IL-13-programmed airway tuft cells produce PGE2, which promotes CFTR-dependent mucociliary function.

Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single-cell RNA sequencing of airway epithelium and mouse modeling, we found that IL-13 expanded and programmed airway tuft cells toward eicosanoid metabolism and that tuft cell deficiency led to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bore a signature of PGE2 activation, and PGE2 activation led to cystic fibrosis transmembrane receptor-dependent ion and fluid secretion and accelerated mucociliary transport. These data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.

Net benefit of ivacaftor during prolonged tezacaftor/elexacaftor exposure in vitro.

BACKGROUND: A decrease in the lumacaftor-mediated increase in F508del-CFTR function and expression upon prolonged exposure to ivacaftor (VX-770) has previously been described. However, the efficacy observed with ivacaftor-containing CFTR modulator therapies in vivo is in conflict with these reports. We hypothesized that a portion of the apparent decrease in CFTR function observed after prolonged ivacaftor exposure in vitro was due to an increase in constitutive CFTR-mediated ion transport. METHODS: Human nasal epithelial (HNE) cells were obtained by brushings from three CF individuals homozygous for the F508del CFTR mutation. Differentiated epithelia were pre-treated with prolonged (24 h) exposure to either lumacaftor (VX-809; 3 µM), tezacaftor (VX-661; 3 µM), elexacaftor (VX-445; 3 µM), and/or ivacaftor (0.1-6.4 µM) or DMSO (vehicle control), and CFTR function was assayed by Ussing chamber electrophysiology. RESULTS: In cells treated with lumacaftor, constitutive CFTR activity was not increased at any concentration of co-treatment with ivacaftor. Constitutive CFTR activity was also unchanged in cells treated with the combination of tezacaftor and elexacaftor. An increase in constitutive CFTR activity above the DMSO controls was only observed in cells treated with the combination of tezacaftor and elexacaftor and co-treated with at least 0.1 µM ivacaftor. CONCLUSIONS: These results demonstrate that ivacaftor is a critical component in the triple combination therapy along with tezacaftor and elexacaftor to increase constitutive CFTR function. This work further elucidates the mechanism of action of the effective triple combination therapeutic that is now the primary clinical tool in treating CF.

Receptor-mediated activation of CFTR via prostaglandin signaling pathways in the airway.

Cystic fibrosis (CF) is a genetic disease caused by mutations of the gene encoding a cAMP-activated Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR modulator therapies consist of small-molecule drugs that rescue mutant CFTR. Regimens of single or combinations of CFTR modulators still rely on endogenous levels of cAMP to regulate CFTR activity. We investigated CFTR activation by the natural mediator prostaglandin E2 (PGE2) and lubiprostone (a Food and Drug Administration-approved drug known to target prostaglandin receptors) and tested the hypothesis that receptor-mediated CFTR activators can be used in combination with currently available CFTR modulators to increase function of mutant CFTR. Primary-cultured airway epithelia were assayed in Ussing chambers. Experimental CFTR activators and established CFTR modulators were applied for 24 h and/or acutely and analyzed for their effect on CFTR activity as measured by changes in short-circuit current (ISC). In non-CF airway epithelia, acute application of lubiprostone and PGE2 activated CFTR to the levels comparable to forskolin (Fsk). Pretreatment (24 h) with antagonists to prostaglandin receptors EP2 and EP4 abolished the ability of lubiprostone to acutely activate CFTR. In F508del homozygous airway epithelia pretreated with the triple combination of elexacaftor, tezacaftor, and ivacaftor (ELEXA/TEZ/IVA; i.e., Trikafta), acute application of lubiprostone was able to maximally activate CFTR. Prolonged (24 h) cotreatment of F508del homozygous epithelia with ELEXA/TEZ/IVA and lubiprostone increased acute CFTR activation by ∼60% compared with the treatment with ELEXA/TEZ/IVA alone. This work establishes the feasibility of targeting prostaglandin receptors to activate CFTR on the airway epithelia and demonstrates that cotreatment with lubiprostone can further restore modulator-rescued CFTR.

The Changing Face of Cystic Fibrosis: An Update for Anesthesiologists.

Cystic fibrosis (CF) is the most common fatal genetic disease in North America. While CF is more common among Whites, it is increasingly being recognized in other races and ethnicities. Although there is no cure, life expectancy has steadily improved, with the median survival exceeding 46 years in the United States. There are now more adults than children with CF in the United States. CF is caused by mutations in a gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein, expressed in many epithelial cells. More than 2100 CFTR mutations have been linked to CF, and newer CFTR modulator drugs are being used to improve the production, intracellular processing, and function of the defective CFTR protein. CF is a multisystem disease that affects primarily the lungs, pancreas, hepatobiliary system, and reproductive organs. Anesthesiologists routinely encounter CF patients for various surgical and medical procedures, depending on the age group. This review article focuses on the changing epidemiology of CF, advances in the classification of CFTR mutations, the latest innovations in CFTR modulator therapies, the impact of the coronavirus disease pandemic, and perioperative considerations that anesthesiologists must know while caring for patients with CF.

Characterizing mucociliary clearance in young children with cystic fibrosis.

BACKGROUND: This research characterized mucociliary clearance (MCC) in young children with cystic fibrosis (CF). METHODS: Fourteen children (5-7 years old) with CF underwent: two baseline MCC measurements (Visits 1 and 2); one MCC measurement approximately 1 year later (Visit 3); and measurements of lung clearance index (LCI), a measure of ventilation inhomogeneity. RESULTS: Median (range) percent MCC through 60 min (MCC60) was similar on Visits 1 and 2 with 11.0 (0.9-33.7) and 12.8 (2.7-26.8), respectively (p = 0.95), and reproducible (Spearman Rho = 0.69; p = 0.007). Mucociliary clearance did not change significantly over 1 year with median percent MCC60 on Visit 3 [12.8 (3.7-17.6)] similar to Visit 2 (p = 0.58). Lower percent MCC60 on Visit 3 was significantly associated with higher LCI scores on Visit 3 (N = 14; Spearman Rho = -0.56; p = 0.04). CONCLUSIONS: Tests of MCC were reproducible and reliable over a 2-week period and stable over a 1-year period in 5-7-year-old children with CF. Lower MCC values were associated with increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF. IMPACT: This is the first study to characterize mucociliary clearance (MCC) in children with cystic fibrosis (CF) who were 5-7 years old. Measurements of mucociliary clearance were reproducible and reliable over a 2-week period and stable over a 1-year period. Variability in MCC between children was associated with differences in ventilation homogeneity, such that children with lower MCC values had increased ventilation inhomogeneity. These results suggest that measurements of MCC could be used in short-term clinical trials of interventions designed to modulate MCC and as a new, non-invasive test to evaluate early lung pathology in children with CF.

Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure.

Quantitation of CFTR function in vitro is commonly performed by acutely stimulating then inhibiting ion transport through CFTR and measuring the resulting changes in transepithelial voltage (Vte) and current (ISC). While this technique is suitable for measuring the maximum functional capacity of CFTR, it may not provide an accurate estimate of in vivo CFTR activity. To test if CFTR-mediated ion transport could be measured in the absence of acute CFTR stimulation, primary airway epithelia were analyzed in an Ussing chamber with treatment of amiloride followed by CFTR(inh)-172 without acute activation of CFTR. Non-CF epithelia demonstrated a decrease in Vte and ISC following exposure to CFTR(inh)-172 and in the absence of forskolin/IBMX (F/I); this decrease is interpreted as a measure of spontaneous CFTR activity present in these epithelia. In F508del/F508del CFTR epithelia, F/I-induced changes in Vte and ISC were ~ fourfold increased after treatment with VX-809/VX-770, while the magnitude of spontaneous CFTR activities were only ~ 1.6-fold increased after VX-809/VX-770 treatment. Method-dependent discrepancies in the responses of other CF epithelia to modulator treatments were observed. These results serve as a proof of concept for the analysis of CFTR modulator responses in vitro in the absence of acute CFTR activation. Future studies will determine the usefulness of this approach in the development of novel CFTR modulator therapies.

Elexacaftor is a CFTR potentiator and acts synergistically with ivacaftor during acute and chronic treatment.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding ("correctors") and/or increase channel function ("potentiators") have proven to be highly effective in the therapeutic treatment of CF. Notably, incorporation of the next-generation CFTR corrector, elexacaftor, into a triple combination therapeutic (marketed as Trikafta) has shown tremendous clinical promise in treating CF caused by F508del-CFTR. Here, we report on a newly-described role of elexacaftor as a CFTR potentiator. We explore the acute and chronic actions, pharmacology, and efficacy of elexacaftor as a CFTR potentiator in restoring function to multiple classes of CFTR mutations. We demonstrate that the potentiating action of elexacaftor exhibits multiplicative synergy with the established CFTR potentiator ivacaftor in rescuing multiple CFTR class defects, indicating that a new combination therapeutic of ivacaftor and elexacaftor could have broad impact on CF therapies.

Downregulation of epithelial sodium channel (ENaC) activity in human airway epithelia after low temperature incubation.

INTRODUCTION: The incubation of airway epithelia cells at low temperatures is a common in vitro experimental approach used in the field of cystic fibrosis (CF) research to thermo-stabilise F508del-CFTR and increase its functional expression. Given that the airway epithelium includes numerous ion transporters other than CFTR, we hypothesised that there was an impact of low temperature incubation on CFTR-independent ionoregulatory mechanisms in airway epithelia derived from individuals with and without CF. METHODS: After differentiation at the air-liquid interface, nasal epithelia were incubated at either 37°C or 29°C (low temperature) for 48 hours prior to analysis in an Ussing chamber. RESULTS: While F508del-CFTR activity was increased after low temperature incubation, activity of CFTR in non-CF epithelia was unchanged. Importantly, cultures incubated at 29°C demonstrated decreased transepithelial potential difference (TEPD) and short-circuit currents (Isc) at baseline. The predominant factor contributing to the reduced baseline TEPD and Isc in 29°C cultures was the reduced activity of the epithelial sodium channel (ENaC), evidenced by a reduced responsiveness to amiloride. This effect was observed in cells derived from both non-CF and CF donors. DISCUSSION: Significant transcriptional downregulation of ENaC subunits ß and γ were observed, which may partially explain the decreased ENaC activity. We speculate that low temperature incubation may be a useful experimental paradigm to reduce ENaC activity in in vitro epithelial cultures.

Effect of apical chloride concentration on the measurement of responses to CFTR modulation in airway epithelia cultured from nasal brushings.

INTRODUCTION: One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber, where the apical and basolateral surfaces are isolated and the potential difference, short-circuit current, and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS: CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661, C4/C18, 4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS: Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity, which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS: Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia, the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride, resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy.

Changes in mucociliary clearance over time in children with cystic fibrosis.

OBJECTIVES: (a) To quantify changes in mucociliary clearance (MCC) over time in children with cystic fibrosis (CF) and the relationship between MCC and rate of infection with Pseudomonas aeruginosa (PA); (b) to determine the impact of MCC on the evolution of CF lung disease; and (c) to explore the role of mucus composition as a determinant of MCC. METHODS: Children with CF, who had previously undergone an MCC measurement (visit 1), underwent the following tests 3 to 10 years later: (a) second MCC measurement (visit 2); (b) multiple breath washout to assess ventilation inhomogeneity, expressed as lung clearance index (LCI); (c) high resolution computed tomography lung scan (HRCT); and (d) induced sputum test. Number of PA + cultures/year between visits was documented and mucus dry weight was quantified in the children and adult controls. RESULTS: Nineteen children completed both visits. Median time between visits was 4.6 years. Clearance declined 30% between visits. Lower MCC on visit 2 was associated with more PA+ cultures/year between visits. Lower MCC values on visit 1 were associated with higher LCI values and higher HRCT scores on visit 2. Mucus dry weight was significantly higher in children with CF compared with controls. Higher dry weights were associated with lower MCC. CONCLUSIONS: Mucociliary clearance declines significantly over time in children with CF. The decline is associated with PA infection rate and is affected by mucus composition. Children with early slowing of MCC appear to be at risk for developing ventilation inhomogeneity and parenchymal lung damage when they are older.

Cystic fibrosis transmembrane conductance regulator function, not TAS2R38 gene haplotypes, predict sinus surgery in children and young adults with cystic fibrosis.

BACKGROUND: Chronic rhinosinusitis symptomatology begins in early childhood individuals with cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) function contributes to sinus development and disease. Genetic variants of the bitter taste receptor TAS2R38 have been suggested to contribute to sinus disease severity in individuals without CF. Our objective was to explore whether functional TAS2R38 haplotypes and CFTR function are associated with sinus disease or the need for sinus surgery in individuals with CF. METHODS: We conducted a retrospective study using prospectively collected data from the CF Twin-Sibling Study. The function of CFTR was assessed via chloride conductance. Genotyping of the TAS2R38 gene identified patients who were homozygous for the functional haplotype, heterozygous, or homozygous for nonfunctional haplotypes. Clustered multivariate logistic regression was performed, controlling for sex and family relationship. RESULTS: A total of 1291 patients were evaluated. Patients with ≤1% CFTR function were 1.56 times more likely to require sinus surgery than those with >1% CFTR function (p = 0.049). CFTR function did not correlate significantly with the presence of sinus disease (p = 0.30). In addition, there were no statistically significant differences in diagnosis of sinus disease or need for sinus surgery between patients with functional and nonfunctional TAS2R38 haplotypes. CONCLUSION: CFTR function correlates with need for sinus surgery, whereas TAS2R38 function does not appear to contribute to sinus disease or the need for sinus surgery in patients with CF.

Impact of sleep opportunity on asthma outcomes in adolescents.

RATIONALE: Insufficient sleep is associated with a number of negative health outcomes; as most adolescents obtain <7 h of sleep per night, it is important to understand how sleep impacts asthma among adolescents. OBJECTIVES: To examine the impact of sleep opportunity on asthma in adolescents. METHODS: In this study, 54 adolescents with asthma (12-17 years, 69% female, 65% Caucasian) participated in a randomized, cross-over sleep manipulation trial, including a sleep stabilization week, five nights of a "Short" sleep opportunity (time in bed: 6.5 h/night), and five nights of a "Long" sleep opportunity (time in bed: 9.5 h/night). Wake times were consistent across all three study weeks. Primary outcomes were lung function (daily peak expiratory flow rate, weekly spirometry) and functional asthma outcomes (daily asthma symptoms, Asthma Control Questionnaire, PROMIS Asthma Impact Scale). Markers of inflammation were also explored. MEASUREMENTS AND MAIN RESULTS: Compared to the Long sleep week, during the Short sleep week, morning FEV1 was lower (p = 0.006), while asthma symptoms and albuterol use was higher (p < 0.05), and asthma showed a trend towards greater negative impact on daily life (p = 0.07). No differences were found for weekly measures of lung function or inflammation. CONCLUSIONS: An insufficient sleep opportunity negatively impacts objective and subjective daily symptoms of asthma in adolescents, as well as health related quality of life. As most adolescents are significantly sleep deprived, it is important to target sleep health in the treatment of asthma.

Benralizumab does not impair antibody response to seasonal influenza vaccination in adolescent and young adult patients with moderate to severe asthma: results from the Phase IIIb ALIZE trial.

BACKGROUND AND OBJECTIVES: Benralizumab is a humanized, afucosylated monoclonal antibody against the IL-5Rα. Initial monthly followed by every-other-month injections result in rapid and nearly complete eosinophil depletion. We evaluated whether three doses of benralizumab modifies antibody response to seasonal influenza vaccination for adolescent/young adult patients with moderate to severe asthma. METHODS: ALIZE (NCT02814643) was a Phase IIIb randomized controlled trial of patients aged 12-21 years receiving medium- to high-dosage inhaled corticosteroids/long-acting ß2-agonists. Patients received benralizumab 30 mg or placebo at Weeks 0, 4, and 8, plus tetravalent influenza vaccination at Week 8. At Week 12, strain-specific antibody responses following vaccination were assessed for four influenza antigens by hemagglutination inhibition (HAI) and microneutralization (MN) assays. RESULTS: A total of 103 patients were randomized and received benralizumab (n=51) or placebo (n=52). There were no consistent differences in HAI or MN antibody responses at Week 12 between patients receiving benralizumab or placebo. HAI geometric mean fold rises (GMFRs) for all influenza strains tested were 3.3-4.2 for benralizumab vs 3.4-3.9 for placebo; MN GMFRs were 2.8-5.1 for benralizumab vs 3.2-4.4 for placebo. A ≥4-fold rise in HAI from Weeks 8 to 12 occurred in 44.0%-56.0% and 30.6%-49.0% of patients receiving benralizumab and placebo, respectively. At Week 12, 78.0%-100% vs 79.6%-100% of patients receiving benralizumab and placebo, respectively, achieved a ≥40 HAI antibody titer. There were no significant safety findings. CONCLUSION: Benralizumab did not impair the antibody response to seasonal virus vaccination in adolescents and young adult patients with moderate to severe asthma.

Effect of ivacaftor on mucociliary clearance and clinical outcomes in cystic fibrosis patients with G551D-CFTR.

BACKGROUND: The ability to restore cystic fibrosis transmembrane regulator (CFTR) function with effective small molecule modulators in patients with cystic fibrosis provides an opportunity to study relationships between CFTR ion channel function, organ level physiology, and clinical outcomes. METHODS: We performed a multisite, prospective, observational study of ivacaftor, prescribed in patients with the G551D-CFTR mutation. Measurements of lung mucociliary clearance (MCC) were performed before and after treatment initiation (1 and 3 months), in parallel with clinical outcome measures. RESULTS: Marked acceleration in whole lung, central lung, and peripheral lung MCC was observed 1 month after beginning ivacaftor and was sustained at 3 months. Improvements in MCC correlated with improvements in forced expiratory volume in the first second (FEV1) but not sweat chloride or symptom scores. CONCLUSIONS: Restoration of CFTR activity with ivacaftor led to significant improvements in MCC. This physiologic assessment provides a means to characterize future CFTR modulator therapies and may help to predict improvements in lung function. TRIAL REGISTRATION: ClinicialTrials.gov, NCT01521338. FUNDING: CFF Therapeutics (GOAL11K1).

Airway microbiota across age and disease spectrum in cystic fibrosis.

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR.

BACKGROUND: Cavosonstat (N91115), an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators. METHODS: A Phase I program evaluated pharmacokinetics, drug-drug interactions and safety of cavosonstat in healthy and cystic fibrosis (CF) subjects homozygous for F508del-CFTR. Exploratory outcomes included changes in sweat chloride in CF subjects. RESULTS: Cavosonstat was rapidly absorbed and demonstrated linear and predictable pharmacokinetics. Exposure was unaffected by a high-fat meal or rifampin-mediated effects on drug metabolism and transport. Cavosonstat was well tolerated, with no dose-limiting toxicities or significant safety findings. At the highest dose, significant reductions from baseline in sweat chloride were observed (-4.1mmol/L; P=0.032) at day 28. CONCLUSIONS: The favorable safety and clinical profile warrant further study of cavosonstat in CF. ClinicalTrials.gov Numbers: NCT02275936, NCT02013388, NCT02500667.

Digitoxin for Airway Inflammation in Cystic Fibrosis: Preliminary Assessment of Safety, Pharmacokinetics, and Dose Finding.

RATIONALE: Cystic fibrosis (CF) lung disease progresses by a combination of airway inflammation, bacterial colonization, and infection. Airway inflammation is predominantly neutrophilic and complicates airway clearance therapies through cellular debris; excessive DNA; excessive and viscous mucus; and high concentrations of neutrophils, IL-8, and related cytokines liberated along the nuclear factor-κB signaling pathway. OBJECTIVES: We conducted a preliminary, single-site, randomized, double-blind, placebo-controlled study to evaluate the effects over 28 days of two dose levels (0.05 mg and 0.1 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on safety, pharmacokinetics, and inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease. METHODS: Patients with CF 18-45 years old with any genotype combination were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure (1) the pharmacokinetics of digitoxin in sera of patients with stable CF; (2) safety indices, including ECG changes and sputum microbiology; (3) the effect of digitoxin on gene expression in nasal epithelial cells of patients with stable CF; and (4) quality-of-life scores using the Cystic Fibrosis Questionnaire-Revised. MEASUREMENTS AND MAIN RESULTS: It took several weeks to achieve a therapeutic serum level of digitoxin in subjects with CF. No safety concerns emerged during the study. Digitoxin treatment showed a trend toward reduction in sputum free neutrophil elastase and neutrophil counts, but not a reduction in sputum IL-8. Digitoxin treatment did not reach statistical significance for the primary or secondary outcome measures over the 28-day study period. However, the nasal mRNA from the group receiving 0.1 mg of digitoxin daily had a distinct distribution of global gene expression levels as compared with either the 0.05-mg dose or placebo treatment. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment, and mucus hypersecretion. CONCLUSIONS: At a dose of 0.1 mg daily for 28 days, digitoxin was safe for adults with CF lung disease, but it did not achieve a significant decrease in sputum inflammatory markers. Clinical trial registered with www.clinicaltrials.gov (NCT00782288).