Chronic inflammation contributes to many human diseases and conditions. Often these may be related to diet suggesting that dietary components may either trigger or modify inflammatory responses. Cell culture systems offer controlled settings in which the effect of specific compounds, including nutrients, can be tested for their effects on inflammatory processes and their mechanisms of action investigated. Human umbilical vein endothelial cells (HUVECs) offer such possibilities. The host lab is currently conducting research with HUVECs. It is proposed to compare the effects of palmitoleic acid, which is not explored in this model, with those of the related fatty acids palmitic acid and oleic acid. The work will identify novel actions and mechanisms of the impact of palmitoleic acid on inflammatory processes. So, HUVECs will be cultured under the standard conditions. They will be exposed to palmitic, oleic or palmitoleic acid at concentrations from 100 to 500 ¼M for a period of 24 hours. This is known to be sufficient time to allow incorporation of fatty acids into the cells; incorporation of the fatty acids will be confirmed through fatty acid analysis of cells using gas chromatography. The modified cells will then be exposed to an inflammatory stimulus. After various periods of times cells will be studied for the appearance of inflammatory markers on their surface using flow cytometry while cell signalling events will be followed by Western blotting of key proteins and confocal microscopy (protein movement within the cell) and changes in inflammatory gene expression will be followed by RT-PCR. Cell culture supernatants will be assayed by ELISA for the appearance of cytokines, chemokines and lipid mediators (eicosanoids, resolvins) involved in the inflammatory response.
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