Gilbert's syndrome leads to elevated bilirubin after initiation of elexacaftor/tezacaftor/ivacaftor in people with cystic fibrosis.

Nine people with cystic fibrosis (pwCF) were found to have isolated elevations in serum total bilirubin after starting elexacaftor/tezacaftor/ivacaftor (ETI) that were associated with Gilbert's Syndrome. In longitudinal examination, total bilirubin levels increased substantially after initiation of ETI without elevations in liver transaminases in those with this syndrome. Because elevated bilirubin levels in Gilbert's Syndrome are benign, ETI was able to be continued in these individuals. Genetic testing for this relatively common syndrome should be strongly considered for pwCF experiencing isolated hyperbilirubinemia after starting ETI, since appropriate diagnosis may help pwCF avoid unnecessary interruption in this therapy with significant health benefits in CF.

Cystic fibrosis airway inflammation enables elexacaftor/tezacaftor/ivacaftor-mediated rescue of N1303K CFTR mutation.

Rescue of N1303K CFTR by highly effective modulator therapy (HEMT) is enabled by CF airway inflammation. These findings suggest that evaluation of HEMT for rare CFTR mutations must be performed under inflammatory conditions relevant to CF airways. https://bit.ly/3tTcoJE.

Effect of elexacaftor/tezacaftor/ivacaftor on mucus and mucociliary clearance in cystic 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 (FEV1) 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.

Changes in fecal elastase-1 following initiation of CFTR modulator therapy in pediatric patients with cystic fibrosis.

BACKGROUND: Improvement in exocrine pancreatic function in persons with CF (pwCF) on cystic fibrosis transmembrane conductance regulator (CFTR) modulators has been documented in clinical trials using fecal pancreatic elastase-1 (FE-1). Our group endeavored to evaluate real-world data on FE-1 in children on CFTR modulator therapy at three pediatric cystic fibrosis (CF) centers. METHODS: Pediatric pwCF were offered FE-1 testing if they were on pancreatic enzyme replacement therapy (PERT) and on CFTR modulator therapy according to their center's guideline. FE-1 data were collected retrospectively. The primary outcome was absolute change in FE-1. RESULTS: 70 pwCF were included for analysis. 53 had baseline and post-modulator FE-1 values. There was a significant increase in FE-1 from median 25 mcg/g (IQR 25-60) at baseline to 57 mcg/g (IQR 20-228) post-modulator (p<0.001 by Wilcoxon matched pairs), with an absolute change in FE-1 of median 28 mcg/g (IQR -5-161) and mean 93.5 ± 146.8 mcg/g. Age was negatively correlated with change in FE-1 (Spearman r=-0.48, p<0.001). 15 pwCF (21%) had post-modulator FE-1 values ≥200 mcg/g, consistent with pancreatic sufficiency (PS). The PS group was significant for younger age at initiation of first CFTR modulator and a higher baseline FE-1. CONCLUSIONS: Most pwCF experienced an increase in FE-1 while receiving CFTR modulator treatment and a small percentage demonstrated values reflective of PS. These data suggest that PS may be attained in those that initiated modulator therapy at a younger age or had a higher baseline FE-1. FE-1 testing is suggested for children on any CFTR modulator therapy.

Prolonged, physiologically relevant nicotine concentrations in the airways of smokers.

Nicotine from cigarette smoke is a biologically active molecule that has pleiotropic effects in the airway, which could play a role in smoking-induced lung disease. However, whether nicotine and its metabolites reach sustained, physiologically relevant concentrations on airway surfaces of smokers is not well defined. To address these issues, concentrations of nicotine, cotinine, and hydroxycotinine were measured by mass spectrometry (MS) in supernatants of induced sputum obtained from participants in the subpopulations and intermediate outcome measures in COPD study (SPIROMICS), an ongoing observational study that included never smokers, former smokers, and current smokers with and without chronic obstructive pulmonary disease (COPD). A total of 980 sputum supernatants were analyzed from 77 healthy never smokers, 494 former smokers (233 with COPD), and 396 active smokers (151 with COPD). Sputum nicotine, cotinine, and hydroxycotinine concentrations corresponded to self-reported smoking status and were strongly correlated to urine measures. A cutoff of ∼8-10 ng/mL of sputum cotinine distinguished never smokers from active smokers. Accounting for sample dilution during processing, active smokers had airway nicotine concentrations in the 70-850 ng/mL (∼0.5-5 µM) range, and concentrations remained elevated even in current smokers who had not smoked within 24 h. This study demonstrates that airway nicotine and its metabolites are readily measured in sputum supernatants and can serve as biological markers of smoke exposure. In current smokers, nicotine is present at physiologically relevant concentrations for prolonged periods, supporting a contribution to cigarette-induced airway disease.

Congenital bilateral cataracts in newborns exposed to elexacaftor-tezacaftor-ivacaftor in utero and while breast feeding.

Elexacaftor-tezacaftor-ivacaftor (ETI) is known to pass through the placenta and into breast milk in mothers who continue on this therapy while pregnant and breast feeding. Toxicity studies of ivacaftor in rats demonstrated infant cataracts, but cataracts were not reported in human infants exposed to ivacaftor. We describe 3 cases of infants exposed to elexacaftor-tezacaftor-ivacaftor (ETI) in utero and while breast feeding who were found to have bilateral congenital cataracts within six months of birth. None of the infants had significant visual impairment from the cataracts nor any report of elevated liver function testing. These data highlight the need to counsel females who continue ETI throughout pregnancy and while breast feeding to consider cataract screen for their infants.

Metabolomics of airways disease in cystic fibrosis.

While discovery metabolomic studies have identified many potential biomarkers of cystic fibrosis (CF) airways disease, relatively few have been validated. We review the recent literature to identify the most promising metabolomic findings as those repeatedly observed over multiple studies. Reproducible metabolomic findings include increased airway amino acids and small peptides in CF airways, as well as changes in phospholipids and sphingolipids. Other commonly altered pathways include adenosine metabolism, polyamine synthesis, and oxidative stress. These pathways represent potential biomarkers and therapeutic targets, though findings require reevaluation in the era of highly effective modulator therapies. Analysis of airway biomarkers in exhaled breath holds promise for non-invasive detection, though technical challenges will need to be overcome.

Prospectively evaluating maternal and fetal outcomes in the era of CFTR modulators: the MAYFLOWERS observational clinical trial study design.

INTRODUCTION: Therapeutic advances have markedly increased life expectancy for those with cystic fibrosis (CF), resulting in a median predicted survival over 50 years. Consequently, people with CF (pwCF) are living through their reproductive years and the rate of pregnancy is rapidly rising. Despite the increased relevance of this topic, multicentre studies investigating the association between maternal health and choices made during pregnancy on maternal and fetal outcomes do not exist. Furthermore, there are very limited data on the outcomes following CF transmembrane conductance regulator (CFTR) modulator use during pregnancy and lactation. METHODS AND ANALYSIS: Maternal and Fetal Outcomes in the Era of Modulators (MAYFLOWERS) is a prospective, multicentre observational clinical trial which will enrol approximately 285 pregnant pwCF including those who are modulator ineligible and those who choose to continue or discontinue CFTR modulator therapy during pregnancy and lactation. The primary aim of this 35-month study is to assess whether lung function changes during pregnancy differ based on the continued use of modulators or other factors such as pre-existing comorbid conditions. Secondary objectives include evaluation of pregnancy related and obstetrical complications and changes in mental health. ETHICS AND DISSEMINATION: The design of this study required special consideration of study burden on pregnant and lactating people with chronic illness in the setting of a substantial number of unanswered questions under these conditions. MAYFLOWERS is the first prospective clinical trial examining pregnancy in CF; the outcomes will guide providers on pregnancy management in pwCF and others with chronic respiratory disease.

Lung Microbiota and Metabolites Collectively Associate with Clinical Outcomes in Milder Stage Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is variable in its development. Lung microbiota and metabolites collectively may impact COPD pathophysiology, but relationships to clinical outcomes in milder disease are unclear. Objectives: Identify components of the lung microbiome and metabolome collectively associated with clinical markers in milder stage COPD. Methods: We analyzed paired microbiome and metabolomic data previously characterized from bronchoalveolar lavage fluid in 137 participants in the SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study), or (GOLD [Global Initiative for Chronic Obstructive Lung Disease Stage 0-2). Datasets used included 1) bacterial 16S rRNA gene sequencing; 2) untargeted metabolomics of the hydrophobic fraction, largely comprising lipids; and 3) targeted metabolomics for a panel of hydrophilic compounds previously implicated in mucoinflammation. We applied an integrative approach to select features and model 14 individual clinical variables representative of known associations with COPD trajectory (lung function, symptoms, and exacerbations). Measurements and Main Results: The majority of clinical measures associated with the lung microbiome and metabolome collectively in overall models (classification accuracies, >50%, P < 0.05 vs. chance). Lower lung function, COPD diagnosis, and greater symptoms associated positively with Streptococcus, Neisseria, and Veillonella, together with compounds from several classes (glycosphingolipids, glycerophospholipids, polyamines and xanthine, an adenosine metabolite). In contrast, several Prevotella members, together with adenosine, 5'-methylthioadenosine, sialic acid, tyrosine, and glutathione, associated with better lung function, absence of COPD, or less symptoms. Significant correlations were observed between specific metabolites and bacteria (Padj < 0.05). Conclusions: Components of the lung microbiome and metabolome in combination relate to outcome measures in milder COPD, highlighting their potential collaborative roles in disease pathogenesis.

Mucus and mucus flake composition and abundance reflect inflammatory and infection status in cystic fibrosis.

BACKGROUND: Mucus hyperconcentration in cystic fibrosis (CF) lung disease is marked by increases in both mucin and DNA concentration. Additionally, it has been shown that half of the mucins present in bronchial alveolar lavage fluid (BALF) from preschool-aged CF patients are present in as non-swellable mucus flakes. This motivates us to examine the utility of mucus flakes, as well as mucin and DNA concentrations in BALF as markers of infection and inflammation in CF airway disease. METHODS: In this study, we examined the mucin and DNA concentration, as well as mucus flake abundance, composition, and biophysical properties in BALF from three groups; healthy adult controls, and two CF cohorts, one preschool aged and the other school aged. BALFs were characterized via refractometry, PicoGreen, immunofluorescence microscopy, particle tracking microrheology, and fluorescence image tiling. RESULTS: Mucin and DNA BALF concentrations increased progressively from healthy young adult controls to preschool-aged people and school-aged people with CF. Notably, mucin concentrations were increased in bronchoalveolar lavage fluid (BALF) from preschool-aged patients with CF prior to decreased pulmonary function. Infrequent small mucus flakes were identified in normal subjects. A progressive increase in the abundance of mucus flakes in preschool and school-aged CF patients was observed. Compositionally, MUC5B dominated flakes from normal subjects, whereas an increase in MUC5AC was observed in people with CF, reflected in a reduced flaked MUC5B/MUC5AC mucin ratio. CONCLUSION: These findings suggest mucus composition and flake properties are useful markers of inflammatory and infection-based changes in CF airways.

Drug exposure to infants born to mothers taking Elexacaftor, Tezacaftor, and Ivacaftor.

Elexacaftor, tezacaftor, ivacaftor (ETI) have been associated with marked clinical improvements in adults with CF, which appears to be associated with increased fertility. However, maternal and fetal effects of therapy continued during pregnancy are not well understood. We collected maternal blood, infant blood, cord blood, and breast milk from 3 mother-infant pairs from women who elected to remain on ETI therapy while pregnant. Our results demonstrated relatively high levels of ETI in cord blood, suggesting placental transfer of these compounds, as well as low levels of ETI in breast milk and infant blood, suggesting further transfer of these compounds to breast-fed infants in the postnatal period. These data underscore the need for larger studies on the effects of modulator surrounding reproduction.

Identification of Sputum Biomarkers Predictive of Pulmonary Exacerbations in COPD.

BACKGROUND: Improved understanding of the pathways associated with airway pathophysiologic features in COPD will identify new predictive biomarkers and novel therapeutic targets. RESEARCH QUESTION: Which physiologic pathways are altered in the airways of patients with COPD and will predict exacerbations? STUDY DESIGN AND METHODS: We applied a mass spectrometric panel of metabolomic biomarkers related to mucus hydration and inflammation to sputa from the multicenter Subpopulations and Intermediate Outcome Measures in COPD Study. Biomarkers elevated in sputa from patients with COPD were evaluated for relationships to measures of COPD disease severity and their ability to predict future exacerbations. RESULTS: Sputum supernatants from 980 patients were analyzed: 77 healthy nonsmokers, 341 smokers with preserved spirometry, and 562 patients with COPD (178 with Global Initiative on Chronic Obstructive Lung Disease [GOLD] stage 1 disease, 303 with GOLD stage 2 disease, and 81 with GOLD stage 3 disease) were analyzed. Biomarkers from multiple pathways were elevated in COPD and correlated with sputum neutrophil counts. Among the most significant analytes (false discovery rate, 0.1) were sialic acid, hypoxanthine, xanthine, methylthioadenosine, adenine, and glutathione. Sialic acid and hypoxanthine were associated strongly with measures of disease severity, and elevation of these biomarkers was associated with shorter time to exacerbation and improved prediction models of future exacerbations. INTERPRETATION: Biomarker evaluation implicated pathways involved in mucus hydration, adenosine metabolism, methionine salvage, and oxidative stress in COPD airway pathophysiologic characteristics. Therapies that target these pathways may be of benefit in COPD, and a simple model adding sputum-soluble phase biomarkers improves prediction of pulmonary exacerbations. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01969344; URL: www. CLINICALTRIALS: gov.

Semiparametric estimation of the proportional rates model for recurrent events data with missing event category.

Proportional rates models are frequently used for the analysis of recurrent event data with multiple event categories. When some of the event categories are missing, a conventional approach is to either exclude the missing data for a complete-case analysis or employ a parametric model for the missing event type. It is well known that the complete-case analysis is inconsistent when the missingness depends on covariates, and the parametric approach may incur bias when the model is misspecified. In this paper, we aim to provide a more robust approach using a rate proportion method for the imputation of missing event types. We show that the log-odds of the event type can be written as a semiparametric generalized linear model, facilitating a theoretically justified estimation framework. Comprehensive simulation studies were conducted demonstrating the improved performance of the semiparametric method over parametric procedures. Multiple types of Pseudomonas aeruginosa infections of young cystic fibrosis patients were analyzed to demonstrate the feasibility of our proposed approach.

Metabolomic profiling of extraesophageal reflux disease in children.

Although respiratory symptoms in children are often attributed to gastroesophageal reflux disease, establishing a clear diagnosis of extraesophageal reflux disease (EERD) can be challenging, as there are no sensitive or specific EERD biomarkers. The aim of this study was to evaluate the metabolite profile in bronchoalveolar (BAL) fluid from children with suspected EERD and assess the impact of reflux treatment on these metabolites. In this prospective pilot study, we performed nontargeted global metabolomic profiling on BAL fluid from 43 children undergoing testing with bronchoscopy, upper endoscopy, and multichannel intraluminal impedance with pH (pH-MII) for evaluation of chronic respiratory symptoms. Twenty-three (54%) patients had an abnormal pH-MII study. Seventeen (40%) patients were on proton pump inhibitors (PPIs) for testing. Levels of histamine, malate, adenosine 5'-monophosphate, and ascorbate were significantly lower in subjects with abnormal pH-MII studies compared to those normal studies. Furthermore, in children off PPI therapy, those with abnormal pH-MII studies had robust increases in a number of glycerophospholipids within phospholipid metabolic pathways, including derivatives of glycerophosphorylcholine, glycerophosphoglycerol, and glycerophosphoinositol, compared to those with normal pH-MII studies. These findings offer insight into the impact of reflux and PPIs on the lungs and provide a foundation for future studies using targeted metabolomic analysis to identify potential biomarkers of EERD.

Airway Epithelial Inflammation In Vitro Augments the Rescue of Mutant CFTR by Current CFTR Modulator Therapies.

In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.

Inverse probability weighted estimation for recurrent events data with missing category.

Modeling recurrent event data with multiple event types has drawn interest in recent biomedical studies due to its flexibility for understanding different risk factors for multiple recurrent event processes. However, in such data type, missing event type appears frequently because of various reasons such as recording ignorance or resource limitation. In this study, we aim to propose an inverse probability weighted estimation that is commonly used in the missing data literature to correct possibly biased estimation by a complete-case analysis. This approach is not limited to a specific form of the recurrent event model. We derive the large sample theory in a general form. We demonstrate that our approach can be applied to either multiplicative or additive rates model with practical sample size via comprehensive simulations. Nonmucoid and mucoid Pseudomonas aeruginosa infections of 14 888 patients in 2016 Cystic Fibrosis Foundation Patient Registry data are analyzed to show that, without including 12% events with missing event type in the analysis, several factors may be misidentified as risk factors for the nonmucoid type of infections.

Novel therapies for treatment of resistant and refractory nontuberculous mycobacterial infections in patients with cystic fibrosis.

Respiratory infections caused by non-tuberculous mycobacteria (NTM) are a major cause of morbidity for patients living with cystic fibrosis (CF), as NTM pulmonary disease (NTM-PD) is challenging to both diagnose and eradicate. Despite the lengthy courses of the established regimens recommended by the Cystic Fibrosis Foundation (CFF) and European Cystic Fibrosis Society (ECFS) consensus guidelines, only about 50% to 60% of patients achieve culture conversion, and treatment regimens are often complicated by antibiotic resistance and toxicities. Since publication of the CFF/ECFS guidelines, several new or alternative antibiotic regimens have been described for patients with CF who have NTM-PD. These regimens offer new options for patients who do not clear NTM with standard therapies or cannot utilize the usual regimens due to toxicities or drug-drug interactions.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.