Figure 7.

Acidic values of extracellular pH increase HIV-1 binding to spermatozoa and the subsequent transmission of HIV-1 to DCs. (A) Spermatozoa (Sp; 1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng of p24 for 60 min at 37°C at pH 7.3 (controls), 6.8, 6.5, or 6.0. Cells were then washed thoroughly, lysed, and assayed for p24 antigen by ELISA. The results are expressed as the mean ± SEM of five experiments performed in triplicate. Asterisks represent statistical significance (P < 0.05 vs. pH 7.3). (B) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with different primary HIV-1 isolates containing 25 ng p24 for 60 min at 37°C at pH 7.3 or 6.5. Cells were then washed thoroughly, lysed, and assayed for p24 antigen by ELISA. A representative experiment performed in duplicate (n = 2–3) is shown. (C) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C at pH 7.3 (controls) or 6.5, in the absence or presence of 10 U/ml heparin. Cells were then washed thoroughly, lysed, and assayed for p24 antigen by ELISA. The results are expressed as the mean ± SEM of four experiments performed in triplicate. *, P < 0.05, heparin vs. controls, at either pH 7.3 or 6.5; **, P < 0.05, controls at pH 6.5 vs. controls at 7.3. (D) Spermatozoa (1.5 × 10⁶/200 µl) were incubated for 60 min at 37°C at pH 7.3 or 6.5, and the expression of HS was then analyzed by flow cytometry. The gray histogram represents isotype control. A representative experiment (n = 3) is shown. (E) 100 µl of whole semen were diluted 1:1 with culture medium, and the pH was adjusted to 7.3 (control) or 6.5. HIV-1 BAL was added containing 25 ng p24, and the samples were incubated for 60 min at 37°C. After centrifugation, cells pellets were washed thoroughly, lysed, and assayed for p24 antigen by ELISA. The results are expressed as the mean ± SEM of four experiments performed in triplicate. The asterisk represents statistical significance (P < 0.05 vs. pH 7.3). (F) DCs (1.5 × 10⁵/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C at pH 7.3 (controls), 6.8, 6.5, or 6.0. Cells were then washed thoroughly, lysed, and assayed for p24 antigen by ELISA. The results are expressed as the mean ± SEM of three experiments performed in triplicate. (G) Spermatozoa (1.5 × 10⁶/200 µl) were incubated with HIV-1 BAL containing 25 ng p24 for 60 min at 37°C, at pH 7.3 or 6.5. Cells were then washed thoroughly and incubated with or without DCs for 7 d at a spermatozoa/DC ratio of 10:1. The infection of DCs was then analyzed by measuring the levels of p24 antigen by ELISA in cell supernatants. The results are expressed as the mean ± SEM of four experiments performed in duplicate. Asterisks represent statistical significance (P < 0.05 vs. spermatozoa cultured without DCs).