Proinflammatory cytokines assay

Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

Immediately after a report confirmed that the promoter for SEPSl was stimulated by proinflammatory cytokines, and also showed that this promoter was the target of the critical inflammatory regulator NF-kB. The data included direct binding of this transcription factor to the promoter region using gel mobility shift assays. However, the stimulation of the promoter by cytokines and activation of NF-kB did not result in synergistic production of the mRNA. The role that the cytokines or inflammation plays in regulation of expression of selenoprotein S is not yet clear. 

Predictive in vitro test methods are as well developed as nonanimal test alternatives, including specific cell-based assays.22 Such cellular assays include the culture of keratinocytes that represent very often the first cells in the skin to encounter potential reactive chemicals. Purified keratinocytes are cultured with a specific test chemical, and the production of proinflammatory cytokines or chemokines are then measured. Another cell population that can be used to test for chemicals inducing contact allergy are Langerhans cells (LCs). LCs are the... 

It is well documented that normal human bronchial epithelial cells release a variety of cytokines and chemokines, and proinflammatory cytokines such as IL-la, IL-1 3, and TNF-a stimulate their production in vitro (Fig. 11) [61], We cultured human normal and transformed bronchial epithelial cells, and studied the effect of EM, CAM, and RXM on IL-6 [17], IL-8 [60, 62] and GM-CSF [63] production. Among the antimicrobes tested, only 14-membered-ring macrolides EM, CAM, and RXM showed an inhibitory action on cytokine release by unstimulated and cytokine-stimulated human bronchial epithelial cells (Fig. 12). In contrast, a 16-membered ring macrolide josamycin (JM) failed to show such effects. LDH release assay, a trypan blue dye exclusion test, and a colorimetric MTT assay showed that this effect was not due to cytotoxicity. Percent inhibition of IL-8 protein release in human primary bronchial epithelial cells was 25.0 5.67% at lO M, which is comparable with clinically observed serum concentration. 

Potential effects on cytokine expression should be determined. The role of cytokine transcription or production should be evaluated as well as the modulation of cytokine receptors. It should also be investigated if cytokine transcription or production is skewed (TH1/TH2). It will require careful determination of which cytokines to measure to obtain most useful information (e.g., proinflammatory, specific immunoregula-tory cytokines). It is recommended to investigate a broader panel of cytokines than is currently used. Both basal and activated cytokine production should be measured, and for activated cytokine production, anti-CD3 and anti-CD28, LPS, or allergen should be used. Whole blood assay is the most promising option due to advanced stage of prevalidation. 

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