ORGAN CULTURE TO TEST MICROBICIDES

体外细胞培养系统已被广泛用作评价杀微生物剂抗HIV-1活性的初级筛选工具。然而，显然需要开发基于阴道和宫颈组织的体外系统，以在完整组织基质的背景下测试细胞毒性和抗病毒活性。我们最近开发了一种宫颈组织来源的器官培养模型，该模型模拟体内条件，并已用于测试杀微生物剂阻断HIV-1传播的能力。我们也有初步的数据表明，该模型可用于监测炎症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，宫颈组织为基础的器官培养， 这是一个理想的系统，用于测试杀微生物剂在生殖器组织中的毒性，以及在阴道中存在常见环境因素（如精液、阴道液和STI相关微生物）的情况下，其阻断具有不同表型特性的HIV-1在宫颈上皮传播的能力。该项目的具体目标是：1）评估项目1（表达Cynanovirin的细菌）和项目2（抗菌肽retrocyclin）的潜在杀微生物剂。这些杀微生物剂将在宫颈组织器官培养中检测其阻断HIV-1通过粘膜传播的能力。此外，将在器官培养中测试混杂物质（如女性精液和阴道液）对这些杀微生物剂抗病毒活性的影响，从而模拟体内条件; 2）评估宫颈组织炎症及其对项目1杀微生物剂反应的变化 2采用器官培养模型。促炎细胞因子如II-1 β、IL-6、IL-8和TNF-α的表达将通过真实的时间PCR测量其在组织中的信息并使用Luminex系统在培养物上清液中分泌来监测; 3）在其他生殖性STI相关微生物如淋病奈瑟氏菌存在下评价杀微生物剂的抗HIV活性。将扩展基于宫颈组织的器官培养模型，以测量阴道和宫颈炎症对HIV-1和这些生殖性STI相关微生物和乳酸杆菌的反应，作为阴性对照。该项目中的拟议研究将通过以下方式补充U19赠款申请中的其他各种项目： 提供了一种有价值的基于体外宫颈组织的检测方法，将在杀微生物剂开发（项目1和2）、猴模型（项目4）、制剂（核心）和FDA批准的其他抗病毒检测核心之间架起桥梁。

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. We also have preliminary data to show that this model can be used to monitor 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 potential microbicides from Projects 1 (Cynanovirin-expressing lactobacillus) and 2 ( antimicrobial peptide retrocyclin). These microbicides will be tested for their ability to block HIV-1 transmission across the mucosa in a cervical tissue-based organ culture. In addition, the effect of confounding substances, such as semen and vaginal fluids from women will be tested on the anti-viral activity of these microbicides in organ culture, thus mimicing the in vivo condition; 2) Assessment of cervical tissue inflammation and their changes in response to microbicides from Project 1 and 2 using the organ culture model. The expression of proinflammatory cytokines, such as II-1beta, IL-6, IL-8 and TNF-alpha 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 microbicides in the presence of other reproductive STI-related microorganisms, such as Neisseria gonorrhoeas. Cervical-tissue based organ culture model will be expanded to measure vaginal and cervical inflammation in response to HIV-1 and these reproductive STI-related microorganisms and lactobacilli, as a negative control. The proposed studies in this project 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), formulation (Core) and FDA approved additional antiviral testing core.