Cytokines production measurement

As stated earlier, activation of endothelial cells by pro-inflammatory stimuli leads to the expression of cell adhesion molecules and cytokines such as IL-6 and IL-8. The expression and hence modulation of surface expressed adhesion molecules by e.g. targeted delivery of inhibitors of NFkB, can be measured using flow cytometric analysis or whole cell ELISA techniques. Cytokine production can be measured in the supernatant of cultured cells or in biological fluids. Furthermore, competitive or quantitative RT-PCR analysis of mRNA levels of cell adhesion molecules or cytokines, allows the transcriptional activity of the genes of interest to be estimated. 

Measuring proliferation or cytokine production Despite oats stimulation of T cell lines, it did not activate a mucosal lesion in most subjects ... 

Cytokines, which are protein mediators produced by immune cells, are involved in the regulation of cell activation, growth and differentiation, inflammation, and immunity. Measurement of cytokine production, as determined by techniques such as bioassay, radioimmunoassay, and enzyme-linked immunosorbent assay, has been used to examine various immune functions. 

In vitro lymphocyte proliferative responses to mitogens such as phytohemagglutinin, concanavalin A, and pokeweed mitogen or to specific antigens of interest. The response may be measured by tritiated thymidine incorporation or by cytokine production (IFN-r, IL-2, TNF, and others). 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

In addition to these simpler cell-based assays, more complex assays can be employed to measure the induction of specific T cell responses to haptens. It is possible to stimulate naive T cells to proliferate in vitro in the presence of antigen-presenting cells such as LCs.27-28 In addition, effector T cells (T cell clones) can be used to investigate T cell stimulation induced by haptens in the presence of the appropriate antigen-presenting cells.29 30 Another complex, but easy to use assay is based on the culture of purified human PBMC in the presence of potential reactive chemicals, and the measurement of proliferation or cytokine production.31-33... 

In the measurement of cytokines in body fluids, samples must be collected and stored in the proper manner. The main problems encountered in the collection process include (1) cytokines can continue to be produced after sample collection by the various cells present in the biological fluid (2) collection tubes can become contaminated by microorganisms, which are a potent stimulus of cytokine production (3) cytokines can be degraded in the collection container and (4) cytokines can bind to cell receptors during storage. 

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. 

The ability of xenobiotics to induce (Dastych et al., 1999) or suppress (O Keefe et al, 1992) cytokine expression has been proposed as a possible mechanism explaining their adverse effects on the immune system. To this end, ICS has been used to measure drug-induced inhibition of cytokine production, inhibition of cytokine release, induction of immunosuppressive cytokines, cytokine homeostatic maintenance, and cytokine-induced alterations in cellular activation or transcriptional mechanisms as reviewed by House (1999). 

What is less clear, at present, is the role that other traditional immune evaluations may play in DIT assessment, including cytokine evaluation, flow cytometry, and immunomics. Based on the literature to date and the specific protocols employed, cell population changes, cytokine production alterations, dendritic cell and/or macrophage assessment, and immune histologic manifestation seem to be accessory parameters that are supportive of functional changes but not of themselves sentinel predictors of DIT. As described for adult animals (see Chapter 4), cytokine measurements in serum or peripheral blood may potentially be performed as a functional means to assess... 

In a mouse vaccination model adapted to study the effect of prebiotics, the animals were vaccinated twice with the Influvac Orthomyxovirus influenza) vaccine (booster vaccination after 21 days). The response to the vaccination was measured at day 30 after the first vaccination. Parameters used to identify the response to vaccination were DTH response (skin response after local subcutaneous vaccine injection as an in vivo measure for Thl-mediated immunity), plasma titers of specific antibodies, ex vivo lymphocyte stimulation, T-cell prohferation, cytokine production, and natural killer cell activity. A specific prebiotic mixture (GOS/lcFOS) significantly stimulated the vaccination response in a dose-dependent manner and increased the DTH response indicating a modulation of the immune system toward a Thl-dominated immune response. This effect only... 

Surcel H, Syrjala H, Karttunen R, Tapaninaho S, Herva E. Development of Francisella tularensis antigen responses measured as T-lymphocyte proliferation and cytokine production (tumor necrosis factor alpha, gamma interferon, and interleukin-2 and -4) during human tularemia. Infect Immun. 1991 59 1948-1953. 

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