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Our findings show that low-dose, short-term topical or systemic γ-secretase inhibitor treatment may lead to restoration of multiciliated cells without toxicity and potentially improve epithelial function in a wide range of chronic lung diseases.

The association between organ laterality abnormalities and ciliary ultrastructural defect or genotype in primary ciliary dyskinesia is poorly understood.To determine if there is an association between presence/type of laterality abnormality and ciliary ultrastructural defect and genotype.Participants with primary ciliary dyskinesia in a multicenter, prospective study were grouped based on ciliary ultrastructural defect or genotype. In a retrospective analysis of this data, the association of ciliary ultrastructural defect or genotype and likelihood of a laterality abnormality was evaluated by logistic regression adjusted for presence of two loss-of-function versus one or more not-loss-of-function variants.Of 559 participants, 286 (51.2%), 215 (38.5%), and 58 (10.4%) were identified as having situs solitus, situs inversus totalis, and situs ambiguus, respectively; heterotaxy, defined as situs ambiguus with complex cardiovascular defects, was present in 14 (2.5%). Compared to group with inner dynein arm defects with microtubular disorganization, laterality defects were more likely in the outer dynein arm defects group (OR 2.07, 95% CI 1.21-3.54, P0.01) and less likely in the normal/near normal ultrastructure group (OR 0.04, 95% CI 0.013-0.151, P0.01). Heterotaxy was present in 11 of 242 (4.5%) in the outer dynein arm defects group but zero of 96 in the inner dynein arm defects with microtubular disorganization group (P=0.038).In primary ciliary dyskinesia, risk of a laterality abnormality differs by ciliary ultrastructure defect. Pathophysiologic mechanisms underlying these differences require further exploration.

Basal airway epithelial cells are a multipotent stem cell population which gives rise to several airway cell types. Basal cells are known to be critical to airway epithelium homeostasis and repair, and altered basal cell phenotypes have been reported in cystic fibrosis and idiopathic pulmonary fibrosis. However, very little is known about how basal cells respond to stimuli in the cystic fibrosis airway environment. Cystic fibrosis patients experience chronic infection with Pseudomonas aeruginosa and contain both high quantities of lipopolysaccharide (LPS) as well as the filamentous bacteriophage Pf produced by biofilm-state P. aeruginosa in the airway. In this study, we sought to investigate the transcriptional responses of human basal cells from both healthy controls and patients with cystic fibrosis to LPS and Pf phage. Basal cells from wildtype and cystic fibrosis donors were cultured in vitro and exposed to LPS and/or Pf phage, followed by single-cell sequencing on the 10x platform. We report that basal cells show strong antiviral responses and neutrophil chemokine production in response to Pf phage. We validate these findings in additional donors by qRT-PCR and show that Pf phage is internalized by basal cells. We also show that Pf decreases basal cell migration and proliferation. We demonstrate that Pf phage, a bacteria-infecting virus which does not replicate in mammalian cells, is taken up by basal cells and activates immune responses. Further studies are needed to determine the impact of this antiviral response to bacterial clearance.Author SummaryWhen we experience a lung infection or injury, the stem cells of the airway—called basal epithelial cells—are crucial for healing the affected tissue. Basal cells proliferate and migrate to close gaps in the epithelium and replace injured and dead cells. We are also learning that basal cells can directly contribute to the immune response against lung pathogens, although little is understood about how basal cells sense viruses and bacteria, and what molecules they secrete in response. In our work, we sought to investigate the role that basal cells play in the course of an infection with the common human pathogen Pseudomonas aeruginosa. We stimulated basal cells with lipopolysaccharide (LPS), a potent immunogen produced by Pseudomonas, as well as the Pseudomonas-infecting virus Pf4. We have previously shown that Pf4, which is produced in high amounts by lung-infecting Pseudomonas, affects the immune response to this bacterium. In our work here, we show that Pf4 is recognized as a virus by basal cells and changes the transcriptional response to LPS, derailing the antibacterial immune response to Pseudomonas. These findings raise the question of whether other bacteria-infecting viruses alter immune responses, and how these viruses interact with other airway cells.

Thick, viscous respiratory secretions are a major pathogenic feature of COVID-19, but the composition and physical properties of these secretions are poorly understood. We characterized the composition and rheological properties (i.e., resistance to flow) of respiratory secretions collected from intubated COVID-19 patients. We found the percentages of solids and protein content were greatly elevated in COVID-19 compared with heathy control samples and closely resembled levels seen in cystic fibrosis, a genetic disease known for thick, tenacious respiratory secretions. DNA and hyaluronan (HA) were major components of respiratory secretions in COVID-19 and were likewise abundant in cadaveric lung tissues from these patients. COVID-19 secretions exhibited heterogeneous rheological behaviors, with thicker samples showing increased sensitivity to DNase and hyaluronidase treatment. In histologic sections from these same patients, we observed increased accumulation of HA and the hyaladherin versican but reduced tumor necrosis factor–stimulated gene-6 staining, consistent with the inflammatory nature of these secretions. Finally, we observed diminished type I interferon and enhanced inflammatory cytokines in these secretions. Overall, our studies indicated that increases in HA and DNA in COVID-19 respiratory secretion samples correlated with enhanced inflammatory burden and suggested that DNA and HA may be viable therapeutic targets in COVID-19 infection.

Background Lumacaftor and ivacaftor combination treatment showed efficacy in patients aged 12 years or older with cystic fibrosis homozygous for F508del-cystic fibrosis transmembrane conductance regulator (CFTR) in placebo-controlled studies and patients aged 6–11 years with cystic fibrosis homozygous for F508del-CFTR in an open-label study. We report efficacy and safety of lumacaftor and ivacaftor in patients with cystic fibrosis aged 6–11 years homozygous for F508del-CFTR. Methods In this phase 3, randomised, double-blind, placebo-controlled, multicentre study, patients were enrolled at 54 hospitals and medical centres in nine countries (the USA, Australia, Belgium, Canada, Denmark, France, Germany, Sweden, and the UK). Eligible patients weighed at least 15 kg, with a confirmed diagnosis of cystic fibrosis, percent predicted forced expiratory volume in 1 s (FEV 1) of 70 or more, and lung clearance index 2·5 (LCI 2·5) of 7·5 or more at screening (values less than these thresholds were permitted at day 1). All patients were tested for CFTR genotype at screening; eligible patients had to have the F508del-CFTR mutation on both alleles. Exclusion criteria included any comorbidity or laboratory abnormality that might confound the study results or pose additional risk to the patient. Patients were stratified by weight (

Examination of ciliary ultrastructure remains the cornerstone diagnostic test for primary ciliary dyskinesia (PCD), a disease of abnormal ciliary structure and/or function. Obtaining a biopsy with sufficient interpretable cilia and producing quality transmission electron micrographs (TEM) is challenging. Methods for processing tissues for optimal preservation of axonemal structures are not standardized. This study describes our experience using a standard operating procedure (SOP) for collecting nasal scrape biopsies and processing TEMs in a centralized laboratory. We enrolled patients with suspected PCD at research sites of the Genetic Disorders of Mucociliary Clearance Consortium. Biopsies were performed according to a SOP whereby curettes were used to scrape the inferior surface of the inferior turbinate, with samples placed in fixative. Specimens were shipped to a central laboratory where TEMs were prepared and blindly reviewed. Four hundred forty-eight specimens were obtained from 107 young children (0-5 years), 189 older children (5-18 years), and 152 adults (18 years), and 88% were adequate for formal interpretation. The proportion of adequate specimens was higher in adults than in children. Fifty percent of the adequate TEMs showed normal ciliary ultrastructure, 39% showed hallmark ultrastructural changes of PCD, and 11% had indeterminate findings. Among specimens without clearly normal ultrastructure, 72% had defects of the outer and/or inner dynein arms (IDA), while 7% had central apparatus defects with or without IDA defects. In summary, nasal scrape biopsies can be performed in the outpatient setting and yield interpretable samples, when performed by individuals with adequate training and experience according to an SOP.

High-frequency chest compression (HFCC) therapy has become the prevailing form of airway clearance for patients with cystic fibrosis (CF) in the United States. The original square waveform was replaced in 1995 with a sine waveform without published evidence of an equality of effectiveness. The recent development of a triangle waveform for HFCC provided the opportunity to compare the functional and therapeutic effects of different waveforms. Clinical testing was done in patients at home with therapy times recorded with all sputum collected in preweighed sealable vials. The eight study patients with CF were regular users of a sine waveform device. They produced sputum consistently and were clinically stable. They used their optimum frequencies for therapy for each waveform and, for one week for each waveform, collected all sputum during their twice-daily timed HFCC therapies. After collection, these vials were reweighed, desiccated, and reweighed to calculate wet and dry weights of sputum per minute of therapy time. Frequency associated vest pressures transmitted to the mouth, and induced airflows at the mouth were measured in healthy volunteers. The pressure waveforms produced in the vest were, in shape, faithfully demonstrable at the mouth. In the healthy subject the transmission occurred in 2 ms and was attenuated to about 75% of the vest pressure for the triangle waveform and 60% for the sine waveform. All patients produced more sputum with the triangle waveform than with the sine waveform. The mean increase was 20%+ range of 4% to 41%. P value was.001. Future studies of HFCC should investigate the other effects of the sine and triangle waveforms, as well as the neglected square waveform, on mucus clearance and determine the best frequencies for each waveform, disease, and patient.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.