• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.78-78
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• 1995

Increasing evidence indicates that the production of nitric oxide (NO) by inducible NO synthase (NOS) is tightely regulated. Transforming growth factor-${\beta}$ (TGF-${\beta}$) is a homodimeric protein secreted during macrophage activation, but several lines of evidence suggest that TGF-${\beta}$ is selectively suppressive for macrophage NO production. We therefore reasoned that a strategy employing oligodeoxynucleotides(ODNs) complemently to TGF-${\beta}$ mRNA (antisense ODNs) might increase NO production in IFN-${\gamma}$-treated murine peritoneal macrophages. To evaluate this concept, we tested the effects of antisense ODNs targeted to TGF-${\beta}$ mRNA (25-mer ODNs complemently to TGF-${\beta}$mRNA sequences) by introducing it into the medium of cultured macrophages. Phosphorothiolation of ODNs were employed to retard their degradation. Antisense ODNs had no effect on NO production by itself, whereas IFN-${\gamma}$ alone had modest effect. When antisense ODNs were used in combination with IFN-${\gamma}$, there was a marked cooperative induction of NO production, These effects of antisense ODNs were associated with decreased TGF-${\beta}$ expression in activated macrophages. ODNs with the same nucleotides but a scrambled sequence had no effect. Adding anti-TGF-${\beta}$ antibodies to the IFN-${\gamma}$-treated macrophages mimicked the positive effect of antisense ODNs on NO production. In addition, the effects of either antisense ODNs or anti-TGF-${\beta}$ antibodies were blocked by adding TGF-${\beta}$ in cultured macrophages. These results indicate that the generation of TGF-${\beta}$ by activated macrophages provides a self-regulating mechanism by which the temporal and perhaps spatial production of NO, a reactive and potentially toxic mediator, can be finely regulated.

• Fisheries and Aquatic Sciences
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• v.10 no.1
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• pp.1-7
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• 2007

The induction of cellular and humoral immunity and cytokine gene expression by synthetic CpG oligodexoynucleotides (CpG-ODNs) has not been investigated systematically in olive flounder Paralichthys olivaceus in vivo. We optimized the proper concentration of CpG-ODNs using an in vitro assay for the superoxide anion $(O_2^-)$. CpG-ODNs induced $O_2^-$ and nitric oxide (NO) production, lysozyme activity, and the proinflammatory cytokine gene expression of $IL-1{\beta}$ and $TNF-{\alpha}$ in olive flounder significantly in vivo, whereas non-CpG-ODNs did not produce these effects or produced them to a lesser extent. This implied that CpG-ODNs could stimulate cellular and humoral immunity and cytokine gene expression in olive flounder. This is the first evidence of NO production and the first study on the mRNA expression of the proinflammatory cytokine genes $IL-1{\beta}$ and $TNF-{\alpha}$ in olive flounder in response to CpG-ODNs. Comparison of the variation in NO production and lysozyme activity to that of other studies led us to postulate that a group-specific difference exists in the immune responses of olive flounder against CpG-ODNs. Furthermore, the detailed immunostimulatory spectrum of CpG-ODNs in olive flounder could be a useful index with which to analyze the effect of CpG-ODNs against the challenge test prior to field applications.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.79-79
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• 1995

Although synthetic antisense oligodeoxynucleotides (ODNs) have been used to dissect gene function in vitro, technical difficulties of targeted delivery prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense transforming growth factor-${\beta}$l (TGF-${\beta}$1) oligonucleotides to decrease the fibrosis in the skin wound. Adult wounds heal with scar-tissue formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We reasoned that strategy emptying antisense TGF-${\beta}$1 ODNs complementary to TGF-${\beta}$1 mRNA might decrease the scarring in dermal wound of mouse. To evaluate this concept, we tested the effects of antisense ODNs targeted to TGF-${\beta}$1 mRNA by topical application of the chemical on the skin wound. Phosphorothioate antisense ODNs was employed to retard their degradation. When antisense TGF-${\beta}$1 ODNs were applied into the wound site, there was a maked reduction of scar compared with control wound site, These effects of antisense TGF-${\beta}$1 ODNs on the scar formation were associated with decreased expression of TGF-${\beta}$1 gene. However sense TGF-${\beta}$l ODNs had no effect on expression of TGF-${\beta}$1 gene. Also, control wounds healed with excessive fibrosis, whereas the antisense treated wounds healed with less fibrosis. In conclusion, our results indicate that antisense TGF-${\beta}$1 ODNs could be used for amelioating scar formation during wound healing.

• International Journal of Industrial Entomology and Biomaterials
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• v.8 no.1
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• pp.95-99
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• 2004

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides in particular base contexts are known to induce immunity in vertebrate cells. In insect, however, it was recent to find out that ODNs induces insect immunity as other immune inducer such as lipopolysaccharide. However, the finding was solely based on one lepidopteran insect, Bombyx mori, and the expression of insect immunity was neither dependent on numbers of CpG repeats nor methylation of CpG repeats within ODNs. Instead, foreignness of DNA has been suggested to be a key factor governing induction of antibacterial peptide. In this study, we expanded our previous understanding to the potentiality of ODNs as an immune inducer for antifungal peptide in Galleria mellonella and B. mori. To do this, a defensin-type antifungal peptide gene, reported from G. mellonella was cloned and partially sequenced from G. mellonella and B. mori successfully and utilized as a probe in the Northern blot analysis. We found out that ODNs also work as an immune inducer for antifungal peptide in the fat body and midgut of G. mellonella and B. mori larvae. Also, induction pattern of antifungal peptide was irrelevant to the numbers of CpG repeats within ODNs as previously reported on the induction pattern of antibacterial peptides.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1285-1288
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• 2008

The purpose of this study was to characterize and investigate the immune activity of CpG oligodeoxynucleotides (ODNs) from Bifidobacterium longum. Bacterial CpG motifs have attracted considerable interests because of their immunomodulatory activities. Genomic DNA from B. longum was prepared and amplified for 4 different 180-188-mer double-stranded ODNs (BLODN1-BLODN4). When immune cells (RAW 264.7 murine macrophages and JAWS II dendritic cells) with these ODNs were treated, BLODN4 induced the highest immune activity. To assess the effectiveness of the CpG sequences within BLODN4, single-stranded 40-mer ODNs containing CpG sequences (sBLODN4-1, sBLODN4-2) were synthesized. sBLODN4-1 induced higher level of cytokines such as interleukin (IL)-12p40 and tumor necrosis factor (TNF)-$\alpha$ by macrophage and IL-6 and TNF-$\alpha$ by dendritic cells than did sBLODN4-2. The results suggest that CpG ODNs-enriched components of B. longum might be useful as an immunomodulatory functional food ingredient.

• BMB Reports
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• v.39 no.6
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• pp.788-793
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• 2006

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express co-stimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-${\kappa}B$ activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-${\kappa}B$-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DF-ODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.

• Clinical and Experimental Pediatrics
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• v.45 no.7
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• pp.875-883
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• 2002

Purpose : Airways eosinophilia and increased IgE, characteristic features of asthma, result from a predominant Th2 response. In this study, we investigated the effect of CpG oligodeoxynucleotides (ODNs) on the inhibition of airways eosinophilia in mice with established airway inflammation. We also investigated the immunological mechanisms involved. Methods : Groups of BALB/c mice were sensitized intradermally with ovalbumin(OVA). At week 10, airway inflammation was induced by intranasal challenge of the mice with OVA. At week 14, the mice were challenged intranasally again with OVA in the presence and without the presence of CpG ODNs. Mice with saline administration served as negative controls. Bronchoalveolar lavage fluids(BALF) were obtained and eosinophils were counted. Th1 and Th2 cytokines in the spleen cell cultures were measured by ELISA. Serum OVA-specific IgE and IgG2a antibodies were also measured by ELISA. Results : BALF eosinophils were significantly inhibited in the CpG ODNs-treated mice(P<0.01). IgE and IgG2a levels increased significantly in both CpG ODNs-treated and untreated groups as compared to the negative control group; there was, however, no significant difference between the two groups four days after intranasal administration of CpG ODNs. Cytokine analysis revealed decreased production of IL-4, IL-5, and IL-13 and increased production of IL-12 in the CpG ODNs-treated group as compared to the untreated group. Interestingly, $IFN-{\gamma}$ levels were not upregulated in the CpG ODNs-treated group. Conclusion : CpG ODNs vaccination is a potentially useful approach for reversing airways eosinophilia in mice with established airways inflammation.

• Fisheries and Aquatic Sciences
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• v.6 no.3
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• pp.125-129
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• 2003

Effects of synthetic oligodeoxynucleotides (ODNs) containing cytidine-phosphate-guanosine(CpG) motif(s) on nonspecific immune responses of olive flounder (Paralichthys olivaceus) and on protection against lethal infection with Edwardsiella tarda were investigated. Respiratory bunt activities of the head kidney phagocytes in the fish injected either 0.25 or 0.5 ${\mu}g/fish$of ODNs containing CpG motifs (ODN 1826 and ODN 1670) were significantly higher than those injected with an ODN containing a guanosine-phosphate-cytidine (GpC) motif (ODN 1720) or with hanks balanced salt solution (HBSS, control) at 3, 5 and 7 days after injection. The serum lysozyme activities of fish injected with 0.25${\mu}g$ of ODN 1826 were significantly higher than those injected with ODN 1720 or HBSS at 1 and 7 days after injection. At 7 days after injection, the group of fish injected with CpG ODNs showed higher serum lysozyme activities than fish injected with ODN 1720 or control. The group of fish injected 0.25 or 0.5${\mu}g$ of CpG ODNs showed higher survival rates than those treated with GpC ODN and the control group after challenge with Edwardsiella tarda. The present study proved the ability of synthetic CpG ODN to increase nonspecific immune responses and disease resistance in olive flounder.

• Bulletin of the Korean Chemical Society
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• v.27 no.7
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• pp.1020-1024
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• 2006

In this report, we describe the synthesis of mono- and di-valent cationic $3\beta$ [L-Lysinamide-carbamoyl] cholesterol (K-Chol) derivatives by solid-phase peptide synthesis method and the characteristics of K-Chol/antisense oligodeoxynucleotide (ODN) complexes. K-Chol was able to interact with antisense ODNs electrostatically and constructed nanometer-sized complexes of 50-100 nm in diameter. The formation of K-Chol/antisense ODN complexes was demonstrated by non-denaturing polyacrylamide gel electrophoresis assay and atomic force microscopy. The cell-associated radioactivity was measured to monitor the cellular uptake of the complexes containing radioactive antisense ODNs using HL 60 cells.

• Microbiology and Biotechnology Letters
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• v.34 no.4
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• pp.329-334
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• 2006

Cationic liposome has been studied as one of the most promising non-viral gene delivery systems. In this report, we describe a new synthesized cholesterolic cationic lipid (2-aminoethylcarbamate-cholesterol) and dioleylphosphatidylethanolamine (DOPE) improve the cellular uptake properties of antisense ODNs, as well as plasmid DNA with low toxicity. This formulation of cholesterolic cationic lipid termed Chol-E, efficiently transfects ODNs and plasmids into many cell types in the presense or absence of 10% serum in the medium.