iNOS is induced by gp41 in cocultures of uninfected MDM and astrocytes. (A) Levels of iNOS mRNA were analyzed by RT-PCR. Total RNA was extracted from MDM, HFA, and HAA cultures or cocultures incubated for 7 days with rgp41 (10 ng/mL) and rgp120 (10 ng/mL). Total RNA from cells stimulated by cytokine mixture containing IFNγ (10 ng/mL), IL-1β (100 ng/mL), and TNF- (100 ng/mL) was extracted after 8 hours of treatment. The cytokine-stimulated A172 cell line was used as a positive control. Cells are shown above the lanes. Lane 1, positive control; lanes 2, 6, 10, 14, and 18, no treatment; lanes 3, 7, 11, 15, and 19, treatment with rgp41; lanes 4, 8, 12, 16, and 20, treatment with rgp120; lanes 5, 9, 13, 17, and 21, treatment with cytokine mixture. (B) HFA were cocultured with uninfected MDM in the presence and absence of rgp41 (10 ng/mL), anti-gp41 Ab (1 mg/mL), and L-NMMA (500 μmol/L) for 9 days. At 3-day intervals, cultures were fed with fresh DMEM medium, and rgp41, L-NMMA and anti-gp41 Ab were replenished. Nitrite in culture supernatants at day 9 was measured by the Griess reaction. Each bar represents the mean ± SD in triplicate samples of supernatants.