Arteriovenous metabolomics in pigs reveals CFTR regulation ofmetabolism inmultiple organs.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a multiorgan disease that exhibits diverse metabolic defects. However, other than specific CFTR mutations, the factors that influence disease progression and severity remain poorly understood. Aberrant metabolite levels have been reported, but whether CFTR loss itself or secondary abnormalities (infection, inflammation, malnutrition, and various treatments) drive metabolic defects are uncertain. Here, we implemented comprehensive arteriovenous metabolomics in newborn CF pigs, and the results revealed CFTR as a bona fide regulator of metabolism. CFTR loss impaired metabolite exchange across organs, including disrupted lung uptake of fatty acids yet enhanced uptake of arachidonic acid, a precursor of pro-inflammatory cytokines. CFTR loss also impaired kidney reabsorption of amino acids and lactate and abolished renal glucose homeostasis. These and additional unexpected metabolic defects prior to disease manifestations reveal a fundamental role for CFTR in controlling multi-organ metabolism. Such discovery informs a basic understanding of CF, provides a foundation for future investigation, and has implications for developing therapies targeting only a single tissue.

Pancreatic enzymes digest obstructive meconium from cystic fibrosis pig intestines.

Introduction: Meconium ileus (MI) is a life-threatening obstruction of the intestines affecting ∼15% of newborns with cystic fibrosis (CF). Current medical treatments for MI often fail, requiring surgical intervention. MI typically occurs in newborns with pancreatic insufficiency from CF. Meconium contains mucin glycoprotein, a potential substrate for pancreatic enzymes or mucolytics. Our study aim was to determine whether pancreatic enzymes in combination with mucolytic treatments dissolve obstructive meconium using the CF pig model. Methods: We collected meconium from CF pigs at birth and submerged it in solutions with and without pancreatic enzymes, including normal saline, 7% hypertonic saline, and the reducing agents N-acetylcysteine (NAC) and dithiothreitol (DTT). We digested meconium at 37 °C with agitation, and measured meconium pigment release by spectrophotometry and residual meconium solids by filtration. Results and discussion: In CF pigs, meconium appeared as a solid pigmented mass obstructing the ileum. Meconium microscopically contained mucus glycoprotein, cellular debris, and bile pigments. Meconium fragments released pigments with maximal absorption at 405 nm after submersion in saline over approximately 8 h. Pancreatic enzymes significantly increased pigment release and decreased residual meconium solids. DTT did not improve meconium digestion and the acidic reducing agent NAC worsened digestion. Pancreatic enzymes digested CF meconium best at neutral pH in isotonic saline. We conclude that pancreatic enzymes digest obstructive meconium from CF pigs, while hydrating or reducing agents alone were less effective. This work suggests a potential role for pancreatic enzymes in relieving obstruction due to MI in newborns with CF.

Standardization of flow cytometry and cell sorting to enable a transcriptomic analysis in a multi-site sarcoidosis study.

The contribution and regulation of various CD4+ T cell lineages that occur with remitting vs progressive courses in sarcoidosis are poorly understood. We developed a multiparameter flow cytometry panel to sort these CD4+ T cell lineages followed by measurement of their functional potential using RNA-sequencing analysis at six-month intervals across multiple study sites. To obtain good quality RNA for sequencing, we relied on chemokine receptor expression to identify and sort lineages. To minimize gene expression changes induced by perturbations of T cells and avoid protein denaturation caused by freeze/thaw cycles, we optimized our protocols using freshly isolated samples at each study site. To accomplish this study, we had to overcome significant standardization challenges across multiple sites. Here, we detail standardization considerations for cell processing, flow staining, data acquisition, sorting parameters, and RNA quality control analysis that were performed as part of the NIH-sponsored, multi-center study, BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints (BRITE). After several rounds of iterative optimization, we identified the following aspects as critical for successful standardization: 1) alignment of PMT voltages across sites using CS&T/rainbow bead technology; 2) a single template created in the cytometer program that was used by all sites to gate cell populations during data acquisition and cell sorting; 3) use of standardized lyophilized flow cytometry staining cocktails to reduce technical error during processing; 4) development and implementation of a standardized Manual of Procedures. After standardization of cell sorting, we were able to determine the minimum number of sorted cells necessary for next generation sequencing through analysis of RNA quality and quantity from sorted T cell populations. Overall, we found that implementing a multi-parameter cell sorting with RNA-seq analysis clinical study across multiple study sites requires iteratively tested standardized procedures to ensure comparable and high-quality results.

Comorbidity of Lifetime History of Abuse and Trauma With Opioid Use Disorder: Implications for Nursing Assessment and Care.

BACKGROUND: Opioid use disorder (OUD) is a public health crisis and is challenging to treat. Previous research has shown correlations between OUD, abuse/trauma, and chronic pain. AIMS: The purpose of this study was to investigate history of lifetime sexual, physical, and/or emotional abuse among participants in a medication-assisted treatment (MAT) program for OUD, and to investigate associations between abuse history and chronic pain. METHODS: This is a secondary analysis of intake data from a 2-year, nonexperimental cohort treatment program of patients with OUD in rural Colorado. De-identified data were provided by 476 adult MAT patients using the Adult Addiction Severity Index (ASI-6). The ASI-6 includes three yes/no questions about history of abuse (emotional, physical, and sexual), with separate scoring for "past 30 days" and "lifetime" abuse. RESULTS: Lifetime history among MAT program for OUD patients was 23% for sexual abuse, 43% for physical abuse, and 58% for emotional abuse. History of physical abuse was significantly associated with having a chronic pain diagnosis, χ2 = 4.49, p = .03, and also with higher reported pain levels, t(460) = 2.71, p = .007. CONCLUSION: Lifetime history of physical abuse was associated with OUD and chronic pain, yet standard pain assessments do not assess these factors. In health care settings, the implementation of standardized trauma-informed screening tools, prompt recognition of abuse/trauma history, and adjunct psychological interventions may reduce stigma, reduce opioid use escalation, and help patients overcome OUD.

Cellular and molecular architecture of submucosal glands in wild-type and cystic fibrosis pigs.

Submucosal glands (SMGs) protect lungs but can also contribute to disease. For example, in cystic fibrosis (CF), SMGs produce abnormal mucus that disrupts mucociliary transport. CF is an ion transport disease, yet knowledge of the ion transporters expressed by SMG acini, which produce mucus, and SMG ducts that carry it to the airway lumen is limited. Therefore, we isolated SMGs from newborn pigs and used single-cell messenger RNA sequencing, immunohistochemistry, and in situ hybridization to identify cell types, gene expression, and spatial distribution. Cell types and transcript levels were the same in non-CF and CF SMGs, suggesting that loss of epithelial anion secretion rather than an intrinsic cell defect causes CF mucus abnormalities. Gene signatures of acinar mucous and acinar serous cells revealed specialized functions in producing mucins and antimicrobials, respectively. However, surprisingly, these two cell types expressed the same ion transporters and neurohumoral receptors, suggesting the importance of balancing mucin and liquid secretion to produce optimal mucus properties. SMG duct cell transcripts suggest that they secrete HCO3- and Cl-, and thus have some similarity to pancreatic ducts that are also defective in CF. These and additional findings suggest the functions of the SMG acinus and duct and provide a baseline for understanding how environmental and genetic challenges impact their contribution to lung disease.

Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is considered to be an important risk factor for the development of cardiovascular disease (CVD). This study aimed to develop and evaluate a machine learning approach with a set of features for assessing the 10-year CVD mortality risk of the OSA population. METHODS: This study included 2,464 patients with OSA who met study inclusion criteria and were selected from the Sleep Heart Health Study. We evaluated the importance of potential features by mutual information. The top 9 features were selected to develop a random forest model. RESULTS: We evaluated the model performance on a test set (n = 493) using the area under the receiver operating curve with 95% confidence interval and confusion matrix. A random forest model awarded the highest area under the receiver operating curve of 0.84 (95% confidence interval: 0.78-0.89). The specificity and sensitivity were 73.94% and 81.82%, respectively. Sixty-three years old was a threshold for increased risk of 10-year CVD mortality. Persons with severe OSA had higher risk than those with mild OSA. CONCLUSIONS: This study demonstrated that a random forest model can provide a quick assessment of the risk of 10-year CVD mortality. Our model may be more informative for patients with OSA in determining their future CVD mortality risk. CITATION: Li A, Roveda JM, Powers LS, Quan SF. Obstructive sleep apnea predicts 10-year cardiovascular disease-related mortality in the Sleep Heart Health Study: a machine learning approach. J Clin Sleep Med. 2022;18(2):497-504.

Defining CD4 T helper and T regulatory cell endotypes of progressive and remitting pulmonary sarcoidosis (BRITE): protocol for a US-based, multicentre, longitudinal observational bronchoscopy study.

INTRODUCTION: Sarcoidosis is a multiorgan granulomatous disorder thought to be triggered and influenced by gene-environment interactions. Sarcoidosis affects 45-300/100 000 individuals in the USA and has an increasing mortality rate. The greatest gap in knowledge about sarcoidosis pathobiology is a lack of understanding about the underlying immunological mechanisms driving progressive pulmonary disease. The objective of this study is to define the lung-specific and blood-specific longitudinal changes in the adaptive immune response and their relationship to progressive and non-progressive pulmonary outcomes in patients with recently diagnosed sarcoidosis. METHODS AND ANALYSIS: The BRonchoscopy at Initial sarcoidosis diagnosis Targeting longitudinal Endpoints study is a US-based, NIH-sponsored longitudinal blood and bronchoscopy study. Enrolment will occur over four centres with a target sample size of 80 eligible participants within 18 months of tissue diagnosis. Participants will undergo six study visits over 18 months. In addition to serial measurement of lung function, symptom surveys and chest X-rays, participants will undergo collection of blood and two bronchoscopies with bronchoalveolar lavage separated by 6 months. Freshly processed samples will be stained and flow-sorted for isolation of CD4 +T helper (Th1, Th17.0 and Th17.1) and T regulatory cell immune populations, followed by next-generation RNA sequencing. We will construct bioinformatic tools using this gene expression to define sarcoidosis endotypes that associate with progressive and non-progressive pulmonary disease outcomes and validate the tools using an independent cohort. ETHICS AND DISSEMINATION: The study protocol has been approved by the Institutional Review Boards at National Jewish Hospital (IRB# HS-3118), University of Iowa (IRB# 201801750), Johns Hopkins University (IRB# 00149513) and University of California, San Francisco (IRB# 17-23432). All participants will be required to provide written informed consent. Findings will be disseminated via journal publications, scientific conferences, patient advocacy group online content and social media platforms.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model.

Hepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl- or HCO3- was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl-/HCO3--free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.

A deep learning-based algorithm for detection of cortical arousal during sleep.

STUDY OBJECTIVES: The frequency of cortical arousals is an indicator of sleep quality. Additionally, cortical arousals are used to identify hypopneic events. However, it is inconvenient to record electroencephalogram (EEG) data during home sleep testing. Fortunately, most cortical arousal events are associated with autonomic nervous system activity that could be observed on an electrocardiography (ECG) signal. ECG data have lower noise and are easier to record at home than EEG. In this study, we developed a deep learning-based cortical arousal detection algorithm that uses a single-lead ECG to detect arousal during sleep. METHODS: This study included 1,547 polysomnography records that met study inclusion criteria and were selected from the Multi-Ethnic Study of Atherosclerosis database. We developed an end-to-end deep learning model consisting of convolutional neural networks and recurrent neural networks which: (1) accepted varying length physiological data; (2) directly extracted features from the raw ECG signal; (3) captured long-range dependencies in the physiological data; and (4) produced arousal probability in 1-s resolution. RESULTS: We evaluated the model on a test set (n = 311). The model achieved a gross area under precision-recall curve score of 0.62 and a gross area under receiver operating characteristic curve score of 0.93. CONCLUSION: This study demonstrated the end-to-end deep learning approach with a single-lead ECG has the potential to be used to accurately detect arousals in home sleep tests.

Age-associated B Cells Appear in Patients with Granulomatous Lung Diseases.

Rationale: A subpopulation of B cells (age-associated B cells [ABCs]) is increased in mice and humans with infections or autoimmune diseases. Because depletion of these cells might be valuable in patients with certain lung diseases, the goal was to find out if ABC-like cells were at elevated levels in such patients.Objectives: To measure ABC-like cell percentages in patients with lung granulomatous diseases.Methods: Peripheral blood and BAL cells from patients with sarcoidosis, beryllium sensitivity, or hypersensitivity pneumonitis and healthy subjects were analyzed for the percentage of B cells that were ABC-like, defined by expression of CD11c, low levels of CD21, FcRL 1-5 (Fc receptor-like protein 1-5) expression, and, in some cases, T-bet.Measurements and Main Results: ABC-like cells in blood were at low percentages in healthy subjects and higher percentages in patients with sarcoidosis as well as at high percentages among BAL cells of patients with sarcoidosis, beryllium disease, and hypersensitivity pneumonitis. Treatment of patients with sarcoidosis led to reduced percentages of ABC-like cells in blood.Conclusions: Increased levels of ABC-like cells in patients with sarcoidosis may be useful in diagnosis. The increase in percentage of ABC-like cells in patients with lung granulomatous diseases and decrease in treated patients suggests that depletion of these cells may be valuable.

Sleep patterns and sleep deprivation recorded by actigraphy in 4th-grade and 5th-grade students.

OBJECTIVE: This study investigates sleep patterns of fourth- and fifth-grade students using actigraphy. METHODS: The study included 257 students enrolled in a Southwestern US school district who participated in a novel sleep science curriculum during the Spring 2016-17 and Fall 2017-18 semesters and met the study inclusion criteria. As part of this curriculum, participants underwent 5-7 days of continuous wrist actigraphy and completed an online sleep diary. RESULTS: Approximately two-thirds of the 9-11-year-old fourth- and fifth-grade students slept less than the minimum 9 h per night recommended by both the American Academy of Sleep Medicine/Sleep Research Society and the National Sleep Foundation. The sleep midpoint time on weekends was about 1 h later than on weekdays. There was a significant effect of age on sleep duration. Compared to 9-year old students, a larger proportion of 10-year old students had a sleep duration less than 8.5 h. Boys had shorter sleep duration than girls, and a larger percentage of boys obtained less than 9 h of sleep compared to girls. CONCLUSIONS: Insufficient sleep is a highly prevalent condition among 9-11-year-old fourth- and fifth-grade elementary students. Importantly, there is a difference between sleep patterns on weekdays and weekends which may portend greater problems with sleep in adolescence and young adulthood.

Subpontic Osseous Hyperplasia: Comprehensive Review of the Literature and Presentation of New Case History.

PURPOSE: To increase awareness of subpontic osseous hyperplasia (SOH), an uncommon benign mass found underneath the pontics of fixed partial dentures (FPDs) and occasionally in implant-supported dental prostheses. MATERIALS AND METHODS: A PubMed search in the English-language literature was conducted for case reports and case series of SOH. Demographic information gleaned from these publications included patient age and gender, lesion sites, outcomes, comorbidities, symptomatology, and periodontal involvement. To exemplify the findings of SOH, a clinical investigation of a 73-year-old affected woman has been detailed. RESULTS/CONCLUSION: With the inclusion of this featured case, 71 patients with 80 affected sites were identified with SOH and served as the basis for the provided database. To date, this aggregation of cases represents the largest collection to undergo clinicopathologic review. SOHs appeared as dome-shaped radiopacities and tended to exhibit increased osteosclerosis with increased duration. The average age at discovery was 57 years, and SOH was found somewhat more often in women. Lesions were more likely to occur in the left posterior mandible. Affected patients may experience increased difficulty maintaining adequate oral hygiene, potentially leading to periodontal disease or discomfort. Overgrowths should be surgically removed when satisfactory oral hygiene measures have been compromised, when there are atypical clinical or radiographic presentations, or with incident symptomatology.

Single-molecule long-read sequencing reveals the chromatin basis of gene expression.

Genome-wide chromatin accessibility and nucleosome occupancy profiles have been widely investigated, while the long-range dynamics remain poorly studied at the single-cell level. Here, we present a new experimental approach, methyltransferase treatment followed by single-molecule long-read sequencing (MeSMLR-seq), for long-range mapping of nucleosomes and chromatin accessibility at single DNA molecules and thus achieve comprehensive-coverage characterization of the corresponding heterogeneity. MeSMLR-seq offers direct measurements of both nucleosome-occupied and nucleosome-evicted regions on a single DNA molecule, which is challenging for many existing methods. We applied MeSMLR-seq to haploid yeast, where single DNA molecules represent single cells, and thus we could investigate the combinatorics of many (up to 356) nucleosomes at long range in single cells. We illustrated the differential organization principles of nucleosomes surrounding the transcription start site for silent and actively transcribed genes, at the single-cell level and in the long-range scale. The heterogeneous patterns of chromatin status spanning multiple genes were phased. Together with single-cell RNA-seq data, we quantitatively revealed how chromatin accessibility correlated with gene transcription positively in a highly heterogeneous scenario. Moreover, we quantified the openness of promoters and investigated the coupled chromatin changes of adjacent genes at single DNA molecules during transcription reprogramming. In addition, we revealed the coupled changes of chromatin accessibility for two neighboring glucose transporter genes in response to changes in glucose concentration.

Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus.

Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30-50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.

Evaluation of Well Designs to Improve Access to Safe and Clean Water in Rural Tanzania.

The objective of this study was to examine three well designs: drilled wells (20-30 m deep), closed dug wells (>5 m deep), and hand-dug open wells (<5 m deep), to determine the water quality for improving access to safe and clean water in rural communities. Heterotrophic plate count (HPC), total coliforms (TC), Escherichia coli (E. coli) and turbidity, were used to assess the water quality of 97 wells. Additionally, the study looked at the microflora diversity of the water, focusing on potential pathogens using outgrowth, PCR, and genome sequencing for 10 wells. Concentrations of TC for the open dug wells (4 × 104 CFU/100 mL) were higher than the drilled (2 × 10³ CFU/100 mL) and closed dug wells (3 × 10³ CFU/100 mL). E. coli concentration for drilled and closed dug wells was <22 MPN (most probable number)/100 mL, but higher for open wells (>154 MPN/100 mL). The drilled well turbidity (11 NTU) was within the standard deviation of the closed well (28 NTU) compared to open dug wells (49 NTU). Drilled and closed wells had similar microbial diversity. There were no significant differences between drilled and closed dug wells. The covering and lining of hand-dug wells should be considered as an alternative to improve access to safe and clean water in rural communities.

Engineering safer care coordination from hospital to home: lessons from the USA.

The safe transition of a patient from hospital into the community requires effective coordination between healthcare professionals across organisational boundaries. Preventing transition-associated failures can be especially challenging when multiple disciplines are involved and the patient has extensive care needs. The field of systems engineering is increasingly recognised as useful to help understand, improve and redesign such complex healthcare processes to improve patient experience and outcomes. To illustrate this approach, we describe how a partnership between healthcare professionals, systems engineers, and health services researchers used a series of engineering methods at a large suburban hospital to analyse and address deficiencies in a hospital-to-home transition process. Using this approach, the team designed a new process to perform more reliably despite inherent system complexity, demonstrating the value of systems engineering approaches and clinician-engineer collaborations.

Postnatal airway growth in cystic fibrosis piglets.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel cause CF. The leading cause of death in the CF population is lung disease. Increasing evidence suggests that in utero airway development is CFTR-dependent and that developmental abnormalities may contribute to CF lung disease. However, relatively little is known about postnatal CF airway growth, largely because such studies are limited in humans. Therefore, we examined airway growth and lung volume in a porcine model of CF. We hypothesized that CF pigs would have abnormal postnatal airway growth. To test this hypothesis, we performed CT-based airway and lung volume measurements in 3-wk-old non-CF and CF pigs. We found that 3-wk-old CF pigs had tracheas of reduced caliber and irregular shape. Their bronchial lumens were reduced in size proximally but not distally, were irregularly shaped, and had reduced distensibility. Our data suggest that lack of CFTR results in aberrant postnatal airway growth and development, which could contribute to CF lung disease pathogenesis.NEW & NOTEWORTHY This CT scan-based study of airway morphometry in the cystic fibrosis (CF) postnatal period is unique, as analogous studies in humans are greatly limited for ethical and technical reasons. Findings such as reduced airway lumen area and irregular caliber suggest that airway growth and development are CF transmembrane conductance regulator-dependent and that airway growth defects may contribute to CF lung disease pathogenesis.

Gel-forming mucins form distinct morphologic structures in airways.

Gel-forming mucins, the primary macromolecular components of airway mucus, facilitate airway clearance by mucociliary transport. In cystic fibrosis (CF) altered mucus properties impair mucociliary transport. Airways primarily secrete two closely related gel-forming mucins, MUC5B and MUC5AC. However, their morphologic structures and associations in airways that contain abundant submucosal glands and goblet cells are uncertain. Moreover, there is limited knowledge about mucins in airways not affected by inflammation, infection, or remodeling or in CF airways. Therefore, we examined airways freshly excised from newborn non-CF pigs and CF pigs before secondary manifestations develop. We found that porcine submucosal glands produce MUC5B, whereas goblet cells produce predominantly MUC5AC plus some MUC5B. We found that MUC5B emerged from submucosal gland ducts in the form of strands composed of multiple MUC5B filaments. In contrast, MUC5AC emerged from goblet cells as wispy threads and sometimes formed mucin sheets. In addition, MUC5AC often partially coated the MUC5B strands. Compared with non-CF, MUC5B more often filled CF submucosal gland ducts. MUC5AC sheets also accumulated in CF airways overlying MUC5B strands. These results reveal distinct morphology and interactions for MUC5B and MUC5AC and suggest that the two mucins make distinct contributions to mucociliary transport. Thus, they provide a framework for understanding abnormalities in disease.

Acid-Sensing Ion Channel 1a Contributes to Airway Hyperreactivity in Mice.

Neurons innervating the airways contribute to airway hyperreactivity (AHR), a hallmark feature of asthma. Several observations suggested that acid-sensing ion channels (ASICs), neuronal cation channels activated by protons, might contribute to AHR. For example, ASICs are found in vagal sensory neurons that innervate airways, and asthmatic airways can become acidic. Moreover, airway acidification activates ASIC currents and depolarizes neurons innervating airways. We found ASIC1a protein in vagal ganglia neurons, but not airway epithelium or smooth muscle. We induced AHR by sensitizing mice to ovalbumin and found that ASIC1a-/- mice failed to exhibit AHR despite a robust inflammatory response. Loss of ASIC1a also decreased bronchoalveolar lavage fluid levels of substance P, a sensory neuropeptide secreted from vagal sensory neurons that contributes to AHR. These findings suggest that ASIC1a is an important mediator of AHR and raise the possibility that inhibiting ASIC channels might be beneficial in asthma.