SIGNIFICANCE OF CORNEAL CELL INVASION BY BACTERIA

描述：(从抽象意义上讲)这一宏伟的长期目标 研究是为了了解细菌入侵角膜细胞的作用 假单胞菌所致细菌性感染性角膜炎的发病机制 铜绿假单胞菌，并制定治疗和/或预防战略 这种疾病。由于铜绿假单胞菌被认为是一种胞外 病原、致病机制的研究一直是本病研究的重点。 细菌对角膜的黏附、细菌毒素和宿主 炎症反应。现在已经证明，铜绿假单胞菌 可侵袭角膜细胞；在角膜感染期间和在各种情况下 体外模型。细胞内的细菌可以逃避宿主的防御和一些 抗生素，因此可以解释为什么铜绿假单胞菌角膜炎 顽固且难以治疗。这项提议的第一个目的是 为了测试铜绿假单胞菌对角膜细胞的侵袭是否有助于 感染性角膜炎的发展。建议的方法包括 确定抑制铜绿假单胞菌侵袭角膜的方法 细胞，然后使用这些方法来抑制入侵，同时 监测对疾病发展的影响。铜绿假单胞菌不能 感染完整健康的角膜，感染是最常见的 与角膜损伤或戴隐形眼镜有关。因此，两个 研究铜绿假单胞菌角膜的不同实验模型 将利用感染；一个涉及擦伤的小鼠模型 角膜，和一只兔子模型的隐形眼镜外套。级别 细菌对细胞的侵袭将取决于庆大霉素的存活率 化验，以及通过肉眼检查评估疾病的严重程度 在眼角膜上。隐形眼镜佩戴是引起铜绿假单胞菌的主要原因 人类的角膜感染。需要检验的第二个假设是 随着铜绿假单胞菌进入眼睛，隐形眼镜 磨损最终会增加细胞内细菌的总数 角膜与不戴镜片的眼睛相比，相差一个或多个 有三种机制。它们是：1)环境条件 发生在隐形眼镜下，它改变了人的代谢功能 角膜细胞也改变了这些细胞对P。 铜绿假单胞菌侵袭；2)戴隐形眼镜增加铜绿假单胞菌 细胞入侵继发于细菌对这些细胞的粘附性增加 细胞；以及3)隐形眼镜佩戴阻碍清除 细胞内铜绿假单胞菌通过干扰 浅层上皮细胞脱落和/或细胞停滞 在隐形眼镜下面。这一假设将通过以下方式进行验证：1) 环境调控对铜绿假单胞菌的影响 体外培养的角膜上皮细胞和小鼠全角膜 2)比较铜绿假单胞菌对浅层角膜的侵袭 人戴隐形眼镜上皮细胞的去除及对照 受试者；以及3)评估细胞内细菌是否停滞 在兔眼戴隐形眼镜期间。感染性角膜炎是 配戴隐形眼镜最严重的并发症。因为隐形眼镜是 最常出现在健康的眼睛里，即使这种情况的发生率很低 严重的疾病是不可接受的。使某些患者更容易 易受感染和新的预防和/或方法 感染的治疗可以通过拟议的研究来确定。

DESCRIPTION: (from the abstract) The broad long term goal of this research is to understand the role of corneal cell invasion by bacteria in the pathogenesis of bacterial infectious keratitis due to Pseudomonas aeruginosa, and to develop strategies for treatment and/or prevention of this disease. Since P. aeruginosa is believed to be an extracellular pathogen, research into the pathogenesis of this disease has focused on bacterial adherence to the cornea, bacterial toxins, and the host inflammatory response. It has now been demonstrated that P. aeruginosa can invade corneal cells; during corneal infection and in various in vitro models. Intracellular bacteria can evade host defenses and some antibiotics, and could thus explain why P. aeruginosa keratitis is often persistent and difficult to treat. The first aim of this proposal is to test whether corneal cell invasion by P. aeruginosa contributes to the development of infectious keratitis. The proposed methods involve identification of means to inhibit P. aeruginosa invasion of corneal cells, and then use of these methods to inhibit invasion whilst monitoring the effect on development of disease. P. aeruginosa cannot infect an intact healthy cornea and infection is most commonly associated with either corneal injury or contact lens wear. Thus, two different experimental models for studying P. aeruginosa corneal infection will be utilized; a murine model involving abrasion injury to the cornea, and a rabbit model for contact lens overwear. Levels of bacterial invasion into cells will be determined by gentamicin survival assays, and the severity of disease assessed by macroscopic examination of the cornea. Contact lens wear is the leading cause of P. aeruginosa corneal infection in humans. The second hypothesis to be tested is that following the introduction of P. aeruginosa into the eye, contact lens wear eventually increases the total number of intracellular bacteria on the cornea in comparison to eyes that do not wear lenses, by one or more of three mechanisms. These are: 1) that environmental conditions occurring under a contact lens which alter the metabolic function of corneal cells also alter the susceptibility of these cells to P. aeruginosa invasion; 2) that contact lens wear increases P. aeruginosa invasion of cells secondarily to increased bacterial adherence to these cells; and 3) that contact lens wear impedes the clearance of intracellular P. aeruginosa from the ocular surface by interfering with superficial epithelial cell exfoliation and/or by stagnation of cells under the contact lens. This hypothesis will be tested by: 1) examining the effect of environmental manipulations on P. aeruginosa entry into cultured corneal epithelial cells and whole mouse cornea in vitro; 2) comparing P. aeruginosa invasion into superficial corneal epithelial cells removed from human contact lens wearing and control subjects; and 3) evaluating whether intracellular bacteria are stagnated in the rabbit eye during contact lens wear. Infectious keratitis is the most serious complication of contact lens wear. Since contact lenses are most frequently worn in healthy eyes, even a low incidence of this serious disease is unacceptable. Factors that make certain patients more susceptible to infection and new approaches for the prevention and/or treatment of infection could be identified by the proposed studies.