Stimulation of exosome release by extracellular DNA is conserved across multiple cell types.

Exosomes are distinguished from other types of extracellular vesicles by their small and relatively uniform size (30-100 nm) and their composition which reflects their endo-lysosomal origin. Involvement of these extracellular organelles in intercellular communication and their implication in pathological conditions has fuelled intensive research on mammalian exosomes; however, currently, very little is known about exosomes in lower vertebrates. Here we show that, in primary cultures of head kidney leukocytes from Atlantic salmon (Salmo salar), phosphorothioate CpG oligodeoxynucleotides induce secretion of vesicles with characteristics very similar to these of mammalian exosomes. Further experiments revealed that the oligonucleotide-induced exosome secretion did not depend on the CpG motifs but it relied on the phosphorothioate modification of the internucleotide linkage. Exosome secretion was also induced by genomic bacterial and eukaryotic DNA in toll-like receptor 9-negative piscine and human cell lines demonstrating that this is a phylogenetically conserved phenomenon which does not depend on activation of immune signaling pathways. In addition to exosomes, stimulation with phosphorothioate oligonucleotides and genomic DNA induced secretion of LC3B-II, an autophagosome marker, which was associated with vesicles of diverse size and morphology, possibly derived from autophagosome-related intracellular compartments. Overall, this work reveals a previously unrecognized biological activity of phosphorothioate ODNs and genomic DNA - their capacity to induce secretion of exosomes and other types of extracellular vesicles. This finding might help shed light on the side effects of therapeutic phosphorothioate oligodeoxynucleotides and the biological activity of extracellular genomic DNA which is often upregulated in pathological conditions.

Kinetics of nuclear factor ?B in LPS stimulated rat alveolar macrophages and its regulative effect on tumor necrosis factor ? expression / 中华创伤杂志

Objective To investigate the activation of nuclear factor ?B (NF-?B) induced by lipopolysaccharides (LPS) in rat alveolar macrophages (AM) and its regulative role in tumor necrosis factor (TNF-?) expression. Methods The dynamic activity changes of NF-?B DNA induced by LPS (E.coli 026:B6) were determined with electrophoretic mobility shift assay (EMSA). The phosphorothioate oligodeoxynucleotides (S-ODN) decoy was transfected into AM 12 hours prior to LPS stimulation. The effect of NF-?B S-ODN decoy on expression of TNF-? in AM stimulated by LPS were measured with enzyme-linked immunosorbent assay (ELISA) kit. Results NF-?B could be activated remarkably after 0.5 hour of LPS stimulation at concentration of 100 ng/ml, reached the highest level 1 hour after LPS stimulation and gradually decreased. But the activation of NF-?B could last at least 8 hours. The dose for LPS stimulation was related to activation of NF-?B in a dose-dependent fashion, ie, the activation of NF-?B gradually strengthened with dose increase of LPS. Supershift assays proved that p50 and p65 were involved in the activation of NF-?B. NF-?B S-ODN decoy could markedly (not completely) inhibit LPS-induced TNF-? production. Conclusions NF-?B plays an important role in LPS induced inflammatory response. However, entire inhibition of the activity of NF-?B can not completely prevent TNF-? expression induced by LPS in rat AM, which implies that other nuclear factors may participate in TNF-? expression.

Effect of allicin on the regulation of VEGF mRNA expression in human hepatocellular carcinona cells / 中国药理学通报

AIM To investigate the effect of allicin on the regulation of VEGF mRNA expression in human hepatocellular carcinoma cells. METHODS Hepatocellular carcinoma cells were treated with the concentration 10 ?g?L -1 allicin in culture medium,and then the relative VEGF mRNA level at 8 h in human hepatocellular carcinoma cells was evaluated by reverse transcriptase polymerase chain reaction using HPRT(hypoxanthine phosphoribosyltransferase)as an internal control standard. RESULTS The expression of VEGF gene mRNA was inhibited obviously by allicin. Compared with control group, the relative expression level of VEGF gene mRNA was decreased by about 66 36%( P

KINETICS OF AP-1 IN LPS-STIMULATED RAT ALVEOLAR MACROPHAGES AND ITS REGULATION ON TNF-? EXPRESSION / 解放军医学杂志

To investigate the kinetics of activation of the activator protein-1(AP-1) and elucidate its role in TNF-? expression induced by lipopolysaccharide(LPS) in rat alveolar macrophages(AM), dynamic changes of the activity of AP-1 were detected with electrophoretic mobility shift assay(EMSA). The phosphorothioate oligodeoxynucleotide (S-ODN) decoy was transfected into AM prior to LPS stimulation. The level of TNF-? in culture supernatants was measured with an ELISA kit. The results showed that after LPS stimulation for 0.5 hour, remarkable activation of AP-1 could be detected and reached the highest level. The activation of AP-1 rapidly decreased at 1 hour, then increased at 3 hours again and reduced at 5 and 8 hours after LPS stimulation. The activation of AP-1 could persist at least 8 hours and showed a dose-dependent manner to LPS within 1000ng/ml. AP-1 S-ODN decoys could markedly inhibit the LPS-induced TNF-? production by rat AM, but it could not completely inhibit the production of TNF-? induced by LPS in rat AM. It is suggested that LPS could induce activation of AP-1 in rat AM; AP-1 played an important role in LPS-induced inflammatory response. It is also suggested that AP-1 involved in the regulation on LPS-induced TNF-? production by rat AM, however, entirely inhibition of the activity of AP-1 could not completely prevent TNF-? production by rat AM. It is also implied that other nuclear factors might also play an important role in the regulation on LPS-induced TNF-? expression by rat AM.