brasiliensis. In C. neoformans, PLB is necessary for the early events of pulmonary infection and for dissemination from the lung via the lymphatic system and blood [9, 17]. Specifically, adhesion to alveolar macrophage cells is reduced in a PLB deletion mutant of C. neoformans and also in
the MM-102 presence of selective chemical inhibitors of PLB and a specific anti-PLB antibody. The extent of adhesion was correlated with PLB activity, but not with lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activity . Lack of established protocols for conducting experiments that might lead to gene disruption or silencing in P. brasiliensis hinders the validation of the plb gene functionality in this pathogen. In view of this fact, we decided to investigate the role of PLB using an in-vitro model of ARS-1620 mw host-pathogen interaction, i.e. the yeast
cells of P. brasiliensis infecting MH-S cells. The use of a specific inhibitor and/or an activator of PLB could be an effective strategy for investigating the possible role of this enzyme during host-pathogen interaction. Effects of alexidine dihydrochloride and pulmonary surfactant EX 527 nmr on cell viability, adhesion, internalization, and PLB activity during co-cultivation of P. brasiliensis and MH-S cells In order to verify whether the treatment with alexidine dihydrochloride and pulmonary surfactant interferes with cell viability, colony-forming unit (CFU) analysis was performed after co-cultivation of P. brasiliensis Non-specific serine/threonine protein kinase and MH-S cells. Cell viability of P. brasiliensis was evaluated by CFU analysis after treatment with the PLB inhibitor (0.25 μM alexidine dihydrochloride) and 100 μg mL-1 pulmonary surfactant. The percentage of cell viability was not significantly altered 6 h post-infection (Figure 1A). Figure 1 Paracoccidioides brasiliensis isolate Pb18 yeast cell viability and infection index after co-culture with alveolar macrophage (MH-S) cells. (A) CFU of P. brasiliensis isolate Pb18 yeast cells; (B) Infection index of in-vitro MH-S cells in the presence of alexidine dihydrochloride
(0.25 μM) and pulmonary surfactant (100 μg.mL-1). Percentage of MH-S cells infected with P. brasiliensis yeast cells – adhered (black bar) or internalized (white bar). In all experiments, MH-S cells and opsonized yeast cells were incubated at a yeast-to-macrophage ratio of 1:5, at 37°C in an atmosphere of 5% CO2 as described in the Materials and Methods. Data shown are derived from two in-vitro independent experiments performed in triplicate (mean ± SEM, with *significance assumed in the range of P < 0:05); ns = non-significantly (P < 0.05); **Significantly different from the untreated control P < 0.001 by the paired 2-tailed Student’s t-test. To further investigate the role of PLB we evaluated the percentage of P. brasiliensis yeast cells adhered to or internalized by MH-S cells after pulmonary surfactant and alexidine dihydrochloride treatments.