In the proposed model, the genes related to phagocytosis and oxidative Rabusertib in vitro burst are up-regulated providing an efficient mechanism
for fungal survival. The increase in IL-12 and decrease in IL-10 after inhibition of PLB participate in the enhancement of IFN-γ activity, which is capable of inducing a cellular immune response. These data confirm the participation of PLB in the mechanism of fungal evasion, interfering with an adequate immune response by the host. Conclusions Based on these data, we conclude that P. brasiliensis PLB is important for adhesion and internalization of yeast cells by MH-S cells. Whether PLB activity results from the production of eicosanoids or leukotrienes or not remains unknown, although studies are in progress to investigate this possibility.
Nevertheless, our study clearly identified activities of fungal PLB that may enhance virulence and subsequent down-regulation of macrophage activation. Methods Strains, cultures and reagents P. brasiliensis Pb18 (ATCC 32069) yeast cells were cultivated in Fava-Netto semisolid medium for 7 days at 37°C and used in in-vitro infection. Alveolar macrophage lineage MH-S (ATCC CRL-2019) was grown in RPMI-1640 tissue culture medium (Sigma-Aldrich, Inc., St. Louis, MO, USA) supplemented with 20 mM HEPES, 1.5 g L-1 BAY 11-7082 cost sodium bicarbonate, 2.5 GW3965 datasheet mg mL-1 gentamicin, and 10 U mL-1 heparin. The viability of MH-S cells was determined by trypan blue exclusion. All assays used the bovine pulmonary surfactant Survanta (Abbott Laboratories, Inc., Columbus, OH, USA), which is an extract of bovine lung containing about N-acetylglucosamine-1-phosphate transferase 75% DPPC and 45% phosphatidylcholine (PC), generating substrates for
phospholipases. The specific inhibitor of PLB – alexidine dihydrochloride (Toronto Research Chemicals, Inc., Toronto, Ontario, Canada) – was prepared as a stock solution at 10 mM in dimethyl sulfoxide (DMSO), which was then diluted to the required concentration with RPMI medium. Infection of MH-S cells with P. brasiliensis yeast cells Phagocytic test MH-S cells were seeded in 24-well (0.2 × 105 cells/well) or in 150 cm2 (0.4 × 107 cells/well) cells culture flasks and incubated at 37°C for 6 h. Non-adherent cells were removed by washing, whereas the adherent cells were incubated in RPMI supplemented as stated above, with 10% heat-inactivated fetal calf serum, at 37°C. P. brasiliensis yeast cells were suspended in RPMI medium containing 20% fresh mouse serum. The opsonization protocol was carried out by incubation of yeast cell suspension at 37°C for 30 min. MH-S cell monolayers were infected with 4 × 106 yeast cells, representing a yeast-to-macrophage ratio of 1:5 . Incubation was carried out at 37°C in a humidified 5% CO2 atmosphere.