Cervical Tissue Derived Organ Culture to Test Microbicides

体外细胞培养系统已被广泛用作评估抗HIV-1的主要筛选工具 杀微生物剂的活性。然而，显然有必要开发一种基于 体外系统，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 最近开发了一种模拟活体条件的宫颈组织衍生器官培养模型，并已 已被用于测试杀菌剂阻断HIV-1传播和测量炎症的能力 细胞因子对杀菌剂和性传播感染相关细菌的反应。我们的假设是 宫颈组织器官培养是检测生殖器组织中杀菌剂毒性的理想系统 以及它阻止HIV-1传播的能力，这种病毒具有不同的表型特征 在存在于阴道中的常见环境因素的情况下，如精液， 阴道液和性传播感染相关微生物。该项目的具体目标是：1)评价-核苷 逆转录酶抑制剂(NNRTI)5-氯-3-苯磺酰吲哚-2-甲酰胺(CSIC)，来自项目1 和来自项目2单独或组合的进入抑制抗菌肽RC101和 他们的配方能够阻止HIV-1在宫颈组织中的粘膜传播 器官培养。2)评价宫颈组织炎症反应及其在CSIC和 RC101，分别来自项目1和2，使用器官培养模型。的表达方式 促炎细胞因子，如11-13，IL-6，IL-8和肿瘤坏死因子-α，将通过测量其 利用Luminex实时定量聚合酶链式反应检测组织中的信息和培养上清液中的分泌物 3)生理相关条件下CSIC和RC101的抗HIV活性评价。CSIC 和RC101单独或组合，它们的制剂将在 有精液和阴道液。此外，器官培养模型将扩展到测量与STI相关的 淋球菌和产黑素普氏杆菌等微生物的检测 促炎细胞因子反应。然后将评估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研究进展 Droject将通过提供有价值的体外试验来补充U19赠款申请中的其他各种项目 宫颈组织化验将在杀菌剂开发(项目1和2)之间架起桥梁， Money模型(项目4)和公式核心(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).