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Immunomodulation Through Excretory/Secretory Products of the Parasitic Helminth Echinococcus Multilocularis

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Immunomodulation Through Excretory/Secretory Products of the Parasitic Helminth Echinococcus Multilocularis Immunomodulation through Excretory/Secretory Products of the parasitic Helminth Echinococcus multilocularis Immunmodulation durch Exkretorisch/Sekretorischen Produkten der parasitären Helminthen Echinococcus multilocularis Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Infection and Immunity submitted by Justin Nono From Bafoussam, Cameroon Würzburg 2012 Submitted on: …………………………………………………………..…….. Office stamp Members of the Promotionskomitee: Chairperson: Prof. Dr. Thomas Hünig Primary Supervisor: Prof. Dr. Klaus Brehm Supervisor (Second): Prof. Dr. Manfred Lutz Supervisor (Third): Prof. Dr. Joachim Morschhäuser Supervisor (Fourth): Prof. Dr. Frank Brombacher Date of Public Defence: …………………………………………….…………… Date of receipt of Certificates: ………………………………………………. 2 AFFIDAVIT (EIDESSTATTLICHE ERKLÄRUNG) I hereby confirm that my thesis entitled Immunomodulation through Excretory/Secretory Products of the parasitic Helminth Echino- coccus multilocularis is the result of my own work. I did not receive any help or support from commercial consultants. All sources and / or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Würzburg……………………………………………….………………………………………… Date Signature 3 CONTENTS ACKNOWLEDGEMENTS .............................................................................................................................. 7 SUMMARY ...................................................................................................................................................... 9 ZUSAMMENFASSUNG .................................................................................................................................. 11 1-INTRODUCTION ........................................................................................................................................ 14 1.1 OVERVIEW OF HELMINTH INFECTIONS. ............................................................................................................... 14 1.2 THE FOX-TAPEWORM ECHINOCOCCUS MULTILOCULARIS. .................................................................................... 14 1.2.1 Phylogeny. .................................................................................................................................................. 14 1.2.2 Epidemiology. ............................................................................................................................................ 16 1.2.3 Life cycle. ................................................................................................................................................... 17 1.2.4 Clinical manifestations of AE. ................................................................................................................... 20 1.2.5 Prevention and control of alveolar echinococcosis. .................................................................................. 21 1.3 HOST IMMUNE RESPONSE TO ECHINOCOCCUS MULTILOCULARIS. ......................................................................... 25 1.3.1 The host immune system ............................................................................................................................. 25 1.3.2 Immunity and susceptibility to Echinococcus multilocularis. .................................................................... 33 1.4 MAINTENANCE OF E. MULTILOCULARIS LARVAE IN THE LABORATORY. .............................................................. 36 1.5 OBJECTIVES. ....................................................................................................................................................... 37 2-MATERIALS AND METHODS ................................................................................................................... 38 2.1 MATERIAL. ......................................................................................................................................................... 38 2.1.1 Equipment. ................................................................................................................................................. 38 2.1.2 Consumables. ............................................................................................................................................. 39 2.1.3 Chemicals, media, commercially available kits and enzymes. ................................................................... 40 2.1.4 Antibodies. ................................................................................................................................................. 42 2.1.5 Softwares, online bioinformatics tools and databases. .............................................................................. 43 2.1.6 Animals. ..................................................................................................................................................... 43 2.2 EFFECTS OF E/S PRODUCTS OF ECHINOCOCCUS MULTILOCULARIS LARVAE ON MURINE DC. ............................... 44 2.2.1 Ethics statement. ........................................................................................................................................ 44 2.2.2 In vivo maintenance of Echinococcus multilocularis larval tissue. ........................................................... 44 2.2.3 In vitro cultivation of E. multilocularis larval tissue. ................................................................................ 45 2.2.4 PCR-based assessment of host cell remnants. ........................................................................................... 47 2.2.5 Normalization of Echinococcus multilocularis larvae biomasses. ............................................................. 47 2.2.6 Preparation of Echinococcus multilocularis E/S products. ....................................................................... 48 2.2.7 Generation of murine bone marrow-derived dendritic cells. ..................................................................... 49 2.2.8 Isolation of murine splenocytes. ................................................................................................................. 49 2.2.9 Human red blood cells preparation. .......................................................................................................... 50 2.2.10 Assessment of the influence of Echinococcus multilocularis larvae E/S products on host cells. ............. 50 2.2.11 Statistical analysis. .................................................................................................................................. 52 2.3. INFLUENCE OF E/S PRODUCTS OF E. MULTILOCULARIS LARVAE ON FOXP3+ TREG EXPANSION. ......................... 53 2.3.1 Ethics statement. ........................................................................................................................................ 53 2.3.2 Parasite material. ...................................................................................................................................... 53 2.3.3 Mice. .......................................................................................................................................................... 53 2.3.4 Peritoneal lavage. ...................................................................................................................................... 54 4 2.3.5 Flow cytometric analyses. .......................................................................................................................... 54 2.3.6 In vitro regulatory T-cell suppression assay. ............................................................................................. 55 2.3.7 In vitro regulatory T-cell conversion assay. .............................................................................................. 56 2.3.8 In vitro IL-10 secretion assay. ................................................................................................................... 56 2.3.9 Statistical analyses. .................................................................................................................................... 57 2.4. E. MULTILOCULARIS LARVAE E/S PRODUCTS AND FOXP3+ TREGS. .................................................................... 57 2.4.1 Animals and Ethics statement. ................................................................................................................... 57 2.4.2 Parasite material. ...................................................................................................................................... 58 2.4.3 Echinococcus multilocularis Genome sequencing project. ........................................................................ 58 2.4.4 Identification of parasite factors, cloning and sequence analysis. ............................................................ 58 2.4.5 Recombinant expression, protein purification and antiserum generation. ................................................ 60 2.4.6 Assessment of factor secretion by in vitro cultivated metacestodes and transfected HEK cells. ............... 63 2.4.7 Treatment of murine BMDC. ..................................................................................................................... 63 2.4.8 Treg conversion assay. ..............................................................................................................................
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