Biology, Immunology & therapy of Acanthamoeba Keratitis

描述（申请人提供）：棘阿米巴角膜炎是由致病性自由生活的阿米巴虫引起的一种危及视力的角膜疾病。本课题的研究基于以下观察结果：1)先天免疫系统在棘阿米巴角膜炎中起重要作用，多形核中性粒细胞（PMN）是棘阿米巴角膜炎病变中突出的炎症细胞；2)中性粒细胞在棘阿米巴感染的初步解决中起重要作用；3)棘阿米巴角膜炎的发病机制因中性粒细胞浸润而加重；4)角膜上皮上toll样受体（TLRs）启动棘阿米巴感染的炎症反应。这可以通过在感染部位固定PMN来实现；5)角膜炎是棘阿米巴滋养虫介导的因子（初步的蛋白酶）介导的组织损伤的结果；6)寄生源致病分子在滋养体成囊或死亡后仍然存在；7)寄生虫传播的致病分子与角膜表面的几种分子发生反应，诱导上皮细胞释放趋化因子和细胞因子；寄生虫衍生的分子具有免疫原性，可用于诱导粘膜抗体的产生，这些抗体将中和致病分子，从而减轻组织损伤并减少中性粒细胞浸润。因此，pmn的保护和破坏作用分别影响棘阿米巴感染和疾病过程的结果。第一个具体目的是验证角膜上皮上toll样受体（TLRs）启动眼棘阿米巴感染的炎症反应的假设。这可以通过固定pmn来实现，这些pmn要么最初杀死棘阿米巴，要么有助于疾病的发病机制。第二个特定目标将验证甘露糖诱导的棘阿米巴细胞病变蛋白（MIP-133）与角膜上皮细胞上的磷脂相互作用，并通过涉及磷脂酶A2 （PLA2）激活的新途径诱导细胞凋亡和花生四烯酸释放的假设。第三个具体目标将验证棘阿米巴滋养体组成性表达PLA2的假设，该PLA2直接诱导角膜上皮细胞的细胞病变作用或激活磷脂并诱导花生四烯酸释放。第四个特异性目的是验证PLA2抑制剂治疗和MIP-133免疫将减轻棘阿米巴角膜炎发病机制的假设。该项目的长期目标是评估棘阿米巴角膜炎的致病机制，这可能对棘阿米巴角膜炎患者设计改进的治疗方法产生巨大影响，这些治疗方法代表了最严重的治疗困境。公共卫生相关性：该项目的长期目标是评估棘阿米巴角膜炎的致病机制，这可能对棘阿米巴患者设计改进的治疗方法产生巨大影响，这些治疗方法代表了最严重的治疗困境。

DESCRIPTION (provided by applicant): Acanthamoeba keratitis is a sight-threatening corneal disease caused by pathogenic free-living amoebae. The rationale for this research project is based on the following observations: 1) The innate immune system plays an important role in Acanthamoeba keratitis and polymorphonuclear neutrophils (PMN) are the prominent inflammatory cells in Acanthamoeba keratitis lesions; 2) neutrophils are important in initial resolution of Acanthamoeba infection; 3) pathogenesis of Acanthamoeba keratitis is exacerbated by the protease elaborated by infiltrating neutrophils; 4 ) Toll-like receptors (TLRs) on the corneal epithelium initiate the inflammatory responses to Acanthamoeba infections. This may be achieved by immobilizing PMN at the site of infection; 5) keratitis is the consequence of tissue damage mediated by factors (preliminary proteases) elaborated by Acanthamoeba trophozoites; 6) parasite- derived pathogenic molecules persist even after trophozoites encyst or die; 7) parasite-borne pathogenic molecules react with several molecules on the surface of the cornea and induce the release of chemokines and cytokines by the epithelial cells; 8) parasite-derived molecules are immunogenic and can be used to elicit the production of mucosal antibodies that will neutralize the pathogenic molecules and thus, mitigate tissue damage and reduce neutrophil infiltration. Thus, the protective and destructive roles of PMNs influence the outcome of Acanthamoeba infection and disease processes respectively. The first specific aim will test the hypothesis that Toll-like receptors (TLRs) on the corneal epithelium initiate the inflammatory responses to ocular Acanthamoeba infections. This may be achieved by immobilizing PMNs that either initially kill Acanthamoeba or contribute to the pathogenesis of the disease. The second specific aim will test the hypothesis that the mannose induced- Acanthamoeba cytopathic protein (MIP-133) interacts with phospholipids on the corneal epithelial cells and induces both apoptosis and arachidonic acid release through a novel pathway involving phospholipase A2 (PLA2) activation. The Third specific aim will test the hypothesis that Acanthamoeba trophozoites constitutively express PLA2 that either directly induces cytopathic effects on the corneal epithelial cells or activates phospholipids and induces arachidonic acid release. The forth specific aim will test the hypothesis that treatment with PLA2 inhibitors and immunization with MIP-133 will mitigate the pathogenesis of Acanthamoeba keratitis. The long-range goal of this project is to evaluate the pathogenic mechanisms of Acanthamoeba keratitis that could have an enormous impact on designing improved therapies for Acanthamoeba patients that represent the most serious therapeutic dilemmas. PUBLIC HEALTH RELEVANCE: The long-range goal of this project is to evaluate the pathogenic mechanisms of Acanthamoeba keratitis that could have an enormous impact for designing improved therapies for Acanthamoeba patients that represent the most serious therapeutic dilemmas.