NF-κB Decoy Oligodeoxynucleotides Research Articles

Efficient Liposomal Nanocarrier‐mediated Oligodeoxynucleotide Delivery Involving Dual Use of a Cell‐Penetrating Peptide as a Packaging and Intracellular Delivery Agent

The effective use of oligonucleotide therapeutics, such as antisense oligodeoxynucleotides (ODNs) and small interfering RNAs (siRNAs), requires efficient delivery systems capable of intracellular penetration. Cell-penetrating peptides (CPPs), including arginine-rich peptides, have been extensively studied as tools for enhancing intracellular uptake efficiency of various bioactive molecules, including nanoparticles and liposomes. CPPs also have an ability to form tight complexes with nucleic acids, such as ODNs and siRNAs, making CPPs effective as packaging agents. Here, we constructed a CPP-modified liposome loaded with complexes of nona-arginine (9R) and NF-κB decoy ODNs, and evaluated intracellular uptake and anticancer activity in vitro. We found that 9R/ODN complexes were efficiently loaded into liposomes that were effectively internalized into U87MG glioblastoma cells and sensitized cells to the effects of paclitaxel. To the best of our knowledge, this is the first report describing the dual use of 9R CPP as a cell penetrating and a complexing agent within a single nanoparticle.

Suppression of Nuclear Factor-κB Activity in Kupffer Cells Protects Rat Liver Graft From Ischemia-Reperfusion Injury

Objective The objective of this study was to investigate the protective effect and mechanisms of nuclear factor (NF)-κB decoy oligodeoxynucleotides (ODN) on rat liver grafts following ischemia-reperfusion injury (IRI). Methods Animals were randomly divided into 3 groups (n = 8): control ischemia-reperfusion (IR) and decoy ODN groups; in the last cohort donor grafts were transfected with 120 μg NF-κB decoy ODN before procurement. Following 2 hours of reperfusion, NF-κB binding activity was detected in isolated Kupffer cells (KCs) using electrophoretic mobility shift assays (EMSA). Tumor necrosis factor (TNF)-α and interleukin (IL)-6 messenger RNA (mRNA) expressions were analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) methods. Liver tissue and blood serum were collected for histopathologic examination and liver function test, respectively. Results The NF-κB binding activity, TNF-α and IL-6 mRNA expression as well as serum ALT and total bilirubin levels were significantly increased compared with the control group following reperfusion ( P < .01). A large number of hepatocytes showed degeneration and necrosis. However, these indices were significantly ameliorated among the decoy ODN group ( P < .01) with preserved hepatic lobule architecture. Conclusion KCs NF-κB activation following reperfusion plays an important role in IRI after liver transplantation. The decoy strategy showed an apparent effect to suppress NF-κB activation and inhibit production of downstream cytokines, thereby protecting liver grafts from IRI.

New Treatment of Periodontal Diseases by Using NF-κB Decoy OligodeoxynucleotidesviaPrevention of Bone Resorption and Promotion of Wound Healing

Nuclear factor-kappa B (NF-kappaB) is involved in osteoclast differentiation and activation. Thus, the blockade of the NF-kappaB pathway might be a novel therapeutic strategy for treating bone metabolic diseases. Periodontitis is subgingival inflammation caused by bacterial infection; this disease also is thought to be a chronic focal point responsible for systemic diseases. In this study, NF-kappaB decoy oligodeoxynucleotides (ODNs) were topically applied for experimental periodontitis in a debris-accumulation model and wound healing in a bone-defect model of beagle dogs to investigate the effect of decoy ODN on bone metabolism. Application of NF-kappaB decoy ODN significantly reduced interleukin-6 activity in crevicular fluid and improved alveolar bone loss in the analysis of dental radiographs and DEXA. Direct measurement of exposed root that lost alveolar bone support revealed that NF-kappaB decoy treatment dramatically protected bone from loss. In a bone-defect model, NF-kappaB decoy ODN promoted the healing process as compared with control scrambled decoy in micro-CT analysis. Overall, inhibition of NF-kappaB by decoy strategy prevented the progression of bone loss in periodontitis and promoted the wound healing in bone defects through the inhibition of osteoclastic bone resorption. Targeting of NF-kappaB might be a potential therapy in various bone metabolic diseases.

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.

Reduced Collagen Biosynthesis Is the Hallmark of Cerebral Aneurysm

Reduced extracellular matrix is a prominent feature of cerebral aneurysms (CAs). We previously reported excessive ECM degradation in CA walls. In the present study, we examined collagen biosynthesis in CA walls and the molecular mechanisms underlying it in CA progression. RT-PCR and immunohistochemistry showed reduced expression of procollagen type I, III, and lysyl oxidase (LOX) in CA walls. Treatment with the LOX inhibitor beta-aminopropionitrile resulted in enhanced progression of CA. Expression of procollagen type I, III, and LOX was inhibited by interleukin-1beta (IL-1beta) in cultured rat aortic smooth muscle cells (RASMCs) in vitro. Nuclear factor kappa-B (NF-kappaB) was activated in IL-1beta-stimulated RASMCs, and treatment with NF-kappaB decoy oligodeoxynucleotides (ODN) restored reduced expression of procollagen type I, III, and LOX in vitro. NF-kappaB decoy ODNs ameliorated the expression of procollagen type I, III, and LOX in CA walls in vivo. Collagen biosynthesis was significantly inhibited at the transcriptional level and in the posttranscriptional enzymatic modification in CA walls through upregulated expression of IL-1beta and the NF-kappaB pathway. Reduced collagen biosynthesis may contribute to CA progression, and inhibition of this process may lead to the prevention of the progression and rupture of CAs.

Nanoparticle-Mediated Drug Delivery and Pulmonary Hypertension

Nanotechnology offers great potential benefits for drug delivery and therapy of respiratory and systemic diseases. Nanoparticles (NPs) have been of significant interest for some time because they can be designed to simultaneously carry a drug payload, specifically target features of diseased tissues, and carry an imaging molecule to track drug accumulation and clearance in tissues. Moreover, they can be engineered to tailor drug delivery and improve pharmacokinetics. A variety of NPs have been investigated in experimental animal models as tools to improve the delivery and therapeutic efficacy of drugs or genes delivered to the lung or other organ systems.1 The nanotechnology platform for drug delivery contains a number of very different types of nanostructures with widely varying properties. Examples of these NPs include dendrimers, fullerenes, carbon nanotubes, and polymeric NPs. In this issue of Hypertension , Kimura et al2 report that nuclear factor κB (NF-κB) decoy oligodeoxynucleotides (ODNs) encapsulated in poly-(ethylene glycol)- block -lactide/glycolide copolymer NPs and delivered to the lungs of rats by intratracheal instillation reduced pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). They showed that NP-encapsulated NF-κB decoy, visualized by fluorescein isothiocyanate labeling, reached the distal regions of the lungs and was present in alveolar macrophages and small pulmonary arteries for ≤14 days after a single instillation. The small pulmonary arteries were also found to be a site of NF-κB activation and NF-κB–dependent inflammatory cytokine production (monocyte chemoattractant protein 1, interleukin 1, and tumor necrosis factor α) in patients with PAH and in rats with MCT-induced PAH. The decoy ODNs, unlike antisense ODNs, which bind specific regions in mRNA, bind directly to the transcription factor and inhibit transcription factor binding to target DNA and initiation of gene transcription (Figure). It was speculated by the authors that cellular uptake of the NPs might slowly release encapsulated …

The Effects of Nuclear Factor-κB Decoy Oligodeoxynucleotide on Lipopolysaccharide-Induced Direct Acute Lung Injury

Background: The pathophysiologic mechanisms of early acute lung injury (ALI) differ according to the type of primary insult. It is important to differentiate between direct and indirect pathophysiologic pathways, and this may influence the approach to treatment strategies. NF-κB decoy oligodeoxynucleotide (ODN) is a useful tool for the blockade of the expression of NF-κB-dependent proinflammatory mediators and has been reported to be effective in indirect ALI. The purpose of this study was to investigate the effect of NF-κB decoy ODN in the lipopolysaccharide (LPS)-induced direct ALI model. Methods: Five-week-old specific pathogen-free male BALB/c mice were used for the experiment. In the preliminary studies, tumor necrosis factor (TNF)-α, interleukine (IL)-6 and NF-κB activity peaked at 6 hours after LPS administration. Myeloperoxidase (MPO) activity and ALI score were highest at 36 and 48 hours, respectively. Therefore, it was decided to measure each parameter at the time of its highest level. The study mice were randomly divided into three experimental groups: (1) control group which was administered 50 μL of saline and treated with intratracheal administration of 200 μL DW containing only hemagglutinating virus of Japan (HVJ) vector (n=24); (2) LPS group in which LPS-induced ALI mice were treated with intratracheal administration of 200 μL DW containing only HVJ vector (n=24); (3) LPS＋ODN group in which LPS-induced ALI mice were treated with intratracheal administration of 200 μL DW containing 160 μg of NF-κB decoy ODN and HVJ vector (n=24). Each group was subdivided into four experimental subgroups: (1) tissue subgroup for histopathological examination for ALI at 48 hours (n=6); (2) 6-hour bronchoalveolar lavage (BAL) subgroup for measurement of TNF-α and IL-6 in BAL fluid (BALF) (n=6); (3) 36-hour BAL subgroup for MPO activity assays in BALF (n=6); and (4) tissue homogenate subgroup for measurement of NF-κB activity in lung tissue homogenates at 6 hours (n=6). Results: NF-κB decoy ODN treatment significantly decreased NF-κB activity in lung tissues. However, it failed to improve the parameters of LPS-induced direct ALI, including the concentrations of tumor necrosis factor-α and interleukin-6 in BALF, myeloperoxidase activity in BALF and histopathologic changes measured by the ALI score. Conclusion: NF-κB decoy ODN, which has been proven to be effective in indirect models, had no effect in the direct ALI model.

Transcriptional regulation of NF-κB by ring type decoy oligodeoxynucleotide in an animal model of nephropathy

Inflammation of the tubulointerstitial compartment, leading to fibrosis, is a major factor in the progressive loss of renal function in a wide variety of kidney diseases. In order to develop a therapeutic approach for nephropathy, we examined the simultaneous inhibition of transcription factor nuclear factor-κB (NF-κB), which is responsible for a wide range of cellular processes, especially inflammation, in a mouse model of unilateral ureteral obstruction. In this study, we employed a ring-type NF-κB (R-NF-κB) decoy oligodeoxynucleotide (ODN), containing consensus promoter sequences of NF-κB. This R-NF-κB decoy ODN is more highly resistant to degradation by nucleases than is the current phosphothiolated double stranded NF-κB decoy ODN. The inhibitory effect of R-NF-κB decoy ODN on nephropathy was confirmed by molecular and histological examinations. In addition, treatment with R-NF-κB decoy ODN reduced the activities of inflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. Interestingly, the treatment with R-NF-κB decoy ODN also suppressed the gene expression of transforming growth factor-β1 and fibronectin, resulting in the inhibition of fibrotic changes. These results suggest that the inhibition of NF-κB using R-NF-κB decoy ODN has potential therapeutic application in the prevention of renal fibrosis.

Decoy oligodeoxynucleotide targeting activator protein-1 (AP-1) attenuates intestinal inflammation in murine experimental colitis

Various therapies are used for inflammatory bowel diseases (IBD), though none seem to be extremely effective. AP-1 is a major transcription factor that upregulates genes involved in immune and proinflammatory responses. We investigated decoy oligodeoxynucleotide (ODN) targeting AP-1 to prevent dextran sulfate sodium (DSS)-induced colitis in mice. Functional efficacies of synthetic decoy and scrambled ODNs were evaluated in vitro by a reporter gene luciferase assay and measuring flagellin-induced IL-8 expression by HCT-15 cells transfected with ODNs. Experimental colitis was induced in mice with a 2.5% DSS solution in drinking water for 7 days, and decoy or scrambled ODNs were intraperitoneally injected from days 2 to 5. Colitis was assessed by weight loss, colon length, histopathology, and detection of myeloperoxidase (MPO), IL-1β, and TNF-α in colon tissue. Therapeutic effects of AP-1 and NF-κB decoy ODNs were compared. Transfection of AP-1 decoy ODN inhibited AP-1 transcriptional activity in reporter assays and flagellin-induced IL-8 production in vitro. In mice, AP-1 decoy ODN, but not scrambled ODN, significantly inhibited weight loss, colon shortening, and histological inflammation induced by DSS. Further, AP-1 decoy ODN decreased MPO, IL-1β, and TNF-α in colonic tissue of mice with DSS-induced colitis. The AP-1 decoy therapeutic effect was comparable to that of NF-κB decoy ODN, which also significantly decreased intestinal inflammation. Double-strand decoy ODN targeting AP-1 effectively attenuated intestinal inflammation associated with experimental colitis in mice, indicating the potential of targeting proinflammatory transcription factors in new therapies for IBD.

Induction of regulatory dendritic cells by topical application of NF-κB decoy oligodeoxynucleotides

Dendritic cells (DC) have a key role in inducing an immune response, but DC in different maturation states are responsible for inducing tolerance. Topical application of nuclear factor (NF)-κB decoy oligodeoxynucleotides (ODN) induces antigen-specific peripheral tolerance in delayed-type hypersensitivity (DTH) to ovalbumin (OVA) by expanding CD4 +CD25 + regulatory T cells and by inhibiting DC migration. Herein we describe how topical NF-κB decoy ODN modulate DC maturation with respect to their migration, phenotype, and cytokine profiles. Topical application of NF-κB decoy ODN after OVA sensitization delayed the migration of Langerhans cells (LC) into draining lymph nodes, and morphologically mature LC remained in the peripheral tissue 2 days longer than in OVA-sensitized mice without application of NF-κB decoy ODN. During migration, NF-κB decoy-treated DC preferentially expressed inhibitory B7 molecules (i.e., B7-H1, B7-DC, and B7-H3) compared to OVA-sensitized DC without NF-κB decoy ODN, whereas co-stimulatory molecules (MHC class II, B7-1 and B7-2) were upregulated. Adoptive transfer of NF-κB decoy-treated DC inhibited DTH induction in prophylactic and therapeutic experiments. Inhibition of DTH by DC transfer was antigen-specific in vivo. This decoy ODN strategy might be useful for regulating immunity through DC.

Long‐term follow up of initial clinical cases with NF‐κB decoy oligodeoxynucleotide transfection at the site of coronary stenting

An essential transcription factor for inflammation, nuclear factor-kappa B (NF-kappaB), plays a pivotal role for restenosis after percutaneous coronary intervention (PCI). To evaluate the safety and effectiveness of NF-kappaB decoy oligodeoxynucleotides (ODN) to prevent restenosis, we initiated an open-label phase I/IIa clinical trial. Seventeen patients who were suffering from angina with organic coronary stenosis were treated with NF-kappaB decoy ODN after PCI using bare metal stents. Although the average coronary stenosis was 90.8 +/- 7.0% before the stent implantation, the stenosis improved to 1.4 +/- 5.9% after the intervention. Serum MCP-1 levels were significantly suppressed in NF-kappaB decoy ODN treated patients compared to those in non-treated patients on day 3 after the PCI. Ticlopidine treatment was employed, since clopidogrel was not launched in Japan. Six months after the PCI and decoy ODN transfection, significant restenosis was found in only 1 of the 17 patients, and the average restenosis rate was 39.6 +/- 22.3%. No in-stent thrombosis was found and no significant systemic adverse effect occurred in any of the patients in this observation period. These results suggest the clinical usefulness and safety of the NF-kappaB decoy ODN transfer after PCI, although further placebo control trials are necessary.

NF-κB Is a Key Mediator of Cerebral Aneurysm Formation

Subarachnoid hemorrhage caused by the rupture of cerebral aneurysm (CA) remains a life-threatening disease despite recent diagnostic and therapeutic advancements. Recent studies strongly suggest the active participation of macrophage-mediated chronic inflammatory response in the pathogenesis of CA. We examined the role of nuclear factor-kappaB (NF-kappaB) in the pathogenesis of CA formation in this study. In experimentally induced CAs in rats, NF-kappaB was activated in cerebral arterial walls in the early stage of aneurysm formation with upregulated expression of downstream genes. NF-kappaB p50 subunit-deficient mice showed a decreased incidence of CA formation with less macrophage infiltration into the arterial wall. NF-kappaB decoy oligodeoxynucleotide also prevented CA formation when it was administered at the early stage of aneurysm formation in rats. Macrophage infiltration and expression of downstream genes were dramatically inhibited by NF-kappaB decoy oligodeoxynucleotide. In human CA walls, NF-kappaB also was activated, especially in the intima. Our data indicate that NF-kappaB plays a crucial role as a key regulator in the initiation of CA development by inducing some inflammatory genes related to macrophage recruitment and activation. NF-kappaB may represent a therapeutic target of a novel medical treatment for CA.

Role of platelet activating factor in pathogenesis of acute respiratory distress syndrome

Role of platelet activating factor in pathogenesis of acute respiratory distress syndrome

High glucose enhances MMP-2 production in adventitial fibroblasts via Akt1-dependent NF-κB pathway

To understand the role of adventitial fibroblasts (AF) in diabetic vascular diseases, the importance of high glucose (HG, 25mM) on matrix metalloproteinase-2 (MMP-2) production in AF was determined. HG enhanced mRNA, protein and gelatinolytic activity of MMP-2. The enhanced MMP-2 activity was significantly attenuated not only by a PI3K inhibitor but also by an Akt inhibitor. These HG-induced MMP-2 responses were markedly reduced in Akt1-deficient (1KO) cells. The diminished HG-induced MMP-2 responses were completely restored by re-expression of Akt1. Both the reporter activity and electrophoretic mobility shift assay for activator protein-1 and nuclear factor-kappa B (NF-κB) were enhanced by HG, but NF-κB were not increased in 1KO cells. Furthermore, HG-induced MMP-2 responses were markedly suppressed by NF-κB decoy oligodeoxynucleotides. Based on these results, it is suggested that HG augments MMP-2 production via PI3K/Akt1/NF-κB pathway.

Inhibitory effects of NFκB decoy oligodeoxynucleotides on neointimal hyperplasia in a rabbit vein graft model

Autologous vein remains the commonly used conduit for bypass grafts; however, neointimal hyperplasia is known to be one of the major disease processes in vein graft failure. In this study, we focused on the important role of NFκB which controls the expression of numerous genes for various cytokines and adhesion molecules in the mechanism of graft failure. Thus, we investigated the inhibitory effect of NFκB decoy oligodeoxynucleotides (ODN) on vein graft failure in a rabbit hypercholesterolemic model. Jugular vein to carotid artery interposition grafts in rabbits were transfected intraoperatively with NFκB decoy ODN (40 μmol/l) by ex-vivo pressure-mediated transfection (300 mm Hg, 10 min). Treatment with NFκB decoy ODN significantly suppressed intimal hyperplasia 4 weeks after vein implantation as compared to scrambled decoy ODN, and increased medial thickness, leading to a significant reduction in intima-to-media ratio. Treatment with NFκB decoy ODN significantly inhibited the recruitment of macrophages and the proliferation of vascular smooth muscle cells (VSMC), while apoptosis in VSMC was significantly increased by NFκB decoy ODN. In addition, a study of vascular reactivity demonstrated that transfection of grafts with NFκB decoy ODN significantly improved endothelium-mediated vasorelaxation as compared to scrambled decoy ODN. Here, we demonstrated that inhibition of NFκB activation using decoy ODN inhibited the development of neointimal hyperplasia, followed by suppression of inflammatory changes and accumulation of VSMC in the neointima of rabbit vein grafts. The present study raises the possibility of a novel strategy for prevention of graft failure.

NFκB decoy oligodeoxynucleotides ameliorates osteoporosis through inhibition of activation and differentiation of osteoclasts

The transcription factor, nuclear factor-kappa B (NFkappaB), is believed to play a pivotal role in osteoclast formation. In this study, we focused on NFkappaB decoy oligodeoxynucleotides (ODN) as a new therapeutic strategy to attenuate osteoporosis. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts formed in mononuclear cells including osteoclast precursors from neonatal rabbit bone marrow were increased in the presence of 1,25-dihydroxyvitamin D3, whereas transfection of NFkappaB decoy ODN decreased the number of TRAP-positive cells and attenuated RANKL and M-CSF-induced osteoclast formation. NFkappaB decoy ODN also inhibited the activity of osteoclasts, as assessed by pit formation. In rat ovariectomized model of estrogen deficiency, continuous administration of NFkappaB decoy ODN attenuated the increase of TRAP activity, accompanied by a significant increase in calcium concentration in tibia and femur and decrease in urinary deoxypyridinoline. In additional osteoporosis model using vitamin C-deficient rat, inhibition of NFkappaB by decoy ODN dramatically improved the bone length, weight, density as assessed by dual-energy X-ray absorptiometry. Overall, inhibition of NFkappaB by decoy strategy prevented osteoporosis through the inhibition of bone resorption. Targeting of NFkappaB might be potential therapy in various bone metabolic diseases.

Inhibitory effect of ribbon-type NF-κB decoy oligodeoxynucleotides on osteoclast induction and activity in vitro and in vivo

In this study we examined the effect of ribbon-type (circular-type) NF-κB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-κB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-κB were both greater than those of phosphorothionated NF-κB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.

Antigen-Specific Peripheral Tolerance Induced by Topical Application of NF-κB Decoy Oligodeoxynucleotide

Activation and maturation of dendritic cells (DC) are crucial for the establishment of delayed-type hypersensitivity (DTH). However, antigen presentation by immature DC (iDC) might lead to antigen-specific peripheral tolerance. NF-kappaB plays significant roles in upregulation of co-stimulatory molecules and cytokines in DC and therefore we investigated whether NF-kappaB decoy oligodeoxynucleotide (ODN) might induce tolerance to DTH. NF-kappaB decoy ODN suppressed ovalbumin (OVA)-induced DTH responses not only in naïve but also in presensitized mice. The suppressive effect was found to be antigen-specific. NF-kappaB decoy ODN-induced tolerance involved CD4(+)CD25(+) regulatory T cells (Treg), because in vivo depletion of CD25(+) T cells abrogated the tolerance, whereas adoptive transfer of such T cell population from tolerant mice induced tolerance. Furthermore, the induction of Treg was related to insufficient migration and/or maturation of DC, because a sizable DC population still remained in peripheral tissue even after exposure to exogenous antigen in NF-kappaB decoy ODN-treated mice. Even if they migrated into lymph nodes, they showed insufficient upregulation of co-stimulatory molecules and impaired antigen-specific activation of T cells. Topical application of NF-kappaB decoy ODN might thus be a new approach to induce antigen-specific peripheral tolerance.

Treatment of murine Th1- and Th2-mediated inflammatory bowel disease with NF- B decoy oligonucleotides

The Th1 and Th2 T cell responses that underlie inflammatory bowel diseases (IBDs) are likely to depend on NF-kappaB transcriptional activity. We explored this possibility in studies in which we determined the capacity of NF-kappaB decoy oligodeoxynucleotides (decoy ODNs) to treat various murine models of IBD. In initial studies, we showed that i.r. (intrarectal) or i.p. administration of decoy ODNs encapsulated in a viral envelope prevented and treated a model of acute trinitrobenzene sulfonic acid-induced (TNBS-induced) colitis, as assessed by clinical course and effect on Th1 cytokine production. In further studies, we showed that NF-kappaB decoy ODNs were also an effective treatment of a model of chronic TNBS-colitis, inhibiting both the production of IL-23/IL-17 and the development of fibrosis that characterizes this model. Treatment of TNBS-induced inflammation by i.r. administration of NF-kappaB decoy ODNs did not inhibit NF-kappaB in extraintestinal organs and resulted in CD4+ T cell apoptosis, suggesting that such treatment is highly focused and durable. Finally, we showed that NF-kappaB decoy ODNs also prevented and treated oxazolone-colitis and thus affect a Th2-mediated inflammatory process. In each case, decoy administration led to inflammation-clearing effects, suggesting a therapeutic potency applicable to human IBD.

P89. The intra-discal injection of naked NFκB decoy oligonucleotide is effective in restoring disc degeneration in the rabbit annular needle puncture model

BACKGROUND CONTEXT: The transcription factor, nuclear factor kappaB (NFκB), is a heterodimeric DNA-binding protein that plays a critical role in the regulation of pro-inflammatory mediators, such as IL-1 and TNF, which might be involved in the pathology of degenerated disc disease. Recently, the transfection of decoy oligodeoxynucleotide (ODN) containing the NFκB cis element has been reported to be a potential therapeutic approach for inflammatory diseases. The application of naked NFκB decoy ODN for intervertebral disc (IVD) degeneration has not been examined.