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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZRG1-AARR-A (50))
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University of Pittsburgh
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Gupta, Phalguni; Lackman-Smith, Carol; Snyder, Beth et al. (2013) Antiviral activity of retrocyclin RC-101, a candidate microbicide against cell-associated HIV-1. AIDS Res Hum Retroviruses 29:391-6
Gupta, Phalguni; Ratner, Deena; Ding, Ming et al. (2012) Retrocyclin RC-101 blocks HIV-1 transmission across cervical mucosa in an organ culture. J Acquir Immune Defic Syndr 60:455-61
Martellini, Julie A; Cole, Amy L; Svoboda, Pavel et al. (2011) HIV-1 enhancing effect of prostatic acid phosphatase peptides is reduced in human seminal plasma. PLoS One 6:e16285
Li, Ming; Patton, Dorothy L; Cosgrove-Sweeney, Yvonne et al. (2011) Incorporation of the HIV-1 microbicide cyanovirin-N in a food product. J Acquir Immune Defic Syndr 58:379-84
Sassi, A B; Cost, M R; Cole, A L et al. (2011) Formulation development of retrocyclin 1 analog RC-101 as an anti-HIV vaginal microbicide product. Antimicrob Agents Chemother 55:2282-9
Cole, Alexander M; Patton, Dorothy L; Rohan, Lisa C et al. (2010) The formulated microbicide RC-101 was safe and antivirally active following intravaginal application in pigtailed macaques. PLoS One 5:e15111
Venkataraman, Nitya; Cole, Amy L; Ruchala, Piotr et al. (2009) Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol 7:e95
Sivaraman, Karthikeyan; Seshasayee, Aswinsainarain; Tarwater, Patrick M et al. (2008) Codon choice in genes depends on flanking sequence information--implications for theoretical reverse translation. Nucleic Acids Res 36:e16
Sorensen, Ole E; Borregaard, Niels; Cole, Alexander M (2008) Antimicrobial peptides in innate immune responses. Contrib Microbiol 15:61-77
Cole, Alexander M; Cole, Amy Liese (2008) Antimicrobial polypeptides are key anti-HIV-1 effector molecules of cervicovaginal host defense. Am J Reprod Immunol 59:27-34

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