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Steven M Rowe,1 Ieuan Jones,2 Mark T Dransfield,1 Nazmul Haque,2 Stephen Gleason,2 Katy A Hayes,2 Kenneth Kulmatycki,2 Denise P Yates,2 Henry Danahay,3 Martin Gosling,3,4 David J Rowlands,2 Sarah S Grant2 1University of Alabama at Birmingham, Department of Medicine, Birmingham, AL, USA; 2Novartis Institutes for BioMedical Research, Cambridge, MA, USA; 3Enterprise Therapeutics, Brighton, UK; 4Sussex Drug Discovery Centre, University of Sussex, Brighton, UKCorrespondence: Sarah S GrantNovartis Institutes for BioMedical Research, 250 Massachusetts Ave, Cambridge, MA 02139, USATel +16178717812Email sarah.grant@novartis.comRationale: Excess mucus plays a key role in COPD pathogenesis. Cigarette smoke-induced cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to disease pathogenesis by depleting airway surface liquid and reducing mucociliary transport; these defects can be corrected in vitro by potentiating CFTR.Objective: To assess the efficacy of the CFTR potentiator icenticaftor in improving airflow obstruction in COPD patients with symptoms of chronic bronchitis.Methods: In this double-blind, placebo-controlled study, COPD patients were randomized (2:1) to either icenticaftor 300 mg or placebo b.i.d. This non-confirmatory proof of concept study was powered for lung clearance index (LCI) and pre-bronchodilator FEV1, with an estimated sample size of 90 patients. The primary endpoint was change from baseline in LCI for icenticaftor versus placebo at Day 29; key secondary endpoints included change from baseline in pre- and post-bronchodilator FEV1 on Day 29. Key exploratory endpoints included change from baseline in sweat chloride, plasma fibrinogen levels, and sputum colonization.Results: Ninety-two patients were randomized (icenticaftor, n=64; placebo, n=28). At Day 29, icenticaftor showed no improvement in change in LCI (treatment difference: 0.28 [19% probability of being better than placebo]), an improvement in pre-bronchodilator FEV1 (mean: 50 mL [84% probability]) and an improvement in post-bronchodilator FEV1 (mean: 63 mL [91% probability]) over placebo. Improvements in sweat chloride, fibrinogen and sputum bacterial colonization were also observed. Icenticaftor was safe and well tolerated.Conclusion: The CFTR potentiator icenticaftor increased FEV1 versus placebo after 28 days and was associated with improvements in systemic inflammation and sputum bacterial colonization in COPD patients; no improvements in LCI with icenticaftor were observed.Keywords: chronic obstructive pulmonary disease, chronic bronchitis, cystic fibrosis transmembrane conductance regulator potentiator, CFTR potentiator, mucociliary clearance, icenticaftor; QBW251

Rationale: High-mobility group box 1 (HMGB1) is a potent inflammatory mediator elevated in sepsis and rheumatoid arthritis, although its role in cystic fibrosis (CF) lung disease is unknown.Objectives: To determine whether HMGB1 contributes to CF lung inflammation, including neutrophil chemotaxis and lung matrix degradation.Methods: We used sputum and serum from subjects with CF and a Scnn1b-transgenic (Scnn1b-Tg) mouse model that overexpresses beta-epithelial Na(+) channel in airways and mimics the CF phenotype, including lung inflammation. Human secretions and murine bronchoalveolar lavage fluid (BALF) was assayed for HMGB1 by Western blot and ELISA. Neutrophil chemotaxis was measured in vitro after incubation with human neutrophils. The collagen fragment proline-glycine-proline (PGP) was measured by tandem mass spectroscopy.Measurements and Main Results: HMGB1 was detected in CF sputum at higher levels than secretions from normal individuals. Scnn1b-Tg mice had elevated levels of HMGB1 by Western blot and ELISA. We demonstrated that dose-dependent chemotaxis of human neutrophils stimulated by purified HMGB1 was partially dependent on CXC chemokine receptors and that this could be duplicated in CF sputum and BALF from Scnn1b-Tg mice. Neutralization by anti-HMGB1 antibody, in both the sputum and BALF-reduced chemotaxis, which suggested that HMGB1 contributed to the chemotactic properties of these samples. Intratracheal administration of purified HMGB1 induced neutrophil influx into the airways of mice and promoted the release of PGP. PGP was also elevated in Scnn1b-Tg mice and CF serum.Conclusions: HMGB1 expression contributes to pulmonary inflammation and lung matrix degradation in CF airway disease and deserves further investigation as a biomarker and potential therapeutic target. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the outcome of 30 femoral head fractures. We modified Pipkin's classification into 4 types: I (5 cases) small fracture of head distal to fovea centralis, which was too small or too fragmented to be fixed with screws; II (18 cases), larger fracture of head distal to fovea centralis; III (4 cases), large fracture of head proximal to fovea centralis, and IV (3 cases), comminuted fracture of head. Excision of the head fragment was done in all 5 cases of type I and in 9 type II fractures. Fixation of the head fragment was performed in 9 type II and in all 4 type III cases. The femoral head was replaced in all 3 type IV fractures. After a mean follow-up of 3-10 years, the clinical outcome, according to Epstein et al.'s critieria, were excellent in 7, good in 15, fair in 4 and poor in 1, except in type IV, and the radiographic outcome was excellent in 15, good in 7, fair in 4 and poor in 1. On the basis of our findings, we conclude that excision of the small fragment is a good choice of treatment in type 1. Early accurate reduction with stable internal fixation in type II or III permits bony union. Arthroplasty seems to be indicated in type IV. [ABSTRACT FROM AUTHOR]

In March 1998, an outbreak of acute gastroenteritis occurred among students at a Texas university. Overall, 125 ill students sought medical care. Case-control studies revealed that illness was significantly associated with eating foods from the university's main cafeteria deli bar on 9 and 10 March. Stool specimens from 9 (50%) of 18 ill students and samples of deli ham showed evidence of Norwalk-like viruses (NLVs) by reverse-transcriptase (RT) polymerase chain reaction (PCR) assay. A food handler who prepared sandwiches for lunch on 9 March reported that her infant had been sick with watery diarrhea since just before the outbreak. A stool sample from the infant was positive for NLV by RT-PCR, and the sequence of the amplified product was identical to that of amplified product from deli ham and students' stool specimens. This is the first time RT-PCR and sequence analysis have successfully confirmed viral contamination of a food item likely to have been contaminated by a food handler. Copyright © 2000 The University of Chicago [ABSTRACT FROM AUTHOR]

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/).)

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.

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).)

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.)

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.)

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).)

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.)

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.

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.

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.

Our current understanding of the spectrum of TB and COVID-19 lesions in the human lung is limited by a reliance on low-resolution imaging platforms that cannot provide accurate 3D representations of lesion types within the context of the whole lung. To characterize TB and COVID-19 lesions in 3D, we applied micro/nanocomputed tomography to surgically resected, postmortem, and paraffin-embedded human lung tissue. We define a spectrum of TB pathologies, including cavitary lesions, calcium deposits outside and inside necrotic granulomas and mycetomas, and vascular rearrangement. We identified an unusual spatial arrangement of vasculature within an entire COVID-19 lobe, and 3D segmentation of blood vessels revealed microangiopathy associated with hemorrhage. Notably, segmentation of pathological anomalies reveals hidden pathological structures that might otherwise be disregarded, demonstrating a powerful method to visualize pathologies in 3D in TB lung tissue and whole COVID-19 lobes. These findings provide unexpected new insight into the spatial organization of the spectrum of TB and COVID-19 lesions within the framework of the entire lung., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant., Competing Interests: Declaration of Competing Interest All the authors have declared their conflict of interests., (Copyright © 2022. Published by Elsevier B.V.)

Airway dehydration causes mucus stasis and bacterial overgrowth in cystic fibrosis (CF), resulting in recurrent respiratory infections and exacerbations. Strategies to rehydrate airway mucus including inhibition of the epithelial sodium channel (ENaC) have the potential to improve mucosal defense by enhancing mucociliary clearance (MCC) and reducing the risk of progressive decline in lung function. In the current work, we evaluated the effects of AZD5634, an ENaC inhibitor that shows extended lung retention and safety profile as compared with previously evaluated candidate drugs, in healthy and CF preclinical model systems. We found that AZD5634 elicited a potent inhibition of amiloride-sensitive current in non-CF airway cells and airway cells derived from F508del-homozygous individuals with CF that effectively increased airway surface liquid volume and improved mucociliary transport (MCT) rate. AZD5634 also demonstrated efficacious inhibition of ENaC in sheep bronchial epithelial cells, translating to dose-dependent improvement of mucus clearance in healthy sheep in vivo. Conversely, nebulization of AZD5634 did not notably improve airway hydration or MCT in CF rats that exhibit an MCC defect, consistent with findings from a first single-dose evaluation of AZD5634 on MCC in people with CF. Overall, these findings suggest that CF animal models demonstrating impaired mucus clearance translatable to the human situation may help to successfully predict and promote the successful translation of ENaC-directed therapies to the clinic.

Background: Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator for people with CF and the G551D mutation. We aimed to investigate the biology of CFTR modulation and systemic effects of CFTR restoration by examining changes in circulating measurements of inflammation and growth and novel proteins with ivacaftor treatment., Methods: Blood samples from 64 CF subjects with G551D-CFTR were analyzed for inflammatory and growth-related proteins at baseline, 1 and 6 months after ivacaftor initiation. In 30 subjects, plasma was assayed for 1,322 proteins using the SomaScan proteomic platform at baseline and 6 months post-ivacaftor. Correlations with clinical outcomes were assessed., Measurements and Main Results: Significant reductions in high mobility group box-1 protein (HMGB-1), calprotectin, serum amyloid A, and granulocyte colony-stimulating factor (G-CSF), and an increase in insulin-like growth factor (IGF-1) occurred 1 month after ivacaftor. This treatment effect was sustained at 6 months for HMGB-1 and calprotectin. Correcting for multiple comparisons in the proteomic analysis, 9 proteins (albumin, afamin, leptin, trypsin, pancreatic stone protein [PSP], pituitary adenylate cyclase-activating polypeptide-38, repulsive guidance molecule A [RGMA], calreticulin, GTPase KRas) changed significantly with ivacaftor. Proteins changing with treatment are involved in lipid digestion and transport and extracellular matrix organization biological processes. Reductions in calprotectin and G-CSF and increases in calreticulin, and RGMA correlated with improved lung function, while increasing IGF-1, leptin and afamin and decreasing PSP correlated with increased weight., Conclusions: Ivacaftor led to changes in inflammatory, lipid digestion, and extracellular matrix proteins, lending insights into the extrapulmonary effects of CFTR modulation., Competing Interests: Conflict of interest statement JEH reports grants from Cystic Fibrosis Foundation, outside the submitted work, SMR reports grants, personal fees, non-financial support and other from Vertex Pharmaceuticals Inc., during the conduct of the study; grants and personal fees from Novartis, grants and personal fees from Bayer, grants from Translate Bio, non-financial support from Proteostasis, grants, personal fees and non-financial support from Galapagos/Abbvie, grants, personal fees and other from Synedgen/Synspira, grants from Eloxx, grants and personal fees from Celtaxsys, grants, personal fees, non-financial support and other from Vertex Pharmaceuticals Inc, personal fees from Renovion, grants and personal fees from Arrowhead, grants and other from Ionis, grants from Astra Zenica, personal fees from Cystetic Medicines, personal fees from Arcturus, outside the submitted work; EMD reports other from EvoEndoscopy (formerly Triple Endoscopy), outside the submitted work and is a consultant for Boehringer Ingelheim, not related to this work. SDS reports grants from the Cystic Fibrosis Foundation that funds the work under consideration, and grants from the Cystic Fibrosis Foundation and National Institutes of Health funding activities outside the submitted work. BDW, JKH and SLH have nothing to disclose., (Copyright © 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Chronic obstructive pulmonary disease (COPD) is a progressive lung disease with poor treatment options. However, most mouse models of COPD produce a primarily emphysematous disease not recapitulating clinically meaningful COPD features like chronic bronchitis., Methods: Wild-type ferrets (Mustela putorius furo) were divided randomly into two groups: whole body cigarette smoke exposure and air controls. Ferrets were exposed to smoke from 1R6F research cigarettes, twice daily for six months. RNA-sequencing was performed on RNA isolated from lung tissue. Comparative transcriptomics analyses of COPD in ferrets, mice, and humans were done to find the uniquely expressed genes. Further, Real-time PCR was performed to confirmed RNA-Seq data on multiple selected genes., Results: RNA-sequence analysis identified 420 differentially expressed genes (DEGs) that were associated with the development of COPD in ferrets. By comparative analysis, we identified 25 DEGs that are uniquely expressed in ferrets and humans, but not mice. Among DEGs, a number were related to mucociliary clearance (NEK-6, HAS1, and KL), while others have been correlated with abnormal lung function (IL-18), inflammation (TREM1, CTSB), or oxidative stress (SRX1, AHRR). Multiple cellular pathways were aberrantly altered in the COPD ferret model, including pathways associated with COPD pathogenesis in humans. Validation of these selected unique DEGs using real-time PCR demonstrated > absolute 2-fold changes in mRNA versus air controls, consistent with RNA-seq analysis., Conclusion: Cigarette smoke-induced COPD in ferrets modulates gene expression consistent with human COPD and suggests that the ferret model may be uniquely well suited for the study of aspects of the disease., (© 2022. The Author(s).)

Cystic fibrosis airway disease is characterised by chronic Pseudomonas aeruginosa infection. Successful eradication strategies have been hampered by a poor understanding of the mechanisms underlying conversion to chronicity. The cystic fibrosis transmembrane conductance receptor (CFTR)-knockout (KO) rat harbours a progressive defect in mucociliary transport and viscosity. KO rats were infected before and after the appearance of the mucus defect, using a clinical mucoid-isolate of P. aeruginosa embedded in agarose beads. Young KO rats that were exposed to bacteria before the development of mucociliary transport defects resolved the infection and subsequent tissue damage. However, older KO rats that were infected in the presence of hyperviscous and static mucus were unable to eradicate bacteria, but instead had bacterial persistence through 28 days post-infection that was accompanied by airway mucus occlusion and lingering inflammation. Normal rats responded to infection with increased mucociliary transport to supernormal rates, which reduced the severity of a second bacterial exposure. We conclude that the aberrant mucus present in the CF airway permits persistence of P. aeruginosa in the lung., Competing Interests: Conflict of interest: A.G. Henderson has nothing to disclose. Conflict of interest: J.M. Davis has nothing to disclose. Conflict of interest: J.D. Keith has nothing to disclose. Conflict of interest: M.E. Green has nothing to disclose. Conflict of interest: A.M. Oden has nothing to disclose. Conflict of interest: S.M. Rowe has a patent “Use of μOCT as a tool for diagnosis and drug discovery” issued. Conflict of interest: S.E. Birket reports grants from NIH NHLBI and Cystic Fibrosis Foundation, during the conduct of the study., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

Rationale: The majority of chronic obstructive pulmonary disease (COPD) patients have chronic bronchitis, for which specific therapies are unavailable. Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction is observed in chronic bronchitis, but has not been proven in a controlled animal model with airway disease. Furthermore, the potential of CFTR as a therapeutic target has not been tested in vivo, given limitations to rodent models of COPD. Ferrets exhibit cystic fibrosis-related lung pathology when CFTR is absent and COPD with bronchitis following cigarette smoke exposure., Objectives: To evaluate CFTR dysfunction induced by smoking and test its pharmacological reversal by a novel CFTR potentiator, GLPG2196, in a ferret model of COPD with chronic bronchitis., Methods: Ferrets were exposed for 6 months to cigarette smoke to induce COPD and chronic bronchitis and then treated with enteral GLPG2196 once daily for 1 month. Electrophysiological measurements of ion transport and CFTR function, assessment of mucociliary function by one-micron optical coherence tomography imaging and particle-tracking microrheology, microcomputed tomography imaging, histopathological analysis and quantification of CFTR protein and mRNA expression were used to evaluate mechanistic and pathophysiological changes., Measurements and Main Results: Following cigarette smoke exposure, ferrets exhibited CFTR dysfunction, increased mucus viscosity, delayed mucociliary clearance, airway wall thickening and airway epithelial hypertrophy. In COPD ferrets, GLPG2196 treatment reversed CFTR dysfunction, increased mucus transport by decreasing mucus viscosity, and reduced bronchial wall thickening and airway epithelial hypertrophy., Conclusions: The pharmacologic reversal of acquired CFTR dysfunction is beneficial against pathological features of chronic bronchitis in a COPD ferret model., Competing Interests: Conflict of interest: S.M. Rowe has received consulting fees from Abbvie related to the design and conduct of clinical trials in cystic fibrosis. M. Borgonovi and K. Conrath are employees of Galapagos. All other authors declare no competing interests., (Copyright ©The authors 2022. For reproduction rights and permissions contact permissions@ersnet.org.)

The human lung differs substantially from its mouse counterpart, resulting in a distinct distal airway architecture affected by disease pathology in chronic obstructive pulmonary disease. In humans, the distal branches of the airway interweave with the alveolar gas-exchange niche, forming an anatomical structure known as the respiratory bronchioles. Owing to the lack of a counterpart in mouse, the cellular and molecular mechanisms that govern respiratory bronchioles in the human lung remain uncharacterized. Here we show that human respiratory bronchioles contain a unique secretory cell population that is distinct from cells in larger proximal airways. Organoid modelling reveals that these respiratory airway secretory (RAS) cells act as unidirectional progenitors for alveolar type 2 cells, which are essential for maintaining and regenerating the alveolar niche. RAS cell lineage differentiation into alveolar type 2 cells is regulated by Notch and Wnt signalling. In chronic obstructive pulmonary disease, RAS cells are altered transcriptionally, corresponding to abnormal alveolar type 2 cell states, which are associated with smoking exposure in both humans and ferrets. These data identify a distinct progenitor in a region of the human lung that is not found in mouse that has a critical role in maintaining the gas-exchange compartment and is altered in chronic lung disease., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon., (The Authors. Published by Elsevier Inc. and Fass 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/).)

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer that has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity, which can normalize the rheological properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro , and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, and may warrant further evaluation as a therapeutic.

CFTR function is required for normal mucociliary clearance (MCC) and cough-assisted clearance (CC). Lumacaftor-ivacaftor is approved for use in people with cystic fibrosis (CF) carrying two copies of F508del-CFTR. In this observational study performed at four study sites, we characterized the effect of lumacaftor-ivacaftor on mucociliary and cough clearance and related this to other clinical and research endpoints after one month of treatment. Twenty-five adolescents and adults were enrolled. No effect on whole lung MCC was observed, but CC was significantly increased. Sweat chloride improved by 18 mEq/L in this group, indicating a modest restoration of CFTR activity, but no demonstrable change in FEV 1 or lung clearance index was observed. We speculate that the modest effect of lumacaftor-ivacaftor on CFTR function was insufficient to yield an improvement in MCC., (Copyright © 2021 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The ABC multidrug exporter MsbA mediates the translocation of lipopolysaccharides and phospholipids across the plasma membrane in Gram-negative bacteria. Although MsbA is structurally well characterised, the energetic requirements of lipid transport remain unknown. Here, we report that, similar to the transport of small-molecule antibiotics and cytotoxic agents, the flopping of physiologically relevant long-acyl-chain 1,2-dioleoyl (C18)-phosphatidylethanolamine in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and the chemical proton gradient as sources of metabolic energy. In contrast, the flopping of the large hexa-acylated (C12-C14) Lipid-A anchor of lipopolysaccharides is only ATP dependent. This study demonstrates that the energetics of lipid transport by MsbA is lipid dependent. As our mutational analyses indicate lipid and drug transport via the central binding chamber in MsbA, the lipid availability in the membrane can affect the drug transport activity and vice versa., (© 2021. The Author(s).)

Background: The Characterizing CFTR Modulated Changes in Sweat Chloride and their Association with Clinical Outcomes (CHEC-SC) study is a large epidemiologic study designed to determine the relationship between sweat chloride response and clinical outcomes in people with cystic fibrosis (CF) on commercially approved CFTR modulators. A challenge to study feasibility was capturing sweat chloride measurements before modulator initiation. We tested the hypothesis that historic sweat chloride approximated contemporary pre-modulator values to estimate CFTR modulator-induced changes, allowing a single-visit study design., Methods: GOAL and PROSPECT were multi-center prospective studies of individuals initiating ivacaftor or lumacaftor-ivacaftor. At enrollment, pre-modulator sweat chloride was measured and historic results recorded. Post-modulator sweat chloride was measured at 1, 3 and 6 months. For this analysis, differences between historic and pre-modulator sweat chloride were estimated. CFTR modulator-induced sweat chloride mean changes were compared using historic and pre-modulator sweat chloride., Results: Paired historic and pre-modulator sweat chloride (n=406 participants) revealed a non-significant mean change of -1.0 mmol/L (95% CI: -2.71, 0.66) over an average of 17.2 years. Calculating sweat response to ivacaftor or lumacaftor-ivacaftor using historic or pre-modulator values resulted in similar estimates of modulator response. Based on these results, the CHEC-SC study was designed with a single, post-modulator sweat chloride measurement., Conclusions: Historic sweat chloride values provide a reliable estimate of pre-modulator sweat chloride for people starting on modulator therapy. The CHEC-SC study anticipates capturing approximately 5,000 sweat chloride values, providing an unprecedented understanding of sweat chloride across the CF population in the era of CFTR modulators., Competing Interests: Declaration of Competing Interest ETZ, NMH, DRV, SMR, MWK: grants or consulting CFF; DRV consults for aMoon, Arrevus, Eloxx, Enbiotix, Felix, Ionis, Matinas, Merck, Polyphor, Respirion, Savara; SMR consults for Vertex; MWK consults for Anthera, AzurRx, Celtaxsys, Chiesi, Ionis, Kala, Laurent, Merck, Paranta, pH Pharma, Santhera, Vertex; JPC, KOD and MS: none., (Copyright © 2021. Published by Elsevier B.V.)

Background: Riociguat is a first-in-class soluble guanylate cyclase stimulator for which preclinical data suggested improvements in cystic fibrosis transmembrane conductance regulator (CFTR) function., Methods: This international, multicenter, two-part, Phase II study of riociguat enrolled adults with cystic fibrosis (CF) homozygous for Phe508del CFTR. Part 1 was a 28-day, randomized, double-blind, placebo-controlled study in participants not receiving CFTR modulator therapy. Twenty-one participants were randomized 1:2 to placebo or oral riociguat (0.5 mg three times daily [tid] for 14 days, increased to 1.0 mg tid for the subsequent 14 days). The primary and secondary efficacy endpoints were change in sweat chloride concentration and percent predicted forced expiratory volume in 1 second (ppFEV 1 ), respectively, from baseline to Day 14 and Day 28 with riociguat compared with placebo., Results: Riociguat did not alter CFTR activity (change in sweat chloride) or lung function (change in ppFEV 1 ) at doses up to 1.0 mg tid after 28 days. The most common drug-related adverse event (AE) was headache occurring in three participants (21%); serious AEs occurred in one participant receiving riociguat (7%) and one participant receiving placebo (14%). This safety profile was consistent with the underlying disease and the known safety of riociguat for its approved indications., Conclusions: The Rio-CF study was terminated due to lack of efficacy and the changing landscape of CF therapeutic development. The current study⁠, within its limits of a small sample size, did not provide evidence that riociguat could be a valid treatment option for CF., Clinical Trial Registration Number: NCT02170025., Competing Interests: Declaration of Competing Interest N. Derichs received a speaker honorarium from Vertex Pharmaceuticals, Inc. for participation in a symposium, and served as a consultant for Vertex Pharmaceuticals, Inc. at educational activities and advisory boards. J.L. Taylor-Cousar reports grants paid to her institution from the Cystic Fibrosis Foundation; grants paid to her institution and consulting and speaking fees from Vertex Pharmaceuticals, Inc.; grants paid to her institution from Bayer; grants, consulting, and speaking fees from Celtaxys; grants paid to her institution and consulting fees from Proteostasis Therapeutics, Inc.; consulting fees from Santhera, 4DMT, AbbVie, and Polarean; and grants paid to her institution from Eloxx Pharmaceuticals. J.C. Davies reports grants from the CF Trust and other funding from Algipharma AS, Bayer AG, Boehringer Ingelheim Pharma GmbH & Co. KG, Galapagos NV, ImevaX GmbH, Nivalis Therapeutics, Inc., ProQR Therapeutics III B.V., Proteostasis Therapeutics, Inc., Raptor Pharmaceuticals, Inc., Vertex Pharmaceuticals, Inc., Enterprise, Novartis, Pulmocide, Flatley, Nivalis Therapeutics, Inc., and Teva. I. Fajac received speaker honoraria from Vertex Pharmaceuticals, Inc. for participation in symposia and served as a consultant for Novartis, Proteostasis Therapeutics, Inc., and Vertex Pharmaceuticals, Inc. E. Tullis reports grants paid to her institution from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Bayer AG, Boehringer Ingelheim, AbbVie, Celtaxis, Corbus, and Spyryx, and consultancy fees and honoraria from Vertex Pharmaceuticals, Proteostasis Therapeutics, Astra Zeneca and Horizon. D. Nazareth has nothing to disclose. D.G. Downey reports grants from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Inc., Gilead, and Chiesi; and honoraria or speaker fees from Vertex Pharmaceuticals, Inc., Proteostasis Therapeutics, Inc., and Chiesi. D. Rosenbluth has nothing to disclose. A. Malfroot has nothing to disclose. C. Saunders has nothing to disclose. R. Jensen has nothing to disclose. G.M. Solomon reports work on advisory boards for Bayer and Electromed. F. Vermeulen has nothing to disclose. A. Kaiser is an employee of Bayer AG. S. Willmann is an employee of Bayer AG. S. Saleh is an employee of Bayer AG. K. Droebner is an employee of Bayer AG. P. Sandner is an employee of Bayer AG. C.E. Bear has nothing to disclose. A. Hoffmann is an employee of Bayer AG. F. Ratjen reports other consultancy fees from Vertex Pharmaceuticals, Inc., Bayer, Talecris, CSL Behring, Roche, and Gilead; grants and consultancy from Novartis; and developed speaker bureau for Genentech outside the submitted work. S.M. Rowe reports grants and consulting fees from Arrowhead, Bayer, Celtaxsys, Galapagos/AbbVie, Novartis, Renovion, Synedgen/Synspira, andVertex Pharmaceuticals Inc.; consulting fees from Arcturus, and Cysteic Medicines; and grants from AstraZeneca, Eloxx, N30 Pharmaceuticals, PTC Therapeutics, Translate Bio, and Proteostasis Therapeutics, Inc., (Copyright © 2021. Published by Elsevier B.V.)

Background: Cystic fibrosis (CF) is characterized by neutrophilic inflammation in the airways. Leukotriene B4 (LTB 4 ) is a neutrophil chemoattractant and has been implicated in CF pathogenesis. Acebilustat, a novel, synthetic, small-molecule leukotriene A4 hydrolase inhibitor, reduces LTB 4 production. We report findings from a randomized placebo-controlled trial of acebilustat in adult subjects with mild-to-moderate lung disease., Methods: Subjects were randomized (1:1:1) to once-daily acebilustat 50 mg, 100 mg or placebo for 48 weeks, concomitantly with their current therapeutic regimen. Subjects were stratified by use of concomitant CF transmembrane conductance regulator (CFTR) modulators, baseline percent predicted forced expiratory volume in 1 second (ppFEV 1 ) 50-75 and >75, and number of pulmonary exacerbations in the past year (1 or >1). Primary endpoints were the change from baseline in ppFEV 1 and safety. Secondary endpoints included the rate of pulmonary exacerbations., Results: Overall, 199 subjects were randomized and dosed (acebilustat 50 mg, n=67; acebilustat 100 mg, n=66; placebo, n=66). Baseline demographics and disease profile were well balanced among treatment groups. Acebilustat had no statistically significant effect on the primary endpoint of change in ppFEV 1 at week 48 or the secondary endpoint pulmonary exacerbations. There was a trend towards reduced pulmonary exacerbations in subjects receiving acebilustat in pre-specified populations with ppFEV 1 >75 (35% rate reduction) and those on concomitant CFTR modulator therapy (20% rate reduction). Acebilustat was well tolerated., Conclusions: Acebilustat did not improve lung function. A trend towards reduced pulmonary exacerbations in subjects with an earlier stage of lung disease suggests a potential effect in this population., Competing Interests: Declaration of Competing Interest Dr. Aaron has nothing to disclose. Dr. Ahuja reports personal fees from Celtaxsys, Inc., during the conduct of the study; In addition, Dr. Ahuja has a patent PCT/US19/034810 pending, and a patent US16/427,571 pending. Dr. Daines has nothing to disclose. Dr. Downey reports and Honoraria from Vertex, Proteostasis and Chiesi. Dr. Elborn reports other from Celtaxsys, during the conduct of the study; grants and other from Corbus, grants from Novartis, grants from polyphor, outside the submitted work. Dr. Fajac reports grants from Celtaxsys, during the conduct of the study; grants and personal fees from Boehringer Ingelheim, grants and personal fees from Proteostasis Therapeutics, grants and personal fees from Vertex Pharmaceuticals, outside the submitted work. Mr. Grosswald reports personal fees from Celtaxsys, Inc., during the conduct of the study; In addition, Dr. Grosswald has a patent PCT/US19/034810 pending, and a patent US16/427,571 pending. Dr. Horsley reports other from Celtaxys pharmaceuticals, during the conduct of the study; grants from NIHR, grants from CF Trust, grants from CF Foundation, personal fees from Vertex pharmaceuticals, personal fees from Mylan, outside the submitted work. Dr. Konstan reports personal fees from Celtaxsys, during the conduct of the study; grants and personal fees from Vertex Pharmaceuticals, personal fees from Chiesi, personal fees from Ionis Pharmaceuticals, grants and personal fees from Laurent Pharmaceuticals, grants and personal fees from Anthera, personal fees from Merck, personal fees from Paranta Biosciences, personal fees from Santhera, personal fees from pH Pharma, grants and personal fees from Cystic Fibrosis Foundation, grants from National Institutes of Health, grants and personal fees from AzurRx, personal fees from Kala Pharmaceuticals, outside the submitted work. John Mershon has nothing to disclose. Dr. Rowe reports grants from Celtaxsys, during the conduct of the study; grants and personal fees from Novartis, grants and personal fees from Bayer, grants from Translate Bio, non-financial support from Proteostasis, grants, personal fees and non-financial support from Galapagos/Abbvie, grants, personal fees and other from Synedgen/Synspira, grants from Eloxx, grants from Celtaxsys, grants, personal fees, non-financial support and other from Vertex Pharmaceuticals Inc, personal fees from Renovion, grants and personal fees from Arrowhead, grants and other from Ionis, grants from Astra Zenica, outside the submitted work. Dr. Springman reports personal fees from Celtaxsys, Inc., during the conduct of the study; In addition, Dr. Springman has a patent PCT/US19/034810 pending, and a patent US16/427,571 pending. Dr. Sutharsan reports personal fees personal Proteostasis Therapeutics, personal fees from Novartis Pharma GmbH, personal fees from Vertex Pharmaceuticals Incorporated, personal fees from Teva GmbH, personal fees from Chiesi GmbH, outside the submitted work; . SS has served as an investigator in clinical trials for Galapagos NV, Proteostasis, Celtaxsys, Flatley Discovery Lab, Novartis and Vertex. Dr. Taylor-Cousar reports grants and personal fees from Celtaxys, during the conduct of the study; grants and personal fees from Gilead, grants from N30, grants and personal fees from Vertex, grants and personal fees from Proteostasis, grants from Bayer, personal fees from Novartis, personal fees from Genentech, personal fees from Protalix, personal fees from Santhera, personal fees from 4DMT, personal fees from Polarean Imaging, personal fees from Insmed, personal fees from Abbvie, outside the submitted work; and Service on the CF TDN Clinical Research Executive CommitteeÐervice as the Chair of the ATS Clinical Problems Assembly Program Committee. Dr. Uluer has served in an advisory capacity with Vertex and Eloxx but these relationships are in no way associated with the work submitted in this manuscript, (Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)

The objective of this observational study was to compare 4 cow-level algorithms to predict cow-level intramammary infection (IMI) status (culture and MALDI-TOF) in late-lactation US dairy cows using standard measures of test performance. Secondary objectives were to estimate the likely effect of each algorithm, if used to guide selective dry cow therapy (SDCT), on dry cow antibiotic use in US dairy herds, and to investigate the importance of including clinical mastitis criteria in algorithm-guided SDCT. Cows (n = 1,594) from 56 US dairy herds were recruited as part of a previously published cross-sectional study of bedding management and IMI in late-lactation cows. Each herd was visited twice for sampling. At each farm visit, aseptic quarter-milk samples were collected from 20 cows approaching dry-off (>180 d pregnant), which were cultured using standard bacteriological methods and MALDI-TOF for identification of isolates. Quarter-level culture results were used to establish cow-level IMI status, which was considered the reference test in this study. Clinical mastitis records and Dairy Herd Improvement Association test-day somatic cell count data were extracted from herd records and used to perform cow-level risk assessments (low vs. high risk) using 4 algorithms that have been proposed for SDCT in New Zealand, the Netherlands, United Kingdom, and the United States. Agreement between aerobic culture (reference test; IMI vs. no-IMI) and algorithm status (high vs. low risk) was described using Cohen's kappa, test sensitivity, specificity, negative predictive value, and positive predictive value. The proportion of cows classified as high risk among the 4 algorithms ranged from 0.31 to 0.63, indicating that these approaches to SDCT could reduce antibiotic use at dry-off by 37 to 69% in the average US herd. All algorithms had poor agreement with IMI status, with kappa values ranging from 0.05 to 0.13. Sensitivity varied by pathogen, with higher values observed when detecting IMI caused by Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, and Lactococcus lactis. Negative predictive values were high for major pathogens among all algorithms (≥0.87), which may explain why algorithm-guided SDCT programs have been successfully implemented in field trials, despite poor agreement with overall IMI status. Removal of clinical mastitis criteria for each algorithm had little effect on the algorithm classification of cows, indicating that algorithms based on SCC alone may have similar performance to those based on SCC and clinical mastitis criteria. We recommend that producers implementing algorithm-guided SDCT use algorithm criteria that matches their relative aspirations for reducing antibiotic use (high specificity, positive predictive value) or minimizing untreated IMI at dry-off (high sensitivity, negative predictive value)., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10 kb C-to-T splicing mutation in the CFTR gene., Methods: We have screened, in FRT cells expressing the 3849+10 kb C-to-T splicing mutation, ~30 2'-O-Methyl-modified phosphorothioate ASOs, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The effect of highly potent ASO candidates on the splicing pattern, protein maturation and CFTR function was further analyzed in well differentiated primary human nasal and bronchial epithelial cells, derived from patients carrying at least one 3849+10 kb C-to-T allele., Results: A highly potent lead ASO, efficiently delivered by free uptake, was able to significantly increase the level of correctly spliced mRNA and completely restore the CFTR function to wild type levels in cells from a homozygote patient. This ASO led to CFTR function with an average of 43% of wild type levels in cells from various heterozygote patients. Optimized efficiency of the lead ASO was further obtained with 2'-Methoxy Ethyl modification (2'MOE)., Conclusion: The highly efficient splicing modulation and functional correction, achieved by free uptake of the selected lead ASO in various patients, demonstrate the ASO therapeutic potential benefit for CF patients carrying splicing mutations and is aimed to serve as the basis for our current clinical development., Competing Interests: Declaration of Competing Interest Batsheva Kerem has equity in SpliSense and is paid for consultancy. All other authors have no financial conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Diseases such as celiac disease, environmental enteric dysfunction, infectious gastroenteritis, type II diabetes and inflammatory bowel disease are associated with increased gut permeability. Dual sugar absorption tests, such as the lactulose to rhamnose ratio (L:R) test, are the current standard for measuring gut permeability. Although easy to administer in adults, the L:R test has a number of drawbacks. These include an inability to assess for spatial heterogeneity in gut permeability that may distinguish different disease severity or pathology, additional sample collection for immunoassays, and challenges in carrying out the test in certain populations such as infants and small children. Here, we demonstrate a minimally invasive probe for real-time localized gut permeability evaluation through gut potential difference (GPD) measurement., Materials and Methods: The probe has an outer diameter of 1.2 mm diameter and can be deployed in the gut of unsedated subjects via a transnasal introduction tube (TNIT) that is akin to an intestinal feeding tube. The GPD probe consists of an Ag/AgCl electrode, an optical probe and a perfusion channel all housed within a transparent sheath. Lactated Ringer's (LR) solution is pumped through the perfusion channel to provide ionic contact between the electrodes and the gut lining. The optical probe captures non-scanning (M-mode) OCT images to confirm electrode contact with the gut lining. A separate skin patch probe is placed over an abraded skin area to provide reference for the GPD measurements. Swine studies were conducted to validate the GPD probe. GPD in the duodenum was modulated by perfusing 45 ml of 45 mM glucose., Results: GPD values of -13.1 ± 2.8 mV were measured in the duodenum across four swine studies. The change in GPD in the duodenum with the addition of glucose was -10.5 ± 2.4 mV ( p < 0.001). M-mode OCT images provided electrode-tissue contact information, which was vital in ascertaining the probe's proximity to the gut mucosa., Conclusion: We developed and demonstrated a minimally invasive method for investigating gastrointestinal permeability consisting of an image guided GPD probe that can be used in unsedated subjects., Competing Interests: Conflict of Interest: GT has a financial/fiduciary interest in SpectraWave, a company developing an OCT-NIRS intracoronary imaging system and catheter. His financial/fiduciary interest was reviewed and is managed by the Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. GT receives sponsored research from VivoLight, iLumen, Boston Scientific, Vertex, Hamamatsu, AstraZeneca, CN USA Biotech Holdings, Wayvector, and Canon Inc. GT receives catheter materials from Terumo Corporation. GT additional receives royalties from Terumo Corporation, iLumen, and MIT The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.