Kirby L. Zeman

Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration

Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS‐internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent‐resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome‐like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS‐exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.—Clunes, L. A., Davies, C. M., Coakley, R. D., Aleksandrov, A. A., Henderson, A. G., Zeman, K. L., Worthington, E. N., Gentzsch, M., Kreda, S. M., Cholon, D., Bennett, W. D., Riordan, J. R., Boucher, R. C., Tarran, R. Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration. FASEB J. 26, 533–545 (2012). www.fasebj.org

Targeting Delivery of Aerosols to Different Lung Regions

With the increasing use of aerosolized drugs, there is a need to understand the means by which these drugs can most effectively be targeted to desired regions of the lung. Several attempts have been made at targeting aerosols in the lung by changing particle sizes and breathing patterns with varying degrees of success. Recent use of such techniques as shallow, aerosol bolus delivery and extremely slow inhalations of aerosols in diagnostic lung tests may also prove beneficial for targeting drug delivery to the conducting airways. This review discusses the potential for utilizing aerosol delivery techniques for selectively targeting aerosol deposition along both serial and parallel pathways in the lung. Based on a review of previous studies concerning factors that determine aerosol and gas distribution in the lung, the potential for utilizing various breathing techniques in concert with variations in particle sizes are considered. Further research on the factors that determine distribution of aerosol in the diseased lung may help in designing successful targeting strategies for the future.

Deposition of fine particles in children spontaneously breathing at rest

Recent epidemiological studies suggest that children may be more susceptible than adults to effects of inhaled particulate matter. To determine if children receive an increased lung dose of particles compared to adults we measured fractional deposition (DF) of fine particles in children, age 7-14 yr (n = 16), adolescents, age 14-18 yr (n = 11), and adults, age 19-35 yr (n = 12). Each subject inhaled 2-mum monodisperse Carnauba wax particles while following a breathing pattern previously determined by respiratory inductance plethysmography for that subject (i.e., that subjects spontaneous pattern at rest). Breath-by-breath DF (ratio of particles not exhaled/total particles inhaled) was determined by photometry at the mouth. Among the children there was no variation in DF with subject age or height, but DF was dependent on intersubject variation in tidal volume (Vt) (p < .001). DF for the children versus the adolescents was 0.22 +/- 0.08(sd) and 0.20 +/- 0.03, respectively (NS), also not different from ...

Regional deposition of coarse particles and ventilation distribution in healthy subjects and patients with cystic fibrosis

The efficacy of inhaled pharmaceuticals depends, in part, on their site of respiratory deposition. Markedly nonuniform ventilation distribution may occur in persons with obstructive airways diseases and may affect particle deposition. We studied the relationship between regional deposition (RDep) and regional ventilation (RVent) in a group of 12 cystic fibrosis (CF) patients with mild to moderate airway obstruction (63 +/- 8% predicted FEV1) and 11 healthy nonsmoking volunteers (104 +/- 13% predicted FEV1) using planar scintigraphic methods. RDep was assessed from initial deposition and 24-h retention images for monodisperse technetium-99m-labeled iron oxide particles (5-microm MMAD). Regional volumes and RVent were assessed from xenon-133 equilibrium and washout, respectively. Six regions of interest per lung were established by dividing each lung into thirds by height and approximately half by width. The two lower regions of the left lung were not analyzed due to activity in the stomach. Remaining regions were categorized as central (two interior-most regions) and peripheral (eight exterior regions). RDep and RVent were computed for the eight peripheral regions. Tracheobronchial (TB) deposition was estimated for each of the peripheral regions as the difference between initial activity and decay-corrected 24-h retention or parenchymal deposition. RDep was computed as the fraction of material within a region normalized to regional volume. RVent for each region was determined by normalizing the xenon washout rate for that region by the total washout rate for the eight peripheral regions. Significant linear associations were found between RDep and RVent in both the healthy subjects and CF patients. In healthy subjects, RDep in the TB airways was positively associated with RVent (p = 0.03). In CF patients, RDep in the TB airways was negatively associated with RVent (p = 0.04) and RDep in the parenchyma was positively associated with RVent (p < 0.001). The initial pattern of RDep in the lung was not significantly associated with RVent in either group. These data suggest that significant coarse particle deposition may occur in the TB airways of poorly ventilated lung regions in CF patients, whereas, particle deposition in the TB airways of the healthy subjects follows ventilation.

Nasal Contribution to Breathing and Fine Particle Deposition in Children Versus Adults

Both the route of breathing, nasal versus oral, and the effectiveness of the nose to filter inhaled, fine particles may differ between children and adults. This study compared (1) the nasal contribution to breathing at rest and during mild to moderate exercise in children (age 6–10 yr) versus young adults and (2) the nasal deposition efficiency (NDE) of fine particles (1 and 2 μm MMAD, GSD < 1.2) under resting and light exercise breathing conditions in the same children and adults. Nasal contribution to breathing was assessed by respiratory inductance plethysmography and a nasal mask with flow meter during incremental exercise on a bicycle ergometer. Fine particle deposition fractions for nasal and oral breathing were assessed by inhalation of monodisperse carnauba wax particles and laser photometry to determine inhaled/exhaled concentrations. There was a trend for children to have a lesser nasal contribution to breathing at rest and during exercise, but the differences from adults were not statistically significant. Children did, however, have significantly decreased NDE for 2-μm particles under light exercise breathing conditions compared to adults, suggesting less efficient nasal filtering for larger particles and higher flow conditions. These results suggest that the lungs of children may be exposed to higher concentrations of inhaled, ambient particles than adults.

ENHANCED DEPOSITION OF FINE PARTICLES IN COPD PATIENTS SPONTANEOUSLY BREATHING AT REST

Particulate air pollution has been linked to acute increases in mortality among individuals with preexisting cardiorespiratory disease. While these individuals may be predisposed to acute toxic effects, they may also receive an increased dose of particles to their lungs compared to healthy subjects. We measured fractional deposition (DF) of inhaled, fine particles in subjects with moderate-severe chronic obstructive pulmonary disease (COPD; n = 13) and an age-matched group of subjects (n = 11) with normal pulmonary function, mean age = 62 versus 67 yr, and FEV1 (% pred) = 33 versus 90, respectively. Each subject inhaled 2- m monodisperse, Carnauba wax particles while following a breathing pattern previously determined by respiratory inductance plethysmography (RIP) for that subject (i.e., that subjects spontaneous pattern at rest). Breath-by-breath DF (ratio of particles not exhaled to total particles inhaled) was determined by photometry at the mouth. The COPD patients had greater DF than normals, 0.40 ...

The relationship of mucus concentration (hydration) to mucus osmotic pressure and transport in chronic bronchitis

RATIONALE Chronic bronchitis (CB) is characterized by persistent cough and sputum production. Studies were performed to test whether mucus hyperconcentration and increased partial osmotic pressure, in part caused by abnormal purine nucleotide regulation of ion transport, contribute to the pathogenesis of CB. OBJECTIVES We tested the hypothesis that CB is characterized by mucus hyperconcentration, increased mucus partial osmotic pressures, and reduced mucus clearance. METHODS We measured in subjects with CB as compared with normal and asymptomatic smoking control subjects indices of mucus concentration (hydration; i.e., percentage solids) and sputum adenine nucleotide/nucleoside concentrations. In addition, sputum partial osmotic pressures and mucus transport rates were measured in subjects with CB. MEASUREMENTS AND RESULTS CB secretions were hyperconcentrated as indexed by an increase in percentage solids and total mucins, in part reflecting decreased extracellular nucleotide/nucleoside concentrations. CB mucus generated concentration-dependent increases in partial osmotic pressures into ranges predicted to reduce mucus transport. Mucociliary clearance (MCC) in subjects with CB was negatively correlated with mucus concentration (percentage solids). As a test of relationships between mucus concentration and disease, mucus concentrations and MCC were compared with FEV1, and both were significantly correlated. CONCLUSIONS Abnormal regulation of airway surface hydration may slow MCC in CB and contribute to disease pathogenesis.

In vivo uptake of inhaled particles by airway phagocytes is enhanced in patients with mild asthma compared with normal volunteers

Background: The uptake of inhaled particulate matter by airway phagocytes is an important defence mechanism contributing to the clearance of potentially toxic substances, including aeroallergens, from the lung. Since airway monocytes and macrophages can also function as antigen presenting cells, their ability to engulf materials deposited on the airway surface is of particular interest in patients with allergic asthma. To determine whether airway mononuclear phagocytes of patients with allergic asthma might have enhanced phagocytic activity, the in vivo uptake of inhaled radiolabelled particles was compared in 10 patients with mild allergic asthma and 8 healthy (non-allergic) individuals. Methods: Phagocyte function was assessed by quantifying the proportion of radioactivity associated with cellular and supernatant fractions of induced sputum 2 h after inhalation of radiolabelled sulfur colloid particles. All subjects were pretreated with albuterol before sputum induction. A standardised breathing pattern was used to target aerosol deposition in the bronchial airways. Results: In vivo particle uptake by airway cells was significantly greater in patients with asthma than in healthy volunteers (57.2% (95% CI 46.5% to 67.9%) vs 22.3% (95% CI 4.9% to 39.6%), p<0.01), as was in vitro phagocytosis of opsonised zymosan-A bioparticles. There was also a significant correlation (r = 0.85, p<0.01) between the percentage of sputum mononuclear phagocytes and the percentage uptake of particles in the patients with asthma but not in the control subjects. Conclusions: In vivo particle uptake by airway macrophages is enhanced in persons with mild asthma. Enhanced uptake and processing of particulate antigens could contribute to the pathogenesis and progression of allergic airways disease and may contribute to the increased risk of disease exacerbation associated with particulate exposure.

PROLONGED AIRWAY RETENTION OF INSOLUBLE PARTICLES IN CYSTIC FIBROSIS VERSUS PRIMARY CILIARY DYSKINESIA

Patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) have been shown to have impaired large airway clearance of radiolabelled particles as measured by external gamma camera scanning up to 6 hours post deposition. Recent studies suggest that 24-hour retention of particles may reflect some airway retention in addition to alveolar retention. In a retrospective study, we analyzed the relationship between the deposition pattern and 24-hour retention (Ret24 hr) of technetium 99-radiolabelled iron oxide (99Tc-Fe2O3) particles in 20 patients with CF, 12 patients with PCD, and 17 normal subjects. By gamma camera analysis, initial aerosol deposition was analyzed in terms of central-peripheral (C/P) activity within the lungs. Gamma camera scanning was performed immediately following deposition and again at 24 hours to assess residual retention (Ret24 hr) as a percent of initial deposition. C/P analysis was also performed on the 24-hour scan (C/P24). For all subjects, initial deposition pattern (C/P) was inversely related to lung function (forced expiratory volume in 1 second [FEV1]%pred vs. C/P, r = -.54). Ret24 hr was also inversely related to initial deposition pattern for all subjects (Ret24 hr vs. C/P ratio, r = -.42). Analysis of covariance showed that for a given C/P ratio, CF patients had significantly greater Ret24 hr compared to normal subjects (9.8 +/- 2.8 [SE]%). In addition, the CF patients had similar C/P24 as the normal subjects (1.35 +/- 0.40 [SD] vs. 1.10 +/- 0.39, respectively). These results suggest that small airway clearance is compromised in CF patients compared to normal subjects. On the other hand, PCD patients had C/P24 similar to their initial deposition C/P ratios (2.78 +/- 1.72 vs. 2.45 +/- 0.87, respectively), significantly greater than 1.0, and significantly greater than CF or normal subjects, suggesting that PCD patients have prolonged particle retention associated with their large bronchial airways.Patients with cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) have been shown to have impaired large airway clearance of radiolabelled particles as measured by external gamma camera scanning up to 6 hours post deposition. Recent studies suggest that 24-hour retention of particles may reflect some airway retention in addition to alveolar retention. In a retrospective study, we analyzed the relationship between the deposition pattern and 24-hour retention (Ret24 hr) of technetium 99 radiolabelled iron oxide (99Tc-Fe2O3) particles in 20 patients with CF, 12 patients with PCD, and 17 normal subjects. By gamma camera analysis, initial aerosol deposition was analyzed in terms of central peripheral (C/P) activity within the lungs. Gamma camera scanning was performed immediately following deposition and again at 24 hours to assess residual retention (Ret24hr) as a percent of initial deposition. C/P analysis was also performed on the 24-hour scan (C/P24). For all subjects, initial deposition pattern (C/P) was inversely related to lung function (forced expiratory volume in 1 second \[FEV1]%pred vs. C/P, r =-.54). Ret24hr was also inversely related to initial deposition pattern for all subjects (Ret24hr vs. C/P ratio, r=-.42). Analysis of covariance showed that for a given C/P ratio, CF patients had significantly greater Ret24hr compared to normal subjects (9.8 +/- 2.8\[SE]%). In addition, the CF patients had similar C/P24 as the normal subjects (1.35 +/- 0.40\[SD] vs. 1.10 +/- 0.39, respectively). These results suggest that small airway clearance is compromised in CF patients compared to normal subjects. On the other hand, PCD patients had C/P24 similar to their initial deposition C/P ratios (2.78 +/- 1.72 vs. 2.45 +/- 0.87, respectively), significantly greater than 1.0, and significantly greater than CF or normal subjects, suggesting that PCD patients have prolonged particle retention associated with their large bronchial airways.

Multisite Comparison of Mucociliary and Cough Clearance Measures Using Standardized Methods

BACKGROUND A standardized protocol for measuring mucociliary (MCC) and cough clearance (CC) was developed and tested at the University of North Carolina at Chapel Hill, NC (UNC), Johns Hopkins University (JHU), and the University of Pittsburgh (Pitt). METHODS A total of 50 healthy nonsmoking adults with normal lung function were studied at the three sites: 30 [21 males/9 females (21M/9F)] at UNC, 10 (6M/4F) at JHU, and 10 (4M/6F) at Pitt. Subjects inhaled an aerosol of (99m)technetium sulfur colloid in 0.9% saline (mass median aerodynamic diameter=5.4 μm) under controlled breathing conditions (500 mL/sec, 30 breaths/min) by following a metronome and flow signal from a commercial dosimeter. Following inhalation, subjects sat in front of a gamma camera as sequential lung images were acquired for 60 min. Subjects then coughed 60 times, and images were acquired after each set of 20 coughs, i.e., at 70, 80, and 90 min. Subjects returned to the laboratory approximately 24 hr later for a final image of residual lung activity. Initial aerosol distribution was measured as a central/peripheral (C/P) ratio of activity. MCC/CC was expressed as the area under the retention versus time curve over 90 min (AUC90). RESULTS A multivariate analysis of clearance versus time with site and C/P as covariates showed no significant site-specific differences. Interestingly, MCC/CC was greater in females (n=19) versus males (n=31), with AUC90=0.84 ± 0.11 and 0.90±0.07, respectively (p=0.03), for the combined data set from all sites (not significant for any given site). There were no gender differences for either C/P ratio or 24-hr clearance. CONCLUSIONS This standardized protocol may prove beneficial in multicenter trials for testing new therapies that are designed to improve MCC/CC.