UCLA Department of Medicine for Treatment of Normal PHA Stimulated Peripheral Blood Lymphocytes Infected with HIV_1 JRCFS

Acquired Human Immune-Deficiency Syndrome (AIDS) is a disease which targets and depletes the body’s T helper cells. It is caused by the Human Immunodeficiency Virus type 1 (HIV_1). Viral load, as measured in Peripheral Blood Mononuclear Cells (PBMCs) cultures, have been shown to correlate with early disease progression and loss of CD4 cells. Similarly suppression of viral replication by BACO in Vitro is a clear indication that such an agent is a potential candidate for treatment of HIV_1 infection.

In this experiment, PHA stimulated PBLs infected with HIV_1 JRCSF were treated with BACO. Supernatants were harvested on day 4 and day 7 tested for P24, to evaluate the inhibitory effect of BACO on the virus. Peripheral Blood Lymphocytes from a normal donor were stimulated in a PHA containing medium for 72 hours.

Cells were washed in RPMI 1640 serum free, re-suspended in a growth medium (RPMI+20% PBS and 10 unites/ml IL_2). Cells were counted. 30 million cells were inoculated with HIV_1 JRCSF, at a dose of 10ng P24 virus/ 10 million cells. Add 15ul polybrene. Incubate at 37 degrees Celsius, for two hours. Wash 2* in serum-free RPMI. Re-suspended in growth medium at a density of one million cells/ml. Distribute in a 24-well plate: 1ml cell suspension per well. Add BACO at different concentrations in triplicate wells. Use the first three wells as controls. On day 4, culture was microscopically observed, the cells all looked healthy. There appeared to be no negative reaction due to the addition of BACO at any concentration: 10ul, 20ul, 40ul, 80ul, 160ul, or 200ul. Cells in control wells looked no different from those in treated well, indicating a positive dose response has not affected the conditions of the cells. On day 7, cells were microscopically examined again; similar observations were made as on day 4. BACO had successfully destroyed 30 million diseased cultures, most of which were HIV/AIDS.