NO is produced and iNOS is expressed in cocultures of HIV-1–infected MDM and human astrocytes. (A) MDM precultured for 7 days in DMEM medium were infected with HIV-1ADA. At day 1 postinfection, infected MDM were cocultured with HFA and HAA at a 2:3 ratio and were fed with fresh DMEM medium every 3 days. Nitrite was measured in culture supernatants by the Griess reaction. Each bar represents the mean ± SD in triplicate samples of supernatants. (B) Levels of iNOS mRNA in cocultures with infected MDM and astrocytes were analyzed by RT-PCR. Amplification cycles used were 35 cycles for iNOS and 25 cycles for β-actin. The cytokine-stimulated A172 cell line was used as a positive control. Days after infection are shown above the lanes. (C) Results of the RT-PCR analysis of different cultures at day 18 postinfection are presented. Lane 1, positive control (PC); lane 2, uninfected MDM; lane 3, infected MDM; lane 4, HFA; lane 5, HAA; lane 6, HFA cocultured with uninfected MDM; lane 7, HAA cocultured with uninfected MDM; lane 8, HFA cocultured with infected MDM; lane 9, HAA cocultured with infected MDM.
