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Robert A. Sack, A Beaton, B. Cooper, S. Sathe, A Mezentsev, M Schwartzman, L Bellner, P. Istroavich; Diurnal-distribution-selective Down- Regulation Of Cytokines but Not Chemokines In Tears. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1134.
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Overnight eye closure results in a stagnant tear layer extremely enriched in pro-inflammatory and immune-modulating cytokines. This study was designed to further characterize this environment and to determine how cytokine bioactivity is down-regulated.
; Open (O) and Closed (C) tears from Ns were subjected to multiple micro well array assays to obtain quantitative data on >80 low abundance proteins. Samples were also separated by molecular sieve HPLC; the eluents were bioassayed for net angiogenic activity and the elution profiles of chemokines, cytokines and interactive proteins established. Cytokine-protein complexes and other protein-protein complexes were characterized using a novel laboratory-developed multiplex micro well assay.
Protein array analysis reveal statistically significant selective >10x increases in many pro-inflammatory and immune-modulating cytokines, chemokines and several integrins in C fluid, accompanied by a parallel build-up of soluble (s)-decoy receptors and other interacting proteins with the levels of the latter greatly exceeding that of the targeted cytokines (pg versus ng/ml range). HPLC separation followed by array analysis, bioassay, and protein-protein binding assays reveals that the vast majority of pro-inflammatory and immune-modulating cytokines in C are devoid of bioactivity and elute in a common high molecular weight fraction (>360 kDa) in which they can be shown to be bound to specific soluble cytokine-receptors as well as to a yet to be fully characterized complexing protein(s). The pattern of distribution suggests a 2-step binding process that inactivates and tags the cytokines for cleavage. In contrast, nearly all of the accumulating chemokines (IP-10, GROα, MCP-1 and most IL-8), which lack soluble receptors, elute as bioactive monomers or oligomers.
Pro-inflammatory and immune- modulating cytokines accumulating in C fluid are inactivated by a novel 2-step co-operative binding process that maybe involved in controlling inflammation elsewhere. Formation of these complexes can also mask epitopes employed in cytokine sandwich ELISAs and array assays thereby becoming a source of error.
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