| Project/Area Number |
12670308
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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 |
Immunology
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| Research Institution | JUNTENDO UNIVERSITY |
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Principal Investigator |
KAYAGAKI Nobuhiko Juntendo University, instructor, 医学部, 助手 (80317403)
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| Co-Investigator(Kenkyū-buntansha) |
MAEDA Keiko Juntendo University, instructor, 医学部, 助手 (20053374)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | Apoptosis / TWEAK / Fas ligand / monocyte / humanization / Molecular modeling / アポトーシス / TRAIL / TWEAK / TNF / 単球 / 生体防御 / IFN-γ |
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| Research Abstract |
TWEAK, a new member of the tumor necrosis factor (TNF) family, induces cell death in some tumor cell lines, but its physiological functions are largely unknown. In this study, we investigated the expression and function of TWEAK in human peripheral blood mononuclear cells (PBMCs) by using newly generated anti-human TWEAK mAbs. Although freshly isolated PBMCs expressed no detectable level of TWEAK on their surfaces, a remarkable TWEAK expression was rapidly observed on monocytes upon stimulation with interferon (IFN)-gamma but not with IFN-alpha or lipopolysaccharide. Cytotoxic activity of IFN-gamma-stimulated monocytes against human squamous carcinoma cell line HSC3 was inhibited partially by anti-TWEAK mAb alone and almost completely by combination with anti-TRA! L (TNF-related apoptosis-inducing ligand) mAb. These results revealed a novel pathway of monocyte cytotoxicity against tumor cells that is mediated by TWEAK and potentiated by IFN-gamma. We furthermore, generated a humanized anti-human Fas ligand (L)/CD95L, a proadoptolic member of the TNF family, mAb and characterized the epitopes of neutralizing mAbs by extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E loop and another near the top ofFasL. Both of these sites on FasL are critically involved in the direct interaction with the corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these FasL/Fas contact sites.
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