Cervical Tissue Derived Organ Culture to Test Microbicides

体外细胞培养系统已被广泛用作评价抗HIV-1的初级筛选工具 杀微生物剂的活性。然而，显然需要开发一种阴道和宫颈组织， 体外系统以在完整组织基质的背景下测试细胞毒性和抗病毒活性。我们有 最近开发了一种宫颈组织来源的器官培养模型，其模拟体内条件， 用于测试杀微生物剂阻断HIV-1传播的能力， 细胞因子对杀微生物剂和性传播感染相关细菌的反应。我们的假设是 基于宫颈组织的器官培养是测试杀微生物剂在生殖器组织中毒性的理想系统 以及其阻断具有不同表型特性的HIV-1在宫颈间传播的能力 上皮细胞在阴道中存在的常见环境因素，如精液， 阴道分泌物和性传播感染相关微生物本项目的具体目标是：1）评价-核苷 逆转录酶抑制剂（NNRTI）来自项目1的5-氯-3-苯磺酰基吲哚-2-甲酰胺（CSIC） 和来自项目2的进入抑制剂抗微生物肽逆转录细胞周期蛋白RC 101单独或组合， 它们的制剂能够阻断HIV-1在基于宫颈组织的 器官培养2)宫颈组织炎症反应的评估及其对CSIC和 分别来自项目1和2的RC101，使用器官培养模型。的表达 促炎细胞因子，如IL-13、IL-6、IL-8和TNF-α将通过测量它们的浓度来监测。 通过真实的时间PCR检测组织中的信息，并使用Luminex 3）在生理相关条件下评价CSIC和RC 101的抗HIV活性。CSIC 和RC101单独或组合使用，并且将评价它们的制剂的抗病毒活性。 精液和阴道分泌物此外，将扩大器官培养模型，以测量STI相关 微生物，如淋病奈瑟氏菌和产黑色素普雷沃氏菌，通过测量 促炎细胞因子反应。然后，将评价杀微生物剂的抗病毒活性， 这些微生物诱导的促炎细胞因子的存在。 Droject将通过提供一种有价值的体外细胞培养技术， 基于宫颈组织的检测，将在杀微生物剂开发（项目1和2）之间架起桥梁， 猴模型（项目4）和制剂核心（核心B）。

The in vitro cell culture system has been widely used as a primary screening tool for evaluating anti-HIV-1 activity of microbicides. However, there is clearly a need to develop a vaginal and cervical tissue based in vitro system to test the cytotoxicity and antiviral activity in the context of the complete tissue matrix. We have recently developed a cervical tissue-derived organ culture model which mimics in vivo conditions and has been used to test microbicides for their ability to block HIV-1 transmission and measure inflammatory cytokines in response to microbicides and sexually transmitted infection-related bacteria. Our hypothesis is that a cervical tissue-based organ culture is an ideal system to test microbicides for toxicity in genital tissue and for its ability to block transmission of HIV-1 with varying phenotypic properties across the cervical epithelium in the presence of common environmental factors that are present in vagina, such as semen, vaginal fluid, and STI-related microorganisms. Specific aims of the project are: 1) Evaluation of -nucleoside reverse transcriptase inhibitor (NNRTI) 5-chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) from Project 1 and the entry inhibitor antimicrobial peptide retrocyclin RC101 from Project 2 alone or in combination and their formulations for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. 2) Assessment of cervical tissue inflammation and their changes in response to CSIC and RC101 from Projects 1 and 2, respectively, using the organ culture model. The expression of proinflammatory cytokines, such as 11-13, IL-6, IL-8 and TNF-a will be monitored by measuring their messages in the tissues by the real time PCR and secretion in the culture supernatant using the Luminex system; 3) Evaluation of anti-HIV activity of CSIC and RC101 under physiologically relevant conditions. CSIC and RC101 alone or in combination and their formulations will be evaluated for their antiviral activities in the presence of semen and vaginal fluid. In addition the organ culture model will be expanded to measure STIrelated microorganisms, such as Neisseria gonorrhoeae and Prevotella melaninogenica by measuring proinflammatory cytokine response. Microbicides will then be evaluated for their antiviral activity in the aresence of the proinflammatory cytokines induced by these microorganisms.The proposed studies in this Droject will complement various other projects in this U19 grant application by providing a valuable in vitro cervical tissue-based assay which will bridge between the microbicide development (Projects 1 and 2), monkey model (Project 4) and Formulation Core (Core B).