title: Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production.
abstract: The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells. At the mRNA level, no change in the IL-2 gene expression level was detected by real-time PCR analysis. At the protein level, various experimental conditions were checked in order to improve the irritant versus sensitizer discrimination with a restricted set of prototypic compounds. In particular, the use of phytohemagglutinin A (PHA) in an ex vivo cell culture step showed an improvement of both signal and discrimination. In these optimised conditions, a panel of irritants and potency-graded sensitizers was used to assess the performance of the modified method. IFN-gamma production was used as a positive control. For each compound, a dose-response was performed and stimulation indexes (SI) were determined. Effective concentrations (EC) for each sensitizers were then extracted and compared to the literature data of the regular LLNA. The IL-2-based LLNA showed similar performances at both qualitative and quantitative levels compared to regular LLNA.
authors.affiliation: AFSSAPS, Unité BCM, 635 Rue de la Garenne, 34740 Vendargues, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Azam
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Peiffer
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Ourlin
authors.forename: Pierre-Antoine
authors.initials: PA
authors.lastname: Bonnet
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Tissier
authors.forename: Laurence
authors.initials: L
authors.lastname: Vian
authors.forename: Isabelle
authors.initials: I
authors.lastname: Fabre
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D017449:Dermatitis, Allergic Contact / Q000209:etiology / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D007509:Irritants / Q000633:toxicity*; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007376:Interleukin-2; D007509:Irritants; D012333:RNA, Messenger; D007371:Interferon-gamma