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Successful Development of a Flow Cytometric Assay to Analyze Cytokine-Induced Phosphorylation Pathways [CIPP] Within Peripheral Blood Leukocytes

Montag, David Thomas (2006) Successful Development of a Flow Cytometric Assay to Analyze Cytokine-Induced Phosphorylation Pathways [CIPP] Within Peripheral Blood Leukocytes. Master's Thesis, University of Pittsburgh. (Unpublished)

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Current strategies designed to assess cells in the peripheral blood are limited to evaluation of phenotype or delayed measurement [>6 hours] of function, usually quantifying cytokine production, cytolytic activity, or response to antigens. Furthermore, these assays often do not assess the individual cell types comprising peripheral blood, instead providing a bulk-readout for the total population. We reasoned that functional abnormalities in distinct peripheral blood cells, which act as immune sentinels capable of rapidly reacting to various injurious agents while circulating throughout the body, could serve as migratory biomarkers, reflecting pathological environs that cells experience in the setting of inflammatory states, including cancer. Two major pathways regulating immune responses are the JAK/STAT and MAPK pathways. These pathways are initiated by ligand-receptor binding, and are rapidly propagated by subsequent protein phosphorylation cascades. Additionally, these pathways are often abnormally activated within cancer cells themselves. We applied flow cytometric strategies, evaluating the brief application of cytokines in vitro to interrogate the early phosphorylation events of these signaling pathways in normal peripheral blood mononuclear cells (PBMC). Individual cytokine doses and time intervals of treatment were assessed to identify conditions useful in a clinical laboratory and as an initial goal, to induce maximal phosphorylation. Surprisingly, all of the STAT proteins assessed and ERK1/2 are maximally phosphorylated within 15 minutes in human PBMC, simply following addition of cytokines alone. Within two hours, cells typically return to their basal phosphorylation states. Confirmation of these results was achieved by Western blotting. Increased phosphorylation usually correlated with increased concentrations of individual cytokines. Furthermore, we developed conditions to enable simultaneous staining of cell surface proteins, identifiable with distinct PBMC subpopulations: CD4, CD8, CD14, CD19, and CD56, together with intracellular phosphorylated proteins. Of the permeabilizing conditions tested, 75% methanol enabled superior simultaneous detection of both cell surface and intracellular epitopes. Using this technique, we were able to identify differential responsiveness between individual cell types. These strategies will enable robust development of simple blood analyses to identify normal activation as well as impairments in STAT and MAPK signaling pathways associated with various human disease states including acute and chronic inflammatory conditions throughout clinical immunology.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Montag, David Thomasdtm7@pitt.eduDTM7
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairLotze, Michael Tlotzemt@upmc.eduMTL5
Committee MemberDonnenberg, Albert Ddonnenbergad@upmc.eduDONNAL
Committee MemberRoy, Parthaproy@engr.pitt.eduPAR19
Date: 27 September 2006
Date Type: Completion
Defense Date: 6 July 2006
Approval Date: 27 September 2006
Submission Date: 18 July 2006
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Bioengineering
Degree: MSBeng - Master of Science in Bioengineering
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: cell signaling; immunity; lymphocytes; mitogen; monocytes
Other ID:, etd-07182006-004114
Date Deposited: 10 Nov 2011 19:51
Last Modified: 19 Dec 2016 14:36


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