Dennis W. Nielson

Airway Microbiota and Pathogen Abundance in Age- Stratified Cystic Fibrosis Patients

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective “early” and “late” colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Submucosal gland dysfunction as a primary defect in cystic fibrosis

It has been proposed that defective submucosal gland function in CF airways is a major determinant of CF airway disease. We tested the hypothesis that submucosal gland function is defective early in CF subjects with minimal clinical disease. Functional assays of gland fluid secretion rate and viscosity were performed on freshly obtained nasal biopsies from 6 CF subjects and 5 non‐CF controls (age range 2–22 years). Secretions from individual submucosal glands were visualized by light/fluorescence microscopy after orienting and immobilizing biopsy specimens in a custom chamber. The viscosity of freshly secreted gland fluid after pilocarpine, measured by fluorescence recovery after photobleaching of microinjected FITC‐dextran, was 4.9 ± 0.2‐ vs. 2.2 ± 0.2‐fold greater than water viscosity in CF vs. non‐CF specimens, respectively (se, P<10−4). Gland fluid secretion rate in CF specimens, measured by video imaging (4.5±0.5 nL/min/gland, n=6), was 2.7–fold reduced compared to non‐CF specimens (n=3, P<0.05). Quantitative histology revealed similar size and morphology of submucosal glands in CF and non‐CF specimens. Our results suggest that defective airway submucosal gland function is an early, primary defect in CF. Therapies directed at normalizing gland fluid secretion early in CF may thus reduce lung disease.

Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl− channel function

The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to approximately 1-microm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 +/- 0.5 microm (SE 10 cultures) under basal conditions, 8.4 +/- 0.4 microm following ENaC inhibition by amiloride, and 14.5 +/- 1.2 microm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 +/- 0.7 microm under basal conditions, 11.0 +/- 1.7 microm following amiloride, 17.0 +/- 3.4 microm following cAMP agonists, and 7.1 +/- 0.5 microm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth.

Antibiotic Prescribing During Pediatric Ambulatory Care Visits for Asthma

OBJECTIVE: National guidelines do not recommend antibiotics as an asthma therapy. We sought to examine the frequency of inappropriate antibiotic prescribing during US ambulatory care pediatric asthma visits as well as the patient, provider, and systemic variables associated with such practice. PATIENTS AND METHODS: Data from the National Ambulatory Medical Care Surveys and National Hospital Ambulatory Medical Care Survey were examined to assess office and emergency-department asthma visits made by children (aged <18 years) for frequencies of antibiotic prescription. International Classification of Diseases, Ninth Revision (ICD-9) codes were used to assess the presence of coexisting conditions warranting antibiotics. Multivariable logistic regression models assessed associations with the prescription of antibiotics. RESULTS: From 1998 to 2007, an estimated 60.4 million visits occurred for asthma without another ICD-9 code justifying antibiotic prescription. Antibiotics were prescribed during 16% of these visits, most commonly macrolides (48.8%). In multivariate analysis, controlling for patient age, gender, race, insurance type, region, and controller medication use, systemic corticosteroid prescription (odds ratio [OR]: 2.69 [95% confidence interval (CI): 1.68–4.30]) and treatment during the winter (OR: 1.92 [95% CI: 1.05–3.52]) were associated with an increased likelihood of antibiotic prescription, whereas treatment in an emergency department was associated with decreased likelihood (OR: 0.48 [95% CI: 0.26–0.89]). A second multivariate analysis of only office-based visits demonstrated that asthma education during the visits was associated with reduced antibiotic prescriptions (OR: 0.46 [95% CI: 0.24–0.86]). CONCLUSIONS: Antibiotics are prescribed during nearly 1 in 6 US pediatric ambulatory care visits for asthma, ∼1 million prescriptions annually, when antibiotic need is undocumented. Additional education and interventions are needed to prevent unnecessary antibiotic prescribing for asthma.

Hypertonic saline therapy in cystic fibrosis : Evidence against the proposed mechanism involving aquaporins

Recent data indicate the clinical benefit of nebulized hypertonic saline in cystic fibrosis lung disease, with a proposed mechanism involving sustained increase in airway surface liquid volume. To account for the paradoxical observation that amiloride suppresses the beneficial effect of hypertonic saline, it has been previously concluded (Donaldson, S. H., Bennett, W. D., Zeman, K. L., Knowles, M. R., Tarran, R., and Boucher, R. C. (2006) N. Engl. J. Med. 354, 241-250) that amiloride-inhibitable aquaporin (AQP) water channels in airway epithelia modulate airway surface liquid volume. Here, we have characterized water permeability and amiloride effects in well differentiated, primary cultures of human airway epithelial cells, stably transfected Fisher rat thyroid epithelial cells expressing individual airway/lung AQPs, and perfused mouse lung. We found high transepithelial water permeability (Pf, 54 ± 5 μm/s) in airway epithelial cells that was weakly temperature-dependent and inhibited by >90% by reduced pH in the basal membrane-facing solution. Reverse transcription-PCR and immunofluorescence suggested the involvement of AQPs 3, 4, and 5 in high airway water permeability. Experiments using several sensitive measurement methods indicated that amiloride does not inhibit water permeability in non-cystic fibrosis (non-CF) or CF airway epithelia, AQP-transfected Fisher rat thyroid cells, or intact lung. Our data provide evidence against the mechanism proposed by Donaldson et al. to account for the effects of amiloride and hypertonic saline in CF lung disease, indicating the need to identify alternate mechanisms.

Improving transition from paediatric to adult cystic fibrosis care: programme implementation and evaluation

Background The paradigm of cystic fibrosis (CF) care has changed as effective therapies extend the lives of patients well into adulthood. Preparing for and maintaining high quality CF care into the adult healthcare setting is critical for prolonged survival. Unfortunately, this transfer process from the paediatric to the adult CF centre is met with a variety of challenges. Objective and methods The objective of this quality improvement (QI) project was to develop, implement and evaluate a theory-based programme for transition from paediatric to adult CF care. In a multi-phase process, the paediatric and adult programmes developed a transition curriculum, addressed care standards and standardised patient transfer protocols. We evaluated the impact of this process through staff surveys, review of field notes from QI meetings, tracking transfers and responses of patients to the Transition Readiness Assessment Questionnaire (TRAQ) at the start of the programme and 18 months after initiation. Results The collaboration between the paediatric and adult teams continued through quarterly meetings over the past 4 years. This has provided a forum that sustained our transition programme, harmonised care across CF centres and addressed other needs of our CF centre. Discussion of transition with families in the paediatric centre increased twofold (35% to 73% p<0.001), and resulted in a trend towards improved patient TRAQ self-advocacy scores and decreased in-hospital transfer. Conclusions We successfully created a curriculum and process for transition from paediatric to adult CF care at our centres. This collaboration shapes the communication between our paediatric and adult CF care teams and enables ongoing feedback among patients, families and providers. The impact of our transition programme on long-term patient morbidity will require future evaluation.

Management of Plastic Bronchitis With Topical Tissue-type Plasminogen Activator

Plastic bronchitis or cast bronchitis is a rare disease of unclear etiology characterized by formation of airway casts that can lead to life-threatening airway obstruction. There is currently limited data regarding optimal treatment of plastic bronchitis. Several therapies have been suggested, but recurrences are common and mortality remains high. We report the case of a 6-year-old boy with refractory eosinophilic bronchial casts, unresponsive to low-dose systemic corticosteroids, inhaled corticosteroids, azithromycin, and dornase alfa, who was treated successfully and safely with direct instillation of tissue-type plasminogen activator (tPA) to the obstructing casts during flexible bronchoscopy and inhaled tPA. Our case illustrates that the current therapy for plastic bronchitis remains inadequate. To our knowledge, this case is the first to show that direct instillation of tPA can be used safely for treatment of this disease. The use of tPA via direct administration into the airways during bronchoscopy and via a nebulizer appeared to be a safe and effective therapy for plastic bronchitis and should be considered early in the course of the disease to prevent complications of severe airway obstruction.

Correctors and Potentiators Rescue Function of the Truncated W1282X-Cystic Fibrosis Transmembrane Regulator (CFTR) Translation Product

W1282X is the fifth most common cystic fibrosis transmembrane regulator (CFTR) mutation that causes cystic fibrosis. Here, we investigated the utility of a small molecule corrector/potentiator strategy, as used for ΔF508-CFTR, to produce functional rescue of the truncated translation product of the W1282X mutation, CFTR1281, without the need for read-through. In transfected cell systems, certain potentiators and correctors, including VX-809 and VX-770, increased CFTR1281 activity. To identify novel correctors and potentiators with potentially greater efficacy on CFTR1281, functional screens were done of ∼30,000 synthetic small molecules and drugs/nutraceuticals in CFTR1281-transfected cells. Corrector scaffolds of 1-arylpyrazole-4-arylsulfonyl-piperazine and spiro-piperidine-quinazolinone classes were identified with up to ∼5-fold greater efficacy than VX-809, some of which were selective for CFTR1281, whereas others also corrected ΔF508-CFTR. Several novel potentiator scaffolds were identified with efficacy comparable with VX-770; remarkably, a phenylsulfonamide-pyrrolopyridine acted synergistically with VX-770 to increase CFTR1281 function ∼8-fold over that of VX-770 alone, normalizing CFTR1281 channel activity to that of wild type CFTR. Corrector and potentiator combinations were tested in primary cultures and conditionally reprogrammed cells generated from nasal brushings from one W1282X homozygous subject. Although robust chloride conductance was seen with correctors and potentiators in homozygous ΔF508 cells, increased chloride conductance was not found in W1282X cells despite the presence of adequate transcript levels. Notwithstanding the negative data in W1282X cells from one human subject, we speculate that corrector and potentiator combinations may have therapeutic efficacy in cystic fibrosis caused by the W1282X mutation, although additional studies are needed on human cells from W1282X subjects.

Normalization of sweat chloride concentration and clinical improvement with ivacaftor in a patient with cystic fibrosis with mutation S549N.

The cystic fibrosis (CF) protein forms an anion channel in epithelial cells, and the absence or defective function of this channel results in the clinical manifestations of CF. CF is an autosomal recessive disorder, and its many disease-causing mutations divide into five or six classes. There are 10 known class 3 gating mutations, the most common of which is G551D. Ivacaftor is a drug that in vitro increases open time and transepithelial chloride transport in all 10 gating mutations, but it is approved for use only in patients with the G551D mutation. We report complete normalization of sweat chloride concentration and rapid clinical improvement over 6 weeks of treatment with ivacaftor in a patient with CF with the gating mutation S549N. The findings suggest that ivacaftor should be considered for use in patients with any of the known gating mutations.

In vivo and in vitro ivacaftor response in cystic fibrosis patients with residual CFTR function: N-of-1 studies.

Ivacaftor, a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator, decreases sweat chloride concentration, and improves pulmonary function in 6% of cystic fibrosis (CF) patients with specific CFTR mutations. Ivacaftor increases chloride transport in many other CFTR mutations in non‐human cells, if CFTR is in the epithelium. Some CF patients have CFTR in the epithelium with residual CFTR function. The effect of ivacaftor in these patients is unknown.