Heat inactivation of any of the serums led to the partial decrease of the expression of both of the tested defensins by cells exposed either to A. fumigatus conidia, HF, or to Il-1β (Figure 2C, D). Kinetics of defensin expression by cells exposed selleck chemical to A. fumigatus organisms To analyse the kinetics of defensin expression, cells were exposed to A. fumigatus for 4, 8 and 18 hours, and the expression of hBD2 and hBD9 was examined. As a positive control, Il-1β-treated cells were examined. As a negative control, untreated cells or cells exposed to 5 × 106 latex beads were analysed as well. According to the results presented on Figure 3, the expression of both defensins, hBD2 and hBD9, were

induced in the 16HBE cells treated with Il-1β either

for 4, 8 or 18 hours. No hBD2 expression was detected after a 4-h exposure by 16HBE to SC, RC or HF of A. fumigatus, in contrast to hBD9 expression by cells exposed to all morphotypes of A. fumigatus for the same period. Incubation of the cells with both types of conidia or HF for 8 h see more resulted in a low level of hBD2 expression and a high level of hBD9 expression, comparable to expression by the cells treated with the positive control, Il-1β. Exposure of the cells to conidia or HF for 18 h led to the high expression of both defensins, hBD2 and hBD9. Exposure of the cells to the latex beads did not induce the defensin expression in any of the experiments. The constitutive expression of hBD1 by the cells exposed either to the different morphotypes of A. fumigatus or to the latex beads for the various periods was observed in the current experiment. Figure 3 Kinetics of defensin mRNA expression by 16HBE human epithelial

bronchial cells exposed to A. fumigatus organisms. 16HBE human epithelial tracheal cells (5 × 106) were grown in six well plates for 24 hours. The cells were then exposed to the different morphotypes of A. fumigatus or latex beads for the different periods: 4 h, 8 h and 18 h. After incubation, the cells were washed Urocanase with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in CT99021 molecular weight Materials and Methods. Specific primer pairs and the conditions of RT-PCR are described in Table 1. The sizes of amplified products are indicated and were as predicted: hBD2, 199-bp product; hBD9, 174 bp product and human GAPDH, 473-bp product. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. Cells were cultivated in a control well in the absence of A. fumigatus. GAPDH was uniformly expressed. One of the four results is shown. Similar kinetics of hBD2 and hBD9 expression was observed with A549 cells (data not shown). Real time PCR The relative level of hBD2 and hBD9 expression in 16HBE and A549 cells exposed to different A. fumigatus morphotypes for 18 hours was quantified by real time PCR.