Tobacco smoking and somatic mutations in human bronchial epithelium.

Tobacco smoking causes lung cancer1-3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6-10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0-6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Suspension laryngoscopy experiences in a tertiary airway service: A prospective study of 150 procedures.

OBJECTIVES: In most cases, suspension laryngoscopy (SL) is efficient, bloodless and with minimal post-procedure discomfort. We aimed to identify predictive patient factors for acceptable surgical views at SL as well as quantify our tertiary airway unit's complication rates. DESIGN: Prospective cohort study of 150 consecutive microlaryngoscopy procedures involving SL over an 8-month period between November 2019 and July 2020. Patients were assessed preoperatively for pre-existing oral, temporomandibular, dental, pharyngeal or laryngeal pathology, interincisor distance and qualitative gross limitations to neck extension and forward head posture. Intraoperatively, the laryngoscopic view was graded by anaesthetic and surgical teams, and complications were recorded on patient interview in recovery. SETTING: Tertiary adult airway service for predominantly benign pathology. RESULTS: Adequate surgical views were obtained in 149/150 procedures. BMI had a weak positive correlation with a more difficult view (r = .22, p = .008) but did not correlate with a statistically significant increase in any complication. There was a weak negative correlation between age and interincisor gap (r = -.20, p = .014), and wider mouth opening correlated very weakly with a lower incidence of sore throat (r = -.19, p = .023). Gross macroglossia showed a significant moderate positive correlation with tongue symptoms (r = .45, p = 1.611 × 10-8 ). CONCLUSION: In the context of an experienced airway unit with a high caseload of predominantly benign pathology, SL is very effective and safe with low associated morbidity and no mortality. The most common complication of SL is temporary sore throat and there remain recognised risks of temporary tongue and dental symptoms.

Sarcoidosis of the ear, nose and throat: A review of the literature.

OBJECTIVES: Sarcoidosis is a multisystemic inflammatory disease with extrathoracic manifestations, most commonly affecting the young and middle-aged, female and Black populations. Diagnosis usually requires evidence of non-caseating granulomata and, when treated, prognosis is usually favourable. We aim to establish the incidence, clinical features and optimal treatment of ENT manifestations of this disease. DESIGN: We performed a PubMed literature review to determine the evidence base supporting this. RESULTS: ENT manifestations are present in 5%-15% of patients with sarcoidosis, often as a presenting feature, and require vigilance for swift recognition and coordinated additional treatment specific to the organ. Laryngeal sarcoidosis presents with difficulty in breathing, dysphonia and cough, and may be treated by speech and language therapy (SLT) or intralesional injection, dilatation or tissue reduction. Nasal disease presents with crusting, rhinitis, nasal obstruction and anosmia, usually without sinus involvement. It is treated by topical nasal or intralesional treatments but may also require endoscopic sinus surgery, laser treatment or even nasal reconstruction. Otological disease is uncommon but includes audiovestibular symptoms, both sensorineural and conductive hearing loss, and skin lesions. CONCLUSIONS: The consequences of ENT manifestations of sarcoidosis can be uncomfortable, disabling and even life-threatening. Effective management strategies require good diagnostic skills and use of specific therapies combined with established treatments such as corticosteroids. Comparisons of treatment outcomes are needed to establish best practice in this area.

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Lung viral infection modelling in a bioengineered whole-organ.

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

Lessons learned from pre-clinical testing of xenogeneic decellularized esophagi in a rabbit model.

Decellularization of esophagi from several species for tissue engineering is well described, but successful implantation in animal models of esophageal replacement has been challenging. The purpose of this study was to assess feasibility and applicability of esophageal replacement using decellularized porcine esophageal scaffolds in a new pre-clinical model. Following surgical replacement in rabbits with a vascularizing muscle flap, we observed successful anastomoses of decellularized scaffolds, cues of early neovascularization, and prevention of luminal collapse by the use of biodegradable stents. However, despite the success of the surgical procedure, the long-term survival was limited by the fragility of the animal model. Our results indicate that transplantation of a decellularized porcine scaffold is possible and vascular flaps may be useful to provide a vascular supply, but long-term outcomes require further pre-clinical testing in a different large animal model.

Cell-intrinsic differences between human airway epithelial cells from children and adults.

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

Primary Immunoglobulin G4-Related Laryngeal Disease: A Case Series and Review of Literature.

OBJECTIVE: Immunoglobulin G4-related disease (IgG4-RD) is an increasingly recognised cause of various systemic fibro-inflammatory conditions. However, laryngeal involvement as a primary feature is extremely rare. We aimed to report on a case series of such patients and examine the global literature relating to laryngeal involvement. METHODS: Having previously reported a case of IgG4-RD laryngeal pseudotumour, we describe a case series of further 4 patients with primary laryngeal IgG4-RD managed by our UK quaternary airway service and provide a brief overview of laryngeal IgG4-RD. RESULTS: Including our cases, 14 cases of primary laryngeal IgG4-RD have been reported. Vocal cord involvement is relatively uncommon. Repeat biopsies may be required to achieve histological diagnosis. Remission is achievable by commencement of immunomodulatory treatment, following which laryngeal reconstruction may be necessary. CONCLUSION: Laryngeal involvement is a rare presentation of IgG4-RD, itself a rare and difficult-to-diagnose condition. A high and prolonged index of suspicion is necessary from both surgical and pathological specialists for correct diagnosis and management.

Ex utero intrapartum treatment (EXIT) for upper airway obstruction.

PURPOSE OF REVIEW: Improvements in the antenatal diagnosis of congenital malformations have led to increased detection of fetal airway obstructing lesions, and pediatric ear, nose, and throat surgeons are increasingly involved in these cases. RECENT FINDINGS: This article outlines the typical range of pathology seen, the logistics in providing support for anticipated deliveries and the multidisciplinary management of complex airway cases. SUMMARY: Traditionally, difficulty in obtaining a patent airway at delivery was a major factor in the dismal prognosis of these pregnancies. The ex utero intrapartum treatment procedure, which involves controlled partial delivery of the fetus whilst maintaining placental circulation, allows various airway maneuvers to be performed to secure the airway in a controlled fashion.

Autologous Cell Seeding in Tracheal Tissue Engineering.

PURPOSE OF REVIEW: There is no consensus on the best technology to be employed for tracheal replacement. One particularly promising approach is based upon tissue engineering and involves applying autologous cells to transplantable scaffolds. Here, we present the reported pre-clinical and clinical data exploring the various options for achieving such seeding. RECENT FINDINGS: Various cell combinations, delivery strategies, and outcome measures are described. Mesenchymal stem cells (MSCs) are the most widely employed cell type in tracheal bioengineering. Airway epithelial cell luminal seeding is also widely employed, alone or in combination with other cell types. Combinations have thus far shown the greatest promise. Chondrocytes may improve mechanical outcomes in pre-clinical models, but have not been clinically tested. Rapid or pre-vascularization of scaffolds is an important consideration. Overall, there are few published objective measures of post-seeding cell viability, survival, or overall efficacy. SUMMARY: There is no clear consensus on the optimal cell-scaffold combination and mechanisms for seeding. Systematic in vivo work is required to assess differences between tracheal grafts seeded with combinations of clinically deliverable cell types using objective outcome measures, including those for functionality and host immune response.

A comparison of tracheal scaffold strategies for pediatric transplantation in a rabbit model.

OBJECTIVES/HYPOTHESIS: Despite surgical advances, childhood tracheal stenosis is associated with high morbidity and mortality. Various tracheal scaffold strategies have been developed as the basis for bioengineered substitutes, but there is no consensus on which may be superior in vivo. We hypothesized that there would be no difference in morbidity and mortality between three competing scaffold strategies in rabbits. STUDY DESIGN: Pilot preclinical study. METHODS: Tracheal scaffolds were prepared by three methods that have been applied clinically and reported: preserved cadaveric ("Herberhold") allografts, detergent-enzymatically decellularized allografts, and synthetic scaffolds (nanocomposite polymer [polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU)]). Scaffolds were implanted into cervical trachea of New Zealand White rabbits (n = 4 per group) without cell seeding. Control animals (n = 4) received autotransplanted tracheal segments using the same technique. Animals underwent bronchoscopic monitoring of the grafts for 30 days. Macroscopic evaluation of tissue integration, graft stenosis, and collapsibility and histological examinations were performed on explants at termination. RESULTS: All surgical controls survived to termination without airway compromise. Mild to moderate anastomotic stenosis from granulation tissue was detected, but there was evidence suggestive of vascular reconnection with minimal fibrous encapsulation. In contrast, three of the four animals in the Herberhold and POSS-PCU groups, and all animals receiving decellularized allografts, required early termination due to respiratory distress. Herberhold grafts showed intense inflammatory reactions, anastomotic stenoses, and mucus plugging. Synthetic graft integration and vascularization were poor, whereas decellularized grafts demonstrated malacia and collapse but had features suggestive of vascular connection or revascularization. CONCLUSIONS: There are mirror-image benefits and drawbacks to nonrecellularized, decellularized, and synthetic grafts, such that none emerged as the preferred option. Results from prevascularized and/or cell-seeded grafts (as applied clinically) may elucidate clearer advantages of one scaffold type over another. LEVEL OF EVIDENCE: NA. Laryngoscope, 127:E449-E457, 2017.

Congenital tracheobronchial stenosis.

Congenital tracheobronchial stenosis is a rare disease characterized by complete tracheal rings that can affect variable lengths of the tracheobronchial tree. It causes high levels of morbidity and mortality both due to the stenosis itself and to the high incidence of other associated congenital malformations. Successful management of this complex condition requires a highly individualized approach delivered by an experienced multidisciplinary team, which is best delivered within centralized units with the necessary diverse expertise. In such settings, surgical correction by slide tracheoplasty has become increasingly successful over the past 2 decades such that long-term survival now exceeds 88%, with normalization of quality of life scores for patients with non-syndrome-associated congenital tracheal stenosis. Careful assessment and planning of treatment strategies is of paramount importance for both successful management and the provision of patients and carers with accurate and realistic treatment counseling.

Heterotopic ossification in chronic fibrosing otitis externa.

Acquired external auditory canal atresia is a rare complication of chronic inflammatory otitis, and is generally fibrous or soft tissue in nature. Here, we present the first reported case of heterotopic ossification within chronic fibrosing otitis externa in a 25-year-old male patient with a childhood history of granular myringitis and failed tympanoplasty. A calcified mass was demonstrated adjacent to the tympanic membrane on CT imaging, and surgical exploration revealed a cohesive bar of bone traversing the medial external auditory canal. Drill canaloplasty and split-thickness skin graft coverage of the lateral tympanic membrane resulted in an improvement in the pure tone average from 79 to 55 dB. As the treatment for chronic fibrosing otitis externa involves the surgical widening of the external auditory canal, we alert surgeons to the possibility of cohesive bone formation as a potential cause of navigational confusion and inadvertent over- or under-drilling of the canal stenosis.