In Vitro Nickel-Specific T-Lymphocyte Proliferation: Methodological Aspects
The conditions necessary for optimal and reproducible test results in the nickel specific in vitro T-lymphocyte proliferation assay (NiPA) were studied. For this purpose peripheral blood mononuclear cells (PBMCs) of nickel-allergic subjects were isolated by density-gradient centrifugation and, when necessary, further separated into T cells and non-T cells. The influence of basic variables such as the number of cells per well, culture medium, serum-type and the type of culture dishes (round- or flat-bottomed) on the result of this assay, were investigated in parallel experiments. Furthermore, in this study enhancement of Ni-specific T-lymphocyte proliferation was attempted by the addition of epidermal cells from normal or patch-tested skin areas, recombinant IL-2 after antigen priming or pre-treatment of the cells with neuraminidase. Optimal and reproducible proliferation was obtained in cultures set up with 2 × 105 PBMCs or with purified T cells in the presence of 4–8 × 104 epidermal cells from patch-tested skin, using RPMI 1640 with HEPES and 15% pooled human AB serum in round-bottomed wells. Under the established optimal conditions the NiPA showed a 100% sensitivity and a specificity of 93%. Pre-treatment of PBMCs with neuraminidase and the addition of epidermal cells or rIL-2 after nickel priming enhanced the proliferative response in low-grade sensitized individuals.
Key wordsIn vitro nickel-proliferation assay Nickel-contact dermatitis
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