Pha m

Heparinized whole venous blood, 2 mL was collected (LH 68 IU BD-Plymouth, United Kingdom, 5 mL) from each subject and was separated equally into two tubes - control tube and a PHA-M stimulated sample. To the stimulated test tube 20 μg/mL PHA-M (Roche Diagnostics GmbH, Germany) was added and the two samples were incubated for 4 h at 37 °C, 5% CO₂. The samples were gently shacked, at regular intervals, on a multispeed vortex (MSV-3500 BioSan LV). Afterwards, 100 μL blood from each tube was labeled with monoclonal anti-CD3 FITC (for determination of the T lymphocytes) and anti-CD69 PE, an early activation marker for T cells (BD Biosciences, United States) and incubated for 30 min, at room temperature (RT) in the dark. Followed a lysis of erythrocytes (BD FACS Lysing Solution, BD Biosciences, United States), then centrifuging at 1300 rpm for 10 min and double washing (CellWash, BD Biosciences, United States). Subsequently, cells were fixed with 200 μL CellFIX (BD Biosciences, United States) and were analyzed with BD FACSCalibur flowcytometer using the Cell Quest software for data acquisition and analysis. Then 20000 lymphocytes were counted and analyzed for expression of CD69. The results obtained for each patient and healthy subject were analyzed for PHA-stimulated and unstimulated lymphocytes.

Isolated PBMCs were stimulated with 3 μg ml⁻¹ of PHA-M (Roche) and 20 U ml⁻¹ of human-IL 2 (hIL-2; Roche) for 3 days; viral supernatant from transfection of 293T cells was used to infect pooled mixed stimulated PBMCs prepared from two healthy donors. The supernatant from HIV-infected PBMCs was collected at day 7 and day 11. Viral titers were determined by HIV-1 p24 ELISA (PerkinElmer).