1998 Fiscal Year Final Research Report Summary
Production of recombinant autoantibodies
| Project/Area Number |
08557150
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Laboratory medicine
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
AKAMIZU Takashi Kyoto Univ.Grad Sch of Med, Assistant, 医学研究科, 助手 (20231813)
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| Co-Investigator(Kenkyū-buntansha) |
KANDA Heditoshi Eiken Kagaku, Dept of Immunol, Researcher, 免疫センター, 研究員
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| Project Period (FY) |
1996 – 1998
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| Keywords | Immunoglobuilin gene / TSH receptor / anti-TSH receptor antibody / Graves*f disease / recombinant antibody |
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| Research Abstract |
[Purpose] 1)Anti-thyrotropin receptor autoantibodies are known to be involved in Graves' disease. To elucidate the molecular mechanism of the pathogenesis of Graves' disease, we previously isolated and reconstituted the immunoglobulin genes of two B cell clones producing a monoclonal thyroid stimulating antibody (TSAb), a stimulating type of TSHRAb, obtained from patients with Graves' disease. In the present study, we produced a large amount of recombinant monoclonal TSAbs in eukariotic cells using these genes and applied them to several further investigations 2) We made a DNA construct for transgenic mouse bearing anti-TSH .receptor antibody genes.
[Method] 1) We tried to identify their epitopes in the TSHR by using a panel of mutants of the extracellular domain of TSHR.Purified antibodies were radiolabeled and tested for binding to cells expressing high levels of TSHR.2) We tried to isolate heavy and light chain genes containing their promotors and 3'-enhancers from genome DNAs of B c
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ell clones producing monoclonal anti-TSH receptor antibodies by the PCR amplification system. 3) We fried to identify dinucleotide repeat polymorphism near the TSHR gene and performed association studies between genetic markers and Japanese patients with autoimmune thyroid disease (AITD).
[Results] 1) Mutations in the N-terminal but not C-terminal region of the extracellular domain of TSHR abrogated or reduced TSAb activities of both antibodies, while they had opposite effects on TSH activity. Although their affinities were lower than that of TSH, their bindings were not displaced by TSH.The antibody bindings were not mutually competitive, either. 2) We isolated heavy and light chain genes containing their promotors and 3'-enhancers from genome DNAs of B cell clones producing monoclonal anti-TSH receptor antibodies by the PCR amplification system. They were legated in tandem.
[Conclusion] 1) These findings indicate that these antibodies interact with the multiple conformational arrays of receptor determinants in the N-terminus and transduce a signal through binding sites different from TSH.2) A DNA construct for transgenic mouse bearing anti-TSH receptor antibody genes was preliminary prepared. Less
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[Publications] Niyamoto Y, Saito Y, Kajiyama N, Yoshimura M, Shimasaki Y, Nakamura M, Kamitani S, Harada M, Ishikawa M, Kawahara K, Ogawa E Hamanaka I.Takahashi N, Kaneshige T, Teraoka H, Akamizu T, Azuma N, Yoshimasa Y, Yoshimasa T, Itoh H, Masuda I, Yasue H, Kanao K,: "Endothelial nitric oxide synthase gene is positively associated with essential hypertension." Hypertension. 32. 3-8 (1998)
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「研究成果報告書概要(和文)」より
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[Publications] Akamizu.M, M.Sale, H.Hiratani, M.Miura, M.Saijos, K.Moriyama, J.Y.Noh, S.S.Rich, D.W.Bowdon, K.Nakao.: "Association of autoimmune thyroid disease with microsatellite markers for the thyrotropin receptor gene and CTLA-4 in a new set of Japanese population." Proceedings of The 7Ith meeting of the American Thyroid Association, Portland, Oregon, (abstract). 80 (1998)
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「研究成果報告書概要(和文)」より
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[Publications] Akamizu M, M.Sale, H.Hiratani, M.Miura, M.Saijo, K.Moriyama, J.Y.Noh, S.S.Rieh, D.W.Bowden, K.Nakao: "CTLA-4 in nssociated with autoimmune thyroid disease in a Japanese Population : some alleles mayhave protective effects while others result in disease suseptibility." Proceedings of The second Japan-Korea-China Thyroid Conference, Tokyo, Japan, (abstract). 309 (1998)
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「研究成果報告書概要(和文)」より
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