Search

We trained machine learning models to identify IMI in late-lactation cows at dry-off to guide antibiotic treatment, and compared their performance to a rule-based algorithm that is currently used on dairy farms in the United States. We conducted an observational test characteristics study using a dataset of 3,645 cows approaching dry-off from 68 US dairy herds. The outcome variables of interest were cow-level IMI caused by all pathogens, major pathogens, and Streptococcus and Streptococcus-like organisms (SSLO), which were determined using aerobic culture of aseptic quarter-milk samples and identification of isolates using MALDI-TOF. Individual cow records were extracted from the farm software to create 53 feature variables at the cow and 39 at the herd level, which were derived from cow-level descriptive data, records of clinical mastitis events, results from routine testing of milk for volume, and concentrations of SCC, fat, and protein. Machine learning (ML) algorithms evaluated were logistic regression, decision tree, random forest, light gradient-boosting machine, naive Bayes, and neural networks. For comparison, cows were also classified according to a conventional rule-based algorithm that considered a cow as high risk for IMI if she had one or more high SCC (>200,000 cells/mL) tests or ≥2 cases of clinical mastitis during the lactation of enrollment. Area under the curve (AUC) and Youden's index were used to compare models, in addition to binary classification metrics, including sensitivity, specificity, and predictive values. The ML models had slightly higher AUC and Youden's index values than the rule-based algorithm for all IMI outcomes of interest. However, these improvements in prediction accuracy were substantially less than what we had considered necessary for the technology to be a worthwhile alternative to the rule-based algorithm. Therefore, evidence is lacking to support the wholesale use of ML-guided selective dry cow therapy at the moment. We recommend that producers wanting to implement algorithm-guided selective dry cow therapy use a rule-based method., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Background: Elexacaftor/tezacaftor/ivacaftor (ETI) is a highly effective therapy that improves lung disease in people with cystic fibrosis (pwCF), but its effect on glucose tolerance and insulin secretion is unclear., Methods: PROMISE is a multicenter prospective, observational study of ETI in pwCF ≥12 years and at least one F508del allele. The PROMISE Endocrine sub-study (PROMISE-ENDO) enrolled participants at 10 CF Centers where hemoglobin A1c (HbA1c) was collected and 3-hour oral glucose tolerance tests (OGTT) conducted to examine glucose tolerance, glucose excursions, insulin secretory rates (deconvolution of C-peptide) and sensitivity (oral minimal model) prior to ETI and 12-18 months (mos) and 24-30 mos following ETI initiation. Longitudinal mixed effects models were used to test within-subject ETI effects., Results: At baseline, 79 participants completed OGTTs [39 (49%) male, median (IQR) age 19.6 (14.7, 27.3) years, BMI z-score 0.12 (-0.51, 0.65)]. At 12-18 mos n=68 and at 24-30 mos n=58 completed OGTTs. At 24-30 mos, fasting glucose (mg/dL) decreased [94 (92, 96) to 90 (88, 93), p=0.02] in the subset not on insulin therapy (n=61), but no differences in 1-hr or 2-hr glucose were found. HbA1c (%) decreased from 5.8 (5.6, 5.9) to 5.5 (5.4, 5.6), p<0.001 by 24-30 mos. Although insulin sensitivity (mU/L-1.min-1) decreased [8.4 (7.2, 9.5) vs. 6.8 (5.8, 7.9), p=0.03], no changes in oral disposition index were found, p=0.14., Conclusions: After two years of ETI, fasting glucose and HbA1c showed modest decreases. Glucose tolerance varied, and overall measures of insulin secretion did not deteriorate., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Reactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report an efficient platform for 'direct-to-biology' (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (M Pro ), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a 'switch' to a reversible inhibitor series. This example of ligand discovery for M Pro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The epithelial lining of luminal organs provides an immune barrier against external factors and regulates transport of nutrients, ions, and water into the body. Several conditions are associated with a breakdown or dysfunction of the epithelial lining. Short circuit current (I sc ) measurement using a bulky, expensive, and hard to deploy system known as the Ussing chamber is the gold standard for evaluation of epithelial transport function but requires tissue excision. We demonstrated the ability of the I sc probe to measure I sc in normal wild type (WT) versus reduced CFTR function knockout (KO) rats as a relevant animal model for testing ion channel function.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) exhibit acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. CFTR modulators may improve outcomes in patients with COPD, although recent data regarding magnitude of benefit have been inconclusive and effects on mucociliary clearance are unknown. We conducted a phase 2, randomized, double blind placebo control trial to determine safety and tolerability, and explore the potential mechanism of ivacaftor for the treatment of COPD., Methods: We randomized 40 patients with moderate to severe COPD and symptoms of chronic bronchitis to ivacaftor (N=30) or placebo (N=10) 150mg BID for 12 weeks. Primary endpoints included evaluation of safety of ivacaftor and pharmacokinetics (PK). Secondary endpoints included measures of CFTR activity and clinical outcomes., Results: Ivacaftor was safe and tolerable with similar rates of adverse events rates between groups. Most common adverse event was diarrhea in the ivacaftor group and acute COPD exacerbation in the placebo group. PK analysis found the mean area under the curve over 12 hours (AUC 12 ) to be 72% of the previously reported AUC 12 in cystic fibrosis (CF). Treatment with ivacaftor did not improve sweat chloride, whole lung mucociliary clearance, lung function or respiratory symptoms., Conclusion: Ivacaftor was safe and well tolerated, but did not improve measures of CFTR activity or mucus clearance. As serum concentrations achieved were lower than observed in CF at the same dose, and modulation of wild type CFTR differs from G551D, further dose determination studies are needed to better understand treatment efficacy of CFTR potentiators in COPD. Clinical trial registration available at www., Clinicaltrials: gov, ID: NCT03085485.

Background: Acrolein, an aldehyde in smoke from tobacco products, inhibits CFTR function in vitro. Ivacaftor is an FDA-approved potentiator that improves mutant CFTR function. This human clinical study investigated the relationship between two urinary markers of tobacco smoke exposure - the acrolein metabolite 3-HPMA and the nicotine metabolite NNAL - and sweat chloride response to ivacaftor in the G551D Observational Trial (GOAL)., Methods: 3-HPMA (low: <50th centile; moderate: 50-75th centile; high: >75th centile) and NNAL (detectable/undetectable) in GOAL samples was quantified with LC-MS/MS. Self-report of tobacco smoke exposure (Y/N) served as a subjective measure. Change in sweat chloride from pre- to 6 months post-ivacaftor treatment (ΔSC) was the primary CFTR-dependent readout., Results: The sample included 151 individuals, mean age 20.7 (SD 11.4) years, range 6-59 years. Smoke exposure prevalence was 15 % per self-reports but 27 % based on detectable NNAL. 3-HPMA was increased in those reporting tobacco smoke exposure (607 vs 354 ng/ml, p = 0.008), with a higher proportion of smoke-exposed in the high- vs low-acrolein group (31 % vs 9 %, p=0.040). Compared to low-acrolein counterparts, high-acrolein participants experienced less decrease in sweat chloride (-35.2 vs -48.2 mmol/L; p = 0.020) and had higher sweat chloride values (50.6 vs 37.6 mmol/L; p = 0.020) 6 months post-ivacaftor. The odds of ivacaftor-mediated potentiation to near normative CFTR function (defined as SC 6mo <40 mmol/L) was more than twice as high in the low-acrolein cohort (OR: 2.51, p = 0.026)., Conclusions: Increased urinary 3-HPMA, an acrolein metabolite of tobacco smoke, is associated with a diminished sweat chloride response to ivacaftor potentiation of CFTR function., Competing Interests: Declaration of competing interest None., (Copyright © 2024 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The mucociliary transport apparatus is critical for maintaining lung health via the coordinated movement of cilia to clear mucus and particulates. A metachronal wave propagates across the epithelium when cilia on adjacent multiciliated cells beat slightly out of phase along the proximal-distal axis of the airways in alignment with anatomically directed mucociliary clearance. We hypothesized that metachrony optimizes mucociliary transport (MCT) and that disruptions of calcium signaling would abolish metachrony and decrease MCT. We imaged bronchi from human explants and ferret tracheae using micro-optical coherence tomography (µOCT) to evaluate airway surface liquid depth (ASL), periciliary liquid depth (PCL), cilia beat frequency (CBF), MCT, and metachrony in situ. We developed statistical models that included covariates of MCT. Ferret tracheae were treated with BAPTA-AM (chelator of intracellular Ca 2+ ), lanthanum chloride (nonpermeable Ca 2+  channel competitive antagonist), and repaglinide (inhibitor of calaxin) to test calcium dependence of metachrony. We demonstrated that metachrony contributes to mucociliary transport of human and ferret airways. MCT was augmented in regions of metachrony compared with nonmetachronous regions by 48.1%, P = 0.0009 or 47.5%, P < 0.0020 in humans and ferrets, respectively. PCL and metachrony were independent contributors to MCT rate in humans; ASL, CBF, and metachrony contribute to ferret MCT rates. Metachrony can be disrupted by interference with calcium signaling including intracellular, mechanosensitive channels, and calaxin. Our results support that the presence of metachrony augments MCT in a calcium-dependent mechanism. NEW & NOTEWORTHY We developed a novel imaging-based analysis to detect coordination of ciliary motion and optimal coordination, a process called metachrony. We found that metachrony is key to the optimization of ciliary-mediated mucus transport in both ferret and human tracheal tissue. This process appears to be regulated through calcium-dependent mechanisms. This study demonstrates the capacity to measure a key feature of ciliary coordination that may be important in genetic and acquired disorders of ciliary function.

Mucus stasis is a pathologic hallmark of muco-obstructive diseases, including cystic fibrosis (CF). Mucins, the principal component of mucus, are extensively modified with hydroxyl (O)-linked glycans, which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however, the consequences of reduced sialylation on mucus clearance have not been fully determined. Here, we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin, MUC5B, and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways, and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally, we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall, this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases., (© 2024. The Author(s).)

Background: Cystic Fibrosis causing mutations in the gene CFTR, reduce the activity of the CFTR channel protein, and leads to mucus aggregation, airway obstruction and poor lung function. A role for CFTR in the pathogenesis of other muco-obstructive airway diseases such as Chronic Obstructive Pulmonary Disease (COPD) has been well established. The CFTR modulatory compound, Ivacaftor (VX-770), potentiates channel activity of CFTR and certain CF-causing mutations and has been shown to ameliorate mucus obstruction and improve lung function in people harbouring these CF-causing mutations. A pilot trial of Ivacaftor supported its potential efficacy for the treatment of mucus obstruction in COPD. These findings prompted the search for CFTR potentiators that are more effective in ameliorating cigarette-smoke (CS) induced mucostasis., Methods: Small molecule potentiators, previously identified in CFTR binding studies, were tested for activity in augmenting CFTR channel activity using patch clamp electrophysiology in HEK-293 cells, a fluorescence-based assay of membrane potential in Calu-3 cells and in Ussing chamber studies of primary bronchial epithelial cultures. Addition of cigarette smoke extract (CSE) to the solutions bathing the apical surface of Calu-3 cells and primary bronchial airway cultures was used to model COPD. Confocal studies of the velocity of fluorescent microsphere movement on the apical surface of CSE exposed airway epithelial cultures, were used to assess the effect of potentiators on CFTR-mediated mucociliary movement., Results: We showed that SK-POT1, like VX-770, was effective in augmenting the cyclic AMP-dependent channel activity of CFTR. SK-POT-1 enhanced CFTR channel activity in airway epithelial cells previously exposed to CSE and ameliorated mucostasis on the surface of primary airway cultures., Conclusion: Together, this evidence supports the further development of SK-POT1 as an intervention in the treatment of COPD., (© 2024. The Author(s).)

Cigarette smoking is associated with COVID-19 prevalence and severity, but the mechanistic basis for how smoking alters SARS-CoV-2 pathogenesis is unknown. A potential explanation is that smoking alters the expression of the SARS-CoV-2 cellular receptor and point of entry, angiotensin converting enzyme-2 (ACE-2), and its cofactors including transmembrane protease serine 2 (TMPRSS2). We investigated the impact of cigarette smoking on the expression of ACE-2, TMPRSS2, and other known cofactors of SARS-CoV-2 infection and the resultant effects on infection severity in vitro. Cigarette smoke extract (CSE) exposure increased ACE-2 and TMPRSS2 mRNA expression compared to air control in ferret airway cells, Calu-3 cells, and primary human bronchial epithelial (HBE) cells derived from normal and COPD donors. CSE-exposed ferret airway cells inoculated with SARS-CoV-2 had a significantly higher intracellular viral load versus vehicle-exposed cells. Likewise, CSE-exposure increased both SARS-CoV-2 intracellular viral load and viral replication in both normal and COPD HBE cells over vehicle control. Apoptosis was increased in CSE-exposed, SARS-CoV-2-infected HBE cells. Knockdown of ACE-2 via an antisense oligonucleotide (ASO) reduced SARS-CoV-2 viral load and infection in CSE-exposed ferret airway cells that was augmented by co-administration of camostat mesylate to block TMPRSS2 activity. Smoking increases SARS-CoV-2 infection via upregulation of ACE2 and TMPRSS2.

The distal bronchioles in Idiopathic Pulmonary Fibrosis (IPF) exhibit histopathological abnormalities such as bronchiolization, peribronchiolar fibrosis and honeycomb cysts that contribute to the overall architectural remodeling of lung tissue seen in the disease. Here we describe an additional histopathologic finding of epithelial desquamation in patients with IPF, wherein epithelial cells detach from the basement membrane of the distal bronchioles. To understand the mechanism driving this pathology, we performed spatial transcriptomics of the epithelial cells and spatial proteomics of the basement membrane of the distal bronchioles from IPF patients and patients with no prior history of lung disease. Our findings reveal a downregulation of cell junctional components, upregulation of epithelial-mesenchymal transition signatures and dysregulated basement membrane matrix in IPF distal bronchioles, facilitating epithelial desquamation. Further, functional assays identified regulation between Collagen IV in the matrix, and the junctional genes JUP and PLEC , that is crucial for maintaining distal bronchiolar homeostasis. In IPF, this balanced regulation between matrix and cell-junctions is disrupted, leading to loss of epithelial adhesion, peribronchiolar fibrosis and epithelial desquamation. Overall, our study suggests that in IPF the interplay between the loss of cell junctions and a dysregulated matrix results in desquamation of distal bronchiolar epithelium and lung remodeling, exacerbating the disease., One Sentence Summary: Two-way regulation of cell junctional proteins and matrix proteins drives cellular desquamation and fibrosis in the distal bronchioles of patients with Idiopathic Pulmonary Fibrosis.

Rationale: The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF., Methods: Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence., Results: Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant 'proximalization' repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level., Conclusions: Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Introduction: Hypoxia due to sinus obstruction is a major pathogenic mechanism leading to sinusitis. The objective of the current study is to define the electrophysiologic characteristics of hypoxia in vitro and in vivo., Methods: Cystic fibrosis bronchoepithelial cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and human sinonasal epithelial cells were exposed to 1% or atmospheric O 2 for 24 h. Time-dependent production of cytoplasmic free radicals was measured. Cells were subjected to Ussing chamber and patch clamp technique where CFTR currents were recorded in whole-cell and cell-attached mode for single channel studies. Indices of mucociliary transport (MCT) were measured using micro-optical coherence tomography. In a rabbit hypoxic maxillary sinus model, tissue oxygenation, relative mRNA expression of HIF-1α, pH, sinus potential difference (SPD), and MCT were determined., Results: Ussing chamber (p < 0.05), whole-cell (p < 0.001), and single channel patch-clamp (p < 0.0001) showed significant inhibition of Cl - currents in hypoxic cells. Cytoplasmic free radicals showed time-dependent elevation peaking at 4 h (p < 0.0001). Airway surface liquid (p < 0.0001), periciliary liquid (p < 0.001), and MCT (p < 0.01) were diminished. Co-incubation with the free radical scavenger glutathione negated the impact of hypoxia on single channel currents and MCT markers. In sinusitis rabbits, mucosa exhibited low tissue oxygenation (p < 0.0001), increased HIF1α mRNA (p < 0.05), reduced pH (p < 0.01), and decreased MCT (p < 0.001). SPD measurements demonstrated markedly diminished transepithelial Cl - transport (p < 0.0001)., Conclusion: Hypoxia induces severe CFTR dysfunction via free radical production causing reduced MCT in vitro and in vivo. Improved oxygenation is critical to reducing the impact of persistent mucociliary dysfunction., (© 2023 ARS‐AAOA, LLC.)

Introduction: Cystic fibrosis (CF) airway disease is characterized by thick mucus and impaired mucociliary transport (MCT). Loss of functional cystic fibrosis transmembrane receptor (CFTR) leads to acidification and oxidation of airway surface mucus. Replacing bicarbonate (HCO 3 - ) topically fails due to rapid reabsorption and neutralization, while the scavenging antioxidant, glutathione sulfhydryl (GSH), is also rapidly degraded. The objective of this study is to investigate GSH/NaHCO 3 nanoparticles as novel strategy for CF airway disease., Methods: GSH/NaHCO 3 poly (lactic-co-glycolic acid) nanoparticles were tested on primary CF (F508del/F508del) epithelial cultures to evaluate dose-release curves, surface pH, toxicity, and MCT indices using micro-optical coherence tomography. In vivo tests were performed in three rabbits to assess safety and toxicity. After 1 week of daily injections, histopathology, computed tomography (CT), and blood chemistries were performed and compared to three controls. Fluorescent nanoparticles were injected into a rabbit with maxillary sinusitis and explants visualized with confocal microscopy., Results: Sustained release of GSH and HCO 3 - with no cellular toxicity was observed over 2 weeks. Apical surface pH gradually increased from 6.54 ± 0.13 (baseline) to 7.07 ± 0.10 (24 h) (p < 0.001) and 6.87 ± 0.05 at 14 days (p < 0.001). MCT, ciliary beat frequency, and periciliary liquid were significantly increased. When injected into the maxillary sinuses of rabbits, there were no changes to histology, CT, or blood chemistries. Nanoparticles penetrated rabbit sinusitis mucus on confocal microscopy., Conclusion: Findings suggest that GSH/NaHCO 3 - nanoparticles are a promising treatment option for viscous mucus in CF and other respiratory diseases of mucus obstruction such as chronic rhinosinusitis., (© 2023 ARS‐AAOA, LLC.)

Many people with CF (pwCF) desire a reduction in inhaled treatment burden after initiation of elexacaftor/tezacaftor/ivacaftor. The randomized, open-label SIMPLIFY study showed that discontinuing hypertonic saline (HS) or dornase alfa (DA) was non-inferior to continuation of each treatment with respect to change in lung function over a 6-week period. In this SIMPLIFY substudy, we used gamma scintigraphy to determine whether discontinuation of either HS or DA was associated with deterioration in the rate of in vivo mucociliary clearance (MCC) in participants ≥12 years of age. While no significant differences in MCC endpoints were associated with HS discontinuation, significant improvement in whole and peripheral lung MCC was observed after discontinuing DA. These results suggest that pwCF on ETI with mild lung disease do not experience a subclinical deterioration in MCC that could later impact health outcomes after discontinuing HS, and in fact may benefit from improved MCC after stopping DA treatment., Competing Interests: Declaration of competing interest Unrelated to the submitted work, SHD reports research funding from Chiesi USA, Vertex Pharmaceuticals, Calithera Biosciences, and 4D Molecular Therapeutics, and consulting income from Boehringer Ingelheim and Abbvie. PJM reports grant funding from Vertex Pharmaceuticals and Eloxx. SMR reports grant funding from Vertex Pharmaceuticals, Galapagos/Abbvie, Eloxx, Synspira, Translate Bio, Arcturus, Astra-Zenica, and Ionis, and consulting income from Vertex Pharmaceuticals, Synspira, Renovion, Cystetic Medicines, and Arcturus; all personal conflicts to SMR were resolved or ended in 2022 or before. AHG has clinical trial agreements with AbbVie, 4D Molecular Therapeutics, and Insmed. TEC reports research funding from NIH, Astra Zeneca, Regeneron, Pieris, and the CF Foundation through a Research Development Program award. NMH reports grants from CFF, NIH, and FDA, and DSMB membership for the NIH., (Copyright © 2024. Published by Elsevier B.V.)

Background: Cystic fibrosis associated liver disease (CFLD) carries a significant disease burden with no effective preventive therapies. According to the gut-liver axis hypothesis for CFLD pathogenesis, dysbiosis and increased intestinal inflammation and permeability permit pathogenic bacterial translocation into the portal circulation, leading to hepatic inflammation and fibrosis. Evaluating the effect of CFTR (cystic fibrosis transmembrane conductance regulator) modulation with elexacaftor/tezacaftor/ivacaftor (ETI) may help determine the role of CFTR in CFLD and increase understanding of CFLD pathogenesis, which is critical for developing therapies. We aimed to characterize the fecal microbiota in participants with CF with and without advanced CFLD (aCFLD) before and after ETI., Methods: This is an ancillary analysis of stool samples from participants ages ≥12 y/o enrolled in PROMISE (NCT04038047). Included participants had aCFLD (cirrhosis with or without portal hypertension, or non-cirrhotic portal hypertension) or CF without liver disease (CFnoLD). Fecal microbiota were defined by shotgun metagenomic sequencing at baseline and 1 and 6 months post-ETI., Results: We analyzed 93 samples from 34 participants (11 aCFLD and 23 CFnoLD). Compared to CFnoLD, aCFLD had significantly higher baseline relative abundances of potential pathogens Streptococcus salivarius and Veillonella parvula. Four of 11 aCFLD participants had an initially abnormal fecal calprotectin that normalized 6 months post-ETI, correlating with a significant decrease in S. salivarius and a trend towards decreasing V. parvula., Conclusions: These results support an association between dysbiosis and intestinal inflammation in CFLD with improvements in both post-ETI, lending further support to the gut-liver axis in aCFLD., Competing Interests: Declaration of competing interest JTD – Grant funding from CFF SJS – Grant funding from Vertex, CFF, NIH SMR – Consultant for Vertex; Grant funding from Vertex, CFF, NIH GMS – Consultant for GSK, Genentech, Electromed; Grant funding from Vertex, CFF, NIH DPN – Consultant for Vertex, Genentech; Grant funding from Vertex, CFF, NIH LRH – Grant funding from CFF, NIH MRN – Consultant for UpToDate, Vertex: Grant funding from Gilead, AbbVie, CFF, NIH NG – Grant funding from SCRI-CRSP, (Copyright © 2024. Published by Elsevier B.V.)

Background: Use of elexacaftor/tezacaftor/ivacaftor (ETI) for treatment of cystic fibrosis (CF) has resulted in unprecedented clinical improvements necessitating development of outcome measures for monitoring disease course. Intranasal micro-optical coherence tomography (μOCT) has previously helped detect and characterize mucociliary abnormalities in patients with CF. This study was done to determine if μOCT can define the effects of ETI on nasal mucociliary clearance and monitor changes conferred to understand mechanistic effects of CFTR modulators beyond CFTR activation., Methods: 26 subjects, with at least 1 F508del mutation were recruited and followed at baseline (visit 1), +1 month (visit 2) and +6 months (visit 4) following initiation of ETI therapy. Clinical outcomes were computed at visits 1, 2 and 4. Intranasal μOCT imaging and functional metrics analysis including mucociliary transport rate (MCT) estimation were done at visits 1 and 2., Results: Percent predicted forced expiratory volume in 1 s (ppFEV 1 ) showed a significant increase of +10.9 % at visit 2, which sustained at visit 4 (+10.6 %). Sweat chloride levels significantly decreased by -36.6 mmol/L and -41.3 mmol/L at visits 2 and 4, respectively. μOCT analysis revealed significant improvement in MCT rate (2.8 ± 1.5, visit 1 vs 4.0 ± 1.5 mm/min, visit 2; P = 0.048)., Conclusions: Treatment with ETI resulted in significant and sustained clinical improvements over 6 months. Functional improvements in MCT rate were evident within a month after initiation of ETI therapy indicating that μOCT imaging is sensitive to the treatment effect of HEMT and suggests improved mucociliary transport as a probable mechanism of action underlying the clinical benefits., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Steven M. Rowe and Guillermo J. Tearney has patent pending to Unlicensed patent on the use of optical coherence tomography., (© 2024 The Authors.)

Humans have a long history of transporting and trading plants, contributing to the evolution of domesticated plants. Theobroma cacao originated in the Neotropics from South America. However, little is known about its domestication and use in these regions. In this study, ceramic residues from a large sample of pre-Columbian cultures from South and Central America were analyzed using archaeogenomic and biochemical approaches. Here we show, for the first time, the widespread use of cacao in South America out of its native Amazonian area of origin, extending back 5000 years, likely supported by cultural interactions between the Amazon and the Pacific coast. We observed that strong genetic mixing between geographically distant cacao populations occurred as early as the middle Holocene, in South America, driven by humans, favoring the adaptation of T. cacao to new environments. This complex history of cacao domestication is the basis of today's cacao tree populations and its knowledge can help us better manage their genetic resources., (© 2024. The Author(s).)

Despite progress in elucidation of disease mechanisms, identification of risk factors, biomarker discovery, and the approval of two medications to slow lung function decline in idiopathic pulmonary fibrosis and one medication to slow lung function decline in progressive pulmonary fibrosis, pulmonary fibrosis remains a disease with a high morbidity and mortality. In recognition of the need to catalyze ongoing advances and collaboration in the field of pulmonary fibrosis, the NHLBI, the Three Lakes Foundation, and the Pulmonary Fibrosis Foundation hosted the Pulmonary Fibrosis Stakeholder Summit on November 8-9, 2022. This workshop was held virtually and was organized into three topic areas: 1 ) novel models and research tools to better study pulmonary fibrosis and uncover new therapies, 2 ) early disease risk factors and methods to improve diagnosis, and 3 ) innovative approaches toward clinical trial design for pulmonary fibrosis. In this workshop report, we summarize the content of the presentations and discussions, enumerating research opportunities for advancing our understanding of the pathogenesis, treatment, and outcomes of pulmonary fibrosis.

Background: The cystic fibrosis transmembrane conductance regulator (CFTR) modulator elexacaftor/tezacaftor/ivacaftor (E/T/I) is highly effective clinically for those with at least one F508del-CFTR allele. The effects of E/T/I on mucociliary clearance (MCC) and sputum properties are unknown. We, therefore, sought to characterize the effects of E/T/I on in vivo MCC and sputum characteristics hypothesized to impact mucus transport., Methods: Forty-four participants ≥12 years of age were enrolled into this prospective, observational trial prior to initiation of E/T/I and had baseline measurement of MCC and characterization of induced sputum and exhaled breath condensate (EBC) samples. Study procedures were repeated after 1 month of E/T/I treatment., Results: Average age was 27.7 years with baseline forced expiratory volume in 1 second (FEV 1 ) of 78.2 % predicted. 52 % of subjects had previously been treated with a 2-drug CFTR modulator combination. The average whole lung MCC rate measured over 60 min (WLAveClr60) significantly improved from baseline to post-E/T/I (14.8 vs. 22.8 %; p = 0.0002), as did other MCC indices. Sputum% solids also improved (modeled mean 3.4 vs. 2.2 %; p<0.0001), whereas non-significant reductions in sputum macrorheology (G', G") were observed. No meaningful changes in exhaled breath condensate endpoints (sialic acid:urea ratio, pH) were observed., Conclusions: E/T/I improved the hydration of respiratory secretions (% solids) and markedly accelerated MCC. These data confirm the link between CFTR function, mucus solid content, and MCC and help to define the utility of MCC and mucus-related bioassays in future efforts to restore CFTR function in all people with CF., Competing Interests: Declaration of Competing Interest All authors received research support from the Cystic Fibrosis Foundation for the conduct of this study. In addition, SHD declares research funding from Vertex Pharmaceuticals, Astra Zeneca, Calithera Biosciences, Chiesi, and 4D Molecular Therapeutics, as well as consulting or advisory fees from Boehringer Ingleheim, Abbvie Pharmaceuticals, and Enterprise Therapeutics; PM reports research support from Eloxx Pharmaceuticals, Vertex Pharmaceuticals; CRE declares research funding Tavanta Therapeutics and the NIDDK; DPN reports consulting fees from Vertex Pharmaceuticals, Respirion, and Kither Biotechnology; WDB reports research funding from Vertex Pharmaceuticals; and SMR reports research funding and non-financial support from Vertex Pharmaceuticals. TEC, JMP, BLL, ERB, ESH, AC, LJE, KZ and JW declare no other relevant conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation, respiratory symptoms, inflammation of the airways, and systemic manifestations of the disease. Genetic susceptibility and environmental factors are important in the development of the disease, particularly exposure to cigarette smoke which is the most notable risk factor. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are the cause of cystic fibrosis (CF), which shares several pathophysiological pulmonary features with COPD, including airway obstruction, chronic airway inflammation and bacterial colonization; in addition, both diseases also present systemic defects leading to comorbidities such as pancreatic, gastrointestinal, and bone-related diseases. In patients with COPD, systemic CFTR dysfunction can be acquired by cigarette smoking, inflammation, and infection. This dysfunction is, on average, about half of that found in CF. Herein we review the literature focusing on acquired CFTR dysfunction and the potential role in the pathogenesis of comorbidities associated with COPD and chronic bronchitis., Competing Interests: Declaration of competing interest In the past 36 months, M. Miravitlles has received consulting fees from AstraZeneca, Atriva Therapeutics, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, CSL Behring, Inhibrx, Mereo Biopharma, Verona Pharma, Spin Therapeutics, ONO Pharma, Palobiofarma SL, Takeda, Novartis, Novo Nordisk, Sanofi and Grifols, speaker fees from AstraZeneca, GlaxoSmithKline, Boehringer Ingelheim, Chiesi, Cipla, Janssen, Kamada, Menarini, Takeda, Speciality Therapeutics, Zambon, CSL Behring, Grifols and Novartis, support for attending meetings/travel from Novartis, Boehringer Ingelheim and Menarini, research grants from Grifols, and has participated on a data safety monitoring board for Mereo. G.J. Criner has no declarations. M.A. Mall declares editorial support from Novartis Pharma AG since the initial planning of the work, and declares grants from German Ministry for Education and Research and the German Research Foundation, consulting fees from Abbvie, Antabio, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotech, Pieris Pharmaceuticals, Santhera, Sterna Biologicals and Vertex Paharmaceuticals, lecture fees from Arrowhead Pharmaceuticals, Boehringer Ingelheim and Vertex Pharmaceuticals, travel reimbursement from Boehringer Ingelheim and Vertex Pharmaceuticals, and personal fees for participation on advisory boards from Boehringer Ingelheim, Arrowhead Pharmaceuticals, Vertex Pharmaceuticals, Santhera, Enterprise Therapeutics, Antabio, Kither Biotech, Pari and Abbvie; an elected unpaid member of the ECFS board. S.M. Rowe declares grant support for clinical trials conducted through university grants/contracts from Novartis, TranslateBio, Galapagos/Abbvie, Vertex Pharmaceuticals, research grant through University grants/contracts from Synedgen/Synspira, Eloxx, Ionis and AstraZeneca, consulting fees services on the design and conduct of clinical trials from Arcturus, Cystetic Medicines, Galapagos/Abbvie, Ionis, Novartis, Renovion, Synedgen/Synspira, Vertex Pharmaceuticals, support for travel to attend meetings from Vertex Pharmaceuticals; co-chair of the Next Generation Steering Committee with Vertex Pharmaceuticals, research product for investigator initiated research provided from Synedgen/Synspira and Renovion, consulting services on the design and conduct of clinical trials including stock options for Synedgen/Synspira and Renovion within the past 36 months; MTAs for investigator-initiated and externally funded research efforts from Ionis, Galapagos/Abbvie and Synedgen/Synspira, all in the past 36 months; and declares six patents. C.F. Vogelmeier declares institution grants from German Ministry of Education and Science (BMBF), AstraZeneca, Boehringer Ingelheim, Chiesi, CSL Behring, GlaxoSmithKline, Grifols and Novartis, consulting fees from Aerogen, AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GlaxoSmithKline, Insmed, Menarini, Novartis and Nuvaira, and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Aerogen, AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GlaxoSmithKline, Insmed, Menarini and Novartis, in the past 36 months. B. Hederer was an employee of Novartis Pharma AG at the timing of writing this manuscript. M. Schoenberger is a full-time employee of Novartis Pharma AG and retains Novartis stock. P. Altman was an employee of Novartis Pharmaceutical Corporation at time of writing this manuscript., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: In two pivotal phase 3 trials, up to 24 weeks of treatment with elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) was efficacious and safe in patients with cystic fibrosis (CF) ≥12 years of age who have at least one F508del allele. The aim of this study is to assess long-term safety and efficacy of ELX/TEZ/IVA in these patients., Methods: In this phase 3, open-label, single-arm extension study, participants with F508del -minimal function (from a 24-week parent study; n=399) or F508del - F508del (from a 4-week parent study; n=107) genotypes receive ELX/TEZ/IVA at the same dose (ELX 200 mg once daily, TEZ 100 mg once daily and IVA 150 mg every 12 h). The primary end-point is safety and tolerability. A prespecified interim analysis was conducted when the last participant reached the Week 144 visit., Results: At the Week 144 interim analysis, mean duration of exposure to ELX/TEZ/IVA in the extension study was 151.1 weeks. Exposure-adjusted rates of adverse events (AEs) (586.6 events per 100 participant-years) and serious AEs (22.4 events per 100 participant-years) were lower than in the ELX/TEZ/IVA treatment group in the 24-week parent study (1096.0 and 36.9 events per 100 participant-years, respectively); most participants had AEs classified as mild (16.4% of participants) or moderate (60.3% of participants) in severity. 14 participants (2.8%) had AEs that led to treatment discontinuation. Following initiation of ELX/TEZ/IVA, participants had increases in forced expiratory volume in 1 s (FEV 1 ) percentage predicted, Cystic Fibrosis Questionnaire-Revised respiratory domain score and body mass index, and had decreases in sweat chloride concentration and pulmonary exacerbation rates that were maintained over the interim analysis period. The mean annualised rate of change in FEV 1 % pred was +0.07 (95% CI -0.12-0.26) percentage points among the participants., Conclusions: ELX/TEZ/IVA was generally safe and well tolerated, with a safety profile consistent with the 24-week parent study. Participants had sustained improvements in lung function, respiratory symptoms, CF transmembrane conductance regulator function, pulmonary exacerbation rates and nutritional status. These results support the favourable safety profile and durable, disease-modifying clinical benefits of ELX/TEZ/IVA., Competing Interests: Conflicts of interest: All authors received nonfinancial support (assistance with manuscript preparation) from Nucleus Global, which received funding from Vertex Pharmaceuticals Incorporated. C.L. Daines has nothing further to disclose. E. Tullis has received consulting, speaker and travel fees from Vertex Pharmaceuticals. S. Costa serves on an advisory board for Vertex Pharmaceuticals. R.W. Linnemann serves on an advisory board and reports grants paid to her institution and consulting fees from Vertex Pharmaceuticals. M.A. Mall reports patient recruitment fees paid to his institution and advisory fees from Vertex Pharmaceuticals, consulting fees from Antabio, Arrowhead Pharmaceuticals, Boehringer Ingelheim, Enterprise Therapeutics, Santhera, Sterna Biologicals and Vertex Pharmaceuticals, speaker fees from Arrowhead Pharmaceuticals, Boehringer Ingelheim and Vertex Pharmaceuticals, travel fees from Boehringer Ingelheim and Vertex Pharmaceuticals, advisory fees from Antabio, Arrowhead Pharmaceuticals, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotech, Santhera and Vertex Pharmaceuticals, and serves on the European Cystic Fibrosis Society (ECFS) board. E.F. McKone reports grants, lecture fees and serving on an advisory board for Vertex Pharmaceuticals, lecture fees from Roche, travel fees from A. Menarini, and serving on advisory boards for Janssen, Insmed and CF Storm. D. Polineni reports grants from Laurent Pharmaceuticals, Parion Sciences, Proteostasis Therapeutics and Vertex Pharmaceuticals, consulting fees from Vertex Pharmaceuticals, nonfinancial support for travel to investigator meeting from Vertex Pharmaceuticals, and serves on an advisory board for Sanofi. B.S. Quon reports payments paid to his institution and speaker fees from Vertex Pharmaceuticals. F.C. Ringshausen reports grants paid to his institution from Basilea Pharmaceutica, German Center for Lung Research (DZL), German Center for Infection Research (DZIF), Inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis (iABC)/Innovative Medicines Initiative (IMI), European Federation of Pharmaceutical Industries and Associations (EFPIA), Insmed, Novartis and Polyphor, consulting fees from Grifols, Insmed, Parion, Shionogi and Zambon, speaker fees from AstraZeneca, Boehringer Ingelheim, Chiesi, Grifols, Insmed and Novartis, participation on advisory boards for Grifols, Insmed, Parion, Shionogi and Zambon, unpaid honoraria as co-chair of the German Bronchiectasis Registry (PROGNOSIS), and payments to his institution from AbbVie, AstraZeneca, Boehringer Ingelheim, Corbus, Celtaxsys, Insmed, Novartis, Parion, Polyphor, Vertex and Zambon; and is a steering committee member of the European Bronchiectasis Registry (EMBARC), a steering committee member of the European NTM registry (EMBARC-NTM), a core network lead in ERN-LUNG, a principal investigator for DZL, a chair of the cystic fibrosis working group of the German Respiratory Society (DGP), a steering committee member of the Group of German CF Physicians (AGAM), and co-chair of medical consultants of PCD Patient Advocacy Group (Kartagener Syndrom und Primäre Ciliäre Dyskinesie eV). S.M. Rowe reports grants paid to his institution from AbbVie, Arrowhead Pharmaceuticals, AstraZeneca, Bayer, Celtaxsys, Eloxx, Ionis Pharmaceuticals, Novartis, Proteostasis Therapeutics, Synedgen, Synspira Therapeutics, Translate Bio and Vertex Pharmaceuticals, nonfinancial support from AbbVie, Ionis Pharmaceuticals, Proteostasis Therapeutics, Renovion, Synedgen and Synspira Therapeutics, consulting fees from AbbVie, Arrowhead Pharmaceuticals, Bayer, Cystetic Medicines, Ionis Pharmaceuticals, Novartis, Renovion, Synedgen, Synspira Therapeutics and Vertex Pharmaceuticals, and serves as co-chair for the Next Generation Steering Committee on Vertex Pharmaceuticals. H. Selvadurai has nothing further to disclose. J.L. Taylor-Cousar serves on the board of trustees, clinical research executive committee, clinical research advisory board and Women's Health Research-Working Group for the US Cystic Fibrosis Foundation, serves on the scientific advisory board for Emily's Entourage, serves on the respiratory health awards working group, scientific grants review committee and clinical problems assembly programming committee for the American Thoracic Society; reports consulting fees from 4D Molecular Therapeutics, Celtaxsys, Prolarean Imaging, Protalix Biotherapeutics, Proteostasis Therapeutics and Santhera Pharmaceuticals, grants to her institution from Bayer, Celtaxsys, Eloxx Pharmaceuticals, Gilead, N30 and Proteostasis Therapeutics, speaking fees from Celtaxsys and Gilead, serves on advisory board for AbbVie, Genentech, Insmed and Novartis, and is an associate editor for the Journal of Cystic Fibrosis. N.J. Withers reports lecture fees from Vertex Pharmaceuticals and serves on an advisory board for Vertex Pharmaceuticals and Proteostasis Therapeutics. B. Ramsey reports travel fees and grants from, and serves on advisory board for Vertex Pharmaceuticals, and personal fees from Cystetic Medicines. M. Griese reports grants to his institution from Vertex Pharmaceuticals. N. Ahluwalia, S.M. Moskowitz, V. Prieto-Centurion, Y.V. Tan, S. Tian, T. Weinstock, F. Xuan and Y. Zhang are employees of Vertex and may own stock or stock options in Vertex., (Copyright ©The authors 2023.)

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: We previously discovered that Korean red ginseng aqueous extract (RGAE) potentiates the TMEM16A channel, improved mucociliary transport (MCT) parameters in CF nasal epithelia in vitro, and thus could serve as a therapeutic strategy to rescue the MCT defect in cystic fibrosis (CF) airways. The hypothesis of this study is that RGAE can improve epithelial Cl - secretion, MCT, and histopathology in an in-vivo CF rat model., Methods: Seventeen 4-month old CFTR -/- rats were randomly assigned to receive daily oral control (saline, n = 9) or RGAE (Ginsenosides 0.4mg/kg/daily, n = 8) for 4 weeks. Outcomes included nasal Cl - secretion measured with the nasal potential difference (NPD), functional microanatomy of the trachea using micro-optical coherence tomography, histopathology, and immunohistochemical staining for TMEM16a., Results: RGAE-treated CF rats had greater mean NPD polarization with UTP (control = -5.48 +/- 2.87 mV, RGAE = -9.49 +/- 2.99 mV, p < 0.05), indicating, at least in part, potentiation of UTP-mediated Cl - secretion through TMEM16A. All measured tracheal MCT parameters (airway surface liquid, periciliary liquid, ciliary beat frequency, MCT) were significantly increased in RGAE-treated CF rats with MCT exhibiting a 3-fold increase (control, 0.45+/-0.31 vs. RGAE, 1.45+/-0.66 mm/min, p < 0.01). Maxillary mucosa histopathology was markedly improved in RGAE-treated cohort (reduced intracellular mucus, goblet cells with no distention, and shorter epithelial height). TMEM16A expression was similar between groups., Conclusion: RGAE improves TMEM16A-mediated transepithelial Cl - secretion, functional microanatomy, and histopathology in CF rats. Therapeutic strategies utilizing TMEM16A potentiators to treat CF airway disease are appropriate and provide a new avenue for mutation-independent therapies., Competing Interests: Declaration of Competing Interest Bradford A. Woodworth, M.D. is a consultant for Cook Medical, Smith and Nephew, and Medtronic. All other authors have no conflicts of interest to report., (Copyright © 2023. Published by Elsevier B.V.)

Background: Mucus stasis, a hallmark of muco-obstructive disease, results from impaired mucociliary transport and leads to lung function decline and chronic infection. Although therapeutics that target mucus stasis in the airway, such as hypertonic saline or rhDNAse, show some therapeutic benefit, they do not address the underlying electrostatic defect apparent in mucins in CF and related conditions. We have previously shown poly (acetyl, arginyl) glucosamine (PAAG, developed as SNSP113), a soluble, cationic polymer, significantly improves mucociliary transport in a rat model of CF by normalizing the charge defects of CF mucin. Here, we report efficacy in the CFTR-sufficient, ENaC hyperactive, Scnn1b-Tg mouse model that develops airway muco-obstruction due to sodium hyperabsorption and airway dehydration., Methods: Scnn1b-Tg mice were treated with either 250 µg/mL SNSP113 or vehicle control (1.38% glycerol in PBS) via nebulization once daily for 7 days and then euthanized for analysis. Micro-Optical Coherence Tomography-based evaluation of excised mouse trachea was used to determine the effect on the functional microanatomy. Tissue analysis was performed by routine histopathology., Results: Nebulized treatment of SNSP113 significantly improved mucociliary transport in the airways of Scnn1b-Tg mice, without altering the airway surface or periciliary liquid layer. In addition, SNSP113 significantly reversed epithelial hypertrophy and goblet cell metaplasia. Finally, SNSP113 significantly ameliorated eosinophilic crystalline pneumonia and lung consolidation in addition to inflammatory macrophage influx in this model., Conclusion: Overall, this study extends the efficacy of SNSP113 as a potential therapeutic to alleviate mucus stasis in muco-obstructive diseases in CF and potentially in related conditions., Competing Interests: Declaration of Competing Interest Steven M. Rowe received grant funding and consulting fees from Synspira and Synedgen that ended in 2022. Shenda M. Baker provided SNSP113 (PAAG) used in study. Elex S. Harris, Lea Novak, Courtney M. Fernandez-Petty, and Susan E. Birket have no conflicts of interest to report., (Copyright © 2023. Published by Elsevier B.V.)

Cystic fibrosis (CF) is a genetic disease hallmarked by aberrant ion transport that results in delayed mucus clearance, chronic infection, and progressive lung function decline. Several animal models have been developed to study the airway anatomy and mucus physiology in CF, but they are costly and difficult to maintain, making them less accessible for many applications. A more available CFTR-/- rat model has been developed and characterized to develop CF airway abnormalities, but consistent dosing of pharmacologic agents and longitudinal evaluation remain a challenge. In this study, we report the development and characterization of a novel ex vivo trachea model that utilizes both wild type (WT) and CFTR-/- rat tracheae cultured on a porcine gelatin matrix. Here we show that the ex vivo tracheae remain viable for weeks, maintain a CF disease phenotype that can be readily quantified, and respond to stimulation of mucus and fluid secretion by cholinergic stimulation. Furthermore, we show that ex vivo tracheae may be used for well-controlled pharmacological treatments, which are difficult to perform on freshly excised trachea or in vivo models with this degree of scrutiny. With improved interrogation possible with a durable trachea, we also established firm evidence of a gland secretion defect in CFTR-/- rat tracheae compared to WT controls. Finally, we demonstrate that the ex vivo tracheae can be used to generate high mucus protein yields for subsequent studies, which are currently limited by in vivo mucus collection techniques. Overall, this study suggests that the ex vivo trachea model is an effective, easy to set up culture model to study airway and mucus physiology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Harris et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The growing use of modulator therapies aimed at restoring cystic fibrosis transmembrane conductance regulator (CFTR) protein function in people with cystic fibrosis has fundamentally altered clinical trial strategies needed to advance new therapeutics across an orphan disease population that is now divided by CFTR modulator eligibility. The development of a robust pipeline of nucleic acid-based therapies (NABTs)-initially directed towards the estimated 10% of the cystic fibrosis population who are genetically ineligible for, or intolerant of, CFTR modulators-is dependent on the optimisation of restricted trial participant resources across multiple development programmes, a challenge that will preclude the use of gold standard placebo-controlled trials. Advancement of a full pipeline of symptomatic therapies across the entire cystic fibrosis population will be challenged by smaller effect sizes and uncertainty regarding their clinical importance in a growing modulator-treated population with more mild and stable pulmonary disease. In this Series paper, we aim to lay the foundation for clinical trial strategy and community partnership that must deviate from established and familiar precedent to advance the future pipeline of cystic fibrosis therapeutics., Competing Interests: Declaration of interests NM-H reports grants from the Cystic Fibrosis Foundation (CFF), the National Institutes of Health (NIH; P30 DK 089507 and UL1 TR002319), and the Food and Drug Administration (FDA); consulting fees from Enterprise Therapeutics; and data safety monitoring board (DSMB) membership for the NIH. JPC is an employee of CFF. RJ reports grants from CFF; consulting fees from Boehringer Ingelheim and Recode Therapeutics; honoraria from Vertex Pharmaceuticals; and travel support from CFF. SHD reports contracts from Calithera, CFF, NIH (P30 DK065988), Vertex Pharmaceuticals, 4D Molecular Therapeutics, and Chiesi USA; consulting fees from Polarean, 501 Ventures, Enterprise Therapeutics, and Boehringer Ingelheim; fees for participation on advisory boards for Innova Healthcare and Boehringer Ingleheim; travel fees from Enterprise Therapeutics and CFF; and participation on a board for Abbvie. IF reports grants from AbbVie, Bayer, Boehringer Ingelheim, Insmed, GSK, and Vertex Pharmaceuticals; honoraria from Vertex Pharmaceuticals; board participation for AbbVie, Boehringer Ingelheim, Kither Biotech, and Vertex Pharmaceuticals; and support to her institution from the European Cystic Fibrosis Society (ECFS). CHG reports grants and contracts from CFF, NIH (P30 DK 089507 and UL1 TR000423), and FDA; consulting fees from Enterprise Therapeutics; honoraria from Gilead Sciences and Vertex Pharmaceuticals; travel support from Vertex Pharmaceuticals and Enterprise Therapeutics; participation on the board for Novartis; stock options for Air Therapeutics; and leadership roles for the American Thoracic Society (ATS). DP reports grants from CFF (002805121), NIH, and Aclaris Pharmaceuticals; travel support from CFF; and board participation for Vertex Pharmaceuticals and Translate Bio. FR reports grants from Vertex Pharmaceuticals and consulting fees from Vertex Pharmaceuticals and Calithera. BSQ reports grants from CFF, Cystic Fibrosis Canada, Vertex Pharmaceuticals, and Gilead Sciences; honoraria from Vertex Pharmaceuticals; and travel support from CFF. ETZ reports grants from NIH, Vertex Pharmaceuticals, and CFF (002884121); consulting fees from CFF; travel support from CFF, Vertex Pharmaceuticals, and ECFS; and participation on boards for CFF and Vertex Pharmaceuticals. SCB reports grants from the National Health and Medical Research Council Australia (APP1102494), the Medical Research Futures Fund Australia, and CFF (BELL1480 and BELL19A0); and honoraria from Vertex Pharmaceuticals. JCD reports grants from the UK Cystic Fibrosis Trust (as part of their Clinical Trials Accelerator Platform), CFF, Cystic Fibrosis Ireland, the Engineering and Physical Sciences Research Council, and the National Institute for Health and Care Research; and honoraria from Vertex Pharmaceuticals, Boehringer Ingelheim, Eloxx, Algipharma, Abbvie, Arcturus, Enterprise Therapeutics, Recode, LifeArc, Genentech, and Tavanta. JCD serves as Deputy Editor for the Journal of Cystic Fibrosis. MWK reports grants from NIH (P01HL128192 and UL1TR002548) and CFF; consulting fees from AbbVie, AzurRx, Cystetic Medicines, EnBiotix, First Wave Biopharma, Insmed, Laurent Pharmaceuticals, Mylan, and PBM BC Holdings; board participation for AbbVie, CFF, First Wave Biopharma, Insmed, Laurent Pharmaceuticals, Sionna, and Vertex; and committee membership for CFF. MAM reports grants from the German Research Foundation (CRC 1449 project #431232613), the German Ministry for Education and Research (82DZL009B1), the German Innovation Fund, and Vertex Pharmaceuticals; consulting fees from Abbvie, Antabio, Arrowhead, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotec, Prieris, Recode, Santhera, Splisense, and Vertex Pharmaceuticals; honoraria from Vertex Pharmaceuticals; travel support from Boehringer Ingelheim and Vertex Pharmaceuticals; and participation on boards for Abbvie, Antabio, Arrowhead, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotec, Pari, and Vertex Pharmaceuticals. EM reports grants from Vertex Pharmaceuticals; honoraria from Vertex Phamaceuticals; travel support from Menarini; and board participation for the Cystic Fibrosis Storm Clinical Trial, Vertex Pharmaceuticals, Janssen, Abbvie, and Insmed. JHR reports grants from Cystic Fibrosis Canada, CFF, Vertex Phamaceuticals, the Canada Foundation for Innovation, and the Canadian Institutes of Health Research; consulting fees from Sanofi; and travel support from Vertex Pharmaceuticals. SMR reports grant funding from CFF (P30DK072482), NIH (UL1TR003096), Vertex Pharmaceuticals, Galapagos/Abbvie, Eloxx, Synspira, Translate Bio, Arcturus, Astra-Zenica, and Ionis; and consulting fees from Vertex Pharmaceuticals, Synspira (including stock options), Renovion (including stock options), Cystetic Medicines, and Arcturus. SMR's potential competing interests were resolved or ended in 2022 or earlier. JLT-C reports grants and contracts from CFF, Vertex Pharmaceutics, Eloxx, and 4DMT; consulting fees from Vertex Phamaceuticals, Insmed, and 4DMT; participation on a DSMB for Abbvie; and participation on advisory boards for CFF, ATS, Journal of Cystic Fibrosis, The Lancet Respiratory Medicine, and Emily's Entourage. GR-B reports grants and contracts from Vertex Pharmaceuticals and CFF. DGD reports grants from Chiesi Farmaceutici and CFF; consulting fees from Vertex and Insmed; honoraria from Chiesi and Gilead; travel support from ECFS and CFF; board participation for Momab and CSL Behring; and support from ECFS as director of the Clinical Trials Network. MJ, NRK, TL, KP, JMP, and LQ declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans 1,2 , but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-Cre ERT2 ::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-Cre ERT2 ::CFTR L/L ). By comparing these models with cystic fibrosis ferrets 3,4 , we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-Cre ERT2 ::CFTR L/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl - and HCO 3 - . Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets., (© 2023. The Author(s).)

Cyclopropanes are highly useful motifs that are often incorporated into drug candidates to improve potency, metabolic stability, or pharmacokinetic properties. An expedient method for the α-cyclopropanation of ketones using hydrogen borrowing (HB) catalysis is described. The transformation occurs via HB alkylation of a hindered ketone with subsequent intramolecular displacement of a pendant leaving group affording the cyclopropanated product. The leaving group can be installed in either the ketone or alcohol component of the HB system, giving access to α-cyclopropyl ketones via two complementary approaches. Conversion to the corresponding carboxylic acids can be achieved in a simple two-step sequence to afford synthetically useful 1,1-substituted spirocyclopropyl acid building blocks.

Background: MRT5005, a codon-optimized CFTR mRNA, delivered by aerosol in lipid nanoparticles, was designed as a genotype-agnostic treatment for CF lung disease., Methods: This was a randomized, double-blind, placebo-controlled Phase 1/2 study performed in the US. Adults with 2 severe class I and/or II CFTR mutations and baseline ppFEV1 values between 50 and 90% were randomized 3:1 (MRT5005: placebo). Six dose levels of MRT5005 (4, 8, 12, 16, 20, and 24 mg) or placebo (0.9% Sodium Chloride) were administered by nebulization. The single ascending dose cohort was treated over a range from 8 to 24 mg; the multiple ascending dose cohort received five weekly doses (range 8-20 mg); and the daily dosing cohort received five daily doses (4 mg)., Results: A total of 42 subjects were assigned to MRT5005 [31] or placebo [11]. A total of 14 febrile reactions were observed in 10 MRT5005-treated participants, which were mild [3] or moderate [11] in severity; two subjects discontinued related to these events. Additionally, two MRT5005-treated patients experienced hypersensitivity reactions, which were managed conservatively. The most common treatment emergent adverse events were cough and headache. No consistent effects on FEV1 were noted., Conclusions: MRT5005 was generally safe and well tolerated through 28 days of follow-up after the last dose, though febrile and hypersensitivity reactions were noted. The majority of these reactions resolved within 1-2 days with supportive care allowing continued treatment with MRT5005 and careful monitoring. In this small first-in-human study, FEV1 remained stable after treatment, but no beneficial effects on FEV1 were observed., Competing Interests: Declaration of Competing Interest The authors have completed ICMJE disclosure of interest forms. MJ, JL, KDM, and SZN report no competing interests. JBZ, CB, JCC, DD, NL, SL, SMR, KAM, KSM, and MSS report clinical trial funding from Translate Bio to their institution for this study. MSS, EBM, KAM, and JCC report support for the current manuscript from Emily's Entourage. DD reports research contracts with Insmed, Inc., Aridis Pharmaceuticals, Armata Pharmaceuticals, and 4D Molecular Therapeutics and fees from the CFF for his role on the Data Safety Monitoring Board (DSMB). DD is a member of the DSMB at the University of Pennsylvania. KSM reports research grants to her institution from the CFF in partnership with 4D Molecular Therapeutics, Abbvie, Aridis Pharmaceuticals, Armata Pharmaceuticals, Boeringer-Ingelheim, Corbus, Insmed, Laurent Pharmaceuticals, Novartis, Eloxx, Vertex Pharmaceuticals, Savara, and Proteostasis for unrelated research. SMR reports research grants to his institution from the CFF in partnership with Novartis, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, and Astra Zenica for unrelated research. SMR reports consulting fees for clinical trial design and conduct from Novartis, Galapagos/Abbvie, Synedgen/Synspira, Vertex Pharmaceuticals, Renovion, Ionis, Cystetic Medicines, and Arcturus. JBZ reports fees from the CFF for his role on the DSMB and research grants from the CFF in partnership with Laurent Pharmaceuticals, Savara, AzureRx Biopharma, Aridis Pharmaceuticals, and Vertex Pharmaceuticals for unrelated research but no personal payments. AB, EBM and MV were full-time employees of Translate Bio during the conduct of the study. MV was an employee of Rho, Inc. and is currently an employee of Krystal Biotech. MV reports stock options provided to employees of Translate Bio and Krystal Biotech. MV is former board member of the CFF Central Carolinas Chapter. AB is currently on the Board of Directors for Pieris Pharmaceuticals. CB reports research grants from the CFF and CFF Therapeutic Development Network (CFFTDN) in partnership with Vertex pharmaceuticals for unrelated research but no personal payments. MSS reports research grants to his institution from the CFF in partnership with Vertex Pharmaceuticals and consulting fees from Vertex Pharmaceuticals. MVI reports research grants from the CFF and CFFTDN. To advance drug development and a search for a cure, the CFF has contracts with several companies to help fund the development of potential treatments and/or cures for CF. Pursuant to these contracts, CFF may receive milestone-based payments, equity interests, royalties on the net sales of therapies, and/or other forms of consideration. Resulting revenue received by CFF is used in support of their mission., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Nonsense mutations are the underlying cause of approximately 11% of all inherited genetic diseases 1 . Nonsense mutations convert a sense codon that is decoded by tRNA into a premature termination codon (PTC), resulting in an abrupt termination of translation. One strategy to suppress nonsense mutations is to use natural tRNAs with altered anticodons to base-pair to the newly emerged PTC and promote translation 2-7 . However, tRNA-based gene therapy has not yielded an optimal combination of clinical efficacy and safety and there is presently no treatment for individuals with nonsense mutations. Here we introduce a strategy based on altering native tRNAs into  efficient suppressor tRNAs (sup-tRNAs) by individually fine-tuning their sequence to the physico-chemical properties of the amino acid that they carry. Intravenous and intratracheal lipid nanoparticle (LNP) administration of sup-tRNA in mice restored the production of functional proteins with nonsense mutations. LNP-sup-tRNA formulations caused no discernible readthrough at endogenous native stop codons, as determined by ribosome profiling. At clinically important PTCs in the cystic fibrosis transmembrane conductance regulator gene (CFTR), the sup-tRNAs re-established expression and function in cell systems and patient-derived nasal epithelia and restored airway volume homeostasis. These results provide a framework for the development of tRNA-based therapies with a high molecular safety profile and high efficacy in targeted PTC suppression., (© 2023. The Author(s).)

Ten percent of cystic fibrosis (CF) patients carry a premature termination codon (PTC); no mutation-specific therapies exist for these individuals. ELX-02, a synthetic aminoglycoside, suppresses translation termination at PTCs (i.e., readthrough) by promoting the insertion of an amino acid at the PTC and restoring expression of full-length CFTR protein. The identity of amino acids inserted at PTCs affects the processing and function of the resulting full-length CFTR protein. We examined readthrough of the rare G550X-CFTR nonsense mutation due to its unique properties. We found that forskolin-induced swelling in G550X patient-derived intestinal organoids (PDOs) was significantly higher than in G542X PDOs (both UGA PTCs) with ELX-02 treatment, indicating greater CFTR function from the G550X allele. Using mass spectrometry, we identified tryptophan as the sole amino acid inserted in the G550X position during ELX-02- or G418-mediated readthrough, which differs from the three amino acids (cysteine, arginine, and tryptophan) inserted in the G542X position after treatment with G418. Compared with wild-type CFTR, Fischer rat thyroid (FRT) cells expressing the G550W-CFTR variant protein exhibited significantly increased forskolin-activated Cl - conductance, and G550W-CFTR channels showed increased PKA sensitivity and open probability. After treatment with ELX-02 and CFTR correctors, CFTR function rescued from the G550X allele in FRTs reached 20-40% of the wild-type level. These results suggest that readthrough of G550X produces greater CFTR function because of gain-of-function properties of the CFTR readthrough product that stem from its location in the signature LSGGQ motif found in ATP-binding cassette (ABC) transporters. G550X may be a particularly sensitive target for translational readthrough therapy. NEW & NOTEWORTHY We found that forskolin-induced swelling in G550X-CFTR patient-derived intestinal organoids (PDOs) was significantly higher than in G542X-CFTR PDOs after treatment with ELX-02. Tryptophan (W) was the sole amino acid inserted in the G550X position after readthrough. Resulting G550W-CFTR protein exhibited supernormal CFTR activity, PKA sensitivity, and open probability. These results show that aminoglycoside-induced readthrough of G550X produces greater CFTR function because of the gain-of-function properties of the CFTR readthrough product.

Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Background: Elexacaftor-tezacaftor-ivacaftor has been shown to be safe and efficacious in people with cystic fibrosis and at least one F508del allele. Our aim was to identify a novel cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination capable of further increasing CFTR-mediated chloride transport, with the potential for once-daily dosing., Methods: We conducted two phase 2 clinical trials to assess the safety and efficacy of a once-daily combination of vanzacaftor-tezacaftor-deutivacaftor in participants with cystic fibrosis who were aged 18 years or older. A phase 2 randomised, double-blind, active-controlled study (VX18-561-101; April 17, 2019, to Aug 20, 2020) was carried out to compare deutivacaftor monotherapy with ivacaftor monotherapy in participants with CFTR gating mutations, following a 4-week ivacaftor monotherapy run-in period. Participants were randomly assigned to receive either ivacaftor 150 mg every 12 h, deutivacaftor 25 mg once daily, deutivacaftor 50 mg once daily, deutivacaftor 150 mg once daily, or deutivacaftor 250 mg once daily in a 1:1:2:2:2 ratio. The primary endpoint was absolute change in ppFEV 1 from baseline at week 12. A phase 2 randomised, double-blind, controlled, proof-of-concept study of vanzacaftor-tezacaftor-deutivacaftor (VX18-121-101; April 30, 2019, to Dec 10, 2019) was conducted in participants with cystic fibrosis and heterozygous for F508del and a minimal function mutation (F/MF genotypes) or homozygous for F508del (F/F genotype). Participants with F/MF genotypes were randomly assigned 1:2:2:1 to receive either 5 mg, 10 mg, or 20 mg of vanzacaftor in combination with tezacaftor-deutivacaftor or a triple placebo for 4 weeks, and participants with the F/F genotype were randomly assigned 2:1 to receive either vanzacaftor (20 mg)-tezacaftor-deutivacaftor or tezacaftor-ivacaftor active control for 4 weeks, following a 4-week tezacaftor-ivacaftor run-in period. Primary endpoints for part 1 and part 2 were safety and tolerability and absolute change in ppFEV 1 from baseline to day 29. Secondary efficacy endpoints were absolute change from baseline at day 29 in sweat chloride concentrations and Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain score. These clinical trials are registered with ClinicalTrials.gov, NCT03911713 and NCT03912233, and are complete., Findings: In study VX18-561-101, participants treated with deutivacaftor 150 mg once daily (n=23) or deutivacaftor 250 mg once daily (n=24) had mean absolute changes in ppFEV 1 of 3·1 percentage points (95% CI -0·8 to 7·0) and 2·7 percentage points (-1·0 to 6·5) from baseline at week 12, respectively, versus -0·8 percentage points (-6·2 to 4·7) with ivacaftor 150 mg every 12 h (n=11); the deutivacaftor safety profile was consistent with the established safety profile of ivacaftor 150 mg every 12 h. In study VX18-121-101, participants with F/MF genotypes treated with vanzacaftor (5 mg)-tezacaftor-deutivacaftor (n=9), vanzacaftor (10 mg)-tezacaftor-deutivacaftor (n=19), vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=20), and placebo (n=10) had mean changes relative to baseline at day 29 in ppFEV 1 of 4·6 percentage points (-1·3 to 10·6), 14·2 percentage points (10·0 to 18·4), 9·8 percentage points (5·7 to 13·8), and 1·9 percentage points (-4·1 to 8·0), respectively, in sweat chloride concentration of -42·8 mmol/L (-51·7 to -34·0), -45·8 mmol/L (95% CI -51·9 to -39·7), -49·5 mmol/L (-55·9 to -43·1), and 2·3 mmol/L (-7·0 to 11·6), respectively, and in CFQ-R respiratory domain score of 17·6 points (3·5 to 31·6), 21·2 points (11·9 to 30·6), 29·8 points (21·0 to 38·7), and 3·3 points (-10·1 to 16·6), respectively. Participants with the F/F genotype treated with vanzacaftor (20 mg)-tezacaftor-deutivacaftor (n=18) and tezacaftor-ivacaftor (n=10) had mean changes relative to baseline (taking tezacaftor-ivacaftor) at day 29 in ppFEV 1 of 15·9 percentage points (11·3 to 20·6) and -0·1 percentage points (-6·4 to 6·1), respectively, in sweat chloride concentration of -45·5 mmol/L (-49·7 to -41·3) and -2·6 mmol/L (-8·2 to 3·1), respectively, and in CFQ-R respiratory domain score of 19·4 points (95% CI 10·5 to 28·3) and -5·0 points (-16·9 to 7·0), respectively. The most common adverse events overall were cough, increased sputum, and headache. One participant in the vanzacaftor-tezacaftor-deutivacaftor group had a serious adverse event of infective pulmonary exacerbation and another participant had a serious rash event that led to treatment discontinuation. For most participants, adverse events were mild or moderate in severity., Interpretation: Once-daily dosing with vanzacaftor-tezacaftor-deutivacaftor was safe and well tolerated and improved lung function, respiratory symptoms, and CFTR function. These results support the continued investigation of vanzacaftor-tezacaftor-deutivacaftor in phase 3 clinical trials compared with elexacaftor-tezacaftor-ivacaftor., Funding: Vertex Pharmaceuticals., Competing Interests: Declaration of interests AZU received grants from the Cystic Fibrosis Foundation and the CFF-Therapeutic Development Network for the present work; received payment or honoraria from Vertex Pharmaceuticals for presentations at CF Centers in UK; and participated in advisory boards for Vertex and Eloxx. VI received grant support from CF TDN for the present work; consulting fees from Mylan for CF—TOBI podhaler advisory board; and grant support from CFF for meeting attendance. PA received support from Vertex Pharmaceuticals for lectures, presentations, and materials; meeting attendance; and participation on data safety monitoring boards or advisory boards. MAM received payment from Vertex for the current work and personal fees for serving on an advisory board; grants from Vertex and from the German Ministry for Education and Research; consulting fees from Boehringer Ingelheim, Arrowhead Pharmaceuticals, Vertex Pharmaceuticals, Santhera, Sterna Biologicals, Enterprise Therapeutics, Antabio, and Abbvie; lecture fees from Boehringer Ingelheim, Arrowhead Pharmaceuticals, and Vertex Pharmaceuticals; travel reimbursement from Boehringer Ingelheim and Vertex Pharmaceuticals; personal fees for participation in an advisory board from Boehringer Ingelheim, Arrowhead Pharmaceuticals, Vertex Pharmaceuticals, Santhera, Enterprise Therapeutics, Antabio, Kither Biotech, Abbvie, and Pari; and serves as an ECFS Board member. EFM received grants and other payments or honoraria from Vertex; and support for meetings or travel from Menarini. BWR received payments from Vertex for the present work, and payments for a presentation in Vancouver, BC, Canada in 2019; and participated on data safety monitoring boards or advisory boards for CF Storm Clinical Trial, Vertex Pharmaceuticals, Janssen, Abbvie, and Insmed. SMR received support for a clinical trial; consulting fees on the design and conduct of clinical trials; support for meeting attendance and for his role as Co-Chair of the Next Generation Steering Committee; received grants or contracts from Novartis, TranslateBio, Galapagos–Abbvie, Synedgen–Synspira, Eloxx, Vertex Pharmaceuticals, and Ionis Astra Zenica; consulting fees from Novartis, Galapagos–Abbvie, Synedgen–Synspira, Vertex Pharmaceuticals, Renovion, Ionis, Cystetic Medicines, and Arcturus; support for meeting attendance from Vertex; has patents planned, issued or pending; serves as a Co-Chair of the Next Generation Steering Committee; and owns stock or stock options with Synedgen–Synspira and Renovion. RCR received clinical trial support and consulting fees from Vertex for the present work; grants from CFF, NIDDK, NHLBI, NICHD, and NIDCD; received consulting fees from Guidepoint Global, Gerson Lehrman Group, and Cystic Fibrosis Foundation; participated on a data safety monitoring or advisory board for NHLBI DSMB. JLT-C received personal consulting fees from Vertex for the present work; received grants from Vertex, Eloxx, and 4DMT for the conduct of a research trial; personal fees from Vertex, Insmed, and 4DMT for trial design consulting; personal fees from Vertex for non-branded speaking; and personal fees from AbbVie for her role as DMC Chair; served as the adult patient care representative to the CFF Board of Trustees, on the CF Foundation's Clinical Research Executive Committee, Clinical Research Advisory Board, and Racial Justice Working Group; as immediate past chair of the CF TDN's Sexual Health, Reproduction and Gender Research Working Group; on the scientific advisory board for Emily's Entourage; on the ATS Respiratory Health Awards Working Group; on the ATS Scientific Grant Review and Clinical Problems Assembly Programming Committees; and served as an associate editor for the Journal of Cystic Fibrosis. ET received payment for the present work and grants for doing clinical trials from Vertex Pharmaceuticals; received payment and reimbursement from Vertex for her role on a steering committee and for presentations at educational events. JB received funding from Vertex Pharmaceuticals for the present work. AH received funding from Vertex Pharmaceuticals for the present work; grant support from NIHR, CF Trust, CF Foundation, and Medical Research Council; payments for educational lectures from Vertex Pharmaceuticals and for an advisory board from Mylan; medical writing support from Vertex; served as Chair of the UK CF Clinical Trials Accelerator Platform and as a board member of the UK CF Medical Association. LMY received salary support from mgH TDN for clinical research activity for the present work. DW has patents planned, issued or pending. DW, LTW, CC, APM, NN, PRS, ST, FVG, and YX are Vertex employees and might own stocks or stock options. GM and CK have nothing to disclose. LV was a clinical pharmacology lead at Vertex during the conduct of this study and conducted PK data analysis for the present study., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

BackgroundLung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CF transmembrane conductance regulator (CFTR) modulators improve activity of dysfunctional CFTR channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections.MethodsWe performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI), on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR, and sequencing.ResultsMean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter spp., and Burkholderia spp. decreased by 2-3 log10 CFU/mL after 1 month of ETI. However, most participants remained culture positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria.ConclusionsTreatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment.Trial RegistrationClinicalTrials.gov NCT04038047.FundingThe Cystic Fibrosis Foundation and the NIH.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel for transport of chloride and bicarbonate anions. Functional roles of CFTR have been identified in a broad range of cell types including epithelial, endothelial, immune and structural cells. While CFTR has been investigated largely in the context of inborn dysfunction in cystic fibrosis, recent evidence shows that CFTR is also affected by acquired dysfunction in COPD. In patients with COPD and smokers, CFTR impairment has been demonstrated in the upper and lower airways, sweat glands and intestines, suggesting both pulmonary and systemic defects. Cigarette smoke, a key factor in COPD development, is the major cause of acquired CFTR dysfunction. Inflammation, bacterial byproducts and reactive oxygen species can further impair CFTR expression and function. CFTR dysfunction could contribute directly to disease manifestation and progression of COPD including disturbed airway surface liquid homeostasis, airway mucus obstruction, pathogen colonisation and inflammation. Mucus plugging and neutrophilic inflammation contribute to tissue destruction, development of dysfunction at the level of the small airways and COPD progression. Acquired CFTR dysfunction in extrapulmonary organs could add to common comorbidities and the disease burden. This review explores how CFTR dysfunction may be acquired and its potential effects on patients with COPD, particularly those with chronic bronchitis. The development of CFTR potentiators and the probable benefits of CFTR potentiation to improve tissue homeostasis, reduce inflammation, improve host defence and potentially reduce remodelling in the lungs will be discussed., Competing Interests: Conflict of interest: M.A. Mall declares editorial support from Novartis Pharma AG since the initial planning of the work, and declares grants from German Ministry for Education and Research and the German Research Foundation, consulting fees from Abbvie, Antabio, Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotech, Pieris Pharmaceuticals, Santhera, Sterna Biologicals and Vertex Paharmaceuticals, lecture fees from Arrowhead Pharmaceuticals, Boehringer Ingelheim and Vertex Pharmaceuticals, travel reimbursement from Boehringer Ingelheim and Vertex Pharmaceuticals, and personal fees for participation on advisory boards from Boehringer Ingelheim, Arrowhead Pharmaceuticals, Vertex Pharmaceuticals, Santhera, Enterprise Therapeutics, Antabio, Kither Biotech and Abbvie; until 2020, M.A. Mall was also an elected unpaid member of the ECFS board. G.J. Criner has no declarations. In the past 36 months, M. Miravitlles has received consulting fees from AstraZeneca, Atriva Therapeutics, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Bial, Gebro Pharma, CSL Behring, Inhibrx, Laboratorios Esteve, Ferrer, Mereo Biopharma, Verona Pharma, Spin Therapeutics, ONO Pharma, pH Pharma, Palobiofarma SL, Takeda, Novartis, Sanofi and Grifols, speaker fees from AstraZeneca, GlaxoSmithKline, Boehringer Ingelheim, Chiesi, Cipla, Menarini, Rovi, Bial, Sandoz, Zambon, CSL Behring, Grifols and Novartis, support for attending meetings/travel from Novartis, Boehringer Ingelheim and Menarini, research grants from Grifols, and has participated on a data safety monitoring board for Mereo. S.M. Rowe declares grant support for clinical trials conducted through university grants/contracts, consulting services on the design and conduct of clinical trials and support for travel to attend meetings from Novartis; support for clinical trials conduct through university grants/contracts, consulting services on the design and conduct of clinical trials, support for travel to attend meetings, co-chair of the Next Generation Steering Committee and providing research product for investigator initiated research from Vertex; grant support for clinical trials conducted through university grants/contracts, consulting services on the design and conduct of clinical trials, material transfer agreements (MTAs) for investigator-initiated and externally funded research efforts from Galapagos/Abbvie since the initial planning of the work; grant support for clinical trials conducted through university grants/contracts from Bayer, TranslateBio and Proteostasis; research grants through university grants/contracts from Synedgen/Synspira; research contract through university grants/contracts from Celtaxsys, Arrowhead, Ionis and AstraZeneca; research contract through university grants/contracts from Eloxx; declares consulting services on the design and conduct of clinical trials from Bayer, Renovion, Arrowhead, Ionis, Cystetic Medicines, Arcturus and Synedgen/Synspira, including stock options for Synedgen/Synspira within the past 36 months; receipt of research products for investigator initiated research from Renovion; MTA agreements for research efforts from Proteostasis; MTAs for investigator-initiated and externally funded research efforts from Ionis, Galapagos/Abbvie and Synedgen/Synspira; all in the past 36 months; and declares six patents. C.F. Vogelmeier declares institution grants from German Ministry of Education and Science (BMBF), AstraZeneca, Boehringer Ingelheim, Chiesi, CSL Behring, GlaxoSmithKline, Grifols and Novartis, consulting fees from Aerogen, AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GlaxoSmithKline, Insmed, Menarini, Novartis and Nuvaira, and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Aerogen, AstraZeneca, Boehringer Ingelheim, CSL Behring, Chiesi, GlaxoSmithKline, Insmed, Menarini and Novartis, in the past 36 months. D.J. Rowlands is an employee of Novartis Pharma AG and retains Novartis stock. M. Schoenberger is a full-time employee of Novartis Pharma AG and retains Novartis stock. P. Altman is a full-time employee of Novartis Pharmaceutical Corporation., (Copyright ©The authors 2023.)

The coronavirus disease (COVID-19) pandemic, caused by SARS-CoV-2 coronavirus, is devastatingly impacting human health. A prominent component of COVID-19 is the infection and destruction of the ciliated respiratory cells, which perpetuates dissemination and disrupts protective mucociliary transport (MCT) function, an innate defense of the respiratory tract. Thus, drugs that augment MCT could improve the barrier function of the airway epithelium and reduce viral replication and, ultimately, COVID-19 outcomes. We tested five agents known to increase MCT through distinct mechanisms for activity against SARS-CoV-2 infection using a model of human respiratory epithelial cells terminally differentiated in an air/liquid interphase. Three of the five mucoactive compounds tested showed significant inhibitory activity against SARS-CoV-2 replication. An archetype mucoactive agent, ARINA-1, blocked viral replication and therefore epithelial cell injury; thus, it was further studied using biochemical, genetic, and biophysical methods to ascertain the mechanism of action via the improvement of MCT. ARINA-1 antiviral activity was dependent on enhancing the MCT cellular response, since terminal differentiation, intact ciliary expression, and motion were required for ARINA-1-mediated anti-SARS-CoV2 protection. Ultimately, we showed that the improvement of cilia movement was caused by ARINA-1-mediated regulation of the redox state of the intracellular environment, which benefited MCT. Our study indicates that intact MCT reduces SARS-CoV-2 infection, and its pharmacologic activation may be effective as an anti-COVID-19 treatment.

Background: Elexacaftor/tezacaftor/ivacaftor (ETI) improves pulmonary disease in people with cystic fibrosis (PwCF), but its effect on gastrointestinal symptoms, which also affect quality of life, is not clear., Methods: PROMISE is a 56-center prospective, observational study of ETI in PwCF >12 years and at least one F508del allele. Gastrointestinal symptoms, evaluated by validated questionnaires: Patient Assessment of Upper Gastrointestinal Disorders-Symptom (PAGI-SYM), Patient Assessment of Constipation-Symptom (PAC-SYM), Patient Assessment of Constipation-Quality of Life (PAC-QOL)), fecal calprotectin, steatocrit and elastase-1 were measured before and 6 months after ETI initiation. Mean difference and 95% confidence intervals were obtained from linear regression with adjustment for age and sex., Results: 438 participants fully completed at least 1 questionnaire. Mean (SD) for baseline PAGI-SYM, PAC-SYM, and PAC-QOL total scores were 0.56 (0.59), 0.47 (0.45), and 0.69 (0.53) out of maximum 5, 4, and 5, respectively (higher score indicates greater severity). Corresponding age- and sex-adjusted 6 months mean changes (95% CI) in total scores were -0.15 (-0.21, -0.09) for PAGI-SYM, -0.14 (-0.19, -0.09) for PAC-SYM, and -0.15 (-0.21, -0.10) for PAC-QOL. While statistically significant, changes were small and unlikely to be of clinical importance. Fecal calprotectin showed a change (95% CI) from baseline of -66.2 µg/g (-86.1, -46.2) at 6 months, while fecal elastase and steatocrit did not meaningfully change., Conclusions: After 6 months of ETI, fecal markers of inflammation decreased. Gastrointestinal symptoms improved, but the effect size was small. Pancreatic insufficiency did not improve., Competing Interests: Declaration of Competing Interest SJS-Consultant for UpToDate, Abbvie, Mirium, Nestle; Grant funding from Gilead, Cystic Fibrosis Foundation, NIH. SDF – Consultant for UpToDate, Abbvie, Nestle, Synspira; Grant funding from NIH, Cystic Fibrosis Foundation, Amagma Therapeutics. MRN – Consultant for UpToDate, Vertex: Grant funding from Gilead, AbbVie, Cystic Fibrosis Foundation, NIH SMR: Consultant for Vertex; Grant funding from Vertex, Cystic Fibrosis Foundation, NIH DPN: Consultant for Vertex, Genentech; Grant funding from Vertex, Cystic Fibrosis Foundation, NIH GMS: Consultant for Genentech, Electromed; Grant Funding from Vertex, Cystic Fibrosis Foundation, NIH DG: Consultant for Vertex, Abbvie, Chiesi USA, Eli Lilly AK: Grant funding from NIH, Cystic Fibrosis Foundation, SDS – Grant funding from Cystic Fibrosis Foundation and NIH MSS—Grant funding from Cystic Fibrosis Foundation, (Copyright © 2022. Published by Elsevier B.V.)

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene's diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC., Competing Interests: A.S., M.D., J.R.B., S.M.R., D.M.B. and K.M.K. are named in an unlicensed patent for the use of triamterene as a translational readthrough agent. S.M.R. and D.M.B. provided consulting services and received grants from PTC Therapeutics, Incorporated.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

Cystic fibrosis (CF), first described in 1938, is a common, life-limiting monogenetic disease. The discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 1989 was crucial in advancing our understanding of disease pathogenesis and paving the road for treatment aimed at the fundamental molecular defect. With the delineation of over 2000 variations in the CFTR gene, a sound understanding of the individual variations in cell biology, and electrophysiological abnormalities conferred by the most common defects propelled the advent of targeted disease-modifying therapeutics beginning in 2012. Since then, CF care has transformed beyond just symptomatic treatment to include a variety of small-molecule therapies that address the basic electrophysiologic defect and cause profound improvements in physiology, clinical manifestations, and long-term outcomes, designed to differentially address six genetic/molecular subtypes. This chapter illustrates the progress made toward how fundamental science and translational initiatives enabled personalized, mutation specific treatment. We highlight the importance of preclinical assays and mechanistically-driven development strategies that were coupled with sensitive biomarkers and a clinical trial cooperative to provide a platform for successful drug development. This convergence of academic and private partnerships, and formation of multidisciplinary care teams directed by evidence-based initiatives provide a seminal example of addressing the needs of individuals with a rare, but fatal genetic disease., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ferret systemic coronavirus (FRSCV) causes a highly fatal disease of ferrets ( Mustela putorius furo ). It is believed to be a mutated variant of ferret enteric coronavirus (FRECV) and has a clinical presentation similar to that of feline infectious peritonitis virus (FIPV) in cats. The interplay of infectious diseases and host genetics will become a greater issue in the research environment as genetically modified species other than rodents become available due to advances in gene editing technology. In this case series, we present the clinical and histopathologic features of a FRSCV outbreak that affected 5 out of 10 ferrets with α-1 antitrypsin knockout (AAT KO) over an approximately 1-y period. Clinical features varied, with the affected ferrets presenting with some combination of wasting, hind limb paralysis, incontinence or sudden death. Multiple ferrets had gross pathologic lesions consistent with FRSCV, but the lesions were typically mild. Microscopic pyogranulomatous inflammation was present in 4 ferrets. Immunohistochemistry using an anti-feline coronavirus antibody that cross reacts with ferret coronavirus confirmed infection of intralesional macrophages in 4 out of 5 animals with suspected FRSCV infection. PCR testing of formalin fixed tissue was negative for all ferrets. PCR testing of feces from healthy wild-type ferrets indicated that the endemic presence of FRECV genotype 2, while PCR surveillance testing of other in-house AAT KO ferrets revealed both enteric coronavirus genotypes 1 and 2. This case series highlights the potential for greater disease incidence in the future as genetically modified ferrets are used more often, and may support exclusion of FRECV and similar viruses from highly susceptible ferret genotypes.