Tsuge Ikuya, Okumura Akihisa, Kondo Yasuto, Itomi Seiko, Kakami Michiko, Kawamura Makiko, Nakajima Yoichi, Komatsubara Ryo, Urisu Atsuo
Department of Pediatrics, School of Medicine, Fujita Health University, Toyoake, Aichi, Japan.
Allergol Int. 2007 Jun;56(2):149-55. doi: 10.2332/allergolint.O-06-457. Epub 2007 May 1.
Phenytoin can induce diversified adverse reactions including generalized eruptions and the hypersensitivity syndrome. Delayed-type allergic mechanisms have been postulated to underlie these reactions. The tests most widely used to detect T-cell sensitization to drugs are the patch test and the lymphocyte transformation test (LTT), but their sensitivity is not sufficient. Simultaneous assessment of both the frequencies and the cytokine-producing phenotypes of allergen-specific T cells has become possible with the recently introduced carboxyfluorescein succinimidyl ester (CFSE) assay.
Seven patients who presented with phenytoin-induced maculopapular exanthema with and without fever were included in this study. Peripheral blood mononuclear cells (PBMCs) were labeled with CFSE and cultured with phenytoin for seven days. The cells were stained with anti-CD4 and cytokine-specific monoclonal antibodies (MoAbs), and analyzed with FACSCalibur.
The phenytoin-specific proliferation of CD4+ cells in patients was significantly higher than in the four controls exposed to phenytoin, and in seven healthy children with no previous phenytoin intake. A significant difference in the percentages of CD4+ IFN-gamma+ cells between patients and the seven healthy children was observed. The sensitivity and specificity of proliferation were 100% and 90.9%, and those of IFN-gamma secretion were 71.4% and 100%, respectively.
Phenytoin-specific proliferation may be detected with greater sensitivity by the CFSE dilution assay than the conventional LTT. The assay revealed that both CD4+ and CD4- T cells proliferated and produced IFN-gamma and TNF-alpha after stimulation with phenytoin. The CFSE dilution assay might be useful for the diagnosis and understanding of drug hypersensitivity.
苯妥英可引发多种不良反应，包括全身性皮疹和超敏反应综合征。推测迟发型过敏机制是这些反应的基础。检测T细胞对药物致敏最常用的试验是斑贴试验和淋巴细胞转化试验（LTT），但其敏感性不足。随着最近引入的羧基荧光素琥珀酰亚胺酯（CFSE）检测法，同时评估过敏原特异性T细胞的频率和产生细胞因子的表型已成为可能。
本研究纳入了7例出现苯妥英诱导的伴有或不伴有发热的斑丘疹性皮疹的患者。外周血单个核细胞（PBMC）用CFSE标记，并与苯妥英一起培养7天。细胞用抗CD4和细胞因子特异性单克隆抗体（MoAb）染色，并用FACSCalibur进行分析。
患者中CD4 +细胞的苯妥英特异性增殖明显高于4例接触苯妥英的对照者以及7例既往未服用过苯妥英的健康儿童。观察到患者与7例健康儿童之间CD4 + IFN-γ +细胞百分比存在显著差异。增殖的敏感性和特异性分别为100%和90.9%，IFN-γ分泌的敏感性和特异性分别为71.4%和100%。
与传统的LTT相比，CFSE稀释检测法可能更灵敏地检测到苯妥英特异性增殖。该检测法显示，苯妥英刺激后CD4 +和CD4 - T细胞均增殖并产生IFN-γ和TNF-α。CFSE稀释检测法可能有助于药物超敏反应的诊断和理解。
