Schreiner M, Peiser M, Briechle D, Stahlmann R, Zuberbier T, Wanner R
Institute of Molecular Biology and Bioinformatics, Charité, Berlin, Germany.
Allergy. 2007 Dec;62(12):1419-28. doi: 10.1111/j.1398-9995.2007.01511.x.
Protection against contact allergy begins with the collection of reliable data about the sensitizing potential of chemicals. Today, the local lymph node assay (LLNA) in mice is widely used to identify sensitizing substances. For several reasons, an in vitro assay could be preferable to animal experiments. We propose an in vitro test for the detection of a sensitizing potential of a chemical composed of a single layer of human nondifferentiating keratinocytes and of allogenic floating monocytes which are cocultured in serum-free medium in the presence of a cytokine cocktail. Within days, the coculture develops to an allergen- sensitive system consisting of activated keratinocytes and of mobile dendritic cell-related cells (DC-related cell). The sensitizing potential can be determined by analyzing the expression of the dendritic cell maturation marker CD86. For the model contact allergens tested so far [trinitrobenzenesulfonic acid (TNBS), phenylendiamine, and 4-aminoacetanilide], the strength of the reaction was in concordance with results from the LLNA. Sensitivity of the assay allowed testing at concentrations without general cytotoxicity. Thus, a differentiation between allergens and irritants was possible. Regarding cytokine secretion, the assay distinguished between the allergen TNBS and the Toll-like receptor ligand lipopolysaccharide. The coculture can be set up from cryopreserved cells. The assay is easy to perform and reproducible. Donor-variance is negligible. This in vitro assay based on a loose-fit coculture is a reasonable approach to screen for the sensitizing potential of xenobiotics and might partially replace the LLNA and other animal tests.
预防接触性过敏始于收集有关化学品致敏潜力的可靠数据。如今，小鼠局部淋巴结试验（LLNA）被广泛用于识别致敏物质。出于多种原因，体外试验可能比动物实验更可取。我们提出一种体外试验，用于检测一种化学品的致敏潜力，该试验由单层人未分化角质形成细胞和同种异体悬浮单核细胞组成，它们在无血清培养基中，在细胞因子混合物存在的情况下进行共培养。在数天内，共培养物发展成为一个由活化的角质形成细胞和移动的树突状细胞相关细胞（DC相关细胞）组成的过敏原敏感系统。致敏潜力可以通过分析树突状细胞成熟标志物CD86的表达来确定。对于迄今为止测试的模型接触性过敏原[三硝基苯磺酸（TNBS）、对苯二胺和4-氨基乙酰苯胺]，反应强度与LLNA的结果一致。该试验的灵敏度允许在无一般细胞毒性的浓度下进行测试。因此，可以区分过敏原和刺激物。关于细胞因子分泌，该试验区分了过敏原TNBS和Toll样受体配体脂多糖。共培养物可以从冷冻保存的细胞中建立。该试验易于操作且可重复。供体差异可忽略不计。这种基于松散共培养的体外试验是筛选外源性物质致敏潜力的合理方法，可能会部分取代LLNA和其他动物试验。
