The glutathione-dependent system is one of the key systems regulating cellular redox balance, and thus cell fate. Cysteine, typically present in its oxidized form cystine in the extracellular space, is regarded as the rate-limiting substrate for glutathione (GSH) synthesis. Cystine is transported into cells by the highly specific amino-acid antiporter system x c À . Since Burkitt's Lymphoma (BL) cells display limited uptake capacity for cystine, and are thus prone to oxidative stress-induced cell death, we stably expressed the substrate-specific subunit of system x c À , xCT, in HH514 BL cells. xCT-overexpressing cells became highly resistant to oxidative stress, particularly upon GSH depletion. Contrary to previous predictions, the increase of intracellular cysteine did not affect the cellular GSH pool, but concomitantly boosted extracellular cysteine concentrations. Even though cells were depleted of bulk GSH, xCT overexpression maintained cellular integrity by protecting against lipid peroxidation, a very early event in cell death progression. Our results show that system x c À protects against oxidative stress not by elevating intracellular GSH levels, but rather creates a reducing extracellular environment by driving a highly efficient cystine/cysteine redox cycle. Our findings show that the cystine/cysteine redox cycle by itself must be viewed as a discrete major regulator of cell survival.

Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis.

No abstract

RCT combined with RHT is an efficient salvage therapy showing high efficacy with acceptable toxicity and can be recommended as treatment option for this unfavorable group of preirradiated patients with local recurrence of rectal cancer.

Although antigen-loaded dendritic cells (DC) are being investigated as antitumor vaccines, which DC differentiation state is most effective is not clear. Three DC functions that may be critical for immunization potential are expression of CD80/86, cytokine production following CD40 engagement, and migration to chemokine receptor 7-binding chemokines. We therefore examined highly purified human monocyte-derived immature and mature DC for these properties from normal donors and cancer patients. Although high expression of CD80/86 and migration to 6Ckine + macrophage-inflammatory protein-3beta were properties of mature DC, cytokine production following CD40 ligation was superior by immature DC. Loss of cytokine secretion occurred with multiple maturation conditions, was not apparently reversible, and was also seen with lipopolysaccharide stimulation in correlation with down-regulated Toll-like receptor expression. Our results suggest that the functions thought to contribute to optimal T cell priming are not coexpressed by the same DC population and that immature and mature DC likely possess distinct CD40-mediated signaling events.

Intracytoplasmic delivery of oligonucleotides (ODN) can improve ODN-based strategies such as the antisense approach and the use of immunostimulatory CpG dinucleotide containing ODN. Shock waves are established for the treatment of nephrolithiasis and other diseases. Here we describe the use of shock waves as a new physical method for the direct transport of antisense ODN into the cytoplasm and the nucleus of cells. Human peripheral blood mononuclear cells together with antisense ODN were exposed to shock waves generated by an electrohydraulic lithotripter. ODN uptake was examined by flow cytometry and fluorescence microscopy. By optimization of physical parameters we achieved the transfer of high amounts of ODN which were detected within less than 5 min after shock wave exposure, with viability of cells higher than 95%. Transfection of human peripheral blood mononuclear cells with an antisense ODN directed against tumor necrosis factor (TNF) alpha resulted in a reduction in lipopolysaccharide-induced TNF production by 62% (n=5, P=0.006). Specificity of TNF suppression was confirmed with a four-mismatch oligonucleotide. Positive atmospheric pressure abolished antisense-mediated inhibition of TNF synthesis by blocking shock wave-induced cavitation and formation of oscillating air bubbles. Electroporation was less effective. The use of shock waves is thus an efficient physical tool for ODN delivery to cells. Shock waves may allow the evaluation of target proteins in cell types difficult to transfect with other methods and thus may improve the antisense technique for the analysis of unknown genes.

No abstract

Inhibition of bcl-2 expression by antisense oligodeoxynucleotides (ODN) might render bcl-2 overexpressing malignant B cells more susceptible to chemotherapy. ODN containing unmethylated CG dinucleotides (CpG) are known to activate B cells. We studied the effects of two bcl-2 antisense ODN, with (G3139) or without CG dinucleotides (NOV 2009) within the sequence, and the effects of a nonantisense, CpG-containing ODN (ODN 2006) on activation and apoptosis of malignant B cell lines and primary B-CLL cells. Without cationic lipids, no antisense-mediated inhibition of bcl-2 synthesis was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen-presenting molecules, as well as in bcl-2 and bcl-xL levels of primary B-CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B-CLL cells of patients with high serum thymidine kinase activity (s-TK, marker for proliferative activity of malignant B cells), whereas in patients with low s-TK activity, apoptosis was induced. In conclusion, our results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense-mediated reduction of bcl-2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B-CLL, especially those with low s-TK activity.