| Project/Area Number |
03454275
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Dermatology
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| Research Institution | Keio University |
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Principal Investigator |
NISHIKAWA Takeji Keio University Sch. of Med. Professor, 医学部, 教授 (50051579)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMIZU Hiroshi Keio Univ. Sch. Med. Instructor, 医学部, 助手 (00146672)
HASHIMOTO Takashi Keio Univ. Sch. Med. Assist. Prof, 医学部, 講師 (20129597)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1992: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1991: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | epidermolysis bullosa / immunoelectron microscopy / bullous pemphigoid / colloidal gold / cryofixation / cryosubstitution / image analyzer / hemidesmosome / 基底膜 |
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| Research Abstract |
Many different types of immuno EM techniques have been introduced in dermatological research varing from relatively simple ones which could be used in most laboratories ( pre-embedding peroxidase method) to complex procedure relevant mainly to the specialized research laboratory (post-embedding, cryofixation and cryosubstitution technique). In this project, we have employed up-to date low temperature immunoelectron microscopic techniques for the study of skin blistering disease including bullous pemphigoid antigen and epidermolysis bullosa acquisita antigen. Previous immuno-peroxidase method could demonstrate the ultrastructural localization of the immunoreactants only fairly accurately in relation to the subcomponents of the dermo-epidermal junction. Because the size of peroxidase-diaminobenzidine reaction products is large and prone to diffuse, and inevitably obscure the underlying structure. Applying cryofixation and cryosubstitution techniques without using chemical fixatives in co
… More
njunction with colloidal gold, we have demonstrated that epidermolysis bullosa acquisita antigen, a target molecule of the autoantibodies of the patient, localized to both dermal and lamina densa ends of anchoring fibrils, but not to the central banded portion itself. Anchoring fibrils is a structure of 785nm in length. Colloidal gold but not peroxidase can elucidate the fine ultrastructural localization of the target epitope on such small structure.
Bullous pemphigoid antigen is composed of two major antigenic proteins of the 230-kD antigen and the 180-kD antigen. We have used this new immunoelectron microscopic technique to localize the epitopes for autoantibodies against each antigen. Autoantibodies against each antigen were affinity-purified using nitrocellulose membrane, which was blotted with SDS-PAGE fractionated antigens from human epidermal extract as the immunoabsorbent. Postembedding immunogold electron microscopy was performed using low temperature techniques. The 230-kD antigen only localized to the intracellular domain of hemidesmosome. In contrast, the autoantibodies against the 180-kD antigen localized along the plasma membrane of hemidesmosome. These result suggest that the autoantibodies against the 180 and 230-kD bullous pemphigoid antigen may play different roles in the blister formation in patients with bullous pemphigoid. Less
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