Fig 3.

DCP has no cytolytic effect on monocytes and no direct inhibitory effect on mycobacteria. (A) Monocytes (∼1.5 × 10⁴ cells/well or ∼10% of the initial PBMC numbers), enriched by plastic overnight adherence in round-bottom 96-well plates (Table 1), were incubated with antibiotic-free complete RPMI medium alone or with 1,600 μM DCP for 72 h. At the end of 3 days, cells were gently detached from wells using a 1:1 mixture of 10 mM EDTA in PBS (pH 7.4) and RPMI 1640 medium containing 20% FBS. The cell viability was determined by using 0.4% trypan blue exclusion. Data shown are absolute numbers of viable cells recovered after incubation with medium alone or DCP. There were no differences in the mean cell numbers cultured with and without DCP, indicating that DCP did not have cytolytic effects on monocytes during the 72-h treatment duration. Each bar represents the mean of two different volunteers (P = 0.7792; t test). (B) A total of 450,000 CFU of BCG was cultured in cell-free Middlebrook 7H9 broth in the absence or presence of DCP (1,600 μM) in round-bottom 96-well plates for 3 days. On day 3, BCG was pulsed with [³H]uridine for 3 more days, and incorporation of tritiated uridine was measured. Data shown are averages of disintegrations per minute obtained from 4 separate experiments. There was no difference in the quantity of BCG in cultures with and without DCP. Each bar represents the mean of 4 separate experiments (P = 0.2000; Mann-Whitney U test). n.s., not significant.