Adriana Ariza, Mar ıa Isabel Monta~nez, Monday O. Ogese, Arun Tailor, Tahia D. Fernandez, Mar ıa J. Torres, and Dean J. Naisbitt; Research Laboratory, IBIMA-Regional University Hospital of MalagaUniversity of Malaga, M alaga, Spain, Research Laboratory, IBIMARegional University Hospital of Malaga-UMA, M alaga, Spain, Dept. Molecular & Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, United Kingdom, Allergy Unit, IBIMA-Regional University Hospital of Malaga-University of Malaga, M alaga, Spain. RATIONALE: Amoxicillin (AX) is the most common cause of drug hypersensitivity mediated by a specific immunological mechanism, which is often prescribed alongside clavulanic acid (Clav). Sensitivity of in vitro testing is low, probably due to the use of structures not optimally recognized. In this study we generate and characterize AXand Clav-specific T-cell clones to be used as tool to study the immunological recognition of new structures. METHODS: Drug-specific T-cell clones were generated from peripheral blood mononuclear cells by serial dilution and repetitive mitogen stimulation. Antigen specificity was assessed by measurement of proliferation ([H]-thymidine incorporation) and cytokine release (ELISpot). RESULTS: 110 AX-specific and 96 Clav-specific T-cell clones were generated from 7 patients. Proliferation of AXand Clav-specific clones was dose-dependent, no cross-reactivity between AX and Clav was observed and they required the presence of drug and antigen presenting cells to proliferate. Drugs were presentend to CD4 T-cells byMHC-II and to CD8 by MHC-I. The highest level of cytokine secreted was IFN-g, followed by IL-13, IL-5 and IL-10. CONCLUSIONS: AXand Clav-specific T-cell clones can be generated from AX-Clav hypersensitivity patients. They are activated only in the presence of antigen presenting cells, supporting the hapten hypothesis for the recognition and presentation of betalactam antibiotics. The specific Tcell clones generated are an immunologically characterized tool that can be used for the analysis of new chemical structures to be included in in vitro diagnostic tests.

中文翻译：

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在哺乳动物细胞中表达的重组抗体用于分析螨过敏原的抗原决定簇

Adriana Ariza、Mar ia Isabel Monta~nez、Monday O. Ogese、Arun Tailor、Tahia D. Fernandez、Mar ia J. Torres 和 Dean J. Naisbitt；研究实验室，IBIMA-马拉加地区大学医院马拉加大学，马拉加，西班牙，研究实验室，IBIMA 马拉加地区大学医院，马拉加，西班牙，分子与临床药理学部，MRC 药物安全科学中心，大学利物浦，英国利物浦，过敏科，IBIMA-马拉加地区大学医院-马拉加大学，西班牙马拉加。理由：阿莫西林 (AX) 是由特定免疫机制介导的药物超敏反应的最常见原因，通常与克拉维酸 (Clav) 一起使用。体外测试的敏感性很低，可能是由于使用了未被最佳识别的结构。在这项研究中，我们生成并表征了 AX 和 Clav 特异性 T 细胞克隆，以用作研究新结构的免疫识别的工具。方法：通过连续稀释和重复有丝分裂原刺激，从外周血单个核细胞中产生药物特异性 T 细胞克隆。通过测量增殖（[H]-胸苷掺入）和细胞因子释放（ELISpot）来评估抗原特异性。结果：从 7 名患者中产生了 110 个 AX 特异性和 96 个 Clav 特异性 T 细胞克隆。AX 和 Clav 特异性克隆的增殖是剂量依赖性的，没有观察到 AX 和 Clav 之间的交叉反应，它们需要药物和抗原呈递细胞的存在才能增殖。药物通过 MHC-II 提供给 CD4 T 细胞，通过 MHC-I 提供给 CD8。分泌的细胞因子水平最高的是 IFN-g，其次是 IL-13，IL-5 和 IL-10。结论：AX 和 Clav 特异性 T 细胞克隆可以从 AX-Clav 超敏反应患者中产生。它们仅在存在抗原呈递细胞的情况下才被激活，从而支持半抗原假说用于识别和呈递β内酰胺抗生素。生成的特定 T 细胞克隆是一种具有免疫学特征的工具，可用于分析要包括在体外诊断测试中的新化学结构。