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Visceral Leishmaniasis in Ethiopia: Innate Immune Functions, Biomarkers of Cure and Potential Roles of Cattle for Transmission

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Visceral Leishmaniasis in Ethiopia: Innate Immune Functions, Biomarkers of Cure and Potential Roles of Cattle for Transmission ___________________________________________________________________________________________________ Aus der Klinik für Infektiologie und Mikrobiologie der Universität zu Lübeck Direktor: Prof. Dr. med. Jan Rupp Visceral leishmaniasis in Ethiopia: Innate immune functions, biomarkers of cure and potential roles of cattle for transmission Dissertation zur Erlangung der Doktorwürde Dr. rer. hum. biol. der Universität zu Lübeck - Aus der Sektion Medizin - presented by Geremew Tasew Guma from Arjo, Wellega, Ethiopia Lübeck 2018 ___________________________________________________________________________________________________ 1. Berichterstatter: Prof. Dr. univ. (Univ. Budapest) Tamás Laskay 2. Berichterstatter: Priv.-Doz. Dr. rer. nat. Norbert Reiling Tag der mündlichen Prüfung: 28.8.2018 Zum Druck genehmigt, Lübeck den 28.8.2018 - Promotionskommission der Sektion Medizin- 0 ABBREVIATIONS___________________________________________________________________ ABBREVIATIONS ATP Adenosine triphosphate BSA Bovine serum albumin CaCl2 Calcium chloride CD62L Cluster of differentiation 62L CD66b Cluster of differentiation 66b CGD Chronic granulomatous disease CO2 Carbondioxide CR3 Complement receptor -3 CRP C-reactive protein DAT Direct agglutination test DC Dendritic cells DCL Diffuse cutaneous leishmaniasis DHR 123 Dihydrorhodamine 123 DNA Deoxyribonucleic acid EGFR Epidermal growth factor receptor EHC Endemic healthy control ELISA Enzyme linked immunosorbent assay FACS Fluorescence-activated cell sorting FBS Fetal bovine serum FITC Fluorescein isothiocyanate fMLP Formyl-methionine-leucine-phenylalanine G-CSF Granulocyte colony stimulating factor GIPL glycoinositolphospholipids GM-CSF Granulocyte macrophage colonystimulating factor H2O2 Hydrogen peroxide HCl Hydrochloric acid HIV Human immunodeficiency virus HIV1/2 Human immuno deficiency viruses 1/2 IFN Interferon IFN- Interferon alpha IFN- Interferon beta IFN-γ Interferon gamma IgG Immunoglobulin G IL-1 Interleukin-1 IL-1 Interleukin-1 beta IL-10 Interleukin-10 IL-12 Interleukin-12 IL-2 Interleukin- 2 IL-6 Interleukin- 6 IL-7 Interleukin- 7 IL-8 Interleukin- 8 iNOS Inducible nitric oxide synthase IP-10 IFN-inducible protein-10 KCl Potassium chloride L. aethiopica Leishmania aethiopica L. braziliensis Leishmania braziliensis I ABBREVIATIONS___________________________________________________________________ L. chagasi Leishmania chagasi L. donovani Leishmania donovani L. enrietti Leishmania enrietti L. infantum Leishmania infantum L. major Leishmania major L. mexicana Leishmania mexicana L. tropica Leishmania tropica LCL Localized cutaneous leishmaniasis LN Lymph nodes LPG Lipophosphoglycan LPS Lipopolysaccharide M. tuberculosis Mycobacterium tuberculosis MALP-2 Macrophage-activating lipopeptide-2 MCF Mononuclear cell factor MCL Mucocutaneous leishmaniasis MCP-1 Monocyte chemotactic protein 1 MDM Monocyte derived macrophages MHC Major histocompatability complex MIP-1 Macrophage inflammatory protein-1 alpha MIP-1 Macrophage inflammatory protein-1 beta MMP- 9 Matrix metalloproteinase-9 MPO Myeloperoxidase MR Mannose receptor MyD88 Myeloid differentiation primary response 88 NaCl Sodium chloride NADPH Nicotinamide adenine dinucleotide phosphate NaHCO3 Sodium bicarbonate NETs Neutrophil extracellular traps NF-B Nuclear factor kappa B NK Natural killer NNN Novy-MacNeal-Nicolle NO Nitric oxide O2¯ Superoxide anion P. orientalis Phlebotomus orientalis PAMP Pathogen-associated molecular patterns PBS Phosphate buffered saline PCR Polymerase chain reaction PCR-RFLP Polymerase chain reaction restriction fragment length polymorphism PE Phycoerythrin PKDL Post kala azar dermal leishmaniasis PMN Polymorphonuclear Poly (I: C) Polyinosinic-polycytidylic acid PRR Pattern recognition receptors RANTES Regulated on activation, normal T cell expressed and secreted rK39 Recombinant fragment of kinesin gene 39 RNA Ribonucleic acid ROS Reactive oxygen species RPMI 1640 Roswell Park Memorial Institute Medium 1640 II ABBREVIATIONS___________________________________________________________________ S. aureus Staphylococcus aureus sCD40L Soluble CD40 ligand SD Standard deviation SEM Standard error of the mean TAE buffer Tris-acetate-EDTA buffer TB Tuberculosis TCR T cell receptor TEM Trans-endothelial migration TGF– Transforming growth factor beta TIR Toll/interleukin-1 receptor TLR Toll-like receptor TNF- Tumor necrosis factor-alpha TRIF TIR Toll/interleukin-1 receptor domain containing adapter inducing interferon-beta VEGF Vascular endothelial growth factor VL Visceral leishmaniasis WHO World Health Organization III CONTENTS______________________________________________________________________________________ CONTENTS Pages ABBREVIATIONS ------------------------------------------------------------------------------------ I CONTENTS -------------------------------------------------------------------------------------------- 1 1. INTRODUCTION ---------------------------------------------------------------------------------- 4 1.1. Visceral leishmaniasis --------------------------------------------------------------------------------------------------------------4 1.2. Epidemiology of visceral leishmaniasis ---------------------------------------------------------------------------------------5 1.3. Life cycle of Leishmania donovani ---------------------------------------------------------------------------------------------7 1.4. Modes of L. donovani transmission--------------------------------------------------------------------------------------------9 1.5. Leishmania donovani parasite ------------------------------------------------------------------------------------------------ 10 1.6. Diagnosis of visceral leishmaniasis------------------------------------------------------------------------------------------- 11 1.7. Pathogenesis of visceral leishmaniasis ------------------------------------------------------------------------------------- 14 1.8. Immunity during visceral leishmaniasis ------------------------------------------------------------------------------------ 15 1.8.1. Innate immunity and Leishmania --------------------------------------------------------------------------------------- 16 1.8.1.1. Toll-Like Receptors (TLR) ---------------------------------------------------------------------------------------- 18 1.8.1.2. Phagocytosis ---------------------------------------------------------------------------------------------------------- 20 1.8.1.3. Reactive oxygen species (ROS) --------------------------------------------------------------------------------- 22 1.8.1.4. Neutrophils ------------------------------------------------------------------------------------------------------------ 25 1.8.1.5. Monocytes ------------------------------------------------------------------------------------------------------------- 27 1.8.1.6. Macrophages ---------------------------------------------------------------------------------------------------------- 28 1.8.1.7. Cytokines --------------------------------------------------------------------------------------------------------------- 30 1.8.1.8. Chemokines ----------------------------------------------------------------------------------------------------------- 32 1.9. Can L. donovani have bovine reservoir host cells? ---------------------------------------------------------------------- 35 1.10. Test of cure for visceral leishmaniasis ------------------------------------------------------------------------------------- 36 1.11. Aims of the study ---------------------------------------------------------------------------------------------------------------- 37 2. MATERIALS AND METHODS ---------------------------------------------------------------- 39 2.1. Materials ----------------------------------------------------------------------------------------------------------------------------- 39 2.1.1. Leishmania parasites ------------------------------------------------------------------------------------------------------- 39 2.1.2. Chemicals, antibiotics, media and buffers ---------------------------------------------------------------------------- 39 2.1.3. Antibodies, stimulants, enzymes and fluorescent agents -------------------------------------------------------- 40 2.1.4. ELISAs and ready to use kits ---------------------------------------------------------------------------------------------- 41 2.1.5. Equipments ------------------------------------------------------------------------------------------------------------------- 42 2.1.6. Plastics and consumables ------------------------------------------------------------------------------------------------- 43 2. 1.7. Primers and PCR reaction ------------------------------------------------------------------------------------------------ 43 2. 1.9. Buffers and media prepared in the laboratory --------------------------------------------------------------------- 44 2.1.10. Software --------------------------------------------------------------------------------------------------------------------- 45 2.2. Methods ----------------------------------------------------------------------------------------------------------------------------- 46 2.2.1. Part I. Establishment of a functional laboratory and optimization of methods ---------------------------- 46 2.2.1.1. Establishment of a functional laboratory to carry out the planned studies in the VL endemic area in Ethiopia ---------------------------------------------------------------------------------------------------- 46 2.2.1.2. Optimization
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