Trx-Px from Plasmodium accept reducing equivalents from the NADPH/TrxR/Trx system and subsequently reduce hydrogen peroxide. These data show that the parasites possess a functional thioredoxin dependent detoxification cycle for reactive hydroperoxides and in the light that neither glutathione peroxidase nor catalase have been yet identi??ed in Plasmodium, this system may prove to be crucial for the parasites’ defence against oxidative stress and their survival during the erythrocytic stages of their life cycle.
Selected Publications ?? Krnajski, Z. et al. (2001). Mol Biochem Parasitol 112: 219-228 ?? Krnajski, Z. et al. (2001). Mol Biochem Parasitol 113: 303-308 ?? Becker, K. et al. (2000). Eur J Biochem 267: 6118-6125 ?? Krnajski, Z. et al. (2000). J Biol Chem 275: 40874-40878 ?? Williams, C.H. et al. (2000). Eur J Biochem 267: 6110-6117
Fig. 3: Purification scheme of differentially tagged protein species synthesised by co-transformed bacteria.
Isolation of Plasmodium thioredoxin and thioredoxin dependent peroxidases
The Plasmodium genome database enabled us to identify other members of the thioredoxin redox cycle such as the natural substrate of TrxR, thioredoxin (Trx) and two thioredoxin-dependent peroxidases or peroxiredoxins (Trx-Px). The genes were cloned, recombinantly expressed and characterized. The thioredoxin gene of P. falciparum contains the typical WCGPC motif. The redox active cysteine pair forms a disulphide in the oxidised
Cooperating Partners ?? R.H. Schirmer, University of Heidelberg, Germany ?? C.H. Williams, Jr., University of Michigan, USA Funding Deutsche Forschungsgemeinschaft (DFG) Investigators Sylke Müller
Zita Krnajski
Rolf D. Walter
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Parasitology Section
Epidemiology and Detection of Lymphatic Filariasis on Alor Island, Indonesia
Zusammenfassung
Lymphatische Filariasis ist ein gro??es Gesundheitsproblem auf der indonesischen Insel Alor. W??hrend an der Küste Wuchereria bancrofti vorkommt, wurde in den Reisanbaugebieten im Inland Brugia timori gefunden. In einem Dorf im Hochland wurde eine standardisierte Pr??valenz der B. timori-Mikro??larientr??ger von 25% gefunden. Die mittlere Mikrofilariendichte dieser Personen lag bei 138 mf/ml. Etwa 40% der Untersuchten zeigten Zeichen einer B. timori-Infektion. Das auff??lligste klinische Zeichen der Infektion waren Lymph??deme der Beine, die bei 13% der untersuchten Personen beobachtet wurden. B. timori hat eine identische repetitive HhaI DNA Sequenz wie Brugia malayi und kann daher mit den gleichen PCR-Methoden nachgewiesen werden. Mit Hilfe eines solchen Ansatzes konnte Anopheles barbirostris als ??bertr??ger festgestellt werden. Die Ergebnisse stellen Basisinformationen für die Bek??mpfung der Filariasis auf Alor dar.
Introduction
Lymphatic ??lariasis (LF) has been targeted by the WHO for elimination as a public health problem. In Asia the key strategy for control is the community-based treatment of the population at risk using a single annual dose of the micro??larcidal drug diethylcarbamazine (DEC). This treatment has been evaluated for W. bancrofti and B. malayi, but not for B. timori. Before establishing and evaluating a control programme for B. timori, detailed data on its distribution, its public health relevance and its improved detection are required. Therefore, the island Alor (East Nusa Tenggara Timor) has been selected to determine the distribution of LF. In a selected village prevalence and morbidity have been assessed and tools for monitoring of B. timori infection in humans and vectors have been evaluated.
Project Description and Results
In four villages in the coastal area bancroftian ??lariasis was detected with micro??laria (mf) prevalences of up to 20%. Whereas in a self-selected population only one person among 628 individuals showed lymphoedema of the leg, up to 29% of the men showed scrotal hydrocele or lymphoedema (Fig. 1A). In four rice-farming villages in the centre of Alor B. timori was found. In the highland village Mainang 586 individuals were examined for B. timori and 157 (27%) mf carriers were detected by ??ltration of night blood. The geometric mean for the mf density of micro??laraemics was 138 mf/ml. Among teenagers and adults, male individuals tended to have higher mf prevalence than females. Prevalence of mf increased with the age and a maximum was observed in the ??fth decade of life (Fig. 2). Many persons reported a history of acute ??larial attacks and 77 (13%) of the examined persons showed lymphoedema of one or both legs (Figs. 1B, 2), sometimes presenting as severe elephantiasis, but no genital LF was observed.
Summary
Lymphatic ??lariasis is a major health problem on the Indonesian island Alor. In the coastal areas Wuchereria bancrofti occurs, whereas in the rice-farming areas in the centre of the island Brugia timori is found. A detailed study of B. timori in a selected highland village revealed a standardized prevalence of micro??lariae-mics of about 25% and a mean micro??larial density of these persons of 138 mf/ml. The most prominent clinical sign was lymphoedema of the leg which was found in 13% of the examined individuals. About 40% of the study population showed signs of B. timori infection. B. timori has an identical HhaI DNA repeat as Brugia malayi and can be detected by the same PCR assays. Using a PCR approach the mosquito Anopheles barbirostris was found to be the local vector of B. timori. These results represent baseline information for a control programme to be established on Alor.
Fig. 1: A. Male with genital ??lariasis from a W. bancrofti endemic area. B. Severe lymphoedema of both legs in a person from an area highly endemic for B. timori. C. Local hygiene to reduce the progression of lymphoedema in Mainang village.
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Parasitology Section
B. timori low and elimination of this infection might be more easily achievable. Although most tools for control and monitoring of B. malayi may be also applicable for B. timori, further studies on the epidemiology and treatment of B. timori are required.
Selected Publications
?? Supali T, Wibowo H, Rückert P, Fischer K, Ismid IS, Purnomo, Djuardi Y, Fischer P (2001). High prevalence of Brugia timori infection in the highland of Alor island, Indonesia. Am J Trop Med Hyg, in press ?? Klüber S, Supali T, Williams SA, Liebau E, Fischer P (2001). Rapid PCR-based detection of Brugia malayi DNA from blood spots by DNA Detection Test Strips??. Trans R Soc Trop Med Hyg 95: 169-170 ?? Fischer P, Supali T, Wibowo H, Bonow I, Williams SA (2000). DNA detection of nocturnally periodic Brugia malayi in night and day blood samples by a PCR-ELISA based method including an internal control. Am J Trop Med Hyg 62:291-296
Fig. 2: Prevalence of individuals with micro??laraemia and/or lymphoedema in a B. timori endemic village by age
Since B. timori and its close relative B. malayi have an identical HhaI DNA repeat, PCR assays based on this target were evaluated for the detection of B. timori. The PCR assay was able to detect a single mf of B. timori on a 30 ??l ??lter paper blood spot. No differences in the sensitivity of the detection of both parasite species were recorded. For the identi??cation of the local vector, mosquitoes were pooled and tested by PCR. Since almost all pools of A. barbirostris mosquitoes were positive by PCR (Fig. 3), it could be confirmed that this species is the vector of B. timori. It is concluded that the described PCR assays can be also employed for the monitoring of B. timori in the framework of the Global Programme to Eliminate Lymphatic Filariasis. Pilot studies on a single dose treatment strategy to control B. timori revealed that DEC (6 mg/kg) alone or in combination with albendazole (400 mg) is effective against this parasite. However, more side effects following treatment occur in B. timori as compared to W. bancrofti. We conclude that B. timori is locally still of great public health importance. Nonetheless, in comparison to W. bancrofti and B. malayi is the global prevalence of
Cooperating Partners
?? Dr. Taniawati Supali, University of Indonesia, Jakarta ?? Dr. Paul Rückert, German Agency for Technical Co-operation (GTZ/SISKES), Kupang, Indonesia
Funding
Scholarship Programme Infectiology (BMBF); UNDP/World Bank/WHO/TDR
Investigators
Peter Fischer Simone Klüber Insa Bonow Sven Pischke Kerstin Fischer*
*Department of Helminthology
Fig. 3: Detection of the HhaI repeat of B. timori in pools of mosquitoes by PCR. Agarose gel, lane 1, 2 controls, lanes 3-11 A. barbirostris, lanes 12-14 Culex.
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Parasitology Section
Microsporidia: Investigation of the Chitinous Spore and Use of a Fluorescence-based Stain
mechanism of polar tube extrusion through the chitinous spore wall. Nothing has so far been known about the chitinolytic acitivty in viable spores and the in??uence of competitive inhibitors of chitin synthetase enzymes on the development of these parasites. Faeces of immunocompromised patients are often contaminated with both the chitin-containing spores of Microsporidia and Candida which exclude the use of Calco??uor white M2R for the identi??cation of Microsporidia spores. Salmonids in worldwide distributed ??sh farms are exposed to infections with the microsporidian spores of Nucleospora salmonis which cause immune dysfunction and ??nally death. The diagnosis of these spores in imprints of fresh and defrosted ??sh material is a prerequisite to prevent the distribution of these parasites. Diagnosis of the disease is an important precondition to prevent distribution of the parasites with ??sh exported to other countries.
Zusammenfassung
Mikrosporidien sind als Krankheitserreger von zunehmender Bedeutung für Mensch und Tier, insbesondere als opportunistische Protozoa für Patienten mit Immuninkompetenz. In unseren Untersuchungen konnte in Sporen der Gattung Encephalitozoon erstmalig eine Chitinaseaktivit??t, nachgewiesen werden, aber keine Cellulaseaktivit??t. ??berdies wurde gezeigt, dass die Chitinsynthetasehemmer Polyoxin D und Nikkomycin Z die Entwicklung von E. cuniculi (Humanstamm) in der Gewebekultur signi??kant hemmen. Für die diagnostisch bedeutsame Unterscheidung der chitinhaltigen Sporen von Microsporidia und Candida wurde eine Kombinationsf??rbung aus Calcofluor white M2R, modifizierter Trichromblauf??rbung plus Methylenblauf??rbung entwickelt. Beim Nachweis des ??schpathogenen Erregers Nucleospora salmonis (bedeutsam für Lachsfarmen) mit der herk??mmlichen Gramf??rbung wurde festgestellt, dass die Sporen lediglich in frischem, nicht aber in zuvor tiefgefrorenem Gewebematerial nachgewiesen werden k??nnen. Eine Alternative ist die F??rbung mit Calco??uor white MR2.
Results
By employing 4-methylumbelliferyl-beta-D-NN’,N’’’-triacetylchitotriose substrate in a semi-quantitative assay, chitinolytic activity in viable spores of the genus Encephalitozoon was detected and a dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80°C for 10 min or at 55°C for 20 min the spores lost their chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity was not observed. A signi??cant reduction of the in vitro development of a human-derived strain of Encephalitozoon cuniculi to the chitin synthetase inhibitors polyoxin D and nikkomycin Z was found. Polyoxin D was approximately 10 fold more effective than nikkomycin Z. A combination staining of Calcofluor white M2R with modi??ed trichrome-blue and subsequent methylene-blue incubation was developed, and permits the differentiation between spores of Microsporidia and Candida. Under fluorescence conditions (365nm – 440 nm) Microsporidia spores have a spotted, brilliant white Calco??uor ??uorescence while Candida spores show a reddish purple colour (Fig.1). For the detection of microsporidian spores, Gram staining (positive) is usually being used because the detection of Calcofluor fluorescence is not possible with standard basic microscope equipment. It was found that the detection of spores of Nucleospora salmonis in tissue material of Salmo salar by Gram staining is not possible when this material has been kept deep frozen and then defrosted at 4°C over several hours. The Gram characteristic gets lost. The spores could only be made visible by elucidation of the defrosted ??sh material in Calco??our (Fig. 3).
Summary
Microsporidia are of increasing importance as pathogens for man and animals especially as opportunistic protozoa for immunocompromised patients. In our investigations, chitinase activity could be detected in spores of the genus Encephalitozoon while cellulase activity could not be found. Moreover, we could demonstrate that the chitin synthetase inhibitors polyoxin D and nikkomycin Z signi??cantly inhibit the development of E. cuniculi (human strain) in tissue culture. For a clear distinction between the chitin-containing spores of Microsporidia and Candida, a combination staining of Calco??uor white M2R, modi??ed trichrome-blue staining, and subseqent methylene-blue incubation was developed. Concerning the detection of Nucleospora salmonis (important for ??sh farms) by the routinely used Gram staining, we found that the spores can be detected only in fresh tissue material but not in material that has been kept deep frozen and defrosted. An alternative method for the detection of spores of Nucleospora salmonis is staining with Calco??uor white M2R.
Introduction
Microsporidia, spore-forming, obligate intracellular living protozoa with a worldwide distribution in invertebrates, vertebrates and man, are increasingly recognized as the cause of severe infections mainly in immunocompromised patients and animals. Little is known about the
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Fig 1: Threefold combination staining: The microsporidia have a brilliant white spotted and Candida a reddish purple colour
Fig 2: Nucleospora salmonis: Gram staining of imprint of defrosted kidney material from Atlantic salmon. No spores are visible (light microscope, oil immersion, x 1000)
Fig 3: Nucleospora salmonis: As ??gure 2, but viewed using UV calcofluor fluorescence ( x1000 ), ( Bar = 2,5??m ). The spore show a brilliant white ??uorescence
Selected Publications
?? Guzman O, Enriquez R, Schottelius J (2001). Detection of spores of Nucleospora salmonis in frozen tissue material from Atlantic salmon. Bull Eur Ass Fish Pathol 21: 81-83 ?? Sobottka J, Bartsch K, Schottelius J, Laufs R (2001). In vitro activity of polyoxin D and nikkomycin Z against Encephalitozoon cuniculi. IJMM 291: 183 ?? Schottelius J, Hünger F, Goncalves da Costa SC (2000). Chitinolytic activity in viable spores of Encephalitozoon species. Mem Inst Oswaldo Cruz 95: 701705 ?? Schottelius J., Kuhn AM, Enriquez R (2000). Microsporidia and Candida spores: Their discrimination by calco??uor, trichrome-blue and methylene-blue combination staining. Trop Med Int Hlth 5: 453-458
Cooperating Partners
?? Dr. Ingo Sobottka, Institute of Medical Microbiology and Immunology, University Hospital Eppendorf, Hamburg ?? Dr. Sylvio C. Goncaves da Costa, Lab. de Imunmodulacao, Dep. de Protozoologia, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil ?? Dr. Ricardo Enriquez and Osiel Guzman, Universidad Austral de Chile, Veterinary Science Faculty, Valdivia, Chile ?? Dr. Anna Maria Kuhn, Biomedical Primate Research Centre, Rijswijk, The Netherlands
Investigators
Justus Schottelius Christa Schmetz Frank Hünger Bernhard Fleischer
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Parasitology Section
Control of the Leishmania Life Cycle via HSP90 Homeostasis
amastigote development by a rise of cultivation temperature to 37°C followed by acidification of the culture medium to pH 5.5. Molecular markers have been discovered which are detectable only in fully differentiated axenic amastigote-like forms, e.g. the products of the A2 gene family. The HSP90 genes of L. major, L. donovani and L. mexicana, formerly designated Hsp83, were shown to be arranged in tandem head-to-tail multicopy gene clusters. HSP90 genes are apparently restricted to a single gene locus per haploid genome. HSP90 is the most abundant heat shock protein in Leishmania, accounting for 2.8% of the promastigote’s total protein, and it shows a cytoplasmic distribution. HSP90 synthesis is transiently induced under heat stress. However, this transient induction does not lead to a signi??cant increase of the protein’s large intracellular pool.
Zusammenfassung
Die Differenzierung von Leishmania-Parasiten vom Insekten-Stadium, der Promastigote, zum pathogenen S??ugetierstadium, der Amastigote, wird haupts??chlich durch den Temperaturanstieg w??hrend der ??bertragung vom Insekt zum S??ugetier ausgel??st. Wir konnten zeigen, dass die Inaktivierung von HSP90 durch Geldanamycin oder Radicicol diese ??bertragung simuliert und die Differenzierung von der Promastigote zur Amastigote induziert. Geldanamycin verursacht au??erdem in Promastigoten einen Wachstumsarrest, der durch ??berexpression von HSP90 aufgehoben werden kann. Weiterhin konnten wir zeigen, dass HSP90 in Leishmania als feedback-Inhibitor der zellul??ren Hitzeschockantwort dient. Unsere Resultate legen den Schluss nahe, dass die HSP90Homeostase als zellul??res Thermometer dieser Parasiten dient, und sowohl die Hitzeschockantwort als auch die Morphogenese kontrolliert.
Project Description and Results
Since HSP90 is an essential protein in all organisms, all approaches to generate gene replacement mutants to analyse HSP90 function have failed to date. However, the benzoquinoid ansamycines, originally developed as antitumour drugs, were found to speci??cally inhibit members of the HSP90 family of chaperones. Of these compounds, geldanamycin (GA) is widely used to study the effects of HSP90 inhibition. GA interacts with the ATPbinding site in the N-terminus of HSP90 which serves to regulate the conformation of this chaperone. We found GA to induce a dose-dependent growth arrest in Leishmania donovani that is due to a speci??c inhibition of HSP90. This growth arrest occurs in G2/M phase of the cell cycle and can be forestalled by overexpression of HSP90. Moreover, inhibition of HSP90 by geldanamycin will lead to a lasting induction of heat shock protein synthesis, including HSP90, HSP70, and HSP100. The two phenomena observed following inhibition of HSP90, growth arrest and induced HSP synthesis, are normally observed under heat stress. This prompted us to compare the two stimuli, heat stress and GA treatment, in the context of Leishmania stage differentiation. We analysed the morphology of L. donovani promastigotes after GA treatment. Figure 1a shows elongated, ??agellated promastigotes, cultivated at 25°C. After 24 hours of heat shock at 37°C (Fig. 1b), a reduction of the ??agellum and a differentiation towards the round amastigote-like shape is observed. The same effect is achieved by GAmediated inhibition of HSP90 at normal temperature of 25°C (Fig. 1c). We conclude that pharmacological inhibition of HSP90 is suf??cient to induce morphological differentiation towards axenic amastigote-like forms.
Summary
The differentiation of Leishmania parasites from the insect stage, the promastigote, towards the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We have shown that inactivation of HSP90, using the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes which can be forestalled by overexpression of the cytoplasmic HSP90. Moreover, we demonstrate that HSP90 serves as a feed-back inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with HSP90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation.
Introduction
Protozoan parasites of the genus Leishmania are found in two morphologically distinct life cycle stages, the promastigote and the amastigote. Transmitted as elongated, ??agellated promastigotes by phlebotomic sand??ies, the parasites persist as round, a??agellated amastigotes inside the host’s macrophages causing the various forms of leishmaniasis. The heat shock encountered during the transmission from arthropod vectors into mammalian hosts is known to be an important signal inducing the onset of the cellular differentiation towards the mammalian stage. L. donovani and L. infantum can be induced towards axenic
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Parasitology Section
Fig. 1: Scanning electron micrograph of Leishmania donovani promastigotes (a), axenic amastigotes cultivated at 37°C and pH5.5 (b), and cells treated with 200 ??g/ml geldanamycin for 48 hrs.
Inhibition of Hsp90 also induces the synthesis of at least one group of amastigote speci??c proteins, the A2 proteins. Figure 2 shows that the A2 proteins, normally restricted to tissue amastigotes and axenic amastigote stages, are expressed immediately upon GA-mediated HSP90 inhibition (Fig. 2).
Fig. 2: Immunoblot analysis. The antibody recognises the various members of the amastigote speci??c A2 protein family. L. donovani cells were incubated at various geldanamycin concentrations (lanes 2-6), with the Hsp90 inhibitor radicicol (lane 7), the drug solvent DMSO (8), or at 37°C. Cells were harvested and equal numbers of cells were lysed in SDS sample buffer and subjected to SDS-PAGE and immunoblot.
hibitory effect of GA indicate its importance for general viability and proliferation. In other eukaryotic model systems, the multiprotein HSP90 chaperone complex acts on cell cycle regulators and transcription factors. During a heat stress signal, HSP90 acts as general chaperone and is sequestered by partially denatured and aggregated proteins. In Leishmania spp., too, HSP90 may serve as chaperone to regulatory proteins whose function would then depend on HSP90 homeostasis. As long as HSP90 is in suf??cient supply, the rapidly proliferating promastigote stage is maintained. Sequestration of HSP90 during ??y to mammal transmission may then trigger amastigote differentiation. Pharmacological inhibition of HSP90 may thus mimick the conditions in the parasite after transmission into the mammalian host. The mechanisms by which the amastigote stage is induced and/or maintained are crucial for parasite survival within mammalian host cells. The identification of the client proteins of the HSP90 chaperone complex may therefore lead to the discovery of novel drug target molecules. This is especially promising since the effector molecules of signal transduction pathways in Leishmania spp. should be different from those in mammalian systems. This can be expected due to the absence of gene speci??c transcription control in Leishmania spp. and the concomitant lack of promoter-binding transcription factors, the modification of which is normally affected by signal transduction cascades. In Leishmania, gene expression control takes effect at a post-transcriptional level. Thus, the effector molecules regulating stage-speci??c gene expression may be targeted without affecting crucial host cell proteins. To this end we have entered into a proteome analysis of Leishmania cells before and after HSP90 inhibition to identify proteins which are directly or indirectly dependent on HSP90 function. Candidate proteins will be tested individually for their role in stage differentiation by reverse genetics approaches.
Selected Publications
?? Wiesgigl M, Clos J (2001a). Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani. Mol Biol Cell 12: 3307-3316 ?? Wiesgigl M, Clos J (2001b). Leishmania and the Leishmaniases: the heat shock protein 90 of Leishmania donovani. Med Microbiol Immunol 190: 27-31
In summary, we propose that Hsp90 homeostasis may serve to sense the parasite’s respective environment, ??y gut or mammalian tissue, and to induce the corresponding differentiation programs towards the two stages, promastigote or amastigote. The relatively high level of this chaperone in promastigotes and the growth in-
Investigators
Martina Wiesgigl Andrea MacDonald Joachim Clos
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Parasitology Section
Signal Transduction in Leishmania: Protein Kinases as Potential Drug Targets
Zusammenfassung
Parasitische Protozoen der Gattung Leishmania (Kinetoplastida, Trypanosomatidae) durchlaufen einen digenetischen Lebenszyklus mit zwei morphologisch und biochemisch klar unterscheidbaren Lebensstadien. Sowohl die Differenzierungsvorg??nge des Parasiten, als auch seine Proliferation werden durch Proteinkinasen reguliert. Gegen LeishmaniaKinasen gerichtete, spezi??sche Hemmstoffe k??nnten daher ideale Wirkstoffe darstellen, die die Anpassung des Erregers an den Wirt behindern oder seine Vermehrung unterbinden. Die Isolierung und molekulare Charakterisierung von 9 Proteinkinasen aus Leishmania mexicana schafft die Grundlage zum Studium der Funktion dieser Proteine und ihrer Eignung als Zielstrukturen zur Medikamententwicklung.
Summary
Parasitic protozoa of the genus Leishmania (Kinetoplastida, Trypanosomatidae) have a digenetic life cycle comprising two morphologically and biochemically distinct life cycle stages. Both differentiation and proliferation of the parasite are regulated by protein kinases. Speci??c inhibitors directed against Leishmania kinases could therefore represent ideal agents to hinder the adaptation of the pathogen to its host organism or to prevent parasite proliferation. The isolation and molecular characterization of 9 protein kinases from Leishmania mexicana forms the basis to study the function of these proteins and their suitability as targets for drug development.
Fig. 1: Life cycle of Leishmania
Introduction
Leishmaniasis, an infection caused by the parasitic protozoon Leishmania, has four main manifestations: cutaneous leishmaniasis is characterized by skin lesions that are often self-healing and lead to immunity to re-infection; diffuse cutaneous leishmaniasis with disseminated lesions; mucocutaneous disease affecting the mucous membranes of the nose, mouth and throat; and visceral leishmaniasis, the most severe form of the disease, characterized by irregular fever, pancytopenia, hepatosplenomegaly, hypergammaglobulinaemia, and anaemia. Worldwide, 12 million cases of leishmaniasis have been estimated, with 1.5 – 2 million new cases occuring annually (World Health Organization 1998). The disease is endemic in 88 countries on four continents with a total of 350 million people at risk of infection and is prevalent wherever mammalian reservoirs and phlebotomine sand??ies, the insect vectors, exist in suf??cient numbers to permit frequent transmission.
During their life cycle, the parasites undergo profound morphological and biochemical changes (Fig. 1). The spindle-shaped, flagellated procyclic promastigotes, which proliferate in the gut of the sand??y, differentiate into the non-dividing infective metacyclic cells. When the insect feeds on a mammal, the metacyclics are transmitted into the skin and are rapidly taken up by host macrophages where they reside in phagolysosomes. The low-pH hydrolytic environment and the elevated temperature in the mammal induce the transformation of the promastigotes into the oval, non-motile amastigotes. Amastigotes, the second proliferative stage of the parasite, are smaller than promastigotes and have a rudimentary ??agellum buried in the ??agellar pocket. Amastigote proliferation ultimately leads to the rupture of the macrophages and liberation of the amastigotes which subsequently can enter new host cells. Both, differentiation and proliferation of Leishmania are likely to be regulated by protein kinases and phosphatases. Therefore, these proteins represent potential targets for inhibitors which could be used as drugs to treat Leishmaniasis.
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Parasitology Section Project Description and Results
Mitogen-activated protein (MAP) kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress responses in eukaryotic cells. As such they represent promising targets for inhibitors. We used degenerate oligonucleotide primers deduced from highly conserved amino acid motifs of MAP kinases to amplify DNA fragments from genomic DNA of Leishmania mexicana. Subcloning and sequencing revealed 9 different potential kinase gene fragments, one of which encodes a cdc2-related protein kinase. The entire open reading frames of the corresponding genes were isolated from a genomic DNA library of Leishmania mexicana and sequenced. Moreover, one additional MAP kinase homologue gene was already present in the Leishmania major genome project database which allowed us to clone and sequence the Leishmania mexicana homologue. Fig. 2 schematically shows the amino acid sequence alignment of the 9 MAP kinase homologues found in Leishmania mexicana. The primary structure alignment was improved by comparison of the secondary structure of the mammalian stress-activated protein kinase p38 with predicted secondary structure elements of the individual Leishmania kinases. All 12 kinase subdomains are highly conserved in sequence and localization in the Leishmania MAP kinase homologues. Three sites of extended amino acid insertions were identified. When superimposing the three-dimensional structure of p38 on LmxMPK7 and LmxMPK8, they were localized in loop regions of the molecules. Moreover, long carboxy-terminal extensions have been found in some of the Leishmania kinases. With the kinase genes at hand we started to create deletion mutants for the individual genes to analyse the function of the respective proteins and their suitability as drug targets. In addition to the deletion mutant for LmxMPK1 which has been proven to be non-infectious, we were able to generate a deletion mutant for LmxMPK9. Its phenotype is currently under investigation.
Selected Publications
?? Wiese M, G??rcke I (2001). Homologues of LMPK, a mitogen-activated protein kinase from Leishmania mexicana, in different Leishmania species. Med Microbiol Immunol 190: 19-22 ?? Wiese M (1998). A mitogen-activated (MAP) protein kinase homologue of Leishmania mexicana is essential for parasite survival in the infected host. EMBO J 17: 2619-2628
Investigators
Martin Wiese Eva Kampen Florian Bengs Daniela Kuhn
Fig. 2: Schematic amino acid alignment of Leishmania mexicana MAP kinase homologues.
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Medical Microbiology Section
Selected Scienti??c Projects Ausgew??hlte wissenschaftliche Projekte
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Medical Microbiology Section
Medical Microbiology Section
Chairman’s Summary
The Medical Microbiology Section combines the Departments of Immunology, Virology, and Helminthology, the Central Diagnostic Unit and the animal facilities. Selected projects are described in the following reports. The Department of Immunology is mainly concerned with the immune response against parasites but also performs work in basic immunology. The work on malaria has been extended by the use of the Plasmodium berghei model in mice. In this model the role of the negatively regulating T cell molecule CTLA-4 (CD152) was analysed. The previous finding that in malaria patients CTLA-4 expressing T cells are increased is now followed up in the murine model, that can be used for a blockade of CTLA4 in vivo. Clearly the negative regulation by this molecule is essential to downregulate the T cells, and blockade of CTLA-4 leads to immunopathology (Thomas Jacobs). Alternative pathways besides the T cell receptor can induce activation of the T cell such as CD26, a multifunctional surface protease and receptor. The CD83 molecule was further analysed and evidence for the existence of a ligand was reported. Although the ligand is still unknown, soluble CD83 immunoglobulin chimaeric molecules have an in??uence on T cell activation by antigen, suggesting the possibility that CD83 plays an important role in T cell activation (Arne von Bonin). Also involved in the activation of the T cell system are certain heat shock proteins that are known to bind antigenic peptides but their major role is the activation of antigen presenting cells. These evolutionary conserved proteins provide a link between innate and adaptive immune system (Arne von Bonin). The different immune responses to Trypanosoma cruzi of inbred mouse strains lead to extreme differences in susceptibility. Genes that confer susceptibility of black mice to T. cruzi were mapped by scanning for microsatellite markers, and two loci were de??ned (Sebastian Graefe). The involvement of various immune mechanisms in the resistance of mice to the parasite is still unclear, in particular NO appears to be a prerequisite only in the ??rst days of the infection and it is not necessary to control the chronic stage of the infection (Michael Saeftel). The Research on Lassa virus in the Department of Virology is expanding. Glycoproteins were expressed and peptide mapping of the T cell epitopes was initiated. Three isolates of Lassa virus were obtained from cases imported to Europe. The sequences show three new strains of Lassa virus which display serological differences (Stephan Günther). These differences have to be
taken into account when testing samples for Lassa virus. A new sero-diagnostic test for Dengue fever was developed that can be used in endemic areas. The serotypes can be directly identi??ed by detecting type-speci??c antibodies with glycoprotein domains expressed in E. coli. This assay will allow to determine the prevalence of the different serotypes in endemic regions (Diana Ludolfs). Additional work was performed to de??ne the enzymatic complex of RNA-unwinding enzymes of Flaviviruses with the aim of de??ning inhibitor molecules (Peter Borowski). The impact of glycosylation of the V3 loop of HIV-1 gp120 protein was further investigated. Mutations affecting glycosylation strongly influence the binding of gp120 to chemokine receptors and also may hinder anti-V3 antibodies from binding to the B cell epitopes on the V3 loop which are known to play a role in virus neutralization. A library of V3 loops was synthesized and inserted into expression vectors (Michael Schreiber). In addition to the well established PCR-methods to detect Lassa, Ebola and Marburg viruses (including all substrains), new ??in tube PCRs“ were developed for a number of tropical viruses. These protocols allow a rough quantitative measurement of viral RNA in the blood. By using these RT-PCRs the load of viral RNA could be followed during infection in patients returning to Europe with acute Lassa virus infections, and was determined in several cases from Nigeria. This was also possible in the blood of two patients with yellow fever from Ivory Coast and The Gambia, respectively, and in two patients with Crimean-Congo fever from Prishtina, Kosovo. The Department of Virology received again public attention due to several cases with virus-induced acute haemorrhagic fever (VHF) in Germany and other European Countries. During 2000 and 2001, viral haemorrhagic fever had been suspected in 45 patients from various European countries. For these patients an emergency diagnostic procedure had to be established. On average a ??nal result is obtained within 6 hours after arrival of the blood specimens in the L4 laboratory. Special attention is paid to the fact that frequently new strains of haemorrhagic fever viruses are being described. Because of the immediately required quarantaine and safety measures in suspected cases of VHF, it is extremely important to obtain a result as fast as possible. A 24 hours emergency hotline has been installed and can be reached day and night via 0049-40-42818-0. Since the biosafety level 4 containment laboratory of the Department has been operating without dif??culties for more than 20 years, experts from several other European countries visited the BNI to obtain advice about construction and maintaining of such a laboratory. The re-established Department of Helminthology concentrates on ??lariasis research. Ongoing work char-
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Medical Microbiology Section
acterizes the T cell response and protective mechanisms in an mouse model of ??larisis, the infection of BALB/c mice with Litomosoides sigmodontis. The role of interleukin-4- and –5-dependent mechanisms is analysed. IL4 is a major factor of micro??larial control whereas IL-5-dependent mechanisms act on adult worms (Lars Volkmann). Interleukin-5 has an effect not only on eosinophilic granulocytes but also indirectly on neutrophils that appear to play an important role in the effector phase (Michael Saeftel). An interesting ??nding is that NK cells play a role in the control of the parasite and that there are alterations in the NK cell subsets expressing certain Ly49 receptors during the course of the infection (Simone Korten). Onchocerciasis research deals with the T cell response of patients with different manifestations of the disease, in particular with the characterization of regulatory T cells from patients with generalized disease (Judith Satoguina). An important topic is the new treatment strategy using antibiotics to target Wolbachia endobacteria. Clinical studies in Ghana are performed that show that depletion of endobacteria from O. volvulus leads to longterm depletion of micro??laria due to ??laricidal effects on adult worms (Sabine Mand). The same is apparently valid for lymphatic ??lariasis that is studied in western Ghana. The Central Diagnostic Unit consists of combined laboratories from the Departments of Immunology, Virology and Clinical Medicine. It performs the direct identi??cation of bacteria, parasites and viruses and the serodiagnosis of parasitic, bacterial and viral infections. New diagnostic methods developed by the research laboratories are evaluated and eventually incorporated into the diagnostic routine. In 2001, a special PCR laboratory was established to widen the spectrum of diagnosticable infectious agents which can be diagnosed by use of molecular biology, such as microsporidia, trypanosomes, M. leprae and several fever viruses. The Diagnostic Unit serves as National Expert Laboratory for malaria, amoebiasis, leishmaniasis, trypanosomiasis, ??lariasis and infections with tropical viruses. In 2001, the Federal Ministry of Health appointed the BNI as the National Reference Centre for Tropical Infections for Germany, starting in 2002. Because of its specialization the Unit receives material submitted from all parts of Germany and also from Denmark and Austria. During the period of public fear of anthrax in October 2001 the Unit received numerous samples that urgently had to be tested for the presence of anthrax spores by diagnostic PCR. All of them turned out to be hoaxes. Experimental investigations in animals are an essential component of research in Tropical Medicine. Certain parasites can only be maintained by passage in animals,
and immunization for the production of monoclonal and polyclonal antibodies has to be performed in animals. Studies on defense mechanisms and possible vaccines against Entamoeba histolytica, Trypanosoma cruzi, Leishmania major and a murine ??laria (Litomosoides sigmodontis) were carried out in mice. The Laboratory Animal Facility cooperates in a number of these scienti??c projects and it supports and advises scientists in planning and executing such experimentation in accordance with the regulations by the Animal Protection Law. Bernhard Fleischer
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Medical Microbiology Section
Sektion Medizinische Mikrobiologie
Bericht des Sektionssprechers
In der Sektion Medizinische Mikrobiologie sind die Abteilungen für Immunologie, Virologie, Helminthologie, die Mikrobiologische Zentraldiagnostik (MZD) und das Tierhaus zusammengefasst. Ein Teil der wissenschaftlichen Arbeiten wird in den folgenden Projektbeschreibungen dargestellt. Die Arbeiten der Abteilung für Immunologie besch??ftigen sich vorwiegend mit der Immunantwort gegen parasit??re Infektionserreger. Die Arbeiten zur Immunologie der Malaria wurden durch die Verfügbarkeit des Mausinfektionsmodells mit Plasmodium berghei ausgeweitet. Die früheren Befunde, dass die T-Lymphozyten von Patienten mit schwerer Malaria gro??e Mengen des negativ regulierenden Moleküls CTLA-4 (CD152) exprimieren, wurden im Mausmodell weiterverfolgt. Experimente mit blockierenden Antik??rpern zeigen, dass die Funktion von CTLA-4 auf T-Zellen essentiell für die Vermeidung von immunpathologischen Vorg??ngen ist (Thomas Jacobs). Alternative Aktivierungswege für T-Zellen k??nnen zur Aktivierung der T-Zellen führen, wie etwa das multifunktionelle CD26-Molekül, das im Rahmen eines Projektes im Sonderforschungsbereich 545 untersucht wird. Ein interessantes Molekül ist das CD83-Molekül, dessen Ligand und Funktion noch unbekannt sind. L??sliche CD83-Immunglobulin-Fusionsmoleküle beein??ussen die T-Zellaktivierung, was auf eine wichtige Rolle im Rahmen der TZellfunktion hinweist (Arne von Bonin). Auch Moleküle des angeborenen Immunsystemsn beeinflussen das T-Zellsystem, z. B. die hochkonservierten Hitzeschockproteine, die antigene Peptide binden, und zu noch unbekannten Rezeptoren transportieren (Arne von Bonin). Inzuchtm??use zeigen extreme Unterschiede in der Empf??nglichkeit gegenüber Trypanosoma cruzi, dem Erreger der Chagas-Krankheit des Menschen. In einem Rückkreuzungsexperiment wurden Genorte, die Empf??nglichkeit in schwarzen M??usen hervorrufen, in einer genomweiten Suche auf den Chromosomen 5 und 17 lokalisiert (Sebastian Graefe). Welche Immunmechanismen für die Kontrolle des Parasiten verantwortlich sind, ist noch unklar. Stickoxyd (NO) ist in den ersten Tagen für das ??berleben der Infektion essentiell, spielt aber nach dieser Zeit und auch in der chronischen Phase keine Rolle (Michael Saeftel). Die Arbeiten der Abteilung Virologie zum Lassa-Virus wurden ausgedehnt, die viralen Glykoproteine wurden rekombinant exprimiert und Epitope für T-Zellen mittels Peptiden de??niert. Drei Isolate des Lassa-Virus wurden aus importierten Lassa-Virusf??llen angezüchtet. Ihre Se-
quenzen ergaben drei neue St??mme mit deutlichen serologischen Unterschieden (Stephan Günther). Diese Unterschiede sind für die Serodiagnostik des Lassa-Fiebers sehr bedeutsam. Ein neuer serologischer Test zum Nachweis der Dengue-Virusinfektion wurde entwickelt, der sich für den Einsatz in Endemiegebieten eignet. Subtyp-spezi??sche Antik??rper k??nnen durch die Bindung an nichtkreuzreagierende Dom??nen der Glykoproteine identi??ziert werden. Dieser Test erlaubt die Bestimmung der pr??valenten Serotypen in Endemiegebieten (Diana Ludolfs). Weitere Arbeiten besch??ftigen sich mit der enzymatischen Aktivit??t der RNA Helicase von Flaviviren mit dem Ziel, spezifische Inhibitoren zu entwickeln (Peter Borowski). Die Bedeutung der Glykosilierung des V3Loops des HIV Glykoproteins gp120 wurde in einem Projekt des Sonderforschungsbereiches 470 untersucht. Mutationen dieser Glykosilierungsstellen haben starken Ein??uss auf die Bindung des gp120 an Chemokinrezeptoren und auf die Bindung neutralisierender Antik??rper an den V3 Loops. Eine Genbank mit V3-Sequenzen wurde in einem Expressionsvektor hergestellt (Michael Schreiber). Zus??tzlich zu den bislang vorhandenen PCR-Methoden zur Identifizierung aller bekannten St??mme von Lassa-, Ebola- und Marburg-Virus wurden PCR Assays für weitere tropische Viren entwickelt. Diese erlauben nun auch eine grobe Quanti??zierung der Virusmenge im Blut. So konnte die Viruslast w??hrend der Infektion in einigen F??llen mit importierter Lassa-Virusinfektion verfolgt werden. Sie wurde auch bei einigen F??llen aus Nigeria bestimmt. Au??erdem gelangen diese Bestimmungen bei Patienten mit Gelb??eber von der Elfenbeinküste und aus Gambia und bei Patienten mit Krim-Kongo-Virusinfektion aus dem Kosovo. Die Abteilung Virologie stand wiederum im Interesse der Medien wegen einiger F??lle von viralem h??morrhagischen Fieber in Deutschland und in anderen europ??ischen L??ndern. In den Jahren 2000 und 2001 gab es 45 derartige Verdachtsf??lle. Für diese F??lle musste ein Bereitschaftsdienst der Virologie eingerichtet werden, da durch die aufwendigen Quarant??ne- und Sicherheitsma??nahmen eine schnelle Diagnose innerhalb von Stunden n??tig ist. Im Durchschnitt vergingen nur 6 Stunden vom Eintreffen der Probe bis zur Diagnose. Ein Notruf wurde eingerichtet und ist über die Telefonnummer 040/42818-0 rund um die Uhr zu erreichen. Die Diagnostik wird kompliziert durch die Beschreibung neuer Virusst??mme, die von den herk??mmlichen Tests nicht erfasst werden. Da das Hochsicherheitslabor inzwischen seit mehr als 20 Jahren in Betrieb ist, kamen Experten aus anderen L??ndern, um sich für den Bau solcher Laboratorien beraten zu lassen.
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Medical Microbiology Section
Die wieder eingerichtete Abteilung für Helminthologie ist vornehmlich mit verschiedenen Aspekten der Filarien besch??ftigt. Laufende Arbeiten charakterisieren die TZell Antwort der Maus gegen Litomosoides sigmodontis, eine Mausfilarie, für die in BALB/c M??usen eine vollst??ndige Entwicklung von der infekti??se Larve bis zur Mikro??larie m??glich ist. Die Analyse der Rolle der Interleukine 4 und 5 ergab, dass IL-4 entscheidend für die Kontrolle der Mikro??larien ist (Lars Volkmann) w??hrend IL-5abh??ngige Mechanismen auf die erwachsenen Würmer wirken, nicht nur über die eosinophilen Granulozyten, sondern auch indirekt auf Neutrophile, die anscheinend wichtige Zellen in der Effektorphase darstellen (Michael Saeftel). Ein neuer Befund ist die Beteiligung von NK Zellen an der Abwehr der Parasiten, wobei sich im Verlauf der Infektion auch die Subpopulationen der NKZellen ver??ndern (Simone Korten). Die Onchocerciasis-Forschung steht weiterhin im Mittelpunkt der Arbeiten. Eine Population von regulatorischen T-Zellen wurde bei Patienten mit generalisierter Infektion gefunden, die für die spezi??sche Immunsuppression bei diesen Patienten verantwortlich sein k??nnten (Judith Satoguina). Ein wichtiger Ansatz war die neue Behandlungsstrategie der Filariasis mit Antibiotika, um die endosymbiotischen Wolbachien aus den Würmern zu entfernen. Klinische Studien in Ghana haben ergeben, dass die Abt??tung der Wolbachien zu schweren und langandauernden Sch??den des Wurmes führt (Sabine Mand). Dies gilt anscheinend auch für die lymphatische Filariasis, für die die Antibiotikatherapie ebenfalls eine neue Therapieoption darstellt. Die Mikrobiologische Zentraldiagnostik des BNI führt die direkte Identi??zierung von Erregern bei Patienten mit bakteriellen, parasit??ren und viralen Infektionen durch sowie die Serodiagnose bei Infektionen mit Bakterien, Parasiten, Rickettsien und Viren. Sie ist Nationales Expertenlabor für Plasmodien, Am??ben, Leishmanien, Trypanosomen und Filarien sowie für Dengue-, Arena- und andere importierte Viren. Ende 2001 wurde sie vom Bundesgesundheitsministerium zum Nationalen Referenzzentrum für tropische Infektionserreger ernannt. Wegen ihrer Spezialisierung erh??lt die Zentraldiagnostik Materialien aus allen Teilen Deutschlands sowie aus D??nemark und ??sterreich zugeschickt. Das P4-Labor wurde in mehreren F??llen für diagnostische Untersuchungen bei Verdacht auf h??morrhagisches Fieber in Anspruch genommen. Es ist in das europ??ische Netzwerk zur Diagnostik h??morrhagischer Fieberviren eingebunden. Neue diagnostische Methoden, die in den Forschungslaboratorien entwickelt werden, werden bewertet und schlie??lich in die diagnostische Routine einbezogen, insbesondere molekularbiologische Methoden, wie der
Nachweis von Mikrosporidien und Trypanosomen. Zur Zeit der Anthrax-Anschl??ge in den USA erhielt die Zentraldiagnostik zahlreiche Einsendungen von verd??chtigen Proben, die mit der PCR auf die Anwesenheit von Milzbrandsporen zu testen waren. Alle Proben waren aber negativ. Experimentelle Untersuchungen an Tieren sind ein essentieller Bestandteil der tropenmedizinischen Forschung. Bestimmte Parasiten k??nnen nur am Leben gehalten werden, wenn sie sich in Tieren vermehren. Immunisierungen zur Gewinnung von monoklonalen und polyklonalen Antik??rpern müssen in Tieren durchgeführt werden. Untersuchungen zu Abwehrmechanismen und zur Effektivit??t von Impfstoffen gegen Entamoeba histolytica, Trypanosoma cruzi, Leishmania major und gegen Litomosoides sigmodontis wurden in M??usen vorgenommen. Das Tierhaus arbeitet in mehreren dieser wissenschaftlichen Projekte mit und unterstützt und ber??t die Wissenschaftler bei der Planung und Durchführung solcher Untersuchungen in ??bereinstimmung mit den Vorschriften des Tierschutzgesetzes. Der Gesundheitszustand der Tiere (fast auschlie??lich M??use) ist ausgezeichnet, Untersuchungen auf Tierpathogene werden regelm????ig durchgeführt und waren immer negativ. Bernhard Fleischer
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Medical Microbiology Section
Medical Microbiology Section
Please note that not all persons listed were employed during the entire reporting period.
Department of Immunology
Scienti??c Staff Prof. Dr. Bernhard Fleischer, Head PD Dr. Barbara Br??ker PD Dr. Arne von Bonin PD Dr. Achim H??rauf Dr. Thomas Jacobs Associated External Members Prof. Erich Mannweiler Dr. Thomas F. Fenner Visiting Scientists Dr. Khaled M. Al-Qaoud, Irbid, Jordan Prof. Dr. Paulo Paes de Andrade, Recife, Brazil Prof. Dr. Cynthia Rayol de Andrade, Recife, Brazil Dr. Klaus Radecke, Essen, Germany
Laboratory Jacobs
Dr. Thomas Jacobs Technical Staff Iris Gaworski Doctoral / Graduate Students Thorsten Lieke Sonja Nicknafs Tanja Plate Beate Schmitter Susanne Tartz
Department of Virology
Scienti??c Staff Prof. Dr. Herbert Schmitz, Head Dr. Peter Borowski Dr. Christian Drosten Dipl. Biol. Petra Emmerich-Paloh Dr. Stefan Günther Dr. Michael Schreiber Dr. Jan ter Meulen Visiting Scientists Dr. Violette Agrba, Sotchi, Russia Dr. Hy-Gong Bae, Berlin, Germany Dr. David Olalaye, Ibadan, Nigeria Doctoral / Graduate Students Jan Altenschmidt Diana Ludolfs Stefan Schilling
Laboratory von Bonin
PD Dr. Arne von Bonin Dr. Minka Breloer Technical Staff Svenja Ehrlich Christiane Steeg Doctoral / Graduate Students Sven Cramer Jochen Hühn Natalie Krey Solveig Moré Anke Osterloh Heiko Weyd
Laboratory Br??ker
PD Dr. Barbara Br??ker Technical Staff Ulricke Klauenberg Doctoral / Graduate Students Claudia Fich Timm Greve Oliver Klein Nandita Tanja Majumder Cathrin Pracht Thorsten Schlotmann Uwe Speck Karoline Steiner Athanasios Tsianakas Uta W??chter Matthias Wolenski
Laboratory Borowski
Dr. Peter Borowski Doctoral / Graduate Students Verena Koch Melanie Lang Michael Reinholz Julian Schulze zur Wiesch Sarah Schalinski
Laboratory Günther
Dr. Stephan Günther Technical Staff Annegret Jung Antje Rhode Martina Westerkofsky Doctoral / Graduate Students Marcel Asper Olaf Kühle
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Laboratory Schreiber
Dr. Michael Schreiber Technical Staff Annette Seifert Sonja Ziegelmair Doctoral / Graduate Students Michael Katt Karsten Krieg Harm Müller Svenja Polzer Ingo Thordsen Korden Walter Tobias Wolk Finn Zedler
Central Diagnostic Unit
Medical Microbiology
Prof. Dr. Bernhard Fleischer Dr. Gisela Bretzel Dr. Sebastian Graefe Stefanie Kramme (AiP) Alexandra Schulz (AiP) Technical Staff Sabine K??hler Fatma Firat Britta Liedigk Birgit Mannes Ute Mehlhoop Gerda Nippold Monika Picker Manuela Reisig Martina Winsche Graduate Student Vera Siegmund
Department of Helminthology
Scienti??c Staff PD Dr. Achim Hoerauf, Head Dr. Simone Korten Dr. Kenneth Pfarr Dr. Michael Saeftel Dr. Judith Satoguina Associated Scienti??c Staff Prof. Dr. Dietrich W. Büttner Prof. Dr. Rolf Garms Visiting Scientists Coralie Martin, Paris, France Alex Debrah, University of Kumasi, Ghana Technical Staff Ingeborg Albrecht Marlis Badusche Kerstin Fischer Doctoral Students Sabine Mand Sabine Specht John Larbi Jussuf Kai?? Lars Volkmann
Virology / VHF Diagnostics Group
Prof. Dr. Herbert Schmitz Dipl. Biol. Petra Emmerich-Paloh Dr. Christian Drosten Marcus Panning (AiP) Technical Staff Stephanie Max Angela Parczani-Hartmann Gabriele Rietdorf Corinna Benthien Nadine Petersen
Clinical Laboratory
Prof. Dr. Manfred Dietrich Technical Staff Doreen G??ttsche Manuela Lemke Anja Rademacher Anke Reinkemeier Claudia Sander-Jülch
Animal Facilities
Dr. Thomas Schüler Technical Staff Beate Richter Support Staff Arshad Ali Horst Fasel Yvonne Richter Karin Wojanowski Wanda Ziglow
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Medical Microbiology Section
Heat Shock Protein 60 (Hsp60) as a ??Dangerous“ Link in Innate and Adaptive Immune Responses
Zusammenfassung
Hitzeschock-Proteine (Hsp) wirken aktivierend auf Antigen-pr??sentierende Zellen (APZ) und induzieren z.B. in??ammatorische Zytokine bzw. führen zu einer verst??rkten Ober????chenexpression von kostimulatorischen Molekülen. Insbesondere Hsp60 stimuliert CD4- und CD8-positive T-Zellen in Gegenwart von suboptimalen Antigenkonzentrationen. Antigen-spezifische T Zellen aus T-Zell-Rezeptor transgenen M??usen werden durch die Zugabe von Hsp60 spezi??sch zur IFN-γ Freisetzung stimuliert. Diese starke Induktion von IFN-γ geht bei Verwendung von APZ aus IL-12 Knockout- (IL-12 KO-) M??usen verloren, ebenso wie bei der Blockade von kostimulatorischen Molekülen durch CTLA-4- Fusionsmoleküle. Unsere Ergebnisse legen den Schluss nahe, dass Hsp60 als eine Art molekulares ??Gefahrensignal“ in angeborener und erworbener Immunantwort fungieren kann (Fig. 3).
Summary
Heat shock proteins (Hsp), like Hsp60, Hsp70 and gp96, act directly on antigen presenting cells (APC), e.g. by inducing the secretion of cytokines. We have analysed the impact of Hsp60 on the antigen-specific activation of CD4+and CD8+ T cells in TCR-transgenic systems. Hsp60 induced low amounts of IFN-γ in the absence of antigenic peptide, however, the release of IFN-γ is signi??cantly increased following the addition of Hsp60 to puri??ed populations of T cells and APC. This effect is strictly correlated with the APCs’ ability to produce IL-12 and is dependent on direct cellular contact. Hsp60, thus, may act as a ??danger-signalling“ molecule when released e.g. from necrotic cells, to initiate both innate and adaptive immune responses (Fig. 3), and its presence has direct consequences for the activation of antigen-specific T cells.
Fig. 1: The Hsp60 induced IFN-γ release is dependent on IL-12 and direct cellular contact. (A) Puri??ed DO11.10 T cells and indicated numbers (x-axis) of peptide-pulsed BALB/c PEC or IL12-/- mice-derived PEC were cultured in the absence (open symbols) or presence (??lled symbols) of Hsp60 (LPS as a control) for 24 h. IFN-γ in the supernatants was determined by ELISA after 24 h (y-axis). (B) Inhibition of co-stimulation with CTLA-4-Ig prevents an enhanced IFN-γ-production of T cells induced by HSP60. Peptide pulsed C57Bl/6 PECs were incubated with puri??ed OT-1 T cells in the presence or absence of Hsp60 with titrated amounts of CTLA-4-Ig fusion protein. After 20 hours, supernatants were analysed for IFN-γ.
Project Description and Results Hsp60 induces speci??cally IFN-γ in na??ve CD4 and CD8-positive T cells.
We employed the mouse strains DO11.10 and OT-1 which express transgenic TCRs speci??c for MHC classII- and MHC class-I-restricted peptides, respectively. As shown in Figure 1, titrated amounts of Hsp60 lead to an increased release of IFN-γ in the culture supernatants of puri??ed DO11.10 and OT-1 T cells and PECs pulsed with suboptimal amounts of antigenic peptide. In contrast to the high induction of IFN-γ by Hsp60, the presence of Hsp60 did not change the amounts of detectable IL-2 or the antigen-dependent proliferation signi??cantly (data not shown). These results indicate that recombinant Hsp60 speci??cally in??uences the activation of antigen-speci??c T cells leading to increased amounts of IFN-γ.
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Effector mechanisms in Hsp60-mediated IFN-γ induction
Since it is known that IL-12 is an important cytokine for the induction of IFN-γ in T- and NK cells, we used PECs from IL-12 KO mice which almost completely failed to increase the amount of detectable IFN-γ in Hsp60-containing cultures (Fig. 2A). Moreover, T cells require a second signal to get completely activated. Blockade of CD28/B7 interactions leads to anergy in naive T cells and incomplete T cell stimulation in effector cell populations. We blocked the CD28 pathway with the help of CTLA-4 immunoglobulin (CTLA-4-Ig) fusion-proteins. Figure 2B shows that addition of CTLA-4-Ig leads to the loss of IFN- γ release. These results demonstrate that the Hsp60 induced speci??c stimulation of IFN-γ in T cells requires both soluble mediators, most importantly IL-12, and direct cellular contact of T cells and APC.
Selected Publications
Wallin RPA, Lundqvist A, Moré SH, von Bonin A, Kiessling R, Ljunggren H-G. (2002). Heat-shock proteins as activators of the innate immune system. Trends in Immunol 23: 130 Moré S, Breloer M, von Bonin A (2001). Eukaryotic heat shock proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells. Int Immunol 13: 1121 Breloer M, Dorner B, Moré SH, Roderian T, Fleischer B, von Bonin A (2001). Heat shock proteins as ??danger signals“: eukaryotic Hsp60 enhances and accelerates antigen-speci??c IFN-γ production in T cells. Eur J Immunol 31: 2051
Fig. 2: The effects of Hsp60 are dose- and time-dependent. Antige pulsed PECs were incubated with TCR transgenic T cells, respectively. (A) in the presence of titrated amounts of Hsp60 (xaxis) DO11.10T cells secreted signi??cant amounts of IFN-γ after 24 hours. (B) shown is the amount of IFN-γ in the supernatants of OT-1 T cultures after the indicated culture-periods (x-axis; closed symbols: Hsp-containing cultures; open symbols: medium; circles: antigen-pulsed APC, squares: controls).
Cooperating Partners
?? Brigitte Dorner, Robert-Koch Institute, Berlin, Germany ?? Hans-Gustaf Ljunggren, Karolinska-Institutet, Stockholm, Sweden ?? Ruurd van der Zee, University of Utrecht, The Netherlands
Funding
Deutsche Forschungsgemeischaft (DFG); Studienstiftung des Deutschen Volkes (S. Moré)
Fig. 3: Hypothetical model of Hsp60 mediated T cell activation. Hsp60 released from necrotic cells or LPS as a representative molecule for ??pathogen associated molecular patterns (PAMP)“ engage the ??pattern recognition receptor“ CD14/TLR-4 on professional APC. The APC release IL-12 and other cytokines and upregulate the expression of costimulatory molecules. IL-12 may activate NK and NKT-cells to release IFN-γ. Primary T cells are activated by MHC/peptide complexes (1) in the presence of IL12 (2). The early IFN-γ produced by NK-cells (3) potentially in??uences the differentiation of the activated T cells.
Investigators
Arne von Bonin Solveig Moré Svenja Ehrlich Minka Breloer Anke Osterloh
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Medical Microbiology Section
Murine Susceptibility to Chagas’ Disease Maps to Chromosomes 5 and 17
Zusammenfassung
Der einzellige Parasit Trypanosoma cruzi ist der Erreger der Chagas-Krankheit des Menschen. Die experimentelle Infektion von Inzucht-M??usen dient als geeignetes Modell zum Studium dieser Erkrankung. Der Hybridstamm B6D2F1 (C57BL/6xDBA/2) ist wesentlich resistenter gegenüber einer experimentellen Infektion als seine Parentalst??mme, was vermuten l??sst, dass rezessive Allele die Suszeptibilit??t bedingen. Um diese Genorte zu lokalisieren, wurden resistente B6D2F1-M??use mit suszeptiblen C57BL/6 rückgekreuzt und die Nachkommen in??ziert. Etwa 25 % verstarben w??hrend der akuten Phase (bis Tag 40), was das Vorliegen zweier rezessiver Suszeptibilit??tsallele vermuten lie??. Die DNS dieser Tiere wurden in einem Genom-Scan mit polymorphen Markern (Mikrosatelliten) auf das Vorliegen homozygoter Genorte untersucht. Zwei Regionen auf Chromosomen 5 (suggestiv) und 17 (signi??kant) wurden identi??ziert, die mit Suszeptibilit??t gegenüber einer schweren T. cruzi-Infektion gekoppelt waren. Diese Loci sind zuvor schon mit der Immunregulation gegenüber anderen Kinetoplastida (T. congolense) assoziiert worden. Ein weiterer Suszeptibilit??ts-Lokus k??nnte auf Chromosom 13 vorliegen, aber dieser muss in erweiterten Untersuchungen noch best??tigt werden. Die Lokalisierung dieser Loci erm??glicht weitere Untersuchungen zur Identifizierung der genetischen Mechanismen, die eine schwere Trypanosomen-Infektion bedingen. extended studies. The localization of these regions fosters further efforts to identify genes responsible for severe T. cruzi-related disease.
Introduction
Up to 20 million people are said to be infected by Trypanosoma cruzi on the American continent, and Chagas’ disease is a major cause of morbidity and mortality in its chronic stage. This is characterized by signs of disrupted autonomous neural activity, mainly affecting the heart and intestine and caused by a persistent in??ammation. The pathogenesis of chronic ailment is not fully understood but possibly mediated by parasite persistence. It is preceded by an acute illness soon after infection that accompanies the spread of parasites. The parasite naturally infects a number of rodents such as rats and mice, and in these, the early phase of the disease is similar to that in man. Inbred mouse strains are therefore commonly used to study Chagas’ disease, and the severity of the infection depends on the strains used. Several inbred mouse strains (such as C57BL/6 and DBA/2) are highly susceptible to T. cruzi, succumbing to infection of less than 100 parasites. Hybrid mice in general, and the B6D2F1 cross in particular, are more resistant to experimental infection, resulting in decreased levels of parasitaemia and mortality during the acute stage (Fig. 1). This suggests that recessive alleles may govern the susceptibility of mice to severe infection with T. cruzi. The genetic basis for differential disease severity has not been identi??ed yet. However, the H2 locus (the murine major histocompatibility complex, MHC) was found to be important, with some haplotypes being of greater bene??t to the host than others. Other genomic regions outside the H2 locus were also inferred to contribute but have not been localized.
Summary
The protozoan pathogen Trypanosoma cruzi causes Chagas’ disease in man. There is considerable variance of susceptibility to severe disease, and the host genetic background is believed to in??uence the course of the infection. However, the genes governing this control are unknown. Chagas’ disease is reliably modelled in inbred mice. Surprisingly, the intercross of two highly susceptible mouse strains (C57BL/6 and DBA/2) is able to resist infectious doses three log-fold higher than the parental strains. This suggests that recessive alleles determine susceptibility in mice. In order to map their respective genomic loci, we backcrossed the resistant hybrid strain (B6D2F1) to one of its susceptible parental strains (C57BL/6). Of the offspring, about 25% succumbed to experimental infection with T. cruzi. Their genomes were scanned with microsatellite markers, and two regions linked to susceptibility were found on chromosomes 5 and 17. These loci have previously been found to mediate the immune response to trypanosomes (T. congolense). A further suggestive locus was identi??ed on chromosome 13, but its in??uence has to be con??rmed in
Fig. 1: Course of early T. cruzi infection in mice. Susceptible C57BL/6 mice (solid lines), resistant B6D2F1 mice (dashed lines) and 192 offspring of a C57BL/6xB6D2F1 backcross (N2, blue line) were infected with 104 T. cruzi Tulahuen blood form trypomastigotes.
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Medical Microbiology Section
Project Description and Results
We applied a backcross strategy to identify linkage of C57BL/6 genomic regions with susceptibility to a severe course of T. cruzi infection. Resistant B6D2F1 mice were backcrossed to C57BL/6 females, and 25% of the male offspring 24% died between days 14 and 40 after experimental infection (Fig. 1). This suggested the presence of two recessive loci linked to this phenotype. We performed a genome wide scan at 10-15 cM intervals with polymorphic microsatellite markers on the genomes of these susceptible offspring. A number of chromosomal regions displayed elevated frequencies of homozygosity (Fig. 2), and these were further analysed with markers at smaller intervals. Notably, loci on chromosomes 5, 13 and 17 were suggestive of in??uencing susceptibility. On chromosome 17, ??ne mapping revealed signi??cant linkage of a region between 17 and 22 cM from the centromere, with a maximum χ2 of 17.04 (p = 0.000018). Increased mapping resolution on chromosome 5 con??rmed the presence of a suggestive candidate region between approximately 45 and 52 cM, with χ2 of 10.52 (p = 0.00059). These regions on chromosomes 5 and 17 map to the positions of quantitative trait loci (QTL) designated Tir2 and Tir1, respectively (“trypanosome immune response”). These were found to confer resistance of C57BL/6 mice to Trypanosoma congolense (Kemp 1997, Nat Genet 16:194) a trypanosome pathogenic to mice and livestock. On chromosome 13, an additional region (marker D13Mit35 at approximately 71 cM,
χ2 = 9.80, p = 0.00087) may also be linked to a susceptible phenotype, but this locus has not previously been described as being involved in the regulation of immunity to protozoan infections in mice. The investigation is currently being extended to ascertain its relevance. Other regions with χ2 > 5 could not be substantiated by increasing mapping resolution or by multipoint analysis. The identi??cation of genomic regions that determine susceptibility to T. cruzi infection in mice supports efforts to unravel the underlying genetic mechanisms associated with the development of severe Chagas’ disease in man, which eventually may lead to the development of effective means to prevent it.
Selected Publication
?? Graefe SEB, Meyer BS, Müller-Myhsok B, Drosten C, Laue T, Steeg C, Nürnberg P, Fleischer B 2002. Murine susceptibility to Chagas’ disease maps to chromosomes 5 and 17 (submitted)
Cooperating Partners
?? Birgit S. Meyer, Peter Nürnberg, Max-Delbrück-Center, Gene Mapping Center, Berlin, Germany
Investigators
Sebastian E.B. Graefe Bertram Müller-Myhsok Bernhard Fleischer Christiane Steeg
Fig. 2: Mapping of C57BL/6 genomic regions to susceptibility to a lethal T. cruzi infection. The male offspring of a C57BL/6xB6D2F1 backcross were infected, and those mice (n=46) identi??ed as being susceptible (i.e., having succumbed to infection) were genotyped with polymorphic microsatellite markers. Homozygous chromosomal regions (expressed as χ2) were identi??ed. Fine mapping of candidate regions con??rmed signi??cant linkage on chromosome 17, and suggestive linkage of loci on chromosomes 5 and 13. The dotted lines at χ2 = 16.56 and χ2 = 8.74 indicate threshold values for signi??cant and suggestive linkage of a locus, respectively.
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Medical Microbiology Section
CTLA-4 Expression during Blood-stage Malaria Could Prevent Immune Pathology
Introduction
The role of T cells in malaria is still not well understood. Blood-stage parasites reside within erythrocytes lacking the machinery to present antigen. Therefore, a direct interaction of T cells and parasites is unlikely. However, the contribution of cytokine-secreting CD4+ T cells in acquired immunity to blood-stage malaria could be demonstrated by several groups. Nevertheless, these cytokines are involved in the pathogenesis of cerebral malaria. These data indicate that T cell responses to malaria antigens are a double-edged sword and have to be tightly controlled. In order to understand how the size and reactivity of the malaria speci??c T cell pool is regulated we analyzed the expression of CTLA-4, which belongs to the family of costimulatory molecules such as CD28. CTLA4 is induced upon activation, and its binding to B7 was shown to deliver negative signals to T cells. Several studies demonstrated that a blockade of CTLA-4 improved the immune response against tumour cells and infectious agents, whereas under some circumstances autoimmune diseases could be enhanced. Recently we demonstrated that CTLA-4 expression is a sensitive and dynamic marker for activation of T cells during the course of human malaria and could be positively correlated with disease severity.
Zusammenfassung
CTLA-4 (CD152) ist ein negativer Regulator der T-Zellfunktion. Im Gegensatz zu CD28 wird CTLA-4 aber nur auf aktivierten T-Zellen exprimiert. Kürzlich konnten wir jedoch zeigen, dass es w??hrend der akuten Phase der humanen Malaria zu einer starken Expression von CTLA-4 kommt. Um die Rolle von T-Zellen und deren Regulation durch CTLA-4 w??hrend der Malaria genauer studieren zu k??nnen, haben wir ein Infektionsmodell mit Plasmodium berghei in der Maus etabliert. Auch dort kommt es w??hrend der Infektion zu einer verst??rkten Expression von CTLA-4 auf TZellen. Die Blockade von CTLA-4 mit Hilfe eines Antik??rpers führte zu einer Verschlimmerung der Erkrankung. Histologische Untersuchung der behandelten M??use best??tigten den Verdacht, dass diese im Verlauf der Infektion eine zerebrale Malaria entwickelt hatten. Diese Daten legen nahe, dass die Expression von CTLA-4 die Reaktivit??t von T-Zellen gegen Antigene von Plasmodien reguliert.
Summary
CTLA-4 (CD152) is a negative regulator of T cell function. In contrast to CD28, CTLA-4 expression is induced after T cell activation. Recently we have shown that CTLA-4 expression is strongly increased in acute human malaria. To further study the role of T cells in the pathogenesis of malaria, we examined the function of CTLA-4 expression in a mouse model of malaria using Plasmodium berghei. Our data demonstrate that CTLA-4 is expressed on T cells during the blood-stage of malaria. Treatment of mice with a monoclonal antibody to CTLA-4 led to an exacerbation of disease. Histological analysis revealed that these mice exhibited signs of cerebral malaria. These data suggest that CTLA-4 expression restricts T cell responses to plasmodial antigens, and thus prevent immune pathology associated with blood-stage malaria.
Project Description and Results
In the present study we analyzed CTLA-4 expression in a murine malaria model using Plasmodium berghei. Infection of mice with P. berghei led to a strong increase of CTLA-4 expression on splenic T cells. Expression could be observed on CD4+ T cells, whereas no induction was found on CD8+ cells (Fig. 1A). A detailed kinetic analysis revealed that expression increased on day 5 post infection (p.i.) and showed a peak on day 9 p.i. (Fig. 1B). At this time point up to 10 % of CD4+ T cells expressed CTLA-4. Mice receiving anti-CTLA-4 abtibodies developed symptoms of cerebral malaria and died early after infection (Fig. 2). Comparison of histological changes in the brain revealed that these mice displayed severe vascular changes, which were accompanied by stasis in capillaries and swelling of endothelial cells. Furthermore, perivascular in??ltrates containing macrophages, CD4+ T cells and CD8+ T cells could be detected (not shown). Spleen cells from infected mice treated with anti-CTLA-4 exhibit an increased proliferation (Fig. 3A), whereas cytokine production was comparable in both groups (Fig. 3B). Noteworthy, cells from mice treated with anti-CTLA4 produced more nitric oxide (Fig. 3C).
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Our data indicate that CTLA-4 expression on T cells during the blood-phase of malaria could counter-regulate T activation, which seems to be one important mechanism to prevent immune pathology. Apart from analyzing the role of CTLA-4 expression in controlling T cell activation, our results also have implications for vaccine strategies: Boosting the T cell response could be protective during the hepatic stage of malaria but may lead to immune pathology like cerebral malaria during blood-stage malaria.
Fig. 1. Induction of CTLA-4 on spleen cells from P. berghei infected mice. Cells from control and infected mice were doublestained for CTLA-4 and for the T cell markers CD4 or CD8. CTLA4 was found to be almost exclusively expressed on CD4+ T cells (A). Cells from infected mice were analyzed at different time points for CTLA-4 expression. Whereas in control mice (open circles) only low expression was detectable, in infected mice (black circles) a high percentage of CD4 / CTLA-4 double positive T cells were found (B).
Selected Publications
?? Schlotmann T, Waase I, Juelch C, Klauenberg U, Müller-Myhsok B, Dietrich M, Fleischer B, Br??ker BM (2000). CD4+ αβ T lymphocytes express high levels of the T lymphocyte antigen CTLA-4 (CD152) in acute malaria. J Infect Dis 182: 367-370 ?? Jacobs T, Graefe SEB, Niknafs S, Gaworski I, Fleischer B. Murine malaria blood-stage is exacerbated by CTLA4 blockade. J Immunol, in press
Cooperating Partners
?? Paul Racz, Klara Tenner-Racz, Department of Pathology, BNI
Investigators
Thomas Jacobs Bernhard Fleischer Iris Gaworski Barbara M. Br??ker Sebastian Graefe Sonja Nicknafs
Fig. 2. CTLA-4 blockade during P. berghei infection exacerbated disease. At the day of infection mice received either control antibody IgG (black line) or the monoclonal anti-CTLA-4 antibody (dotted line).
Fig. 3. Ex vivo characterization of spleen cells after CTLA-4 blockade. Mice were treated with 500 ??g anti-CTLA-4 antibody or control IgG and subsequently infected. Spleen cells were isolated at day 5 post infection and stimulated with anti-CD3. Cultures were analyzed for proliferation (A), IFN-γ production (B) and NO (C).
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Medical Microbiology Section
Wolbachia Endobacteria in Filarial Nematodes as Targets for a Novel Chemotherapeutic Approach: From the Lab to the Field
Zusammenfassung
Wolbachien, endosymbiotische Bakterien in Filarien, stellen Ziele für eine neuartige Therapie gegen Filarien dar. Studien an Onchozerkosepatienten mit Doxycyclin in Ghana zeigten, dass die Blockade der Embryogenese der weiblichen Würmer langandauernd ist. Zusammen mit Ivermectin wurde eine langanhaltende Amicro??laridermie erzielt. Die Anwesenheit der Wolbachien ist entscheidend für die Akkumulation von neutrophilen Granulozyten im Onchozerkom und spielt eine kritische Rolle in der Pathogenese der Keratitis im Tiermodell. Somit übertrifft die gegen Wolbachien gerichtete Chemotherapie durch Doxycyclin im Verbund mit Ivermectin bei weitem die Wirkung einer Monotherapie mit Ivermectin und stellt eine Alternative zur Unterbrechung der Transmission in ausgew??hlten Herden dar. Die Elimination der Endobakterien verringert wahrscheinlich auch die Filarien-assoziierte Pathologie. causes pathology, is only reduced for a few months, resulting in the re-emergence of skin micro??lariae and in continued transmission. It is clear that repeated masstreatment with ivermectin alone will not eliminate onchocerciasis. Therefore, a goal of the WHO is the development of additional antifilarial chemotherapies that are either macro??laricidal or have a long-term sterilizing effect on female worms. The rediscovery of the Wolbachia endosymbiotic bacteria (order: Rickettsiales) in ??lariae has provided a new target for the development of potential chemotherapeutic drugs. Our lab has been a leader in de??ning Wolbachia as a target for the control of ??lariae.
Project Description and Results
Previous ??eld studies showed that doxycycline treatment of patients infected with O. volvulus leads to a long term block in embryogenesis with a corresponding reduction in circulating micro??lariae (mf) as older mf die. This effect of anti-Wolbachia treatment on mf density is even more dramatic when doxycycline is combined with ivermectin treatment. Doxycycline was administered to patients for 6 weeks with all patients given ivermectin either 2.5 or 6 months after entering the study to eliminate micro??lariae. As expected, mf counts decreased to near zero after ivermectin treatment. However, those patients that received only ivermectin treatment had a return of micro??laridermia within 2-3 months after drug administration. Patients receiving doxycycline followed by ivermectin had mf counts of zero or near zero (<1/mg skin) at 19 months after the start of the study (Fig. 1). This result indicates that doxycycline treatment leads to a disruption of embryogenesis that may be permanent. Therefore, anti-Wolbachia therapy in combination with ivermectin treatment could provide a way to permanently interrupt transmission of the parasite in selected foci. This question will be strategically addressed together with the Carter Center, Atlanta and members of OEPA (Onchocerciasis Eradication Program of the Americas) in Latin American countries. We also studied how Wolbachia may interact with the mammalian immune system. Wolbachia are responsible for the recruitment of neutrophils into nodules around live O. volvulus worms. Neutrophils were not seen in nodules from patients treated with doxycycline nor were they seen in nodules containing the deer ??laria O. ??exuosa. The chemotaxis of neutrophils was dependent on the presence of Wolbachia and activation of neutrophils occured directly and also indirectly via monocytes. A collaboration with Dr. Eric Pearlman (Case Western University) showed that neutrophil attraction in the mouse model of onchocercal keratitis was critically dependent on Wolbachia and was mediated by Toll-like receptor 4
Summary
Wolbachia endosymbiotic bacteria offer a new target for treatment with the prospect to better contain the transmission of ??larial nematodes. Field studies of patients in Ghana infected with Onchocerca volvulus using doxycycline demonstrated that the embryotoxic effect of antiWolbachia treatment was also seen in human infections and was long-term. Doxycycline treatment in combination with ivermectin achieved a sustained amicro??laridermia as a result of female worm sterility caused by doxycycline. The presence of Wolbachia is required for the attraction of neutrophils to the onchocercomas and plays a critical role in the pathogenesis of onchocerciasis (river blindness) in the animal model. The ef??cacy of anti-Wolbachia treatment with doxycycline in conjunction with ivermectin far surpasses the effects of ivermectin alone, providing an alternate approach to finally interrupting transmission in selected foci. Elimination of the endobacteria may lead to a decrease in pathological effects.
Introduction
Onchocerciasis (river blindness) is an insect borne infection caused by the filarial nematode Onchocerca volvulus, and is endemic in 37 countries in Africa. It af??icts an estimated 17 million people and is a major cause of visual impairment and dermatitis, making the disease a public health problem in Africa. Current mass-treatment programmes (APOC) with the micro??laricidal drug ivermectin have brought about considerable relief from severe consequences of the infection. A major drawback is that the production of micro??lariae (mf), the stage that
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Medical Microbiology Section Selected Publications
?? Saint André A, Blackwell NM, Hall LR, Hoerauf A, Brattig N, Taylor M, Ford L, Hise A, Volkmann L, Lass JH, Diaconu E, Pearlman E (2002). A critical role for endosymbiotic Wolbachia bacteria and TLR4 signaling in the pathogenesis of river blindess. Science 295: 1892-1895 ?? Brattig NW, Büttner DW, Hoerauf A (2001). Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria. Microbes Infect 3: 439-446 ?? Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW (2001). Depletion of Wolbachia endobacteria in Onchocerca volvulus and microfilaridermia after ivermectin treatment. Lancet 357: 1415-1416 ?? Taylor M, Hoerauf A (2001). A new approach to the treatment of ??lariasis. Curr Opin Infect Dis 14: 727-731 ?? Hoerauf A, Volkmann L, Hamelmann C, Adjei O, Authenrieth IB, Fleischer B, Büttner D (2000). Endosymbiotic bacteria in worms as targets for a novel chemotherapy in ??lariasis. Lancet 355: 1242-1243
Cooperating Partners
?? O. Adjei, C. Hamelmann, T. Kruppa, Kumasi Centre for Collaborative Research, Ghana ?? I. Authenrieth, University of Tübingen ?? B. Barrell, Sanger Centre, Cambridge, UK ?? M. Blaxter, University of Edinburgh, Edinburgh, UK ?? P. Fischer, Bernhard Nocht Institute for Tropical Medicine, Hamburg ?? T. Klei, Louisiana State University, Baton Rouge, USA ?? R. Lucius, Humboldt University, Berlin ?? E. Pearlman, Case Western University, Cleveland, USA ?? M. Taylor, Liverpool School of Tropical Medicine, Liverpool, UK
Fig.: Micro??laridermia (mf) after treatment with doxycyline plus ivermectin (open bars) in comparison to ivermectin alone (block bars). Mf load is shown as Williams means (one less than the geometric means of one plus the mf counts). Numbers in brackets are the 10th and the 90th percentiles, respecitively. Mann-Whitney U test (differences from control group)*: p<0.0001; ??: p=0.043.
(TLR4), the primary receptor for bacterial lipopolysaccharide (LPS). Finally, in murine ??lariasis, TLR4 appears to be involved in the inhibition of embryogenesis or the killing of free mf. Mice which do not express TLR4, and therefore do not respond to LPS, have worms which contain all embryonic stages and are released as full length mf. Mice which do respond to LPS rarely have worms with a complete pro??le of embryogenesis and contain no free mf. Our laboratory is continuing to study Wolbachia as a source for novel anti-filarial therapy. Field studies in Ghana are ongoing and have been expanded to Cameroon and Indonesia to include patients infected with the lymphatic ??lariae W. bancrofti and B. malayi. Preliminary data indicate that a combination of a micro??laricide and doxycycline may yield the same long-term sterilization seen in O. volvulus. We are also interested in the genes involved in the symbiosis of Wolbachia with the nematode host. To understand the molecular interaction between the worm and the endosymbiont, differential display is being used to identify changes in gene expression in the worm during anti-Wolbachia treatment.
Funding
Caritas European Union Wellcome Trust Deutsche Forschungsgemeinschaft VW-Foundation
Investigators
Achim H??rauf Norbert Brattig Alex Debrah John Larbi Yeboah Marfo Lars Volkmann Ingeborg Albrecht Dietrich W. Büttner Kerstin Fischer Sabine Mand Kenneth Pfarr
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Medical Microbiology Section
Inhibition of Flaviviridae NTPase/helicase by Ring-Expanded Nucleosides (REN’s). New Class of Antiviral Agents
Zusammenfassung
RNA NTPase/Helikasen repr??sentieren eine Familie von Proteinen, die in fast allen biologischen Systemen, in denen RNA eine zentrale Rolle spielt, nachgewiesen wurde. Auch im Genom der Flaviviridae wurden diese Enzyme gefunden. Die NTPase/Helikasen katalysieren die enzymatische Entwindung der RNAoder DNA-Doppelhelix durch Spaltung der Wasserstoffbrücken, die die beiden Str??nge zusammenhalten. Die Helikaseaktivit??t ist abh??ngig von der Energie, die w??hrend der NTP-Hydrolyse produziert wird. Dies l????t den Schlu?? zu, dass potentielle spezifische Inhibitoren der NTPase/Helikase der Flaviviridae durch die Entkoppelung der NTP-Hydrolyse mit der Entwindungsreaktion agieren k??nnten. Wir pr??sentieren eine Familie von Nukleosid-Analoga, die natürlichen Purinen ??hneln, ??Ring-Expanded Nucleosides“ (REN’s) genannt. Die Extension des Purin-Rings verursacht ein beachtliches Hemmpotential der Substanzen bezüglich der Helikaseaktivit??t der Flaviviridae NTPase/Helikase. Da die Hemmung der Enzyme in einer korrespondierenden Reduktion der Virusreplikation resultiert, repr??sentieren REN’s eine potentielle Gruppe antiviraler Stoffe. agents because of the multiple enzymatic activities associated with this protein. NS3 exhibits serine protease activity residing in the NH2-terminus of the protein and nucleoside triphosphatase (NTPase) and RNA helicase activities located in the COOH-terminus. The NTPase/helicase together with the NS5-associated RNA polymerase is thought to be an essential component of the viral replicase complex. Our experiences with some inhibitors of the helicase activity reported previously and mutational analyses published by other authors show accordingly that inhibition of the enzyme leads to reduction of the replication of the virus. In this context, the inhibition of the unwinding activity of the NS3 associated NTPase/helicase may represent a novel therapeutical concept.
Project Description and Results
Studies performed with halogenated benzimidazoles and benzotriazoles combined with selective screening of chemical libraries indicated that the extension of the heterocyclic ring of the purine base results in inhibitory activity of Flaviviridae NTPase/helicases. We have designed and screened a broad range of REN’s based on the structural skeleton of a REN base with a 5:7-fused ring, as presented in Fig. 1. Systhematic screening combined with rational substitutions revealed following features of the REN molecule that appear to be conditio sine qua non for the inhibiting property. i) 2’-deoxy form or ii) an acyclic form of the sugar with attached large spatial strong hydrophobic moiety. III) long aliphatic chain (longer than 12 carbon atoms) placed in position 6. By a combination of the modi??cations highly selective - in respect of the virus - helicase inhibitors were obtained.
Summary
RNA NTPase/helicases represent a large family of proteins that are detected in almost all biological systems in which RNA plays a central role. The enzymes have also been denti??ed in viral genomes of the members of Flaviviridae family. The NTPase/helicases are capable of enzymatically unwinding duplex RNA or DNA structures by disrupting the hydrogen bonds that keep the two strands together. The helicase activity is dependent on the energy produced in course of the NTP hydrolysis. Thus, potential speci??c inhibitors of NTPase/helicase of Flaviviridae could act by inhibition of the coupling of NTP hydrolysis to the unwinding reaction resulting from an occupation of a nucleoside-binding allosteric site of the enzyme. We present a family of nucleoside analogues that resemble natural purines, so-called “Ring Expanded Nucleosides” (REN’s). The ring-extension generates considerable inhibitory potential of the compounds towards the helicase activity of the Flaviviridae NTPase/helicase. Since it is established that the inhibition of the enzyme results in a corresponding reduction of the virus replication, REN’s represent potential utility as antiviral agents.
Fig.1: The structure of imidazo[4,5-e]diazepine that serves as starting point for the development of inhibitors of Flaviviridae NTPase/helicase
Introduction
Between the structural and non-structural (NS) proteins encoded by the viral genome of Flaviviridae the NS3 protein appears to be the most promising target for antiviral
After analysing of the kinetic data, we have come up with the following working model to explain the observed inhibition. 1. The nucleoside analogues that we screened belong to
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Medical Microbiology Section
Fig.2: Mechanistic model of Flaviviridae NTPase/helicase presenting putative targets for REN within the enzyme molecule
two categories: i) binding to duplex DNA or RNA substrates of helicase either in the minor or major groove and cause the dsDNA and dsRNA stabilizing (or destabilizing) effect, and thus cause inhibition (or activation) of unwinding activity of helicase. ii) not binding at all to DNA or RNA substrate. 2. The enzyme has two binding sites for nucleosides and nucleotides: NTP1 and NTP2. Out of the two, NTP1 is where the ATP normally binds, and is somewhat rigid and speci??c in terms of nucleotide substrate. In contrast, NTP2 is the allosteric binding site, and is somewhat non-speci??c in terms of nucleoside/nucleotide substrate. The REN??s rather occupy the NTP2 site and there is a consequent conformational change of the enzyme, which ultimately leads to its inhibition. A model elucidating the mechanism of action of REN’s is presented in Fig. 2. Since there is a close correlation between the inhibition of the helicase activity in vitro and reduction of the virus replication in vivo the compounds based on the structure of REN may represent a starting point for a novel therapeutic concept.
K, Kulikowski T, Schmitz H (2001). Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme. J Virol 75:3220-3229 ?? Borowski P, Mueller O, Niebuhr A, Kalitzky M, Hwang L-H, Schmitz H, Siwecka M A, Kulikowski T (2000). ATP-binding domain of NTPase/helicase as target for hepatitis C antiviral therapy. Acta Biochim Polon 47:173-180 ?? Borowski P, Kühl R, Mueller O, Hwang L-H, Schulze zur Wiesch J, Schmitz H (1999). Biochemical Properties of a minimal functional domain with ATP binding activity of the NTPase/helicase of hepatitis C virus. Eur J Bioch 266: 715-723
Cooperating Partners
?? Prof. Ramachandra S. Hosmane, Department of Chemistry & Biochemistry, University of Maryland, Baltimore, USA ?? Prof. Tadeusz Kulikowski, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland ?? Prof. Joonho Choe Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejeon, Korea ?? Dr. Winifred Kopf, Drug Hunting e. K., Aalen, Germany
Selected Publications
?? Borowski P, Lang M, Haag A, Schmitz H, Choe J, Chen H-M, Hosmane, RS (2002). Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus NTPase/helicase: Evidence for activity on the level of substrate and/or enzyme. Antimicrob Agents Chemother 46(5):1231-1239 ?? Borowski P, Lang M, Niebuhr A, Haag A, Schmitz H, Schulze zur Wiesch J, Choe J, Siwecka M A, Kulikowski T (2001). Inhibition of the helicase activity of HCV NTPase/helicase by 1-??-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5` triphosphate (ribavirin-TP). Acta Biochim Polon 48:739-744 ?? Borowski, P, Niebuhr A, Mueller O, Bretner M, Felczak
Funding
Deutsche Krebshilfe
Investigators
Peter Borowski Melanie Lang Herbert Schmitz Verena Koch Sarah Schalinski
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Medical Microbiology Section
Imported Cases of Lassa Fever: Molecular Epidemiology and Clinical Virology
kb and 7 kb, respectively. The S RNA encodes the viral glycoprotein precursor protein and the nucleoprotein. The L RNA encodes the viral polymerase and a small, zinc-binding protein.
Zusammenfassung
Vier F??lle von t??dlichem Lassa-Fieber wurden im Jahre 2000 von Afrika nach Europa importiert. Bei drei der Patienten wurde die Labordiagnose am BernhardNocht-Institut gestellt und es wurden weiterführende Untersuchungen durchgeführt. Alle Patienten zeigten unterschiedliche Verl??ufe der Lassa-Virusinfektion. Die Viruslast wurde mit der ??real-time“ PCR gemessen und im Serum wurden Zytokinkonzentrationen bestimmt. Eine schnelle Charakterisierung der LassaVirusst??mme war mittels ??long-distance“ PCR-Technologien m??glich. Es wurden neue Daten hinsichtlich der klinischen Manifestation und der Pathogenese der Lassa-Virusinfektion als auch der molekularen Epidemiologie des Erregers erhalten.
Project Description and Results
During the year 2000, four cases of fatal Lassa fever were imported from Africa to Europe. In three of these patients, the laboratory diagnosis of Lassa fever was established at the Bernhard Nocht Institute and further virological investigations were initiated. Patient 1 was a female traveller who visited Ghana, C??te D’Ivoire, and Burkina Faso and was treated at the Missions??rztliche Klinik Würzburg. Patient 2 was a male surgeon working in rural Sierra Leone who returned to The Netherlands. Patient 3 was a Nigerian male who presented with an unusual course of Lassa fever and was treated at the Dr. Horst SchmidtKliniken Wiesbaden. In patients 1 and 2, consecutive serum samples were available for monitoring of virus load and cytokine levels in addition to standard laboratory data. Patient 1 developed multi-organ failure and died of hemorrhagic shock on day 15 of illness, while patient 2 died without hemorrhage on day 16. Ribavirin was administered to both patients beginning on day 11, but did not prevent clinical deterioration. High serum AST and LDH levels were remarkable in both patients. Patient 1 had an initial virus load of 106 S RNA copies/ml as measured by real-time reverse transcription-PCR (RT-PCR). Viremia increased steadily and reached a plateau of about 108-109 copies/ml four days before death, while IFN-γ and TNF-α rose to extremely high levels only shortly before death. In contrast, in patient 2 the virus load decreased from 107 to 106 copies/ml during the late stage of illness which was paralleled by a decrease in the IFN-γ and TNF-α levels. The IL-10 level increased in parallel with speci??c IgM and IgG. These data suggest that a high virus load and high levels of pro-in??ammatory cytokines in the late stage play an important role in the pathogenesis of hemorrhage, multi-organ failure, and shock in Lassa fever. The pathogenesis of neurological complications of Lassa fever is also poorly understood. It is still obscure whether the encephalopathy during the acute phase is due to infection of the central nervous system (CNS) or whether indirect effects such as metabolic changes play a role. In the Nigerian patient 3, encephalopathy as characterized by disorientation, seizures, and blood-brain barrier dysfunction has been the chief manifestation during the late phase of infection. For this reason, neither in Nigeria nor in Germany Lassa fever was clinically suspected. It was eventually diagnosed by laboratory screening. Several PCR assays demonstrated Lassa virus RNA in cerebrospinal ??uid (CSF) but not in serum, a ??nding which has not yet been reported in Lassa fever patients.
Summary
Four cases of fatal Lassa fever were imported from Africa to Europe in the year 2000. In three of these patients the laboratory diagnosis of Lassa fever was established at the Bernhard Nocht Institute and further virological investigations were initiated. The patients presented with various clinical manifestations of Lassa fever. Viral load was measured by real-time PCR assays and cytokine levels were determined. The causative strains were readily characterized by using long-distance RT-PCR techniques. These studies provide novel insights into the clinical manifestation and pathogenesis of Lassa fever, as well as the molecular epidemiology of Lassa virus.
Introduction
Transmission of Lassa virus (family Arenaviridae) from its natural rodent reservoir to humans can cause haemorrhagic fever, a clinical syndrome which is associated with high mortality. Lassa fever is endemic in West Africa and has to date been reported from Sierra Leone, Guinea, Liberia, and Nigeria. The importation of Lassa virus into other regions of the world, for example by travellers, is a rare event with only a few cases documented. The import of the disease often raises public interest and fears due to the possibility of human-to-human transmission, the highly pathogenic nature of the virus, and the lack of a generally safe therapy. Arenaviruses can be divided phylogenetically, serologically, and geographically into two major complexes, the Old World complex (e.g. Lassa virus, lymphocytic choriomeningitis virus) and the New World complex (e.g. Tacaribe virus, Junin virus, or Machupo virus). There are also genetic, serological, and possibly pathogenic differences among different isolates of Lassa virus. The single-stranded arenavirus genome consists of a small (S) and a large (L) RNA fragment, with a size of 3.4
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Lassa virus was isolated from CSF and quanti??cation by real-time PCR indicates that the virus load in CSF was magnitudes higher than in serum. The RNA level corresponded to 103 plaque forming units per ml. These ??ndings suggest that Lassa virus persisted in the CNS, where it may be directly involved in neuropathogenesis, or via induction of cytokines as suggested for in??uenza virusassociated encephalopathy. This study also shows that Lassa fever should be taken into consideration in West African patients or in those returning from this area who present only with fever and neurological signs. Secondary spread of viral haemorrhagic fever from imported cases is of major concern. Therefore, contact persons of patient 1 were tested serologically (n=149) and evaluated for their level of exposure to the index case. Serologic testing showed no secondary infections in contact persons during the initial phase of symptomatic illness (up to day 8) of the index patient despite numerous high risk exposures. However, Lassa virus-speci??c antibodies were detected in the serum of a physician who had a close contact on day 9. These data suggest a minute risk for transmission during the initial phase of symptomatic disease and a time-dependent increase in the risk parallel to disease progression. In order to facilitate molecular analysis of the new Lassa virus isolates, a long-range RT-PCR was established. The primers bind to highly conserved RNA termini and allow ampli??cation of full-length (3.4 kb) S RNA directly from patient samples. This technique was used to characterize the virus strains isolated from serum of patients 1 and CSF of patient 3. The Lassa virus isolate from patient 1 (strain AV) originated from an area of West Africa where Lassa fever has not yet been reported (Ghana, C??te D’Ivoire, or Burkina Faso) and was found to represent an novel phylogenetic lineage. The coding sequences of strain AV differed from those of all known Lassa prototype strains by about 20%, mainly at third codon positions. The highest degree of variability was seen in parts of the 3’ and 5’-non-coding regions, while the intergenic RNA stem-loop structure was conserved. Consistent with the geographic origin of strain AV, it segregated phylogenetically between strains from Nigeria and strains from Sierra Leone, Guinea, and Liberia. This geographic clustering suggests an East->West evolution of Lassa virus within West Africa. To characterize the Lassa virus in patient 3, the 3.5-kb S RNA puri??ed from CSF was ampli??ed and sequenced. In the S RNA coding sequences, the CSF strain differed between 20% and 24.6% from all known prototype strains. Thus, the CSF isolate can also be regarded as a prototype strain of a phylogenetic cluster based on its considerable genetic differences to all known prototype strains. It is speculative whether strains of this cluster are associated with an increased risk of CNS manifestations. fever. Microbes Infect 4: 43-50 ?? Asper M, Hofmann P, Osmann C, Funk J, Metzger C, Bruns M, Kaup FJ, Schmitz H, Günther S (2001). First outbreak of callitrichid hepatitis in Germany: Genetic characterization of the causative lymphocytic choriomeningitis virus strains. Virology 284: 203-213 ?? Günther S, Kühle O, Rehder D, Odaibo GN, Olaleye DO, Emmerich P, ter Meulen J, Schmitz H (2001). Antibodies to Lassa virus Z protein and nucleoprotein cooccur in human sera from Lassa fever endemic regions. Med Microbiol Immunol 189: 225-229 ?? Günther S, Weisner B, Roth A, Grewing T, Asper M, Drosten C, Emmerich P, Petersen J, Wilczek M, Schmitz H (2001). Lassa fever encephalopathy: Lassa virus in cerebrospinal ??uid but not in serum. J Infect Dis 184: 345-349 ?? Günther S., Emmerich P, Laue T, Kühle O, Asper M, Jung A, Grewing T, ter Meulen J, Schmitz H (2000). Imported Lassa fever in Germany: Molecular characterization of a new Lassa virus strain. Emerg Infect Dis 6: 466-476
Cooperating Partners
?? Prof. Boye Weisner, Dr. Horst Schmidt-Kliniken, Wiesbaden, Germany ?? Dr. Andreas Roth, Life Camp Clinic, Abuja F.C.T., Nigeria ?? Dr. Petra Hofmann, Deutsches Primatenzentrum, G??ttingen ?? Dr. Christine Osmann, Zoo Dortmund, Dortmund ?? Dr. Jürgen Funk, Institut für Veterin??r-Pathologie, Justus-Liebig-Universit??t, Giessen ?? Dr. Christoph Metzger, Labor Prof. Enders und Partner, Stuttgart ?? Dr. Michael Bruns, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universit??t Hamburg ?? Prof. Klaus Fleischer, Missions??rztliche Klinik, Würzburg ?? Dr. Matthias Beersma, Department of Infectious Diseases, Leiden University, Leiden, The Netherlands ?? Dr. David Olaleye, Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria ?? Dr. Walter Haas, Infectious Disease Epidemiology, Robert Koch-Institut, Berlin ?? Dr. Achim Hoerauf, Department of Helminthology, BNI
Funding
Grant E/B31E/M0171/M5916 from the Bundesamt für Wehrtechnik und Beschaffung
Investigators
Stephan Günther Christian Drosten Annegret Jung Antje Rhode Herbert Schmitz Marcel Asper Petra Emmerich Olaf Kühle Martina Westerkofsky
Selected Publications
?? Schmitz H, K??hler B, Laue T, Drosten C, Veldkamp PJ, Günther S, Emmerich P, Geisen HP, Fleischer K, Beersma MF, Hoerauf A (2002). Monitoring of clinical and laboratory data in two cases of imported Lassa
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Medical Microbiology Section
Serological Differentiation of Dengue Virus 1-4 and West Nile Virus Infections using Recombinant Antigens
Zusammenfassung
Wir haben die B-Domaine des ??u??eren Glykoproteins aller vier Dengue-Viren und auch des West Nil-Virus zusammen mit einem His6-tag in E. coli exprimiert. Mit diesen rekombinanten Antigenen konnte nun zum ersten Mal ein einfacher Immunoblot-Test zur Bestimmung von Dengue-typenspezi??schen Antik??rpern erhalten werden. Bei einer Untersuchung von Proben von Patienten mit Dengue-Fieber, bei denen der Serotyp mittels 5??Nuklease- RT-PCR bestimmt worden war, konnte das PCR-Ergebnis durch den Immunoblot mit hoher Spezi??t??t und Sensitivit??t best??tigt werden. The puri??ed recombinant proteins were applied to immunoblot strips to detect dengue virus serotype-speci??c antibodies in human serum samples. A special collection of well characterized serum samples could be used for the tests. Of all 41 patients included in our study early serum samples were available where the serotype had been identi??ed by RT-PCR (Laue et al.) The recombinant proteins were tested against a set of consecutive serum samples of the 41 patients. In the early serum samples of the patients, viral RNA could be detected and the serotype was identi??ed by type-speci??c 5’??-nuclease RTPCR. In PCR positive early serum samples of 33 patients with primary infection no speci??c IgG or IgM antibodies were found. In the eight secondary dengue cases dengue virus-speci??c IgG was detected in the presence of virus RNA while speci??c IgM was absent. In 39 of 41 patients with either primary or secondary dengue infection the serotype identi??ed by RT-PCR was con??rmed by the immunoblot (95% sensitivity). In late serum samples from 10 of the 33 patients with primary infection a slight additional reactivity with heterologous dengue antigens was observed. However, in only one of the 33 patients this slight cross-reactivity caused a misinterpretation of the serotype. In all patients with secondary infection two or more serotype-speci??c bands were detected in late serum samples. Out of 85 control sera of healthy subjects without antidengue antibodies, 82 were negative with all four recombinant dengue antigens (97 % speci??city). The sera of four patients with West Nile infection selectively reacted with the West Nile B domain antigen. As documented in our study, the immunoblot test may provide a simpler alternative to PCR typing. In contrast to serotyping by RT-PCR, which is limited to the short time of viremia, the immunoblot, detecting IgG antibodies, can be applied in healthy seropositive subjects. Besides, the assay is easy to perform, taking only a few hours to complete. Due to the stability of the antigen, the strips can be handled at room temperature without expensive laboratory facilities. As the test can be read with the naked eye it could be used even under ??eld conditions. A simple and reliable serotype-speci??c diagnosis may be of particular interest for frequent travelers, who after having acquired a primary infection want to calculate their risk of future secondary infection. Finally, the immunoblot may help to measure the antibody response to all four dengue serotypes during dengue vaccination programmes. The data presented here suggest, that by including further B domain antigens in our immunoblot system the diagnosis of the various ??avivirus infections in the tropics may be improved.
Summary
The B domains of the glycoprotein E of dengue viruses (serotypes 1-4) and West Nile virus were expressed in E. coli fused to a His6-tag. For the first time a dengue serotype-specific antibody test is now available. The method was evalutated with a unique collection of samples obtained from patients with either primary or secondary dengue virus infection, in whom the serotype had been determined by type-speci??c RT-PCR technology. With this new immunoblot method the serotype, as detected by PCR could be con??rmed with high sensitivity and speci??city.
Introduction
Four different dengue viruses (dengue 1-4) can cause dengue fever. Unfortunately, in infected individuals no cross-protective immunity is seen. Therefore, secondary infections frequently occur in endemic areas. The E glycoproteins of dengue viruses and other closely related ??aviviruses have most of the B-cell epitopes in common. For this reason, anti-dengue IgM and IgG antibodies in human sera cross-react with all four dengue serotypes and even with other ??avivirus antigens. By using routine ELISA techniques, it is impossible to identify the dengue serotype involved. A serotype differentiation has only been possible by testing the immune response using time consuming neutralization tests or type-speci??c RT-PCR technology. A highly type-speci??c immune response seems to be directed to an epitope located in the B-domain of the envelope glycoprotein E of ??aviviruses. We therefore tried to establish a serotype-speci??c assay using B domainspeci??c antigens of dengue and West Nile viruses. The tests were run with well-de??ned serum samples of patients with recent dengue infections and known serotype, determined by RT-PCR.
Project Description and Results
The B domains of dengue virus serotypes 1-4 and West Nile virus were expressed in E. coli fused to a His6-tag.
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Medical Microbiology Section
Selected Publications
?? Laue T, Emmerich P, Schmitz H (1999). Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated ampli??cation system. J Clin Microbiol 37: 2543-2547 ?? Schmitz H, Emmerich P (1984). Detection of speci??c immunoglobulin M antibody to different ??aviviruses by use of enzyme-labeled antigens. J Clin Microbiol 19, No. 5: 664-667
Investigators
Diana Ludolfs Herbert Schmitz
Fig.: Reactivity of B domain antigens on the immunoblot strips with various sera with known serotype speci??city as determined by 5`nuclease RT-PCR. Den 1: dengue 1 antigen, Den 2: dengue 2 antigen, Den 3:dengue 3 antigen, Den 4: dengue 4 antigen; WNA: West Nile antigen, JE: Japanese encephalitis antigen (negative control). 1, 2, 3, and 4: serum samples of patients with dengue 1, dengue 2, dengue 3 and dengue 4 infection, respectively. WN: West Nile antibody positive serum; Neg.: serum without ??avivirus antibodies. For improved identi??cation of the various protein bands, the upper rim of each band was marked with pencil.
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Medical Microbiology Section
Role of N-glycosylation of the HIV-1 Envelope gp120 for Interaction with the Viral Coreceptors CXCR4 und CCR5
Zusammenfassung
Das ??u??ere Hüllprotein des HIV-1, gp120, bewirkt die Bindung des Virus an das CD4-Protein und an die Chemokinrezeptoren CXCR4 und CCR5. Viren, die zus??tzlich zum CD4-Protein den CCR5 Chemokinrezeptor als Korezeptor benutzen, bezeichnet man als R5-trope Viren und Viren, die CXCR4 benutzen, werden als X4-trope Viren bezeichnet. Die gp120-Region, welche entscheidet, welcher Korezeptor benutzt wird, ist der V3 loop. Der V3 loop ist eine 35-37 Aminos??ure grosse Schleife, die durch eine S-S-Bindung geschlossen wird. Im V3 loop befindet sich die N-Glykosylierungsstelle g15. Folgende Erkenntnisse wurden erhalten: Die Infektion von CD4/CXCR4 positiven Zellen durch ein X4-monotropes Virus verbessert sich, wenn die Glycosylierungsstelle g15 fehlt. Neben Untersuchungen mit gezielt hergestellten Virusvarianten +/g15 wurden auch aus Patienten X4-trope Viren isoliert. Bei diesen X4-tropen Viren wurde h??u??g eine mutierte g15 Glycosylierungstelle gefunden. Die Infektion von CD4/CCR5-positiven Zellen durch ein R5-monotropes Virus wird verbessert, wenn die Glycosylierungsstelle g15 vorhanden ist. Auch hier zeigte ein Vergleich mit HIV-Patientenisolaten, dass R5-trope Viren bevorzugt eine g15 Glycosylierungstelle besitzen. Die N-Glycosylierung spielt also für die Bindung an CCR5 eine aktive Rolle, wogegen die Bindung an CXCR4 durch den g15-Zucker im V3 loop des gp120 gest??rt wird. Recently, we have demonstrated that two of these glycans play a role in CXCR4-speci??c infection (Polzer et al., 2001). Infectivity and resistance against SDF-1 inhibition of HIVNL4-3 mutants lacking glycan g15 was enhanced in contrast to mutants containing fully glycosylated V3 loop. Especially the lack of the glycans g15 and g17 markedly enhanced infectivity and SDF-1 resistance for CXCR4 expressing cells. To study the function of V3 loop glycans most site directed mutagenesis studies have been conducted with X4-tropic HIV-1 strains and only limited data are available for R5X4-dualtropic viruses. Therefore, we have investigated the role of V3 loop carbohydrates for viral infectivity using a complete set of X4-, R5X4- but also R5-mono-tropic viruses differing in glycosylation of the V3 loop region. The study presents conclusive evidence that V3 loop glycans are affecting the interaction between gp120 and the coreceptors CCR5 and CXCR4 in a different manner. In CXCR4-speci??c infection glycans are masking the V3 loop and lack of various high mannose (HM) and complex sugars (C) i.e. g14 (HM), g15 (C) and g17 (HM) markedly enhanced CXCR4-dependent infectivity. In CCR5-speci??c infection the lack of g15 was down-regulating the CCR5-speci??c infectivity of R5-tropic viruses. Interestingly, the CCR5speci??city of a R5X4-dualtropic virus was completely lost without g15. Our data demonstrate that especially glycan g15 plays an important role in gp120-coreceptor interaction and in shifting the viral phenotype from R5- to X4-tropism.
Selected Publications
?? Polzer S, Dittmar MT, Schmitz H, Schreiber M (2002) The N-linked glycan g15 within the V3 loop of the HIV1 external glycoprotein gp120 affects coreceptor usage and cellular tropism. Virology, in press ?? Polzer S, Dittmar MT, Schmitz H, Meyer B, Müller H, Kr??usslich HG,Schreiber M (2001). Loss of N-linked glycans in the V3-loop region of gp120 is correlated to an enhanced infectivity of HIV-1. Glycobiology 11: 1119
Introduction
The human immunode??ciency virus type-1 (HIV-1) external glycoprotein gp120 mediates cell entry by binding speci??c receptors on the surface of the target cell. These receptors are CD4 and chemokine receptors. The chemokine receptors CXCR4 and CCR5 are the two major coreceptors used by HIV-1 isolates. During disease progression, viruses can be detected which are using CXCR4 in addition to CCR5. In the late stage of infection, X4-monotropic viruses are emerging. The emergence of the X4-isolates correlates with the transition from the asymptomatic to the symptomatic stage of HIV infection and the development of AIDS.
Cooperating Partners
?? PD M.T. Dittmar, Hygiene-Institut, University of Heidelberg, Germany ?? Prof. B. Meyer, University of Hamburg, Germany ?? Prof T. Peters, University of Lübeck, Germany ?? Prof. M. Dietrich, Dr. C. Manegold, Clinical Department, BNI
Project Description & Results
While the mechanism of coreceptor switching from R5to X4-tropism is still unclear, the interactions between the gp120 and the two coreceptors CXCR4 and CCR5 are more and more understood. The V3 loop is one of the ??ve variable regions of the envelope protein gp120, a extensively glycosylated protein with 24-26 potential glycosylation sites. Five of the glycosylation sites, designated g13, g14, g15, g16 and g17 are located in the V3 loop region.
Funding
Deutsche Forschungsgemeinschaft SFB 470 und Graduiertenkolleg 464 BMBF AIDS Verbund Hamburg
Investigators
Michael Schreiber Heiko Hauser Martin Kirst Svenja Polzer Annette Seifert Ingo Thordsen Markus Vossmann Tobias Wolk
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Medical Microbiology Section
Diagnostic PCR
Zusammenfassung
Ein Labor für eine umfangreiche Routine-Diagnostik durch PCR steht seit Sommer 2000 zur Verfügung. Ihre Aufgabe ist die Etablierung und Durchführung molekularer Verfahren zum Nachweis tropenmedizinisch relevanter Mikroorganismen. Als Einrichtung der Mikrobiologischen Zentraldiagnostik deckt sie Bakterien, Parasiten, Pilze und Viren ab. Seit seiner Einrichtung hat dieses Labor mehr als 40 verschiedene PCR-Testverfahren etabliert, die für die hauseigene Klinik sowie für Einsender aus ganz Deutschland durchgeführt werden. Eine Anzahl weiterer Tests be??ndet sich in Entwicklung oder wird auf spezielle Anfrage durchgeführt. Enwicklungsschwerpunkte im Berichtszeitraum waren h??morrhagische Fieberviren, HIV-1, Leishmanien, Plasmodien und Mycobakterien. Für den Nachweis von RT-PCR Produkten wurde ein neues real-time Detektionsreagenz entwickelt und zum Patent angemeldet. Im Rahmen von Kollaborationen hat das Labor real-time PCR Methoden für Forschungsprojekte in verschiedenen Arbeitsgruppen innerhalb und au??erhalb des Instituts entwickelt. Laufende Projekte befassen sich mit der Etablierung von transportablen Testsystemen und der Detektion von Antigenen mit Hilfe von Aptameren. Das PCR-Labor des BNI wurde zusammen mit der Abteilung für Virologie etwa 35 mal zum Ausschluss eines h??morrhagischen Fiebers konsultiert. Als im Herbst 2001 nach den Terroranschl??gen auf die Vereinigten Staaten Anthrax-verd??chtige Postsendungen auftraten, war das Labor zusammen mit der Universit??t Hohenheim eine der beiden Einrichtungen in Deutschland, die Milzbrand mit der PCR unmittelbar nachweisen konnten.
Table: List of diagnostical PCRs presently available in the PCR laboratory: Plasmodium spp. Leishmania Microsporidia B. melitensis M. ulcerans M. leprae Diphteria toxin gene N. meningitidis Y. pestis B. recurrentis Cholera toxin gene Ebola/Marburg Virus LCMV Crimean-Congo virus Hantaan virus Alphaviruses (general) Dengue viruses 1-4 West Nile virus Poxviruses (general) HBC VZV HIV-1 gag T. cruzi T. brucei B. suis M. tuberculosis cpl. Mycobacteria L. interrogans B. anthracis B. burgdorferi F. tularensis Funghi (general) Lassa Virus Arenaviruses Rift Valley virus Puumala virus Yellow fever virus Dengue virus 1/2/3/4 Flaviviruses (general) HCV HSV CMV HIV-1 LTR HTLV-1/2
Summary
A routine PCR laboratory for a broad spectrum of agents has been established in summer 2000. It is dedicated to the development and conduction of molecular methods for the detection of microbes causing tropical diseases. As a facility of the central diagnostic unit, it covers the diagnosis of viral, bacterial, parasitical, and fungal pathogens. Since the establishment of the laboratory, more than 40 diagnostical PCR/RT-PCR procedures have been made available for the clinical department of the Bernhard Nocht Institute as well as to for submittors from all over Germany (Table 1).
Further tests are under development or available upon special request. Special focuses in the development of new tests have been haemorrhagic fever viruses, HIV-1, Leishmania, Plasmodium, and Mycobacteria. A new detection chemistry has been developed for real-time RT-PCR (patent pending). The laboratory has contributed experimental real-time PCR met