Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Inflammatory Activity of Epithelial Stem Cell Variants from Cystic Fibrosis Lungs Is Not Resolved by CFTR Modulators.

Rationale: CFTR (cystic fibrosis transmembrane conductance regulator) modulator drugs restore function to mutant channels in patients with cystic fibrosis (CF) and lead to improvements in body mass index and lung function. Although it is anticipated that early childhood treatment with CFTR modulators will significantly delay or even prevent the onset of advanced lung disease, lung neutrophils and inflammatory cytokines remain high in patients with CF with established lung disease despite modulator therapy, underscoring the need to identify and ultimately target the sources of this inflammation in CF lungs. Objectives: To determine whether CF lungs, like chronic obstructive pulmonary disease (COPD) lungs, harbor potentially pathogenic stem cell "variants" distinct from the normal p63/Krt5 lung stem cells devoted to alveolar fates, to identify specific variants that might contribute to the inflammatory state of CF lungs, and to assess the impact of CFTR genetic complementation or CFTR modulators on the inflammatory variants identified herein. Methods: Stem cell cloning technology developed to resolve pathogenic stem cell heterogeneity in COPD and idiopathic pulmonary fibrosis lungs was applied to end-stage lungs of patients with CF (three homozygous CFTR:F508D, one CFTR F508D/L1254X; FEV1, 14-30%) undergoing therapeutic lung transplantation. Single-cell-derived clones corresponding to the six stem cell clusters resolved by single-cell RNA sequencing of these libraries were assessed by RNA sequencing and xenografting to monitor inflammation, fibrosis, and mucin secretion. The impact of CFTR activity on these variants after CFTR gene complementation or exposure to CFTR modulators was assessed by molecular and functional studies. Measurements and Main Results: End-stage CF lungs display a stem cell heterogeneity marked by five predominant variants in addition to the normal lung stem cell, of which three are proinflammatory both at the level of gene expression and their ability to drive neutrophilic inflammation in xenografts in immunodeficient mice. The proinflammatory functions of these three variants were unallayed by genetic or pharmacological restoration of CFTR activity. Conclusions: The emergence of three proinflammatory stem cell variants in CF lungs may contribute to the persistence of lung inflammation in patients with CF with advanced disease undergoing CFTR modulator therapy.

Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury.

Cartilaginous airways of larger mammals and the mouse trachea contain at least 3 well-established stem cell compartments, including basal cells of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline with age in mice. Notably, Sox9-lineage glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair, and its deletion significantly reduced proliferation following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation of progenitors in both the SAE and SMG shortly following injury, but these progenitors failed to proliferate in vitro in the absence of Sox9, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not surface airway, progenitors. We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively.

Challenges, highlights, and opportunities in cellular transplantation: A white paper of the current landscape.

Although cellular transplantation remains a relatively small field compared to solid organ transplantation, the prospects for advancement in basic science and clinical care remain bountiful. In this review, notable historical events and the current landscape of the field of cellular transplantation are reviewed with an emphasis on islets (allo- and xeno-), hepatocytes (including bioartificial liver), adoptive regulatory immunotherapy, and stem cells (SCs, specifically endogenous organ-specific and mesenchymal). Also, the nascent but rapidly evolving field of three-dimensional bioprinting is highlighted, including its major processing steps and latest achievements. To reach its full potential where cellular transplants are a more viable alternative than solid organ transplants, fundamental change in how the field is regulated and advanced is needed. Greater public and private investment in the development of cellular transplantation is required. Furthermore, consistent with the call of multiple national transplant societies for allo-islet transplants, the oversight of cellular transplants should mirror that of solid organ transplants and not be classified under the unsustainable, outdated model that requires licensing as a drug with the Food and Drug Administration. Cellular transplantation has the potential to bring profound benefit through progress in bioengineering and regenerative medicine, limiting immunosuppression-related toxicity, and providing markedly reduced surgical morbidity.

Repair of iatrogenic tracheal injury in acute respiratory failure with veno-venous extracorporeal membrane oxygenation.

Iatrogenic tracheal injuries are rare but potentially serious complications of endotracheal intubation that frequently require lung isolation to repair. This is not tolerated in patients with severe respiratory failure. We describe a case in a patient with acute respiratory distress syndrome, repaired using veno-venous extracorporeal membrane oxygenation.

Stem cells and lung regeneration.

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.

Derivation of induced pluripotent stem cells from ferret somatic cells.

Ferrets are an attractive mammalian model for several diseases, especially those affecting the lungs, liver, brain, and kidneys. Many chronic human diseases have been difficult to model in rodents due to differences in size and cellular anatomy. This is particularly the case for the lung, where ferrets provide an attractive mammalian model of both acute and chronic lung diseases, such as influenza, cystic fibrosis, A1A emphysema, and obliterative bronchiolitis, closely recapitulating disease pathogenesis, as it occurs in humans. As such, ferrets have the potential to be a valuable preclinical model for the evaluation of cell-based therapies for lung regeneration and, likely, for other tissues. Induced pluripotent stem cells (iPSCs) provide a great option for provision of enough autologous cells to make patient-specific cell therapies a reality. Unfortunately, they have not been successfully created from ferrets. In this study, we demonstrate the generation of ferret iPSCs that reflect the primed pluripotent state of human iPSCs. Ferret fetal fibroblasts were reprogrammed and acquired core features of pluripotency, having the capacity for self-renewal, multilineage differentiation, and a high-level expression of the core pluripotency genes and pathways at both the transcriptional and protein level. In conclusion, we have generated ferret pluripotent stem cells that provide an opportunity for advancing our capacity to evaluate autologous cell engraftment in ferrets.

Preoperative Patient Expectations of Postoperative Pain Are Associated with Moderate to Severe Acute Pain After VATS.

OBJECTIVE: The goal of this post hoc analysis of subjects from a prospective observational study was to identify the predictors of patients developing moderate to severe acute pain (mean numerical rating scale [NRS] ≥4, 0-10) during the first three days after video-assisted thoracoscopic surgery (VATS) from a comprehensive evaluation of demographic, psychosocial, and surgical factors. METHODS: Results from 82 patients who were enrolled one week before VATS and evaluated during the first three postoperative days are presented. The primary outcome variable of the current study was the presence of moderate to severe acute pain after VATS. RESULTS: Fifty-nine percent (95% confidence interval, 47-69%) of study subjects developed moderate to severe acute pain after VATS. Factors univariately associated with the presence of moderate to severe acute pain were greater average expected postoperative pain, greater pain to a suprathreshold cold stimulus, and longer durations of surgery and hospital stay (P < 0.05). When considered in the multiple logistic regression models, the patients' preoperative average intensity of expected postoperative pain (NRS, 0-10) was the only measure associated with the moderate to severe acute pain. Average intensity of postoperative pain expected by patients when questioned preoperatively mediated the effect of reported intensity of pain to the suprathreshold cold stimulus for moderate to severe acute pain levels. Preoperative patient expectations had greater predictive value than other assessed variables including psychosocial factors such as catastrophizing or anxiety assessed one week before surgery. CONCLUSIONS: None of the preoperative psychosocial measures were associated with the moderate to severe acute pain after VATS. Average expected postoperative pain was the only measure associated with the development of moderate to severe acute pain after VATS.

Depletion of Airway Submucosal Glands and TP63+KRT5+ Basal Cells in Obliterative Bronchiolitis.

RATIONALE: Obliterative bronchiolitis (OB) is a major cause of mortality after lung transplantation. Depletion of airway stem cells (SCs) may lead to fibrosis in OB. OBJECTIVES: Two major SC compartments in airways are submucosal glands (SMGs) and surface airway p63 (also known as TP63 [tumor protein 63])-positive/K5 (also known as KRT5 [keratin 5])-positive basal cells (BCs). We hypothesized that depletion of these SC compartments occurs in OB. METHODS: Ferret orthotopic left lung transplants were used as an experimental model of OB, and findings were corroborated in human lung allografts. Morphometric analysis was performed in ferret and human lungs to evaluate the abundance of SMGs and changes in the expression of phenotypic BC markers in control, lymphocytic bronchiolitis, and OB airways. The abundance and proliferative capacity of proximal and distal airway SCs was assessed using a clonogenic colony-forming efficiency assay. MEASUREMENTS AND MAIN RESULTS: Ferret allografts revealed significant loss of SMGs with development of OB. A progressive decline in p63+/K5+ and increase in K5+/K14+ and K14+ BC phenotypes correlated with the severity of allograft rejection in large and small ferret airways. The abundance and proliferative capacity of basal SCs in large allograft airways declined with severity of OB, and there was complete ablation of basal SCs in distal OB airways. Human allografts mirrored phenotypic BC changes observed in the ferret model. CONCLUSIONS: SMGs and basal SC compartments are depleted in large and/or small airways of lung allografts, and basal SC proliferative capacity declines with progression of disease and phenotypic changes. Global airway SC depletion may be a mechanism for pulmonary allograft failure.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

RATIONALE: Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. OBJECTIVES: To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. METHODS: CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. MEASUREMENTS AND MAIN RESULTS: Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. CONCLUSIONS: These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

Heterogeneity of Pulmonary Stem Cells.

Epithelial stem cells reside within multiple regions of the lung where they renew various region-specific cells. In addition, there are multiple routes of regeneration after injury through built-in heterogeneity within stem cell populations and through a capacity for cellular plasticity among differentiated cells. These processes are important facets of respiratory tissue resiliency and organism survival. However, this regenerative capacity is not limitless, and repetitive or chronic injuries, environmental stresses, or underlying factors of disease may ultimately lead to or contribute to tissue remodeling and end-stage lung disease. This chapter will review stem cell heterogeneity among pulmonary epithelia in the lower respiratory system, discuss recent findings that may challenge long-held scientific paradigms, and identify several clinically relevant research opportunities for regenerative medicine.

Per oral endoscopic myotomy: early experience and safety of a multispecialty approach.

BACKGROUND: Per oral endoscopic myotomy (POEM) has gained increasing popularity for treating achalasia. A multidisciplinary approach may allow safe and early adoption of POEM into clinical practice. MATERIALS AND METHODS: We performed a retrospective review of our initial POEM cases. All procedures were performed by a team of interventional gastroenterologist and thoracic surgeon. We analyzed demographics, comorbidities, achalasia subtypes, length of hospital stay, duration of surgery, morbidity, mortality, length of myotomy, preoperative and postoperative Eckardt scores. RESULTS: Thirty-one consecutive patients underwent POEM during the 24-month period from January 2014 to December 2015. Eighteen patients (58%) had prior non-operative interventions. Average duration of follow-up was 9.6 months. Seventeen patients (66.8%) had follow-up of 12 months and longer. Average preoperative Eckardt score was 6.3 (3-10), median 6. Average postoperative Eckardt score was 1.4 (0-8), median 1, in 1 month and an average 2.2, median 1, in 1 year. Patients with type III achalasia were most refractory to treatment, while patients with type II had the best results. Average LOS was 1.3 days (1-5), median 1 day. Average DOS was 106 min (60-148), median 106. Average LOM was 13 cm (10-15), with median of 13 cm. We had one 30-day mortality secondary to coronary artery disease. Four patients had prior Heller myotomies and underwent a posterior myotomy during POEM, with outcomes similar to patients with no prior myotomy. CONCLUSIONS: We demonstrated safety and efficiency of a multispecialty approach for achalasia with POEM with a low rate of complications.

SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl- conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl- transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl- conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

A Prospective Study of Chronic Pain after Thoracic Surgery.

BACKGROUND: The goal of this study was to detect the predictors of chronic pain at 6 months after thoracic surgery from a comprehensive evaluation of demographic, psychosocial, and surgical factors. METHODS: Thoracic surgery patients were enrolled 1 week before surgery and followed up 6 months postsurgery in this prospective, observational study. Comprehensive psychosocial measurements were assessed before surgery. The presence and severity of pain were assessed at 3 and 6 months after surgery. One hundred seven patients were assessed during the first 3 days after surgery, and 99 (30 thoracotomy and 69 video-assisted thoracoscopic surgery, thoracoscopy) patients completed the 6-month follow-up. Patients with versus without chronic pain related to thoracic surgery at 6 months were compared. RESULTS: Both incidence (P = 0.37) and severity (P = 0.97) of surgery-related chronic pain at 6 months were similar after thoracotomy (33%; 95% CI, 17 to 53%; 3.3 ± 2.1) and thoracoscopy (25%; 95% CI, 15 to 36%; 3.3 ± 1.7). Both frequentist and Bayesian multivariate models revealed that the severity of acute pain (numerical rating scale, 0 to 10) is the measure associated with chronic pain related to thoracic surgery. Psychosocial factors and quantitative sensory testing were not predictive. CONCLUSIONS: There was no difference in the incidence and severity of chronic pain at 6 months in patients undergoing thoracotomy versus thoracoscopy. Unlike other postsurgical pain conditions, none of the preoperative psychosocial measurements were associated with chronic pain after thoracic surgery.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 µg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets.

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

Sox2 modulates Lef-1 expression during airway submucosal gland development.

Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/ß-catenin-dependent induction of lymphoid enhancer factor (Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred posttranscriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/ß-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing ß-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.

Human cystic fibrosis airway epithelia have reduced Cl- conductance but not increased Na+ conductance.

Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.

Resection of the innominate artery to prevent an impending tracheoinnominate fistula and to permit tracheotomy in a patient with subglottic stenosis and high-riding innominate.

OBJECTIVE: This study aimed to determine the long-term viability of innominate artery resection and tracheotomy for a patient at high risk of developing a tracheoinnominate fistula (TIF) in the setting of subglottic stenosis and a high-riding innominate artery. METHODS: Chart review with 2-year follow-up. RESULTS: A 45-year-old diabetic man with obstructive sleep apnea and multiple admissions for coma and delirium tremens associated with alcohol abuse developed subglottic stenosis. He was found to have a palpable supraclavicular pulse during preoperative examination for a tracheotomy. Computed tomography examination revealed a high-riding innominate artery at the level of stenosis along with granulation tissue and disruption of the cartilaginous trachea, suggesting a high risk of impending TIF. The patient underwent a sternotomy-approach resection of the innominate artery with closure of the distal stump with a sternohyoid muscle flap. Intraoperatively, a plane of adhesions between the posterior innominate artery and trachea was dissected. The anterior tracheal wall appeared calcified but without evidence of erosion of either the trachea or the artery. Six weeks later, a tracheotomy was performed. Follow-up at 27 months did not identify complications from the innominate artery resection. CONCLUSION: Resection of the innominate artery is an option for some patients either to address the warning signs of TIF or to permit a tracheotomy to be performed in the presence of a high innominate artery.