Design and development of nanosized DNA assemblies in polypod-like structures as efficient vehicles for immunostimulatory CpG motifs to immune cells.

The immunostimulatory activity of phosphodiester DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides, or CpG motifs, was significantly increased by the formation of Y-, X-, or dendrimer-like multibranched shape. These results suggest the possibility that the activity of CpG DNA is a function of the structural properties of branched DNA assemblies. To elucidate the relationship between them, we have designed and developed nanosized DNA assemblies in polypod-like structures (polypod-like structured DNA, or polypodna for short) using oligodeoxynucleotides (ODNs) containing CpG motifs and investigated their structural and immunological properties. Those assemblies consisting of three (tripodna) to eight (octapodna) ODNs were successfully obtained, but one consisting of 12 ODNs was not when 36-mer ODNs were annealed under physiological sodium chloride concentration. High-speed atomic force microscopy revealed that these assemblies were in polypod-like structures. The apparent size of the products was about 10 nm in diameter, and there was an increasing trend with an increase in ODN length or with the pod number. Circular dichroism spectral data showed that DNA in polypodna preparations were in the B-form. The melting temperature of polypodna decreased with increasing pod number. Each polypodna induced the secretion of tumor necrosis factor-α and interleukin-6 from macrophage-like RAW264.7 cells, with the greatest induction by those with hexa- and octapodna. Increasing the pod number increased the uptake by RAW264.7 cells but reduced the stability in serum. These results indicate that CpG DNA-containing polypodna preparations with six or more pods are a promising nanosized device with biodegradability and high immunostimulatory activity.

Biodegradable CpG DNA hydrogels for sustained delivery of doxorubicin and immunostimulatory signals in tumor-bearing mice.

Immunostimulatory CpG DNA was self-assembled to form DNA hydrogels for use as a sustained delivery system for both intercalated doxorubicin (DXR) and immunostimulatory CpG motifs for cancer treatment. X-shaped DNA (X-DNA) was designed as a building unit, and underwent ligation to form DNA hydrogels. Two types of X-DNA were constructed using four oligodeoxynucleotides each, one containing six potent CpG motifs (CpG X-DNA) and the other with none (CpG-free X-DNA). CpG X-DNA was more effective than its components or the CpG-free counterpart in terms of the production of tumor necrosis factor-α from murine macrophage-like RAW264.7 cells, as well as maturation of the murine dendritic DC2.4 cells. The cytotoxic effects of X-DNA, DXR and their complexes were examined in a co-culture system of colon26/Luc cells, a murine adenocarcinoma clone stably expressing firefly luciferase, and RAW264.7 cells. DXR/CpG X-DNA showed the highest ability to inhibit the proliferation of colon26/Luc cells. DXR was slowly released from CpG DNA hydrogels. Injections of DXR/CpG DNA hydrogels into a subcutaneous colon26 tumor effectively inhibited tumor growth. These results show that CpG DNA hydrogels are an effective sustained system for delivery of immunostimulatory signals to TLR9-positive immune cells and DXR to cancer cells.

Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides.

Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation.

Understanding externalizing behavior from children's personality and parenting characteristics.

A total of 946 Japanese children in the 5th to 9th grades and their parents were studied in order to investigate the extent to which parenting characteristics (measured by the Parental Bonding Instrument) and the personality of the child (measured by the junior version of the Temperament and Character Inventory) would be associated with the two aspects of the externalizing problems--aggression and delinquency--of the child (measured by the Child Behavior Checklist). A series of regression analyses demonstrated that (1) aggressive children were higher in Novelty Seeking, and delinquent children were higher in Novelty Seeking and lower in Harm Avoidance, and (2) both aggressive and delinquent children were characterised by low maternal care, paternal over-protection, and low maternal overprotection. A structural equation model confirmed these findings except for the link between the two externalizing behaviour scores and the maternal care. Moreover, it was suggested that Novelty seeking of the child would be predicted by low parental care and low paternal and high maternal overprotection. The children's aggression and delinquency could, to some extent, be explainable by their temperament patterns and parental characteristics.

Enhanced immunostimulatory activity of oligodeoxynucleotides by Y-shape formation.

DNA containing unmethylated CpG dinucleotides (CpG DNA) is a potent activator of innate and acquired immune responses. Although the sequence-specific immunostimulatory activity of CpG DNA has been extensively explored, little information is available about the importance of the stereochemical properties of CpG DNA. In this study, Y-shaped oligodeoxynucleotides (Y-ODNs) were prepared using three ODNs with the halves of each ODN being partially complementary to a half of the other two ODNs. Y-ODN induced greater amounts of tumour necrosis factor-alpha and interleukin-6 from RAW264.7 macrophage-like cells than did conventional single-stranded ODN (ssODN) or double-stranded ODN (dsODN). The Y-ODN was less stable in serum than dsODN, but greater amounts of Y-ODN were taken up by macrophage-like cells compared with dsODN. A newly designed Y-ODN containing three potent CpG motifs generated significantly higher levels of cytokines compared with dsODN containing the identical sequences. These results indicate that the Y-shaped form of ODN is a novel, reproducible and reliable approach to enhancing the immunostimulatory activity of ODNs.