Generation of MUC1-stimulated mononuclear cells using optimized conditions.

Mucin is a glycoprotein found on the surface of cell membranes of adenocarcinomas. The purpose of these studies was to generate MUC1 multiple tandem repeat (VNTR)-stimulated mononuclear cells (M1SMC). We first determined the optimal conditions to influence the immune response. In these studies, peripheral blood mononuclear cells (PBMC), from patients with adenocarcinomas, were stimulated by different numbers of M1SMC stimulations, various concentrations of MUC1 peptide, washing of PBMC prior to stimulation and days in culture, to determine the optimal conditions to influence the immune response. The results of this study indicate that the mononuclear cells (MC) stimulated twice 1 week apart with MUC1 VNTR1 produced a greater specific killing of the breast cancer cell line MCF-7 than the 0, 1, 3 or 4 weekly stimulations. The optimal molarity for inducing cytotoxicity and cytokines (granulocyte macrophage colony-stimulating factor, gamma-interferon and interleukin-10) was 45 x 10(-8) M (1 microg/ml); except for tumour necrosis factor (TNF)-alpha which was 22 x 10(-8) M (0.5 microg/ml). The unwashed MC were superior to washing them with Ficoll-Hypaque. The optimal number of days in culture for cytotoxicity and cytokine production was after two stimulations (i.e. after day 7). Optimum conditions for generation of M1SMC identified in these studies were two stimulations with peptide, concentration of 45 x 10(-8) M (1 microg/ml) peptide, unwashed cells, and after two stimulations or after 8 days in culture. M1SMC were generated from multiple patients with breast cancer which lysed adenocarcinoma cells.

Characterization of protective immunity induced against Schistosoma mansoni via DNA priming with the large subunit of calpain (Sm-p80) in the presence of genetic adjuvants.

Despite advances in control via snail eradication and large-scale chemotherapy using praziquental, schistosomiasis continues to spread to new geographic areas particularly in sub-Saharan Africa. Presently, there is no vaccine for controlling this disease. We have concentrated on a functionally important schistosome antigen Sm-p80 as a possible vaccine candidate for schistosomiasis. Here we report the proliferation of spleen cells in response to the recombinant Sm-p80 protein and cytokine (IFN-gamma and IL-4) production by the splenocytes. These spleen cells were obtained from groups of mice that were vaccinated with a DNA vaccine formulation containing Sm-p80 and one of the Th-1 (IL-2 or IL-12) or Th-2 (GM-CSF, IL-4) enhancer cytokines. The splenocytes from the groups of mice vaccinated with Sm-p80 DNA in the presence of Th-2 enhancer cytokines showed moderate but detectable proliferation. The splenocytes obtained from mice vaccinated with Sm-p80 DNA with Th-1 enhancer cytokines IL-2 and IL-12 provided the highest proliferation. The IFN-gamma production by splenocytes was found to follow the similar pattern [(Sm-p80) < (Sm-p80 + IL-4) < (Sm-p80 + GMCSF) < (Sm-p80 + IL-12) < (Sm-p80 + IL-2)], as has been observed for the proliferation and protection data. However, the elevated IL-4 production was inversely correlated to Sm-p80-induced splenocyte proliferation or the protection. These results show again that protective immune response induced by Sm-p80 is of Th-1 type.