Cytotoxic Molecules in Invertebrate Immunity

昆虫和其他节肢动物是许多具有医疗和兽医重要性的寄生虫病的媒介，包括疟疾、锥虫病(昏睡病、恰加斯病)、丝虫病(眼线虫、象皮病)和狗心虫病。了解昆虫媒介中调节感染过程的因素对于制定应对策略以保护人类和牲畜免受昆虫媒介寄生虫的不利影响至关重要。昆虫具有有效的先天细胞和体液免疫机制，它们与哺乳动物的先天免疫反应有着惊人的相似之处，这表明这些至关重要的过程具有共同的祖先。除了记录和表征几种昆虫的各种免疫效应反应的研究外，人们对最初的非自我识别反应的潜在机制，或免疫激活细胞使用的杀伤分子的身份和作用模式知之甚少。这项拟议的研究旨在阐明昆虫产生的细胞毒性成分的性质，这些成分专门针对并摧毁昆虫体内的寄生虫。研究人员计划研究果蝇对后生动物寄生虫Leptopilina boulardi的细胞防御反应，以及残存昆虫Rhodnius prolixus对原生动物寄生虫锥虫的细胞毒性反应。使用这两个昆虫-寄生虫模型系统，将监测各种活性氧中间体(超氧阴离子、过氧化氢和羟基自由基)的产生，以及活性氮中间体(例如，一氧化氮和过氧亚硝酸盐)的产生，以及两种重要酶NADPH氧化酶和一氧化氮合酶的活性水平。为了研究果蝇和罗得牛对其后生动物和原生动物寄生虫的免疫应答过程中生物化学和酶活性的变化，研究人员将使用高效液相色谱结合电化学检测、分光光度法、荧光法和分子技术。这种在昆虫免疫反应过程中积极识别特定细胞毒性分子及其产生的酶机制的综合方法将非常有用，因为它将为昆虫的细胞-细胞信号和免疫识别提供重要的见解。

Insects and other arthropods are vectors of numerous parasitic diseases of medical and veterinary importance, including malaria, trypanosomiasis (sleeping sickness, Chagas' disease) filariasis (eye worms, elephantiasis), and dog heartworm. Knowledge of the factors that regulate the infection process in the insect vector is essential for the development of counter strategies to protect humans and livestock from the adverse effects of insect-vectored parasites. Insects possess effective innate cellular and humoral immune mechanisms that exhibit striking parallels between the innate immune responses of mammals, suggesting a common ancestry for these critically important processes. With the exception of studies documenting and characterizing various immune effector responses for several insect species, little or nothing is known of the mechanisms underlying the initial non-self recognition response, or the identity and mode of action of killing molecules used by immune activated cells. The proposed research is designed to elucidate the nature of the cytotoxic components generated by insects that specifically target and destroy their internal parasites. The researchers plan to study the cellular defence responses made by the fruit fly Drosophila melanogaster against the metazoan parasite Leptopilina boulardi, and the cytotoxic responses of the reduviid bug Rhodnius prolixus against the protozoan parasite Trypanosoma. Using these two insect-parasite model systems, the production of various reactive oxygen intermediates (superoxide anion, hydrogen peroxide, and the hydroxyl radical) will be monitored, as will the production of reactive nitrogen intermediates (e.g., nitric oxide and peroxynitrite), and the activity levels of two important enzymes, NADPH oxidase and nitric oxide synthase. To investigate the changes in biochemistry and enzyme activity that occur during the immune responses elicited by Drosophila and Rhodnius against their respective metazoan and protozoan parasites, the investigators will employ high performance liquid chromatography with electrochemical detection, spectrophotometry, fluorometry and molecular techniques. This comprehensive approach for positively identifying specific cytotoxic molecules and the enzymatic mechanisms by which they are produced during insect immune responses will be extremely useful by providing important insights concerning cell-cell signaling and immune recognition in insects.