The data were normalized to Trappin-2/Elafin levels in the Ecx, w

The data were normalized to Trappin-2/Elafin levels in the Ecx, which typically expressed low

amounts of Trappin-2/Elafin mRNA. As shown in Fig. 1a, in all four patients, FT had the highest levels of Trappin-2/Elafin expression – 10–368-fold higher than that seen in Ecx – set at 1. Trappin-2/Elafin mRNA levels in the Cx were also greater than the Ecx, being 2–36-fold higher. UT epithelial cells, however, typically showed very low Trappin-2/Elafin mRNA expression, which was significantly lower than epithelial cells from all the other compartments (FT, Cx, Ecx). In order to determine whether this pattern of mRNA expression would ACP-196 nmr match that of protein expression, we analyzed the CM collected from FRT epithelial cells from FT, UT, Cx and Ecx, by Trappin-2/Elafin ELISA. As shown in Fig. 1b, when CM from multiple patients was analyzed, we found that FT epithelial cells secreted the highest levels of Trappin-2/Elafin, significantly higher than that of UT, Cx and Ecx. The average of three to five patients per tissue is shown in Fig. 1b. Our laboratory has previously reported that the FRT epithelial cells can mount

an antiviral response upon stimulation with Poly(I:C), a synthetic mimic for viral dsRNA.11,12 Therefore, we were interested in determining whether Trappin-2/Elafin, a known antimicrobial, would also be produced in response to Poly(I:C) stimulation. As shown in Fig. 2a, when UT epithelial cells were treated with Poly(I:C) Rapamycin in vitro for 24 hr, Trappin-2/Elafin

mRNA expression was significantly up-regulated by four- to 95-fold when compared with control cells whose expression was set at 1 (six out of six patients). In a time–course experiment where cells were treated with Poly(I:C) and harvested 3, 6 and 24 hr after treatment, we observed that Poly(I:C) treatment up-regulated Trappin-2/Elafin mRNA expression at 6 hr, with continued increases seen at 24 hr (Fig. 2b). To demonstrate whether Poly(I:C) also CHIR-99021 datasheet stimulated secretion of Trappin-2/Elafin protein we analyzed 24 hr CM by ELISA. As shown for a representative patient (Fig. 2c), we found that Trappin-2/Elafin secretion by UT epithelial cells is significantly increased upon Poly(I:C) stimulation. Furthermore, when apical and basolateral secretions were analyzed, we found that the secretion of Trappin-2/Elafin was preferentially apical. The concentration of Trappin-2/Elafin was measurable in basolateral secretions, but very low relative to apical secretions (data not shown). To evaluate more fully the extent of Poly(I:C)-mediated Trappin-2/Elafin secretion throughout the FRT, similar analyses were carried out with FT, Cx and Ecx epithelial cells. Unexpectedly, we found that whereas cells from all compartments constitutively produced Trappin-2/Elafin both at the mRNA and the protein levels (Fig.

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