Post-Implantation Inflammatory Responses to Xenogeneic Tissue-Engineered Cartilage Implanted in Rabbit Trachea: The Role of Cultured Chondrocytes in the Modification of Inflammation.

Immune responses to tissue-engineered grafts made of xenogeneic materials remain poorly studied. The scope of current investigations is limited by the lack of information on orthotopically implanted grafts. A deeper understanding of these processes is of great importance since innovative surgical approaches include the implantation of xenogeneic decellularized scaffolds seeded by cells. The purpose of our work is to study the immunological features of tracheal repair during the implantation of tissue-engineered constructs based on human xenogeneic scaffolds modified via laser radiation in rabbits. The samples were stained with hematoxylin and Safranin O, and they were immunostained with antibodies against tryptase, collagen II, vimentin, and CD34. Immunological and inflammatory responses were studied by counting immune cells and evaluating blood vessels and collagen. Leukocyte-based inflammation prevailed during the implantation of decellularized unseeded scaffolds; meanwhile, plasma cells were significantly more abundant in tissue-engineered constructs. Mast cells were insignificantly more abundant in tissue-engineered construct samples. Conclusions: The seeding of decellularized xenogeneic cartilage with chondrocytes resulted in a change in immunological reactions upon implantation, and it was associated with plasma cell infiltration. Tissue-engineered grafts widely differed in design, including the type of used cells. The question of immunological response depending on the tissue-engineered graft composition requires further investigation.

Analysis of miRNA expression in the trachea of Ri chicken infected with the highly pathogenic avian influenza H5N1 virus.

BACKGROUND: Highly pathogenic avian influenza virus (HPAIV) is considered a global threat to both human health and the poultry industry. MicroRNAs (miRNA) can modulate the immune system by affecting gene expression patterns in HPAIV-infected chickens. OBJECTIVES: To gain further insights into the role of miRNAs in immune responses against H5N1 infection, as well as the development of strategies for breeding disease-resistant chickens, we characterized miRNA expression patterns in tracheal tissues from H5N1-infected Ri chickens. METHODS: miRNAs expression was analyzed from two H5N1-infected Ri chicken lines using small RNA sequencing. The target genes of differentially expressed (DE) miRNAs were predicted using miRDB. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were then conducted. Furthermore, using quantitative real-time polymerase chain reaction, we validated the expression levels of DE miRNAs (miR-22-3p, miR-146b-3p, miR-27b-3p, miR-128-3p, miR-2188-5p, miR-451, miR-205a, miR-203a, miR-21-3p, and miR-200a-3p) from all comparisons and their immune-related target genes. RESULTS: A total of 53 miRNAs were significantly expressed in the infection samples of the resistant compared to the susceptible line. Network analyses between the DE miRNAs and target genes revealed that DE miRNAs may regulate the expression of target genes involved in the transforming growth factor-beta, mitogen-activated protein kinase, and Toll-like receptor signaling pathways, all of which are related to influenza A virus progression. CONCLUSIONS: Collectively, our results provided novel insights into the miRNA expression patterns of tracheal tissues from H5N1-infected Ri chickens. More importantly, our findings offer insights into the relationship between miRNA and immune-related target genes and the role of miRNA in HPAIV infections in chickens.

Wnt signaling regulates ion channel expression to promote smooth muscle and cartilage formation in developing mouse trachea.

Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. Tracheobronchomalacia (TBM) and complete tracheal rings (CTR) are disorders affecting the muscle and cartilage of the trachea and bronchi, whose etiology remains poorly understood. We demonstrated that trachealis muscle organization and polarity are disrupted after epithelial ablation of Wntless (Wls), a cargo receptor critical for the Wnt signaling pathway, in developing trachea. The phenotype resembles the anomalous trachealis muscle observed after deletion of ion channel encoding genes in developing mouse trachea. We sought to investigate whether and how the deletion of Wls affects ion channels during tracheal development. We hypothesize that Wnt signaling influences the expression of ion channels to promote trachealis muscle cell assembly and patterning. Deleting Wls in developing trachea causes differential regulation of genes mediating actin binding, cytoskeleton organization, and potassium ion channel activity. Wnt signaling regulates the expression of Kcnj13, Kcnd3, Kcnj8, and Abcc9 as demonstrated by in vitro studies and in vivo analysis in Wnt5a and ß-catenin-deficient tracheas. Pharmacological inhibition of potassium ion channels and Wnt signaling impaired contractility of developing trachealis smooth muscle and formation of cartilaginous mesenchymal condensation. Thus, in mice, epithelial-induced Wnt/ß-catenin signaling mediates trachealis muscle and cartilage development via modulation of ion channel expression, promoting trachealis muscle architecture, contractility, and cartilaginous extracellular matrix. In turn, ion channel activity may influence tracheal morphogenesis underlying TBM and CTR.NEW & NOTEWORTHY Ion channels play critical roles in the physiology and function of the nervous system and contractile tissue; however, their role in noncontractile tissue and embryonic development has yet to be understood. In this study, we focused on the role of ion channels in the differentiation and patterning of the large airways of the developing respiratory tract. We identify a mechanism by which Wnt-beta-catenin signaling controls levels of ion channel-encoding genes to promote tracheal differentiation.

An ex vivo rat trachea model reveals abnormal airway physiology and a gland secretion defect in cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease hallmarked by aberrant ion transport that results in delayed mucus clearance, chronic infection, and progressive lung function decline. Several animal models have been developed to study the airway anatomy and mucus physiology in CF, but they are costly and difficult to maintain, making them less accessible for many applications. A more available CFTR-/- rat model has been developed and characterized to develop CF airway abnormalities, but consistent dosing of pharmacologic agents and longitudinal evaluation remain a challenge. In this study, we report the development and characterization of a novel ex vivo trachea model that utilizes both wild type (WT) and CFTR-/- rat tracheae cultured on a porcine gelatin matrix. Here we show that the ex vivo tracheae remain viable for weeks, maintain a CF disease phenotype that can be readily quantified, and respond to stimulation of mucus and fluid secretion by cholinergic stimulation. Furthermore, we show that ex vivo tracheae may be used for well-controlled pharmacological treatments, which are difficult to perform on freshly excised trachea or in vivo models with this degree of scrutiny. With improved interrogation possible with a durable trachea, we also established firm evidence of a gland secretion defect in CFTR-/- rat tracheae compared to WT controls. Finally, we demonstrate that the ex vivo tracheae can be used to generate high mucus protein yields for subsequent studies, which are currently limited by in vivo mucus collection techniques. Overall, this study suggests that the ex vivo trachea model is an effective, easy to set up culture model to study airway and mucus physiology.

The proteolysis of ZP proteins is essential to control cell membrane structure and integrity of developing tracheal tubes in Drosophila.

Membrane expansion integrates multiple forces to mediate precise tube growth and network formation. Defects lead to deformations, as found in diseases such as polycystic kidney diseases, aortic aneurysms, stenosis, and tortuosity. We identified a mechanism of sensing and responding to the membrane-driven expansion of tracheal tubes. The apical membrane is anchored to the apical extracellular matrix (aECM) and causes expansion forces that elongate the tracheal tubes. The aECM provides a mechanical tension that balances the resulting expansion forces, with Dumpy being an elastic molecule that modulates the mechanical stress on the matrix during tracheal tube expansion. We show in Drosophila that the zona pellucida (ZP) domain protein Piopio interacts and cooperates with the ZP protein Dumpy at tracheal cells. To resist shear stresses which arise during tube expansion, Piopio undergoes ectodomain shedding by the Matriptase homolog Notopleural, which releases Piopio-Dumpy-mediated linkages between membranes and extracellular matrix. Failure of this process leads to deformations of the apical membrane, tears the apical matrix, and impairs tubular network function. We also show conserved ectodomain shedding of the human TGFß type III receptor by Notopleural and the human Matriptase, providing novel findings for in-depth analysis of diseases caused by cell and tube shape changes.

Integrated analysis of genes and long non-coding RNAs in trachea transcriptome to decipher the host response during Newcastle disease challenge in different breeds of chicken.

Newcastle disease is a highly infectious economically devastating disease caused by Newcastle disease Virus in Chicken (Gallus gallus). Leghorn and Fayoumi are two breeds which show differential resistance patterns towards NDV. This study aims to identify the differentially expressed genes and lncRNAs during NDV challenge which could play a potential role in this differential resistance pattern. A total of 552 genes and 1580 lncRNAs were found to be differentially expressing. Of them, 52 genes were annotated with both Immune related pathways and Gene ontologies. We found that most of these genes were upregulated in Leghorn between normal and challenged chicken but several were down regulated between different timepoints after NDV challenge, while Fayoumi showed no such downregulation. We also observed that higher number of positively correlating lncRNAs was found to be downregulated along with these genes. This shows that although Leghorn is showing higher number of differentially expressed genes in challenged than in non-challenged, most of them were downregulated during the disease between different timepoints. With this we hypothesize that the downregulation of immune related genes and co-expressing lncRNAs could play a significant role behind the Leghorn being comparatively susceptible breed than Fayoumi. The computational pipeline is available at https://github.com/Venky2804/FHSpipe.

Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways.

The acquisition of distinct branch sizes and shapes is a central aspect in tubular organ morphogenesis and function. In the Drosophila airway tree, the interplay of apical extracellular matrix (ECM) components with the underlying membrane and cytoskeleton controls tube elongation, but the link between ECM composition with apical membrane morphogenesis and tube size regulation is elusive. Here, we characterized Emp (epithelial membrane protein), a Drosophila CD36 homolog belonging to the scavenger receptor class B protein family. emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid filled airways. Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain containing proteins. emp mutants also display over elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad, and phosphorylated Src (p-Src). We show that Emp associates with and organizes the ßH-Spectrin cytoskeleton and is itself confined by apical F-actin bundles. Overexpression or loss of its cargo protein Serp lead to abnormal apical accumulations of Emp and perturbations in p-Src levels. We propose that during morphogenesis, Emp senses and responds to luminal cargo levels by initiating apical membrane endocytosis along the longitudinal tube axis and thereby restricts airway elongation.

A succinate/SUCNR1-brush cell defense program in the tracheal epithelium.

Host-derived succinate accumulates in the airways during bacterial infection. Here, we show that luminal succinate activates murine tracheal brush (tuft) cells through a signaling cascade involving the succinate receptor 1 (SUCNR1), phospholipase Cß2, and the cation channel transient receptor potential channel subfamily M member 5 (TRPM5). Stimulated brush cells then trigger a long-range Ca2+ wave spreading radially over the tracheal epithelium through a sequential signaling process. First, brush cells release acetylcholine, which excites nearby cells via muscarinic acetylcholine receptors. From there, the Ca2+ wave propagates through gap junction signaling, reaching also distant ciliated and secretory cells. These effector cells translate activation into enhanced ciliary activity and Cl- secretion, which are synergistic in boosting mucociliary clearance, the major innate defense mechanism of the airways. Our data establish tracheal brush cells as a central hub in triggering a global epithelial defense program in response to a danger-associated metabolite.

Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

Nickel ions influence the transepithelial sodium transport in the trachea, intestine and skin.

Measurements of transepithelial potential and resistance in tissue and organ model systems enable the evaluation of the Ni2+ effect on the epithelial sodium channels, aquaporin 3, and the sodium-potassium pump in the epithelial cells. The aim of the presented study was to assess the immediate and prolonged effect of nickel ions on the transport of sodium ions in tissues exposed to direct contact with nickel, including airways, digestive tract and the skin. The influence of 0.1 mM nickel solution was performed on the trachea (n = 34), intestine (n = 44), and skin (n = 51) samples descended from 16 New Zealand albino rabbits. The electrophysiological parameters were measured in a modified Ussing chamber in stationary conditions and during a 15-s mechanical-chemical stimulation. A statistically significant decrease in the electric resistance values and the smallest range of the measured potential were observed for the Ni-treated trachea specimens. The use of nickel solution did not affect the sodium transport in the intestine epithelium. The skin fragments showed altered sodium ion transport, as demonstrated by the lower range and intensity of the measured potential. The gastrointestinal tract seems to be an organ best adapted to contact with nickel ions. In airways, nickel ions most likely enter epithelial cells and the space between them, modifying proteins and the airway surface liquid. The skin turned out to be the most sensitive tissue to the intensification of sodium ion transport through nickel ions.

Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila.

Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system.

Ultrasound-assisted extraction of collagen from broiler chicken trachea and its biochemical characterization.

Broiler chicken tracheas are a co-product from chicken slaughterhouses which are normally turned into low value animal feed despite their high levels of collagen. Typical collagen extraction by acid and/or pepsin usually results in relatively low yield. Ultrasound-assisted extraction (UAE) could be a means to improve collagen yield. The objectives of this study were to investigate the effects of ultrasonic parameters on the yield and biochemical properties of trachea collagen (TC). Conventional extraction using acetic acid and pepsin for 48 h resulted in acid-soluble (AS) and pepsin-soluble (PS) collagen with a yield of 0.65% and 3.10%, respectively. When an ultrasound with an intensity of 17.46 W·cm-2 was applied for 20 min, followed by acid extraction for 42 h (U-AS), the collagen yield increased to 1.58%. A yield of 6.28% was obtained when the ultrasound treatment was followed by pepsin for 36 h (U-PS). PS and U-PS contained collagen of 82.84% and 85.70%, respectively. Scanning electron microscopy images revealed that the ultrasound did not affect the collagen microstructure. All collagen samples showed an obvious triple helix structure as measured by circular dichroism spectroscopy. Fourier transform infrared spectroscopy indicated that the ultrasound did not disturb the secondary structure of the protein in which approximately 30% of the α-helix content was a major structure for all collagen samples. Micro-differential scanning calorimetry demonstrated that the denaturation temperature of collagen in the presence of deionized water was higher than collagen solubilized in 0.5 M acetic acid, regardless of the extraction method. All collagen comprised of α1 and α2-units with molecular weights of approximately 135 and 116 kDa, respectively, corresponding to the type I characteristic. PS and U-PS collagen possessed higher imino acids than their AS and U-AS counterparts. Based on LC-MS/MS peptide mapping, PS and U-PS collagen showed a high similarity to type I collagen. These results suggest that chicken tracheas are an alternative source of type I collagen. UAE is a promising technique that could increase collagen yield without damaging its structure.

IL-11 drives the phenotypic transformation of tracheal epithelial cells and fibroblasts to enhance abnormal repair after tracheal injury.

Tracheal stenosis (TS) is a multifactorial and heterogeneous disease that can easily lead to respiratory failure and even death. Interleukin-11 (IL-11) has recently received increased attention as a fibrogenic factor, but its function in TS is uncertain. This study aimed to investigate the role of IL-11 in TS regulation based on clinical samples from patients with TS and a rat model of TS produced by nylon brush scraping. Using lentiviral vectors expressing shRNA (lentivirus-shRNA) targeting the IL-11 receptor (IL-11Rα), we lowered IL-11Rα levels in the rat trachea. Histological and immunostaining methods were used to evaluate the effects of IL-11Rα knockdown on tracheal injury, molecular phenotype, and fibrosis in TS rats. We show that IL-11 was significantly elevated in circulating serum and granulation tissue in patients with TS. In vitro, TGFß1 dose-dependently stimulated IL-11 secretion from human tracheal epithelial cells (Beas-2b) and primary rat tracheal fibroblasts (PRTF). IL-11 transformed the epithelial cell phenotype to the mesenchymal cell phenotype by activating the ß-catenin pathway. Furthermore, IL-11 activated the atypical ERK signaling pathway, stimulated fibroblasts proliferation, and transformed fibroblasts into alpha-smooth muscle actin (α-SMA) positive myofibroblasts. IL-11-neutralizing antibodies (IL-11NAb) or ERK inhibitors (U0126) inhibited IL-11 activity and downregulated fibrotic responses involving TGFß/SMAD signaling. In vivo, IL-11Rα knockdown rats showed unobstructed tracheal lumen, relatively intact epithelial structure, and significantly reduced granulation tissue proliferation and collagen fiber deposition. Our findings confirm that IL-11 may be a target for future drug prevention and treatment of tracheal stenosis.

The metabolic and lipidomic profiling of the effects of tracheal occlusion in a rabbit model of congenital diaphragmatic hernia.

PURPOSE: Fetal tracheal occlusion (TO) reverses the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH), but its mechanism of action remains poorly understood. 'Omic' readouts capture metabolic and lipid processing function, which aid in understanding CDH and TO metabolic mechanisms. METHODS: CDH was created in fetal rabbits at 23 days, TO at 28 days and lung collection at 31 days (Term ∼32 days). Lung-body weight ratio (LBWR) and mean terminal bronchiole density (MTBD) were determined. In a cohort, left and right lungs were collected, weighed, and samples homogenized, and extracts collected for non-targeted metabolomic and lipidomic profiling via LC-MS and LC-MS/MS, respectively. RESULTS: LBWR was significantly lower in CDH while CDH + TO was similar to controls (p = 0.003). MTBD was significantly higher in CDH fetuses and restored to control and sham levels in CDH + TO (p < 0.001). CDH and CDH + TO resulted in significant differences in metabolome and lipidome profiles compared to sham controls. A significant number of altered metabolites and lipids between the controls and CDH groups and the CDH and CDH + TO fetuses were identified. Significant changes in the ubiquinone and other terpenoid-quinone biosynthesis pathway and the tyrosine metabolism pathway were observed in CDH + TO. CONCLUSION: CDH + TO reverses pulmonary hypoplasia in the CDH rabbit, in association with a specific metabolic and lipid signature. A synergistic untargeted 'omics' approach provides a global signature for CDH and CDH + TO, highlighting cellular mechanisms among lipids and other metabolites, enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology and recovery. TYPE OF STUDY: Basic Science, Prospective. LEVEL OF EVIDENCE: II.

The Osiris family genes function as novel regulators of the tube maturation process in the Drosophila trachea.

Drosophila trachea is a premier model to study tube morphogenesis. After the formation of continuous tubes, tube maturation follows. Tracheal tube maturation starts with an apical secretion pulse that deposits extracellular matrix components to form a chitin-based apical luminal matrix (aECM). This aECM is then cleared and followed by the maturation of taenidial folds. Finally, air fills the tubes. Meanwhile, the cellular junctions are maintained to ensure tube integrity. Previous research has identified several key components (ER, Golgi, several endosomes) of protein trafficking pathways that regulate the secretion and clearance of aECM, and the maintenance of cellular junctions. The Osiris (Osi) gene family is located at the Triplo-lethal (Tpl) locus on chromosome 3R 83D4-E3 and exhibits dosage sensitivity. Here, we show that three Osi genes (Osi9, Osi15, Osi19), function redundantly to regulate adherens junction (AJ) maintenance, luminal clearance, taenidial fold formation, tube morphology, and air filling during tube maturation. The localization of Osi proteins in endosomes (Rab7-containing late endosomes, Rab11-containing recycling endosomes, Lamp-containing lysosomes) and the reduction of these endosomes in Osi mutants suggest the possible role of Osi genes in tube maturation through endosome-mediated trafficking. We analyzed tube maturation in zygotic rab11 and rab7 mutants, respectively, to determine whether endosome-mediated trafficking is required. Interestingly, similar tube maturation defects were observed in rab11 but not in rab7 mutants, suggesting the involvement of Rab11-mediated trafficking, but not Rab7-mediated trafficking, in this process. To investigate whether Osi genes regulate tube maturation primarily through the maintenance of Rab11-containing endosomes, we overexpressed rab11 in Osi mutant trachea. Surprisingly, no obvious rescue was observed. Thus, increasing endosome numbers is not sufficient to rescue tube maturation defects in Osi mutants. These results suggest that Osi genes regulate other aspects of endosome-mediated trafficking, or regulate an unknown mechanism that converges or acts in parallel with Rab11-mediated trafficking during tube maturation.

GATA6 triggers fibroblast activation and tracheal fibrosis through the Wnt/ß-catenin pathway.

Tracheal fibrosis is a key abnormal repair process leading to fatal stenosis, characterized by excessive fibroblast activation and extracellular matrix (ECM) deposition. GATA6, a zinc finger-containing transcription factor, is involved in fibroblast activation, while its role in tracheal fibrosis remains obscure. The present study investigated the potential role of GATA6 as a novel regulator of tracheal fibrosis. It was found that GATA6 and α-smooth muscle actin (α-SMA) were obviously increased in tracheal fibrotic granulations and in TGFß1-treated primary tracheal fibroblasts. GATA6 silencing inhibited TGFß1-stimulated fibroblast proliferation and ECM synthesis, promoted cell apoptosis, and inactivated Wnt/ß-catenin pathway, whereas GATA6 overexpression showed the reverse effects. SKL2001, an agonist of Wnt/ß-catenin signaling, restored collagen1a1 and α-SMA expression which was suppressed by GATA6 silencing. Furthermore, in vivo, knockdown of GATA6 ameliorated tracheal fibrosis, as manifested by reduced tracheal stenosis and ECM deposition. GATA6 inhibition in rat tracheas also impaired granulation proliferation, increased apoptosis, and inactivated Wnt/ß-catenin pathway. In conclusion, our findings indicate that GATA6 triggers fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/ß-catenin signaling pathway. Targeting GATA6 may represent a promising therapeutic approach for tracheal fibrosis.

Ionic Detergent Under Pressure-Vacuum as an Innovative Strategy to Generate Canine Tracheal Scaffold for Organ Engineering.

Decellularized scaffolds applied in tissue engineering offer improvements, supplying the elevated necessity for organs and tissues for replacement. However, obtaining a functional trachea for autotransplantation or allotransplantation is tricky due to the organ anatomical and structural complexity. Most tracheal decellularization protocols are lengthy, expensive, and could damage the tracheal extracellular matrix (ECM) architecture and functionality. Here, we aimed to evaluate the effectiveness of 3 different decellularization protocols combined with chemical and physical methods to obtain acellular canine tracheal scaffolds. Six adult dog tracheas were incised (tracheal segments) resulting in 28 rings for control tissue and 84 rings for decellularization (5-7 mm thick). Subsequently, decellularized tracheal scaffolds were microscopically/macroscopically characterized by histological analysis (Hematoxylin-Eosin, Masson's trichrome, Picrosirius red, Alcian blue, and Safranin O), immunohistochemistry for ECM components, scanning electron microscopy, and genomic DNA quantification. After decellularization, the tracheal tissue revealed reduced genomic DNA, and maintenance of ECM components preserved (structural proteins, adhesive glycoproteins, glycosaminoglycans and proteoglycans), suggesting ECM integrity and functionality. Comparatively, the combined ionic detergent with high vacuum pressure decellularization protocol revealed superior genomic DNA decrease (13.5 ng/mg) and improvement on glycosaminoglycans and proteoglycans preservation regarding the other decellularized trachea scaffolds and native tissue. Our results indicate that the 3 chemical/physical protocols reduce the decellularization time without ECM proteins damage. Notwithstanding, the use of ionic detergent under vacuum pressure was able to generate an innovative strategy to obtain acellular canine tracheal scaffolds with the highest levels of adhesive proteins that support its potentiality for recellularization and future tissue engineering application.

Association of enteral feeding with microaspiration in critically ill adults.

AIM: This study explored relationships between enteral feeding and tracheal pepsin A. BACKGROUND: Mechanically ventilated (MV) patients receiving enteral feeding are at risk for microaspiration. Tracheal pepsin A, an enzyme specific to gastric cells, was a proxy for microaspiration of gastric secretions. METHODS: Secondary analysis of RCT data from critically ill, MV adults was conducted. Microaspiration prevention included elevated head of bed, endotracheal tube cuff pressure management, and regular oral care. Tracheal secretions for pepsin A were collected every 12 h. Microaspiration was defined as pepsin A ≥ 6.25 ng/mL. Positive pepsin A in >30 % of individual tracheal samples was defined as abundant microaspiration (frequent aspirator). Chi-squared, Fisher's Exact test, and generalized linear model (GLM) were used. RESULTS: Tracheal pepsin A was present in 111/283 (39 %) mechanically ventilated patients and 48 (17 %) had abundant microaspiration. Enteral feeding was associated with tracheal pepsin A, which occurred within 24 h of enteral feeding. Of the patients who aspirated, the majority received some enteral feeding 96/111 (86 %), compared to only 15/111 (14 %) who received no feeding. A greater number of positive pepsin A events occurred with post-pyloric feeding tube location (55.6 %) vs. gastric (48.6 %), although significant only at the event-level. Frequent aspirators (abundant pepsin A) had higher pepsin A levels compared to infrequent aspirators. CONCLUSIONS: Our findings confirmed the stomach as the microaspiration source. Contrary to other studies, distal feeding tube location did not mitigate microaspiration. Timing for first positive pepsin A should be studied for possible association with enteral feeding intolerance.

Airway basal cells show regionally distinct potential to undergo metaplastic differentiation.

Basal cells are multipotent stem cells of a variety of organs, including the respiratory tract, where they are major components of the airway epithelium. However, it remains unclear how diverse basal cells are and how distinct subpopulations respond to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the adult murine trachea. Among these, we found two major subpopulations, collectively comprising the most uncommitted of all the pools, but with distinct gene expression signatures. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to self-renew and initiate a program of differentiation in response to environmental perturbations in primary cultures and in mouse injury models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are still being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of tracheal cartilage-deficient mouse mutants. Finally, we show that features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide novel insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions.

Regulation of Archease by the mTOR-vATPase axis.

Larval terminal cells of the Drosophila tracheal system generate extensive branched tubes, requiring a huge increase in apical membrane. We discovered that terminal cells compromised for apical membrane expansion - mTOR-vATPase axis and apical polarity mutants - were invaded by the neighboring stalk cell. The invading cell grows and branches, replacing the original single intercellular junction between stalk and terminal cell with multiple intercellular junctions. Here, we characterize disjointed, a mutation in the same phenotypic class. We find that disjointed encodes Drosophila Archease, which is required for the RNA ligase (RtcB) function that is essential for tRNA maturation and for endoplasmic reticulum stress-regulated nonconventional splicing of Xbp1 mRNA. We show that the steady-state subcellular localization of Archease is principally nuclear and dependent upon TOR-vATPase activity. In tracheal cells mutant for Rheb or vATPase loci, Archease localization shifted dramatically from nucleus to cytoplasm. Further, we found that blocking tRNA maturation by knockdown of tRNAseZ also induced compensatory branching. Taken together, these data suggest that the TOR-vATPase axis promotes apical membrane growth in part through nuclear localization of Archease, where Archease is required for tRNA maturation.