Role of Toll-Like Receptors in Bacterial Keratitis

项目摘要/摘要 铜绿假单胞菌是一种常见的机会微生物。 与细菌性角膜炎有关，尤其是长期配戴隐形眼镜的人。我们的 目的是确定细菌性角膜炎发生的分子机制， 具体地说，Toll样受体(TLR)4的作用，目前仍然 在眼睛里没有被充分发掘。我们假设TLR4在先天的 角膜对铜绿假单胞菌的免疫应答及其激活和信号转导 通过这一TLR将调节疾病的结局。提出了三个具体目标：1) 验证TLR4调节感染者炎性血管生成的假说 眼角膜。2)验证TLR4调控感染角膜细胞凋亡的假说。 3)验证TLR4调节感染者体内抗菌肽的假设 眼角膜。实验将包括使用实时RT-PCR、Short 干扰RNA(SiRNA)和平板计数，酶联免疫吸附试验 (ELISA)、髓过氧化物酶(MPO)测定中性粒细胞(PMN)双抗体 免疫组织化学染色、组织病理学、TUNEL法、DNA梯状条带、核因子B功能检测 和Western blotting。我们的长期目标是了解 细菌和小鼠角膜的免疫反应使非常规 可以为人类患者开发治疗角膜炎的方法。因为在美国 仅在美国，每年微生物角膜炎的发病率就有25,000-30,000例，其中 治疗费用估计为1500-3000万美元，随着细菌耐药性的出现， 研究与人类健康相关，并具有相当大的医疗和经济价值 冲击力。

Project Summary/Abstract Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic organism associated with bacterial keratitis, especially in extended wear contact lens users. Our goal is to determine the molecular mechanisms for development of bacterial keratitis, specifically, the role of Toll-like receptor (TLR) 4, which at present remains underexplored in the eye. We hypothesize that TLR4 plays a critical role in the innate immune response to P. aeruginosa in the cornea and that the activation and signaling through this TLR will regulate disease outcome. Three specific aims are proposed: 1) To test the hypothesis that TLR4 regulates inflammatory angiogenesis in the infected cornea. 2) To test the hypothesis that TLR4 regulates apoptosis in the infected cornea. 3) To test the hypothesis that TLR4 regulates antimicrobial peptides in the infected cornea. Experiments will include use of techniques such as real time RT-PCR, short interfering RNA (siRNA), as well as plate counts, enyme linked immunosorbant assay (ELISA), myeloperoxidase assay (MPO) for neutrophil (PMN) quantitation, dual antibody immunostaining, histopathology, Tunel assay, DNA laddering, NF¿B functional assays and Western blotting. Our long-term objective is to understand the interactions between bacteria and the immune response in the mouse cornea so that non-conventional therapies against keratitis can be developed for human patients. Since in the United States alone the incidence of microbial keratitis is 25,000-30,000 cases annually, with cost of treatment estimated at $15-30 million, and with emerging bacterial resistance, the studies are of relevance to human health and have considerable medical and economic impact.