Functional evaluation of the cystic fibrosis transmembrane conductance regulator in the endocervix†.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride/bicarbonate ion channel in epithelial cells. Mutations in CFTR cause cystic fibrosis, a disease characterized by thickened mucus secretions and is associated with subfertility and infertility. CFTR function has been well characterized in vitro and in vivo in airway and other epithelia studies. However, little is known about CFTR function in the cervix in health and its contribution to cyclic regulation of fertility from endocervical mucus changes. Contributing to this research gap is the lack of information on the effect of sex steroid hormones on CFTR expression in cervical epithelial cells across the menstrual cycle. Herein, we demonstrate the hormonal regulation of CFTR expression in endocervical cells both in vitro and in vivo, and that conditionally reprogrammed endocervical epithelial cells can be used to interrogate CFTR ion channel function. CFTR activity was demonstrated in vitro using electrophysiological methods and functionally inhibited by the CFTR-specific inhibitors inh-172 and GlyH-101. We also report that CFTR expression is increased by estradiol in the macaque cervix both in vitro and in vivo in Rhesus macaques treated with artificial menstrual cycles. Estrogen upregulation of CFTR is blocked in vivo by cotreatment with progesterone. Our findings provide the most comprehensive evidence to date that steroid hormones drive changes in CFTR expression. These data are integral to understanding the role of CFTR as a fertility regulator in the endocervix.

The Effect of Extended Continuous Positive Airway Pressure on Changes in Lung Volumes in Stable Premature Infants: A Randomized Controlled Trial.

OBJECTIVE: To compare changes in lung volumes, as measured by functional residual capacity (FRC), through to discharge in stable infants randomized to 2 weeks of extended continuous positive airway pressure CPAP (eCPAP) vs CPAP discontinuation (dCPAP). STUDY DESIGN: Infants born at ≤32 weeks of gestation requiring ≥24 hours of CPAP were randomized to 2 weeks of eCPAP vs dCPAP when meeting CPAP stability criteria. FRC was measured with the nitrogen washout technique. Infants were stratified by gestational age (<28 and ≥ 28 weeks) and twin gestation. A linear mixed-effects model was used to evaluate the change in FRC between the 2 groups. Data were analyzed blinded to treatment group allocation. RESULTS: Fifty infants were randomized with 6 excluded, for a total of 44 infants. Baseline characteristics were similar in the 2 groups. The infants randomized to eCPAP vs dCPAP had a greater increase in FRC from randomization through 2 weeks (12.6 mL vs 6.4 mL; adjusted 95% CI, 0.78-13.47; P = .03) and from randomization through discharge (27.2 mL vs 17.1 mL; adjusted 95% CI, 2.61-17.59; P = .01). CONCLUSIONS: Premature infants randomized to eCPAP had a significantly greater increase in FRC through discharge compared with those randomized to dCPAP. An increased change in FRC may lead to improved respiratory health. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02249143.

Lipid Nanoparticle-Delivered Chemically Modified mRNA Restores Chloride Secretion in Cystic Fibrosis.

The promise of gene therapy for the treatment of cystic fibrosis has yet to be fully clinically realized despite years of effort toward correcting the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR). mRNA therapy via nanoparticle delivery represents a powerful technology for the transfer of genetic material to cells with large, widespread populations, such as airway epithelia. We deployed a clinically relevant lipid-based nanoparticle (LNP) for packaging and delivery of large chemically modified CFTR mRNA (cmCFTR) to patient-derived bronchial epithelial cells, resulting in an increase in membrane-localized CFTR and rescue of its primary function as a chloride channel. Furthermore, nasal application of LNP-cmCFTR restored CFTR-mediated chloride secretion to conductive airway epithelia in CFTR knockout mice for at least 14 days. On day 3 post-transfection, CFTR activity peaked, recovering up to 55% of the net chloride efflux characteristic of healthy mice. This magnitude of response is superior to liposomal CFTR DNA delivery and is comparable with outcomes observed in the currently approved drug ivacaftor. LNP-cmRNA-based systems represent a powerful platform technology for correction of cystic fibrosis and other monogenic disorders.

Effect of Vitamin C Supplementation for Pregnant Smokers on Offspring Airway Function and Wheeze at Age 5 Years: Follow-up of a Randomized Clinical Trial.

Importance: Vitamin C supplementation (500 mg/d) for pregnant smokers has been reported to increase offspring airway function as measured by forced expiratory flow (FEF) through age 12 months; however, its effects on airway function at age 5 years remain to be assessed. Objective: To assess whether vitamin C supplementation in pregnant smokers is associated with increased and/or improved airway function in their offspring at age 5 years and whether vitamin C decreases the occurrence of wheeze. Design, Setting, and Participants: This study followed up the Vitamin C to Decrease the Effects of Smoking in Pregnancy on Infant Lung Function (VCSIP) double-blind, placebo-controlled randomized clinical trial conducted at 3 centers in the US (in Oregon, Washington, and Indiana) between 2012 and 2016. Investigators and participants remain unaware of the treatment assignments. Forced expiratory flow measurements at age 5 years were completed from 2018 to 2021. Interventions: Pregnant smokers were randomized to vitamin C (500 mg/d) or placebo treatment. Main Outcomes and Measures: The primary outcome was the prespecified measurement of FEF between 25% and 75% expired volume (FEF25-75) by spirometry at age 5 years. Secondary outcomes included FEF measurements at 50% and 75% of expiration (FEF50 and FEF75), forced expiratory volume in 1 second (FEV1), and occurrence of wheeze. Results: Of the 251 pregnant smokers included in this study, 125 (49.8%) were randomized to vitamin C and 126 (50.2%) were randomized to placebo. Of 213 children from the VCSIP trial who were reconsented into this follow-up study, 192 (90.1%) had successful FEF measurements at age 5 years; 212 (99.5%) were included in the analysis of wheeze. Analysis of covariance demonstrated that offspring of pregnant smokers allocated to vitamin C compared with placebo had 17.2% significantly higher mean (SE) measurements of FEF25-75 at age 5 years (1.45 [0.04] vs 1.24 [0.04] L/s; adjusted mean difference, 0.21 [95% CI, 0.13-0.30]; P < .001). Mean (SE) measurements were also significantly increased by 14.1% for FEF50 (1.59 [0.04] vs 1.39 [0.04] L/s; adjusted mean difference, 0.20 [95% CI, 0.11-0.30]; P < .001), 25.9% for FEF75 (0.79 [0.02] vs 0.63 [0.02] L/s; 0.16 [95% CI, 0.11-0.22]; P < .001), and 4.4% for FEV1 (1.13 [0.02] vs 1.09 [0.02] L; 0.05 [95% CI, 0.01-0.09]; P = .02). In addition, offspring of pregnant smokers randomized to vitamin C had significantly decreased wheeze (28.3% vs 47.2%; estimated odds ratio, 0.41 [95% CI, 0.23-0.74]; P = .003). Conclusions and Relevance: In this follow-up study of offspring of pregnant smokers randomized to vitamin C vs placebo, vitamin C supplementation during pregnancy resulted in significantly increased airway function of offspring at age 5 years and significantly decreased the occurrence of wheeze. These findings suggest that vitamin C supplementation for pregnant smokers may decrease the effects of smoking in pregnancy on childhood airway function and respiratory health. Trial Registration: ClinicalTrials.gov Identifier: NCT03203603.

Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP(4)).

The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP(4) receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP(4) receptor or ß adrenergic receptor (ßAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either ßAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and ßAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the ß adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP(4) did not respond to the presence of 0.1, 1, or 10 µM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and ßAR did not respond to hyperpolarization, acidic pH, or 1 µM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR(inh)172. Co-expression of CFTR and EP(4) resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR(inh)172. The EC(50) for lubiprostone mediated CFTR activation was ~10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP(4) receptor in oocytes.

Engineering Lipid Nanoparticles for Enhanced Intracellular Delivery of mRNA through Inhalation.

Despite lipid nanoparticles' (LNPs) success in the effective and safe delivery of mRNA vaccines, an inhalation-based mRNA therapy for lung diseases remains challenging. LNPs tend to disintegrate due to shear stress during aerosolization, leading to ineffective delivery. Therefore, LNPs need to remain stable through the process of nebulization and mucus penetration, yet labile enough for endosomal escape. To meet these opposing needs, we utilized PEG lipid to enhance the surficial stability of LNPs with the inclusion of a cholesterol analog, ß-sitosterol, to improve endosomal escape. Increased PEG concentrations in LNPs enhanced the shear resistance and mucus penetration, while ß-sitosterol provided LNPs with a polyhedral shape, facilitating endosomal escape. The optimized LNPs exhibited a uniform particle distribution, a polyhedral morphology, and a rapid mucosal diffusion with enhanced gene transfection. Inhaled LNPs led to localized protein production in the mouse lung without pulmonary or systemic toxicity. Repeated administration of these LNPs led to sustained protein production in the lungs. Lastly, mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was delivered after nebulization to a CFTR-deficient animal model, resulting in the pulmonary expression of this therapeutic protein. This study demonstrated the rational design approach for clinical translation of inhalable LNP-based mRNA therapies.

Evaluation of rescue oral glucocorticoid therapy during inpatient cystic fibrosis exacerbations.

An acute pulmonary exacerbation (APE) in cystic fibrosis (CF) is characterized by increased pulmonary symptoms attributed to bacterial colonization, neutrophil recruitment, and inflammation. Antimicrobials, airway clearance, and nutrition are the mainstay of therapy. However, when patients fail to improve, corticosteroids have been added to therapy. We retrospectively examined the use of rescue steroids in a children's hospital from 2013 to 2017 during CF APE treatment following at least 1 week of inpatient therapy without expected clinical improvement. In total, 106 encounters of 53 unique patients, aged 6-20 years, who had FEV1 percent predicted (FEV1pp) data at baseline, admission, midpoint, and discharge, and had admission duration of at least 12 days were studied. Encounters treated with steroids had less improvement at midpoint percent change from admission in FEV1pp (4.9 ± 11.3) than nonsteroid group change in FEV1pp (20.1 ± 24.6; p < .001). Failure to improve as expected was the rationale for steroid use. At discharge, there was no difference in mean FEV1pp (p = .76). Delays in steroid therapy by waiting until the end of the second week increased the total length of stay (LOS). Propensity matching, comparing outcomes in patients without midpoint improvement in FEV1pp, was also evaluated. There was no difference in admission or discharge FEV1pp between groups. Equally, no difference in FEV1pp at follow-up visit or in time until the next APE was detected. Secondary analysis for associations including gender, genotype, fungal colonization, or inhaled antimicrobials was nonsignificant. These data suggest rescue use of corticosteroids during APE does not predictably impact important outcome measures during CF APE treatment.

Engineered mutant α-ENaC subunit mRNA delivered by lipid nanoparticles reduces amiloride currents in cystic fibrosis-based cell and mice models.

Cystic fibrosis (CF) results from mutations in the chloride-conducting CF transmembrane conductance regulator (CFTR) gene. Airway dehydration and impaired mucociliary clearance in CF is proposed to result in tonic epithelial sodium channel (ENaC) activity, which drives amiloride-sensitive electrogenic sodium absorption. Decreasing sodium absorption by inhibiting ENaC can reverse airway surface liquid dehydration. Here, we inhibit endogenous heterotrimeric ENaC channels by introducing inactivating mutant ENaC α mRNA (αmutENaC). Lipid nanoparticles carrying αmutENaC were transfected in CF-based airway cells in vitro and in vivo. We observed a significant decrease in macroscopic as well as amiloride-sensitive ENaC currents and an increase in airway surface liquid height in CF airway cells. Similarly, intranasal transfection of αmutENaC mRNA decreased amiloride-sensitive nasal potential difference in CFTRKO mice. These data suggest that mRNA-based ENaC inhibition is a powerful strategy for reducing mucus dehydration and has therapeutic potential for treating CF in all patients, independent of genotype.

An improved simple method of mouse lung intubation.

Given the ubiquitous use of mice to study lung disease, it is curious that more investigators do not use repeated intubation to study mechanical and cellular changes in individual mice. One of the reasons for this limited use of intubation is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper, we describe a complete procedure, including novel approaches that simplify this intubation, so that it can be routinely accomplished with relatively little training. The technique can also be set up with relatively little expense and expertise. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice and potentially increasing the statistical power by using each mouse as its own control.

Primary snoring and growth failure in a patient with cystic fibrosis.

An 8-year-old female with pancreatic-insufficiency cystic fibrosis presented with recurrent pharyngitis, and reduction in body mass index and height velocity during the previous 2 years. Her symptoms (eg, snoring and restless sleep) suggested obstructive sleep apnea, and physical examination revealed tonsillar hypertrophy. While her respiratory disturbance index on nocturnal polysomnography was normal, there was evidence of prolonged periods of snoring, associated with hypercapnia. Adenotonsillectomy decreased the snoring, improved her sleep, and in the 18-month follow-up period she had substantial weight-gain and growth improvement. This case demonstrates that adenotonsillar hypertrophy associated with recurrent pharyngitis and primary snoring might hinder growth in a patient with cystic fibrosis.

Vitamin C Supplementation Among Pregnant Smokers and Airway Function Trajectory in Offspring: A Secondary Analysis of a Randomized Clinical Trial.

This secondary analysis of a randomized clinical trial analyzes the association of vitamin C supplementation in women who smoked during pregnancy with airway function trajectory in their offspring at 4 to 6 years of age.

Cost effectiveness of vitamin c supplementation for pregnant smokers to improve offspring lung function at birth and reduce childhood wheeze/asthma.

OBJECTIVE: To determine the implications of supplemental vitamin C for pregnant tobacco smokers and its effects on the prevalence of pediatric asthma, asthma-related mortality, and associated costs. STUDY DESIGN: A decision-analytic model built via TreeAge compared the outcome of asthma in a theoretical annual cohort of 480,000 children born to pregnant smokers through 18 years of life. Vitamin C supplementation (500 mg/day) with a standard prenatal vitamin was compared to a prenatal vitamin (60 mg/day). Model inputs were derived from the literature. Deterministic and probabilistic sensitivity analyses assessed the impact of assumptions. RESULT: Additional vitamin C during pregnancy would prevent 1637 cases of asthma at the age of 18 per birth cohort of pregnant smokers. Vitamin C would reduce asthma-related childhood deaths and save $31,420,800 in societal costs over 18 years per birth cohort. CONCLUSION: Vitamin C supplementation in pregnant smokers is a safe and inexpensive intervention that may reduce the economic burden of pediatric asthma.

Cystic fibrosis transmembrane regulator protein mutations: 'class' opportunity for novel drug innovation.

Cystic fibrosis (CF) is the most common autosomal, recessive, life-span shortening disease in Caucasians. Since discovery of the gene for CF (cystic fibrosis transmembrane conductance regulator [CFTR]) in 1989, knowledge of the molecular function of this gene and its interactions has offered new therapeutic targets. New therapeutics aimed at improving mutant CFTR protein function, also known as 'protein repair therapy,' have been proposed but are yet to be successful in clinical trials. Some of the most exciting efforts involve a new field known as small molecule discovery, which entails the identification, evaluation, and optimization of small organic compounds that can alter the function of a selected gene target or cell phenotype. More than 1300 CFTR mutations have been identified. Many of the more common mutations have been organized into five broad classes based on the fate of the mutant CFTR protein. In each of these mutation classes, interventions have been able to restore some level of CFTR function in vitro. While these 'repairs' have yet to be demonstrated clinically, some early clinical trials are underway. Questions regarding the amount of CFTR correction needed, delivery methods, and optimal therapeutic combinations, however, remain outstanding.

Maternal high-fat diet in mice leads to innate airway hyperresponsiveness in the adult offspring.

Maternal obesity prior to and during pregnancy has been associated with an increased incidence of childhood asthma. As diets rich in saturated fat are linked to obesity and inflammation, we created a murine model to investigate the effect of maternal high-fat diet (HFD) on adult offspring airway hyperreactivity (AHR), a cardinal feature of asthma. Balb/cByJ dams were fed a HFD (60% fat Calories) or normal-fat diet (NFD) (10% fat Calories) from 8 weeks prior to first breeding through their pregnancies. Pups were weaned to either a HFD or NFD (at 4 weeks of age). AHR was measured in the 10-week-old offspring following inhaled methacholine challenge by end-inflation technique. Bronchial alveolar lavage fluid (BALF) was analyzed for cell count, total protein, and IL-6. Offspring of HFD dams weaned to NFD had increased AHR compared to offspring of NFD dams weaned to NFD Offspring of HFD dams that remained on HFDs had increased AHR compared to offspring of NFD dams weaned to HFDs. Offspring of HFD dams had higher BALF cell counts, higher neutrophil percentage, greater total protein, and IL-6 in the BALF These results demonstrate that a maternal diet high in saturated fat through pregnancy and lactation plays a key role in programming adult offspring AHR.

One Center's Guide to Outpatient Management of Pediatric Cystic Fibrosis Acute Pulmonary Exacerbation.

Cystic fibrosis (CF) is a chronic disorder characterized by acute pulmonary exacerbations that comprise increased cough, chest congestion, increased mucus production, shortness of breath, weight loss, and fatigue. Typically, severe episodes are treated in the inpatient setting and include intravenous antimicrobials, airway clearance therapy, and nutritional support. Children with less-severe findings can often be managed as outpatients with oral antimicrobials and increased airway clearance therapy at home without visiting the specialty CF center to begin treatment. Selection of specific antimicrobial agents is dependent on pathogens found in surveillance culture, activity of an agent in patients with CF, and the unique physiology of these patients. In this pediatric review, we present our practice for defining acute pulmonary exacerbation, deciding treatment location, initiating treatment either in-person or remotely, determining the frequency of airway clearance, selecting antimicrobial therapy, recommending timing for follow-up visit, and recognizing and managing treatment failures.

Maternal body mass index before pregnancy is associated with increased bronchodilator dispensing in early childhood: A cross-sectional study.

RATIONALE: Maternal prepregnancy obesity has been associated with early wheeze and childhood asthma in their offspring. Some of these studies have been in minority, urban, and disadvantaged populations using parental recall and questionnaires. The association of maternal prepregnancy obesity with bronchodilator dispensing to their offspring, in a primarily insured, non-urban, White population in the United States is unknown. OBJECTIVES AND METHODS: We conducted a retrospective cohort study using pharmacy dispensing data from the electronic medical records of a large United States health maintenance organization to examine the relationship between maternal prepregnancy body mass index (BMI) and inhaled bronchodilator dispensing in the offspring to 4 years of age. We included infants ≥37 weeks' gestation with birth weight ≥2.5 kg which yielded 6,194 mother-baby pairs. Maternal prepregnancy BMI was categorized as underweight (<18.5 kg/m(2) ), normal (18.5-24.9 kg/m(2) ), overweight (25-29.9 kg/m(2) ), or obese (≥30 kg/m(2) ). RESULTS: In the entire cohort, 27.6% of the offspring received a bronchodilator dispensing. This ranged from 19.2% in the offspring of underweight mothers to 31.3% of those born to obese mothers. In the fully adjusted model using normal BMI as the referent, children of obese mothers had a 22% higher rate of bronchodilator dispensing (adjusted OR = 1.22; 95%CI 1.05-1.41; P = 0.008). CONCLUSIONS: In this insured, non-urban, White population, maternal prepregnancy obesity was associated with bronchodilator dispensing in the offspring in early life. These results extend previous data and reaffirm the potential widespread public health impact that prepregnancy obesity may have on subsequent childhood respiratory health. Pediatr Pulmonol. 2016;51:803-811. © 2016 Wiley Periodicals, Inc.

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice.

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.