The Local Lymph Node Assay

3 Toward Nonproliferation-Based In Vitro Assays Cell Surface Activation Markers, Cytokines, Chemokines, and Skin-Homing Receptors 

Activation markers such as CD69, CD25, CD71, and the MHC class II cell surface receptor HLA-DR are expressed and may be upregulated on the T cell surface. CD69, widely used in vitro and in vivo as a marker of T cell activation for 

2 Monitoring of Cytokines from T Cells and the ELISPOT Assay 

The ELISPOT (enzyme-linked immunospot) assay is based on classical immunoassay principles and the detection procedures employed in ELISA assays. Its sensitivity, ease of use, employment of highly reactive, standardized monoclonal antibodies, conunercial availability, and ready application to studies on individual cell types makes it a good choice for research and diagnostic 

3 Granzyme B ELISPOT Assay for the Detection of Drug-Reactive T Cells 

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Kimber, I. et al., The local lymph node assay past, present and future, Contact Dermatitis, 47, 315, 2002. 

Finally, multiparameter flow cytometry has the potential to improve existing methodologies for the evaluation of immunotoxicological endpoints such as the natural killer cell (NK) assay and the local lymph node assay (LLNA) to evaluate innate immunity and chemical-induced allergic disease. Accepted methodologies typically utilize radio-... 

Basketter, D.A., Gerberick, G.F., and Kimber, I., Skin sensitisation, vehicle effects and, the local lymph node assay. Food Chem. Toxicol., 39, 621, 2001. 

Warbrick, E.V., et al., Influence of application vehicle on skin sensitization to methylchlo-rothiazolinone/isothiazolinone An analysis using the local lymph node assay. Contact Dermatitis, 41, 325, 1999. 

Wright, Z.M., et al., Vehicle effects on the skin sensitizing potency of four chemicals assessment using the local lymph node assay. Ini. J. Cosmetic Sci., 23, 75. 

Basketter, D.A., et al., Use of the local lymph node assay for the estimation of relative contact allergenic potency. Contact Dermatitis, 42, 344, 2000. 

Loveless, S.E. et al., Further evaluation of the local lymph node assay in the final phase of an international collaborative trial, Toxicology, 108, 141, 1996. 

Basketter, D.A. et al., Threshold for classification as a skin sensitizer in the local lymph node assay A statistical evaluation, Fd. Chem. Toxicol., 37, 1167, 1999. 

Schneider, K. and Akkan, Z., Quantitative relationship between the local lymph node assay and human skin sensitization assays. Regul. Toxicol. Pharmacol., 39, 245, 2004. 

Gerberick, G.F. et al., Examination of the local lymph node assay for use in contact sensitization risk assessment, Fundam. Appl. Toxicol., 19, 438, 1992. 

Vandebriel, R.J. et al., Assessment of preferential T-helper 1 or T-helper 2 induction by low molecular weight compounds using the local lymph node assay in conjunction with RT-PCR and ELISA for interferon-y and interleukin-4. Toxicol. Appl. Pharmacol., 162, 77, 2000. 

Basketter, D.A., Scholes, E.W., Kimber, I., Botham, RA., Hilton, J., Miller, K., Robbins, M.C., Harrison, P.T.C. and Waite, S.J. (1991). Interlaboratory evaluation of the local lymph node assay with 25 chemicals and comparison with guinea pig test data. Toxicol. Methods 1 30-43. 

Kimber, I., Dearman, R.J., Scholes, E.W. and Basketter, D.A. (1994). The local lymph node assay Developments and applications. Toxicology 93 13-31. 

D. A. Basketter, G. F. Gerberick, I. Kimber, and S. E. Loveless. The local lymph node assay A viable alternative to currently accepted skin sensitization tests. Food Chem. Toxicol. 34 985-997 (1996). 

Basketter, D.A., Lea, L.J., Cooper, K., Stocks, J., Dickens, A., Pate, I., Dearman, R.J. and Kimber, I. (1999) Threshold for classification as a skin sensitizer in the local lymph node assay a statistical evaluation. Food and Chemical Toxicologf An International Journal Published for the British Industrial Biological Research Association, 37, 1167-1174. 

Figure 19.5 Assessing chemicals for potential contact sensitivity. In the local lymph node assay the chemical in question is applied to both ears on three consecutive days. Control mice are treated with vehicle. Radioisotope is injected intravenously on day 6. The draining lymph nodes are removed 5 hours later and the proliferative response is measured by the incorporation of radio isotope. Results are frequently presented as a stimulation index (counts per min (cpm) for the test chemical/cpm for control). (Picture adapted from D. Sailstad, Lab Animal 31 36, 2002.)...

This test uses an in vitro human dendritic cell culture to obtain information of the potential for various chemicals to induce allergic contact dermatitis. This test is used as an alternative to the Local Lymph node assay (LLNA) to minimize or replace the use of live animal testing for predicting skin sensitization (Kimber et al. 2002, see below). The test allows for evaluation of skin sensitization by examining the presence of cell surface markers on Periperal Blood Mononuclear Cell (PBMC)-derived dendritic cells (DC) that are known to be involved in the development of allergic contact dermatitis. 

Aiba S, Terunuma A, Manome H, Tagami H (1997) Dendritic cells differently respond to haptens and irritants by their production of cytokines and expressin of co-stimulatory molecules. Eur J Immunol 27 3031-3038 Hulette B, Gilmour N, Ryan C et al. (2003) Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergens. Toxicol 72 54 Kimber I, Dearman RJ, Basketter DA et al. (2002) The local lymph node assay past, present and future. Contact Dermatitis 47 315-328... 

Kimber I, Hilton J, Dearman RJ et al.(1995) An international evaluation of the murine local lymph node assay and comparison of modified procedures. Toxicology 103 63-73 Schneider K, Akkan Z (2004) Quantitative relationship between the local lymph node assay and human skin sensitization assays. Regul Toxicol Pharmacol 39(3) 245-255 Takeyoshi M, Noda S, Yamazaki S et al. (2004) Assessment of the skin sensitizatio potency of eugenol and its dimmers using a non-radioisotopic modification of the local lymph node assay. J Appl Toxicol 24 77-81... 

Recently, a more economical, less subjective test for contact hypersensitivity has been developed using mice. This test, the local lymph node assay (LLNA), assesses the proliferative response of lymphocytes in the draining lymph node following appli-... 

In addition to the above testing protocols, the local lymph node assay (LLNA) has been validated and accepted to assess the skin sensitization potential of chemicals in animals. This does not replace the guinea pig maximization test but is considered to be an equivalent. This in vivo method helps to reduce the number of animals used for contact sensitization activity. This test is based on the principle that sensitizer can induce a primary proliferation of lymphocytes in the lymph node draining the site of chemical application. This provides a quantitative measurement in which the proliferation is proportional to the dose applied. 

There are several acceptable ways to evaluate DTH responses in nonclinical species. Of these, the most common are the guinea pig assays used to assess contact sensitization. Both the Magnusson and Kligman model (guinea pig maximization test) and the Buehler model measure the elicitation phase of the hypersensitivity response, though the tests vary in their methods of chemical application and utilization of adjuvants. Most recently, the local lymph node assay has been accepted as a stand alone test for chemical hypersensitivity. This assay is conducted in mice and measures the induction phase of sensitization. In humans, the most common methods to assess delayed hypersensitivity are the patch test (contact sensitivity for diagnostic purposes) and the human repeat insult patch test (contact sensitivity for predictive purposes). Additionally, intradermal... 

Piperazine was evaluated in the local lymph node assay (LLNA) and the guinea pig maximization test. In both studies, piperazine was a mild sensitizer. In the LLNA, piperazine did not induce markers indicative of respiratory sensitization. In laboratory studies, piperazine has exhibited cross-sensitization reactions with diethylenetriamine. 

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