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DEVELOPMENT OF A HUMAN ORAL EPITHELIAL TISSUE MODEL TO STUDY HIV TRANSMISSION AND THE ROLE OF BETA-DEFENSINS.
Nittayananta1, W., Coombs1, R.W., Ayehunie2, S., Klausner2, M., Dale1, B.A.
1University of Washington, Seattle, WA; 2MatTek Corporation, Ashland, MA, United States.
Presented at XV International AIDS Conference, Bangkok, Thailand, July 11-16, (2004).
Keywords: Anti-microbial, Anti-microbial peptides, Antimicrobial, Antimicrobial peptides, Beta-defensins, Buccal, EpiOral, Epithelial cells, HBD-2, HBD-3, HBD-3 mRNA, HIV, Human beta-defensin 2 (hBD-2), Human beta-defensin 2 mRNA, Human beta-defensin 3 (hBD-3), Human buccal epithelial, Messenger RNA expression, Mucosal polarity, Mucosal surfaces, Oral HIV transmission, Oral epithelial tissue model, Oral tissue, R5 virus, Submucosal, hBD-2, hBD-3, hBD-3 mRNA
Endpoints: mRNA expression, hBD-2, hBD-3
Materials Tested: HIV-1, R5 virus
Summary:
Background: As part of innate immunity, oral mucosal epithelial cells secrete a class of antimicrobial peptides termed human beta-defensins (hBD). HIV induces expression of hBD-2 and hBD-3 mRNA in human oral epithelial cells; however, the role of hBD in HIV transmission using a human oral tissue model has not been studied.
Methods: We used a human buccal epithelial cell tissue raft system (MatTek Corp., Ashland, MA) to evaluate the effect of in vitro mixed-lymphocyte culture conditions for HIV replication on the expression of hBD-2 and hBD-3. The tissues were cultured for 48 hours under the following conditions; IL-2 alone, IL-2 plus peripheral blood mononuclear cells (PBMC; 1x10^6 versus 10x10^6), either alone or in combination, and presented to either the top or the bottom of the tissue raft. The tissue with culture media alone was used as a control. Expression of hBD-2 and hBD-3 was determined at both transcription (mRNA) and translation levels by reverse transcriptase (RT)-PCR amplification and immunohistochemistry (IHC), respectively.
Results: The expression of hBD-2 and hBD-3 was increased when medium containing IL-2 alone or IL-2 plus PBMC was present on the top compared to the bottom of the tissue raft. The up-regulation of hBD was higher in the presence of both IL-2 and PBMC compared to IL-2 alone. However, increasing the concentration of PBMC from 1x10^6 to 10x10^6 decreased the expression of hBD.
Conclusions: Our preliminary data suggest that this in vitro human epithelial tissue model is biologically plausible as we showed a mucosal polarity to hBD expression under controlled conditions; as such, this model may be useful for studying the effect of beta-defensins on oral HIV transmission. To this end, we have initiated experiments to determine the effects of both cell free- and cell-associated HIV-1 on hBD expression.
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