Mixtures of opsonized Candida in mouse autologous serum (10%) were added to 0.2 mL of macrophage suspension. The mixture was incubated for 30 min at 37°C. The percentage of phagocytosis was expressed as the percentage of phagocytosing macrophages in 200 cells counted using an optical microscope (15). Alveolar and peritoneal macrophages SB525334 cost monolayers were prepared as described above. In order to determinate the influence of lactobacilli on the capacity of macrophages to produce cytokines, alveolar and peritoneal macrophages were challenged in vitro with heat-killed C.

albicans AV4 at a concentration of 107 cells/mL. After incubation at 37°C in 5% CO2, the supernatant was recovered and kept frozen until cytokine analyses.

IL-1β and TNF-α were determined using the corresponding mouse ELISA kits Vemurafenib (R & D Systems). In order to evaluate the influence of lactobacilli treatments on the immune response against C. albicans in vivo, challenges with pathogenic C. albicans AV4 were performed. Yeast cells were grown in Sabouraud broth at 37°C until the log phase was reached. The pathogens were harvested by centrifugation at 3600 g for 10 min at 4°C and washed three times with sterile PBS. Intraperitoneal challenge with C. albicans AV4 was performed on the day after the end of each Lactobacillus treatment (third or sixth days). The mice were challenged with injections of 200 μL of an inoculum containing 108 cells. For yeast cell counts in blood, liver and spleen, mice were killed on day 2 post-infection. The livers and spleens were excised, weighed and homogenized in 5 mL of sterile peptone water. The homogenates were diluted appropriately, plated in duplicate on Sabouraud agar and MRIP incubated at 37°C. C. albicans colonies were counted and the results expressed as log10 CFU/g of organ or mL of blood. Intranasal challenge

with C. albicans AV4 was performed on the day after the end of each Lactobacillus treatment (third or sixth days). The mice were challenged nasally with the pathogen by dripping 25 μL of an inoculum containing 107 cells into each nostril. To facilitate migration of the inoculum to the alveoli, the mice were held in a head-up vertical position for 2 min. For yeast cell counts in lung and blood, mice were killed on day 2 post-infection. The lungs were excised, weighed and homogenized in 5 mL of sterile peptone water. The homogenates were diluted appropriately, plated in duplicate on Sabouraud agar and incubated at 37°C. The C. albicans colonies were counted and the results expressed as log10 CFU/g of organ or ml of blood. In order to evaluate innate immune responses after challenges, the concentrations of TNF-α and IFN-γ and the number of leukocytes and neutrophils were determined in BAL and peritoneal fluid according to techniques described in a previous report (15).