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We broaden the clinical versatility of human nasal epithelial (HNE) cells. HNEs were isolated from 10 participants harboring cystic fibrosis transmembrane conductance regulator ( CFTR ) variants: 9 with rare variants (Q359R [ n  =   2], G480S, R334W [ n  =   5], and R560T) and 1 harboring R117H;7T;TG10/5T;TG12. Cultures were differentiated at the air-liquid interface. CFTR function was measured in Ussing chambers at three conditions: baseline, ivacaftor, and elexacaftor + tezacaftor + ivacaftor (ETI). Four participants initiated modulators. Q359R HNEs had 5.4% (% wild-type) baseline CFTR function and 25.5% with ivacaftor. With therapy, sweat [Cl - ] decreased and symptoms resolved. G480S HNEs had 4.1% baseline and 32.1% CFTR function with ETI. Clinically, forced expiratory volume in 1 second increased and sweat [Cl - ] decreased (119 to 46 mmol/L) with ETI. In vitro cultures derived from 5 participants harboring R334W showed a moderate increase in CFTR function with exposure to modulators. For one of these participants, ETI was begun in vivo ; symptoms and forced expiratory volume in 1 second improved. The c.1679G>C (R560T) HNEs had less than 4% baseline CFTR function and no modulator response. RNA analysis confirmed that c.1679G>C completely missplices. A symptomatic patient harboring R117H;7T;TG10/5T;TG12 exhibited reduced CFTR function (17.5%) in HNEs, facilitating a diagnosis of mild CF. HNEs responded to modulators (ivacaftor: 32.8%, ETI: 55.5%), and, since beginning therapy, lung function improved. We reaffirm HNE use for guiding therapeutic approaches, inform predictions on modulator response (e.g., R334W), and closely assess variants that affect splicing (e.g., c.1679G>C). Notably, functional studies in HNEs harboring R117H;7T;TG10/5T;TG12 facilitated a diagnosis of mild CF, suggesting the use for HNE functional studies as a clinical diagnostic test.

Volatile organic compounds (VOCs) produced by the lung in response to exposure to environmental pollutants can be utilized to study their impact on lung health and function. Previously, we developed a method to measure VOCs emitted from well-differentiated tracheobronchial epithelial cells in vitro. Using this method, we exposed well-differentiated proximal (PECs) and distal airway epithelial cells (DECs) to varying doses of traffic-related air pollutants (TRAP) and wildfire particulates to determine specific VOC signatures after exposure. We utilized PM2.5 TRAP collected from the Caldecott tunnel in Oakland, CA and the 2018 Camp Fire to model "real-life" exposures. The VOCs were collected and extracted from Twisters and analyzed using gas chromatography-mass spectrometry (GC-MS). Exposure to both types of particulate matter (PM) resulted in specific VOC responses grouped by individual subjects with little overlap. Interestingly the VOCs produced by the PECs and DECs were also differentiated from each other. Our studies suggest that PM exposure induces a specific compartmentalized cellular response that can be exploited for future studies. This response is cell-type specific and potentially related to a phenotype we have yet to uncover., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Understanding protein adsorption and aggregation at the air-liquid interfaces of protein solutions is an important open challenge in biopharmaceutical, medical, and biotechnological applications, among others. Proteins, being amphiphilic, adsorb at the surface, partially unfold, and form a viscoelastic film through non-covalent interactions. Mechanical agitation of the surface can break this film up, releasing insoluble protein particles into the solution. These aggregates are usually highly undesirable and even toxic in cases, such as for biopharmaceutical application. Therefore, it is imperative to be able to predict the behavior of such solutions undergoing surface agitation during handling, usually transport or mixing. We apply the findings on the viscoelastic protein film, formed at the air-liquid interface, to the prediction of surface mediated aggregation in selected protein solutions of direct biopharmaceutical relevance. Our broad study of Brewster angle microscopy and aggregation monitoring across multiple size ranges by micro-flow imaging, light scattering, and size exclusion chromatography shows that formation of protein particles is driven by the adsorption rate as compared to the rate of surface turnover and that surface film dynamics in the quiescent phase directly affect aggregation. We demonstrate how these learnings can be directly applied to the design of a novel small scale biopharmaceutical stability study, simulating relevant transport conditions. More generally, we show the impact of adsorption dynamics at the air-liquid interface on the stability of a distinct protein solution, as a general contribution to understanding different colloidal and biological interfacial systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A clear answer on whether vaping is safe and, if not, to what degree it threatens human health and well-being, still needs to be communicated. Such an answer requires collecting, analyzing, and interpreting sometimes conflicting and indeterminate results. This paper reviews the most recently published research articles that examine vaping toxicities. It highlights the differences in the techniques employed from one paper to another. While e-cigarettes do not appear to cause the same degree of harm as cigarettes, they pose a real biological threat regarding inflammation, oxidative stress, mucociliary interference, and membrane damage. The concentration of nicotine present is directly related to these endpoints and is often higher in fourth-generation devices. However, third-generation devices can do more harm than their successors, possibly due to their high voltage and low resistance capabilities. In addition to nicotine, the flavorants used in e-cigarettes have also been shown to relate to biological stress, and the adverse health effects increase in vape formulations with higher concentrations and numbers of flavor types. Different biological models also yield different health effects, especially when comparing bronchial and alveolar cells or tissues. To universalize the results of vape experiments, researchers should seek greater consistency within the experimental design. Key methodological variables must be recognized and disclosed in future research, including puff duration and number, types of e-cigarettes and e-liquids being tested, device settings during aerosolization, and any details of the employed exposure method that may affect dosimetry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Previous research has indicated that ultrafine particles (UFPs, particles less than 100 nm) emitted from desktop three-dimensional (3D) printers exhibit cytotoxicity. However, only a limited number of particles from different filaments and their combinations have been tested for cytotoxicity. This study quantified the emissions of UFPs from a commercially available filament extrusion desktop 3D printer using three different filaments, including acrylonitrile butadiene Styrene (ABS), thermoplastic polyurethane (TPU), and polyethylene terephthalate glycol (PETG). In this study, controlled experiments were conducted where the particles emitted were used to expose cells grown in an air-liquid interface (ALI) system. The ALI exposures were utilized for in vitro characterization of particle mixtures, including UFPs from a 3D printer. Additionally, a lactate dehydrogenase (LDH) assay was used to evaluate the cytotoxic effects of these UFPs. A549 cells were exposed at the ALI to UFPs generated by an operational 3D printer for an average of 45 and 90 min. Twenty-four hours post-exposure, the cells were analyzed for percent cytotoxicity in a 24-well ALI insert (LDH assay). UFP exposure resulted in diminished cell viability, as evidenced by significantly increased LDH levels. The findings demonstrate that ABS has the most significant particle emission. ABS was the only filament that showed a significant difference compared to the high efficiency particulate arrestance (HEPA) following 90 min of exposure (p-value < 0.05). Both ABS and PETG exhibited a significant difference compared to the HEPA control after 45 min of exposure. A preliminary analysis of potential exposure to these products in a typical environment advises caution when operating multiple printer and filament combinations in poorly ventilated spaces or without combined gas and particle filtration systems.

Several oncogenic viruses are associated with approximately 20% of human cancers. Experimental models are crucial for studying the pathogenicity and biological aspects of oncogenic viruses and their potential mechanisms in tumorigenesis. Current cell models have considerable limitations such as: their low yield, genetic and epigenetic modification, and reduction in tumor heterogeneity during long propagation. Cancer cell lines are limited and not appropriate for studying the viral life cycle, for example, natural viral life cycles of HPV and EBV, and their persistence and latency in epithelial cells are poorly understood, since these processes are highly related to epithelial differentiation. Therefore, there is an urgent need of reliable human physiological cell models to study viral life cycle and cancer initiation. Conditional cell reprogramming (CCR) is a rapid and robust cell culture system, where the cells can be established from minimally invasive or noninvasive specimens and their lineage functions preserved during the long-term culture. These CR cells retain their ability to differentiate at air–liquid interface (ALI). Here, we recapitulated the applications of CR and ALI approaches in modeling host–virus interactions and viral-mediated tumorigenesis.

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro models. [ABSTRACT FROM AUTHOR]

This study investigated the inhalation toxicity of the emissions from 3-D printing with acrylonitrile butadiene styrene (ABS) filament using an air-liquid interface (ALI) in vitro model. Primary normal human-derived bronchial epithelial cells (NHBEs) were exposed to ABS filament emissions in an ALI for 4 hours. The mean and mode diameters of ABS emitted particles in the medium were 175 ± 24 and 153 ± 15 nm, respectively. The average particle deposition per surface area of the epithelium was 2.29 × 107 ± 1.47 × 107 particle/cm2, equivalent to an estimated average particle mass of 0.144 ± 0.042 μg/cm2. Results showed exposure of NHBEs to ABS emissions did not significantly affect epithelium integrity, ciliation, mucus production, nor induce cytotoxicity. At 24 hours after the exposure, significant increases in the pro-inflammatory markers IL-12p70, IL-13, IL-15, IFN-γ, TNF-α, IL-17A, VEGF, MCP-1, and MIP-1α were noted in the basolateral cell culture medium of ABS-exposed cells compared to non-exposed chamber control cells. Results obtained from this study correspond with those from our previous in vivo studies, indicating that the increase in inflammatory mediators occur without associated membrane damage. The combination of the exposure chamber and the ALI-based model is promising for assessing 3-D printer emission-induced toxicity. [ABSTRACT FROM AUTHOR]

Photochemical vapor generation (PVG) of germanium (Ge) was first reported in this work. The synergistic effect from cobalt/chloride ions and air-liquid interfaces was found for the PVG of Ge. No obvious signal response was observed from the standard solution of Ge in 10% (v/v) formic acids (FAs) under UV irradiation. The addition of 300 mg L−1 of Co2+ and 30 mmol L−1 of Cl− resulted in enhanced photochemical reduction for Ge, and the introduction of air-liquid interfaces proceeding and succeeding the sample solution caused another 4.6 folds of enhancement in signal response of Ge. Under the selected condition, the limit of detection (LOD, 3σ, n = 11) was obtained to be 0.008 ng mL−1 with inductively coupled plasma mass spectrometry (ICP MS) measurement. A good precision, expressed as a relative standard deviation (RSD, n = 7) of 2.0%, was found from replicated measurements of 2 ng mL−1 of Ge. The generation efficiency was found to be no better than 9 ± 2%. The PVG mechanism of Ge was investigated in this work. The new finding is useful for understanding the principle of PVG, and further exploring the analytical and environmental application of PVG. [ABSTRACT FROM AUTHOR]

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm2, comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment. [ABSTRACT FROM AUTHOR]

The exposure of lung epithelium to environmental hazards is linked to several chronic respiratory diseases. We assessed the ability of an inhaled dry powder (DPI) medical device product (PolmonYDEFENCE/DYFESATM, SOFAR SpA, Trezzano Rosa, Italy), using a formulation of sodium hyaluronate (Na-Hya) as the key ingredient as a defensive barrier to protect the upper respiratory tract. Specifically, it was evaluated if the presence of the barrier formed by sodium hyaluronate present on the cells, reducing direct contact of the urban dust (UD) with the surface of cells can protect them in an indirect manner by the inflammatory and oxidative process started in the presence of the UD. Cytotoxicity and the protection capability against the oxidative stress of the product were tested in vitro using Calu-3 cells exposure to UD as a trigger for oxidative stress. Inflammation and wound healing were assessed using an air-liquid interface (ALI) culture model of the Calu-3 cells. Deposition studies of the formulation were conducted using a modified Anderson cascade impactor (ACI) and the monodose PillHaler® dry powder inhaler (DPI) device, Na-Hya was detected and quantified using high-performance-liquid-chromatography (HPLC). Solubilised PolmonYDEFENCE/DYFESATM gives protection against oxidative stress in Calu-3 cells in the short term (2 h) without any cytotoxic effects. ALI culture experiments, testing the barrier-forming (non-solubilised) capabilities of PolmonYDEFENCE/DYFESATM, showed that the barrier layer reduced inflammation triggered by UD and the time for wound closure compared to Na-Hya alone. Deposition experiments using the ACI and the PillHaler® DPI device showed that the majority of the product was deposited in the upper part of the respiratory tract. Finally, the protective effect of the product was efficacious for up to 24 h without affecting mucus production. We demonstrated the potential of PolmonYDEFENCE/DYFESATM as a preventative barrier against UD, which may aid in protecting the upper respiratory tract against environmental hazards and help with chronic respiratory diseases. [ABSTRACT FROM AUTHOR]

The emergence of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inspired rapid research efforts targeting the host range, pathogenesis and transmission mechanisms, and the development of antiviral strategies. Genetically modified mice, rhesus macaques, ferrets, and Syrian golden hamsters have been frequently used in studies of pathogenesis and efficacy of antiviral compounds and vaccines. However, alternatives to in vivo experiments, such as immortalized cell lines, primary respiratory epithelial cells cultured at an air–liquid interface, stem/progenitor cell-derived organoids, or tissue explants, have also been used for isolation of SARS-CoV-2, investigation of cytopathic effects, and pathogen–host interactions. Moreover, initial proof-of-concept studies for testing therapeutic agents can be performed with these tools, showing that animal-sparing cell culture methods could significantly reduce the need for animal models in the future, following the 3R principles of replace, reduce, and refine. So far, only few studies using animal-derived primary cells or tissues have been conducted in SARS-CoV-2 research, although natural infection has been shown to occur in several animal species. Therefore, the need for in-depth investigations on possible interspecies transmission routes and differences in susceptibility to SARS-CoV-2 is urgent. This review gives an overview of studies employing alternative culture systems like primary cell cultures, tissue explants, or organoids for investigations of the pathophysiology and reverse zoonotic potential of SARS-CoV-2 in animals. In addition, future possibilities of SARS-CoV-2 research in animals, including previously neglected methods like the use of precision-cut lung slices, will be outlined. [ABSTRACT FROM AUTHOR]

The occupational exposure to particles such as crystalline quartz and its impact on the respiratory tract have been studied extensively in recent years. For hazard assessment, the development of physiologically more relevant in-vitro models, i.e., air-liquid interface (ALI) cell cultures, has greatly progressed. Within this study, pulmonary culture models employing A549 and differentiated THP-1 cells as mono-and co-cultures were investigated. The different cultures were exposed to α-quartz particles (Min-U-Sil5) with doses ranging from 15 to 66 µg/cm2 under submerged and ALI conditions and cytotoxicity as well as cytokine release were analyzed. No cytotoxicity was observed after ALI exposure. Contrarily, Min-U-Sil5 was cytotoxic at the highest dose in both submerged mono- and co-cultures. A concentration-dependent release of interleukin-8 was shown for both exposure types, which was overall stronger in co-cultures. Our findings showed considerable differences in the toxicological responses between ALI and submerged exposure and between mono- and co-cultures. A substantial influence of the presence or absence of serum in cell culture media was noted as well. Within this study, the submerged culture was revealed to be more sensitive. This shows the importance of considering different culture and exposure models and highlights the relevance of communication between different cell types for toxicological investigations. [ABSTRACT FROM AUTHOR]

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Abstract Background Exposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air–liquid interface (ALI) and dynamic culture (DC) systems. Methods HPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI–DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI–DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting. Results Scanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI–DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI–DC platform, indicating that the ALI–DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs. Conclusion A novel ALI–DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with unprecedented economic and societal impact. Currently, several vaccines are available and multitudes of antiviral treatments have been proposed and tested. Although many of the vaccines show clinical efficacy, they are not equally accessible worldwide. Additionally, due to the continuous emergence of new variants and generally short duration of immunity, the development of effective antiviral treatments remains of the utmost importance. Since the emergence of SARS-CoV-2, substantial efforts have been undertaken to repurpose existing drugs for accelerated clinical testing and emergency use authorizations. However, drug-repurposing studies using cellular assays often identify hits that later prove ineffective clinically, highlighting the need for more complex screening models. To this end, we evaluated the activity of single compounds that have either been tested clinically or already undergone extensive preclinical profiling, using a standardized in vitro model of human nasal epithelium. Furthermore, we also evaluated drug combinations based on a sub-maximal concentration of molnupiravir. We report the antiviral activity of 95 single compounds and 30 combinations. We show that only a few single agents are highly effective in inhibiting SARS-CoV-2 replication while selected drug combinations containing 10 µM molnupiravir boosted antiviral activity compared to single compound treatment. These data indicate that molnupiravir-based combinations are worthy of further consideration as potential treatment strategies against coronavirus disease 2019 (COVID-19).

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff’s reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.

Abstract Background The asthma-related airway wall remodeling is associated i.a. with a damage of bronchial epithelium and subepithelial fibrosis. Functional interactions between human bronchial epithelial cells and human bronchial fibroblasts are known as the epithelial-mesenchymal trophic unit (EMTU) and are necessary for a proper functioning of lung tissue. However, a high concentration of the transforming growth factor-β1 (TGF-β1) in the asthmatic bronchi drives the structural disintegrity of epithelium with the epithelial-to-mesenchymal transition (EMT) of the bronchial epithelial cells, and of subepithelial fibrosis with the fibroblast-to-myofibroblast transition (FMT) of the bronchial fibroblasts. Since previous reports indicate different intrinsic properties of the human bronchial epithelial cells and human bronchial fibroblasts which affect their EMT/FMT potential beetween cells derived from asthmatic and non-asthmatic patients, cultured separatelly in vitro, we were interested to see whether corresponding effects could be obtained in a co-culture of the bronchial epithelial cells and bronchial fibroblasts. In this study, we investigate the effects of the TGF-β1 on the EMT markers of the bronchial epithelial cells cultured in the air-liquid-interface and effectiveness of FMT in the bronchial fibroblast populations in the EMTU models. Results Our results show that the asthmatic co-cultures are more sensitive to the TGF-β1 than the non-asthmatic ones, which is associated with a higher potential of the asthmatic bronchial cells for a profibrotic response, analogously to be observed in '2D' cultures. They also indicate a noticeable impact of human bronchial epithelial cells on the TGF-β1-induced FMT, stronger in the asthmatic bronchial fibroblast populations in comparison to the non-asthmatic ones. Moreover, our results suggest the protective effects of fibroblasts on the structure of the TGF-β1–exposed mucociliary differentiated bronchial epithelial cells and their EMT potential. Conclusions Our data are the first to demonstrate a protective effect of the human bronchial fibroblasts on the properties of the human bronchial epithelial cells, which suggests that intrinsic properties of not only epithelium but also subepithelial fibroblasts affect a proper condition and function of the EMTU in both normal and asthmatic individuals.

Culturing respiratory epithelial cells at an air–liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.

The mucociliary airway epithelium lines the human airways and is the primary site of host-environmental interactions in the lung. Following virus infection, airway epithelial cells initiate an innate immune response to suppress virus replication. Therefore, defining the virus-host interactions of the mucociliary airway epithelium is critical for understanding the mechanisms that regulate virus infection, including Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Non-human primates (NHP) are closely related to humans and provide a model to study human disease. However, ethical considerations and high costs can restrict the use of in vivo NHP models. Therefore, there is a need to develop in vitro NHP models of human respiratory virus infection that would allow for rapidly characterizing virus tropism and the suitability of specific NHP species to model human infection. Using the olive baboon (Papio anubis), we have developed methodologies for the isolation, in vitro expansion, cryopreservation, and mucociliary differentiation of primary fetal baboon tracheal epithelial cells (FBTECs). Furthermore, we demonstrate that in vitro differentiated FBTECs are permissive to SARS-CoV-2 infection and produce a potent host innate-immune response. In summary, we have developed an in vitro NHP model that provides a platform for the study of SARS-CoV-2 infection and other human respiratory viruses.

Background: Human rhinovirus C (HRV-C) accounts for a large proportion of HRV-related illnesses, but the immune response to HRV-C infection has not been elucidated. Our objective was to assess the effect of HRV-C on cytokine secretion in human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI) and compare it with that of respiratory syncytial virus (RSV). Methods: HBE cells were differentiated at ALI culture and the full-length cDNA clones of HRV-C651 and HRV-C15, clinical isolates of HRV-C79 and HRV-C101, and two RSV isolates were inoculated in the HBE cells. The effect of HRV-C on cytokine secretion was assessed and compared with that of RSV. Results: HRV-Cs infect and propagate in fully differentiated HBE cells and significantly increase the secretion of IFN-λ1, CCL5, IP10, IL-6, IL-8, and MCP-1. The virus loads positively correlated with the levels of the cytokines. HRV-C induced lower secretion of CCL5 (P = 0.048), IL-6 (P = 0.016), MCP-1 (P = 0.008), and IL-8 (P = 0.032), and similar secretion of IP10 (P = 0.214) and IFN-λ1 (P = 0.214) when compared with RSV. Conclusion: HBE ALI culture system supported HRV-C infection and propagation and HRV-C induced relatively weaker cytokine expression than RSV. [ABSTRACT FROM AUTHOR]

Nanoparticle toxicity assessments have moved closer to physiological conditions while trying to avoid the use of animal models. An example of new in vitro exposure techniques developed is the exposure of cultured cells at the air–liquid interface (ALI), particularly in the case of respiratory airways. While the commercially available VITROCELL® Cloud System has been applied for the delivery of aerosolized substances to adherent cells under ALI conditions, it has not yet been tested on lung surfactant and semi-adherent cells such as alveolar macrophages, which are playing a pivotal role in the nanoparticle-induced immune response. Objectives: In this work, we developed a comprehensive methodology for coating semi-adherent lung cells cultured at the ALI with aerosolized surfactant and subsequent dose-controlled exposure to nanoparticles (NPs). This protocol is optimized for subsequent transcriptomic studies. Methods: Semi-adherent rat alveolar macrophages NR8383 were grown at the ALI and coated with lung surfactant through nebulization using the VITROCELL® Cloud 6 System before being exposed to TiO 2 NM105 NPs. After NP exposures, RNA was extracted and its quantity and quality were measured. Results: The VITROCELL® Cloud system allowed for uniform and ultrathin coating of cells with aerosolized surfactant mimicking physiological conditions in the lung. While nebulization of 57 μ L of 30 mg/mL TiO 2 and 114 μ L of 15 mg/mL TiO 2 nanoparticles yielded identical cell delivered dose, the reproducibility of dose as well as the quality of RNA extracted were better for 114 μ L. [ABSTRACT FROM AUTHOR]

Several animal species are susceptible to SARS-CoV-2 infection, as documented by case reports and serological and in vivo infection studies. However, the susceptibility of many animal species remains unknown. Furthermore, the expression patterns of SARS-CoV-2 entry factors, such as the receptor angiotensin-converting enzyme 2 (ACE2), as well as transmembrane protease serine subtype 2 (TMPRSS2) and cathepsin L (CTSL), cellular proteases involved in SARS-CoV-2 spike protein activation, are largely unexplored in most species. Here, we generated primary cell cultures from the respiratory tract of domestic and wildlife animals to assess their susceptibility to SARS-CoV-2 infection. Additionally, the presence of ACE2, TMPRSS2 and CTSL within respiratory tract compartments was investigated in a range of animals, some with unknown susceptibility to SARS-CoV-2. Productive viral replication was observed in the nasal mucosa explants and precision-cut lung slices from dogs and hamsters, whereas culture models from ferrets and multiple ungulate species were non-permissive to infection. Overall, whereas TMPRSS2 and CTSL were equally expressed in the respiratory tract, the expression levels of ACE2 were more variable, suggesting that a restricted availability of ACE2 may contribute to reduced susceptibility. Summarized, the experimental infection of primary respiratory tract cell cultures, as well as an analysis of entry-factor distribution, enable screening for SARS-CoV-2 animal reservoirs. [ABSTRACT FROM AUTHOR]

[Display omitted] • Air-control data from three donors across multiple years of work show donor cross-comparison is possible. • Comparison of adapted Vitrocell VC10 and Borgwaldt LM4E for in vitro e-cigarette exposure in MucilAir. • ePen3 e-cigarette response in MucilAir comparable across both aerosols generating system used. • Allows further comparison of previously published eBox with ePen3 responses in MucilAir. • New e-cigarette technology in ePen3 reduced toxicity compared to eBox and cigarette smoke. In vitro studies play an important role in supporting the toxicological assessment of e-cigarettes, with many current methods reliant on sophisticated in vitro exposure systems designed for conventional cigarette testing. In this study, we have compared two distinct systems; the modified Vitrocell VC10 and Borgwaldt LM4E designed to deliver undiluted e-cigarette aerosol. We assessed the cytotoxicity response of 3D reconstituted lung tissue (MucilAir) exposed to undiluted aerosol from ePen3 (closed modular e-cigarette) using these two exposure systems. As the induced cytotoxicity profiles were comparable, we then compared these responses against historical eBox (open modular e-cigarette) and 3R4F reference cigarette data to show evolution of product technology. This latter approach was deemed possible by monitoring intrinsic donor-to-donor control variability over a three-year period, bridging between exposure systems and observed biological responses. Despite the differences in the technology, on a puff-by-puff basis these machines gave remarkably similar cytotoxicity profiles for ePen3, as determined by MTT, and consistency of pre-cytotoxicity markers: transepithelial electrical resistance (TEER), cilia beat frequency and cilia active area. When responses are compared as a function of exposed nicotine concentration, we see differences due to the dynamics of the exposure systems. The parity of responses between the systems in generated undiluted aerosol has allowed us to compare back to previously published eBox data, irrespective of aerosol generating system and MucilAir donor, showing how evolution from open systems to podmod e-cigarette design can make a step change in the cytotoxicity profile of the product. [ABSTRACT FROM AUTHOR]

The calculation results on the 3-D fluid motion induced by the oscillating circular cylinders through the air-water interface are presented. The boundary immersed method without tracking the gasliquid interface by introducing the pressure divided by the fluid density is used. The calculation results were qualitatively verified by comparing them with the experimental studies. The results imply that the three-dimensional flow induced by the oscillating cylinders is extremely complex despite the simplicity of the system. [ABSTRACT FROM AUTHOR]

Ultrafine particles represent a growing concern in the public health community but their precise role in many illnesses is still unknown. This lack of knowledge is related to the experimental difficulty in linking their biological effects to their multiple properties, which are important determinants of toxicity. Our aim is to propose an interdisciplinary approach to study fine (FP) and ultrafine (UFP) particles, generated in a controlled manner using a miniCAST (Combustion Aerosol Standard) soot generator used with two different operating conditions (CAST1 and CAST3). The chemical characterization was performed by an untargeted analysis using ultra-high resolution mass spectrometry. In conjunction with this approach, subsequent analysis by gas chromatography–mass spectrometry (GC–MS) was performed to identify polycyclic aromatic hydrocarbons (PAH). CAST1 enabled the generation of FP with a predominance of small PAH molecules, and CAST3 enabled the generation of UFP, which presented higher numbers of carbon atoms corresponding to larger PAH molecules. Healthy normal human bronchial epithelial (NHBE) cells differentiated at the air-liquid interface (ALI) were directly exposed to these freshly emitted FP and UFP. Expression of MUC5AC, FOXJ1, OCLN and ZOI as well as microscopic observation confirmed the ciliated pseudostratified epithelial phenotype. Study of the mass deposition efficiency revealed a difference between the two operating conditions, probably due to the morphological differences between the two categories of particles. We demonstrated that only NHBE cells exposed to CAST3 particles induced upregulation in the gene expression of IL-8 and NQO1. This approach offers new perspectives to study FP and UFP with stable and controlled properties. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] Organophosphorus-based ligands represent a versatile set of solvent extraction reagents whose chemical makeup plays an important role in extraction mechanism. We hypothesize that the branching of the extractant hydrophobic tail and its oil-phase solvation affect the liquid/liquid interfacial structure. Understanding the structure mediated adsorption and interfacial ordering becomes key in designing ligands with enhanced selectivity and efficiency for targeted extractions. We employed vibrational sum frequency generation spectroscopy and interfacial tension measurements to extract thermodynamic adsorption energies, map interfacial ordering, and rationalize disparate behaviors of model di-(2-ethylhexyl) phosphoric acid and dioctyl phosphoric acid ligands at the hexadecane water interface. With increased surface loading, ligands with branched hydrophobic tails formed stable interfaces at much lower concentrations than those observed for ligands with linear alkyl tails. The lack of an oil phase and associated solvation results in markedly different interfacial properties, and thus measurements made at air/liquid surfaces cannot be assumed to correlate with the processes occurring at buried liquid/liquid interfaces. We attribute these differences in the surface mediated self-assembly to key variations in hydrophobic interactions and tail solvation taking place in the oil phase demonstrating that interactions in both the polar and nonpolar phases are essential to understand self-assembly and function. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the characteristics of Calu-3 cells as a model to examine the toxicological responses of inhalable substances. Calu-3 cells were grown to the confluence at an air-liquid interface (ALI) using a Transwell® permeable support system. The ALI resulted in biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles. We further characterized and optimized the Calu-3 cell line model using ALI culturing conditions, immunolabeling of protein expression, ultrastructural analysis using scanning electron microscopy (SEM), and transepithelial electrical resistance (TEER) measurements, and then screened for the cytotoxicity of tobacco flavoring extracts. Calu-3 cells displayed dose-dependent responses when treated with the flavoring extract. Within 8–10 days, cell monolayers developed TEER ≥1000 Ω·cm2. During this time, Calu-3 cells exposed to flavoring extracts X01 and X06 exhibited a loss of cellular integrity and decreased ZO-1 and E-cadherin protein expression. In conclusion, we investigated the Calu-3 cell line culture conditions, culture time, and barrier integrity and tested the effect of six new synthetic tobacco flavoring extracts. Our data demonstrate that the Calu-3 human bronchial epithelial cell monolayer system is a potential in vitro model to assess the inhalation toxicity of inhalable substances. [ABSTRACT FROM AUTHOR]

Iota-carrageenan (IC) nasal spray, a medical device approved for treating respiratory viral infections, has previously been shown to inhibit the ability of a variety of respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), to enter and replicate in the cell by interfering with the virus binding to the cell surface. The aim of this study was to further investigate the efficacy and safety of IC in SARS-CoV-2 infection in advanced in vitro models of the human respiratory epithelium, the primary target and entry port for SARS-CoV-2. We extended the in vitro safety assessment of nebulized IC in a 3-dimensional model of reconstituted human bronchial epithelium, and we demonstrated the efficacy of IC in protecting reconstituted nasal epithelium against viral infection and replication of a patient-derived SARS-CoV-2 strain. The results obtained from these two advanced models of human respiratory tract epithelia confirm previous findings from in vitro SARS-CoV-2 infection assays and demonstrate that topically applied IC can effectively prevent SARS-CoV-2 infection and replication. Moreover, the absence of toxicity and functional and structural impairment of the mucociliary epithelium demonstrates that the nebulized IC is well tolerated.

Knowledge of interfacial structures in liquid–liquid systems is imperative, especially for improving two-phase biological and chemical reactions. Therefore, we developed a new sample cell for neutron reflectometry (NR), which enables us to observe the layer structure around the interface, and investigated the adsorption behavior of a typical surfactant, sodium dodecyl sulfate (SDS), on the toluene-d8-D2O interface under the new experimental conditions. The new cell was characterized by placing the PTFE frame at the bottom to produce a smooth interface and downsized compared to the conventional cell. The obtained NR profiles were readily analyzable and we determined a slight difference in the SDS adsorption layer structure at the interface between the toluene-d8-D2O and air-D2O systems. This could be owing to the difference in the adsorption behavior of the SDS molecules depending on the interfacial conditions. [ABSTRACT FROM AUTHOR]

Summary: Soy protein isolate (SPI) is widely used as a foaming agent due to its rich nutritional value and low price. Foaming is one of the key processing characteristics in food processing and is generally used in foam‐type foods. It plays an essential role in the flavour quality and appearance characteristics of food. Generally speaking, the foaming activity and foaming stability of regular SPI are not sufficient enough to meet the needs of practical applications. There is an urgent need to solve this drawback and improve the status quo of the food processing industry. The current research on the foaming properties of SPI across the globe focuses on improving foaming characteristics through optimising influencing factors and the use of different modification methods. Hence, this review aims to sort out the scientific research on the foaming mechanism of SPI and summarises evaluation methods, influencing factors and modification methods to improve the foamability of SPI to explore more applications of SPI. It will provide a theoretical basis for the production of SPI natural green active agents with good performance. [ABSTRACT FROM AUTHOR]

Microscopic analysis of mucus quantity and composition is crucial in research and diagnostics on muco‐obstructive diseases. Currently used image‐based methods are unable to extract concrete numeric values of mucosal proteins, especially on the expression of the key mucosal proteins MUC5AC and MUC5B. Since their levels increase under pathologic conditions such as extensive exposure to cigarette smoke, it is imperative to quantify them to improve treatment strategies of pulmonary diseases. This study presents a simple, image‐based, and high‐processing computational method that allows determining the ratio of MUC5AC and MUC5B within the overall airway mucus while providing information on their spatial distribution. The presented pipeline was optimized for automated downstream analysis using a combination of bright field and immunofluorescence imaging suitable for tracheal and bronchial tissue samples, and air–liquid interface (ALI) cell cultures. To validate our approach, we compared tracheal tissue and ALI cell cultures of isolated primary normal human bronchial epithelial cells derived from smokers and nonsmokers. Our data indicated 18‐fold higher levels of MUC5AC in submucosal glands of smokers covering about 8% of mucosal areas compared to <1% in nonsmoking individuals, confirming results of previous studies. We further identified a subpopulation of nonsmokers with slightly elevated glandular MUC5AC levels suggesting moderate exposure to second‐hand smoke or fine particulate air pollution. Overall, this study demonstrates a novel, user‐friendly and freely available tool for digital pathology and the analysis of therapeutic interventions tested in ALI cell cultures. [ABSTRACT FROM AUTHOR]

Background: Lycium barbarum berries have been utilized in Asia for many years. However, the mechanisms of its lung-defensive properties are indeterminate. Objective: We investigate whether L. barbarum polysaccharide (LBP) could weaken Pseudomonas aeruginosa infection-induced lung injury. Design: Mice primary air-liquid interface epithelial cultures were pretreated with LBP and subsequently treated with pyocyanin (PCN). Lung injury, including apoptosis, inflammation, and oxidative stress, was estimated by western blot, enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction, Real-time qPCR (Q-PCR). Flow cytometry was used to test cell apoptosis. Moreover, Balb/c mice were used to evaluate the tissue injury. We used hematoxylin-eosin staining and immunofluorescence to detect the expression of related proteins and tissue damage in mouse lungs and spleen. Results: The flow cytometric analysis shows the potential of LBP to reduce time-dependent cell death by PCN. Mechanistically, LBP reduces PCN-induced expression of proapoptotic proteins and caspase3 and induces the activation of Bcl-2 in mice bronchial epithelial cells. Similarly, LBP reduces PCN-induced intracellular reactive oxygen species (ROS) production. Moreover, LBP inhibits the production of inflammatory cytokines, Interleukin (IL-1β), Tumor Necrosis Factor (TNF), IL-6, and IL-8. Our study confirms the ability of LBP to retard PCN-induced injury in mice lung and spleen. Conclusions: The inhibition of PCN-induced lung injury by LBP is capable of protecting mice cells from injury. [ABSTRACT FROM AUTHOR]

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ABSTRACT Canine distemper virus (CDV) is a highly contagious pathogen and is known to enter the host via the respiratory tract and disseminate to various organs. Current hypotheses speculate that CDV uses the homologous cellular receptors of measles virus (MeV), SLAM and nectin-4, to initiate the infection process. For validation, here, we established the well-differentiated air-liquid interface (ALI) culture model from primary canine tracheal airway epithelial cells. By applying the green fluorescent protein (GFP)-expressing CDV vaccine strain and recombinant wild-type viruses, we show that cell-free virus infects the airway epithelium mainly via the paracellular route and only after prior disruption of tight junctions by pretreatment with EGTA; this infection was related to nectin-4 but not to SLAM. Remarkably, when CDV-preinfected DH82 cells were cocultured on the basolateral side of canine ALI cultures grown on filter supports with a 1.0-μm pore size, cell-associated CDV could be transmitted via cell-to-cell contact from immunocytes to airway epithelial cultures. Finally, we observed that canine ALI cultures formed syncytia and started to release cell-free infectious viral particles from the apical surface following treatment with an inhibitor of the JAK/STAT signaling pathway (ruxolitinib). Our findings show that CDV can overcome the epithelial barrier through different strategies, including infection via immunocyte-mediated transmission and direct infection via the paracellular route when tight junctions are disrupted. Our established model can be adapted to other animals for studying the transmission routes and the pathogenicity of other morbilliviruses. IMPORTANCE Canine distemper virus (CDV) is not only an important pathogen of carnivores, but it also serves as a model virus for analyzing measles virus pathogenesis. To get a better picture of the different stages of infection, we used air-liquid interface cultures to analyze the infection of well-differentiated airway epithelial cells by CDV. Applying a coculture approach with DH82 cells, we demonstrated that cell-mediated infection from the basolateral side of well-differentiated epithelial cells is more efficient than infection via cell-free virus. In fact, free virus was unable to infect intact polarized cells. When tight junctions were interrupted by treatment with EGTA, cells became susceptible to infection, with nectin-4 serving as a receptor. Another interesting feature of CDV infection is that infection of well-differentiated airway epithelial cells does not result in virus egress. Cell-free virions are released from the cells only in the presence of an inhibitor of the JAK/STAT signaling pathway. Our results provide new insights into how CDV can overcome the barrier of the airway epithelium and reveal similarities and some dissimilarities compared to measles virus.

The effectiveness of inhalation therapy depends on aerosol size distribution, which determines the penetration and regional deposition of drug in the lungs. As the size of droplets inhaled from medical nebulizers varies depending on the physicochemical properties of the nebulized liquid, it can be adjusted by adding some compounds as viscosity modifiers (VMs) of a liquid drug. Natural polysaccharides have been recently proposed for this purpose and while they are biocompatible and generally recognized as safe (GRAS), their direct influence of the pulmonary structures is unknown. This work studied the direct influence of three natural VMs (sodium hyaluronate, xanthan gum, and agar) on the surface activity of the pulmonary surfactant (PS) measured in vitro using the oscillating drop method. The results allowed for comparing the variations of the dynamic surface tension during breathing-like oscillations of the gas/liquid interface with the PS, and the viscoelastic response of this system, as reflected by the hysteresis of the surface tension. The analysis was done using quantitative parameters, i.e., stability index (SI), normalized hysteresis area (HAn), and loss angle (φ), depending on the oscillation frequency (f). It was also found that, typically, SI is in the range of 0.15–0.3 and increases nonlinearly with f, while φ slightly decreases. The effect of NaCl ions on the interfacial properties of PS was noted, which was usually positive for the size of hysteresis with an HAn value up to 2.5 mN/m. All VMs in general were shown to have only a minor effect on the dynamic interfacial properties of PS, suggesting the potential safety of the tested compounds as functional additives in medical nebulization. The results also demonstrated relationships between the parameters typically used in the analysis of PS dynamics (i.e., HAn and SI) and dilatational rheological properties of the interface, allowing for easier interpretation of such data.

In recent years, the use of carbon fibers (CFs) in various sectors of industry has been increasing. Despite the similarity of CF degradation products to other toxicologically relevant materials such as asbestos fibers and carbon nanotubes, a detailed toxicological evaluation of this class of material has yet to be performed. In this work, we exposed advanced air–liquid interface cell culture models of the human lung to CF. To simulate different stresses applied to CF throughout their life cycle, they were either mechanically (mCF) or thermo-mechanically pre-treated (tmCF). Different aspects of inhalation toxicity as well as their possible time-dependency were monitored. mCFs were found to induce a moderate inflammatory response, whereas tmCF elicited stronger inflammatory as well as apoptotic effects. Furthermore, thermal treatment changed the surface properties of the CF resulting in a presumed adhesion of the cells to the fiber fragments and subsequent cell loss. Triple-cultures encompassing epithelial, macrophage, and fibroblast cells stood out with an exceptionally high inflammatory response. Only a weak genotoxic effect was detected in the form of DNA strand breaks in mono- and co-cultures, with triple-cultures presenting a possible secondary genotoxicity. This work establishes CF fragments as a potentially harmful material and emphasizes the necessity of further toxicological assessment of existing and upcoming advanced CF-containing materials.

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air–liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air–liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

Monoclonal antibodies (mAbs) are a successful class of therapeutics, but their development can be challenging due to the risk of degradation that could happen during manufacturing, storage, and clinical use. One of the common causes of degradation is agitation stress from transportation and clinical handling, which increases interfacial stresses to mAbs. For example, the preparation of the dose solution prior to administration often requires diluting therapeutic mAbs in intravenous (IV) infusion bags containing normal saline, which can substantially reduce the level of protective surfactant and increase the level of salt in mAb solutions. Then the interfacial stress in the subsequent transportation of IV bags can cause mAb aggregation or even particle formation. To better understand the complex interplay between dilution, interfacial stress, and salt, we studied the impact of sodium chloride (NaCl) on the aggregation of two mAbs under agitation stress. We found that the presence of NaCl accelerates the aggregation of both mAbs, but the aggregation mechanism, morphology, and reversibility are very different. Our results clearly highlight the impact of salt on mAb stability at the clinical in-use condition. We believe this study further increases our understanding of protein aggregation mediated by interfacial stresses and brings valuable insights to support development of mAb formulations for patients., (© PDA, Inc. 2024.)

The colonic epithelium is comprised of three-dimensional crypts (3D) lined with mucus secreted by a heterogeneous population of goblet cells. In this study, we report the formation of a long-lived, and self-renewing replica of human 3D crypts with a mucus layer patterned in the X-Y-Z dimensions. Primary colon cells were cultured on a shaped scaffold under an air-liquid interface to yield architecturally accurate crypts with a mucus bilayer (605 ± 180 μm thick) possessing an inner (149 ± 50 μm) and outer (435 ± 111 μm) region. Lectins with distinct carbohydrate-binding preferences demonstrated that the mucus in the intercrypt regions was chemically distinct from that above and within the crypts replicating in vivo chemical patterning. Constitutive mucus secretion ejected beads from crypt lumens in 8-10 days, while agonist-stimulated secretion increased mucus thickness by 17-fold in 8 h. The tissue was long-lived, > 50 days, the longest time assessed. In conclusion, the in vitro mucus replicated key physiology of the human mucus, including the bilayer (Z) structure and intercrypt-crypt (X-Y) zones, constitutive mucus flow, spatially complex chemical attributes, and mucus secretion response to stimulation, with the potential to reveal local and global determinants of mucus function and its breakdown in disease., (© 2024. The Author(s).)

The generation of lung epithelial cells through the directed differentiation of human pluripotent stem cells (hPSCs) in vitro provides a platform to model both embryonic lung development and adult airway disease. Here, we describe a robust differentiation protocol that closely recapitulates human embryonic lung development. Differentiating cells progress through obligate intermediate stages, beginning with definitive endoderm formation and then patterning into anterior foregut endoderm that yields lung progenitors (LPs) with extended culture. These LPs can be purified using the cell surface marker CD166 (also known as ALCAM), and further matured into proximal airway epithelial cells including basal cells, secretory cells and multiciliated cells using either an organoid platform or culture at the air-liquid interface (ALI). We additionally demonstrate that these hPSC-derived airway epithelial cells can be used to model Influenza A infection. Collectively, our results underscore the utility of CD166 expression for the efficient enrichment of LPs from heterogenous differentiation cultures and the ability of these isolated cells to mature into more specialized, physiologically relevant proximal lung cell types., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: The role of Akt in nasal immunity is unstudied. Akt phosphorylates and activates endothelial nitric oxide synthase (eNOS) expressed in epithelial ciliated cells. Nitric oxide (NO) production by ciliated cells can have antibacterial and antiviral effects. Increasing nasal NO may be a useful antipathogen strategy in chronic rhinosinusitis (CRS). We previously showed that small-molecule Akt activator SC79 induces nasal cell NO production and suppresses IL-8 via the transcription factor Nrf-2. We hypothesized that SC79 NO production may additionally have antibacterial effects., Methods: NO production was measured using fluorescent dye DAF-FM. We tested effects of SC79 during co-culture of Pseudomonas aeruginosa with primary nasal epithelial cells, using CFU counting and live-dead staining to quantify bacterial killing. Pharmacology determined the mechanism of SC79-induced NO production and tested dependence on Akt., Results: SC79 induced dose-dependent, Akt-dependent NO production in nasal epithelial cells. The NO production required eNOS and Akt. The NO released into the airway surface liquid killed P. aeruginosa. No toxicity (LDH release) or inflammatory effects (IL8 transcription) were observed over 24 h., Conclusions: Together, these data suggest multiple immune pathways are stimulated by SC79, with antipathogen effects. This in vitro pilot study suggests that a small-molecule Akt activator may have clinical utility in CRS or respiratory other infection settings, warranting future in vivo studies., (© 2024 ARS‐AAOA, LLC.)

The lung epithelial lining serves as the primary barrier to inhaled environmental toxins, allergens, and invading pathogens. Pulmonary fungal infections are devastating and carry high mortality rates, particularly in those with compromised immune systems. While opportunistic fungi infect primarily immunocompromised individuals, endemic fungi cause disease in immune competent and compromised individuals. Unfortunately, in the case of inhaled fungal pathogens, the airway epithelial host response is vastly understudied. Furthering our lack of understanding, very few studies utilize primary human models displaying pseudostratified layers of various epithelial cell types at air-liquid interface. In this review, we focus on the diversity of the human airway epithelium and discuss the advantages and disadvantages of oncological cell lines, immortalized epithelial cells, and primary epithelial cell models. Additionally, the responses by human respiratory epithelial cells to invading fungal pathogens will be explored. Future investigations leveraging current human in vitro model systems will enable identification of the critical pathways that will inform the development of novel vaccines and therapeutics for pulmonary fungal infections.

Pseudomonas fluorescens SBW25 is a model soil- and plant-associated bacterium capable of forming a variety of air–liquid interface biofilms in experimental microcosms and on plant surfaces. Previous investigations have shown that cellulose is the primary structural matrix component in the robust and well-attached Wrinkly Spreader biofilm, as well as in the fragile Viscous Mass biofilm. Here, we demonstrate that both biofilms include extracellular DNA (eDNA) which can be visualized using confocal laser scanning microscopy (CLSM), quantified by absorbance measurements, and degraded by DNase I treatment. This eDNA plays an important role in cell attachment and biofilm development. However, exogenous high-molecular-weight DNA appears to decrease the strength and attachment levels of mature Wrinkly Spreader biofilms, whereas low-molecular-weight DNA appears to have little effect. Further investigation with CLSM using an amyloid-specific fluorophore suggests that the Wrinkly Spreader biofilm might also include Fap fibers, which might be involved in attachment and contribute to biofilm strength. The robust nature of the Wrinkly Spreader biofilm also allowed us, using MALDI-TOF mass spectrometry, to identify matrix-associated proteins unable to diffuse out of the structure, as well as membrane vesicles which had a different protein profile compared to the matrix-associated proteins. CLSM and DNase I treatment suggest that some vesicles were also associated with eDNA. These findings add to our understanding of the matrix components in this model pseudomonad, and, as found in other biofilms, biofilm-specific products and material from lysed cells contribute to these structures through a range of complex interactions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells, via its spike protein, and transmembrane protease, serine 2 (TMPRSS2) cleaves the spike-ACE2 complex to facilitate virus entry. As rate-limiting steps for virus entry, modulation of ACE2 and/or TMPRSS2 may decrease SARS-CoV-2 infectivity and COVID-19 severity. In silico modeling suggested the natural bioactive flavonoid quercetin can bind to ACE2 and a recent randomized clinical trial demonstrated that oral supplementation with quercetin increased COVID-19 recovery. A range of cultured human cells were assessed for co-expression of ACE2 and TMPRSS2. Immortalized Calu-3 lung cells, cultured and matured at an air-liquid interface (Calu-3-ALIs), were established as the most appropriate. Primary bronchial epithelial cells (PBECs) were obtained from healthy adult males (N = 6) and cultured under submerged conditions to corroborate the outcomes. Upon maturation or reaching 80% confluence, respectively, the Calu-3-ALIs and PBECs were treated with quercetin, and mRNA and protein expression were assessed by droplet digital PCR and ELISA, respectively. SARS-CoV-2 infectivity, and the effects of pre- and co-treatment with quercetin, was assessed by median tissue culture infectious dose assay. Quercetin dose-dependently decreased ACE2 and TMPRSS2 mRNA and protein in both Calu-3-ALIs and PBECs after 4 h, while TMPRSS2 remained suppressed in response to prolonged treatment with lower doses (twice daily for 3 days). Quercetin also acutely decreased ADAM17 mRNA, but not ACE, in Calu-3-ALIs, and this warrants further investigation. Calu-3-ALIs, but not PBECs, were successfully infected with SARS-CoV-2; however, quercetin had no antiviral effect, neither directly nor indirectly through downregulation of ACE2 and TMPRSS2. Calu-3-ALIs were reaffirmed to be an optimal cell model for research into the regulation of ACE2 and TMPRSS2, without the need for prior genetic modification, and will prove valuable in future coronavirus and respiratory infectious disease work. However, our data demonstrate that a significant decrease in the expression of ACE2 and TMPRSS2 by a promising prophylactic candidate may not translate to infection prevention., (© 2024 The Author(s). BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Langmuir-Blodgett (LB) film technology is an advanced technique for the preparation of ordered molecular ultra-thin films at the molecular level, which transfers a single layer of film from the air/water interface to a solid substrate for the controlled assembly of molecules. LB technology has continually evolved over the past century, revealing its potential applications across diverse fields. In this study, the latest research progress of LB film technology is reviewed, with emphasis on its latest applications in gas sensors, electrochemical devices, and bionic films. Additionally, this review evaluates the strengths and weaknesses of LB technology in the application processes and discusses the promising prospects for future application of LB technology.