Clarification of pathogenesis in pemphigus : Establishment of new techniques for diagnosis.

阐明天疱疮发病机制：建立诊断新技术。

• 批准号: 05404036
• 负责人: NISHIKAWA Takeji
• 金额: $ 17.15万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05404036/
• 关键词: immunoelectron microscopy; crvoultramicrotomy; crvofixation; cryosubstitution; cadherin; 凍結置換; クリオウルトラ切片; カドヘリン; 凍結固定; クリオウルトラ切手; セドヘリン

We analyzed the location of binding sites for pemphigus vulgaris (PV) antigen and pemphigus foliaceus (PF) antigen in the human epidermis using serum samples obtained from 3 patients with PV and 3 patients with PF.Confocal laser scanning microscopyimmunofluorescent examination of ultrathin cryosections, and immunoperoxidase EM demonstrated discontinuous dots along the epidermal cell surfaces. Immunogold EM of ultrathin cryosections showed specific binding of PV and PF autoantibodies only to desmosomes. Post-embedding immunogold EM using cryofixation and cryosubstitution enabled the whole depth of the epidermis to be examined and the binding of PV and PF autoantibodies to be quantitated by counting gold particles. Both PV and PF sutoantibodies bound to all desmosomes in the epidermis, but not to the surface of the non-desmosomal keratinocytes. The majority of autoantibody binding occurred in the extracellular domain (PV : 62% ; PF : 69%). The statistical analysis of two-way analysis of variance regarding the number of gold particles labeling a single desmosome confirmed a significant interaction between subtypes of pemphigus (PV and PF) and the different epidermal cell layrs (p<0.044). The results indicate that the number of gold particles bound to individual desmosomes with PV sera was significantly higher in the lower epidermis than in the upper epidermis, and that of PF sera showed reciprocal pattern. This inversely graded binding pattern suggests that the heterogeneity of the composition of the desmosomes, which may explain the differences in level of acantholysis between PV and PF.

我们分析了寻常型天疱疮（PV）抗原和叶状天疱疮（PF）抗原在人表皮中的结合位点。共聚焦激光扫描显微镜、超薄冷冻切片免疫荧光检查和免疫过氧化物酶电镜显示表皮细胞表面不连续的点。超薄冷冻切片的免疫金电镜显示PV和PF自身抗体仅与桥粒特异性结合。采用冷冻固定和冷冻替代的免疫金电镜包埋后，可以检测表皮的整个深度，并通过计数金颗粒来定量PV和PF自身抗体的结合。PV抗体和PF抗体都能结合到表皮的所有桥粒上，但不结合到非桥粒角质形成细胞的表面。大多数自身抗体结合发生在细胞外结构域（PV: 62%; PF: 69%）。单桥粒标记金颗粒数量的双向方差分析证实天疱疮亚型（PV和PF）与不同表皮细胞层之间存在显著的相互作用（p<0.044）。结果表明，PV血清与单个桥粒结合的金粒子数量在下表皮显著高于上表皮，而PF血清与单个桥粒结合的金粒子数量呈反比关系。这种反向分级结合模式表明桥粒组成的异质性，这可能解释PV和PF之间棘层溶解水平的差异。

项目成果

Shimizu,H.: "Immunogoldsilver staining Ples, methods, and applications" CRC Press, 6 (1995)

Shimizu,H.：“免疫金银染色方法、方法和应用”CRC Press，6 (1995)

Amagai, M.: "Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calsium dependent and glycosylation independent." Journal of Investigative Dermatology. 105. 243-247 (1995)

Amagai, M.：“天疱疮抗原（Dsg1 和 Dsg3）的构象表位依赖于钙且不依赖于糖基化。”

Shimizu, H.: "Pemphigus vulgaris and pemphigus foliaceus sera show an inversely graded binding pattern to extracellular regions of desmosomes in different layrs of human epidermis." Journal of Investigative Dermatology. 105. 153-159 (1995)

Shimizu, H.：“寻常型天疱疮和落叶型天疱疮血清与人类表皮不同层桥粒的细胞外区域显示出反向分级的结合模式。”

Ide, A.: "Two patients with unusual skin lesions and circulating antikera-tinocyte cell surface antibodies : detection of antibodies to the intracelular domain of the pemphigus foliaceus antigen (desmoglein by studies using fusion proteins." British Journ

Ide, A.：“两名具有异常皮肤损伤和循环抗角质形成细胞表面抗体的患者：检测落叶型天疱疮抗原（桥粒芯糖蛋白，通过使用融合蛋白的研究）细胞内结构域的抗体。”英国杂志

Shimizu H: "Demonstration of Desniosonral antigens by electron microscopy using ayofixed and cryosubstituted skin with sitver‐enhanced gold probe." J Histochem Cytochem. 42. 687-691 (1994)

Shimizu H：“通过电子显微镜演示 Desniosonral 抗原，使用 sitver 增强金探针固定和冷冻替代皮肤。”J Histochem Cytochem 42. 687-691 (1994)