Search Results (183)

Tracheal reconstruction remains a formidable clinical challenge, particularly for long-segment defects that are not amenable to standard surgical resection or primary anastomosis. Tissue engineering has emerged as a promising strategy for restoring the tracheal structure and function through the integration of biomaterials, stem cells, and bioactive molecules. This review provides a comprehensive overview of recent advances in tissue-engineered tracheal grafts, particularly in scaffold design, cellular sources, fabrication technologies, and early clinical experience. Innovations in biomaterial science, three-dimensional printing, and scaffold-free fabrication approaches have broadened the prospects for patient-specific airway reconstruction. However, persistent challenges, including incomplete epithelial regeneration and mechanical instability, have hindered its clinical translation. Future efforts should focus on the design of modular biomimetic scaffolds, the enhancement of immunomodulatory strategies, and preclinical validation using robust large animal models. Sustained interdisciplinary collaboration among surgical, engineering, and biological fields is crucial for advancing tissue-engineered tracheal grafts for routine clinical applications. Within this context, biomimetic approaches, including three-dimensional bioprinting, hybrid materials, and scaffold-free constructs, are gaining prominence as strategies to replicate the trachea’s native architecture and improve graft integration. Full article

Therapeutic gene editing strategies utilize endogenous DNA repair pathways—nonhomologous end joining (NHEJ) or homology-directed repair (HDR)—to introduce targeted genomic modifications. Because HDR is restricted to dividing cells, whereas NHEJ functions in both dividing and non-dividing cells, NHEJ-based approaches are better suited for in vivo gene editing in the largely post-mitotic airway epithelium. Homology-independent targeted insertion (HITI), an NHEJ-based method, offers a promising strategy for cystic fibrosis (CF) gene therapy. Here, we applied HITI to drive the expression of a promoterless reporter through an exon trap strategy in both proliferating airway basal cells and well-differentiated primary airway epithelial cultures derived from transgenic ROSA^mTmG ferrets. We also established a versatile human gene editing reporter (GER) airway basal cell line capable of multipotent differentiation, enabling real-time visualization of editing outcomes and the quantitative assessment of HDR- and NHEJ-based editing efficiencies. Together, these platforms provide easily accessible tools for optimizing genome editing strategies in the respiratory epithelium and advancing clinically relevant delivery strategies for CF gene therapy. Full article

Delta-9-tetrahydrocannabinol (Δ-9-THC or THC), the primary psychoactive constituent of cannabis, can lead to adverse health conditions, including mental health issues, brain impairment, and cardiac and respiratory problems. The amount of THC in cannabis has steadily climbed over the past few decades, with today’s cannabis having three times the concentration of THC compared to 25 years ago. Inhalation is a major route of exposure, allowing substances to enter the body via the respiratory tract. THC exposure causes cell death in the airway epithelium; however, the molecular underpinning of THC exposure-induced bronchial epithelial cell death is not clearly understood. To address the mechanisms involved in this process, the present study examined the cell viability, oxidative stress, lipid peroxidation, and transcriptional alterations caused by various concentrations of Δ-9-THC (0, 800, 1000, 1200, and 1500 ng/mL) in a human bronchial epithelial cell line (BEAS-2B) in vitro. Δ-9-THC exposure caused a significant dose-dependent decrease in cell viability after 24 h exposure. Transcriptome analysis showed a distinct dose-dependent response. HIF-1 signaling, ferroptosis, AMPK signaling, and immunogenic pathways were activated by Δ-9-THC-upregulated genes. Glutathione and fatty acid metabolic pathways were significantly altered by Δ-9-THC-dependent downregulated genes. Ingenuity Pathway Analysis (IPA) revealed several top canonical pathways altered by Δ-9-THC exposure, including ferroptosis, NRF-2-mediated oxidative stress response, caveolar-mediated endocytosis (loss of cell adhesion to the substrate), tumor microenvironment, HIF1alpha signaling, and the unfolded protein response pathway. Δ-9-THC-induced cell death was ameliorated by inhibiting the ferroptosis pathway, whereas treatments with ferroptosis agonist exacerbated the cell death process, suggesting that Δ-9-THC-induced bronchial epithelial cell death potentially involves the ferroptosis pathway. Full article

Pollen allergy is rated as a major public health problem, causing significant morbidity and adversely affecting the quality of people’s lives. The airway epithelium serves as the first line of defense in the respiratory system, playing a crucial role in orchestrating immune responses to allergens. In this work, we studied the important transport steps in the major alder pollen allergen Aln g 1 through the human airway epithelium in comparison with those of the birch pollen allergen Bet v 1. Using fluorescence spectroscopy, we showed that both allergens can destroy liposomes with a composition modeling the adult human pulmonary surfactant. Using a polarized Calu-3 monolayer, we showed similar efficiencies of Aln g 1 and Bet v 1 transport through the artificial epithelial barrier. Using qPCR, we showed that Aln g 1 upregulates the expression of IL-33, TSLP, IL-1β, CXCL8 in epithelial cells, playing an important role in the sensitization process. The obtained results may improve our understanding of the primary sensitization mechanisms with the involvement of the PR-10 family of lipid-binding allergens. Full article

Respiratory syncytial virus (RSV) is the leading cause of respiratory infections in children. Extracellular vesicles (EVs), released by airway epithelial cells, contain proteins and different families of non-coding RNAs (EV cargo) that can modulate the responses of target cells to viral infection. Nasal mucosa is a primary site of viral entry and the source of EVs present in the upper airway secretions. In this study we characterized proteins, including inflammatory mediators and cytokines, and the piwi-interacting RNA (piRNAs) cargo of EVs isolated from pediatric human nose organoids (HNO) and nasopharyngeal secretions (NPS) positive for RSV. Using Proximity Extension Assay (PEA) and Luminex multi-target arrays, we found significant enrichment in several chemokines and other mediators/biomarkers, including CCL2, CCL20, CXCL5, CX3CL1, CXCL6, MMP-1, MMP-10, uPA, Flt3L, ARNT and CD40 in EVs secreted by RSV-infected HNO compared to control mock HNO. Analysis of NPS samples from RSV infected children revealed that CCL3, CCL20, CXCL8, uPA, VEGFA, were concentrated in the NPS-EV fraction. LC-MS/MS and Gene Ontology indicated that RSV positive NPS-EVs originate from different cellular sources, with the most abundant proteins from neutrophils and epithelial cells. A total of 490 piRNAs were detected by NGS sequencing of small RNA libraries obtained from NPS-EVs, which has not been reported prior to this study. Identification of inflammatory mediators and small non-coding RNAs which are compartmentalized in EVs contributes to understanding mechanisms of virus-mediated pathogenesis in RSV infections. Full article

Current animal and in vitro cell culture models do not fully recapitulate the physiological and pathophysiological characteristics of the human lung. As a result, the translation of these models to clinical practice is very limited, and clinical trials initiated on the extrapolation of such data fail. Although current models are beneficial in fundamental research, there is a need to constantly improve models to more accurately predict outcomes in clinical trials and personalized medicine. Here, we report important strategies to develop a 3D lung model with human primary lung cells. Starting from the well-established air-liquid interface (ALI) culture system, we describe a gradual increase in the complexity of the system by co-culturing different primary cell types, by testing different coatings, and by adding a three-dimensional matrix. As a result, we have established a reproducible 3D in vitro model of the airway consisting of human primary cells representing a differentiated mucociliary airway epithelium, an underlying submucosa with fibroblasts, and an endothelial interface. Full article

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract viral infection in infants and causes around 60,000 in-hospital deaths annually. Emerging evidence suggests that RSV induces metabolic changes in host cells to support viral replication, presenting a potential target for therapeutic intervention. To investigate RSV-driven metabolic changes in situ, we combined positron emission tomography (PET), live-cell bioenergetics, and metabolomic profiling in the upper and lower airways of children. PET imaging revealed persistent, hyper-glycolytic regions in the lungs of RSV-infected children. Bioenergetic analysis of freshly collected nasopharyngeal aspirates from infants showed live upper respiratory cells (URCs) infected with RSV in situ exhibited significantly higher levels of glycolysis, glycolytic capacity, glycolytic reserves, and mitochondrial respiration than uninfected controls. Metabolomic analysis of nasopharyngeal fluids from these patients revealed distinct metabolic signatures, including increased citrate and malate, and decreases in taurine. In vitro infection of pediatric nasopharynx tissue-derived multicellular epithelial cultures (TEpiCs) and bronchial epithelial cells further confirmed RSV-induced increases in glycolysis. Together, these findings demonstrate that RSV infection induces hypermetabolism in both upper and lower primary airways in situ, supporting the potential of host-targeted metabolic interventions as a therapeutic strategy—particularly in vulnerable populations such as infants for whom vaccines are not currently available. Full article

Background and Objectives: Oral appliances (OAs) are commonly used to manage sleep-disordered breathing conditions, including primary snoring, and offer an alternative treatment for individuals with obstructive sleep apnea (OSA) who cannot tolerate continuous positive airway pressure (CPAP) therapy. Our study analyzed the possible factors associated with higher snoring rates compared with those associated with lower snoring rates. Materials and Methods: A customized dental brace with a tongue compressor was the essential part of the Lin OA (LOA). The compressor is available in various lengths, ranging from 0.5 to 3.0 cm across different versions. The participants wore the LOA throughout the night while sleeping. Their snoring rates were recorded using the SnoreClock app on their cell phones. Results: The analysis included 36 participants, comprising 30 males and 6 females. The participants had a mean age of 44.91 ± 9.96 years, a mean BMI of 26.18 ± 3.50 kg/m², and an average recording duration of 398.27 ± 77.56 min per session. In total, 4052 sleep recordings were analyzed. The number of files for females was less than that for males (563 vs. 3489). In this study, individuals belonging to the highest one-third based on the baseline snoring rate (H group) experienced a significant reduction in snoring, approximately 84.8%, when using the LOA-3 cm device equipped with a 3 cm tongue compressor. The individuals who belonged to the lower two-thirds based on the baseline snoring rate were classified as the L group. Among male participants, snoring decreased by approximately 66.4%. Similarly, among female participants in the L group, snoring decreased by approximately 69.3% when using the LOA-3 cm. Conclusions: In this study, we observed a significant reduction in snoring for the two groups of participants wearing the LOA-3 cm, with the rate decrements ranging from 66.4% to 84.8%. This reduction was more pronounced in the H group than in the male participants in the L group. Further studies are needed to explore the reasons for these findings. Full article

Lassa mammarenavirus (LASV), a member of the family Arenaviridae, is a highly pathogenic virus capable of causing severe systemic infections in humans. The primary host reservoir is the Natal multimammate mouse (Mastomys natalensis), with human infections typically occurring through mucosal exposure to virus-containing aerosols from rodent excretions. To better understand the molecular mechanisms underlying LASV replication in the respiratory tract, we utilized differentiated primary human airway epithelial cells (HAECs) grown under air–liquid interface conditions, closely mimicking the bronchial epithelium in vivo. Our findings demonstrate that HAECs are permissive to LASV infection and support productive virus replication. While LASV entry into polarized HAECs occurred through both apical and basolateral surfaces, progeny virus particles were predominantly released from the apical surface, consistent with an intrinsic apical localization of the envelope glycoprotein GP. This suggests that apical virus shedding from infected bronchial epithelia may facilitate LASV transmission via airway secretions. Notably, limited basolateral release at later stages of infection was associated with LASV-induced rearrangement of the actin cytoskeleton, resulting in compromised epithelial barrier integrity. Finally, we demonstrate that LASV-infected HAECs exhibited a pronounced type III interferon response. A detailed understanding of LASV replication and host epithelial responses in the respiratory tract could facilitate the development of targeted future therapeutics. Full article

Background/Objectives: In vitro models play a crucial role in preclinical respiratory research, enabling the testing and screening of mucosal formulations, dosage forms, and inhaled drugs. Mucociliary clearance (MCC) is an essential defense mechanism in mucosal drug delivery but is often impaired in respiratory diseases. Despite its importance, standardized in vitro MCC assays are rarely reported. Furthermore, many published methods primarily measure cilia beat frequency (CBF), which requires high-speed cameras that are not accessible to all laboratories. Therefore, this study aimed to develop a physiologically relevant, differentiated in vitro model of the respiratory epithelium that incorporates both beating cilia and functional MCC. We chose porcine airway mucosa as an alternative to human tissue due to ethical considerations and limited availability. The established model is designed to provide a reproducible and accessible method for a broad range of research laboratories. Methods: The previously published tracheal mucosal primary cell (TMPC DS) model, derived from porcine tissue, lacked the presence of beating cilia, which are crucial for effective MCC analysis. For accurate MCC assessment, beating cilia are essential as they play a key role in mucus clearance. To address this limitation, the here-described ciliated tracheal mucosal primary cell (cTMPC) model was developed. cTMPCs were isolated from porcine tissue and cultured under air–liquid interface (ALI) conditions for 21 days to promote differentiation. This model was evaluated for cell morphology, tight junction formation, ciliated and mucus-producing cells, barrier function, gene expression, and tracer/IgG transport. MCC and the model’s suitability for standardized MCC assays were assessed using an inverted microscope. In contrast to the TMPC DS model, which lacked beating cilia and thus could not support MCC analysis, the cTMPC model allows for comprehensive MCC studies. Results: The developed differentiated in vitro model demonstrated key structural and functional features of the respiratory epithelium, including well-differentiated cell morphology, tight junction integrity, ciliated and mucus-producing cells, and effective barrier function. Functional MCC was observed, confirming the model’s potential for standardized clearance assays. Conclusions: This differentiated in vitro model closely replicates the structural and functional characteristics of in vivo airways. It provides a valuable platform for studying mucociliary clearance, toxicology, drug uptake, and evaluating mucosal formulations and dosage forms in respiratory research. Full article

The pandemic threat from newly emerging viral diseases constitutes a major unsolved issue for global health. Antiviral therapy can play an important role in treating and preventing the spread of unprecedented viral infections. A repository of compounds exhibiting broad-spectrum antiviral activity against a series of different viral families would be an invaluable asset to be prepared for future pandemic threats. Utilizing an open innovation crowd-sourcing paradigm, we were able to identify a compound class of diphenylureas that exhibits in vitro antiviral activity against multiple viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), adenovirus, dengue virus, herpes, and influenza viruses. Compound 4 among the series exhibits strong activity against dengue virus, a growing global health problem with high medical need and no approved antiviral drug. The compounds are active against SARS-CoV-2 in a primary human stem cell-based mucociliary airway epithelium model and also active in vivo, as shown in a murine SARS-CoV-2 infection model. These results demonstrate the potential of the chemical class as antivirals on the one hand and the power of open innovation, crowd-sourcing, and repurposing on the other hand. Full article

The nasal epithelium is the primary site for entry of respiratory viruses. In comparison to oral administration, nasal drug applications directed locally to the site of infection can serve as early interventional barriers against respiratory virus pathogenesis by limiting viral spread in the upper airway. Experiments on the diffusion of methylene blue and nanoparticles in both water and low pH conditions revealed that hydroxypropyl methylcellulose (HPMC) can act as an effective physical barrier. This study also evaluated the activity of HPMC as a barrier against common respiratory viruses, i.e., rhinovirus (RV) and influenza A virus (IAV) using the in vitro human nasal epithelial cell (hNEC) model. Utilizing the hNEC infection model, we assessed the protective effects of HPMC in pH 3.5 and pH 7 buffers against RV and IAV. Acidic and pH-neutral buffers and HPMC dissolved in acidic and pH-neutral buffers were administered for 4 h prior to virus infection and at 4 h post-infection (hpi). The apical supernatant was harvested at 24 hpi to determine the viral loads of RV and IAV (H1N1 and H3N2). HPMC was demonstrated to exert protective effects in the infected hNECs independent of acidic pH. Pre-treatment with HPMC in acidic buffer significantly diminished viral loads for both RV and IAV infections of hNECs. Similarly, direct treatment of HPMC in acidic buffer after infection (4 hpi) also effectively decreased viral loads of both RV and IAV. Moreover, treatment using HPMC in acidic buffer before or after infection did not affect the epithelial integrity and ciliary function of hNECs. This study demonstrates the protective effects of HPMC in acidic buffer against RV and IAV infections of the human nasal epithelium. Full article

Particulate matter (PM) is a major component of ambient air pollution. PM exposure is linked to numerous adverse health effects, including chronic lung diseases. Air quality guidelines designed to regulate levels of ambient PM are currently based on the mass concentration of different particle sizes, independent of their origin and chemical composition. The objective of this study was to assess the relative hazardous effects of carbonaceous particles (soot), ammonium nitrate, ammonium sulfate, and copper oxide (CuO), which are standard components of ambient air, reflecting contributions from primary combustion, secondary inorganic constituents, and non-exhaust emissions (NEE) from vehicular traffic. Human epithelial cells representing bronchial (BEAS-2B) and alveolar locations (H441 and A549) in the airways, human lung fibroblasts (HFL-1), and rat precision-cut lung slices (PCLS) were exposed in submerged cultures to different concentrations of particles for 5–72 h. Following exposure, cell viability, metabolic activity, reactive oxygen species (ROS) formation, and inflammatory responses were analyzed. CuO and, to a lesser extent, soot reduced cell viability in a dose-dependent manner, increased ROS formation, and induced inflammatory responses. Ammonium nitrate and ammonium sulfate did not elicit any significant cytotoxic responses but induced immunomodulatory alterations at very high concentrations. Our findings demonstrate that secondary inorganic components of PM have a lower hazard cytotoxicity compared with combustion-derived and indicative NEE components, and alveolar epithelial cells are more sensitive to PM exposure. This information should help to inform which sources of PM to target and feed into improved, targeted air quality guidelines. Full article

Background: Chronic obstructive pulmonary disease (COPD) is characterized by progressive and incurable airflow obstruction and chronic inflammation. Both TGF-β1 and CXCL8 have been well described as fundamental to COPD progression. DNA methylation and histone acetylation, which are well-understood epigenetic mechanisms regulating gene expression, are associated with COPD progression. However, a deeper understanding of the complex mechanisms associated with DNA methylation, histone post-translational changes and RNA methylation in the context of regulatory pathways remains to be elucidated. We here report on how DNA methylation and histone acetylation inhibition differentially affect CXCL8 signaling in primary human non-COPD and COPD airway cells. Methods: Airway smooth muscle (ASM) cells, a pivotal cell type in COPD, were isolated from the small airways of heavy smokers with and without COPD. Histone acetylation and DNA methylation were inhibited before the TGF-β1 stimulation of cells. Subsequently, CXCL8 production and the abundance and activation of pertinent transcription regulatory proteins (NF-κB, p38 MAPK and JNK) were analyzed. Results: TGF-β1-stimulated CXCL8 release from ASM cells from ‘healthy’ smoker subjects was significantly modulated by DNA methylation (56.32 pg/mL and 56.60 pg/mL) and acetylation inhibitors (27.50 pg/mL and 48.85 pg/mL) at 24 and 48 h, respectively. However, modulation via the inhibition of DNA methylation (34.06 pg/mL and 43.18 pg/mL) and acetylation (23.14 pg/mL and 27.18 pg/mL) was observed to a lesser extent in COPD ASM cells. These changes were associated with differences in the TGF-β1 activation of NF-κB and MAPK pathways at 10 and 20 min. Conclusions: Our findings offer insight into differential epigenetics in controlling COPD ASM cells and provide a foundation warranting future studies on epigenetic differences associated with COPD diagnosis. This would provide a scope for developing therapeutic interventions targeting signaling and epigenetic pathways to improve patient outcomes. Full article

Bovine viral diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, is a major livestock pathogen. Horizontal transmission leads to acute transient infections via the oronasal route, whereas vertical transmission might lead to the birth of immunotolerant, persistently infected animals. In both cases, BVDV exerts an immunosuppressive effect, predisposing infected animals to secondary infections. E^rns, an immunomodulatory viral protein, is present on the envelope of the virus and is released as a soluble protein. In this form, it is taken up by cells and, with its RNase activity, degrades single- and double-stranded (ds) RNA, thus preventing activation of the host’s interferon system. Here, we show that E^rns of the pestiviruses BVDV and Bungowannah virus effectively inhibit dsRNA-induced IFN synthesis in well-differentiated airway epithelial cells cultured at the air–liquid interface. This activity was observed independently of the side of entry, apical or basolateral, of the pseudostratified, polarized cell layer. Virus infection was successful from both surfaces but was inefficient, requiring several days of incubation. Virus release was almost exclusively restricted to the apical side. This confirms that primary, well-differentiated respiratory epithelial cells cultured at the air–liquid interface are an appropriate model to study viral infection and innate immunotolerance in the bovine respiratory tract. Furthermore, evidence is presented that E^rns might contribute to the immunosuppressive effect observed after BVDV infections, especially in persistently infected animals. Full article