PMID- 12700420
OWN - NLM
STAT- MEDLINE
DCOM- 20031205
LR  - 20210702
IS  - 1096-6080 (Print)
IS  - 1096-0929 (Linking)
VI  - 73
IP  - 1
DP  - 2003 May
TI  - Assessment of cumulative allergen-activated lymph node cell proliferation using 
      flow cytometry.
PG  - 80-9
AB  - The murine local lymph node assay (LLNA) is a method for the prospective 
      identification of chemical contact allergens. The current validated protocol 
      assesses lymphocyte proliferation induced in the draining lymph node as a 
      function of in situ incorporation of radiolabeled thymidine. We have explored the 
      potential utility of an alternative nonradioisotopic marker of cell division, the 
      cytoplasmic dye carboxyfluoresein succinimidyl ester (CFSE). Using this method, 
      the cell phenotype and the number of divisions each cell has undergone can be 
      tracked using flow cytometry. BALB/c strain mice were exposed topically to 
      various concentrations of the contact allergens 2,4-dinitrochlorobenzene (DNCB), 
      oxazolone (ox) or hexyl cinnamic aldehyde (HCA), or to the nonsensitizing skin 
      irritant methyl salicylate (MS). Five days later, lymph node cells (LNC) were 
      labeled with CFSE, cultured for 96 h, then incubated with fluorescent labeled 
      anti-CD4 (T helper) and -CD8 (T cytotoxic) cell antibodies, and proliferating 
      CD4+ and CD8+ cells analyzed by flow cytometry. In LNC populations derived from 
      vehicle-treated animals, less than 1% of either cell population had undergone one 
      cell division or more. Topical exposure to MS (2.5 to 20%) did not increase the 
      frequencies of proliferating cells. Exposure to all three allergens, however, 
      resulted in a marked increase in the percentages of both CD4+ and CD8+ cells 
      undergoing division, with up to 5% and 3% of these cells, respectively, 
      proliferating in response to DNCB and oxazolone, and with lower levels of 
      proliferation stimulated by HCA. These preliminary data suggest that this method 
      may be applied to provide the basis of a nonradioisotopic end point for the LLNA, 
      particularly for the identification of potent contact allergens.
FAU - Humphreys, Neil E
AU  - Humphreys NE
AD  - Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, 
      SK10 4TJ, United Kingdom.
FAU - Dearman, Rebecca J
AU  - Dearman RJ
FAU - Kimber, Ian
AU  - Kimber I
LA  - eng
PT  - Journal Article
DEP - 20030415
PL  - United States
TA  - Toxicol Sci
JT  - Toxicological sciences : an official journal of the Society of Toxicology
JID - 9805461
RN  - 0 (5-(6)-carboxyfluorescein diacetate succinimidyl ester)
RN  - 0 (Allergens)
RN  - 0 (Dinitrochlorobenzene)
RN  - 0 (Fluoresceins)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Mitogens)
RN  - 0 (Succinimides)
RN  - 15646-46-5 (Oxazolone)
RN  - 7864XYD3JJ (Acrolein)
RN  - SR60A3XG0F (cinnamaldehyde)
RN  - VC2W18DGKR (Thymidine)
SB  - IM
MH  - Acrolein/*analogs & derivatives/toxicity
MH  - Allergens/*toxicity
MH  - Animals
MH  - CD4-Positive T-Lymphocytes/drug effects
MH  - CD8-Positive T-Lymphocytes/drug effects
MH  - Cell Division/physiology
MH  - Dinitrochlorobenzene/toxicity
MH  - Female
MH  - Flow Cytometry
MH  - Fluoresceins
MH  - Fluorescent Dyes
MH  - In Situ Hybridization
MH  - *Local Lymph Node Assay
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Mice, Inbred CBA
MH  - Mitogens/pharmacology
MH  - Oxazolone/toxicity
MH  - Succinimides
MH  - Thymidine/metabolism
EDAT- 2003/04/18 05:00
MHDA- 2003/12/06 05:00
CRDT- 2003/04/18 05:00
PHST- 2003/04/18 05:00 [pubmed]
PHST- 2003/12/06 05:00 [medline]
PHST- 2003/04/18 05:00 [entrez]
AID - kfg056 [pii]
AID - 10.1093/toxsci/kfg056 [doi]
PST - ppublish
SO  - Toxicol Sci. 2003 May;73(1):80-9. doi: 10.1093/toxsci/kfg056. Epub 2003 Apr 15.