Poster: Cynthia Boardman, Yu Suen, Keith Roby and Scott Boyer
The pattern of cytokine production by helper T cells directs the body’s response to disease and infection. Helper cells are divided into classes based on the cytokines they produce. Th1 cells produce IFNγ, IL-2, TNFα, and TNFβ and mediate a pro-inflammatory (cell-mediated) response while Th2 cells produce IL-4, IL-5, IL-6, IL-10 and TNFβ and mediate an anti-inflammatory (humoral or antibody-mediated) response.
The type of response stimulated by an infection or disease informs treatment choices and influences treatment efficacy. For example, many drugs are designed to target specific immune-system mechanisms while others may solicit unintended and adverse immunotoxic effects such as immunosupression, hypersensitivity (allergenicity) and inflammation. Recently, the United States FDA has issued guidelines to the pharmaceutical industry recommending nonclinical immunotoxicity testing for all new human pharmaceuticals.** The ability to monitor the expression of mediators of inflammation and immune response is therefore critical to the study of disease and of pharmacodynamics.
We show here the potential of the GenomeLab™ GeXP genetic analysis system to become a powerful ally in responding to the challenge to monitor immune response. Specifically, we have used the GenomeLab GeXP system to develop a multiplexed RT-PCR§ assay that can analyze the mRNA expression profile of 10 Th1/Th2 cytokines simultaneously (IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNFα and TNFβ). We then used this multiplex to analyze changes in the mRNA expression of these genes in peripheral blood mononuclear cells upon stimulation with lipopolysaccharide or a combination of phorbol-13-myristate-12-acetate and phytohemagglutinin. Supernatants from the same cell samples were analyzed for cytokine protein content via the Th1/Th2 10-Plex quantitative fluorescent bead immunoassay using the Cytomics FC500 MPL Flow Cytometry System with the Biomek® FX laboratory automation workstation. Results from both systems are presented.
**U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Biologics Evaluation and Research (CBER) (April 2006) Guidance for Industry S8 Immunotoxicity Studies for Human
Pharmaceuticals (http://www.fda.gov/cder/guidance/6748fnl.htm)
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