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Lung surfactant is a complex mixture of phospholipids and surfactant proteins that is produced in alveolar type 2 cells. It prevents lung collapse by reducing surface tension and is involved in innate immunity. Exogenous animal-derived and, more recently, synthetic lung surfactant has shown clinical efficacy in surfactant-deficient premature infants and in critically ill patients with acute respiratory distress syndrome (ARDS), such as those with severe COVID-19 disease. COVID-19 pneumonia is initiated by the binding of the viral receptor-binding domain (RBD) of SARS-CoV-2 to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Inflammation and tissue damage then lead to loss and dysfunction of surface activity that can be relieved by treatment with an exogenous lung surfactant. Surfactant protein B (SP-B) is pivotal for surfactant activity and has anti-inflammatory effects. Here, we study the binding of two synthetic SP-B peptide mimics, Super Mini-B (SMB) and B-YL, to a recombinant human ACE2 receptor protein construct using molecular docking and surface plasmon resonance (SPR) to evaluate their potential as antiviral drugs. The SPR measurements confirmed that both the SMB and B-YL peptides bind to the rhACE2 receptor with affinities like that of the viral RBD–ACE2 complex. These findings suggest that synthetic lung surfactant peptide mimics can act as competitive inhibitors of the binding of viral RBD to the ACE2 receptor.

Graphical abstract, Highlights • We describe how inhaled substances can inhibit lung surfactant function. • The inhibition of lung surfactant function leads to alveolar collapse. • We present the weight of evidence that supports the adverse outcome pathway. • We give an overview of the methods available to measure each key event of the pathway., Inhaled substances, such as consumer products, chemicals at the workplace, and nanoparticles, can affect the lung function in several ways. In this paper, we explore the adverse outcome pathway (AOP) that starts when inhaled substances that reach the alveoli inhibit the function of the lung surfactant, and leads to decreased lung function. Lung surfactant covers the inner surface of the alveoli, and regulates the surface tension at the air–liquid interface during breathing. The inhibition of the lung surfactant function leads to alveolar collapse because of the resulting high surface tension at the end of expiration. The collapsed alveoli can be re-opened by inspiration, but this re-opening causes shear stress on cells covering the alveoli. This can damage the alveolar-capillary membrane integrity, allowing blood components to enter the alveolar airspace. Blood components, such as albumin, can interact with the lung surfactant and further inhibit its function. The collapse of the alveoli is responsible for a decrease in the surface area available for blood oxygenation, and it reduces the volume of air that can be inhaled and exhaled. These different key events lead to decreased lung function, characterized by clinical signs of respiratory toxicity and reduced blood oxygenation. Here we present the weight of evidence that supports the AOP, and we give an overview of the methods available in vitro and in vivo to measure each key event of the pathway, and how this AOP can potentially be used in screening for inhalation toxicity.

Graphical abstract, Highlights • OECD TGs for acute inhalation toxicity testing rely on animal studies. • OECD TGs for acute inhalation toxicity testing focus on systemic effects (LC50). • The in vitro lung surfactant bioassay correlates with decreased lung function. • This bioassay predicts effects caused by lung surfactant function inhibition., To date there are no OECD validated alternative approaches to study toxicity following inhalation exposure to airborne chemicals. The available OECD test guidelines for acute inhalation toxicity aim to estimate a value of the lethal air concentration of the test chemical leading to the death of 50% of the exposed animals (LC50), to satisfy hazard classification and labelling requirements. This paper explores the view that alternative approaches must compare to outcomes of existing guideline methods to become accepted and implemented in a regulatory context. This case study describes the initiatives taken to validate the lung surfactant bioassay, an in vitro cell-free method, and discusses the challenges faced. While the lung surfactant bioassay could not predict the GHS classification for acute inhalation toxicity of 26 chemicals, the assay successfully predicted the clinical signs of respiratory toxicity observed during or shortly after exposure in vivo as reported in registration dossiers. The lung surfactant bioassay is a promising alternative approach to assess the potential of chemicals to cause changes to respiration remaining after exposure (indicating decreased lung function), and can be combined with other test methods in an integrated approach to testing and assessment of inhaled substances.

Identifying the vital states of trapped survivors during post-disaster rescue missions can result in improved rescue strategies and provide injury pre-diagnosis information. The most effective rescue method is the use of bio-radar based non-contact measurements. Presently, bio-radar techniques focus on detecting and locating. Herein, a method to identify vital states with an ultra-wideband bio-radar is proposed, while simulating a trapped condition with Beagle dogs. This investigation revealed three vital stages under the trapped condition: normal, transitioning, and agonal stages. Upon entering the transitioning stage, the heartrates were apparently high, and the respiratory rates increased sharply. The temperatures dropped rapidly once passing this stage. In particular, the respiratory waveforms from the bio-radar frequently change from a normal sine like curve to an “M” like curve within the transitioning stage. The accurate beginning and ending of the transitioning stage are defined by a newly proposed indicator of relative occurrence frequency. Pathological observations indicated that the fragmentation of lamellar bodies within type II alveolar cells caused the insufficiency of the lung surfactant, and further resulted in the occurrence of the “M” like curves. This pioneering work realizes the vital states identification only using a non-contact ultra-wideband bio-radar, thereby enables to infer the health conditions, life expectancy, and appropriate subsequent treatment of victims in the trapped condition. Therefore, it has the potential to promote the welfare of post-disaster trapped human victims.

Les poumons présentent de nombreux avantages en tant que voie d’administration de médicaments. Ils possèdent une grande surface (70-100 m2) pour l’adsorption de molécules et de particules, une mince barrière épithéliale, une faible acidité ainsi qu’un système vasculaire sous-jacent abondant. L’administration par inhalation est une approche prometteuse pour le traitement du cancer des poumons et des infections microbiennes comorbides dans 33% des cas puisqu’elle permet la livraison ciblée d’agents chimiothérapeutiques. Les nanoparticules sont des vecteurs idéaux d’acheminements ciblés de médicaments avec des avantages tels qu’une stabilité élevée/une longue durée de conservation ainsi qu’une capacité de transport élevée. Les nanoparticules inhalées atteignant les alvéoles pulmonaires interagissent avec le surfactant pulmonaire. Ce mélange de lipides et de protéines tapisse l’interface eau/air des alvéoles servant ainsi de barrière. L’interaction physique et chimique des nanoparticules avec le surfactant pulmonaire déterminera leur clairance, rétention et translocation. Nous proposons l’utilisation de nanoparticules de phytoglycogène, extraites de maïs sans OGM, pour l’administration pulmonaire d’un peptide anticancéreux et antimicrobien à double action dont l’administration par voie orale ou par injection est problématique. Le nanophytoglycogène, composé de molécules de glucose, est non-biopersistant, non-toxique et est certifié GRAS (Generally Recognized as Safe) par le Food and Drug Administration pour l’ingestion. Cependant, son innocuité pour l’inhalation reste à déterminer. Avant de déterminer l’efficacité du nanophytoglycogène à des fins de nanotransporteur organique pour la délivrance par aérosol de peptides thérapeutiques, son impact sur les propriétés biophysiques et sur la structure de phase du surfactant pulmonaire doit premièrement être caractérisé. L’objectif du projet est d’étudier les effets de nanophytoglycogène de différentes charges sur les propriétés physicochimiques de modèles du surfactant pulmonaire en utilisant les monocouches Langmuir. Plus précisément, il est question d’étudier les effets des nanoparticules sur l’activité de surface, la morphologie, la réversibilité ainsi que l’épaisseur du film du surfactant pulmonaire. L’imagerie par microscopie à angle de Brewster (BAM, Brewster Angle Microscopy), les isothermes (pression de surface vs aire moléculaire) ainsi que l’ellipsométrie à l’interface eau-air permettent une conjecture des effets néfastes potentielles du nanophytoglycogène sur les poumons. À l’aide de ces techniques, il a été possible d’étudier des monocouches de phospholipides et de protéines, représentant le surfactant pulmonaire. En présence de nanoparticules anioniques et quasi-neutres, les différentes monocouches ne subissaient aucune perturbation. Cependant, les résultats ont démontré que les nanoparticules cationiques se lient aux phospholipides anioniques, ce qui augmente l’épaisseur de la monocouche et ainsi le travail requis pour effectuer un cycle respiratoire. Ces travaux ont démontré l’importance de la charge des nanomatériaux lors de leur interaction avec le surfactant pulmonaire. De plus, les résultats de cette étude ont aussi permis de classer les nanophytoglycogènes quasi-neutre et anionique comme étant des vecteurs de médicaments potentiels., The human lungs present many advantages as a drug delivery route, namely a high surface area (70-100 m2) for the adsorption of molecular species and particles, a thin epithelial barrier, an abundant underlying vasculature, and low acidity. Inhalation delivery is expected to be an ideal approach for the treatment of lung cancer and associated pulmonary infection (33% of cases) as it allows the site-specific physical delivery of chemotherapeutic. Nanoparticle carriers broaden the options for targeted drug delivery systems with advantages including high stability/long shelf life and high carrier capacity. In the alveoli, inhaled nanoparticles interact with lung (pulmonary) surfactant, a lipid/protein mixture that lines the alveolar air/fluid interface and serves as a primary barrier to uptake. The physical/chemical interaction of the nanoparticles with the surfactant determines their clearance, retention, and translocation. We propose to use novel phytoglycogen nanoparticles, extracted from non-GMO corn, for the pulmonary delivery of a dual action anticancer and antimicrobial peptide that is problematic to deliver orally or by injection. Nanophytoglycogen, composed of glucose molecules, is non-biopersistent, non-toxic and is GRAS (Generally Recognized as Safe) for oral ingestion. However, its safety for inhalation remains to be determined. Before evaluating the efficacy of nanophytoglycogen to serve as an organic nanocarrier for the aerosol delivery of peptide therapeutics, their impact on the biophysical properties and phase structure of lung surfactant must first be characterized. The objective of the research is to investigate the effect of nanophytoglycogens of different surface charge on the physicochemical properties of pulmonary surfactant model systems using Langmuir monolayers. More specifically, the effect of the nanoparticles on the surface activity, morphology, reversibility, and film thickness of pulmonary surfactant is studied. Isotherms (surface pressure vs. molecular area), BAM (Brewster Angle Microscopy) imaging, and ellipsometry at the air-water interface allow a surmise of the potential adverse effects of nanophytoglycogen on the lungs. Using these techniques, it was possible to study monolayers of phospholipids and proteins, representing the pulmonary surfactant. In the presence of anionic and quasi-neutral iv nanoparticles, the different monolayers didn’t undergo any disturbance. However, the results demonstrated that cationic nanoparticles bind to anionic phospholipids, which increases the thickness of the monolayer and thus the work required to complete a respiratory cycle. This study has demonstrated the importance of nanoparticle’s surface charge during their interaction with pulmonary surfactant. In addition, the results of this study also made it possible to classify the quasi-neutral and anionic nanophytoglycogens as being potential drug vectors.

The main function of the lung is gas exchange. This function is essential for life and optimized by combining a large surface area in contact with air and a thin barrier for gas (O2/CO2) diffusion. An array of conductive tubes (conductive airways: trachea and bronchi) leads atmospheric air through convection to the distal (alveolar) side of the lung, where gas exchange happens by diffusion (through a pressure gradient). At the alveoli level, many physical forces also play a vital role during the respiratory cycle. To the elastic recoil of the lung, due to the pleura and chest recoil forces, elastic properties of the connective tissue of the alveolar septa should also be taken into account. In addition, alveoli should also overcome interfacial forces from the air-liquid interface they are exposed to. This is mainly the surface tension at the liquid lining layer or hypophase. This force is primarily counteracted by lung surfactant, a specific mix of lipids and proteins synthesized and secreted by specialized cells, the alveolar epithelial type 2 cell (AE2C). On the other hand, the focus of this work is a specific lung disease, lung fibrosis. is a severe disease characterized by chronic inflammation, myofibroblast accumulation, and excessive extracellular matrix deposition, resulting in the damage of lung structure and respiratory failure. In Europe alone, approximately 40,000 new cases are diagnosed each year; however, its prognosis is overall poor, with a median survival of 3–4 years. We hypothesized that: 1) surfactant dysfunction contributes to the early stages and progression of the disease and; 2) we could target lung surfactant dysfunction as a therapeutic approach. With the use of animal models, we concluded that lung surfactant dysfunction is an early event, where AE2C injury plays a key role and alveolar macrophages play an important role during the progression of the disease. In addition, knowing the timing of lung surfactant dysfunction, we performed surfactant replacement therapies in order to prevent or slow down the onset or progression of the disease. From the results presented in this work, we can conclude that exogenous surfactant has an anti-fibrotic effect, increasing the surface area of opened alveoli and reducing the septal wall thickness. We could observe the same effect when transplanting healthy AE2C in diseased animal lungs, in order to replace the inactivated lung surfactant with healthy synthesizing cells.

Fluid interfaces, i.e. the boundary layer of two liquids or a liquid and a gas, play a vital role in physiological processes as diverse as visual perception, oral health and taste, lipid metabolism, and pulmonary breathing. These fluid interfaces exhibit a complex composition, structure, and rheology tailored to their individual physiological functions. Advances in interfacial thin film techniques have facilitated the analysis of such complex interfaces under physiologically relevant conditions. This allowed new insights on the origin of their physiological functionality, how deviations may cause disease, and has revealed new therapy strategies. Furthermore, the interactions of physiological fluid interfaces with exogenous substances is crucial for understanding certain disorders and exploiting drug delivery routes to or across fluid interfaces. Here, we provide an overview on fluid interfaces with physiological relevance, namely tear films, interfacial aspects of saliva, lipid droplet digestion and storage in the cell, and the functioning of lung surfactant. We elucidate their structure-function relationship, discuss diseases associated with interfacial composition, and describe therapies and drug delivery approaches targeted at fluid interfaces. Statement of Significance: Fluid interfaces are inherent to all living organisms and play a vital role in various physiological processes. Examples are the eye tear film, saliva, lipid digestion & storage in cells, and pulmonary breathing. These fluid interfaces exhibit complex interfacial compositions and structures to meet their specific physiological function. We provide an overview on physiological fluid interfaces with a focus on interfacial phenomena. We elucidate their structure-function relationship, discuss diseases associated with interfacial composition, and describe novel therapies and drug delivery approaches targeted at fluid interfaces. This sets the scene for ocular, oral, or pulmonary surface engineering and drug delivery approaches., Acta Biomaterialia, 130, ISSN:1742-7061, ISSN:1878-7568

Neonatal respiratory distress syndrome (NRDS) is a type of newborn disorder caused by the deficiency or late appearance of lung surfactant, a mixture of lipids and proteins. Studies have shown that lung surfactant replacement therapy could effectively reduce the morbidity and mortality of NRDS, and the therapeutic effect of animal-derived surfactant preparation, although with its limitations, performs much better than that of protein-free synthetic ones. Plasmalogens are a type of ether phospholipids present in multiple human tissues, including lung and lung surfactant. Plasmalogens are known to promote and stabilize non-lamellar hexagonal phase structure in addition to their significant antioxidant property. Nevertheless, they are nearly ignored and underappreciated in the lung surfactant-related research. This report will focus on plasmalogens, a minor yet potentially vital component of lung surfactant, and also discuss their biophysical properties and functions as anti-oxidation, structural modification, and surface tension reduction at the alveolar surface. At the end, we boldly propose a novel synthetic protein-free lung surfactant preparation with plasmalogen modification as an alternative strategy for surfactant replacement therapy.

Idiopathic pulmonary fibrosis (IPF) is a progressively and ultimately fatal lung disease. Previously it has been shown that intratracheal administration of alveolar epithelial type II cells (AE2C) in the animal model of bleomycin-induced pulmonary fibrosis is able to reverse fibrosis and restore surfactant protein levels. However, to date, it has not been evaluated whether these changes involve any improvement in alveolar dynamics. Consequently, the aim of the present work was to study lung physiology after AE2C transplantation at different time points during the development of injury and fibrosis. Lung fibrosis was induced by intratracheal instillation of bleomycin (4U/kg) in rat lungs. The animals were transplanted with AE2C (2.5 × 106 cells/animal) 3 or 7 days after bleomycin instillation. Assessments were done at day 7 and 14 after the induction of fibrosis to plot time dependent changes in lung physiology and mechanics. To assess the pressures and rates at which closed alveoli reopens invasive pulmonary tests using a small-animal mechanical ventilator (Flexivent®, Scireq, Canada) including de-recruitability tests and forced oscillation technique as well as quasi-static pressure volume loops were performed. Afterwards lungs were fixed by vascular perfusion and subjected to design-based stereological evaluation at light and electron microscopy level. AE2C delivered during the lung injury phase (3 days) of the disease are only able to slightly recover the volume of AE2C and volume fraction of LB in AE2C. However, it did not show either positive effects regarding ventilated alveolar surface nor any increase of lung compliance. On the other hand, when AE2C are delivered at the beginning of the fibrotic phase (7 days after bleomycin instillation), an increased ventilated alveolar surface to control levels and reduced septal wall thickness can be observed. Moreover, transplanted animals showed better lung performance, with increased inspiratory capacity and compliance. In addition, a detailed analysis of surfactant active forms [mainly tubular myelin, lamellar body (LB)-like structures and multilamellar vesicles (MLV)], showed an effective recovery during the pro-fibrotic phase due to the healthy AE2C transplantation. In conclusion, AE2C transplantation during fibrogenic phases of the disease improves lung performance, structure and surfactant ultrastructure in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressively and ultimately fatal lung disease. Previously it has been shown that intratracheal administration of alveolar epithelial type II cells (AE2C) in the animal model of bleomycin-induced pulmonary fibrosis is able to reverse fibrosis and restore surfactant protein levels. However, to date, it has not been evaluated whether these changes involve any improvement in alveolar dynamics. Consequently, the aim of the present work was to study lung physiology after AE2C transplantation at different time points during the development of injury and fibrosis. Lung fibrosis was induced by intratracheal instillation of bleomycin (4U/kg) in rat lungs. The animals were transplanted with AE2C (2.5 × 10 cells/animal) 3 or 7 days after bleomycin instillation. Assessments were done at day 7 and 14 after the induction of fibrosis to plot time dependent changes in lung physiology and mechanics. To assess the pressures and rates at which closed alveoli reopens invasive pulmonary tests using a small-animal mechanical ventilator (Flexivent®, Scireq, Canada) including de-recruitability tests and forced oscillation technique as well as quasi-static pressure volume loops were performed. Afterwards lungs were fixed by vascular perfusion and subjected to design-based stereological evaluation at light and electron microscopy level. AE2C delivered during the lung injury phase (3 days) of the disease are only able to slightly recover the volume of AE2C and volume fraction of LB in AE2C. However, it did not show either positive effects regarding ventilated alveolar surface nor any increase of lung compliance. On the other hand, when AE2C are delivered at the beginning of the fibrotic phase (7 days after bleomycin instillation), an increased ventilated alveolar surface to control levels and reduced septal wall thickness can be observed. Moreover, transplanted animals showed better lung performance, with increased inspiratory capacity and compliance. In addition, a detailed analysis of surfactant active forms [mainly tubular myelin, lamellar body (LB)-like structures and multilamellar vesicles (MLV)], showed an effective recovery during the pro-fibrotic phase due to the healthy AE2C transplantation. In conclusion, AE2C transplantation during fibrogenic phases of the disease improves lung performance, structure and surfactant ultrastructure in bleomycin-induced lung fibrosis., GG-J was supported by the European respiratory Society/Sociedad Española de Neumología y Cirugía Torácica, ERS/SEPAR Short-Term Research Training Fellowship (STRTF 2014-6502). This work was supported by grants from Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (PI13/00282), Cofinanciado por el Fondo Europeo de Desarrollo Regional (FEDER), Unión Europea, Una manera de hacer Europa, Fundació la Marató de TV3 (MTV3 122410). Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Cluster of Excellence REBIRTH (Regenerative Biology to Reconstructive Therapy), and the German Research Foundation (DFG KN 916 1-1).

Understanding and controlling the interaction between nanoparticles and biological entities is fundamental to the development of nanomedicine applications. In particular, the possibility to realize nanoparticles capable of directly targeting neutral lipid membranes would be advantageous to numerous applications aiming at delivering nanoparticles and their cargos into cells and biological vesicles. Here, we use experimental and computational methodologies to analyze the interaction between liposomes and gold nanoparticles (AuNPs) featuring cationic headgroups in their protecting monolayer. We find that in contrast to nanoparticles decorated with other positively charged headgroups, guanidinium-coated AuNPs can bind to neutral phosphatidylcholine liposomes, inducing nondisruptive membrane permeabilization. Atomistic molecular simulations reveal that this ability is due to the multivalent H-bonding interaction between the phosphate residues of the liposome’s phospholipids and the guanidinium groups. Our results demonstrate that the peculiar properties of arginine magic, an effect responsible for the membranotropic properties of some naturally occurring peptides, are also displayed by guanidinium-bearing functionalized AuNPs. Guanidinium-containing macromolecules, such as arginine-rich peptides, have peculiar properties in their interactions with membranes, including efficient translocation. Here the authors show that cationic gold nanoparticles functionalized with guanidinium groups can specifically and nondisruptively interact with phospholipid bilayer membranes.

Alveolar macrophage (AM) is a mononuclear phagocyte key to the defense against respiratory infections. To understand AM’s role in airway disease development, we examined the influence of Secretoglobin family 1a member 1 (SCGB1A1), a pulmonary surfactant protein, on AM development and function. In a murine model, high-throughput RNA-sequencing and gene expression analyses were performed on purified AMs isolated from mice lacking in Scgb1a1 gene and were compared with that from mice expressing the wild type Scgb1a1 at weaning (4 week), puberty (8 week), early adult (12 week), and middle age (40 week). AMs from early adult mice under Scgb1a1 sufficiency demonstrated a total of 37 up-regulated biological pathways compared to that at weaning, from which 30 were directly involved with antigen presentation, anti-viral immunity and inflammation. Importantly, these pathways under Scgb1a1 deficiency were significantly down-regulated compared to that in the age-matched Scgb1a1-sufficient counterparts. Furthermore, AMs from Scgb1a1-deficient mice showed an early activation of inflammatory pathways compared with that from Scgb1a1-sufficient mice. Our in vitro experiments with AM culture established that exogenous supplementation of SCGB1a1 protein significantly reduced AM responses to microbial stimuli where SCGB1a1 was effective in blunting the release of cytokines and chemokines (including IL-1b, IL-6, IL-8, MIP-1a, TNF-a, and MCP-1). Taken together, these findings suggest an important role for Scgb1a1 in shaping the AM-mediated inflammation and immune responses, and in mitigating cytokine surges in the lungs.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Corona virus SARS-CoV-2 has already spread around the whole world and is currently, with no vaccine available yet, unstoppable. As per today, COVID-19 affects more than 3,000,000 confirmed patients globally. First line medications are antiviral drugs and multiple urgent clinical trials are under way. However, a recent clinical trial testing the HIV protease inhibitor combination lopinavir and ritonavir showed no significant antiviral activity against SARS-CoV-2 in patients with severe disease (1). As long as we do not have specific antiviral therapies against SARS-CoV-2, we need to provide supportive symptomatic therapies to prevent pulmonary failure, the most common cause of COVID-19 mortality.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is a common clinical problem, leading to significant morbidity and mortality, and no effective pharmacotherapy exists. The problem of ARDS causing mortality became more apparent during the COVID-19 pandemic. Biotherapeutic products containing multipotent mesenchymal stromal cell (MMSC) secretome may provide a new therapeutic paradigm for human healthcare due to their immunomodulating and regenerative abilities. The content and regenerative capacity of the secretome depends on cell origin and type of cultivation (two- or three-dimensional (2D/3D)). In this study, we investigated the proteomic profile of the secretome from 2D- and 3D-cultured placental MMSC and lung fibroblasts (LFBs) and the effect of inhalation of freeze-dried secretome on survival, lung inflammation, lung tissue regeneration, fibrin deposition in a lethal ALI model in mice. We found that three inhaled administrations of freeze-dried secretome from 2D- and 3D-cultured placental MMSC and LFB protected mice from death, restored the histological structure of damaged lungs, and decreased fibrin deposition. At the same time, 3D MMSC secretome exhibited a more pronounced trend in lung recovery than 2D MMSC and LFB-derived secretome in some measures. Taking together, these studies show that inhalation of cell secretome may also be considered as a potential therapy for the management of ARDS in patients suffering from severe pneumonia, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, their effectiveness requires further investigation.

Air pollutants generate reactive oxygen species on lung surfaces. Here we report how hydroxyl radicals (·OH) injected on the surface of water react with SP-B_(1–25), a 25-residue polypeptide surrogate of human lung surfactant protein B. Our experiments consist of intersecting microjets of aqueous SP-B_(1–25) solutions with O_3/O_2/H_2O/N_2(g) gas streams that are photolyzed into ·OH(g) in situ by 266 nm laser nanosecond pulses. Surface-sensitive mass spectrometry enables us to monitor the prompt (

Background Despite their growing popularity, the potential respiratory toxicity of electronic cigarettes (e-cigarettes) remains largely unknown. One potential aspect of e-cigarette toxicity is the effect of e-cigarette vapor on lung surfactant function. Lung surfactant is a mixture of lipids and proteins that lines the alveolar region. The surfactant layer reduces the surface tension of the alveolar fluid, thereby playing a crucial role in lung stability. Due to their small size, particulates in e-cigarette vapor can penetrate the deep lungs and come into contact with the lung surfactant. The current study sought to examine the potential adverse effects of e-cigarette vapor and conventional cigarette smoke on lung surfactant interfacial properties. Methods Infasurf®, a clinically used and commercially available calf lung surfactant extract, was used as lung surfactant model. Infasurf® films were spread on top of an aqueous subphase in a Langmuir trough with smoke particulates from conventional cigarettes or vapor from different flavors of e-cigarettes dispersed in the subphase. Surfactant interfacial properties were measured in real-time upon surface compression while surfactant lateral structure after exposure to smoke or vapor was examined using atomic force microscopy (AFM). Results E-cigarette vapor regardless of the dose and flavoring of the e-liquid did not affect surfactant interfacial properties. In contrast, smoke from conventional cigarettes had a drastic, dose-dependent effect on Infasurf® interfacial properties reducing the maximum surface pressure from 65.1 ± 0.2 mN/m to 46.1 ± 1.3 mN/m at the highest dose. Cigarette smoke and e-cigarette vapor both altered surfactant microstructure resulting in an increase in the area of lipid multilayers. Studies with individual smoke components revealed that tar was the smoke component most disruptive to surfactant function. Conclusions While both e-cigarette vapor and conventional cigarette smoke affect surfactant lateral structure, only cigarette smoke disrupts surfactant interfacial properties. The surfactant inhibitory compound in conventional cigarettes is tar, which is a product of burning and is thus absent in e-cigarette vapor.

Background: Damage to lung surfactant (LS) enabling the lung local immunity may contribute to the development of bronchial inflammation in patients with bronchial asthma (BA). Methods and Results: A 70-day course of 21 LS (Surfactant-BL) inhalations at the dose of 25 mg was added to inhaled corticosteroids (ICS) and short/long-acting bronchodilators or combined inhalers in 13 BA patients. After 21 inhalations, many patients reported lower frequency of cough and profuse expectoration, fewer night and day attacks, resolution of wheezing, resolution or lower frequency of bronchospasm episodes under moderate exercise, and termination (1 patient) or double reduction (8 patients out of 13) of the ICS dose. The values of PFPs in patients at Days 250 and 340 did not differ significantly from the values achieved at Day 41. The mean values at Days 250 and 340 were as follows: FVC =86.4±6.43 (%), FEV1=61.4±8.41 (%), FEV1/FVC =66.5±8.87 (%). Conclusion: LS inhalations improve the condition of patients with BA, allow ICS dose reduction by 2 times, and improve pulmonary function parameters. A total of 70% of patients had quite a long remission of BA symptoms following a 21-inhalation therapy course of the formulation.

Background Rab38 small GTPase regulates intracellular transport in melanocytes and alveolar type II epithelial cells. Ruby rats carrying Rab38 and other gene mutations exhibit oculocutaneous albinism, bleeding diathesis, and hence, are a rat model of human Hermansky-Pudlak syndrome (HPS). We previously showed that Long Evans Cinnamon (LEC) rats, one strain of the Ruby rats, developed aberrant lung surfactant homeostasis with remarkably enlarged lamellar bodies in alveolar type II cells. Methods A replication-deficient recombinant adenovirus expressing rat Rab38 (Ad-Rab38) was constructed. Alveolar type II cells were isolated from the LEC rats and tested for lung surfactant phosphatidylcholine secretion. The rats were also examined whether exogenous expression of Ad- Rab38 could rescue the altered lung surfactant homeostasis in the lungs. Results Isolated type II cells infected with Ad-Rab38 exhibited improved secretion patterns of [3H]phosphatidylcholine, i.e. increased basal hyposecretion and decreased agonist-induced hypersecretion. Endobronchial administration of Ad-Rab38 improved the morphology of type II cells and lamellar bodies, reducing their sizes close to those of wild-type rats. The increased amounts of phosphatidylcholine and surfactant protein B in the lamellar body fractions were decreased in the Ad-Rab38 infected lungs. Conclusions These results provide strong evidence that the aberrant lung surfactant homeostasis in the LEC rats is caused by Rab38 deficit, and suggest that endobronchial delivery of the responsive transgene could be an effective method to ameliorate the abnormal lung phenotype in the animal model of HPS.

Measurements of dynamic surface tension were carried out in aqueous systems (water or 0.1 mM Triton X-100) comprising nanoparticles formed from chemically modified polyaldehyde dextran (PAD). The nanostructures, considered as potential drug carriers in aerosol therapy, were obtained from biocompatible polysaccharides by successive oxidation and reactive coiling in an aqueous solution. The dynamic surface tension of the samples was determined by the maximum bubble pressure (MBP) method and by the axisymmetric drop shape analysis (ADSA). Experiments with harmonic area perturbations were also carried out in order to determine surface dilatational viscoelasticity. PAD showed a remarkable surface activity. Ward-Tordai equation was used to determine the equilibrium surface tension and diffusion coefficient of PAD nanoparticles (D = 2.3×10-6 m2/s). In a mixture with Triton X-100, PAD particles showed co-adsorption and synergic effect in surface tension reduction at short times (below 10 s). Tested nanoparticles had impact on surface rheology in a mixed system with nonionic surfactant, suggesting their possible interactions with the lung surfactant system after inhalation. This preliminary investigation sets the methodological approach for further research related to the influence of inhaled PAD nanoparticles on the lung surfactant and mass transfer processes in the respiratory system.

Aim To develop a fast bedside lung maturity test. Methods Gastric aspirates obtained from premature infants contain lamellar bodies, carrying lung surfactant. To estimate lung maturity, we isolated lamellar bodies from fresh gastric aspirates by centrifugation. Erythrocytes and other cells were lysed by adding water and discarded subsequently with the supernatant. Mid-infrared spectroscopy was then performed to measure the lung maturity as lecithin-sphingomyelin ratio. Lecithin was determined as dipalmitoylphosphatidylcholine, the most surface-active phospholipid. Algorithms to measure lecithin and sphingomyelin concentrations in fresh gastric aspirates were developed on aspirates from 140 premature infants. Each gastric aspirate sample was divided into two samples: one for mass spectrometry as reference and one for spectroscopy. Development of the algorithm is described in detail in Appendix S1. Results Gastric aspirates stored at 4-5°C avoid flocculation of proteins and phospholipids in contrast to when the aspirates were frozen and thawed. Omission of freezing and concentration of the lung surfactant by centrifugation combined with diminished influence of proteins improves the spectroscopic measurement of lecithin-sphingomyelin ratio. Measurement of lecithin-sphingomyelin ratio by the new method was performed within 10-15 minutes. Conclusion We present a new fast bedside lung maturity test on fresh gastric aspirate for early targeted surfactant treatment.

Surfactant replacement therapy, commonly consisting of bovine or porcine lung surfactant extracts, is a lifesaving treatment for preterm newborns that suffer for respiratory distress syndrome (RDS). Recently, a synthetic surfactant, CHF5633, has been developed by Chiesi Farmaceutici (Italy) for the treatment of neonatal RDS. CHF5633 contains two phospholipids forming multi lamellar vesicles and two peptides which are analogues of surfactant protein B and surfactant protein C. By conjugating the solvatochromic fluorophore dansyl to the peptides we aimed at characterizing their environment and distribution within the formulation. The fluorescence properties of dansyl-conjugated SP-B Analogue and SP-C Analogue were characterized in different solvents and compared with their emission properties when individually formulated in CHF5633. Both peptides exhibited a close interaction with the phospholipid matrix, as observed for the natural counterparts. In particular, SP-C Analogue is more deeply inserted in the phospholipid bilayer compared to SP-B Analogue. The localization of the fluorescent peptides within CHF5633 did not experience significant changes over a storage period of nine months. (C) 2019 Elsevier B.V. All rights reserved.

In dieser Arbeit wurde das Adsorptionsverhalten verschiedener Substanzen aus der Gas- oder Flüssigphase an Modellmembranen untersucht. Lipidmembranen spielen in lebenden Organismen eine wesentliche Rolle. In Form von Doppelschichten organisieren sie die Abgrenzung der Zelle und der intrazellulären Kompartimente. In der Lunge reguliert eine komplexe Lipid-Protein Monolage, das sogenannte pulmonale Surfactant, den störungsfreien Gasaustausch. Heutzutage ist die menschliche Lunge vielen potenziell schädlichen Molekülen ausgesetzt, wie z.B. Lösungsmitteln und flüchtigen Substanzen, die in Farben, Lacken, Weichmachern, Waschmitteln und Anästhesiemitteln verwendet werden. Andererseits haben Substanzen wie Perfluorkohlenstoffe vielversprechende Eigenschaften, die geschädigtes Surfactant der Lunge unterstützen könnten. Um diese Wechselwirkungen besser zu verstehen, wurde der Einfluss von Alkanen und perfluorierten Substanzen auf Modellmembranen und ihre Wirkung auf pathophysiologische Proteinadsorptionsprozesse untersucht. Die natürliche Umgebung einer zellulären Membran ist eine Elektrolytlösung. Ionen spielen eine wichtige Rolle in Membran-assoziierten Prozessen, wie Signaltransduktion, Fusion, Endozytose und beeinflussen Protein/Membran-Wechselwirkungen. Ein weiterer Schwerpunkt dieser Arbeit ist die Untersuchung des Einflusses von Kationen auf die Membranstruktur und auf Proteinadsorptionsprozesse. Mit den oberflächensensitiven Röntgenstreumethoden Röntgenreflektivität und Röntgendiffraktion unter streifenden Einfall ist es möglich, Strukturen an Grenzflächen mit sub-Ångström Auflösung in-situ zu analysieren. Als Modellsysteme wurden Langmuirfilme an der Flüssigkeits/Gas-Grenzfläche sowie substratgebundene Lipidschichten an der Festkörper/Flüssigkeits- bzw. an der Festkörper/Gas-Grenzfläche untersucht., In this work the adsorption behaviour of different substances from the gas or liquid phase at model membranes was investigated. Lipid membranes play an essential role in living organisms. In the form of bilayers they organise the boundary of the cell and of the intracellular compartments. In the lung, a complex lipid-protein monolayer, the so-called pulmonary surfactant, regulates the proper gas exchange. Nowadays, the human lung is exposed to various potentially harmful molecules, e.g. solvents and volatile substances used in paints, varnishes, plasticiser, detergents and anaesthetics. On the other hand, substances such as perfluorocarbons have promising properties that could support injured lung surfactant. For a deeper understanding of these interactions, the influence of alkanes and perfluorinated substances on model membranes and their effect on pathophysiological protein adsorption processes was investigated. The natural environment of a cellular membrane is an electrolyte solution. Ions play an important role in membrane-associated processes, such as signal transduction, fusion, endocytosis and affect protein/membrane-interactions. Therefore, another point of emphasis of this work is the investigation of the influence of cations on membrane structures and protein adsorption processes. With the surface-sensitive x-ray scattering techniques x-ray reflectivity and grazing-incidence x-ray diffraction it is possible to analyse structures at interfaces with sub-angstrom resolution in-situ. As model systems Langmuir films at the liquid/gas-interface and substrate-bound lipid layers at the solid/liquid- and solid/gas-interface were investigated.

Carbon nanomaterials are widespread in the atmospheric aerosol as a result of the combustion processes and their extensive industrial use. This has raised many question about the potential toxicity associated with the inhalation of such nanoparticles, and its incorporation into the lung surfactant layer. In order to shed light on the main physical bases underlying the incorporation of carbon nanomaterials into lung surfactant layers, this work has studied the interaction at the water/vapor interface of carbon nanosheets (CN) with Langmuir monolayers of 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), with this lipid being the main component of lung surfactant layers and responsible of some of the most relevant features of such film. The incorporation of CN into DPPC Langmuir monolayers modifies the lateral organization of the DPPC at the interface, which is explained on the basis of two different effects: (i) particles occupy part of the interfacial area, and (ii) impoverishment of the lipid composition of the interface due to lipid adsorption onto the CN surface. This results in a worsening of the mechanical performance of the monolayers which may present a negative impact in the physiological performance of lung surfactant. It would be expected that the results obtained here can be useful as a step toward the understanding of the most fundamental physico-chemical bases associated with the effect of inhaled particles in the respiratory cycle.

The rare lysosomal storage disorder Niemann-Pick disease type C1 (NPC1) arises from mutation of NPC1, which encodes a lysosomal transmembrane protein essential for normal transport and trafficking of cholesterol and sphingolipids. NPC1 is highly heterogeneous in both clinical phenotypes and age of onset. Previous studies have reported sub-Mendelian survival rates for mice homozygous for various Npc1 mutant alleles but have not studied the potential mechanisms underlying this phenotype. We performed the first developmental analysis of a Npc1 mouse model, Npc1em1Pav, and discovered significant fetal growth restriction in homozygous mutants beginning at E16.5. Npc1em1Pav/em1Pav mice also exhibited cyanosis, increased respiratory effort, and over 50% lethality at birth. Analysis of neonatal lung tissues revealed lipid accumulation, notable abnormalities in surfactant, and enlarged alveolar macrophages, suggesting that lung abnormalities may be associated with neonatal lethality in Npc1em1Pav/em1Pav mice. The phenotypic severity of the Npc1em1Pav model facilitated this first analysis of perinatal lethality and lung pathology in an NPC1 model organism, and this model may serve as a useful resource for developing treatments for respiratory complications seen in NPC1 patients.

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B&rsquo, s structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B&rsquo, s structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B&rsquo, s &ldquo, central loop&rdquo, and &ldquo, hinge&rdquo, regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B&rsquo, s essential functions.

Background Respiratory Distress Syndrome (RDS) is a prematurity-related breathing disorder caused by a quantitative deficiency of pulmonary surfactant. Surfactant replacement therapy is effective for RDS newborns, although treatment failure has been reported. The aim of this study is to trace exogenous surfactant by 13C variation and estimate the amount reaching the lungs at different doses of the drug. Methods Forty-four surfactant-depleted rabbits were obtained by serial bronchoalveolar lavages (BALs), that were merged into a pool (BAL pool) for each animal. Rabbits were in nasal continuous positive airway pressure and treated with 0, 25, 50, 100 or 200 mg/kg of poractant alfa by InSurE. After 90 min, rabbits were depleted again and a new pool (BAL end experiment) was collected. Disaturated-phosphatidylcholine (DSPC) was measured by gas chromatography. DSPC-Palmitic acid (PA) 13C/12C was analyzed by isotope ratio mass spectrometry. One-way non-parametric ANOVA and post-hoc Dunn’s multiple comparison were used to assess differences among experimental groups. Results Based on DSPC-PA 13C/12C in BAL pool and BAL end experiment, the estimated amount of exogenous surfactant ranged from 61 to 87% in dose-dependent way (p

Background: Damage to lung surfactant (LS) enabling the lung local immunity may contribute to the development of bronchial inflammation in patients with bronchial asthma. Methods and Results: A 40-day course of 16 LS (Surfactant-BL) inhalations at the dose of 25mg was added to inhaled corticosteroids (ICS) and short/long-acting bronchodilators or combined inhalers in 14 patients with bronchial asthma. After 7 inhalations, patients demonstrated a significant decrease in shortness of breath and bronchospasm attacks, double reduction of ICS dose (p=0.01), and improvement of pulmonary function. Forced vital capacity (FVC) increases during treatment in a linear fashion (y=62.9+5.60•x; p

Lung surfactant is a complex mixture of lipids and proteins which plays a major role in the respiratory cycle. This makes necessary to understand the effects of different external factors or agents, for example, inhaled particles, as a potential source of alteration of the normal physiological response of lung surfactant. However, in most cases, in vivo studies are difficult to perform, and preliminary studies based in model systems are required. Films of lipids or mixtures of lipids and proteins at the water–vapor interface are accounted as one of the most useful methodologies for initial assessments of the potential toxicity of inhaled particles. Thus, the study of the modifications induced by the incorporation of colloidal particles in the interfacial properties of layers mimicking some of the physicochemical features of lung surfactant might provide a first evaluation of the risks and hazards associated with the inhalation of particulate matter. Considering the importance of particles in technology and industry, it is mandatory to develop strategies providing information about toxicological aspects of these widespread materials. This review focuses its interest on the recent advancements on the application of studied bases on monolayers at the fluid interface as preliminary assay for deepening on a complex situation with biological interest.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cells bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome. Funding Agencies|ZON-MW MKMD program (The Netherlands Organisation for Health Research and Development); Swedish Research Council/Swedish International Cooperation Agency (SIDA) [2012-3349, 2015-02593]; Swedish Heart Lung/the Oskar II Jubilee Foundations [20130685, 20150709]; County Council of Ostergotland

Background The well-known inflammatory and fibrogenic changes of the lung upon crystalline silica are accompanied by early changes of the phospholipid composition (PLC) as detected in broncho-alveolar lavage fluid (BALF). Amorphous silica nanoparticles (NPs) evoke transient lung inflammation, but their effect on PLC is unknown. Here, we compared effects of unmodified and phosphonated amorphous silica NP and describe, for the first time, local changes of the PLC with innovative bioimaging tools. Methods Unmodified (SiO2-n), 3-(trihydroxysilyl) propyl methylphosphonate coated SiO2-n (SiO2-p) as well as a fluorescent surrogate of SiO2-n (SiO2-FITC) nanoparticles were used in this study. In vitro toxicity was tested with NR8383 alveolar macrophages. Rats were intratracheally instilled with SiO2-n, SiO2-p, or SiO2-FITC, and effects on lungs were analyzed after 3 days. BALF from the right lung was analyzed for inflammatory markers. Cryo-sections of the left lung were subjected to fluorescence microscopy and PLC analyses by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MS), Fourier transform infrared microspectroscopy (FT-IR), and tandem mass spectrometry (MS/MS) experiments. Results Compared to SiO2-p, SiO2-n NPs were more cytotoxic to macrophages in vitro and more inflammatory in the rat lung, as reflected by increased concentration of neutrophils and protein in BALF. Fluorescence microscopy revealed a typical patchy distribution of SiO2-FITC located within the lung parenchyma and alveolar macrophages. Superimposable to this particle distribution, SiO2-FITC elicited local increases of phosphatidylglycerol (PG) and phosphatidylinositol (PI), whereas phoshatidylserine (PS) and signals from triacylgyceride (TAG) were decreased in the same areas. No such changes were found in lungs treated with SiO2-p or particle-free instillation fluid. Conclusions Phosphonate coating mitigates effects of silica NP in the lung and abolishes their locally induced changes in PLC pattern. Bioimaging methods based on MALDI-MS may become a useful tool to investigate the mode of action of NPs in tissues. Electronic supplementary material The online version of this article (10.1186/s12989-018-0267-z) contains supplementary material, which is available to authorized users.

Pulmonary surfactant protein D (SP-D) is a glycoprotein from the collectin family that takes part in the lung surfactant system. It exhibits host defense and immune regulatory functions within the lung. It is known that SP-D monomer forms trimers, which associate into higher order oligomers. However, the pathway and the interactions involved in the assembly of SP-D oligomers are not clearly understood. In the current study, a recombinant form of human SP-D (rhSP-D) has been studied by Atomic Force Microscopy (AFM) and electrophoresis, with the goal of analyzing, qualitative and quantitatively, the conformational diversity and the determinants defining the oligomerization pathway of the protein. The studied rhSP-D is a mixture of trimers, hexamers, dodecamers and higher order oligomeric species, with dodecamers accounting for more than 50% of the protein by mass. Similar structures were also found in hSP-D obtained from proteinosis patients. The proportion of dodecamer is increased under acidic conditions, accompanied by a conformational change into more compact configurations. Dodecamers were found to be necessarily formed by two hexamers joined through non-covalent ionic and hydrophobic interactions between the N-terminal domains and the nearby area of SP-D collagen stems., MINECO. Spanish Ministry of Economy (BIO2015-67930-R). Regional Government of Madrid (S2013/MlT-2807). Spanish Ministry of Education. European Research Council (ERC). European Union’s Horizon2020 Research and Innovation Program (grant agreement No 681299).

Many pathologies of the respiratory tract are inadequately treated with existing small molecule-based therapies. The emergence of RNA interference (RNAi) enables the post-transcriptional silencing of key molecular disease factors that cannot readily be targeted with conventional small molecule drugs. Pulmonary administration of RNAi effectors, such as small interfering RNA (siRNA), allows direct delivery into the lung tissue, hence reducing systemic exposure. Unfortunately, the clinical translation of RNAi is severely hampered by inefficient delivery of siRNA therapeutics towards the cytoplasm of the target cells. In order to have a better control of the siRNA delivery process, both extra- and intracellular, siRNAs are typically formulated in nanosized delivery vehicles (nanoparticles, NPs). In the lower airways, which are the targeted sites of action for multiple pulmonary disorders, these siRNA-loaded NPs will encounter the pulmonary surfactant (PS) layer, covering the entire alveolar surface. The interaction between the instilled siRNA-loaded NPs and the PS at this nano-bio interface results in the adsorption of PS components onto the surface of the NPs. The formation of this so-called biomolecular corona conceals the original NP surface and will therefore profoundly determine the biological efficacy of the NP. Though this interplay has initially been regarded as a barrier towards efficient siRNA delivery to the respiratory target cell, recent reports have illustrated that the interaction with PS might also be beneficial for local pulmonary siRNA delivery.

The interaction of carbon black particles (CB) with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) at the water/air interface has been investigated by means of a pool of surface sensitive techniques, in order to analyze the thermodynamic and rheological aspects of these mixed systems. The incorporation of carbonaceous particles to the lipid monolayers induces changes on the surface pressure-area isotherm, as revealed by the shifting to higher surface area of the maximum packing degree of the monolayer, and the decrease of the collapse pressure. These changes are strongly dependent on the DPPC:CB weight ratio at the interface and can be explained by the disruption of the monolayer structure due to the particle incorporation that provokes the modification of the cohesive interactions along the monolayer. Measurements of dilational viscoelasticity against frequency, at different degrees of monolayer compression, have been performed by means of the Oscillatory Barrier method. The rheological response of the monolayer is only slightly affected by the presence of CB, even if a modification of the quasi-equilibrium dilational elasticity, as well as of the frequency dependence of the viscoelastic modulus, is appreciable increasing the particle concentration. Being DPPC the major component of many systems with biological interest (cell membranes, lung surfactant), the results obtained here are expected to contribute to the understanding of the carbon particle interaction with biological relevant systems.

Background: Damage to lung surfactant, which is responsible for the lung local immunity, may contribute to the development of bronchial inflammation in patients with bronchial asthma. Different doses of glucocorticoids produce a stimulating or inhibiting effect on the synthesis of the surfactant protein (SP-A) mRNA. Lung surfactant disorders may negatively influence bronchial homeostasis and aggravate the condition of patients with bronchial asthma and COPD. The objective of this study was to evaluate the influence of long-term inhaled corticosteroids on the phospholipid levels of the lung surfactant in rats. Methods and Results: Inhalations of prednisolone hemisuccinate (PH) were given to white non-pedigree rats weighing 180-200g at a dose of 0.3mg/kg daily for 30 days. Already by the end of the first study period (10 days), lung surfactant phospholipid levels were found to decrease significantly from 1.35±0.060mg to 1.02±0.045mg (P

The lungs have potential as a means of systemic drug delivery of macromolecules. Systemic delivery requires crossing of the air-blood barrier, however with molecular size-dependent limitations in lung absorption of large molecules. Systemic availability after inhalation can be improved by absorption enhancers, such as bile salts. Enhancers may potentially interfere with the different constituents of the lungs, e.g. the lung surfactant lining the alveoli or the lung epithelium. We used two in vitro models to investigate the potential effects of bile salts on lung surfactant function (with the constrained drop surfactometer) and on the epithelium in the proximal airways (with the MucilAirTM cell system), respectively. In addition, we measured direct effects on respiration in mice inhaling bile salt aerosols. The bile salts inhibited lung surfactant function at different dose levels, however they did not affect the integrity of ciliated cells at the tested doses. Furthermore, the bile salt aerosols induced changes in the breathing pattern of mice indicative of pulmonary irritation. The bile salts were ranked according to potency in vitro for surfactant function disruption and in vivo for induction of pulmonary irritation. The ranking was the same, suggesting a correlation between the interference with lung surfactant and the respiratory response.

The lipid–protein mixture that covers the lung alveoli, lung surfactant, ensures mechanical robustness and controls gas transport during breathing. Lung surfactant is located at an interface between water-rich tissue and humid, but not fully saturated, air. The resulting humidity difference places the lung surfactant film out of thermodynamic equilibrium, which triggers the buildup of a water gradient. Here, we present a millifluidic method to assemble multilamellar interfacial films from vesicular dispersions of a clinical lung surfactant extract used in replacement therapy. Using small-angle X-ray scattering, infrared, Raman, and optical microscopies, we show that the interfacial film consists of several coexisting lamellar phases displaying a substantial variation in water swelling. This complex phase behavior contrasts to observations made under equilibrium conditions. We demonstrate that this disparity stems from additional lipid and protein gradients originating from differences in their transport properties. Supplementing the extract with cholesterol, to levels similar to the endogenous lung surfactant, dispels this complexity. We observed a homogeneous multilayer structure consisting of a single lamellar phase exhibiting negligible variations in swelling in the water gradient. Our results demonstrate the necessity of considering nonequilibrium thermodynamic conditions to study the structure of lung surfactant multilayer films, which is not accessible in bulk or monolayer studies. Our reconstitution methodology also opens avenues for lung surfactant pharmaceuticals and the understanding of composition, structure, and property relationships at biological air–liquid interfaces., Composition gradients develop across lung surfactant films under physiologically relevant nonequilibrium conditions, yielding a structural heterogeneity that is suppressed by cholesterol addition.

Lung surfactant and a tear film lipid layer are examples of biologically relevant macromolecular structures found at the air–water interface. Because of their complexity, they are often studied in terms of simplified lipid layers, the simplest example being a Langmuir monolayer. Given the profound biological significance of these lipid assemblies, there is a need to understand their structure and dynamics on the nanoscale, yet there are not many techniques able to provide this information. Atomistic molecular dynamics simulations would be a tool fit for this purpose; however, the simulation models suggested until now have been qualitative instead of quantitative. This limitation has mainly stemmed from the challenge to correctly describe the surface tension of water with simulation parameters compatible with other biomolecules. In this work, we show that this limitation can be overcome by using the recently introduced four-point OPC water model, whose surface tension for water is demonstrated to be quantitatively consistent with experimental data and which is also shown to be compatible with the commonly employed lipid models. We further establish that the approach of combining the OPC four-point water model with the CHARMM36 lipid force field provides nearly quantitative agreement with experiments for the surface pressure–area isotherm for POPC and DPPC monolayers, also including the experimentally observed phase coexistence in a DPPC monolayer. The simulation models reported in this work pave the way for nearly quantitative atomistic studies of lipid-rich biological structures at air–water interfaces. acceptedVersion

This thesis explores lipid self-assembly and aims to give a broad picture of self-assembly structures in simple and complex lipid-protein systems. The systems studied are lung surfactant, stratum corneum and simple model membranes. The lung surfactant mixture lines the alveolus in our lungs and stabilises the air- tissue interface. The lung surfactant lipid phase behaviour was here investigated with respect to the effects of cholesterol concentration and changes in the external conditions of temperature and a water gradient. Taken together the studies of lung surfactant give a comprehensive picture of the phase behaviour of a clinical lung surfactant extract, showing the importance of cholesterol and non-equilibrium conditions. It is a recurrent observation for all studies that the addition of physiologically relevant levels of cholesterol to the clinical lung surfactant forms a single robust liquid ordered phase under both equilibrium and non- equilibrium conditions.Conditions such as draught, high salinity or freezing, exerts the lipid systems to osmotic stress, which can lead to phase changes between different self-assembled structures. The outer layer of the skin, the stratum corneum is most of the time exposed to osmotic stress from dry air in the environment. The stratum corneum contains small polar molecules, to counteract phase changes due to osmotic stress. We study how the self-assembly in stratum corneum and model membranes are affected by the presence of osmolytes under conditions of osmotic stress. It is shown that these compounds under dry conditions act to replace the water in both stratum corneum and model membranes and they may stabilize the fluid lipid phases at lower humidities.In plants, there are several strategies for protection against osmotic stress. One strategy involves the expression of specific proteins. We have studied how one such protein from the family of dehydrins, influences lipid self-assembly, aiming at molecular understanding of how these proteins can protect membranes against osmotic stress. The dehydrin protein, Lti30, is shown to stabilise the liquid crystalline lamellar phases over a large range of hydration conditions, preventing phase transitions at low water contents, and extensive swelling of the lamellar phase at high water contents.

Physicochemical Problems of Mineral Processing; ISSN 2084-4735

According to REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), testing for acute inhalation toxicity is required for chemicals manufactured or imported at tonnages ≥ 10 tons per year. Three OECD test guidelines for acute inhalation toxicity in vivo are adopted (TG 403, TG 436, and TG 433). Since animal testing is ethically, scientifically and economically questionable, adoption of alternative methods by the European Union and the OECD is needed. An in vitro system based on the study of lung surfactant function is introduced.

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to development of mammalian lungs, and great efforts have been done to understand its structure–function relationships. Previous evidence has outlined the importance of SP-C palmitoylation to sustain the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification aids SP-C to stabilize the interfacial surfactant film along the compression–expansion breathing cycles, is still unrevealed. In this work we have compared the structure, orientation and lipid–protein interactions of a native palmitoylated SP-C with those of a non-palmitoylated recombinant SP-C (rSP-C) form in air-exposed multilayer membrane environments, by means of ATR-FTIR spectroscopy. Palmitoylation does not affect the secondary structure of the protein, which exhibits a full α-helical conformation in partly dehydrated phospholipid multilayer films. However, differences between the Amide I band of the IR spectrum of palmitoylated and non-palmitoylated proteins suggest subtle differences affecting the environment of their helical component. These differences are accompanied by differential effects on the IR bands from phospholipid phosphates, indicating that palmitoylation modulates lipid–protein interactions at the headgroup region of phospholipid layers. On the other hand, the relative dichroic absorption of polarized IR has allowed calculating that the palmitoylated protein adopts a more tilted transmembrane orientation than the non-palmitoylated SP-C, likely contributing to more compact, dehydrated and possibly stable multilayer lipid–protein films. As a whole, the behavior of multilayer films containing palmitoylated SP-C may reflect favorable structural properties for surfactant reservoirs at the air–liquid respiratory interface.

V doktorskem delu nas je zanimal biološki vpliv izbranih nanomaterialov, ki se lahko uporabljajo za medicinske namene. Vpliv nanomaterialov smo testirali s pasjimi ledvičnimi celicami MDCK in s človeškimi pljučnimi celicami A549 z lastnostmi alveolarnih celic tipa II. Glavni namen doktorskega dela je bil preveriti, ali lahko nanomateriali v izbranih celicah sprožijo nastanek fosfolipidoze (prekomerno znotrajcelično kopičenje fosfolipidov v lamelarnih telesih) kot enega zgodnjih znakov škodljivega vpliva nanomaterialov. Poleg tega smo želeli preveriti, ali so biološki vplivi nanomaterialov odvisni od njihove velikosti ter od njihovega raztapljanja. Na celicah MDCK, ki smo jih izpostavili necitotoksičnim koncentracijam nanodelcev ZnO, mikrodelcev ZnO in cinkove soli ZnCl2, smo zaznali poškodbe DNA le pri nanodelcih ZnO. Z merjenjem raztapljanja ZnO smo ugotovili, da sproščanje cinkovih ionov v gojišče za celice ni zadostno, da bi z njim lahko razložili škodljive vplive nanodelcev ZnO na celice. Z barvilom LipidTOX™ phospholipidosis, ki v celici obarva s fosfolipidi bogate organele, in ki se uporablja za testiranje potenciala za sprožitev fosfolipidoze, smo ugotovili, da kljub kvarnemu delovanju nanodelci ZnO ne sprožijo fosfolipidoze v celicah MDCK, kakor tudi ne v človeških pljučnih celicah A549. V celicah MDCK ni nobeden od izbranih nanomaterialov sprožil povečane količine s fosfolipidi bogatih organelov. V celicah A549 pa so nanodelci γ-Fe2O3+SiO2 sprožili povečano vsebnost s fosfolipidi bogatih organelov, povečano vsebnost fosfatidilholina in povečano vsebnost organelov, ki sodelujejo pri biogenezi lamelarnih teles. Vendar pa se je količina lamelarnih teles v izpostavljenih celicah A549 zmanjšala, povečala pa se je vsebnost avtofagnih vakuol, ki so pogosto vsebovale poškodovana lamelarna telesa, namenjena razgradnji. Rezultati na celicah A549 so pokazali, da nanodelci γ-Fe2O3+SiO2 vplivajo na metabolizem lipidov, na biogenezo lamelarnih teles in na zniževanje površinske napetosti kapljevine nad celicami. Kljub temu, da so naši rezultati pokazali, da pojav fosfolipidoze ni ustrezen indikator zgodnjih kvarnih vplivov nanomaterialov v celicah MDCK in A549, smo pokazali, da lahko nanomateriali pri necitotoksičnih koncentracijah sprožijo poškodbe DNA ter da lahko ovirajo pomembne funkcije celic. We investigated the biological effects of selected nanomaterials that have potential for medical use. Effects of nanomaterials were evaluated on the canine kidney MDCK cells and on the human lung A549 cells with properties of alveolar type II cells. The main purpose of the doctoral work was to determine whether nanomaterials can induce phospholipidosis (excessive intracellular accumulation of phospholipids in lamellar bodies) as early sign of adverse effects of nanomaterials. In addition, we wanted to check if biological effects of nanomaterials depend on their size and on their dissolution. On MDCK cells that were exposed to sub-cytotoxic concentrations of ZnO nanoparticles, ZnO microparticles and to zinc salt ZnCl2, we observed increased DNA damage only in cells exposed to nanoparticles. By measuring the ZnO nanoparticle dissolution, we found that the zinc ion release in the cell culture medium is insufficient to be the main cause of ZnO nanoparticle toxicity. By staining cells with LipidTOX™ phospholipidosis dye that is used to test potential for phospholipidosis induction, we found that ZnO nanoparticles as well as all other selected nanomaterials did not increased the amount of phospholipid rich organelles in MDCK cells. In contrast, in A549 cells, we observed that silica coated maghemite nanoparticles (γ-Fe2O3+SiO2) caused increased cellular content of phospholipids rich organelles, increased content of phosphatidylcholine (PC) and increased content of organelles that are involved in lamellar body biogenesis. Despite these results, indicating intensive lamelar body biogenesis in the exposed A549 cells, the amount of lamellar bodies had been decreased. In γ-Fe2O3+SiO2 treated A549 cells, we observed an increased amount of autophagic vacuoles that often contained defective lamellar bodies destined for degradation. Results on A549 cells showed that γ-Fe2O3+SiO2 affect lipid metabolism, interfered with the lamellar body biogenesis and reduced cell capacity to lower the surface tension of hypophase. Our results show that phospholipidosis is not good indicator of nanotoxicity in MDCK and A549 cells. Nevertheless we have shown that nanomaterials can induce DNA damage and may interfere with important functions of the cell, even at non-cytotoxic concentrations.

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

The toxicity of some per- and polyfluoroalkyl substances (PFASs), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) has been studied thoroughly, showing that systemic PFASs targets the lungs. However, regulators lack data to assess the impact of other PFASs on the lungs and alternative methods to test substances for lung toxicity are needed. We combined two in vitro models to assess toxicity to the respiratory system; i) a lung surfactant (LS) function assay to assess the acute inhalation toxicity potential, and ii) a cell model with human bronchial epithelial cells to study pro-inflammatory potential and modulation of inflammatory responses. We tested salts of four PFASs: perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), PFOS, and PFOA as well as the fluorotelomer 8:2 FTOH. The results show that PFHxS, PFOA and PFOS can inhibit LS function. High PFOS concentrations induced a pro-inflammatory response, measured as increased IL-1α/β release. Moderate concentrations of PFOS suppressed release of the chemokines CXCL8 and CXCL10, whereas both PFOS and PFOA stimulated the release of the pro-inflammatory cytokine IL-1β in immune stimulated human bronchial epithelial cells. These findings support the concern that some PFASs may increase the risk of acute lung toxicity and of airway infections.

Part 2 of the review considers the problem of surfactant therapy for acute respiratory distress syndrome (ARDS) in adults and young and old children. It gives information on the results of surfactant therapy and prevention of ARDS in patients with severe concurrent trauma, inhalation injuries, complications due to complex expanded chest surgery, or severe pneumonias, including bilateral pneumonia in the presence of A/H1N1 influenza. There are data on the use of a surfactant in obstetric care and prevention of primary graft dysfunction during lung transplantation. The results of longterm use of surfactant therapy in Russia, suggesting that death rates from ARDS may be substantially reduced (to 20%) are discussed. Examples of surfactant therapy for other noncritical lung diseases, such as permanent athelectasis, chronic obstructive pulmonary diseases, and asthma, as well tuberculosis, are also considered.

OBJECTIVE: To compare the use of drugs for pulmonary diseases in children who were exposed to antidepressants in utero with non-exposed children. Some studies suggest an association between in utero exposure to antidepressants and the occurrence of pulmonary diseases later in life. Antidepressants are thought to be involved in the development of the respiratory rhythm generator and the maturation of surfactant formation. DESIGN AND METHODS: The pharmacy prescription database IADB.nl was used for a cohort study. The use of drugs for pulmonary diseases in children after in utero exposure to antidepressants was compared to children with no antidepressant exposure in utero. Drugs for pulmonary diseases were applied as a proxy for disturbed development of the respiratory tract. RESULTS: A small but significant increase in the incidence risk ratio (IRR) of the use of drugs for pulmonary diseases was found after anytime in utero exposure to serotonin reuptake inhibitors (SSRIs) (1.17; CI951.16-1.18). IRR also was increased for specific use of SSRIs in the first trimester (1.18; CI951.17-1.20). IRR increase for children who were exposed to tricyclic antidepressants was not statistically significant. As sample size was rather small in both groups, the effect size is modest and may also be confounded by maternal smoking. CONCLUSIONS: Children exposed to SSRIs in utero use significantly more drugs for pulmonary diseases, especially when exposure occurred during the first trimester of pregnancy. However, the increased use of such drugs may also be related to other factors.

OBJECTIVE: To compare the use of drugs for pulmonary diseases in children who were exposed to antidepressants in utero with non-exposed children. Some studies suggest an association between in utero exposure to antidepressants and the occurrence of pulmonary diseases later in life. Antidepressants are thought to be involved in the development of the respiratory rhythm generator and the maturation of surfactant formation. DESIGN AND METHODS: The pharmacy prescription database IADB.nl was used for a cohort study. The use of drugs for pulmonary diseases in children after in utero exposure to antidepressants was compared to children with no antidepressant exposure in utero. Drugs for pulmonary diseases were applied as a proxy for disturbed development of the respiratory tract. RESULTS: A small but significant increase in the incidence risk ratio (IRR) of the use of drugs for pulmonary diseases was found after anytime in utero exposure to serotonin reuptake inhibitors (SSRIs) (1.17; CI951.16-1.18). IRR also was increased for specific use of SSRIs in the first trimester (1.18; CI951.17-1.20). IRR increase for children who were exposed to tricyclic antidepressants was not statistically significant. As sample size was rather small in both groups, the effect size is modest and may also be confounded by maternal smoking. CONCLUSIONS: Children exposed to SSRIs in utero use significantly more drugs for pulmonary diseases, especially when exposure occurred during the first trimester of pregnancy. However, the increased use of such drugs may also be related to other factors.

The photophysical behavior of 8-anilino-1-naphthalenesulphonate (ANS) in vesicles of dipalmitoylphosphatidylcholine (DPPC), a pulmonary surfactant, has been carried out in a detailed manner. ANS shows notable variations in fluorescence intensity, lifetime, and anisotropy parameters as it gets into the vesicle. It was found that ANS partitions well into the DPPC bilayer membrane with an estimated partition coefficient of ?2.0 � 105. Among the various fluorescence parameters of ANS, fluorescence anisotropy was found to be most responsive to the temperature induced phase change of the bilayer membrane. These interesting fluorescence parameters of ANS were then used to study the hydration of lipid bilayer membrane by submicellar concentration of bile salts. From the steady-state fluorescence intensity and dynamic fluorescence lifetime analyses it is clear that ANS is able to probe the submicellar concentration (?1 mM) of bile salt induced hydration of lipid bilayer membrane that accompanies expulsion of ANS from the bilayer to the aqueous bulk phase. Lower-temperature shift in the phase transition of DPPC bilayer indicates that fluorescence anisotropy of ANS is sensitive enough to the bile salt induced perturbation in the packed acyl chains of DPPC bilayer and modification in the membrane fluidity. In presence of sodium deoxycholate (NaDC) and sodium cholate (NaC) in DPPC vesicles, ANS experiences restriction in rotational mobility which is evident from the variation in steady-state fluorescence anisotropy and fluorescence anisotropy decay parameters. � 2013 American Chemical Society.