abortus rough strain RB51 and smooth strain 2308 to stimulate murine bone marrow-derived DC (BMDC) activation and function based on the cell surface expression of costimulatory molecule and cytokine production. This study assessed simultaneously, for the first time, the differential ability of live, HK and IR rough and smooth strains of B. abortus KU-57788 chemical structure at the same doses to stimulate DC activation and function. Female 6–8-week-old BALB/c mice were obtained from Charles River Laboratories Inc. (Wilmington, MA). Mice were used under animal care protocols approved by Institutional Animal Care and Use Committee at Virginia Tech. BMDCs were generated,
as described previously (Inaba et al., 1992). Briefly, tibias and fibulas of 7–8-week-old BALB/c mice were incised and bone marrow (BM) cells were removed. Following red blood cell lysis and filtration, the cells were resuspended and plated in RPMI 1640 complete media with 10% non-heat-inactivated fetal bovine serum and 20 ng mL−1 rGM-CSF (recombinant Granulocyte colony stimulating factor; Invitrogen, Carlsbad, CA). The cells were incubated at 37 °C in 5% CO2. Fresh media containing rGM-CSF was added at days 2, 4 and 5 and harvested on day 6. The cells harvested on day 6 were typically 70% CD11c+ and displayed low levels of major
histocompatibility complex (MHC) class II, CD40 and CD86 expression, consistent with immature DCs. Flow cytometry was performed to confirm DC activation status (Inaba et al., 1992). Stock cultures of live-attenuated rough B. abortus vaccine strain RB51 and virulent smooth selleck chemical strain 2308 from our culture collection (Schurig et al., 1991; Vemulapalli et al., 2000) were stored at −80 °C. An aliquot each of strain RB51 and strain
2308 were subjected to γ-irradiation using a 60Co source irradiator with a radiation output of 2200 rads min−1 (Model 109-68R by J.L. Shepherd and Associates, San Fernando, CA) for 3 h (396 krads SDHB of γ-radiation). Aliquots of strain RB51 and strain 2308 were subjected to heat killing by incubating in an 80 °C water bath for 60 min. IR and HK bacterial preparations were confirmed to be nonviable by plating aliquots on TSA plates and confirming lack of growth following 4 days of incubation. All experiments with Brucella were performed in our CDC-approved (C2003 1120-0016) Biosafety Level-3 facility. On day 6, DCs were harvested and plated at 5 × 105 cells per well in 24-well plates and stimulated with live, IR or HK strain RB51 or strain 2308 at 1 : 10 (DC : Brucella) or 1 : 100 CFUs per well (i.e. 5 × 106 or 5 × 107 CFU equivalents per well of IR or HK B. abortus). Stimulation was enhanced by a short spin at 400 g for 5 min at room temperature. The stimulated cells were incubated for 4 h at 37 °C in 5% CO2. Then cells were washed with media containing gentamicin (Sigma, St. Louis, MO) 30 μg mL−1. The stimulated cells were incubated for an additional 20 h in complete media with 10 ng mL−1 rGM-CSF and 30 μg mL−1 gentamicin.