1996 Fiscal Year Annual Research Report

レーザー・蛍光色素法による細胞の可視化計測とエイズ免疫,癌診断,治療への応用

Research Project

Project/Area Number	06044027
Research Institution	Ibaraki University
Principal Investigator	藤井寛一茨城大学, 工学部, 教授 (00054354)
Co-Investigator(Kenkyū-buntansha)	WHINNERY J.R カルフォルニア大学, 工学部, 教授 GUSTAFSON T. カルフォルニア大学, 工学部, 教授 AMANO M. スタンフォード大学, 医学部, 助手 TOWNS C.H. カルフォルニア大学, 理学部, 教授 MIROV S. アラバマ大学, 病理学部, 教助授 SCHAWLOW A.L スタンフォード大学, 病理学部, 教授 MCCUTCHEON M アラバマ大学, 工学部, 教授 MCGHEE J.R. アラバマ大学, 免疫ワクチンセンター, 所長教授石川悟日立製作所日立総合病院, 泌尿器科主任医長岡裕爾日立製作所日立総合病院, 副院長 (20160658) 妻鹿純一日本大学, 松戸歯学部, 講師 (40190946) 亀丸俊一北見工業大学, 工学部, 教授 (60175289) 清野宏大阪大学, 微生物病研究所, 教授 (10271032) 久保田俊夫茨城大学, 工学部, 講師 (40143143) 辻龍助茨城大学, 工学部, 教助授 (40188537) 白石昌武茨城大学, 工学部, 教授 (10091860)
Keywords	ILISPOT / 免疫検査法 / サイトカイン / IDIP / 自動化 / パターン認識 / レーザー / 蛍光色素
Research Abstract	本研究は茨城大学工学部・アラバマ大学バ-ミングハム校・スタンフォード大学医学部を中核として,平成6年度に3年計画で開始した。当初の研究計画に記したように本研究は,平成2年度から4年度にかけて実施された大学間協力研究の成果を基に新しい免疫検査システムの構築と実用化を目指して逐次検討を進めた。総合的に判断して,当初の目的はおおむね達成できたと判断しているが,最終目標であるILISPOT-IDIP法の完全自動化と実用化には今一歩およばなかった。以下に,具体的に進捗状況および成果をまとめる。 1.ILISPOT-IDIP法については,ハード部分のコンピュータによる自動化を目標として検討を進めた。その結果,顕微鏡試料台の自動アドレッシング制御を達成した。ソフト的には画像処理系が未だに半自動であるが,アドレッシング制御用ソフトと画像処理系との連携も達成している。さらに,画像処理系では擬似スポットと真スポットとを自動的に判別することも可能となり,システム全体の完成度はかなり高いレベルに到達していると判断している。 2.ILISPOT-IDIP法用の新しい光源として,広帯域カラーセンターレーザーは非常に有力であるが,現在の装置は大型であるため,装置の小型化が実用化の鍵となると判断し,波長選択技術とともに実用化に向けた検討を行った。しかし,水銀ランプを使用した現行のILISPOT-IDIP法の確立と実用化が先決であるとの判断から,波長選択技術と広帯域カラーセンターレーザーのそれぞれの基礎技術の確立につとめそれぞれの分野で大きな成果を達成した。これらの成果のILISPPT-IDIP法への適用については今後検討を続行することになる。 3.ILISPOT-IDIP法用蛍光色素として,脂溶性,細胞膜透過性,水溶性,強い蛍光強度などの特性を満足するものが望まれていたが,そのような色素として,含フッ素色素が期待された。含フッ素色素の合成を検討し,三種類の新規な含フッ素BODIPY系蛍光色素の合成に成功した。この成果については,日米化学会の合同シンポジウムで公表された。ILISPOT-IDIP法への適用に関しては抗体やDNAとの結合法など染色プロトコールについて解決すべき問題点を残す結果となった。 4.免疫系研究担当者は,ILISPOT-IDIP法を用いた極微量タンパクであるサイトカイン類の定性・定量の概念の構築を行い,エイズや癌の免疫学への応用を実装置で検証する段階に到達している。

Research Products
(30 results)

All Other

All Publications (30 results)

[Publications] 藤井寛一: "Mode Control of Semiconductor Laser with Diffraction and Dispersion Feedback" Procceeding of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 849-853 (1995)
[Publications] 藤井寛一: "Pulse Broadning and of Copper Vapor in Copper-Halide Lasing System" Procceeding of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 872-877 (1995)
[Publications] 藤井寛一: "Enhancement of Population Inversion by Effective Use of Potential Energy in self-Termination Laser" Procceedin of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 878-882 (1995)
[Publications] 藤井寛一: "Comparison of A. C. Driven Barrier Discharge with D. C. Driven Malter Effect Corona Discharges for Plasma Reactor" Procceeding of 11th International Conference on Gas Discharges and Their Applications. 1. 164-167 (1995)
[Publications] 藤井寛一: "A Proposal of Modifying the Quittner Method for Gamma-Ray Peak Area Determination" Radio Isotopes. 45. 92-94 (1996)
[Publications] Takahashi,I.,Nakagawa,I.,Kiyono,H.,McGhee,J.R.,Clements,J.D.,and Hamada,S.: "Mucosal T cells induce systemic anergy for oral tolerance" Biochem. Biophys. Res. Commun.296. 414-420 (1995)
[Publications] Beagley,K.W.,Yamamoto,M.,McGhee,J.R.and Kiyono,H.: "Differences in intraepithelial lymphocyte T cell subsets isolated from murine samll versus large intestine" J.Immunol.154. 5611-5619 (1995)
[Publications] Hiroi,T.,Fujihashi,K.,McGhee,J.R. and Kiyono,H.: "Polarized Th2 cytokine expression by both mucosal γδ and αβ T cells." Eur.J.Immunol.25. 2743-2751 (1995)
[Publications] Bukawa.H.,Sekigawa,K-I.,Hamajima,K.,Fukuoka,J.,Yamada,Y.,Kiyono,H.and Okuda,K.: "Neutralization of HIV-1 secretory IgA induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate." Nature Medicine. 1. 681-685 (1995)
[Publications] Marinaro,M.,Staats,H.F.,Hiroi T.,Kiyono,H.,Jackson,R.J.,Coste,M. and McGhee,J.R.: "The Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4 whith supports IgE responses" J.Immunol.155 : 4621-4629.155. 4621-4629 (1995)
[Publications] Takahashi,I.,Kiyono,H.,Marinaro,M.,Jackson,R.J.,Nakagawa,I.,Fujihashi,K.,Hamada,S.,Clements,Bost,K.L., and McGhee,J.R.: "Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin" J.Infect.Dis.173. 627-635 (1996)
[Publications] Fujihashi,K.,Yamamoto,M.,Hiroi,T.,McGhee,J.R.,and Kiyono,H.: "Selected Th1 and Th2 cyotkine mRNA expression by CD4^+ T cells isolated from inflamed human gingival tissues" Clin.Exp.Immunol.103. 422-428 (1996)
[Publications] Takahashi,I.,Kiyono,H.,Jackson,R.J.,Fujihashi,K.,Stats,H.F.,Hamada,S.,Clements,J.D.,Bost,K.L.,and McGhee,J.R.: "Epitope maps of Escherichia coli labile toxin B subunit for development of a synthetic oral vaccine." Infect.Immune.64. 1290-1298 (1996)
[Publications] VanCott,J.L.,Kobayashi,T.,Pillai,S.,McGhee,J.R.,and Kiyono,H.: "Induction of polysaccharide specific mucosal immune responses by oral immunization" Vaccine. 14. 392-398 (1996)
[Publications] Yamamoto,M.,Kawabata,K.,Fujihashi,K.,McGhee,J.R.,Van Dyke,T.E.,Bamberg,T.V.and Kiyono,H.: "Lach of IL-4 contributes to persistence of IL-4 receptor positive macrophage in inflamed gingival tissues." Am.J.Pathol.148. 331-339 (1996)
[Publications] Vancott,J.L.,Staats,H.F.,Roberts,M.,Chatfield,S.N.,Hiroi,T.,Yamamoto,M.,Carter,P.B.,Kiyono,H. and McGhee,J.R.: "Regulation of mucosal and systemic antibody responses by T helper subsets,macrophages and derived cytokines following oral immunization with live recombinant Salmonella" J.Immunol.156. 1504-1514 (1996)
[Publications] Okahashi,N.,Yamanoto.M.Vancott,J.L.,Charfield,S.N.,Roberts,M.,Bluethmann,H.,Hiroi,T.,Kiyono,H.and McGhee,J.R.: "Mucosal immunity in IL-4 knockout mice : oral administration of recombinant Salmonella or cholea toxin elicits CD4^+ Th2 cells producing IL-6 and IL-10 and IgA responses" Infect. Immun.64. 1516-1525 (1996)
[Publications] Jackson,R.J.,Fujihashi,K.,Kiyono,H.and McGhee,J.R.: "Luminometry : a novel bioluminescent method enhances the quantitation of mucosal and systemic antibody responses" J.Immunol.Methods. 190. 189-197 (1996)
[Publications] Fujihashi,K.,Kawabata,S.,Hiroi,T.,Yamamoto,M.,McGhee,J.R.,Nishikawa,S-I.and Kiyono,H.: "IL-2 and IL-7 reciprocally induce IL-7 and IL-2 receptors on γδ TCR^+ intraepithelial lymphocytes." Proc.Natl.Acad.Sci.USA. 93. 3613-3618 (1996)
[Publications] Fujihashi,K.,McGhee,J.R.,Kweon,M-N,Cooper,M.D.,Tonegawa,S.,Takahashi,I.,Hiroi,T.,Mestechy,J. and Kiyono,H.: "γδ T cell deficient mice have imparied mucosal IgA responses" J.Exp.Med.183. 1929-1935 (1996)
[Publications] Elson,C.O.,Beagley,K.W.,Sharmanov,A.T.,Kiyono,H.,Tennyson,G.S.,Ridwan,B.,Fujihashi,K. and McGhee,J.R.: "Hapten-induced model of murine IBD : mucosal immune responses and protection by tolerance." J.Immunol.157 : 2174-2185.157. 2174-2185 (1996)
[Publications] Nakagawa,I.,Takahashi,I.,Kiyono,H.,McGhee,J.R. and Hamada,S.: "Oral Immunization with the B subnit of the heat-labile entretotoxin of Escherichia coil induces early Th1 and Th2 cytokines expression in Peyer's patches." J.Infect.Dis.173. 1426-1428 (1996)
[Publications] Jackson,R.J.,Marinaro,M.,VanCott,J.L.,Yamamoto,M.,Okahashi,N.,Fujihashi,K.,Kiyono,H.,Chatfield,S.N.,and McGhee,J.R.: "Mucosal immunity : regulation by helper T cells and a novel method for datection" J.Biotech.44. 209-216 (1996)
[Publications] 伊藤,亀丸: "特徴抽出に基づくマッチトフィルタリングとニューラルネットワークを用いたパターン認識" 光学. 24. 32-39 (1995)
[Publications] S.Kamemaru,K.Tanaka,M.Nakazawa: "Universal patern recognition by matched filters synthesized by primitive patterns and by the algorithm for uniquely selecting the opitmum reference patterns" Proc.of SPIE. 232-239 (1995)
[Publications] T.Kubota: "Fluorine-containing Fluorescent Dye for Immunoassay and Optical Resolving Agent from Trifluoropropene Oxide" Proc.of Pacifichem '95. 09N904 (1995)
[Publications] T.Kubota: "Separation of an Enantionmorph and its Racemate by Distillation : Strong Chiral Recognition Ability of Trifluorolactates" Chemistry Letters. 115-116 (1996)
[Publications] Kiyono,H: "Mucosal vaccine : New Trends in Immunization." Academic Press,San Diego, 479 (1996)
[Publications] Kagnoff,M: "Essential of Mucosal Immunology," Academic Press,San Diego, 597 (1996)
[Publications] Kiyono,H: "Igaku-no-Ayumi,Current view of host protection-mucosal immune system." Igakushoin Pub.Co.Ltd.,Tokyo, 206 (1996)

1996 Fiscal Year Annual Research Report

レーザー・蛍光色素法による細胞の可視化計測とエイズ免疫,癌診断,治療への応用

Principal Investigator

藤井 寛一 茨城大学, 工学部, 教授 (00054354)

Research Products

[Publications] 藤井寛一: "Mode Control of Semiconductor Laser with Diffraction and Dispersion Feedback" Procceeding of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 849-853 (1995)

[Publications] 藤井寛一: "Pulse Broadning and of Copper Vapor in Copper-Halide Lasing System" Procceeding of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 872-877 (1995)

[Publications] 藤井寛一: "Enhancement of Population Inversion by Effective Use of Potential Energy in self-Termination Laser" Procceedin of 12th International Conference on Laser Interaction and Related Plasma Phenomena. part2. 878-882 (1995)

[Publications] 藤井寛一: "Comparison of A. C. Driven Barrier Discharge with D. C. Driven Malter Effect Corona Discharges for Plasma Reactor" Procceeding of 11th International Conference on Gas Discharges and Their Applications. 1. 164-167 (1995)

[Publications] 藤井寛一: "A Proposal of Modifying the Quittner Method for Gamma-Ray Peak Area Determination" Radio Isotopes. 45. 92-94 (1996)

[Publications] Takahashi,I.,Nakagawa,I.,Kiyono,H.,McGhee,J.R.,Clements,J.D.,and Hamada,S.: "Mucosal T cells induce systemic anergy for oral tolerance" Biochem. Biophys. Res. Commun.296. 414-420 (1995)

[Publications] Beagley,K.W.,Yamamoto,M.,McGhee,J.R.and Kiyono,H.: "Differences in intraepithelial lymphocyte T cell subsets isolated from murine samll versus large intestine" J.Immunol.154. 5611-5619 (1995)

[Publications] Hiroi,T.,Fujihashi,K.,McGhee,J.R. and Kiyono,H.: "Polarized Th2 cytokine expression by both mucosal γδ and αβ T cells." Eur.J.Immunol.25. 2743-2751 (1995)

[Publications] Bukawa.H.,Sekigawa,K-I.,Hamajima,K.,Fukuoka,J.,Yamada,Y.,Kiyono,H.and Okuda,K.: "Neutralization of HIV-1 secretory IgA induced by oral immunization with a new macromolecular multicomponent peptide vaccine candidate." Nature Medicine. 1. 681-685 (1995)

[Publications] Marinaro,M.,Staats,H.F.,Hiroi T.,Kiyono,H.,Jackson,R.J.,Coste,M. and McGhee,J.R.: "The Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4 whith supports IgE responses" J.Immunol.155 : 4621-4629.155. 4621-4629 (1995)

[Publications] Takahashi,I.,Kiyono,H.,Marinaro,M.,Jackson,R.J.,Nakagawa,I.,Fujihashi,K.,Hamada,S.,Clements,Bost,K.L., and McGhee,J.R.: "Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin" J.Infect.Dis.173. 627-635 (1996)

[Publications] Fujihashi,K.,Yamamoto,M.,Hiroi,T.,McGhee,J.R.,and Kiyono,H.: "Selected Th1 and Th2 cyotkine mRNA expression by CD4^+ T cells isolated from inflamed human gingival tissues" Clin.Exp.Immunol.103. 422-428 (1996)

[Publications] Takahashi,I.,Kiyono,H.,Jackson,R.J.,Fujihashi,K.,Stats,H.F.,Hamada,S.,Clements,J.D.,Bost,K.L.,and McGhee,J.R.: "Epitope maps of Escherichia coli labile toxin B subunit for development of a synthetic oral vaccine." Infect.Immune.64. 1290-1298 (1996)

[Publications] VanCott,J.L.,Kobayashi,T.,Pillai,S.,McGhee,J.R.,and Kiyono,H.: "Induction of polysaccharide specific mucosal immune responses by oral immunization" Vaccine. 14. 392-398 (1996)

[Publications] Yamamoto,M.,Kawabata,K.,Fujihashi,K.,McGhee,J.R.,Van Dyke,T.E.,Bamberg,T.V.and Kiyono,H.: "Lach of IL-4 contributes to persistence of IL-4 receptor positive macrophage in inflamed gingival tissues." Am.J.Pathol.148. 331-339 (1996)

[Publications] Jackson,R.J.,Fujihashi,K.,Kiyono,H.and McGhee,J.R.: "Luminometry : a novel bioluminescent method enhances the quantitation of mucosal and systemic antibody responses" J.Immunol.Methods. 190. 189-197 (1996)

[Publications] Fujihashi,K.,Kawabata,S.,Hiroi,T.,Yamamoto,M.,McGhee,J.R.,Nishikawa,S-I.and Kiyono,H.: "IL-2 and IL-7 reciprocally induce IL-7 and IL-2 receptors on γδ TCR^+ intraepithelial lymphocytes." Proc.Natl.Acad.Sci.USA. 93. 3613-3618 (1996)

[Publications] Fujihashi,K.,McGhee,J.R.,Kweon,M-N,Cooper,M.D.,Tonegawa,S.,Takahashi,I.,Hiroi,T.,Mestechy,J. and Kiyono,H.: "γδ T cell deficient mice have imparied mucosal IgA responses" J.Exp.Med.183. 1929-1935 (1996)

[Publications] Elson,C.O.,Beagley,K.W.,Sharmanov,A.T.,Kiyono,H.,Tennyson,G.S.,Ridwan,B.,Fujihashi,K. and McGhee,J.R.: "Hapten-induced model of murine IBD : mucosal immune responses and protection by tolerance." J.Immunol.157 : 2174-2185.157. 2174-2185 (1996)

[Publications] Nakagawa,I.,Takahashi,I.,Kiyono,H.,McGhee,J.R. and Hamada,S.: "Oral Immunization with the B subnit of the heat-labile entretotoxin of Escherichia coil induces early Th1 and Th2 cytokines expression in Peyer's patches." J.Infect.Dis.173. 1426-1428 (1996)

[Publications] Jackson,R.J.,Marinaro,M.,VanCott,J.L.,Yamamoto,M.,Okahashi,N.,Fujihashi,K.,Kiyono,H.,Chatfield,S.N.,and McGhee,J.R.: "Mucosal immunity : regulation by helper T cells and a novel method for datection" J.Biotech.44. 209-216 (1996)

[Publications] 伊藤,亀丸: "特徴抽出に基づくマッチトフィルタリングとニューラルネットワークを用いたパターン認識" 光学. 24. 32-39 (1995)

[Publications] S.Kamemaru,K.Tanaka,M.Nakazawa: "Universal patern recognition by matched filters synthesized by primitive patterns and by the algorithm for uniquely selecting the opitmum reference patterns" Proc.of SPIE. 232-239 (1995)

[Publications] T.Kubota: "Fluorine-containing Fluorescent Dye for Immunoassay and Optical Resolving Agent from Trifluoropropene Oxide" Proc.of Pacifichem '95. 09N904 (1995)

[Publications] T.Kubota: "Separation of an Enantionmorph and its Racemate by Distillation : Strong Chiral Recognition Ability of Trifluorolactates" Chemistry Letters. 115-116 (1996)

[Publications] Kiyono,H: "Mucosal vaccine : New Trends in Immunization." Academic Press,San Diego, 479 (1996)

[Publications] Kagnoff,M: "Essential of Mucosal Immunology," Academic Press,San Diego, 597 (1996)

[Publications] Kiyono,H: "Igaku-no-Ayumi,Current view of host protection-mucosal immune system." Igakushoin Pub.Co.Ltd.,Tokyo, 206 (1996)

藤井寛一茨城大学, 工学部, 教授 (00054354)