Biology, Immunology & therapy of Acanthamoeba Keratitis

描述(申请人提供)：棘阿米巴角膜炎是由致病的自由生活的阿米巴引起的一种威胁视力的角膜疾病。本研究基于下列观察结果：1)天然免疫系统在棘阿米巴角膜炎中起重要作用，中性粒细胞(PMN)是棘阿米巴角膜炎病变中最重要的炎症细胞；2)中性粒细胞在棘阿米巴感染的最初消退中起重要作用；3)中性粒细胞渗入的蛋白酶加剧了棘阿米巴角膜炎的发病；4)角膜上皮细胞上的Toll样受体(TLRs)启动了对棘阿米巴感染的炎症反应。这可通过将PMN固定在感染部位来实现；5)角膜炎是由棘阿米巴滋养体所阐述的因子(初步蛋白酶)介导的组织损伤的结果；6)寄生虫衍生的致病分子即使在滋养体包囊或死亡后仍存在；7)寄生虫携带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；8)寄生虫衍生的分子具有免疫原性，可用于诱导产生粘膜抗体，从而中和致病分子，从而减轻组织损伤和减少中性粒细胞的渗透。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转归。第一个特定目标将检验角膜上皮细胞上的Toll样受体(TLRs)启动对眼棘阿米巴感染的炎症反应的假说。这可以通过固定PMN来实现，PMN最初可以杀死棘阿米巴，或者有助于疾病的发病。第二个特定目的是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说。第三个特定目标将检验这一假设，即棘阿米巴滋养体结构性地表达PLA2，该PLA2要么直接诱导对角膜上皮细胞的细胞病变作用，要么激活磷脂并诱导花生四烯酸释放。第四个特定目标将检验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，这可能对设计代表最严重治疗困境的棘阿米巴患者的改进疗法产生巨大影响。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，这可能对设计代表最严重治疗困境的棘阿米巴患者的改进疗法产生巨大影响。

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.