Moira L. Aitken

Sputum induction as a research tool for sampling the airways of subjects with cystic fibrosis

BACKGROUND Sputum induction (SI) has proved to be a reliable non-invasive tool for sampling inflammatory airway contents in asthma, with distinct advantages over collection of expectorated sputum (ES) and bronchoalveolar lavage (BAL). A study was undertaken to evaluate the safety of SI and to assess if it might be an equally valuable outcome tool in patients with cystic fibrosis (CF). METHODS The safety of the procedure was examined and sample volume, cell counts, cytokine concentrations, and bacterial culture results obtained by SI, spontaneous ES, and fibreoptic bronchoscopy were compared in 10 adults with CF. RESULTS SI was well tolerated and was preferred to BAL by all subjects. The mean (SE) sample volume obtained by SI was significantly greater than ES (6.74 (1.46) ml v 1.85 (0.33) ml, p = 0.005). There was no significant difference in the number of cells per ml of sample collected. There was a difference in the mean (SD) percentage of non-epithelial, non-squamous cells collected (67 (28)%, 86 (21)%, and 99 (1)% for ES, SI, and BAL, respectively). These percentage counts were different between ES and both SI and BAL (p=0.03 and p=0.006, respectively). Cell differential counts (excluding squamous cells) from all collection methods were similar (mean (SD) 84 (9)%, 87 (7)%, and 88 (11)% polymorphonuclear cells for ES, SI, and BAL, respectively). The concentrations of interleukin (IL)-8 and tumour necrosis factor (TNF)-α were the same in all three samples when corrected for dilution using urea concentration. The test specific detection rate for recovery of bacteriological pathogens was 79% for SI, 76% for ES, and 73% for BAL. CONCLUSION SI offers safety advantages over BAL and may be a more representative airway outcome measurement in patients with CF.

Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation

BACKGROUND A new approach in the treatment of cystic fibrosis involves improving the function of mutant cystic fibrosis transmembrane conductance regulator (CFTR). VX-770, a CFTR potentiator, has been shown to increase the activity of wild-type and defective cell-surface CFTR in vitro. METHODS We randomly assigned 39 adults with cystic fibrosis and at least one G551D-CFTR allele to receive oral VX-770 every 12 hours at a dose of 25, 75, or 150 mg or placebo for 14 days (in part 1 of the study) or VX-770 every 12 hours at a dose of 150 or 250 mg or placebo for 28 days (in part 2 of the study). RESULTS At day 28, in the group of subjects who received 150 mg of VX-770, the median change in the nasal potential difference (in response to the administration of a chloride-free isoproterenol solution) from baseline was -3.5 mV (range, -8.3 to 0.5; P=0.02 for the within-subject comparison, P=0.13 vs. placebo), and the median change in the level of sweat chloride was -59.5 mmol per liter (range, -66.0 to -19.0; P=0.008 within-subject, P=0.02 vs. placebo). The median change from baseline in the percent of predicted forced expiratory volume in 1 second was 8.7% (range, 2.3 to 31.3; P=0.008 for the within-subject comparison, P=0.56 vs. placebo). None of the subjects withdrew from the study. Six severe adverse events occurred in two subjects (diffuse macular rash in one subject and five incidents of elevated blood and urine glucose levels in one subject with diabetes). All severe adverse events resolved without the discontinuation of VX-770. CONCLUSIONS This study to evaluate the safety and adverse-event profile of VX-770 showed that VX-770 was associated with within-subject improvements in CFTR and lung function. These findings provide support for further studies of pharmacologic potentiation of CFTR as a means to treat cystic fibrosis. (Funded by Vertex Pharmaceuticals and others; ClinicalTrials.gov number, NCT00457821.).

Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation

Background VX-809, a cystic fibrosis transmembrane conductance regulator (CFTR) modulator, has been shown to increase the cell surface density of functional F508del-CFTR in vitro. Methods A randomised, double-blind, placebo-controlled study evaluated the safety, tolerability and pharmacodynamics of VX-809 in adult patients with cystic fibrosis (n=89) who were homozygous for the F508del-CFTR mutation. Subjects were randomised to one of four VX-809 28 day dose groups (25, 50, 100 and 200 mg) or matching placebo. Results The type and incidence of adverse events were similar among VX-809- and placebo-treated subjects. Respiratory events were the most commonly reported and led to discontinuation by one subject in each active treatment arm. Pharmacokinetic data supported a once-daily oral dosing regimen. Pharmacodynamic data suggested that VX-809 improved CFTR function in at least one organ (sweat gland). VX-809 reduced elevated sweat chloride values in a dose-dependent manner (p=0.0013) that was statistically significant in the 100 and 200 mg dose groups. There was no statistically significant improvement in CFTR function in the nasal epithelium as measured by nasal potential difference, nor were there statistically significant changes in lung function or patient-reported outcomes. No maturation of immature F508del-CFTR was detected in the subgroup that provided rectal biopsy specimens. Conclusions In this study, VX-809 had a similar adverse event profile to placebo for 28 days in F508del-CFTR homozygous patients, and demonstrated biological activity with positive impact on CFTR function in the sweat gland. Additional data are needed to determine how improvements detected in CFTR function secondary to VX-809 in the sweat gland relate to those measurable in the respiratory tract and to long-term measures of clinical benefit. Clinical trial number NCT00865904

Effect of genotype on phenotype and mortality in cystic fibrosis: a retrospective cohort study.

BACKGROUND Over 1000 mutations of the cystic fibrosis transmembrane conductance regulator gene (CFTR) that cause cystic fibrosis have been identified. We examined the effect of CFTR genotype on mortality and disease phenotype. METHODS Using the US Cystic Fibrosis Foundation National Registry, we did a retrospective cohort study to compare standardised mortality rates for the 11 most common genotypes heterozygous for DeltaF508 with those homozygous for DeltaF508. Of the 28455 patients enrolled in the registry at the time of our analysis, 17853 (63%) were genotyped. We also compared the clinical phenotype, including lung function, age at diagnosis, and nutritional measures, of 22 DeltaF508 heterozygous genotypes. Mortality rates and clinical phenotype were also compared between genotypes classified into six classes on the basis of their functional effect on CFTR production. FINDINGS Between 1991 and 1999, genetic and clinical data were available for 17853 patients with cystic fibrosis, which was 63% of the total cohort. There were 1547 deaths during the 9 years of follow-up. In the analysis of the 11 most common genotypes, DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/3849+10kbC-->T, and DeltaF508/2789+5G-->A had a significantly lower mortality rate (4.7, 8.0, 11.9, and 4.4, respectively) than the genotype homozygous for DeltaF508 (21.8, p=0.0060). DeltaF508/R117H, DeltaF508/DeltaI507, DeltaF508/ 3849+10 kbC-->T, DeltaF508/2789+5G-->A, and DeltaF508/A455E have a milder clinical phenotype. Outcomes for all functional classes were compared with that of class II (containing DeltaF508 homozygotes) and classes IV and V had a significantly lower mortality rate and milder clinical phenotype. INTERPRETATION Patients with cystic fibrosis have distinct genetic subgroups that are associated with mild clinical manifestations and low mortality. These differences in phenotype are also related to the functional classification of CFTR genotype.

Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.

A Phase I Study of Aerosolized Administration of tgAAVCF to Cystic Fibrosis Subjects with Mild Lung Disease

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota

Recent work using culture-independent methods suggests that the lungs of cystic fibrosis (CF) patients harbor a vast array of bacteria not conventionally implicated in CF lung disease. However, sampling lung secretions in living subjects requires that expectorated specimens or collection devices pass through the oropharynx. Thus, contamination could confound results. Here, we compared culture-independent analyses of throat and sputum specimens to samples directly obtained from the lungs at the time of transplantation. We found that CF lungs with advanced disease contained relatively homogenous populations of typical CF pathogens. In contrast, upper-airway specimens from the same subjects contained higher levels of microbial diversity and organisms not typically considered CF pathogens. Furthermore, sputum exhibited day-to-day variation in the abundance of nontypical organisms, even in the absence of clinical changes. These findings suggest that oropharyngeal contamination could limit the accuracy of DNA-based measurements on upper-airway specimens. This work highlights the importance of sampling procedures for microbiome studies and suggests that methods that account for contamination are needed when DNA-based methods are used on clinical specimens.

Molecular Mechanism of Mucin Secretion: I. The Role of Intragranular Charge Shielding

Mucus is an ubiquitous polymer hydrogel that functions as a protective coat on the surface of integument and mucosa of species ranging from simple animals (such as coelenterates) to mammals. The polymer matrix of mucus is made out of long-chain glycoproteins called mucins that are tangled together, forming a randomly woven, highly polyionic network (Lee et al., 1977; Verdugo et al., 1983). Mucin-containing granules, produced by mammalian goblet cells in vitro, undergo massive post-exocytotic swelling. Their swelling kinetics is similar to the swelling of condensed artificial polymer gels (Verdugo, 1984; Tanaka and Fillmore, 1979). We had proposed that mucins must be condensed in the secretory granule and expand by hydration during or after exocytosis (Verdugo, 1984; Tam and Verdugo, 1981). However, the polyionic charges of mucins prevents condensation unless they (the mucins) are appropriately shielded. The present experiments were designed to assert the presence of an intragranular shielding cation and its role in secretion. Giant mucin granules of the slug (Ariolimax columbianus) are released intact from mucus-secreting cells of the slugs skin. They burst spontaneously outside the cell, forming, upon hydration, the typical slug mucus (Deyrup-Olsen et al., 1983). We report here that these granules contain from 2.5 to 3.6 moles calcium/kg dry material, and that calcium is released from the granules immediately before the burst that discharges their secretory product. Therefore, we propose that calcium functions as a shielding cation of poly ionic mucins, and that the bursting discharge of mucins from secretory granules must result from the release of calcium from the intragranular compartment. Calcium release would unshield the polyionic charges of mucins, driving the mutual repulsion of polymer chains and triggering a quick expansion of the mucin network (resembling a Jack-in-the-box mechanism). The existence of a poly ion associated with a shielding cation seems to be a common feature in a large variety of secretory granules. Thus, the proposed spring-loaded release system based on the unshielding of a condensed polyion may serve as a general model for explaining the molecular mechanism of product release in secretion.

Association between Stenotrophomonas maltophilia and lung function in cystic fibrosis

Background:Stenotrophomonas maltophilia (SM) is a Gram-negative non-fermenting bacteria cultured from the sputum of patients with cystic fibrosis (CF). To date, no information is available regarding the effect of this organism on lung function in CF. Methods: A cohort study was conducted to assess the effect of SM on lung function among CF patients aged ⩾6 years in the CF Foundation National Patient Registry from 1994 to 1999. Repeated measures regression was used to assess the association between SM and lung function. Results: The cohort consisted of 20 755 patients with median age at entry of 13.8 years and median follow up time of 3.8 years; 2739 patients (13%) were positive at least once for SM and 18 016 (87%) were never positive. After adjusting for sex, height and age, patients with SM had a mean forced expiratory volume in 1 second which was 0.09 l less (95% CI 0.05 to 0.14) than those without SM. The mean rate of decline associated with SM positivity was 0.025 l/year (95% CI 0.012 to 0.037) but, after adjusting for confounders (sex, height, weight, intravenous antibiotic courses, hospital admissions, pancreatic insufficiency, and Pseudomonas aeruginosa and Burkholderia cepacia status), the mean rate of decline decreased to 0.008 l/year (−0.008, 95% CI −0.019 to 0.003). Conclusions: Although CF patients with SM have worse lung function at the time of positivity, no association was found between SM and increased rate of decline after controlling for confounders.

Osteoporosis in patients with cystic fibrosis.

Decreased bone density and increased risk of fractures are seen in patients with cystic fibrosis. Suboptimal vitamin D levels, nutrition problems, hypogonadism, inactivity, corticosteroid use, and cytokines may contribute to the low bone mass seen in these patients. Treatment recommendations must be individualized and may include nutrition, vitamin D, estrogen or testosterone, and exercise. In high-risk patients calcitonin or growth hormone could be considered.