Figure 1

Inhibition of TNF by LMP-420. A) Dose response of LMP-420 on human PBMC stimulated with LPS. PBMC were resuspended to 3.75 × 10⁶ total cells/ml in complete RPMI 1640 medium (containing 5% heat-inactivated human AB serum) and 0.4 ml of cell suspension put into each well of a 48-well tissue culture plate. To each well was added 0.1 ml of media or media containing LMP-420 (diluted from a stock solution of 338 mM in DMSO) to give the indicated final concentration. The cell cultures were incubated for 2 h at 37°C in humidified 5% CO₂ and then 55 μl of media or LPS (S. typhosa, 1 μg/ml of media) was added to each well. The cultures were incubated 20 h at 37°C, the contents of each well removed to a 5-ml polypropylene centrifuge tube and centrifuged for 20 min at 400 g. The supernatants were removed to a fresh tube and frozen at -20°C until assayed by solid phase ELISA (R & D Systems). B) Dose response of LMP-420 on human PBMC stimulated with anti-CD3 or SEB. PBMC were resuspended to 3.75 × 10⁶ total cells/ml in complete RPMI 1640 medium (containing 5% heat-inactivated human AB serum) and 0.4 ml of cell suspension put into each well of a 48-well tissue culture plate. To each well was added 0.1 ml of media or media containing LMP-420 (diluted from a stock solution of 338 mM in DMSO) to give the indicated final concentration. The cell cultures were incubated for 2 h at 37°C in humidified 5% CO₂ and then 55 μl of media or anti-CD3 (25 ng/ml final concentration) or SEB (100 ng/ml final concentration) was added to each well. The cultures were incubated for 48 h at 37°C, the contents of each well removed to a 5-ml polypropylene centrifuge tube and centrifuged for 20 min at 400 g. The supernatants were removed to a fresh tube and frozen at -20°C until assayed by solid phase ELISA (R & D Systems). C) RT-PCR of samples prepared from LPS-stimulated human PBMC. PBMC (1 × 10⁶/well) were incubated for 2 h at 37°C with the indicated concentration of LMP-420 and then stimulated for 3 h at 37°C with LPS (S. typhosa; 1 μg/ml). Cells were harvested, total RNA extracted, cDNA prepared and RT-PCR performed for 30 cycles using primers for TNF-α and β-actin obtained from R & D Systems.