TRICHOMONAS VAGINALIS AND SLPI MUCOSAL DEFENSES

这项资助的长期目标是了解健康女性生殖道的粘膜环境，了解其对阴道毛滴虫和其他性病（包括艾滋病毒感染）的非免疫保护机制以及炎症反应。实验将集中在分泌性白细胞蛋白酶抑制剂（SLPI）提供的保护机制，一种主要的丝氨酸蛋白酶抑制剂，发现在阴道上皮细胞和确定T。迷走蛋白酶（TVP）对SLPI功能的影响。我们推测，阴道毛滴虫（TV）半胱氨酸蛋白酶切割分泌性白细胞蛋白酶抑制剂（SLPI），并损害其粘膜抗菌防御功能。我们计划：1）确定SLPI对STD微生物包括阴道毛滴虫（TV）、淋病奈瑟菌（GC）和沙眼衣原体（CT）的正常抗菌功能。2)确定SLPI在TV的不同肉汤生长和代谢条件下的抗微生物功能，包括需氧和厌氧生长;不同浓度的游离铁、人转铁蛋白和乳铁蛋白;以及在存在和不存在半胱氨酸蛋白酶抑制剂E-64的情况下。3)分离并表征导致SLPI降解的特异性TVP活性。通过诱导上清液的离子交换色谱分离三种TVP（220、92和68 kDa），并通过SDS-PAGE和Western印迹检测其对SLPI的单独降解作用。4)将通过气相微测序对所得肽片段进行测序。将TVP切割位点和肽映射到已知的序列数据和功能结构域。5)将测试肽的残余抗微生物功能。6）我们将测定来自青春期女性生殖道的正常和感染分泌物中的SLPI水平。将对14-19岁的青春期女孩进行阴道液采样，并通过ELISA测定确定SLPI水平。这些患者将被纵向随访一年，并将SLPI水平与卷曲乳杆菌携带率进行比较。这里阐明的机制将与我们对体内平衡的天然阴道机制以及在感染简单或复杂生殖道疾病期间的理解相关。这些发现也可能与其他粘膜性病病原体的感染机制有关，包括HIV感染和其他寄生虫病。

The long term objectives of this grant are to understand the mucosal environment of the healthy female genital tract in terms of its non-immune protective mechanisms against Trichomonas vaginalis and other STDs including HIV infection, and the inflammatory response. Experiments will focus on protective mechanisms provided by secretory leukocyte protease inhibitor (SLPI), a predominant serine protease inhibitor found on the vaginal epithelium and determine the deleterious effects of T. vaginalis proteases (TVPs) on SLPI function. We hypothesize that Trichomonas vaginalis (TV) cysteine proteases cleave secretory leukocyte protease inhibitor (SLPI) and impair its mucosal antimicrobial defense function. We plan to: 1) determine the normal antimicrobial function of SLPI against STD organisms including Trichomonas vaginalis (TV), Neisseria gonorrhoeae (GC), and Chlamydia trachomatis (CT). 2) determine the antimicrobial function of SLPI under different broth growth and metabolic conditions of TV including aerobic and anaerobic growth; different concentrations of free iron, and human transferrin and lactoferrin; and in the presence and absence of cysteine protease inhibitor E-64. 3) isolate and characterize the specific TVP activity which causes the degradation of SLPI. Three TVPs (220, 92 and 68 kDa) will be isolated by ion exchange chromatography of induction supernatants and tested for their individual degradative effects on SLPI by SDS-PAGE and Western blot. 4) Resulting peptide fragments will be sequenced by gas phase microsequencing. The TVP cleavage sites and peptides will be mapped to known sequence data and functional domains. 5) Residual antimicrobic function of peptides will be tested, 6) We will determine SLPI levels in normal and infected secretions from the adolescent female genital tract. Adolescent girls, ages 14-19, will have vaginal fluid samples taken and SLPI levels determined by ELISA assay. These patients will be followed longitudinally lover a year and SLPI levels compared to Lactobacillus crispatus carriage rates, The mechanisms elucidated here will be relevant to our understanding of native vaginal mechanisms of homeostasis and during infection with simple or complicated genital tract disease. These findings may also be relevant to mechanisms of infection of other mucosal STD pathogens, including HIV infection, and other parasitic diseases.