Pathogenesis of P. aeruginosa corneal infection

描述：这项应用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，并开发化疗和免疫治疗干预措施来治疗这种严重的眼部感染。P. aeruginosa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。目的1的目标包括探索CFTR-P的作用。绿脓杆菌在角膜病理中的相互作用表现为宿主趋化因子和细胞因子反应。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，铜绿假单胞菌诱导和CFTR依赖的细胞因子释放与发病机制和感染抗性有关，特别是IL-1， IL-6， IL-18和ifn - γ，将被检测。我们将在适当的WT和转基因小鼠中，使用角膜划伤眼感染模型，或在抗体中和该因子的小鼠中，测试所确定的因子在眼睛感染和病理中的作用。铜绿假单胞菌- CFTR的相互作用依赖于脂筏的形成，初步数据表明，脂筏的破坏可防止角膜病变并促进感染眼睛的细菌清除。在目标2中，这些筏在发病机制中的作用将通过WT和CF角膜细胞系以及由于酸性鞘磷脂酶基因破坏而无法形成脂筏的小鼠，以及用环糊精处理的小鼠进一步评估。环糊精治疗铜绿假单胞菌眼部感染具有高效的潜力，成为治疗的重要组成部分。最后，针对P. aeruginosa alginate （P. aeruginosa alginate，一种保守的细胞表面多糖，在角膜分离物中低水平表达），制备了一种完整的人源单克隆抗体（MAb）。重要的是，在P. aeruginosa感染小鼠的角膜中检测到藻酸盐表达，并且单抗对P. aeruginosa感染具有高度保护作用。在目标3中，将在小鼠角膜感染模型中评估单抗对6种不同铜绿假单胞菌菌株的治疗效果，目的是获得一种适用于大多数临床分离株的单一免疫治疗试剂，作为铜绿假单胞菌溃疡性角膜炎的辅助治疗方法。综上所述，基于对宿主-病原体相互作用的细胞和分子研究，铜绿假单胞菌角膜炎治疗的重大进展应该是显而易见的。

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.