Attacking the Global Problem of Antimicrobial Resistance

项目摘要 除了对医疗保健的影响外，抗菌素耐药性的经济影响也很大。超过 每年有200万例感染是由至少对一线抗生素有抗药性的细菌引起的， 美国医疗保健系统每年花费200亿美元。铜绿假单胞菌（PA）导致超过51，000健康 在美国，每年发生的护理相关感染，其中13%为多药耐药（MDR）。非抗生 预防和治疗细菌感染的方法为抗生素提供了有吸引力的替代品 对MDR PA分离株的有效性更长。其中之一是甘草素（GLY），一种从甘草根中提取的提取物 （光果甘草），在治疗败血症、结肠炎、肺和脑损伤的动物模型中是有效的;并且 用于慢性肝炎的临床管理。感染后，GLY减少细胞外释放高 移动族蛋白1（HMGB 1），一种激活细胞表面先天免疫受体的原型警报蛋白 影响宿主的炎症反应。甘草酸苷对PA实验性角膜炎有直接抗菌作用 由非MDR临床（角膜炎）分离株诱导（例如，KEI 1025）和MDR 9（一种非眼部分离株）。 初步/最近发表的数据支持在MDR 9诱导的PA角膜炎中，GLY：a）渗透细菌 膜，B）降低外排泵活性，增加细菌杀灭，和d）与环丙沙星组合， 体内，最佳地减少嗜中性粒细胞浸润和平板计数。因此，我们的首要假设是， GLY对角膜和眼附属器无毒，在PA感染后，与HMGB 1结合， 先天免疫受体的激活。为了检验这一假设，提出了两个具体的目标。 具体目标1：检验GLY耐受性良好且不改变细胞结构或细胞凋亡的假设。 正常角膜、结膜和眼附属器的生理参数。为此，我们将测试 局部给予GLY对未感染的正常角膜的影响，以及如果眼内压（IOP），泪液量， 角膜神经模式、角膜敏感性、常驻免疫细胞和/或结膜改变。 具体目的2：检验GLY对PA角膜炎的保护作用是通过抑制 HMGB 1扩增髓系细胞中的TLR 4/TLR 4信号通路。这一目标将测试GLY是否与 HMGB 1抑制HMGB 1-TLR 4（-TLR 4）相互作用和信号传导通路，下游作用是 未成熟髓样树突状细胞（DC）成熟，巨噬细胞产生促炎细胞因子， 趋化因子和中性粒细胞浸润和功能。目标2a将在GLY处理的小鼠中测试这一假设， 用KEI 1025感染，KEI 1025是一种使用TLR 4和ESTK 0小鼠分离的非MDR PA角膜炎， HMGB 1 KO小鼠。目的2b是翻译的，并将在GLY治疗的小鼠中检验这一假设。 MDR-PA感染后18 h开始联合莫西沙星治疗。

Project Summary Besides the impact on health care, the economic impact of antimicrobial resistance is significant. More than two million infections a year are caused by bacteria that are resistant to at least first-line antibiotics, costing the US health care system 20 billion dollars each year. Pseudomonas aeruginosa (PA) causes over 51,000 health care associated infections per year in the US, of which 13% are multi-drug resistant (MDR). Non-antibiotic approaches to prevent and treat bacterial infections provide attractive alternatives/adjuncts to antibiotics no longer effective against MDR PA isolates. One of these, glycyrrhizin (GLY), an extract from the licorice root (Glycyrrhiza glabra), is effective in treatment of animal models of sepsis, colitis, lung and brain injury; and is used in the clinical management of chronic hepatitis. After infection, GLY reduces extracellularly released high mobility group box 1 (HMGB1), a prototypic alarmin that activates cell surface innate immune receptors affecting host inflammatory responses. GLY has direct antimicrobial effects in PA experimental keratitis induced by non-MDR clinical (keratitis) isolates (e.g., KEI 1025) and by MDR9, a non-ocular isolate. Preliminary/recently published data support that in MDR9 induced PA keratitis, GLY: a) permeabilizes bacterial membranes, b) reduces efflux pump activity, increases bacterial killing and d) combined with Ciprofloxacin in vivo, reduces the neutrophil infiltrate and plate count optimally. Therefore, our overarching hypothesis is that GLY is non-toxic to the cornea and ocular adnexa and after PA infection, binds to HMGB1 preventing activation of innate immune receptors. To test this hypothesis two Specific aims are proposed. Specific Aim 1: Tests the hypothesis that GLY is well-tolerated and does not alter the cytoarchitectue nor the physiological parameters of the normal cornea, conjunctiva and ocular adnexa. In this aim, we will test the effects of GLY given topically on the uninfected, normal cornea, and if intraocular pressure (IOP), tear volume, corneal nerve pattern, corneal sensitivity, resident immune cells and/or conjunctiva are altered. Specific Aim 2: Tests the hypothesis that GLY's protective effect on PA keratitis is mediated by inhibiting HMGB1 amplification of TLR4/RAGE signaling pathways in myeloid cells. This aim will test if GLY binding to HMGB1, inhibits HMGB1-TLR4 (-RAGE) interactions and signaling pathways, with downstream effects on immature myeloid dendritic cell (DC) maturation, macrophage production of proinflammatory cytokines and chemokines and neutrophil infiltration and function. Aim 2a will test this hypothesis in GLY treated mice after infection with KEI1025, a non-MDR PA keratitis isolate using TLR4 and RAGE KO mice and myeloid specific HMGB1 KO mice. Aim 2b is translational and will test this hypothesis in mice in which GLY treatment is combined with Moxifloxacin and initiated 18h after infection with MDR PA.