Growth inhibition of SK-MEL-30 human melanoma cells by antisense c-myc oligonucleotides delivered by poly(N-isopropylacrylamide)/ poly(ethyleneimine) copolymer.

The c-myc oncogene has been shown to be overexpressed in a number of malignancies and plays a key role in the abnormal growth regulation of melanoma cells. This study aimed to provide an efficient system for the in vitro manipulation of c-myc expression by antisense oligonucleotides. Therefore, we used poly(NIPA)/PEI2B copolymer as vector in order to improve the intracellular availability and stability of AS ODNs. We targeted oligonucleotide sequences within the human c-myc mRNA as free AS ODNs or conjugated with a thermosensitive copolymer, in an effort to inhibit the growth of human melanoma cells. The conjugates adopted more positive charge and smaller size at 37 degrees C and they had no toxic effects on human fibroblast cells. The conjugated AS ODNs showed increased antiproliferative effect on melanoma cells as compared to free AS ODNs. At a concentration of 100 ng, AS ODNs inhibited SK-MEL 30 human melanoma cell line proliferation maximally by 18.6%, whereas the same amount of conjugated AS ODN provided 52% inhibition. The greatest inhibition was obtained by conjugates having a polymer:AS ODN ratio of 9. Greatest inhibition was detected at 48 h and decreased after 96 h, which may be due to the depletion of AS ODNs. The results confirm the enhanced antiproliferative effects of poly(NIPA)/PEI2B-conjugated AS ODNs, which may provide improved intracellular availability for c-myc-directed antisense strategies.

Effects of some antigenic fractions of Leishmania major on nitric oxide production and IL-12 secretion by murine peritoneal macrophages.

Nitric oxide (NO) and IL-12 are important mediators of the immune response to Leishmania major. In this study, the effects of L. major promastigotes, crude antigenic fraction (CAF) and its subfractions on NO production and IL-12 secretion by BALB/c mice peritoneal macrophages is investigated. The subfractions of CAF, namely, fractions 1, 2 and 3, that were in the molecular weight range of 97.4-66, 66-45 and below 45 kDa, respectively, were separated by SDS-PAGE. NO production was determined by using Griess reagent and IL-12 was measured by ELISA. It was found that NO production was stimulated by promastigotes but not by CAF or its subfractions. IL-12 secretion was stimulated by promastigotes, CAF and fraction 1 while fractions 2 and 3 did not have any effect.

Defibrotide augments the anticandidial activity of NK cells.

1. The mechanism of action of Defibrotide, a fibrinolytic agent on Natural Killer (NK) cell cytotoxicity, was investigated through verapamil, TMB-8 cells and pertussis toxin. 2. Defibrotide increased the activity against Candida albicans cells (anticandidial activity), and it is determined that the calcium channels have a role in this effect. 3. Blockage of calcium channels reduced the anticandidial effect by 39.2%. Pertussis toxin led to a 10.7% inhibition, whereas the application of TMB-8 resulted in the stimulation of anticandidial activity. 4. It is concluded that defibrotide is a potent activator of NK cells.

Effects of crude antigenic fractions of Leishmania major on natural killer cell cytotoxicity, interferon-gamma and interleukin-4 secretion from peripheric blood lymphocytes of unexposed individuals.

The crude antigenic fraction (CAF) isolated from Leishmania major was fractionated into three subfractions by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The effects of CAF and its subfractions on NK cell cytotoxicity is investigated by chromium release assay. These subfractions designated as fractions 1, 2 and 3 correspond to 97.4-66 kD, 66-45 kD and 29 kD and below respectively. Although both CAF and its subfractions have inhibited the cytotoxicity of natural killer (NK) cells, the effects of fractions 2 and 3 were more pronounced. The effect of the fractions on the Interferon-gamma (IFN-gamma) and Interleukin-4 (IL-4) secretion by peripheric blood lymphocytes was also analyzed. It was found that CAF and fraction 1 induce IFN-gamma secretion while on the other hand IL-4 secretion was mostly suppressed by fraction 2. Therefore, further research is being executed which focuses on the effects of CAF, fractions 1 and 2 on macrophage effector functions.