Human bronchial epithelial cells express and secrete MMP-12.

Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins, which may be responsible for enlargement of alveoli in chronic obstructive pulmonary disease (COPD) and remodeling of pulmonary tissue associated with chronic asthma. Here, we provide novel evidence that MMP-12 is expressed and secreted by normal human bronchial epithelial cell cultures (NHBECs) and reveal the regulation of MMP-12 gene expression by tumor necrosis factor-alpha (TNF-alpha), epidermal growth factor (EGF), and interferon gamma (IFN-gamma). Reverse transcription-polymerase chain reaction analyses demonstrated MMP-12 mRNA presence in unstimulated differentiated NHBEC cultures. Cultures stimulated independently with EGF or IFN-gamma failed to alter MMP-12 mRNA abundance, while TNF-alpha, TNF-alpha+EGF, or TNF-alpha+IFN-gamma elicited relatively early (6 h) peak increases in MMP-12 mRNA levels. Western blot analyses specifically indicated the presence of MMP-12 in differentiated NHBEC-conditioned media. These findings indicate that the bronchial epithelium may be an important source of elastolytic activity in COPD and tissue remodeling in chronic asthma.

Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14alpha-demethylase.

To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10(8) and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [(14)C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14alpha-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.