CAP37 and ocular inflammation

描述(申请人提供)：本申请旨在研究炎症介质CAP37在眼部炎症中的作用。CAP37是最近在眼睛中发现的对细菌感染的反应。令人惊讶的是，它的定位并不局限于中性粒细胞(PMN)，在那里它被认为是结构性表达的，但它也被发现诱导在角膜上皮细胞，以及角膜缘和睫状血管的血管内皮细胞。有许多炎症相关的生物活性受CAP37调控，赋予该分子在天然宿主防御入侵病原体方面的作用。CAP37是一种有效的抗生素，可促进单核细胞趋化，并对包括角膜上皮细胞在内的宿主细胞具有免疫调节作用。宿主防御分子在眼睛最外层和血管内皮细胞对感染的反应中的诱导，表明它可能在调节角膜的炎症和愈合中发挥关键作用，并从长远来看，形成了开发某些眼睛感染的新疗法的基础。我们的假设是，CAP37在角膜对感染的炎症反应中起关键作用，它是由PMN释放的或在感染部位的角膜上皮细胞中诱导的。我们假设它通过一种新颖的双重作用机制来实现这一点，这种双重作用机制基于其抗菌/抗炎和免疫调节特性。目的：为了验证CAP37对角膜上皮细胞增殖、趋化和黏附的调控依赖于细胞内信号分子蛋白激酶C的假说，我们将使用免疫组织化学、药物抑制剂和RNA干扰等技术来验证这一假说。目的：为了验证CAP37是眼内宿主防御的生物相关介质的假设，我们将检测CAP37及其生物活性多肽和中和抗体进入角膜基质后对炎症细胞、介质、细胞因子和黏附分子的诱导和识别。采用流式细胞术、酶联免疫吸附试验、免疫组织化学和RT-PCR等方法对数据进行分析。目的：为了验证CAP37的抗炎和免疫调节特性在眼部感染的宿主防御中的重要作用，我们将CAP37及其生物活性多肽应用于铜绿假单胞菌角膜炎小鼠模型，以确定其体内疗效。与公众健康相关：一旦角膜完整性被破坏，需要及时使用抗生素治疗，以阻止感染和有害后遗症的发展，如溃疡、穿孔、结疤和失明。不幸的是，目前可用于眼科的抗生素有严重的局限性。我们相信，我们的发现将有助于开发基于生物分子CAP37及其生物活性多肽的治疗眼睛创伤和感染的新疗法。

DESCRIPTION (provided by applicant): This application is designed to investigate the role of the inflammatory mediator, CAP37, in ocular inflammation. CAP37 was recently identified in the eye in response to bacterial infection. Surprisingly, its localization was not confined to the neutrophil (PMN), where it is known to be constitutively expressed, but it was also found induced in corneal epithelial cells, and vascular endothelial cells of the limbal and ciliary vessels. There are many inflammatory-associated biological activities regulated by CAP37 that confer on this molecule a role in innate host defense against invading pathogens. CAP37 is a potent antibiotic, promotes monocyte chemotaxis, and has immunomodulatory effects on host cells including corneal epithelial cells. The induction of a host defense molecule in the outermost layers of the eye and in the vascular endothelium in response to infection, suggests that it could play a key role in regulating inflammation and healing in the cornea and in the long term form the basis for developing new therapeutics for certain eye infections. It is our hypothesis that CAP37, either released by PMNs or induced in corneal epithelial cells at the site of infection, plays a pivotal role in the inflammatory response of the cornea to infection. We hypothesize that it accomplishes this through a novel, dual mechanism of action based on its antibiotic/anti-inflammatory and immunomodulatory properties. Aim 1: To test the hypothesis that the regulation of proliferation, chemotaxis, and adhesion of corneal epithelial cells in ocular inflammation is dependent on CAP37 and is mediated through the intracellular signaling molecule protein kinase C, we will use techniques that include immunohistochemistry, pharmacological inhibitors, and RNA interference. Aim 2: To test the hypothesis that CAP37 is a biologically relevant mediator in host defense in the eye, we will measure the induction and identity of inflammatory cells, mediators, cytokines, and adhesion molecules in response to the administration of CAP37, its bioactive peptides, and neutralizing antibodies into the corneal stroma. Flow cytometry, ELISA, immunohistochemistry, and RT-PCR will be used to analyze data. Aim 3: To test the hypothesis that the anti-inflammatory and immunomodulatory properties of CAP37 are important for host defense in ocular infection we will use CAP37 and its bioactive peptides in a murine model of Pseudomonas aeruginosa keratitis to determine in vivo efficacy. Relevance to public health: Once corneal integrity is breached, prompt treatment with antibiotics is required to halt the development of infection and harmful sequelae such as ulceration, perforation, scarring, and loss of vision. Unfortunately, the antibiotics currently available for ophthalmic use have serious limitations. We believe our findings will help develop new therapies for treating wounds and infections of the eye based on the biological molecule CAP37 and its bioactive peptides.