Samples and staining selleck Enzalutamide Blood samples were collected after clinical evaluation at each follow-up visit (immediately before HAART initiation and 4, 8, 12, 24, 39, 52 and 104 weeks after the initiation of treatment). Peripheral blood mononuclear cells (PBMCs) were obtained by density sedimentation (Lymphoprep, Oslo, Norway), cryopreserved, and thawed following the ACTG Consensus Protocol [25]. Cells were washed in medium and incubated overnight at 37��C and 5% CO2 prior to staining. A total of 106 cells were washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (EMD Biosciences, Darmstadt, Germany) and 0.1% sodium azide (JT Baker, Mexico). Cells were surface stained with either APC-Cy7-anti-CD4 or APC-Cy7-anti-CD8 plus APC-anti-CD45RA, PE-Cy7-anti-CCR7, PerCP-Cy5.

5-anti-CD38, FITC-anti-HLADR (all from BD Biosciences, San Jose, CA), and biotin-anti-CD28 followed by Streptavidin-PE-Texas Red (BD Biosciences, San Jose, CA). Control cells were stained with anti-CD4 or anti-CD8 antibodies plus fluorochrome-conjugated isotype controls (all from BD Biosciences). Cells were re-suspended in PBS with 1% paraformaldehyde (Sigma Aldrich, Steinheim, Germany) and analyzed on a FACSAria flow cytometer (Becton Dickinson, San Jose, CA). Data analysis Data were analyzed using FACSDiva (Becton Dickinson, San Jose, CA). CD4+ and CD8+ T cells were identified according to their light-scattering properties and high CD4- or CD8-associated fluorescence. Naive (CD45RA+ CCR7+), central memory (CM) (CD45RA- CCR7+) [Lanzavecchia, Sallusto 1990, Schwendeman, Lederman], and effector memory (EM) (CD45RA- CCR7-) cells were delineated as defined elsewhere [26-32] (Additional file 4: Figure S3).

The CCR7+ gate was additionally verified using the FACSDiva auto-gate tool and by back-gating CD28+ cells into CCR7+ populations. Frequencies of activated subsets were determined according to their patterns of HLA-DR and CD38+ expression (CD38+ HLADR-, CD38+ HLADR+, and CD38- HLADR+). (Additional file 4: Figure S3). Absolute counts of cells from different subpopulations were calculated using their proportions and the corresponding absolute CD4+ or CD8+ T lymphocyte counts. Three-group comparisons were determined with the Kruskal-Wallis test. If the Kruskal-Wallis test found overall significant differences between groups (tied p value<0.

05), post-hoc two-group comparisons were performed with the Mann�CWhitney test. Tests were performed using StatView. Results Differential frequencies of activated CD8+ T cell subsets during HAART according to CD38 and HLADR expression The frequency of CD8+ T cells consistently tended to smaller decreases in the TB-IRIS group under HAART, even though all groups had comparable Brefeldin_A %CD8+ T cell values at week 0 (Additional file 2: Figure S2A). Groups�� %CD8+ T cells tended to differ at weeks 8, 12, and 24 (p=0.0194, p=0.062, p=0.101, correspondingly), and showed significant differences at weeks 39 and 52 (p=0.