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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI065430-04
Application #
7658727
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
4
Fiscal Year
2008
Total Cost
$158,632
Indirect Cost
Name
University of Pittsburgh
Department
Type
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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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