Pathogenesis of P. aeruginosa corneal infection

产品说明：本申请的长期目标是了解角膜上皮细胞对铜绿假单胞菌的分子和细胞反应，并开发用于治疗这种严重眼部感染的化疗和免疫干预。 铜绿假单胞菌通过囊性纤维化跨膜传导调节因子（CFTR）进入角膜上皮细胞，这些细胞内细菌能够在组织中持续存在并避开宿主防御，导致严重的角膜病理学。目的1的目标包括探索CFTR-P. aeruginosa相互作用在角膜病理学中的作用，如通过宿主趋化因子和细胞因子应答所表现的。使用表达突变型或野生型（WT）CFTR的等基因人角膜上皮细胞系，将检查铜绿假单胞菌诱导的和CFTR依赖性的与发病机制和感染抗性有关的细胞因子（特别是IL-1、IL-6、IL-18和IFN-γ）的释放。将在适当的WT和转基因小鼠中使用角膜划痕损伤的眼感染模型或在用抗体中和该因子的小鼠中测试所鉴定的因子在眼感染和病理学中的作用。 铜绿假单胞菌- CFTR相互作用取决于脂质筏的形成，并且初步数据表明筏的破坏防止角膜病理学并促进细菌从感染的眼睛中清除。 在目标2中，将使用WT和CF角膜细胞系以及由于酸性鞘磷脂酶基因破坏而不能形成脂筏的小鼠和用环糊精处理的小鼠（其破坏脂筏）进一步评价这些筏在发病机制中的作用。环糊精治疗铜绿假单胞菌眼部感染具有高度有效的潜力，并成为治疗的重要组成部分。最后，已经开发了针对铜绿假单胞菌藻酸盐的全人单克隆抗体（MAb），铜绿假单胞菌藻酸盐是由角膜分离株以低水平表达的保守的细胞表面多糖。重要的是，在铜绿假单胞菌感染的小鼠的角膜中检测到藻酸盐表达，并且MAb对铜绿假单胞菌感染具有高度保护性。在目标3中，将在鼠角膜感染模型中评价Mab对6种不同铜绿假单胞菌菌株的治疗功效，目的是获得可用于对抗大多数临床分离株的单一免疫抑制剂，作为铜绿假单胞菌溃疡性角膜炎当前治疗方式的辅助手段。总体而言，在这些目标的结论中，基于对宿主-病原体相互作用的细胞和分子研究的使用，铜绿假单胞菌角膜炎的治疗开发的显著进展应该变得明显。

DESCRIPTION: The long-term goal of this application is to understand the molecular and cellular responses of corneal epithelial cells to Pseudomonas aeruginosa and develop chemotherapeutic and immunotherapeutic interventions for treatment of this serious eye infection. P. aeruginosa enters corneal epithelial cells via the cystic fibrosis transmembrane conductance regulator (CFTR), and these intracellular bacteria are able to persist in the tissue and avoid host defenses, leading to serious corneal pathology. Goals of aim 1 include exploring the role of the CFTR-P. aeruginosa interactions in corneal pathology as manifest by host chemokine and cytokine responses. Using isogenic human corneal epithelial cell lines expressing either mutant or wild-type (WT) CFTR, P. aeruginosa induced and CFTR-dependent release of cytokines implicated in pathogenesis and resistance to infection, notably IL-1, IL-6, IL-18 and IFN-gamma, will be examined. The role of the identified factors in eye infection and pathology will be tested in appropriate WT and transgenic mice using a corneal scratch-injured eye infection model or in mice in which the factor is neutralized with antibody. P. aeruginosa- CFTR interactions depend on formation of lipid rafts, and preliminary data indicate disruption of rafts prevents corneal pathology and promotes bacterial clearance from infected eyes. In aim 2, the role of these rafts in pathogenesis will be further evaluated using the WT and CF corneal cell lines as well as mice unable to form lipid rafts due to disruption of the acid sphingomyelinase gene, and in mice treated with cyclodextrin, which disrupts lipid rafts. Cyclodextrin treatment of P. aeruginosa eye infections has the potential to be highly efficacious and become an important component of therapy. Finally, a fully human monoclonal antibody (MAb) has been developed to P. aeruginosa alginate a conserved, cell surface polysaccharide that is expressed at low levels by corneal isolates. Importantly, alginate expression is detected n the corneas of P. aeruginosa infected mice and the MAb is highly protective against P. aeruginosa infection. In aim 3, the Mab will be evaluated for therapeutic efficacy against 6 different P. aeruginosa strains in the murine corneal infection model with the goal of having a single immunotherapeutic reagent useful against most clinical isolates as an adjunct to current treatment modalities for P. aeruginosa ulcerative keratitis. Overall at the conclusion of these aims, significant advances in the development of therapies for P. aeruginosa keratitis should become apparent based on use of cellular and molecular studies on host-pathogen interactions.