Transcription factor decoy oligodeoxynucleotides preclude Th2 cytokines expression and airways hyperreactivity in experimental asthma
| Project/Area Number |
16590759
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Respiratory organ internal medicine
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| Research Institution | Iwate Medical University |
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Principal Investigator |
NAKAMURA Yutaka Iwate Medical University, School of Medicine, Lecturer, 医学部, 講師 (60328614)
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| Co-Investigator(Kenkyū-buntansha) |
YAMUCHI Kohei Iwate Medical University, Associate professor, 医学部, 助教授 (20200579)
INOUE Hiroshi Iwate Medical University, Professor, 医学部, 教授 (40133962)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Bronchial asthma / gene therapy / decoy oligodeoxynucleotides / transcription factor / GATA-3 / NF-AT / Th2-type cytokine / HVJ-envelope / decoy oligodeoxynuclotides / CD3陽性T細胞 / decoy oligodeoxynucleotides / IL-5 / 好酸球 / CD3陽性Tリンパ球 |
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| Research Abstract |
2005 : RATIONALE : Transcription factor GATA-3 plays a significant role in the coordinated transactivation of Th2 cytokines. We hypothesized that synthetic ribbon-type double-stranded DNA with high affinity for GATA-3 may be introduced as a "decoy" cis element to bind the transcription factor, and block gene activation, resulting in an effective therapeutic agent for treating bronchial asthma.
METHODS : Primary cultures of freshly isolated CD3^+ T cells were stimulated with CD3 and CD28 after transfection of GATA-3 decoy oligodeoxynucleotides (ODNs). We employed electrophoretic mobility shift assay (EMSA) to determine whether ODNs specifically inhibit the DNA-binding activity of GATA-3. Culture supernatants were analyzed for Th1 and Th2 cytokines concentrations by ELISA. mRNA expression in CD3^+T cells was determined by RT-PCR. RESULTS : Transfection of GATA-3 decoy ODNs significantly attenuated GATA-3 DNA-binding activity in CD3^+ T cells (p<0.01). Notably, GATA-3 decoy ODNs led to a s
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ignificant reduction in IL-5 production of CD3^+ T cells under conditions of CD3 and CD28 stimulation (p<0.01). CONCLUSIONS : These data indicate a critical role for GATA-3 in the effector phase of IL-5 related inflammation and suggest that GATA-3 decoy ODNs may be a novel approach for the treatment of allergic inflammation such as in asthma.
2006 : GATA-3 decoy oligodeoxynucleotides could not suppress the airway hyperreactivity in experimental asthma, therefore we used NF-AT decoy oligodeoxynucleotides. RATIONALE : NF-ATc1 is required for the differentiation of Th2 responses. We examined the role of NF-AT in the mouse experimental asthma and tested the hypothesis that NF-AT blockade with a decoy against the cis element of NF-AT can prevent the progression of asthma. METHODS : To determine the effects of NF-AT decoy on the development of airway inflammation and airway hyperreactivity (AHR), we adoptively transferred NF-AT transfected T cells into mice. Ovalbumin (OVA)-sensitized 1x10^7 T cells were derived from spleen of the mice that had been immunized with OVA. Previously, we designed a novel ribbon-type oligodeoxynucleotides containing two binding sites for NF-AT in a single decoy molecule without an open end, which was more stable than conventional decoy, and largely preserving its structural integrity in the presence of nucleuses and sera. Ribbon-type NF-AT decoy (Ri NF-AT) was transfected into the sensitized 1x10^7 T cells in vitro. OVA-immunized mice received these Ri NF-AT transfected OVA-specific T cells by intraperitoneal injection. Twenty four hours after the last challenge, AHR was measured and transfected T cell responses to the airways were characterized. RESULTS : Transfer of Ri NF-AT transfected T cells significantly reduce the number of eosinophils and the concentration of IL4, IL-5. and IL-13 induced by control group in BAL fluid of OVA-treated mice. Transfer of Ri NF-AT did not induce AHR to methacholine. CONCLUSIONS : We found that NF-AT is a key regulator in the progression of asthma and that in vivo transfection of NF-AT decoy precludes Th2 inflammation and AHR in experimental asthma. Less
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Report
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Research Products
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