Saccharomyces cerevisiae expressing surface-displayed ApxIIA#5 was prepared as previously described [9]. Briefly, the yeast was

cultured in a selective medium (uracil-deficient medium: casamino acid 5 g, yeast nitrogen base 6.7 g, glucose 20 g, adenine 0.03 g and tryptophan 0.03 g in 1 L of DW) for 16 hrs at 30°C and then transferred and cultured in basic medium (YEPD: yeast extract 10 g, bacto peptone 20 g and glucose 20 g in 1 L of DW) for 3 days at 30°C. Yeast harboring a control vector or yeast expressing surface-displayed ApxIIA#5 was washed in saline and diluted to a titer of 5 × 108 cells/mL in PBS. Five-week-old female C57BL/6 buy Selisistat mice (Central Lab Animal Inc., Seoul, Korea) were used in this study, which was conducted in accordance with the policies and regulations of the care and use of laboratory animals of the Institute of Laboratory Animal Resources, Seoul National University, Korea. All the animals were provided with standard mouse chow and water ad libitum. 1.5 × 109

cells/day per mouse of surface-displayed ApxIIA#5 expressed on S. cerevisiae (vaccinated group) and vector-only S. cerevisiae (vector control group) were administered by oral gavage for two days on each occasion at 10-day intervals. Nontreated mice were also maintained as a mock control. Specimens and serum samples were collected 3 days after each immunization. Murine DCs were isolated from bone marrow progenitors according to previously described procedures [15]. The bone marrow cells were cultured in RPMI 1640 medium (Gibco Invitrogen, HSP inhibitor Karlsruhe, Germany) in the presence of 10% heat-inactivated FBS (Gibco Invitrogen), 10 ng/mL recombinant murine GM-CSF (PeproTech, London, UK) and 5 ng/mL recombinant IL-4 (PeproTech). Non-adherent cells were collected

and used for further experiments on Day 10. The purity of the cells, assessed by flow cytometry using phycoerythrin-conjugated anti-CD11c mAb (Abcam, Cambridge, UK), was 91.1 ± 0.92%. Single cell suspensions were obtained Diflunisal from samples of SP, intestinal LP and PP for T-cell proliferation and ELISPOT assays, as previously described [16, 17]. To examine the in vitro activation of the DCs by transgenic S. cerevisiae, immature DCs (1 × 106 cells/mL) were stimulated with surface-displayed ApxIIA#5 expressed on S. cerevisiae or vector-only S. cerevisiae (1 × 106 cells/mL). After 48 hrs, the cells were harvested for flow cytometry, and supernatants collected and stored at −80°C until the analysis of cytokine secretion by quantitative ELISA. The secreted concentrations of TNF-α, IL-1β, IL-10 and IL-12p70 were measured using the ELISA method (eBioscience, San Diego, CA, USA). The activation and upregulation of costimulatory molecules in the DCs were examined using a FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA).