ERWEITERTE ZUSAMMENFASSUNG
6.2. Erweiterte Zusammenfassung
6.2.2. Ergebnisse und Schlussfolgerungen
Als Verfahren zur Gewinnung von granulozytenhaltigen Zellsuspensionen für den Gebrauch in der Mikro-Chemotaxis-Kammer hat sich vor allem die Separation dieser Zellen aus Schafsblut bewährt. Hier wurden routinemäßig Suspensionen mit mehr als 80% Granulozyten, davon bis zu 28% eosinophile Granulozyten, erzielt. Eine weitere Anreicherung dieser Granulozytenfraktion aus Schafsblut war jedoch mit keiner der erprobten Methoden möglich. Die Injektion von Parasitenextrakt in die nicht laktierende Milchdrüse führte zur Ansammlung eines Exsudats mit bis zu 90% eosinophilen Granulozyten. Bei der Anwendung dieser Exsudatzellen in Chemotaxis-Experimenten wurde jedoch festgestellt, dass sie verstärkt aneinander hafteten und zur Klumpenbildung auf dem Filter neigten, was ihre Nutzbarkeit in der Mikro-Chemotaxis-Kammer einschränkte.
Die Injektion parasitärer Produkte in die subkutan implantierten Kammern hingegen resultierte nicht in einem verstärkten Einstrom eosinophiler Granulozyten, obwohl sie zu einer signifikanten Erhöhung der Gesamtleukozytenzahl führte. Die in vitro Kultur von Knochenmarkszellen war nicht erfolgreich, weil die vorhandenen Cytokine, die essentiell sind für die Heranreifung der Granulozyten, nicht den Anforderungen entsprachen.
Die in vitro Experimente mit der Mikro-Chemotaxis-Kammer demonstrierten die Präsenz chemotaktischer Substanzen sowohl für neutrophile als auch für eosinophile Granulozyten in Extrakten und E/S Produkten von H. contortus und O. circumcincta Larven und adulten Parasiten. In mehreren Experimenten gab es außerdem Anhaltspunkte für das Vorhandensein von Substanzen mit chemokinetischer Wirkung (die ungerichtete Bewegungsaktivität von Zellen beeinflussend). Insgesamt war die chemotaktische Reaktion neutrophiler
6. Summary / Erweiterte Zusammenfassung
insgesamt 35 Experimenten für neutrophile Chemotaxis waren nur 7 Tests negativ, während eosinophile Chemotaxis in 20 von insgesamt 38 Experimenten nachgewiesen werden konnte. Die E/S Produkte adulter Parasiten hatten eine ausgeprägtere chemotaktische Aktivität sowohl für neutrophile als auch eosinophile Granulozyten als die E/S Produkte von H. contortus und O.
circumcincta Larven. Dabei konnte in den meisten Experimenten, in denen verschiedene Verdünnungsstufen der parasitären Produkte getestet wurden, eine Konzentrationsabhängigkeit der chemotaktischen Aktivität für neutrophile und weniger ausgeprägt auch für eosinophile Granulozyten demonstriert werden.
Wie auch in der SDS-Gel Elektrophorese gezeigt wurde, war die Trennung der Extrakte und E/S Produkte in Fraktionen mit unterschiedlichem Molekulargewicht mittels Ultrafiltration nur bedingt erfolgreich und es gelang nicht, die chemotaktische Aktivität auf bestimmte Fraktionen einzugrenzen. Es wäre möglich, dass es sich bei den chemotaktischen Faktoren um sehr kleine Moleküle handelt, die die Poren aller verwendeten Filter passieren können oder die an größeren Proteinen haften und so in den Fraktionen höherer Molekulargewichte verbleiben.
Die molekularbiologische Untersuchung der aus Parasitenextrakten isolierten DNA ergab keinen Hinweis auf das Vorhandensein von Wolbachia DNA in diesen Proben. Die Möglichkeit, dass andere symbiotische Bakterien in den Parasiten vorhanden und an der Bildung chemotaktischer Faktoren beteiligt sind, ist jedoch nicht auszuschließen.
Die Ergebnisse dieser Studie unterstützen die These, dass H. contortus und O.
circumcincta, wie für verschiedene andere Parasiten bereits nachgewiesen, Substanzen mit einer direkten chemotaktischen Wirkung auf neutrophile und eosinophile Granulozyten produzieren und durch die Ausscheidung dieser Stoffe für die Akkumulation von Leukozyten im Rahmen der parasitären Gastritis direkt mitverantwortlich sind.
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