Figure 4

A. Effect of polymyxin B incorporation on rApxIIIA-containing crude toxin preparation cytotoxicity towards porcine PBMCs. The fraction of PI-positive cells was measured by flow cytometry after a two-hour exposition (i) to 50 μl RPMI-1640 (C-), (ii) to 50 μg/ml or 100 μg/ml polymyxin B alone (Pol50 and Pol100 respectively), (iii) to 50 μg/ml or 100 μg/ml polymyxin B supplemented with 5 μg rApxIIIA during the second hour (Pol50/Apx and Pol100/Apx), or after a one-hour exposition either (iii) to 5 μg rApxIIIA alone (Apx) or (iv) to paraformaldehyde 10% (C+). Values are means ± SDs from three representative experiments. Asterisks (1): PI-positive cell densities significantly higher than that recorded in C-, Pol50 and Pol100 sets of experiments (P < 0.0001). PI-positive cell densities retrieved from Pol50/Apx, Pol100/Apx and rApxIIIA groups were not statistically different from each other (P > 0.5). B. Effect of anti-ApxIIIA antibodies incorporation on rApxIIIA-containing crude toxin preparation cytotoxicity towards porcine PBMCs. The fraction of PI-positive cells was measured by flow cytometry after a one-hour exposition (i) to 50 μl RPMI-1640 (C-), (ii) to a monospecific anti-ApxIIIA polyclonal antiserum diluted 1/1,000 (Ab-Apx), (iii) to a mix of rApxIIIA (0.31 or 0.16 μg) and the anti-ApxIIIA polyclonal antiserum diluted 1/1,000 (Ab-Apx + Apx), and (iv) to rApxIIIA alone (Apx) (0.31 or 0.16 μg). Values are means ± SDs from three representative experiments. Asteriks (2): positive cell densities significantly higher than that recorded in Ab-Apx + Apx sets of experiments (P < 0.01).