Leukocyte activation evaluation

Lymphocyte proliferation and cytokine production form the basis of progression to an immunocompetent response, and drug-induced alterations in either or both parameters can indicate a potential for impaired host defense or selftolerance. Furthermore, ex vivo functional assays have the potential to elucidate mechanisms of drug-induced immunotoxicity and may provide essential information to address human risk (i.e., relevant host defense models). Finally, state-of-the-art methodologies enable rapid, highly sensitive and comprehensive evaluations of leukocyte activity, and implementation in preclinical evaluations may assist in the identification and/or characterization of drug-induced immunotoxicity. 

Shandelya, S. M., P. Kuppusamy, M. L. Weisfeldt, and J. L. Zweier. 1993. Evaluation of the role of polymorphonuclear leukocytes on contractile function in myocardial reperfusion injury. Evidence for plasma-mediated leukocyte activation. 87(2) 536 6. 

A variety of assays have been developed to quantify phagocytic activity. These include direct microscopic visualization (2,3), spectrophotometric evaluation of phagocytized paraffin droplets containing dye (4), scintillation counting of radiolabeled bacteria (5), fluorometric (6), and flow cytometric analysis of fluorescent particles (7-13). The flow cytometric assay offers the advantage of rapid analysis of thousands of cells and quantification of the internalized particle density for each analyzed cell. The assay may be performed with purified leukocyte preparations (7-13) or anficoagulated whole blood (14,15). 

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

Human leukocyte elastase (40mU/ml) and 0.1% of the experimental agent were applied to the substrate methyl-O-succinate alanine alanine proline valine-/ -nitroanilide, then incubated at 37°C for 60 minutes. Elastase activity was then evaluated by spectrophotometry. Testing results are provided in Tables 1 and 2. 

The use of PBMCs is usually reserved for those few compounds that show good activity in the primary screen. PBMCs are more difficult to infect than lymphoblastoid cells and more virus is used 100 TCID50. The release of sufficient p24 for measurement usually takes at least 5 d. If only a few compounds are to be evaluated at one time it is possible to increase the number of replicates at each concentration (5-10) since more variation is observed between wells. PBMCs are prepared from human blood donations either from a transfusion center or from a donor panel of healthy volunteers. Transfusion centers can provide a "buffy coat," which is a leukocyte concentrate of one unit of blood containing red blood cells (RBCs) and platelets. It is usually necessary to obtain ethical permission to work with PBMCs from donors because their blood will be tested for p24. The method describes the separation of PBMCs from a buffy coat or a heparinized whole-blood donation from a healthy volunteer. 

Many biological processes take place under dynamic rather than static conditions. For example, the ability of the selectins to mediate the dynamic process of leukocyte rolling is a critical aspect of their function yet most selectin inhibitors have been evaluated in static assays. A smdy evaluating multivalent L-selectin inhibitors revealed dramatic differences in their activities in static cell free versus dynamic cell rolling assays. 

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