Additionally, exposure to TNF IFN did not markedly alter transcellular permeability. We observed an eight percent increase in FITC dextran flux at 37 C when compared to the 4 C group. The modest increase in FITC dextran http://www.selleckchem.com/products/Vandetanib.html recovery due to elevated temper ature was expected, however, was not significantly differ ent from the 4 C treatment. In order to examine the effect of dose of TNF IFN on epithelial barrier function confluent MDCK cultures were treated for 24 hours then fluorescein flux was determined. Fluorescein recovery was markedly elevated with increas ing dose of TNF IFN. The lowest dose tested resulted in a two fold elevation in flux, doses of TNF IFN produced a dose dependent significant elevation in flux. In Figure 2B, we report the mannitol flux using MDCK cultures treated for 24 hours with either TNF or IFN.

mannitol was added to the apical chamber and recovery was measured in the basolateral chamber following a 120 minute incubation at 37 C. By in large MDCK cell flux is resistant to effects of these cytokines when administered alone, only the highest dose of TNF produced a significant 40% eleva tion of paracellular flux compared to control. IFN alone had no detectable effect on MDCK flux. In Figure 3B, we report the mannitol flux using MDCK cultures treated for 72 hours with TNF IFN. In control cells, approximately 2% of the mannitol is recovered in the basolateral chamber following a 120 minute assay. MDCK cells treated with TNF IFN produce a two fold increase in mannitol flux permitting 4% of the label to enter the basolateral com partment.

In this model of inflammatory stress using MDCK cells, TER and flux appear not to be inversely related. We investigated the effect of MAP kinase pathway inhibi tors on paracellular flux using MDCK cells exposed to TNF IFN for 24 hr. Following analysis of TER, mannitol flux assays were performed and reported as percent of control. TNF IFN resulted in a 66% increase in flux compared to control cells, U0126 significantly decreased flux, SB202190 decreased flux dose dependently, the combination of U0126 and SB202190 signifi claudin 1, claudin 2 and claudin 3 expression were exam ined in MDCK cells in response to TNF IFN dose fol lowing a twenty four hour exposure. In general, low dose of TNF IFN produces an initial eleva tion in tight junction protein expression with the excep tion of claudin 2.

the intensity and patterns observed vary. In Figure 5B, exposure to TNF IFN for twenty four hours induced a significant 37% 6% eleva tion Brefeldin_A in claudin 1 expression when compared to control as measured by densitometric analysis. The occludin expres sion pattern is comparable to claudin 1, ho ever the magnitude of elevation is modest, only a 22% 6. 5% was observed at the lowest TNF IFN dose. Clau din 2 levels decrease markedly in a dose dependent man ner to 45% 7% of control values.