![]() |
![]() |
||||||||||||
|
02395 CHARACTERIZATION OF AN EX VIVO/IN VITRO INTESTINAL EXPLANT MODEL FOR RECTAL MICROBICIDE DEVELOPMENT. McGowan, Ian* Introduction: In vitro models of rectal mucosal HIV-1 transmission are required for the preclinical evaluation of candidate rectal microbicides. Here we describe the use of endoscopic biopsies for this purpose. Methods: Endoscopic biopsies were collected 30cm from the anal margin from three HIV-1 seronegative subjects. Five of the biopsies were immediately processed for histology and flow cytometry and the remaining ten were each set up on Gelfoam rafts. Explants were harvested after 24 hours and one week of culture. Intestinal mononuclear cells were isolated from the remaining four explants by collagenase digestion. Cell viability was assessed using trypan blue exclusion and flow cytometry was performed after staining for CD45, CD3, CD4, and CD8. Trucount beads were used to quantify individual cell populations. Results: Intestinal biopsies processed immediately had normal morphology. After 24 hours of culture, significant deterioration of the mucosal architecture occurred with subtotal loss of intestinal crypts and epithelial detachment. At one week, stromal elements remained but intestinal crypts were absent. The mean cell viability at baseline was 91% (range 89 96), 63% at 24 hours (range 60 70) and 36% at 1 week (range 25 50). At 24 hours mean CD3+ lymphocyte count was 61% of baseline (range 59 63) and 50% of baseline (range 30-70) at 1 week. No preferential loss of CD4/CD8 subsets was seen. Challenge studies at Day 0 with R5 (SF162), X4 (LAV.04) and dual tropic R5/X4 (89.6) HIV-1 variants resulted in productive HIV -1 infection at Day 7 that was evidenced by increasing p24 levels in culture supernatant and by the accumulation of intracellular p24. Conclusions: These data suggest that endoscopic intestinal explants are a useful ex vivo/in vitro model for the evaluation of candidate rectal microbicides. Dr. Ian McGowan |
|||||||||||||