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 |
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Authors: | Jung Kyoung-Mi Jang Won-Hee Lee Yong-Kyoung Yum Young Na Sohn Soojung Kim Bae-Hwan Chung Jin-Ho Park Young-Ho Lim Kyung-Min |
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Affiliation: | a Medical Beauty Research Institute, Amorepacific CO R&D Center,Republic of Korea b National Institute of Food and Drug Safety Evaluation/Korea Food and Drug Administration,Republic of Korea c Department of Public Health, Keimyung University,Republic of Korea d College of Pharmacy, Seoul National University, Seoul 151-742,Republic of Korea |
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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, 3H-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. |
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Keywords: | LLNA, local lymph node assay tLLNA, traditional local lymph node assay BrdU, 5-bromo-2&prime -deoxyuridine FCM, flow cytometry |
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