Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. Results:We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. (Continued on next page)Conclusions: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF.

Inhaled mannitol, 400 mg twice a day, resulted in improved lung function over 26 weeks, which was sustained after an additional 26 weeks of treatment. The safety profile was also acceptable, demonstrating the potential role for this chronic therapy for CF. Clinical trial registered with www.clinicaltrials.gov (NCT 00630812).

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na ؉ currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, ␣-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and ␣-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, ␣␤␥ mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and ␣-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

Limited information is available regarding domains within the epithelial Na؉ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278 -283) within the extracellular domain of ␣rENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant ␣rENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type ␣rENaC was inhibited by amiloride with a K i of 169 nM. Deletion of the entire WYRFHY tract (␣rENaC ⌬278 -283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K i ‫؍‬ 26.5 M). Similar results were obtained when either ␣rENaC or ␣rENaC ⌬278 -283 were co-expressed with wt ␤-and ␥rENaC (K i values of 155 nM and 22.8 M, respectively). Moreover, ␣rENaC H282D was insensitive to submicromolar concentrations of amiloride (K i ‫؍‬ 6.52 M), whereas ␣rENaC H282R was inhibited by amiloride with a K i of 29 nM. These mutations do not alter ENaC Na ؉ :K ؉ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of ␣rENaC and mutations preceding the second membrane-spanning domains of ␣-, ␤-, and ␥rENaC alters amiloride's K i , suggest that selected regions of the extracellular loop of ␣rENaC may be in close proximity to residues within the channel pore.The diuretic amiloride is a prototypic inhibitor of epithelial Na ϩ channels (ENaCs) 1 (1), although amiloride and its various derivatives inhibit many Na ϩ -selective transport proteins. Several laboratories have recently identified domains within the epithelial Na ϩ channel and the Na ϩ /H ϩ exchanger that appear to participate in amiloride binding. Residues within the second membrane-spanning domain of ␣rENaC may interact with amiloride, as mutations of a serine residue at position 589 result in a large decrease of the apparent K i for amiloride and the amiloride analog benzamil, as well as alter cation selectivity (2). Selected mutations of residues within a hydrophobic region, termed H2 (3), immediately preceding the second membrane-spanning domains of the ␣-, ␤-, and ␥-subunits of rENaC (i.e. Trp-␣582, Ser-␣583, Gly-␤525, Gly-␥537) and the ␣-subunit of bovine ENaC (Lys-504, Lys-515) affect the K i for amiloride, and several of these mutations affect single-channel conductance (4, 5). Snyder and co-workers have identified splice variants of ␣rENaC in which the C-terminal 199 or 216 amino acid residues, including the second membrane-spanning domain, are truncated (6). These splice variants are not functional when expressed in Xenopus oocytes, but retain amiloride and phenamil binding activity, s...

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Sustained six-month improvements in lung function and decreased pulmonary exacerbation incidence indicate that inhaled mannitol is an important additional drug in the treatment of CF.

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.