title: B cell increases and ex vivo IL-2 production as secondary endpoints for the detection of sensitizers in non-radioisotopic local lymph node assay using flow cytometry.
abstract: Non-radioisotopic local lymph node assay (LLNA) using 5-bromo-2'-deoxyuridine (BrdU) with flow cytometry (FCM) is gaining attention since it is free from the regulatory issues in traditional LLNA (tLLNA) accompanying in vivo uses of radioisotope, (3)H-thymidine. However, there is also concern over compromised performance of non-radioisotopic LLNA, raising needs for additional endpoints to improve the accuracy. With the full 22 reference substances enlisted in OECD Test Guideline No. 429, we evaluated the performance of LLNA:BrdU-FCM along with the concomitant measurements of B/T cell ratio and ex vivo cytokine production from isolated lymph node cells (LNCs) to examine the utility of these markers as secondary endpoints. Mice (Balb/c, female) were topically treated with substances on both ears for 3 days and then, BrdU was intraperitoneally injected on day 5. After a day, lymph nodes were isolated and undergone FCM to determine BrdU incorporation and B/T cell sub-typing with B220+ and CD3e+. Ex vivo cytokine production by LNCs was measured such as IL-2, IL-4, IL-6, IL-12, IFN-γ, MCP-1, GM-CSF and TNFα. Mice treated with sensitizers showed preferential increases in B cell population and the selective production of IL-2, which matched well with the increases in BrdU incorporation. When compared with guinea pig or human data, BrdU incorporation, B cell increase and IL-2 production ex vivo could successfully identify sensitizers with the accuracy comparable to tLLNA, suggesting that these markers may be useful for improving the accuracy of LLNA:BrdU-FCM or as stand-alone non-radioisotopic endpoints.
authors.affiliation: Medical Beauty Research Institute, Amorepacific CO R&D Center, Republic of Korea.
authors.forename: Kyoung-Mi
authors.initials: KM
authors.lastname: Jung
authors.forename: Won-Hee
authors.initials: WH
authors.lastname: Jang
authors.forename: Yong-Kyoung
authors.initials: YK
authors.lastname: Lee
authors.forename: Young Na
authors.initials: YN
authors.lastname: Yum
authors.forename: Soojung
authors.initials: S
authors.lastname: Sohn
authors.forename: Bae-Hwan
authors.initials: BH
authors.lastname: Kim
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Chung
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Park
authors.forename: Kyung-Min
authors.initials: KM
authors.lastname: Lim
mesh_terms: D000485:Allergens / Q000276:immunology / Q000494:pharmacology; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002478:Cells, Cultured; D003627:Data Interpretation, Statistical; D017449:Dermatitis, Allergic Contact; D023381:Endpoint Determination; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006168:Guinea Pigs; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000276:immunology; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology*; D018655:Lymphocyte Count; D051379:Mice; D008807:Mice, Inbred BALB C; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology
chemical_list: D000485:Allergens; D007376:Interleukin-2; D001973:Bromodeoxyuridine