01 IU/mL, susceptibility Galunisertib mouse to the disease [1] and [16]. Cellular immune response against tetanus toxoid was determined by the percentages of CD4+ T and CD8+ T cells expressing intracellular interferon-gamma after in vitro stimulation with tetanus toxoid by flow cytometry. A culture was done using full blood diluted to 1:10 in RPMI 1640 culture medium (Gibco, NY, USA) supplemented with l-glutamine, penicillin and streptomycin. The diluted blood was then distributed in polystyrene tubes in volumes of 1 mL. Following the addition of the antigen, the tubes

were sealed and incubated at 37 °C for 72 h in an atmosphere with 5% CO2. For all tests, one tube of blood stimulated with phytohemagglutinin was used as the positive control and another of non-stimulated blood was used as the negative control. Tetanus toxoid was obtained from Butantã Institute (São Paulo, Brazil). In the last 4 h of incubation, brefeldin A was added at a concentration of 10 μg/mL to all tubes (Sigma, St. Louis, USA). CD3-APC and CD8-PerCP conjugated monoclonal antibodies (BD Biosciences) were used for cell-surface staining. Cells were fixed, washed, resuspended with permeabilization

learn more buffer, and incubated for 10 min in the dark at room temperature. IFNγ-FITC-conjugated monoclonal antibody was then added. Finally, the cells were washed and kept at +4 °C in the dark until data acquisition. Sample acquisition was performed with FACSCalibur Cytometer (BD Biosciences) using Cell Quest software (BD Biosciences). The analysis was performed using FlowJo software (Tree Star, Ashland, USA). Fifty thousand events were acquired in the lymphocyte gate based on the forward scatter and side scatter dot plot.

CD3+ T cells were selected based on the side Cytidine deaminase scatter profile and CD3-APC fluorescence. CD8+ T lymphocytes were defined as CD3+/CD8+; due to down regulation of CD4 molecules during activation, CD4+ T lymphocytes were defined as CD3+/CD8−. Intracellular IFN-γ production was evaluated in CD3+/CD8+ and CD3+/CD8− cells. The final value of positive cells to each stimulus was obtained by subtracting the percentage of positive cells of the culture without stimulus (negative control) from the culture in the presence of stimulus. The numerical variables were compared using either the Student’s t-test (normal distribution) or the Mann–Whitney test (non-normal distribution). The categorical variables were compared using either the chi-squared (χ2) test or Fisher’s exact test. Multiple linear regression analysis was performed to determine factors associated with tetanus antibody levels measured at 18 months. Variables associated with optimal protective antibody level (≥0.1 IU/mL) against tetanus at 15 months of age were studied by multiple logistic regression analysis. The statistical analysis was carried out using the Statistical Package for Social Sciences for Windows, version 17.0 (SPSS, Chicago, IL, USA), with the level of significance set to 5% (p < 0.05).