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Entwicklung Eines Funktionellen in Vitro Test (FIT) Zur Prüfung Der

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Entwicklung Eines Funktionellen in Vitro Test (FIT) Zur Prüfung Der Aus dem Institut für Parasitologie und der Arbeitsgruppe Immunologie der Tierärztlichen Hochschule Hannover ___________________________________________________________________________ Entwicklung eines funktionellen in vitro Test (FIT) zur Prüfung der Sensibilisierung feliner basophiler Granulozyten von Katzen mit kontrollierter Flohexposition (Ctenocephalides felis felis) INAUGURAL – DISSERTATION zur Erlangung des Grades eines Doctor of Philosophy (PhD) durch die Tierärztliche Hochschule Hannover Vorgelegt von Kristin Stuke aus Emsbüren ___________________________________________________________________________ Hannover 2005 Wissenschaftliche Betreuung: Prof. Dr. med. vet. G. von Samson-Himmelstjerna Prof. Dr. med. vet. Dr. h. c. W. Leibold Prof. Dr. med. vet. P. Valentin-Weigand Prof. Dr. med. vet. A. Daugschies 1. Gutachten: Prof. Dr. med. vet. Dr. h. c. W. Leibold Prof. Dr. med. vet. G. von Samson-Himmelstjerna Prof. Dr. med. vet. P. Valentin-Weigand Prof. Dr. med. vet. A. Daugschies 2. Gutachten: PD Dr. med. vet. R. Straubinger, PhD Tag der öffentlichen Disputation: 10. Novermber 2005 Diese Arbeit entstand mit der Unterstützung der Bayer AG, Leverkusen „Do what you can, with what you have, right where you are.” (Theodore Roosevelt) Teile der vorliegenden Dissertation wurden bereits mündlich oder schriftlich veröffentlicht: Stuke K, von Samson-Himmelstjerna G, Mencke N, Hansen O, Schnieder T, Leibold W. Flea Allergy Dermatitis in the Cat: Establishment of a Functional In vitro Test. Parasitol Res. 2003 Jul; 90 (3): 129-31. Stuke K, von Samson-Himmelstjerna G, Mencke N, Hansen O, Schnieder T, Leibold W. Flea Allergy Dermatitis in the Cat: Establishment of a Functional In vitro Test. The 19th International conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP), New Orleans, Louisiana, USA, 10.-14.08.2003 Stuke K, von Samson-Himmelstjerna G, Mencke N, Hansen O, Schnieder T, Leibold. Flea Allergy in the Cat: Kinetics of in vivo Sensitization of Basophilc Granuloytes monitored by a Functional In vitro Test (FIT). 1st Short Course for Young Parasitologists, Würzburg, 15.- 17.03.2004 Stuke K, von Samson-Himmelstjerna G, Mencke N, Hansen O, Schnieder T, Leibold W. Prinzipielle Entwicklung eines Funktionellen In-vitro-Tests (FIT). Tagung der Deutschen Veterinärmedizinischen Gesellschaft e. V. (DVG), Starnberg⁄München, 09.-11.06.2004 Stuke K, von Samson-Himmelstjerna G, Mencke N, Hansen O, Schnieder T, Leibold W. Flea Hypersensitivity in Cats: What can we learn from their Basophils?. 8th International Symposium on Ectoparasites of Pets (ISEP), Hannover, 08.-11.05.2005 Inhaltsverzeichnis 1 EINLEITUNG UND ZIELSETZUNG 19 2 LITERATURÜBERSICHT 22 2.1 Allergien bei Mensch und Katze 22 2.1.1 Übersicht über die verschiedenen Allergietypen 22 2.1.2 Mechanismen der Typ I Allergie 26 2.1.3 Immunglobulin-Isotypen der Katze und ihre Bedeutung für die Typ I Allergie 31 2.1.4 Überblick über Hypersensibilitätsreaktionen von Katzen gegen Arthropoden 32 2.1.5 Die Flohspeichelallergie 34 2.2 Allergiediagnostik bei der Katze 40 2.2.1 In vivo Allergietests 41 2.2.2 In vitro Allergietests 41 2.3 Untersuchungen zur Chemotaxis 44 2.3.1 Chemotaktische Substanzen 45 3 GERÄTE, MATERIAL UND METHODEN 48 3.1 Geräte 48 3.2 Material 49 3.2.1 Klinikbedarf und Medikamente 49 3.2.2 Laborbedarf und Verbrauchsmaterialien 50 3.2.3 Reagenzien 51 3.2.4 Puffer und Lösungen 53 3.2.5 Antikörper und Seren 55 3.2.6 Allergen-Präparationen 56 3.2.7 Bestandteile des Funktionellen In vitro Test (FIT) 56 3.2.8 Bestandteile des Radio Immuno Assay (RIA) 58 3.2.9 Bestandteile des Intrakutantest (IKT) 59 3.2.10 Lösungen für die Dichtezentrifugation und Membranimmunfluoreszenz (MIF) 60 3.2.11 Lösungen für die Durchflusszytometrie 61 3.2.12 Bestandteile des Transmigrationstests 62 3.2.13 Versuchstiere 65 3.3 Methoden 67 3.3.1 Durchführung der Verlaufsuntersuchung 67 3.3.2 Blutentnahme 67 3.3.3 Serumgewinnung 68 3.3.4 Aufbereitung der Allergen-Präparationen 68 3.3.5 Funktioneller In vitro Test (FIT) 70 3.3.6 Radio Immuno Assay (RIA) 72 3.3.7 Intrakutantest (IKT) 75 3.3.8 Allercept™ FcεRI-Test 77 3.3.9 Beurteilung der klinischen Reaktion auf Flohstiche 78 3.3.10 Parasitologische Untersuchungen 79 3.3.11 Blutbildanalyse 79 3.3.12 Dichtezentrifugation 80 3.3.13 Zellquantifizierung und Vitalitätsbestimmung 80 3.3.14 Durchflusszytometrie 81 3.3.15 Transmigrationstest 83 3.3.16 Bestimmung der Reaktion angereicherter feliner Basophiler im FIT 87 3.3.17 Immunphänotypisierung peripherer Blutlymphozyten 89 3.3.18 Statistische Auswertungen 91 4 ERGEBNISSE 96 4.1 Adaptierung des funktionellen in vitro Test (FIT) für die Katze 96 4.1.1 Durchführung des FIT mit basophilen Granulozyten aus gewaschenem Vollblut der Katze 96 4.1.2 Versuch der Anreicherung feliner Basophiler zur Prüfung im FIT 114 4.2 Verlaufsuntersuchung zur Erfassung der Sensibilisierung von Katzen gegen C. felis während experimentell kontrollierter Flohexpositionen 130 4.2.1 Allgemeine Kontrolluntersuchungen 130 4.2.2 Prüfung des Reaktionsverhaltens feliner Basophiler mit dem FIT 136 4.2.3 Nachweis der Sensibilisierung von felinen Mastzellen gegen eine Ctenocephalides-Präparation im Intrakutantest 174 4.2.4 Nachweis von Anti-Flohantigen-spezifischem Immunglobulin E im Katzenserum 177 4.3 Weitere Untersuchungen mit dem funktionellen in vitro Test 186 4.3.1 Untersuchung der Beständigkeit der Floh-spezifischen Sensibilisierung nach Flohexposition 186 4.3.2 Untersuchung auf eine Sensibilisierung gegen weitere Allergen-Präparationen 188 4.3.3 Untersuchung der Reaktionsbereitschaft basophiler Granulozyten von SPF-Katzen gegen Arthropodenantigene 190 4.3.4 Untersuchung der Histaminfreisetzung durch ein Ziegenantiserum gegen Hunde Immunglobulin E 191 5 DISKUSSION 194 5.1 Methodische Aspekte 194 5.1.1 Schwankungen im Gesamthistamingehalt in der untersuchten Katzenpopulation 194 5.1.2 Untersuchung der Antikörper-vermittelten Histaminfreisetzung 195 5.1.3 Untersuchung der Antigen-induzierten Histaminfeisetzung 199 5.1.4 Histaminfreisetzung aus Basophilen der Katze in Abhängigkeit von Lagerungsdauer und Lagerungstemperatur der Blutprobe 201 5.1.5 Versuch der Anreicherung feliner Basophiler mittels Dichtezentrifugation und Transmigrationsverfahren 203 5.1.6 Auswirkung der Anreicherungsverfahren auf die Histaminfreisetzung feliner Basophiler im FIT 205 5.1.7 Beurteilung der spontanen Histaminfreisetzung 206 5.1.8 Beurteilung des Sensibilisierungsgrades 207 5.2 Untersuchung der Auswirkungen wiederholter Flohexpostionen auf das Reaktionsverhalten feliner Basophiler im Zeitverlauf 208 5.2.1 Einfluss wiederholter Flohexpositionen auf Grad und Verlauf der Basophilen- Sensibilisierung in der untersuchten Katzenpopulation 208 5.2.2 Einfluss wiederholter Flohexpositionen auf die Blutwerte in der untersuchten Katzenpopulation 212 5.3 Vergleich des FIT mit anderen Testmethoden zum Nachweis allergischer Hauterkrankungen bei Katzen 212 5.4 Interpretation der klinischen Ausprägung der Flohspeichelallergie bei der untersuchten Katzenpopulation 215 6 ZUSAMMENFASSUNG 218 7 SUMMARY 220 8 LITERATURVERZEICHNIS 222 9 ANHANG I GLOSSAR Verzeichnis der in Text und Bild verwendeten Abkürzungen, Termini Technici und Symbolik ♂ männlich ♀ weiblich α anti (gerichtet gegen) ε epsilon °C Grad Celsius λ lambda µ mikro (x 10-6) Abb. Abbildung ADCC Antibody Dependent Cellular Cytotoxicity (antikörperabhängige zelluläre Zytotoxizität) AK Antikörper Aliquots gleiche Anteile einer Probe AO Akridinorange APC Antigen Presenting Cell(s) (antigenpräsentierende Zelle(n)) Aqua dest. Aqua destillata (destilliertes Wasser) Aqua tridest. Aqua tridestillata (dreifach destilliertes Wasser) bzgl. bezüglich B0 Histaminfreie Probe im RIA; da es sich bei dem verwendeten System um einen kompetitiven RIA handelt, wird hier durch den spezifischen Antikörper die maximal erfassbare Menge an 125J- markiertem Histamin bestimmt, ohne dass ein Probenhistamin konkurriert; dient als Bezugsgröße (100%) für die Probenwerte, in denen das zu bestimmende konkurrierende Histamin enthalten ist Bq Becquerel, SI-Einheit für den Zerfall einer radioaktiven Substanz, 1 Bq = 1 Zerfall pro Sekunde; alte Einheit Curie, 1 Ci = 3,7 x 1010 Bq bzw. beziehungsweise ca. circa CD Cluster of Differentiation (Zelldifferenzierungsantigene) CD4+ Zellen, die das Differenzierungsantigen CD4 an der Oberfläche exprimieren CD8+ Zellen, die das Differenzierungsantigen CD8 an der Oberfläche exprimieren GLOSSAR CD40 Zellen, die das Differenzierungsantigen CD40 an der Oberfläche exprimieren CD40L Zellen, die den Liganden für das Differenzierungsantigen CD40 an der Oberfläche exprimieren CO2 Kohlenstoffdioxid cpm counts per minute (Zählimpulse pro Minute) cm Zentimeter Cn Culicoides nubeculosus Ct Ctenocephalides felis felis / C. canis Cte f 1; rCte f 1 erstes Hauptallergen des Katzenflohs C. felis felis (18 kDa); rekombinante Form des Proteins Da Dalton (Molekulargewicht; 1,66 x 10-24g) DF Domestic fly (Musca domestica) Dfa Dermatophagoides farinae DMSO Dimethylsulfoxid DNA Desoxyribonucleic Acid (Desoxyribonucleinsäure) Dpt Dermatophagoides pteronyssinus EA Units ELISA Absorbance Units (Intensitätseinheiten der Farbänderung im ELISA) EDTA Ethylendiamintetraacetat EKH Europäische Kurzhaarkatze ELISA Enzyme-Linked Immunosorbent Assay (Enzym gekoppelter Immunadsorptionstest) engl. englisch et al. et alii (und andere) Events Messereignisse in der Durchflusszytometrie f femto (x 10-15) FACScan® Fluorescence Activated Cell Scanner (Fluoreszenzaktiviertes Durchflusszytometer der Firma Becton Dickinson, Heidelberg)
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