In a second experiment, CD11 and CD8 were and / or CD4 T cells isolated from a pool of 10 rats and CD49b and CD45R and / or CD4 were isolated from a second pool of 10 rats. Peripheral blood leukocytes halothane anesthetized blood cultures C57Bl / 6 Mice were aseptically by cardiac puncture into heparinized R Hrchen collected. Blood of 50 M were usen For the first experiment Hedgehog Pathway and 30 Mice Pooled for the second attempt. The blood of healthy donors were obtained from the blood of New Zealand. The blood of 12 donors were processed in batches of two to three installation. Mononuclear Ren murine or human blood cells were isolated by centrifugation on Ficoll Paque density and were cultured in 96-flat bottom and with 10 or 300 g / ml in a final volume of 200 DMXAA liter of culture medium. Cured hands of humans and mice PBL cultures were harvested after 16 hours and 4 hours and stored at  0 to the test.
Multiplex cytokine assay kit multiplex cytokine murine complex complex 22 and 32, and seven complex MDV3100 human complexes 30 and 42 complexes were used according to the instructions of the manufacturer. Serum samples were diluted 1:5, and samples of the tumor and spleen were were diluted 1:10 with diluent matrix with the kits, and the Kulturberst Provided ligands tested undiluted. The concentration of each cytokine in the samples was measured using the Luminex 100 instrument. Each sample was tested in duplicate and the results were expressed as mean  SEM of three Mice per group and triplicate cultures per experimental group. Data between treated and untreated groups were compared with DMXAA tests st students or ANOVA when multiple comparisons were made.
Paired t-test types performed to compare the concentrations of cytokines in treated and untreated cultures for all 12 donors. The data were considered significant when P .05. Results Effect of DMXAA on leukocytes in c Lon 38 The tumor CD45 leukocyte infiltrate tumors c Lon to 38 was shown by FACS analysis to understand CD3CD8a cells 43%, 20% CD3CD4 T cells, lymphocytes, B 12%, 14% CD19CD45R CD11bF4/80 Macrophages / monocytes, immature mature macrophages CD11bF4/80 11% and 12% CD49b NK cells. Weight Changes in the content of leukocytes groups Colon 38 tumors before and 1, 3, 5, 7 and 10 days after a single injection of DMXAA at its maximum tolerable Adjusted dose of 25 mg / kg, was monitored. Tumor weight of almost 70% in the first 3 days of rose, then something about the n Next 4 days before a second phase of tumor reduction was observed on day 7.
CD45 leukocytes per gram of tumor in the first 24 hours after treatment, tripled when Tumorgr It sinks. CD45 leukocytes then fell to 16,106 × a low point of 3103 cells per tumor weight × program on day 3, then increased Ht and 10,106 cells × stabilized after 7 days. The increase in leukocyte content w During the first 24 hours was not An influx of lympho Of. CD19CD45R B lymphocytes, NK cells and CD49b and CD3CD8a CD3CD4 subsets all the numbers fell during the first 3 days, then increased pretreatment levels after 7 days Ht, then stabilized. Myelomonocytic cells CD11bF4/80 Followed change one Hnlichen trend as the cell. CD11bF4/80 of untreated tumors have the appearance of mature macrophages.