Automated quantitation of cell-mediated HIV type 1 infection of human syncytiotrophoblast cells by fluorescence in situ hybridization and laser scanning cytometry

Infection of human placental syncytiotrophoblast cells with HIV requires di rect contact with infected leukocytes. In vitro investigations into mechani sms regulating placental HIV transmission and into the development of thera peutic interventions have been hampered by difficulties inherent in quantit ating HIV levels in cocultures of infected lymphocytes and adherent multinu cleated syncytiotrophoblast cells. Here, we have used fluorescence in situ hybridization (FISH) for the direct detection of HIV-1 RNA within syncytiot rophoblast cells combined with laser scanning cytometry (LSC) to quantitate HIV levels exclusively in the syncytiotrophoblast cells. HIV-1-infected ly mphocytic MOLT-4 cells were cocultured with primary human syncytiotrophobla st cells. Lymphocytic cells were identified with an anti-vimentin antibody and Cy5. HIV RNA was localized by in situ hybridization, using a digoxigeni n-labeled riboprobe detected by Oregon Green, and nuclei were stained with 7-aminoactinomycin D. The three-color cocultures were analyzed by LSC to re move unwanted cell populations and quantitate HIV expression levels. The to tal HIV RNA level (green fluorescence integral) in each colony was normaliz ed for cell size by dividing by the total DNA content (red fluorescence int egral). The nuclear-normalized fluorescence integral was 2.3 times higher i n infected cocultures than in uninfected cultures. When cocultures were inc ubated with 10 muM AZT, the green/red fluorescence integral value was signi ficantly lower than that of cocultures incubated in the absence of AZT, cor responding to a 78% reduction in fluorescence. Laser scanning cytometry can be used to quantitate cell-mediated HIV infection in syncytiotrophoblast c ells and should allow drug assessment studies and studies aimed at understa nding the mechanism of virus entry into trophoblast cells to be carried out .