Briefly, 96-well Millipore polyvinylidene difluoride plates were coated with anti-mouse IFN-γ or IL-2 Ab (BD Pharmingen) diluted in PBS and incubated overnight at 4°C. Plates were then washed and blocked with 10% MLC media (DMEM supplemented
with 10−6 M of 2-mercaptoethanol and 10% FBS) for 2 h at 37°C. Lymphocytes were added to plates at 2×105 cells per well in see more triplicates, and stimulated with the 9-mer peptide AMQMLKETI or the total pool of 123 15-mer peptides derived from consensus Gag clade B, in the presence of anti-mouse CD28 and CD49d (BD Pharmingen) for 18–20 h at 37°C in 10% CO2. Cells were removed and plates incubated with biotin-labeled Ab (BD Pharmingen) for 2 h at room temperature. Streptavidin alkaline phosphatase (Mabtech AB) was added for 1 h, and the spots developed by adding BCIP/NBT
(Pierce) for 5 min. Plates were washed in water and dried before counting using the C.T.L. Series 3A Analyzer and ImmunoSpot 3.2 (Cellular Technology). Data from unstimulated cells www.selleckchem.com/products/PF-2341066.html were used as background control, and values were subtracted from sample values before plotting. In parallel, cells were stained with an Ab to CD8α and analyzed by flow cytometry to determine the frequencies of this cell subset. These results were used to normalize the data obtained by ELISpot assays and data show numbers of SFU/106 CD8+ cells. Samples that resulted in less than 55 SFU/106 cells were considered negative. Each Verteporfin molecular weight experiment was conducted repeatedly with 5–20 mice and figures show means and standard deviations based on the independent experiments. Statistical significance of differences between groups was calculated by unpaired two-sample Student’s t-test using
GraphPad Prism (GraphPad Software, La Jolla, CA, USA). The p-values of <0.05 or <0.01 were considered statistically significant. The authors thank Christina Cole for assistance with preparation of the manuscript. This work was supported by grant AI074078-01 from the National Institutes of Health, by Wistar Cancer Center Support Grant P30 CA 010815 from the National Cancer Institute, and by the Gates Foundation (GCGH). Partial support was also provided by CAPES and PNDST/Aids, Brazil. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. "
“3M-003, like related imidazoquinoline immunomodulators, interacts with Toll-like receptor-7 (TLR-7) and TLR-8. TLRs are important in the defense against fungal pathogens. The effect of 3M-003 on killing of Candida was evaluated on mouse (BALB/c) effector cell lineages: monocytes, neutrophils, and macrophages. After direct application, 3M-003 (1–80 μg mL−1) enhanced (P<0.05–0.