Contaminating endotoxins were removed from the HmuY sample using Detoxi-Gel Endotoxin Removing Columns (Thermo Scientific, Rockford, IL, USA). HmuY was prepared at a final concentration of 2.5 μg/mL. Twenty milliliters
of Selleck Linsitinib peripheral venous blood were drawn from each individual and collected in heparin tubes. Mononuclear cells (PBMC) were obtained from peripheral blood samples and purified by density centrifugation in accordance with manufacturer guidelines (SepCell, StemCell Technologies Inc., USA). All cells were washed twice in RPMI (Roswell Park Memorial Institute) medium (LGCBio, São Paulo, SP, Brazil) and PBMCs were cultured in flat-bottom 24-well plates (106 cells/well) in RPMI medium containing 10% fetal calf serum (complement proteins inactivated by heat) and 1% antibiotic/antimycotic solution (R&D Systems, Minneapolis, MN, USA). All cultures XMU-MP-1 were grown for 48 h at 37°C under 5% CO2 in humid conditions. Cells were also incubated
with 5 μg/mL of pokeweed mitogen (PWM) as a positive control, 0.5 μg/mL of P. gingivalis extract (ATCC 33277), 2.5 μg/mL of HmuY, or in the absence of antigens (Cells). All PBMCs were collected by centrifugation and resuspended in 500 μL of 1×binding buffer, then incubated with fluorescently labeled antibodies in accordance with manufacturer instructions (Life Science, Carlsbad, CA, USA). To identify the expression of the anti-apoptotic protein Bcl-2 and the Fas death receptor, mouse anti-human Bcl-2 (IgG1 kappa) conjugated with PE CY, mouse anti-human CD95 (IgG1) conjugated with fluorescein isothiocyanate
(FITC), mouse anti-human CD3 conjugated with PerCP CY (IgG2a), or isotype-matched controls antibodies were used. The triple expression of CD3, CD4 and CD8 was identified by flow cytometry using the FITC, PE CY and PerCP CY signal detectors and BD FACSCalibur equipment (BD Facscalibur, Franklin Lakes, NJ, USA). Clinical variables were described in terms of means±standard deviations (mean±SD). Student’s t-test was used to compare clinical features among groups. The Mann–Whitney test was used to assess differences among groups with respect to immunological data in the absence of normal distribution. Statistical significance was considered when p < 0.05. SPSS 17.0 (Statistical Package for Social Science, USA) software was used to perform all statistical nearly analyses. Acknowledgments This study was supported by grant no. 20100291 from the Coordination for the Improvement of Higher Education Personnel in Brazil (awarded to Paulo Cirino de Carvalho Filho), and no. N303 518438 from the Polish Ministry of Science and Higher Education in Poland (Tereza Olzack). The authors would like to thank the Laboratory of Immunology and Molecular Biology at the Health Sciences Institute of the Federal University of Bahia (UFBA) and the Research Support Foundation of the State of Bahia for providing assistance with student fellowships. The authors would also like to thank Andris K.