IMPROVED EX-VIVO IN-VITRO LYMPH-NODE CELL-PROLIFERATION ASSAY IN GUINEA-PIGS FOR A SCREENING-TEST OF CONTACT HYPERSENSITIVITY OF CHEMICAL-COMPOUNDS

Simple and efficient ex vivo/in vitro screening systems For contact al lergens are developed for alternative to conventional animal tests. We have previously proposed an ex vivo/in vitro proliferation assay as a first stage screening method with advantages over existing alternativ es, using lymph node cells (LNC) from sensitized guinea pigs of the Ha rtley strain. In this study, we have first confirmed, by histochemical analysis using in vivo bromodeoxyuridine (BrdU) and pyronin staining, that the ex vivo/in vitro LNC proliferation reflects in vivo response of lymph nodes to contact allergens. Furthermore, to improve the LNC assay, we then have investigated several experimental conditions for t heir influences on the LNC assay, demonstrating that, (1) the subscapu lar and the cervical LNC responded highly to contact allergens, (2) am ong three cervical lymph nodes the superficial dorsal cervical lymph n odes were the most reactive, (3) several vehicles alone used for anima l sensitization exhibited little influence on the LNC proliferation, ( 4) employment of stimulation index offset the inter-batch fluctuation of the LNC proliferation in the control animals as baseline proliferat ion. Under optimized experimental conditions as above, experimentally determined stimulation indexes of several contact allergens correlated well with their sensitizing potential estimated by conventional anima l tests. Therefore, the ex vivo/in vitro LNC proliferation assay shoul d be a simple and efficient alternative to conventional guinea pig tes tings including the guinea pig maximization test (GPMT).