Evaluating FEV1 decline in diagnosis and management of pulmonary exacerbations in children with cystic fibrosis.

RATIONALE: Forced expiratory volume in 1 s (FEV1) decline (ΔFEV1) is associated with pulmonary exacerbation (PEx) diagnosis in cystic fibrosis (CF). Spirometry may not be available during telehealth visits and could impair clinician ability to diagnose PEx. This study aims to (1) identify the associations between degrees of ΔFEV1 (decrease of <5% predicted vs. 5%-9% predicted vs. ≥10% predicted from baseline), clinical symptoms, and clinician-diagnosed PEx and (2) evaluate the correlation between respiratory symptoms, ΔFEV1, and antibiotic treatment. METHODS: Retrospective, descriptive study of PEx diagnosis and management in 628 outpatient clinical encounters with spirometry in 178 patients with CF ages 6-17 years at Riley Hospital for Children during 2019. Odds ratios (OR) of symptoms associated with clinician-defined PEx diagnosis and antibiotic management stratified by ΔFEV1 decline were determined. RESULTS: Clinician-diagnosed PEx occurred at 199 (31.7%) visits; increased cough (77.4%) and sputum/wet cough (57.8%) were the most frequently reported symptoms. Compared to no ΔFEV1, the odds of a clinician-diagnosed PEx were increased when ΔFEV15%-9% and ΔFEV1≥10% was present with increased cough (OR 1.56, 95% confidence interval [CI] 1.25-1.94 and OR 1.82, 95% CI 1.52-2.19, respectively), increased sputum (OR 1.59, 95% CI 1.20-2.12 and OR 1.78, 95% CI 1.37-2.32, respectively), and increased cough and sputum together (OR 1.51, 95% CI 1.08-2.13 and OR 1.68, 95% CI 1.22-2.31, respectively). CONCLUSIONS: ΔFEV1 is associated with increased likelihood that cough and sputum are diagnosed as a PEx. Spirometry is essential for PEx diagnosis and treatment and is a necessary component of all clinical encounters.

Early Lung Disease Exhibits Bacteria-Dependent and -Independent Abnormalities in Cystic Fibrosis Pigs.

Rationale: Although it is clear that cystic fibrosis (CF) airway disease begins at a very young age, the early and subsequent steps in disease pathogenesis and the relative contribution of infection, mucus, and inflammation are not well understood. Objectives: As one approach to assessing the early contribution of infection, we tested the hypothesis that early and continuous antibiotics would decrease the airway bacterial burden. We believed that, if they do, this might reveal aspects of the disease that are more or less sensitive to decreasing infection. Methods: Three groups of pigs were studied from birth until ∼3 weeks of age: 1) wild-type, 2) CF, and 3) CF pigs treated continuously with broad-spectrum antibiotics from birth until study completion. Disease was assessed with chest computed tomography, histopathology, microbiology, and BAL. Measurements and Main Results: Disease was present by 3 weeks of age in CF pigs. Continuous antibiotics from birth improved chest computed tomography imaging abnormalities and airway mucus accumulation but not airway inflammation in the CF pig model. However, reducing bacterial infection did not improve two disease features already present at birth in CF pigs: air trapping and submucosal gland duct plugging. In the CF sinuses, antibiotics did not prevent the development of infection or disease or the number of bacteria but did alter the bacterial species. Conclusions: These findings suggest that CF airway disease begins immediately after birth and that early and continuous antibiotics impact some, but not all, aspects of CF lung disease development.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

Postnatal airway growth in cystic fibrosis piglets.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis.NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl- transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Lentiviral-mediated phenotypic correction of cystic fibrosis pigs.

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (CFTR), resulting in defective anion transport. Regardless of the disease-causing mutation, gene therapy is a strategy to restore anion transport to airway epithelia. Indeed, viral vector-delivered CFTR can complement the anion channel defect. In this proof-of-principle study, functional in vivo CFTR channel activity was restored in the airways of CF pigs using a feline immunodeficiency virus-based (FIV-based) lentiviral vector pseudotyped with the GP64 envelope. Three newborn CF pigs received aerosolized FIV-CFTR to the nose and lung. Two weeks after viral vector delivery, epithelial tissues were analyzed for functional correction. In freshly excised tracheal and bronchus tissues and cultured ethmoid sinus cells, we observed a significant increase in transepithelial cAMP-stimulated current, evidence of functional CFTR. In addition, we observed increases in tracheal airway surface liquid pH and bacterial killing in CFTR vector-treated animals. Together, these data provide the first evidence to our knowledge that lentiviral delivery of CFTR can partially correct the anion channel defect in a large-animal CF model and validate a translational strategy to treat or prevent CF lung disease.

Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility.

RATIONALE: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. OBJECTIVES: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. METHODS: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. MEASUREMENTS AND MAIN RESULTS: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. CONCLUSIONS: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing.

Early airway structural changes in cystic fibrosis pigs as a determinant of particle distribution and deposition.

The pathogenesis of cystic fibrosis (CF) airway disease is not well understood. A porcine CF model was recently generated, and these animals develop lung disease similar to humans with CF. At birth, before infection and inflammation, CF pigs have airways that are irregularly shaped and have a reduced caliber compared to non-CF pigs. We hypothesized that these airway structural abnormalities affect airflow patterns and particle distribution. To test this hypothesis we used computational fluid dynamics (CFD) on airway geometries obtained by computed tomography of newborn non-CF and CF pigs. For the same flow rate, newborn CF pig airways exhibited higher air velocity and resistance compared to non-CF. Moreover we found that, at the carina bifurcation, particles greater than 5-µm preferably distributed to the right CF lung despite almost equal airflow ventilation in non-CF and CF. CFD modeling also predicted that deposition efficiency was greater in CF compared to non-CF for 5- and 10-µm particles. These differences were most significant in the airways included in the geometry supplying the right caudal, right accessory, left caudal, and left cranial lobes. The irregular particle distribution and increased deposition in newborn CF pig airways suggest that early airway structural abnormalities might contribute to CF disease pathogenesis.

Air trapping and airflow obstruction in newborn cystic fibrosis piglets.

RATIONALE: Air trapping and airflow obstruction are being increasingly identified in infants with cystic fibrosis. These findings are commonly attributed to airway infection, inflammation, and mucus buildup. OBJECTIVES: To learn if air trapping and airflow obstruction are present before the onset of airway infection and inflammation in cystic fibrosis. METHODS: On the day they are born, piglets with cystic fibrosis lack airway infection and inflammation. Therefore, we used newborn wild-type piglets and piglets with cystic fibrosis to assess air trapping, airway size, and lung volume with inspiratory and expiratory X-ray computed tomography scans. Micro-computed tomography scanning was used to assess more distal airway sizes. Airway resistance was determined with a mechanical ventilator. Mean linear intercept and alveolar surface area were determined using stereologic methods. MEASUREMENTS AND MAIN RESULTS: On the day they were born, piglets with cystic fibrosis exhibited air trapping more frequently than wild-type piglets (75% vs. 12.5%, respectively). Moreover, newborn piglets with cystic fibrosis had increased airway resistance that was accompanied by luminal size reduction in the trachea, mainstem bronchi, and proximal airways. In contrast, mean linear intercept length, alveolar surface area, and lung volume were similar between both genotypes. CONCLUSIONS: The presence of air trapping, airflow obstruction, and airway size reduction in newborn piglets with cystic fibrosis before the onset of airway infection, inflammation, and mucus accumulation indicates that cystic fibrosis impacts airway development. Our findings suggest that early airflow obstruction and air trapping in infants with cystic fibrosis might, in part, be caused by congenital airway abnormalities.

Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs.

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid-binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR-/-;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.