Lab. in the gate of blast lymphocytes. Gamma interferon, tumor necrosis factor alpha, interleukin-4 (IL-4), and IL-10 levels in supernatants MSI-1436 and serum anti-PT IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). The net percentage of CD3+ blasts in cultures with in the group vaccinated with wP was higher than that in the group vaccinated with the wPlow vaccine (medians of 6.2% for the wP vaccine and 3.9% for the wPlow vaccine; = 0.029). The frequencies of proliferating Rabbit Polyclonal to EGFR (phospho-Ser1071) CD4+, CD8+, and + cells, cytokine concentrations in supernatants, and the geometric mean titers of anti-PT IgG were similar for the two vaccination groups. There was a significant difference between the T-cell subpopulations for and PHA cultures, with a higher percentage of + cells in the cultures ( 0.001). The overall data did suggest that wP vaccination resulted in modestly better specific CD3+ cell proliferation, and + cell expansions were similar with the two vaccines. Efforts to immunize against whooping cough began almost as soon as Bordet discovered the causative bacterium, cells were treated with MSI-1436 an organic solvent and washed in order to perform LPS extraction. The culture was then detoxified by the addition of 0.2% formalin, and the bacterial biomass was obtained by tangential flow filtration to formulate the wPlow vaccine. Each 0.5-ml dose of either vaccine contained four protective units of pertussis toxin and two protective units of diphtheria and tetanus toxoids. The antigens were adsorbed onto 1.25 mg of aluminum hydroxide, and 0.2 mg of thimerosal was used as a preservative. All infants received vaccine from the same batch. The vaccines were visually indistinguishable and identically packaged and were administered intramuscularly with the use of standard techniques. Study design. This prospective, randomized, double-blind comparative trial was conducted between August 2006 and July 2007 and followed the principles outlined in the Declaration of Helsinki. Written informed consent was obtained from all participants’ parents or legal guardians before study procedures were initiated. The study protocol was approved by the Committee for Ethics in Research of the State University of Campinas, S?o Paulo, Brazil. Antigens used in cell culture assays. A suspension of heat-killed (lot IB-CIIn/P14/06; Butantan Institute), without thimerosal, was used at a concentration of 5 106 cells/ml. Phytohemagglutinin (PHA; Sigma) was used as a positive control at 7.5 g/ml. T-cell proliferation assay. Ten milliliters of heparinized peripheral blood was collected and used for the evaluation of immune responses to pertussis vaccination at 7 months of age. The protocol for the proliferation assay was adapted from the method of Gaines and Biberfeld (20). Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Ficoll-Hypaque (Amersham Biosciences), washed, diluted to 1 1 106 cells/ml in RPMI 1640 medium (Sigma) supplemented with 10% human AB serum (Sigma), 1% glutamine (Sigma), and 0.1% gentamicin, and stimulated for 6 days with heat-killed value of 0.05 were considered statistically significant. Prism software (version 4.0; GraphPad Software) was employed for the figures. To evaluate the anti-PT titers, calculation of the geometric mean titers (GMTs) of Abs was performed on log10-transformed data, and we report the antilogarithms. For each group, GMTs and 95% CIs were calculated. For comparison of the logarithm of the titers, Student’s test was applied for independent samples. Comparisons MSI-1436 manifesting a two-tailed value of 0.05 were considered statistically significant (25). The differences in the proportions of subjects with seroprotection against diphtheria and tetanus and 90% CI were calculated as recommended for noninferiority studies (10, 36). Differences or ratios equal to or lower than 10% were accepted as the limit for defining noninferiority of the wPlow vaccine. The null hypothesis of noninferiority of the wPlow vaccine was accepted when the lower limit of the CI was not.