Division of Immunology, Department of Infection and Immunity, Institute Research Center (Health Research Course), Kagoshima University Graduate School of Medical and Dental Sciences.
Acta Histochem Cytochem. 2012 Apr 26;45(2):83-106. doi: 10.1267/ahc.11034. Epub 2012 Apr 21.
Antigen retrieval (AR) and ultra-super sensitive immunohistochemistry (ultra-IHC) have been established for application to archival human pathology specimens. The original ultra-IHC was the ImmunoMax method or the catalyzed signal amplification system (ImmunoMax/CSA method), comprising the streptavidin-biotin complex (sABC) method and catalyzed reporter deposition (CARD) reaction with visualization of its deposition. By introducing procedures to diminish non-specific staining in the original ultra-IHC method, we developed the modified ImmunoMax/CSA method with AR heating sections in an AR solution (heating-AR). The heating-AR and modified ImmunoMax/CSA method visualized expression of the predominantly simple present form of HTLV-1 proviral DNA pX region p40Tax protein (Tax) in adult T-cell leukemia/lymphoma (ATLL) cells in archival pathology specimens in approximately 75% of cases. The simple present form of Tax detected exhibited a close relation with ATLL cell proliferation. We also established a new simplified CSA (nsCSA) system by replacing the sABC method with the secondary antibody- and horse radish peroxidase-labeled polymer reagent method, introducing the pretreatments blocking non-specific binding of secondary antibody reagent, and diminishing the diffusion of deposition in the CARD reaction. Combined with AR treating sections with proteinase K solution (enzymatic-AR), the nsCSA system visualized granular immunostaining of the complex present form of Tax in a small number of ATLL cells in most cases, presenting the possibility of etiological pathological diagnosis of ATLL and suggesting that the complex present form of Tax-positive ATLL cells were young cells derived from ATLL stem cells. The heating-AR and ultra-IHC detected physiological expression of the p53 protein and its probable phosphorylation by Tax in peripheral blood mononuclear cells of peripheral blood tissue specimens from HTLV-1 carriers, as well as physiological and pathological expression of the molecules involved with G1 phase progression and G1-S phase transition (E2F-1, E2F-4, DP-1, and cyclin E) in ATLL and peripheral T-cell lymphoma cells. The ultra-IHC with AR is useful for etiological pathological diagnosis of ATLL since HTLV-1 pathogenicity depends on that of Tax, and can be a useful tool for studies translating advanced molecular biology and pathology to human pathology.
抗原修复（AR）和超敏免疫组化（ultra-IHC）已被应用于存档的人体病理学标本。最初的 ultra-IHC 是 ImmunoMax 方法或催化信号放大系统（ImmunoMax/CSA 方法），包括链霉亲和素-生物素复合物（sABC）方法和催化报告分子沉积（CARD）反应及其沉积的可视化。通过引入减少原始 ultra-IHC 方法中非特异性染色的程序，我们开发了改良的 ImmunoMax/CSA 方法，其中包括在 AR 溶液中加热切片（加热-AR）。加热-AR 和改良的 ImmunoMax/CSA 方法在存档的病理学标本中大约 75%的成人 T 细胞白血病/淋巴瘤（ATLL）细胞中可视化表达 HTLV-1 前病毒 DNA pX 区域 p40Tax 蛋白（Tax）的主要简单现在形式。检测到的简单现在形式的 Tax 与 ATLL 细胞增殖密切相关。我们还通过用二抗和辣根过氧化物酶标记的聚合物试剂法取代 sABC 法，引入预处理以阻断二抗试剂的非特异性结合，并减少 CARD 反应中沉积的扩散，建立了一种新的简化 CSA（nsCSA）系统。结合用蛋白酶 K 溶液处理切片（酶切-AR），nsCSA 系统在大多数情况下能够在少数 ATLL 细胞中可视化 Tax 的复杂现在形式的颗粒免疫染色，提示 ATLL 的病因病理诊断的可能性，并表明 Tax 阳性 ATLL 细胞的复杂现在形式是来自 ATLL 干细胞的年轻细胞。加热-AR 和 ultra-IHC 检测到 HTLV-1 携带者外周血组织标本外周血单个核细胞中 p53 蛋白及其可能被 Tax 磷酸化的生理表达，以及 ATLL 和外周 T 细胞淋巴瘤细胞中与 G1 期进展和 G1-S 期转变相关的分子（E2F-1、E2F-4、DP-1 和细胞周期蛋白 E）的生理和病理表达。AR 的 ultra-IHC 对 ATLL 的病因病理诊断有用，因为 HTLV-1 的致病性取决于 Tax，并且可以成为将先进的分子生物学和病理学转化为人体病理学的有用工具。
