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.mucus | cystic fibrosis | lung | asthma | COPD

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.

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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.

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.

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.

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.

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 ΔFEV1 5%−9% and ΔFEV1 ≥10% was present with increased cough (OR