FIG 6.

Activities of HMW chitosan against L. major-infected BMMs after 4 h at pH 6.5 (A), 24 h at pH 6.5 (B), or 24 h at pH 7.5 (C), with or without phagocytosis inhibitor or pinocytosis (CME) inhibitor. We found that chitosan requires pinocytosis (CME) and not phagocytosis by BMMs for killing of L. major amastigotes at pH 6.5 and 7.5. BMMs were infected with stationary-phase promastigotes. Some of the infected macrophages were preincubated with cytochalasin D (phagocytosis inhibitor) or dynasore (pinocytosis [CME] inhibitor) and exposed to various concentrations (1.64, 4.9,14.8, 44.4, 133.3, or 400 μg/ml) of chitosan for 4 h and 24 h, followed by microscopic counting of the number of infected macrophages. There was no significant difference in the activities of HMW chitosan after inhibition of phagocytosis (P > 0.05 by t test). In contrast, significant inhibition of chitosan-mediated parasite killing occurred in the presence of dynasore at the two pH values (P < 0.05 by t test). Values are expressed as percentages of inhibition of infection relative to the results for untreated controls. Experiments were conducted in quadruplicate cultures, and data are expressed as mean values ± SD. Experiments were reproduced a further two times and the results confirmed (data not shown). Initial macrophage infection rate was >80% after 24 h.