Synthetic oligodeoxynucleotides (ODNs) containing cytosine-guanosine (CpG) motifs stimulate B and plasmacytoid dendritic cells of the vertebrate immune system. We found that in primates strong stimulation of these cells could also be achieved using certain non-CpG ODNs. The immunostimulatory motif in this case is a sequence with the general formula PyNTTTTGT in which Py is C or T, and N is A, T, C, or G. Assays performed on purified cells indicated that the immunostimulatory activity is direct. The use of a nuclease-resistant phosphorothioate backbone is not a necessary condition, since phosphodiester PyNTTTTGT ODNs are active. It was also demonstrated that ODN 2006, a widely used immunostimulant of human B cells, possess two kinds of immunostimulatory motifs: one of them mainly composed of two successive TCG trinucleotides located at the 5′ end and another one (duplicated) of the PyNTTTTGT kind here described. Even though PyNTTTTGT ODNs are mainly active on primate cells, some of them, bearing the CATTTTGT motif, have a small effect on cells from other mammals. This suggests that the immunostimulatory mechanism activated by these ODNs was present before, but optimized during, evolution of primates. Significant differences in the frequency of PyNTTTTGT sequences between bacterial and human DNA were not found. Thus, the possibility that PyNTTTTGT ODNs represent a class of pathogen-associated molecular pattern is unlikely. They could, more reasonably, be included within the category of danger signals of cell injury.

ABSTRACT

Aims/hypothesis IMT504 is an oligonucleotide that promotes tissue repair in bone injury and neuropathic pain models by stimulating progenitor cells. Here we evaluated the effect of IMT504 on the recovery of islet function in a streptozotocin (STZ)-induced model of diabetes in the rat. Methods Male Sprague-Dawley rats were injected with STZ (60 mg/kg, i.p., day1) or citrate buffer (Control). Animals with glycaemia between 11 and 20 mmol/l on day 4 were injected with IMT504 (4 mg/animal in saline, s.c., STZ-IMT504) or with saline (STZ-Saline) for 10 days. Glycaemia and water and food intake were recorded for 33 days. Intraperitoneal glucose tolerance tests (IPGTTs) were performed on day30. On day35, overnight-fasted animals were killed and blood samples and pancreases collected for hormonal and histological studies. A second group of STZ-IMT504 rats was killed, together with Control and STZ-Saline rats, after two consecutive days of blood glucose decreases after the beginning of IMT504 treatment. Pancreases were collected and proliferating cell nuclear antigen (PCNA), nestin and neurogenin 3 (NGN3) detected by immunohistochemistry. Results IMT504 greatly improved blood glucose and food and water intakes in STZ-IMT504 rats by day8, as well as IPGTTs on day30. Significant increases in islet number and beta cell content were observed in STZ-IMT504 rats (day 33). Furthermore, after two to five IMT504 injections, blood glucose decreased, and an increase in pancreatic nestin (mainly in endothelial cells), PCNA and NGN3 production (in islets) was observed in STZ-IMT504 rats. Conclusions/interpretation IMT504 induced a marked recovery of STZ-induced diabetes that correlated with early production of progenitor cell markers, such as nestin and NGN3.

Nucleic acid immunization is a new vaccination technology. DNA vaccines do not only carry the genetic information for the antigen of interest but also deliver an adjuvant effect due to the presence of immunostimulatory sequences within the plasmid backbone. It is generally assumed that the adjuvant properties of plasmid DNA are equal to those described for oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs. To challenge this hypothesis we have carried out a series of experiments comparing the ability of single-and double-stranded ODN containing CpG motifs to induce the activation of mouse spleen cells. Moreover, we compared the immunostimulatory properties of plasmids that were modified by the addition of two to four CpG motifs. Our results establish that plasmid DNA express their adjuvanticity as either double or single strands, and no differences were observed between modified and unmodified plasmids. On the other hand, the strongest stimulatory ODN sequences lost their adjuvant properties when administered as double-strand DNA. Furthermore, the profile of cytokines induced on spleen cells by plasmid DNA and ODN is different. Strikingly, plasmid DNA induces a moderate synthesis of IL-6 and a strong synthesis of IFN-+ , whereas stimulation with ODN showed an inverse profile with a higher increase in the synthesis of IL-6 but a moderate increase in IFN-+ . Finally, in vivo studies were consistent with the results obtained in vitro. Mice immunized with modified or unmodified plasmids encoding the glycoprotein D of HSV showed similar levels of cellular and humoral immune responses.