brasiliensis (Figure 1B), at least a 3-fold increase in comparison with the control. Also, the proportion of internalized yeast cells (23%) was higher than the proportion of yeast cells adhered to macrophage surfaces (6%). In contrast, we found that 0.25 μM alexidine JNK-IN-8 datasheet dihydrochloride caused an 8-fold inhibition in the levels of phagocytosis by MH-S cells compared with the control (Figure 1B). No effects of alexidine dihydrochloride or pulmonary surfactant on adhesion and internalization of heat-killed P. brasiliensis were observed (data not shown).
Table 1 Phospholipase B activities secreted under the experimental conditions used for Phagocytic test Treatment Specific activity of PLB (μmol min-1mg-1protein) Untreated control 1.21 ± 0.02 Pulmonary surfactant (100 μg mL-1) check details 1.55 ± 0.06* (28% activation) Alexidine dihydrochloride (0.25 μM) 0.41 ± 0.08* (66% inhibition) Phospholipase B activities were assayed after 6 h of co-cultivation
of alveolar macrophage (MH-S) cells with P. brasiliensis yeast cells with pulmonary surfactant (100 μg mL-1) and alexidine dihydrochloride (0.25 μM), as well as without treatment (untreated control), as described in Materials and Methods. *Significantly different from the untreated control, P < 0.05 by the paired 2-tailed Student's t-test. Results are means ± SEM of triplicate assays. A role for CH5424802 cost PLB activity in adhesion of C. neoformans to lung epithelial cells has already been proposed ; DPPC is predicted to be the favored lipid substrate for PLB, leading to the production of glycerophosphocholine and free palmitic acid. In this context, it is hypothesized that the addition of pulmonary surfactant (rich in DPPC) would increase the adhesion of P. brasiliensis yeast cells to MH-S cells. These results strongly suggest that PLB activity is important in P. brasiliensis adhesion to and/or internalization by MH-S cells. In the present study, enzyme activities were tested under conditions
used for adhesion Cytidine deaminase (Table 1). P. brasiliensis produced high levels of PLB at 6 h post-infection. 0.25 μM Alexidine dihydrochloride selectively inhibited PLB activity by 66%. In contrast, PLB activity in the presence of 100 μg mL-1 pulmonary surfactant was significantly increased (28%) compared to the control experiment. Modulation of P. brasiliensis and MH-S genes in the host-pathogen interaction Real-time quantitative reverse-rranscriptase-polymerase chain reaction (qRT-PCR) analysis confirmed that the plb1 (PLB), sod3 (Cu, Zn superoxide dismutase – SOD), and icl1 (isocitrate lyase) genes were up-regulated in P. brasiliensis yeast cells during 6 h of interaction with MH-S cells in the presence of pulmonary surfactant. The sod3 gene presented a 4.1-fold increase in expression (Figure 2) and under these conditions a higher percentage of yeast cell internalization was observed (Figure 1B).