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 with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in 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.