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The mouse drug allergy model (MDAM) was developed as a tool to predict the potential of systemically administered drugs to produce hypersensitivity reactions (HR). Drugs associated with HR in the clinic produce a marked increase in the cellularity of the draining lymph nodes (DLN) in the MDAM. The objective of this study was to characterize the changes in the DLN following exposure to drugs associated with HR and to investigate whether lymphocyte migration and/or proliferation play a role in the response. These endpoints were also investigated in the local lymph node assay (LLNA) to determine whether responses between the two assays occur via similar mechanisms. Results demonstrated that total numbers of T- and B-cells were proportionally increased in the DLN of mice treated with positive control drugs (i.e. abacavir, amoxicillin, ofloxacin, and sulfamethoxazole) compared to animals administered the vehicle or negative control drugs (metformin and cimetidine). In contrast, a significant increase in the B-cell population of the DLN was observed for 2,4-dinitrofluorobenzene (DNFB) following the LLNA protocol. Down-regulation of CD62L and up-regulation of CCR7 were observed for T-cells from the DLN of the positive control treated mice in the MDAM, but not with DNFB in the LLNA. A mild increase in T-cell proliferation was observed in the MDAM with positive control drugs, while DNFB in the LLNA induced proliferation within the B-cell population only. Anti-CD40L antibody administration inhibited MDAM responses to positive control drugs, but did not affect DNFB-induced increases in total cell number in the LLNA. These results suggest that the increased cellularity of the DLN in the MDAM may be the result of drug-induced alterations in lymphocyte migration and/or effects on lymphocyte proliferation. Moreover, it appears that different mechanisms may be involved in driving the MDAM and LLNA responses. [ABSTRACT FROM PUBLISHER]

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell-Dutton assay ( in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4+ and CD8+ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment. [ABSTRACT FROM AUTHOR]