RT-PCR analysis of various defensin expression levels in human 16HBE epithelial bronchial cells exposed to A. fumigatus organisms. 16HBE human epithelial tracheal cells (5 × 106) were grown in six well plates for 24 hours. After exposing the cells to RC, SC, HF or latex beads for 18 hours, 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 (Table 1) were used for RNA amplification: hBD1, 273 bp product; hBD2, 199 bp product; hBD8, 176 bp product; hBD9, 174 bp product; hBD18, 400 bp product and human GAPDH, which was used as an internal control, 473-bp product. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus. As a positive control for defensin expression, exposure to human Il-1β was used in all experiments. The hBD1, hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. One of the four experiments is shown. Abbreviations: resting conidia (RC), swollen conidia (SC), hyphal fragments (HF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (Il–1β).