The integrated system with Tm mapping and the FCS-based NF-kB assay for sepsis patients

• Project/Area Number: 25670268
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory medicine
• Research Institution: University of Toyama
• Principal Investigator: KITAJIMA Isao 富山大学, 大学院医学薬学研究部(医学), 教授 (50214797)
• Co-Investigator(Kenkyū-buntansha): NIIMI Hideki 富山大学, 大学病院, 助教 (50401865)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 感染症 / 微生物 / 敗血症 / 遺伝子検査 / 転写因子 / 1分子計測 / 一分子計測

Outline of Final Research Achievements

The earliest possible identification of pathogenic microorganisms is critical for selecting appropriate antimicrobial therapy. We developed a nested-PCR-based assay for detecting Mycoplasma and other bacteria in amniotic fluid samples using eukaryotic, thermostable DNA polymerase. Next, a "melting temperature (Tm) mapping method" was developed for identifying a broad range of bacteria. Employing primer sets for a 16S ribosomal gene, more than 100 bacterial species can be identified. Nuclear transcription factor-kappa B (NF-kB) activation plays a key role in the regulation of immune responses to inflammation. We developed an experimental apparatus for detection of NF-kB activation based on fluorescence correlation spectroscopy (FCS), which permits analysis of DNA-protein binding in the liquid phase. FCS was performed using the single-molecule fluorescence detention system. We have integrated Tm mapping and the FCS based NF-kB assay and are conducting clinical trials in sepsis patients.

Research Products

• [Journal Article] Post-maketing surveillance of thrombomodulin alfa, a novel drug for treatment of disseminated intravascular coagulation treatment- Safety and efficacy in 1,032 patients assosiated with hematologic malignancy (2014)
  • Author(s): Asakura H, Takahashi H, Matsushita T, Ninomya H, Honda G, Mimuro J, Eguchi Y, Kitajima I, Sakata Y
  • Journal Title: Thromｂ Res. Volume: 133 Pages: 364-370
  • Peer Reviewed
• [Journal Article] Thrombomodulin alfa treatment in patients with acute promyelocytic leukemia and disseminated intravascular coagulation: a retrospective analysis of a open label multicenter postmarketing surveillance study cohort. (2014)
  • Author(s): Mastusita T, Watanabe J, Honda G, Mimuro J, Tsuji H, Eguchi Y, Kitajima I, Sakata Y
  • Journal Title: Thromb Res. Volume: 133 Pages: 772-781
  • Peer Reviewed
• [Journal Article] Post-marketing surveillance data of the thrombomodulin alfa: sub-analysis in patients with sepsis-induced disseminated intravascular coagulation. (2014)
  • Author(s): Eguchi Y, Gando S, Ishikura H, Saitoh D, Mimuro J, Takahashi H, Kitajima I, Tshuji H, Matsushita T, Tsujita R, Nagao O, Sakata Y
  • Journal Title: Journal of Intensive Care. Volume: 2 Pages: 30-30
  • Peer Reviewed / Open Access
• [Journal Article] Post-marketing surveillance of thrombomodulin alfa, a novel treatment of disseminated intravascular coagulation - Safety and efficacy in 1,032 patients with hematologic malignancy (2014)
  • Author(s): Asakura H, Takahashi H, Tsuji H, Matsushita T, Ninomiya H, Honda G, Mimuro J, Eguchi Y, Kitajima I, Sakata Y
  • Journal Title: Thromb Res Volume: 133 Issue: 3 Pages: 364-370
  • DOI: 10.1016/j.thromres.2013.12.033
  • Peer Reviewed / Open Access
• [Journal Article] N-terminal pro-Brain Natriuretic Peptide as a Predictor of Reoperation in Children With Surgically Corrected Tetralogy of Fallot (2014)
  • Author(s): Hirono K1, Sekine M, Shiba N, Hayashi S, Nakaoka H, Ibuki K, Saito K, Watanabe K, Ozawa S, Higuma T, Yoshimura N, Kitajima I, Ichida F.
  • Journal Title: Circ J Volume: ７８（３） Pages: 693-700
  • NAID: 130003382234
  • Peer Reviewed
• [Journal Article] A rapid and high-throughput quantitation assay of the nuclear factor κB activity using fluorescence correlation spectroscopy in the setting of clinical laboratories. (2013)
  • Author(s): Harada K, Mikuni S, Beppu H, Niimi H, Abe S, Hano N, Yamagata K, Kinjo M, Kitajima I.
  • Journal Title: PLoS One Volume: 8 Issue: 10 Pages: e75579-e75579
  • DOI: 10.1371/journal.pone.0075579
  • Peer Reviewed
• [Journal Article] Distinct reactivity of the commercially available monoclonal antibodies of D-dimer and plasma FDP testing to the molecular variants of fibrin degradation products. (2013)
  • Author(s): Madoiwa S, Kitajima I, Ohmori T, Sakata Y, Mimuro J.
  • Journal Title: Thromb Res. Volume: 132 Issue: 4 Pages: 457-464
  • DOI: 10.1016/j.thromres.2013.08.006
  • Peer Reviewed
• [Presentation] Tm mapping法（感染症起炎菌迅速同定法）の院内試験運用とその評価 (2014)
  • Author(s): 仁井見英樹、上野智浩、森正之、多葉田誉、南洋、杉江和茂、齋藤滋、北島勲
  • Organizer: 第61回日本臨床検査医学会学術集会
  • Place of Presentation: 福岡
  • Year and Date: 2014-11-20 – 2014-11-23
• [Presentation] 感染症起炎菌迅速同定法(Tm mapping法)の学内試験運用における正確性の評価 (2014)
  • Author(s): 仁井見英樹、上野智浩、森正之、杉江和茂、北島勲
  • Organizer: 第54回日本臨床化学会年次学術集会
  • Place of Presentation: 東京
  • Year and Date: 2014-09-03 – 2014-09-05
• [Presentation] The Tm mapping method. A noel rapid, easy, and cost-effective method that identifies unknown pathogenic microorganisms within 3 hours of patietnt sample collection. (2014)
  • Author(s): Niimi H, Kitajima I
  • Organizer: Microbiology and Infectious Diseases Asia Congress. 2014
  • Place of Presentation: シンガポール
  • Year and Date: 2014-06-10 – 2014-06-11
  • Invited
• [Presentation] A Novel Method That Enables Rapid Identification of Unknown Pathogenic Bacteria In Sepsis Within Three Hours of Whole Blood Collection. (2014)
  • Author(s): Niimi H, Ueno T, Hayashi S, Abe A, Tsurue T, Mori M, Tabata H, Minami H, Goto M, Saito S and Kitajima I. The Melting Temperature (Tm) Mapping Method:
  • Organizer: ECCMID 2014
  • Place of Presentation: バルセロナ
  • Year and Date: 2014-05-10 – 2014-05-13
• [Presentation] I: The Melting Temperature (TM) Mapping Method: A Novel Method That Enables Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Patient Sample Collection. (2014)
  • Author(s): Niimi H, Ueno T, Kitajima Ｉ．
  • Organizer: The 9th International Conference of Clinical Laboratory Automation and Robotics
  • Place of Presentation: 横浜
  • Year and Date: 2014-04-17 – 2014-04-19
• [Presentation] Novel haemostatic biomarkers in acute cardioembolic stroke. (2013)
  • Author(s): Kitajima I, Hirano K, Tani M, Tanaka K
  • Organizer: Novel haemostatic biomarkers in acute cardioembolic stroke, 2013 American Association for Clinical Chemistry Annaul Meeting,
  • Place of Presentation: Houston, TX ,USA
• [Presentation] A New rapid, easyand cost-effective method that identifies unknown pathogenic microorganisms within 3 hours from sample collection (2013)
  • Author(s): Niimi H, Ueno T, Hayashi S, Mori M, Tabata H, Minami H, Saito S, Kitajima
  • Organizer: 20th European Congress of Clinical Chemistry and Laboratory Medicine
  • Place of Presentation: Milano、Itaria
  • Invited
• [Presentation] Thrombomodulin Alfa Treatment For Disseminated Intravascular Coagulation In Acute Promyelocytic Leukemia: A Retrospective Analysis Of The Open-Label, Multicenter Study Cohort. (2013)
  • Author(s): Matsushita T, Watanabe J, Goichi Honda, Mimuro J, Takahashi H, Tsuji H, Eguchi Y,, Kitajima I, Sakata Y.
  • Organizer: 55th American Society of Hematology Annual Meeting and Exposition
  • Place of Presentation: New Orleans, LA, USA.
• [Presentation] 血栓・止血分子マーカーの最前線 (2013)
  • Author(s): 北島勲
  • Organizer: .第60回日本臨床検査医学会学術集会
  • Place of Presentation: 神戸
  • Invited
• [Presentation] Tm mappng法：検体採取3時間以内で不特定の起因菌を同定する迅速検査法 (2013)
  • Author(s): 仁井見英樹, 上野智浩, 森正之, 多葉田誉, 南洋, 北島勲.
  • Organizer: 第53回日本臨床化学会年次学術集会
  • Place of Presentation: 徳島