CFTR expression and activity from the human CFTR locus in BAC vectors, with regulatory regions, isolated by a single-step procedure.

We have assembled two BAC vectors containing a single fragment spanning the entire CFTR locus and including the upstream and downstream regions. The two vectors differ in size of the upstream region, and were recovered in Escherichia coli, with intact BAC DNAs prepared for structural and functional analyses. Sequence analysis allowed precise mapping of the inserts. We show that the CFTR gene was wild type and is categorized as the most frequent haplotype in Caucasian populations, identified by the following polymorphisms: (GATT)7 in intron 6a; (TG)11T7 in intron 8; V470 at position 470. CFTR expression and activity were analyzed in model cells by RT-PCR, quantitative real-time PCR, western blotting, indirect immunofluorescence and electrophysiological methods, which show the presence of an active CFTR Cl ⁻ channel. Finally, and supporting the hypothesis that CFTR functions as a receptor for Pseudomonas aeruginosa, we show that CFTR-expressing cells internalized more bacteria than parental cells that do not express CFTR. Overall, these data demonstrate that the BAC vectors contain a functional CFTR fragment and have unique features, including derivation from a single fragment, availability of a detailed genomic map and the possibility to use standard extraction procedures for BAC DNA preparations.

Inflammatory cytokines and biofilm production sustain Staphylococcus aureus outgrowth and persistence: a pivotal interplay in the pathogenesis of Atopic Dermatitis.

Individuals with Atopic dermatitis (AD) are highly susceptible to Staphylococcus aureus colonization. However, the mechanisms driving this process as well as the impact of S. aureus in AD pathogenesis are still incompletely understood. In this study, we analysed the role of biofilm in sustaining S. aureus chronic persistence and its impact on AD severity. Further we explored whether key inflammatory cytokines overexpressed in AD might provide a selective advantage to S. aureus. Results show that the strength of biofilm production by S. aureus correlated with the severity of the skin lesion, being significantly higher (P < 0.01) in patients with a more severe form of the disease as compared to those individuals with mild AD. Additionally, interleukin (IL)-ß and interferon γ (IFN-γ), but not interleukin (IL)-6, induced a concentration-dependent increase of S. aureus growth. This effect was not observed with coagulase-negative staphylococci isolated from the skin of AD patients. These findings indicate that inflammatory cytokines such as IL1-ß and IFN-γ, can selectively promote S. aureus outgrowth, thus subverting the composition of the healthy skin microbiome. Moreover, biofilm production by S. aureus plays a relevant role in further supporting chronic colonization and disease severity, while providing an increased tolerance to antimicrobials.