Publications

[No abstract available]

JTD Keywords: Breathing, Cost, Developed country, Developing countries, Developing country, Health care facility, Home monitoring, Human, Humans, Internet, Internet of things, Letter, Lowest income group, Lung function, Lung mechanics, Lung resistance, Mathematical model, Middle income country, Mobile applications, Non invasive procedure, Open source technology, Oscillometry, Pneumotachygraphy, Telemedicine

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with in vitro and in vivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors.

JTD Keywords: Cancer-associated fibroblast,fibrosis,nintedanib,non-small-cell lung cancer,smad3,therapy resistance,timp-, Cell carcinoma,breast-cancer,expression,progression,inhibitor,blockade,efficac

While having already killed more than 7 million of people worldwide in 4 years, SARS-CoV-2, the etiological agent of COVID-19, is still circulating and evolving. Understanding the pathogenesis of the virus is of capital importance. It was shown that in vitro and in vivo infection with SARS-CoV-2 can lead to cell cycle arrest but the effect of the cell cycle arrest on the virus infection and the associated mechanisms are still unclear. By stopping cells in the G1 phase as well as targeting several pathways involved using inhibitors and small interfering RNAs, we were able to determine that the cell cycle arrest in the late G1 is beneficial for SARS-CoV-2 replication. This cell cycle arrest is independent of p53 but is dependent on the CDC25A-CDK2/cyclin E pathway. These data give a new understanding in SARS-CoV-2 pathogenesis and highlight some possible targets for the development of novel therapeutic approaches.

JTD Keywords: Cell lung-cancer,exchanger nhe,g1 phase,proliferation,inhibitio, Covid-19,coronavirus,pathogenicity,replication,cdk2,cyclin e,cdc25a,treatment

A fundamental task of the respiratory system is to operate as a mechanical gas pump ensuring that fresh air gets in close contact with the blood circulating through the lung capillaries to achieve O2 and CO2 exchange. To ventilate the lungs, the respiratory muscles provide the pressure required to overcome the viscoelastic mechanical load of the respiratory system. From a mechanical viewpoint, the most relevant respiratory system properties are the resistance of the airways (R aw), and the compliance of the lung tissue (C L) and chest wall (C CW). Both airflow and lung volume changes in spontaneous breathing and mechanical ventilation are determined by applying the fundamental mechanical laws to the relationships between the pressures inside the respiratory system (at the airway opening, alveolar, pleural, and muscular) and R aw, C L, and C CW. These relationships also are the basis of the different methods available to measure respiratory mechanics during spontaneous and artificial ventilation. Whereas a simple mechanical model (R aw, C L, and C CW) describes the basic understanding of ventilation mechanics, more complex concepts (nonlinearity, inhomogeneous ventilation, or viscoelasticity) should be employed to better describe and measure ventilation mechanics in patients.Thieme. All rights reserved.

JTD Keywords: airway-resistance, alveolar, compliance, dilution, elastance, flow, inhomogeneous ventilation, input impedance, lung-volume, mechanical ventilation, monitoring, pendelluft, pleural pressure, respiratory-distress-syndrome, viscoelasticity, Chest-wall mechanics, Resistance

JTD Keywords: Bacterial biofilms, Device, Growth, In-vitro, Infections, Laboratory method, Lung model, Responses, Temperature, Time

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

JTD Keywords: drug delivery, icam-1, inflammation, lung injury, messenger-rna, migration, model, nanoparticles, neutrophils, pharmacokinetics, t-cells, white bloodcells, Adhesion molecules, Brain, Drug delivery, Inflammation, Nanoparticles, Pharmacokinetics, White blood cells

Fibrillar collagens are the most abundant extracellular matrix components in non-small cell lung cancer (NSCLC). However, the potential of collagen fiber descriptors as a source of clinically relevant biomarkers in NSCLC is largely unknown. Similarly, our understanding of the aberrant collagen organization and associated tumor-promoting effects is very scarce. To address these limitations, we identified a digital pathology approach that can be easily implemented in pa-thology units based on CT-FIRE software (version 2; https://loci.wisc.edu/software/ctfire) analysis of Picrosirius red (PSR) stains of fibrillar collagens imaged with polarized light (PL). CT-FIRE set-tings were pre-optimized to assess a panel of collagen fiber descriptors in PSR-PL images of tissue microarrays from surgical NSCLC patients (106 adenocarcinomas [ADC] and 89 squamous cell carcinomas [SCC]). Using this approach, we identified straightness as the single high-accuracy diagnostic collagen fiber descriptor (average area under the curve 1/4 0.92) and fiber density as the single descriptor consistently associated with a poor prognosis in both ADC and SCC inde-pendently of the gold standard based on the TNM staging (hazard ratio, 2.69; 95% CI, 1.55-4.66; P < .001). Moreover, we found that collagen fibers were markedly straighter, longer, and more aligned in tumor samples compared to paired samples from uninvolved pulmonary tissue, particularly in ADC, which is indicative of increased tumor stiffening. Consistently, we observed an increase in a panel of stiffness-associated processes in the high collagen fiber density patient group selectively in ADC, including venous/lymphatic invasion, fibroblast activation (a-smooth muscle actin), and immune evasion (programmed death-ligand 1). Similarly, a transcriptional correlation analysis supported the potential involvement of the major YAP/TAZ pathway in ADC. Our results provide a proof-of-principle to use CT-FIRE analysis of PSR-PL images to assess new collagen fiber-based diagnostic and prognostic biomarkers in pathology units, which may improve the clinical management of patients with surgical NSCLC. Our findings also unveil an aberrant stiff micro -environment in lung ADC that may foster immune evasion and dissemination, encouraging future work to identify therapeutic opportunities. (c) 2023 THE AUTHORS. Published by Elsevier Inc. on behalf of the United States & Canadian Academy of Pathology. This is an open access article under the CC BY-NC-ND license (http://creativecommo ns.org/licenses/by-nc-nd/4.0/).

JTD Keywords: biomarkers, collagen, ct-fire, lung cancer, mechanobiology, Adenocarcinoma, Association, Biomarkers, Collagen, Ct-fire, Differentiation, Expression, Extracellular-matrix, I collagen, Invasion, Lung cancer, Mechanobiology, Microenvironment, Signature, Survival, Tumor microenvironment

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.Copyright © 2023 Liang, Nilsson, Kragh, Hedal, Alcàcer-Almansa, Kiilerich, Andersen and Tolker-Nielsen.

JTD Keywords: aggregates, antibiotic tolerance, biofilm formation, extracellular matrix, genome, growth, lungs, molecular-mechanisms, mutations, polysaccharide, pseudomonas aeruginosa, psl, system, Aggregates, Antibiotic tolerance, Biofilm, Extracellular matrix, Pseudomonas aeruginosa, Small-colony variants

Mechanical changes in tumors have long been linked to increased malignancy and therapy resistance and attributed to mechanical changes in the tumor extracellular matrix (ECM). However, to the best of our knowledge, there have been no mechanical studies on decellularized tumors. Here, we studied the biochemical and mechanical progression of the tumor ECM in two models of lung metastases: lung carcinoma (CAR) and melanoma (MEL). We decellularized the metastatic lung sections, measured the micromechanics of the tumor ECM, and stained the sections for ECM proteins, proliferation, and cell death markers. The same methodology was applied to MEL mice treated with the clinically approved anti-fibrotic drug nintedanib. When compared to healthy ECM (~0.40 kPa), CAR and MEL lung macrometastases produced a highly dense and stiff ECM (1.79 ± 1.32 kPa, CAR and 6.39 ± 3.37 kPa, MEL). Fibronectin was overexpressed from the early stages (~118%) to developed macrometastases (~260%) in both models. Surprisingly, nintedanib caused a 4-fold increase in ECM-occupied tumor area (5.1 ± 1.6% to 18.6 ± 8.9%) and a 2-fold in-crease in ECM stiffness (6.39 ± 3.37 kPa to 12.35 ± 5.74 kPa). This increase in stiffness strongly correlated with an increase in necrosis, which reveals a potential link between tumor hypoxia and ECM deposition and stiffness. Our findings highlight fibronectin and tumor ECM mechanics as attractive targets in cancer therapy and support the need to identify new anti-fibrotic drugs to abrogate aberrant ECM mechanics in metastases.

JTD Keywords: atomic force microscopy, basement membrane, breast-cancer, decellularization, expression, extracellular matrix, extracellular-matrix, fibronectin, intermittent hypoxia, lung carcinoma, lung metastases, melanoma, metastatic niche formation, micromechanical properties, nintedanib, signature, stiffness, tumor-growth, Colorectal-cancer progression, Lung metastases, Stiffness

The extracellular matrix (ECM) of the lung is a filamentous network composed mainly of collagens, elastin, and proteoglycans that provides structural and physical support to its populating cells. Proliferation, migration and overall behaviour of those cells is greatly determined by micromechanical queues provided by the ECM. Lung fibrosis displays an aberrant increased deposition of ECM which likely changes filament organization and stiffens the ECM, thus upregulating the profibrotic profile of pulmonary cells. We have previously used AFM to assess changes in the Young’s Modulus (E) of the ECM in the lung. Here, we perform further ECM topographical, mechanical and viscoelastic analysis at the micro- and nano-scale throughout fibrosis development. Furthermore, we provide nanoscale correlations between topographical and elastic properties of the ECM fibres. Firstly, we identify a softening of the ECM after rats are instilled with media associated with recovery of mechanical homeostasis, which is hindered in bleomycin-instilled lungs. Moreover, we find opposite correlations between fibre stiffness and roughness in PBS- vs bleomycin-treated lung. Our findings suggest that changes in ECM nanoscale organization take place at different stages of fibrosis, with the potential to help identify pharmacological targets to hinder its progression.

JTD Keywords: atomic force microscopy, cells, deposition, extracellular matrix, idiopathic pulmonary fibrosis, mechanisms, mechanosensing, membranes, micromechanical properties, pathogenesis, stiffness, tissues, viscoelasticity, Extracellular matrix, Induced lung fibrosis, Mechanosensing

The use of physiomimetic decellularized extracellular matrix-derived hydrogels is attracting interest since they can modulate the therapeutic capacity of numerous cell types, including mesenchymal stromal cells (MSCs). Remarkably, extracellular vesicles (EVs) derived from MSCs display similar functions as their parental cells, mitigating tissue damage in lung diseases. However, recent data have shown that ECM-derived hydrogels could release other resident vesicles similar to EVs. Here, we aim to better understand the contribution of EVs and ECM-vesicles released from MSCs and/or lung-derived hydrogel (L-HG) in lung repair by using an in vitro lung injury model. L-HG derived-vesicles and MSCs EVs cultured either in L-HG or conventional plates were isolated and characterized. The therapeutic capacity of vesicles obtained from each experimental condition was tested by using an alveolar epithelial wound-healing assay. The number of ECM-vesicles released from acellular L-HG was 10-fold greater than EVs from conventional MSCs cell culture revealing that L-HG is an important source of bioactive vesicles. MSCs-derived EVs and L-HG vesicles have similar therapeutic capacity in lung repair. However, when wound closure rate was normalized by total proteins, the MSCs-derived EVs shows higher therapeutic potential to those released by L-HG. The EVs released from L-HG must be considered when HG is used as substrate for cell culture and EVs isolation.

JTD Keywords: cell, extracellular vesicles, hydrogel, lung epithelial cells, lung repair, mesenchymal stem cells, Extracellular matrix, Extracellular vesicles, Hydrogel, Lung epithelial cells, Lung repair, Mesenchymal stem cells, Respiratory-distress-syndrome

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy.

JTD Keywords: alveolar epithelial cells, ards, extracellular matrix, hydrogels, inflammation, lung-on-a-chip, Acute lung injury, Alveolar epithelial cells, Ards, Dexamethasone, Epithelial-mesenchymal transition, Extracellular matrix, Extracellular-matrix, Hydrogels, Inflammation, Lung-on-a-chip, Mesenchymal stromal cells, Oxygen, Stem-cells

Assessing tidal volume during mechanical ventilation is critical to improving gas exchange while avoiding ventilator-induced lung injury. Conventional flow and volume measurements are usually carried out by built-in pneumotachographs in the ventilator or by stand-alone flowmeters. Such flow/volume measurement devices are expensive and thus usually unaffordable in low-resource settings. Here, we aimed to design and test low-cost and technically-simple calibration and assembly pneumotachographs. The proposed pneumotachographs are made by manual perforation of a plate with a domestic drill. Their pressure-volume relationship is characterized by a quadratic equation with parameters that can be tailored by the number and diameter of the perforations. We show that the calibration parameters of the pneumotachographs can be measured through two maneuvers with a conventional resuscitation bag and by assessing the maneuver volumes with a cheap and straightforward water displacement setting. We assessed the performance of the simplified low-cost pneumotachographs to measure flow/volume during mechanical ventilation as carried out under typical conditions in low-resource settings, i.e., lacking gold standard expensive devices. Under realistic mechanical ventilation settings (pressure- and volume-control; 200-600 mL), inspiratory tidal volume was accurately measured (errors of 2.1% on average and <4% in the worst case). In conclusion, a simple, low-cost procedure facilitates the construction of affordable and accurate pneumotachographs for monitoring mechanical ventilation in low- and middle-income countries.

JTD Keywords: calibration, flow measurement, low- and middle-income countries, mechanical ventilation, pneumotachograph, Calibration, Flow, Flow measurement, Low- and middle-income countries, Lung injury, Mechanical ventilation, Pneumotachograph, Pressure-drop, Resistance, Tidal volume

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

JTD Keywords: 3dna nanocarrier, acid sphingomyelinase, antibody, carrier design parameters, carriers, dna nanostructures, doxorubicin, drug type, icam-1, inflammation, lung targeting, multiparametric hierarchy, nanoparticles, size, 3dna nanocarrier, Intracellular delivery, Multiparametric hierarchy

Pulmonary fibrosis (PF) is characterized by aberrant extracellular matrix (ECM) deposition, activation of fibroblasts to myofibroblasts and parenchymal disorganization, which have an impact on the biomechanical traits of the lung. In this context, the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors of metalloproteinases (TIMPs) is lost. Interestingly, several MMPs are overexpressed during PF and exhibit a clear profibrotic role (MMP-2, -3, -8, -11, -12 and -28), but a few are antifibrotic (MMP-19), have both profibrotic and antifibrotic capacity (MMP7), or execute an unclear (MMP-1, -9, -10, -13, -14) or unknown function. TIMPs are also overexpressed in PF; hence, the modulation and function of MMPs and TIMP are more complex than expected. EMMPRIN/CD147 (also known as basigin) is a transmembrane glycoprotein from the immunoglobulin superfamily (IgSF) that was first described to induce MMP activity in fibroblasts. It also interacts with other molecules to execute non-related MMP aactions well-described in cancer progression, migration, and invasion. Emerging evidence strongly suggests that CD147 plays a key role in PF not only by MMP induction but also by stimulating fibroblast myofibroblast transition. In this review, we study the structure and function of MMPs, TIMPs and CD147 in PF and their complex crosstalk between them.

JTD Keywords: basigin, cd147, emmprin, mmps, timps, Basigin, Cd147, Cell-surface, Emmprin, Extracellular-matrix, Gelatinase-b, Gene-expression profiles, Growth-factor-beta, Immunoglobulin superfamily, Induced lung injury, Inducer emmprin, Mmps, Pulmonary fibrosis, Timps, Tissue inhibitor, Transforming growth-factor-beta-1

Background: Opioid-related deaths are increasing globally. Respiratory complications of opioid use and underlying respiratory disease in people with Opioid Use Disorder (OUD) are potential contributory factors. Individual variation in susceptibility to overdose is, however, incompletely understood. This study investigated the prevalence of respiratory depression (RD) in OUD treatment and compared this to patients with chronic obstructive pulmonary disease (COPD) of equivalent severity. We also explored the contribution of opioid agonist treatment (OAT) dosage, and type, to the prevalence of RD. Methods: There were four groups of participants: 1) OUD plus COPD (‘OUD-COPD’, n = 13); 2) OUD without COPD (‘OUD’, n = 7); 3) opioid-naïve COPD patients (‘COPD'n = 13); 4) healthy controls (‘HC'n = 7). Physiological indices, including pulse oximetry (SpO2%), end-tidal CO2 (ETCO2), transcutaneous CO2 (TcCO2), respiratory airflow and second intercostal space parasternal muscle electromyography (EMGpara), were recorded continuously over 40 min whilst awake at rest. Significant RD was defined as: SpO2%< 90% for > 10 s, ETCO2 per breath > 6.6 kPa, TcCO2 overall mean > 6 kPa, respiratory pauses > 10 s Results: At least one indicator was observed in every participant with OUD (n = 20). This compared to RD episode occurrence in only 2/7 HC and 2/13 COPD participants (p < 0.05,Fisher's exact test). The occurrence of RD was similar in OUD participants prescribed methadone (n = 6) compared to those prescribed buprenorphine (n = 12). Conclusions: Undetected RD is common in OUD cohorts receiving OAT and is significantly more severe than in opioid-naïve controls. RD can be assessed using simple objective measures. Further studies are required to determine the association between RD and overdose risk. © 2022 Elsevier B.V.

JTD Keywords: buprenorphine, comorbidity, deaths, drive, heroin, lung disease, opioid substitution treatment, opioids, overdose, pulse oximetry, respiratory depression, risk, Acute exacerbations, Comorbidity, Lung disease, Opioid substitution treatment, Opioids, Overdose, Respiratory depression

Decellularization procedures have been developed and optimized for the entire organ or tissue blocks, by either perfusion of decellularizing agents through the tissue’s vasculature or submerging large sections in decellularizing solutions. However, some research aims require the analysis of native as well as decellularized tissue slices side by side, but an optimal protocol has not yet been established to address this need. Thus, the main goal of this work was to develop a fast and efficient decellularization method for tissue slices—with an emphasis on lung—while attached to a glass slide. To this end, different decellularizing agents were compared for their effectiveness in cellular removal while preserving the extracellular matrix. The intensity of DNA staining was taken as an indicator of remaining cells and compared to untreated sections. The presence of collagen, elastin and laminin were quantified using immunostaining and signal quantification. Scaffolds resulting from the optimized protocol were mechanically characterized using atomic force microscopy. Lung scaffolds were recellularized with mesenchymal stromal cells to assess their biocompatibility. Some decellularization agents (CHAPS, triton, and ammonia hydroxide) did not achieve sufficient cell removal. Sodium dodecyl sulfate (SDS) was effective in cell removal (1% remaining DNA signal), but its sharp reduction of elastin signal (only 6% remained) plus lower attachment ratio (32%) singled out sodium deoxycholate (SD) as the optimal treatment for this application (6.5% remaining DNA signal), due to its higher elastin retention (34%) and higher attachment ratio (60%). Laminin and collagen were fully preserved in all treatments. The SD decellularization protocol was also successful for porcine and murine (mice and rat) lungs as well as for other tissues such as the heart, kidney, and bladder. No significant mechanical differences were found before and after sample decellularization. The resulting acellular lung scaffolds were shown to be biocompatible (98% cell survival after 72 h of culture). This novel method to decellularize tissue slices opens up new methodological possibilities to better understand the role of the extracellular matrix in the context of several diseases as well as tissue engineering research and can be easily adapted for scarce samples like clinical biopsies. Copyright © 2022 Narciso, Ulldemolins, Júnior, Otero, Navajas, Farré, Gavara and Almendros.

JTD Keywords: biocompatibility, bioscaffold recellularization, decellularization, extracellular matrix, flow, impact, lung, scaffolds, tissue slices, Ammonia, Bio-scaffolds, Biocompatibility, Biological organs, Bioscaffold recellularization, Cell removal, Cells, Collagen, Cytology, Decellularization, Dna, Dna signals, Elastin, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Glycoproteins, Laminin, Lung, Mammals, Recellularization, Scaffolds (biology), Sodium deoxycholate, Sulfur compounds, Tissue, Tissue slice, Tissue slices

The mechanical signals sensed by the alveolar cells through the changes in the local matrix stiffness of the extracellular matrix (ECM) are determinant for regulating cellular functions. Therefore, the study of the mechanical response of lung tissue becomes a fundamental aspect in order to further understand the mechanosensing signals perceived by the cells in the alveoli. This study is focused on the development of a finite element (FE) model of a decellularized rat lung tissue strip, which reproduces accurately the mechanical behaviour observed in the experiments by means of a tensile test. For simulating the complex structure of the lung parenchyma, which consists of a heterogeneous and non-uniform network of thin-walled alveoli, a 3D model based on a Voronoi tessellation is developed. This Voronoi-based model is considered very suitable for recreating the geometry of cellular materials with randomly distributed polygons like in the lung tissue. The material model used in the mechanical simulations of the lung tissue was characterized experimentally by means of AFM tests in order to evaluate the lung tissue stiffness on the micro scale. Thus, in this study, the micro (AFM test) and the macro scale (tensile test) mechanical behaviour are linked through the mechanical simulation with the 3D FE model based on Voronoi tessellation. Finally, a micro-mechanical FE-based model is generated from the Voronoi diagram for studying the stiffness sensed by the alveolar cells in function of two independent factors: the stretch level of the lung tissue and the geometrical position of the cells on the extracellular matrix (ECM), distinguishing between pneumocyte type I and type II. We conclude that the position of the cells within the alveolus has a great influence on the local stiffness perceived by the cells. Alveolar cells located at the corners of the alveolus, mainly type II pneumocytes, perceive a much higher stiffness than those located in the flat areas of the alveoli, which correspond to type I pneumocytes. However, the high stiffness, due to the macroscopic lung tissue stretch, affects both cells in a very similar form, thus no significant differences between them have been observed. © 2021 The Authors

JTD Keywords: rat, scaffolds, stiffness, Afm, Animal cell, Animal experiment, Animal model, Animal tissue, Article, Biological organs, Cell function, Cells, Computational geometry, Cytology, Extracellular matrices, Extracellular matrix, Extracellular-matrix, Geometry, High stiffness, Human, Lung alveolus cell type 1, Lung alveolus cell type 2, Lung parenchyma, Lung tissue, Male, Mechanical behavior, Mechanical modeling, Mechanical simulations, Mechanosensing, Model-based opc, Nonhuman, Physical model, Rat, Rigidity, Stiffness, Stiffness matrix, Tensile testing, Thin walled structures, Three dimensional finite element analysis, Tissue, Type ii, Voronoi tessellations

Background and aims: In this work, breath samples from clinically stable bronchiectasis patients with and without bronchial infections by Pseudomonas Aeruginosa- PA) were collected and chemically analysed to determine if they have clinical value in the monitoring of these patients. Materials and methods: A cohort was recruited inviting bronchiectasis patients (25) and controls (9). Among the former group, 12 members were suffering PA infection. Breath samples were collected in Tedlar bags and analyzed by e-nose and Gas Chromatography-Mass Spectrometry (GC-MS). The obtained data were analyzed by chemometric methods to determine their discriminant power in regards to their health condition. Results were evaluated with blind samples. Results: Breath analysis by electronic nose successfully separated the three groups with an overall classification rate of 84% for the three-class classification problem. The best discrimination was obtained between control and bronchiectasis with PA infection samples 100% (CI95%: 84–100%) on external validation and the results were confirmed by permutation tests. The discrimination analysis by GC-MS provided good results but did not reach proper statistical significance after a permutation test. Conclusions: Breath sample analysis by electronic nose followed by proper predictive models successfully differentiated between control, Bronchiectasis and Bronchiectasis PA samples. © 2021 The Author(s)

JTD Keywords: biomarkers, breath analysis, bronchiectasis, diagnosis, e-nose, fingerprints, gc-ms, identification, lung-cancer, partial least-squares, pseudomonas-aeruginosa, signal processing, validation, volatile organic-compounds, Airway bacterial-colonization, Breath analysis, Bronchiectasis, E-nose, Gc-ms, Signal processing

Obstructive sleep apnea (OSA) is a sleep disorder in which repetitive upper airway obstructive events occur during sleep. These events can induce hypoxia, which is a risk factor for multiple cardiovascular and cerebrovascular diseases. Chronic obstructive pulmonary disease (COPD) is a disorder which induces a persistent inflammation of the lungs. This condition produces hypoventilation, affecting the blood oxygenation, and leads to an increased risk of developing lung cancer and heart disease. In this study, we evaluated how COPD affects the severity and characteristics of OSA in a multivariate demographic database including polysomnographic signals. Results showed SpO2 subtle variations, such as more non-recovered desaturations and increased time below a 90% SpO2 level, which, in the long term, could worsen the risk to suffer cardiovascular and cerebrovascular diseases.Clinical Relevance - COPD increases the OSA risk due to hypoventilation and altered SpO2 behavior. © 2021 IEEE.

JTD Keywords: Chronic obstructive lung disease, Complication, Epidemiologic studies, Epidemiology, Human, Humans, Oxygen saturation, Pulmonary disease, chronic obstructive, Sleep apnea, obstructive, Sleep disordered breathing, Syndrome

Pulmonary fibrosis (PF) is a progressive disease that disrupts the mechanical homeostasis of the lung extracellular matrix (ECM). These effects are particularly relevant in the lung context, given the dynamic nature of cyclic stretch that the ECM is continuously subjected to during breathing. This work uses an in vivo model of pulmonary fibrosis to characterize the macro-and micromechanical properties of lung ECM subjected to stretch. To that aim, we have compared the micromechanical properties of fibrotic ECM in baseline and under stretch conditions, using a novel combination of Atomic Force Microscopy (AFM) and a stretchable membrane-based chip. At the macroscale, fibrotic ECM displayed strain-hardening, with a stiffness one order of magnitude higher than its healthy counterpart. Conversely, at the microscale, we found a switch in the stretch-induced mechanical behaviour of the lung ECM from strain-hardening at physiological ECM stiffnesses to strain-softening at fibrotic ECM stiffnesses. Similarly, we observed solidification of healthy ECM versus fluidization of fibrotic ECM in response to stretch. Our results suggest that the mechanical behaviour of fibrotic ECM under stretch involves a potential built-in mechanotransduction mechanism that may slow down the progression of PF by steering resident fibroblasts away from a pro-fibrotic profile. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: atomic force microscopy, extracellular matrix, fibrosis, mechanics, mechanosensing, strain, system, viscoelasticity, Atomic force microscopy, Extracellular matrix, Fibrosis, Lung fibrosis, Mechanosensing

Lung cancer is the leading cause of cancer-related death worldwide. The desmoplastic stroma of lung cancer and other solid tumors is rich in tumor-associated fibroblasts (TAFs) exhibiting an activated/myofibroblast-like phenotype. There is growing awareness that TAFs support key steps of tumor progression and are epigenetically reprogrammed compared to healthy fibroblasts. Although the mechanisms underlying such epigenetic reprogramming are incompletely understood, there is increasing evidence that they involve interactions with either cancer cells, pro-fibrotic cytokines such as TGF-β, the stiffening of the surrounding extracellular matrix, smoking cigarette particles and other environmental cues. These aberrant interactions elicit a global DNA hypomethylation and a selective transcriptional repression through hypermethylation of the TGF-β transcription factor SMAD3 in lung TAFs. Likewise, similar DNA methylation changes have been reported in TAFs from other cancer types, as well as histone core modifications and altered microRNA expression. In this review we summarize the evidence of the epigenetic reprogramming of TAFs, how this reprogramming contributes to the acquisition and maintenance of a tumor-promoting phenotype, and how it provides novel venues for therapeutic intervention, with a special focus on lung TAFs.

JTD Keywords: cancer-associated fibroblasts, desmoplasia, dna methylation, epigenetics, expression, genomic dna, lung cancer, mechanical memory, myofibroblast differentiation, pulmonary fibroblasts, smoking, stromal fibroblasts, tgf-?, tgf-beta, tgf-β, transforming growth-factor-beta-1, tumor stroma, Cancer-associated fibroblasts, Carcinoma-associated fibroblasts, Desmoplasia, Epigenetics, Lung cancer, Smoking, Tgf-β, Tumor stroma

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell–matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.

JTD Keywords: 3d bioprinting, acute lung injury, adhesion, collagen, differentiation, dimension, elastic properties, extracellular matrix, hydrogels, in-vitro, mechanical-properties, mesenchymal stromal cells, microenvironment, potentiate, tissue engineering, 3d bioprinting, Acute lung injury, Extracellular matrix, Hydrogels, Mesenchymal stromal cells, Stem-cells, Tissue engineering

Asthma is the most common inflammatory disease affecting the lungs, which can be caused by intrauterine or postnatal insults depending on the exposure to environmental factors. During early life, the exposure to different risk factors can influence the microbiome leading to undesired changes to the immune system. The modulations of the immunity, caused by dysbiosis during development, can increase the susceptibility to allergic diseases. On the other hand, immune training approaches during pregnancy can prevent allergic inflammatory diseases of the airways. In this review, we focus on evidence of risk factors in early life that can alter the development of lung immunity associated with dysbiosis, that leads to asthma and affect childhood and adult life. Furthermore, we discuss new ideas for potential prevention strategies that can be applied during pregnancy and postnatal period.

JTD Keywords: asthma, dysbiosis, early life immunity, lung microbiome, Adulthood, Antibiotic exposure, Asthma, Childhood, Disease, Disease exacerbation, Dysbiosis, Early life immunity, Gut microbiome, Human, Immunity, Intestine flora, Lung development, Lung microbiome, Lung microbiota, Nonhuman, Perinatal period, Pregnancy, Prevention, Prevention strategies, Review, Risk, Risk factor, Sensitization, Supplementation, Vitamin-d, Wheeze

© 2021 Large cell carcinoma (LCC) is a rare and aggressive lung cancer subtype with poor prognosis and no targeted therapies. Tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we identify MMP1 as overexpressed specifically in LCC cell lines, and we show that expression of MMP1 by LCC cells is necessary for induction of fibroblast senescence and consequent tumor promotion in both cell culture and mouse models. We also show that MMP1, in combination with TGF-β1, is sufficient to induce fibroblast senescence and consequent LCC promotion. Furthermore, we implicate PAR-1 and oxidative stress in MMP1/TGF-β1-induced TAF senescence. Our results establish an entirely new role for MMP1 in cancer, and support a novel therapeutic strategy in LCC based on targeting senescent TAFs.

JTD Keywords: cancer-associated fibroblasts, lung cancer, mmp1, senescence, tgf-?, tgf-beta, tgf-β, Cancer-associated fibroblasts, Lung cancer, Mmp1, Senescence, Tgf-β

Asthma is a widespread disease characterized by chronic airway inflammation. It causes substantial disability, impaired quality of life, and avoidable deaths around the world. The main treatment for asthmatic patients is the administration of corticosteroids, which improves the quality of life; however, prolonged use of corticosteroids interferes with extracellular matrix elements. Therefore, cell-based therapies are emerging as a novel therapeutic contribution to tissue regeneration for lung diseases. This study aimed to summarize the advancements in cell therapy involving mesenchymal stromal cells, extracellular vesicles, and immune cells such as T-cells in asthma. Our findings provide evidence that the use of mesenchymal stem cells, their derivatives, and immune cells such as T-cells are an initial milestone to understand how emergent cell-based therapies are effective to face the challenges in the development, progression, and management of asthma, thus improving the quality of life.

JTD Keywords: asthma treatments, cell-based therapies, chronic lung diseases, extracellular vesicles, immune cells, mesenchymal stromal cells, Asthma treatments, Cell-based therapies, Chronic lung diseases, Extracellular vesicles, Immune cells, Mesenchymal stromal cells

© 2020 Elsevier Inc. Severe asthma is associated with an increased airway smooth muscle (ASM) mass and altered composition of the extracellular matrix (ECM). Studies have indicated that ECM-ASM cell interactions contribute to this remodeling and its limited reversibility with current therapy. Three-dimensional matrices allow the study of complex cellular responses to different stimuli in an almost natural environment. Our goal was to obtain acellular bronchial matrices and then develop a recellularization protocol with ASM cells. We studied equine bronchi as horses spontaneously develop a human asthma-like disease. The bronchi were decellularized using Triton/Sodium Deoxycholate. The obtained scaffolds retained their anatomical and histological properties. Using immunohistochemistry and a semi-quantitative score to compare native bronchi to scaffolds revealed no significant variation for matrixial proteins. DNA quantification and electrophoresis revealed that most DNA was 29.6 ng/mg of tissue ± 5.6, with remaining fragments of less than 100 bp. Primary ASM cells were seeded on the scaffolds. Histological analysis of the recellularizations showed that ASM cells migrated and proliferated primarily in the decellularized smooth muscle matrix, suggesting a chemotactic effect of the scaffolds. This is the first report of primary ASM cells preferentially repopulating the smooth muscle matrix layer in bronchial matrices. This protocol is now being used to study the molecular interactions occurring between the asthmatic ECMs and ASM to identify effectors of asthmatic bronchial remodeling.

JTD Keywords: 2d, airway smooth muscle cells, asthma, decellularization, disease, elastin, extracellular matrix, lung scaffolds, migration, peptide, recellularization, tissues, Airway smooth muscle cells, Asthma, Culture-systems, Decellularization, Extracellular matrix, Recellularization

Many studies have focused on novel noninvasive techniques to monitor respiratory rate such as bioimpedance. We propose an algorithm to detect respiratory phases using wearable bioimpedance to compute time parameters like respiratory rate, inspiratory and expiratory times, and duty cycle. The proposed algorithm was compared with two other algorithms from literature designed to estimate the respiratory rate using physiological signals like bioimpedance. We acquired bioimpedance and airflow from 50 chronic obstructive pulmonary disease (COPD) patients during an inspiratory loading protocol. We compared performance of the algorithms by computing accuracy and mean average percentage error (MAPE) between the bioimpedance parameters and the reference parameters from airflow. We found similar performance for the three algorithms in terms of accuracy (>0.96) and respiratory time and rate errors (<3.42 %). However, the proposed algorithm showed lower MAPE in duty cycle (10.18 %), inspiratory time (10.65 %) and expiratory time (8.61 %). Furthermore, only the proposed algorithm kept the statistical differences in duty cycle between COPD severity levels that were observed using airflow. Accordingly, we suggest bioimpedance to monitor breathing pattern parameters in home situations.Clinical relevance - This study exhibits the suitability of wearable thoracic bioimpedance to detect respiratory phases and to compute accurate breathing pattern parameters. © 2021 IEEE.

JTD Keywords: algorithms, copd, signals, Algorithm, Algorithms, Bioimpedance, Breathing rate, Chronic obstructive lung disease, Electronic device, Human, Humans, Lung, Pulmonary disease, chronic obstructive, Respiratory rate, Wearable electronic devices

Intracellular invasion is an advantageous mechanism used by pathogens to evade host defense and antimicrobial therapy. In patients, the intracellular microbial lifestyle can lead to infection persistence and recurrence, thus worsening outcomes. Lung infections caused by Pseudomonas aeruginosa, especially in cystic fibrosis (CF) patients, are often aggravated by intracellular invasion and persistence of the pathogen. Proliferation of the infectious species relies on a continuous deoxyribonucleotide (dNTP) supply, for which the ribonucleotide reductase enzyme (RNR) is the unique provider. The large genome plasticity of P. aeruginosa and its ability to rapidly adapt to different environments are challenges for studying the pathophysiology associated with this type of infection. Using different reference strains and clinical isolates of P. aeruginosa independently combined with alveolar (A549) and bronchial (16HBE14o- and CF-CFBE41o-) epithelial cells, we analyzed host–pathogen interactions and intracellular bacterial persistence with the aim of determining a cell type-directed infection promoted by the P. aeruginosa strains. The oscillations in cellular toxicity and oxygen consumption promoted by the intracellular persistence of the strains were also analyzed among the different infectious lung models. Significantly, we identified class II RNR as the enzyme that supplies dNTPs to intracellular P. aeruginosa. This discovery could contribute to the development of RNR-targeted strategies against the chronicity occurring in this type of lung infection. Overall our study demonstrates that the choice of bacterial strain is critical to properly study the type of infectious process with relevant translational outcomes.

JTD Keywords: Pseudomonas aeruginosa, Intracellular persistence, Lung, Epithelial cells, Clinical isolates, Host-pathogen interactions, Intracellular lifestyle, Chronic infections, Cystic fibrosis, Ribonucleotide reductase

Lung sound (LS) signals are often contaminated by impulsive artifacts that complicate the estimation of lung sound intensity (LSI) using conventional amplitude estimators. Fixed sample entropy (fSampEn) has proven to be robust to cardiac artifacts in myographic respiratory signals. Similarly, fSampEn is expected to be robust to artifacts in LS signals, thus providing accurate LSI estimates. However, the choice of fSampEn parameters depends on the application and fSampEn has not previously been applied to LS signals. This study aimed to perform an evaluation of the performance of the most relevant fSampEn parameters on LS signals, and to propose optimal fSampEn parameters for LSI estimation. Different combinations of fSampEn parameters were analyzed in LS signals recorded in a heterogeneous population of healthy subjects and chronic obstructive pulmonary disease patients during loaded breathing. The performance of fSampEn was assessed by means of its cross-covariance with flow signals, and optimal fSampEn parameters for LSI estimation were proposed.

JTD Keywords: Large scale integration, Lung, Estimation, Entropy, Loading, Robustness, Diseases

DNA nanostructures hold great potential for drug delivery. However, their specific targeting is often compromised by recognition by scavenger receptors involved in clearance. In our previous study in cell culture, we showed targeting specificity of a 180 nm, 4-layer DNA-built nanocarrier called 3DNA coupled with antibodies against intercellular adhesion molecule-1 (ICAM-1), a glycoprotein overexpressed in the lungs in many diseases. Here, we examined the biodistribution of various 3DNA formulations in mice. A formulation consisted of 3DNA whose outer-layer arms were hybridized to secondary antibody-oligonucleotide conjugates. Anchoring IgG on this formulation reduced circulation and kidney accumulation vs. non-anchored IgG, while increasing liver and spleen clearance, as expected for a nanocarrier. Anchoring anti-ICAM changed the biodistribution of this antibody similarly, yet this formulation specifically accumulated in the lungs, the main ICAM-1 target. Since lung targeting was modest (2-fold specificity index over IgG formulation), we pursued a second preparation involving direct hybridization of primary antibody-oligonucleotide conjugates to 3DNA. This formulation had prolonged stability in serum and showed a dramatic increase in lung distribution: the specificity index was 424-fold above a matching IgG formulation, 144-fold more specific than observed for PLGA nanoparticles of similar size, polydispersity, ζ-potential and antibody valency, and its lung accumulation increased with the number of anti-ICAM molecules per particle. Immunohistochemistry showed that anti-ICAM and 3DNA components colocalized in the lungs, specifically associating with endothelial markers, without apparent histological changes. The degree of in vivo targeting for anti-ICAM/3DNA-nanocarriers is unprecedented, for which this platform technology holds great potential to develop future therapeutic applications.

JTD Keywords: 3DNA, DNA nanostructure, Drug nanocarrier, Endothelial and lung targeting, ICAM-1, In vivo biodistribution

The extracellular matrix (ECM) of the lung provides physical support and key mechanical signals to pulmonary cells. Although lung ECM is continuously subjected to different stretch levels, detailed mechanics of the ECM at the scale of the cell is poorly understood. Here, we developed a new polydimethylsiloxane (PDMS) chip to probe nonlinear mechanics of tissue samples with atomic force microscopy (AFM). Using this chip, we performed AFM measurements in decellularized rat lung slices at controlled stretch levels. The AFM revealed highly nonlinear ECM elasticity with the microscale stiffness increasing with tissue strain. To correlate micro- and macroscale ECM mechanics, we also assessed macromechanics of decellularized rat lung strips under uniaxial tensile testing. The lung strips exhibited exponential macromechanical behavior but with stiffness values one order of magnitude lower than at the microscale. To interpret the relationship between micro- and macromechanical properties, we carried out a finite element (FE) analysis which revealed that the stiffness of the alveolar cell microenvironment is regulated by the global strain of the lung scaffold. The FE modeling also indicates that the scale dependence of stiffness is mainly due to the porous architecture of the lung parenchyma. We conclude that changes in tissue strain during breathing result in marked changes in the ECM stiffness sensed by alveolar cells providing tissue-specific mechanical signals to the cells. Statement of Significance: The micromechanical properties of the extracellular matrix (ECM) are a major determinant of cell behavior. The ECM is exposed to mechanical stretching in the lung and other organs during physiological function. Therefore, a thorough knowledge of the nonlinear micromechanical properties of the ECM at the length scale that cells probe is required to advance our understanding of cell-matrix interplay. We designed a novel PDMS chip to perform atomic force microscopy measurements of ECM micromechanics on decellularized rat lung slices at different macroscopic strain levels. For the first time, our results reveal that the microscale stiffness of lung ECM markedly increases with macroscopic tissue strain. Therefore, changes in tissue strain during breathing result in variations in ECM stiffness providing tissue-specific mechanical signals to lung cells.

JTD Keywords: AFM, ECM micromechanics, Multiscale lung mechanics, Tensile testing

Objectives: Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear. Material and Methods: We conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient. Results: Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3 years of 129% and 94% for fibrillar collagens imaged with bright-field (p = 0.004) and polarized light (p = 0.003), respectively, and of 89% for α-SMA (p = 0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia. Conclusions: Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMA + TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy.

JTD Keywords: Cancer associated fibroblast, Collagen, Lung cancer, Necrosis, Survival, α-SMA

Increasing evidence shows that lungs can be damaged by inhalation of nanoparticles (NPs) at environmental and occupational settings. Recent findings have associated the exposure to iron oxide (Fe2O3) and titanium dioxide (TiO2) – NPs widely used in biomedical and clinical research – with pulmonary oxidative stress and inflammation. Although changes on cellular mechanics could contribute to pulmonary inflammation, there is no information regarding the effects of Fe2O3 and TiO2 on alveolar epithelial cell biomechanics. The aim was to investigate the NPs-induced biomechanical effects in terms of cell stiffness and traction forces exerted by human alveolar epithelial cells. Cell Young’s modulus (E) measured by atomic force microscopy in alveolar epithelial cells significantly decreased after exposure to Fe2O3 and TiO2 (-28 and -25%, respectively) compared to control conditions. Moreover, both NPs induced a similar reduction in the traction forces exerted by the alveolar epithelial cells in comparison to the control conditions. Accordingly, immunofluorescence images revealed a reduction of actomyosin stress fibers in response to the exposure to NPs. However, no inflammatory response was detected. In conclusion, an acute exposure of epithelial pulmonary cells to Fe2O3 and TiO2 NPs, which was mild since it was non-cytotoxic and did not induce inflammation, modified cell biomechanical properties which could be translated into damage of the epithelial barrier integrity, suggesting that mild environmental inhalation of Fe2O3 and TiO2 NPs could not be innocuous.

JTD Keywords: Actomyosin fibers, Air pollution, Cell biomechanics, Lung epithelium, Nanoparticles

Continuous adventitious sounds (CAS) are commonly observed in obstructive pulmonary diseases and are of great clinical interest. However, their evaluation is generally subjective. We have previously developed an automatic CAS segmentation and classification algorithm for CAS recorded on the chest surface. The aim of this study is to establish whether these pulmonary CAS can be identified in a similar way using a tracheal microphone. Respiratory sounds were originally recorded from 25 participants using five contact microphones, four on the chest and one on the trachea, during three progressive respiratory maneuvers. In this work CAS component detection was performed on the tracheal channel using our automatic algorithm based on the Hilbert spectrum. The tracheal CAS detected were then compared to the previously analyzed pulmonary CAS. The sensitivity of CAS identification was lower at the tracheal microphone, with CAS that appeared simultaneously in all four pulmonary recordings more likely to be identified in the tracheal recordings. These observations could be due to the CAS being obscured by the lower SNR present in the tracheal recordings or not being transmitted through the airways to the trachea. Further work to optimize the algorithm for the tracheal recordings will be conducted in the future.

JTD Keywords: Microphones, Lung, Diseases, Time-frequency analysis, Spectrogram, Sensitivity

Introduction: The role of lung mesenchymal stem cells (L-MSCs) in pulmonary diseases remain to be fully elucidated. A relevant open question is to understand the crosstalk between L-MSCs and lung extracellular matrix (L-ECM). To this end, a suitable 3D model including MSCs and L-ECM is of high interest. Aim: To study how L-MSCs can be 3D bioprinted, cultured and harvested from L-ECM hydrogels. Methods: L-MSCs were isolated from Sprague-Dawley rats following established protocols. Porcine lungs were decellularized by a detergent-based procedure. The resulting L-ECM was freeze-dried, milled in liquid nitrogen and enzymatically digested by pepsin. After pH neutralization, resulting pre-gels were mixed with L-MSCs and 3D bioprinted by using F-127 as structural and sacrificial hydrogel. Cells were harvested from the 3D hydrogel by digestion with collagenase after 7 days of 3D culture and reseeded in standard plastic 2D culture plates. Cell viability and spatial distribution within the hydrogel was evaluated by live/dead (Thermo Scientific, MA, USA) staining and laser scanning confocal imaging. Biological activity was evaluated by hydrogel contraction assays. Results: Viability higher than 90% and homogenous 3D spatial distribution of L-MSCs were observed. Cells contracted the hydrogel up to 75% of their original size, showing that L-MSCs had an active interaction with the L-ECM. Recovered L-MSCs from the bioprinted structures were able to spread and proliferate when reseeded in plastic. Conclusion: Cell-laden hydrogels based on L-ECM can be used as bioink to build realistic 3D models for studying cell-matrix crosstalk in respiratory diseases.

JTD Keywords: Lung mechanics, Experimental approaches

Lung biofabrication is a new tissue engineering and regenerative development aimed at providing organs for potential use in transplantation. Lung biofabrication is based on seeding cells into an acellular organ scaffold and on culturing them in an especial purpose bioreactor. The acellular lung scaffold is obtained by decellularizing a non-transplantable donor lung by means of conventional procedures based on application of physical, enzymatic and detergent agents. To avoid immune recipient's rejection of the transplanted bioengineered lung, autologous bone marrow/adipose tissue-derived mesenchymal stem cells, lung progenitor cells or induced pluripotent stem cells are used for biofabricating the bioengineered lung. The bioreactor applies circulatory perfusion and mechanical ventilation with physiological parameters to the lung during biofabrication. These physical stimuli to the organ are translated into the stem cell local microenvironment - e.g. shear stress and cyclic stretch - so that cells sense the physiological conditions in normally functioning mature lungs. After seminal proof of concept in a rodent model was published in 2010, the hypothesis that lungs can be biofabricated is accepted and intense research efforts are being devoted to the topic. The current experimental evidence obtained so far in animal tests and in ex vivo human bioengineered lungs suggests that the date of first clinical tests, although not immediate, is coming. Lung bioengineering is a disrupting concept that poses a challenge for improving our basic science knowledge and is also an opportunity for facilitating lung transplantation in future clinical translation.

JTD Keywords: Tissue engineering, Regenerative medicine, Lung transplantation, Lung repair, Lung regeneration

JTD Keywords: Airway obstruction, Lung function, Non-invasive technique, Respiratory resistance, Vocal cord dysfunction

Bioengineering of functional lung tissue by using whole lung scaffolds has been proposed as a potential alternative for patients awaiting lung transplant. Previous studies have demonstrated that vascular resistance (Rv) could be altered to optimize the process of obtaining suitable lung scaffolds. Therefore, this work was aimed at determining how lung inflation (tracheal pressure) and perfusion (pulmonary arterial pressure) affect vascular resistance. This study was carried out using the lungs excised from 5 healthy male Sprague-Dawley rats. The trachea was cannulated and connected to a continuous positive airway pressure (CPAP) device to provide a tracheal pressure ranging from 0 to 15cmH2O. The pulmonary artery was cannulated and connected to a controlled perfusion system with continuous pressure (gravimetric level) ranging from 5 to 30cmH2O. Effective Rv was calculated by ratio of pulmonary artery pressure (P PA) by pulmonary artery flow (V'PA). Rv in the decellularized lungs scaffolds decreased at increasing V' PA, stabilizing at a pulmonary arterial pressure greater than 20cmH2O. On the other hand, CPAP had no influence on vascular resistance in the lung scaffolds after being subjected to pulmonary artery pressure of 5cmH2O. In conclusion, compared to positive airway pressure, arterial lung pressure markedly influences the mechanics of vascular resistance in decellularized lungs.

JTD Keywords: Decellularized lung, Scaffolds, Vascular resistance

Organ biofabrication is a potential future alternative for obtaining viable organs for transplantation. Achieving intact scaffolds to be recellularized is a key step in lung bioengineering. Perfusion of decellularizing media through the pulmonary artery has shown to be effective. How vascular perfusion pressure and flow vary throughout lung decellularization, which is not well known, is important for optimizing the process (minimizing time) while ensuring scaffold integrity (no barotrauma). This work was aimed at characterizing the pressure/flow relationship at the pulmonary vasculature and at how effective vascular resistance depends on pressure- and flow-controlled variables when applying different methods of media perfusion for lung decellularization. Lungs from 43 healthy mice (C57BL/6; 7-8 weeks old) were investigated. After excision and tracheal cannulation, lungs were inflated at 10cmH2O airway pressure and subjected to conventional decellularization with a solution of 1% sodium dodecyl sulfate (SDS). Pressure (PPA) and flow (V'PA) at the pulmonary artery were continuously measured. Decellularization media was perfused through the pulmonary artery: (a) at constant PPA=20cmH2O or (b) at constant V'PA=0.5 and 0.2ml/min. Effective vascular resistance was computed as Rv=PPA/V'PA. Rv (in cmH2O/(ml/min)); mean±SE) considerably varied throughout lung decellularization, particularly for pressure-controlled perfusion (from 29.1±3.0 in baseline to a maximum of 664.1±164.3 (p<0.05), as compared with flow-controlled perfusion (from 49.9±3.3 and 79.5±5.1 in baseline to a maximum of 114.4±13.9 and 211.7±70.5 (p<0.05, both), for V'PA of 0.5 and 0.2ml/min respectively. Most of the media infused to the pulmonary artery throughout decellularization circulated to the airways compartment across the alveolar-capillary membrane. This study shows that monitoring perfusion mechanics throughout decellularization provides information relevant for optimizing the process time while ensuring that vascular pressure is kept within a safety range to preserve the organ scaffold integrity.

JTD Keywords: Acellular lung, Fluid mechanics, Lung bioengineering, Lung scaffold, Organ biofabrication, Tissue engineering, Vascular resistance

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1. kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7. kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6. kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8. kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

JTD Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering

Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.

JTD Keywords: Gamma irradiation, Lung bioengineering, Lung decellularization, Organ scaffold, Pulmonary mechanics, Decellularization, Gamma irradiation, Mouse lung, Pulmonary mechanics, dodecyl sulfate sodium, animal tissue, Article, artificial ventilation, bioengineering, bioreactor, compliance (physical), controlled study, freezing, gamma irradiation, lung, lung mechanics, lung resistance, male, mouse, nonhuman, room temperature, scanning electron microscopy, tissue scaffold, trachea pressure

Lung bioengineering based on decellularized organ scaffolds is a potential alternative for transplantation. Freezing/thawing, a usual procedure in organ decellularization and storage could modify the mechanical properties of the lung scaffold and reduce the performance of the bioengineered lung when subjected to the physiological inflation-deflation breathing cycles. The aim of this study was to determine the effects of repeated freezing/thawing on the mechanical properties of decellularized lungs in the physiological pressure-volume regime associated with normal ventilation. Fifteen mice lungs (C57BL/6) were decellularized using a conventional protocol not involving organ freezing and based on sodium dodecyl sulfate detergent. Subsequently, the mechanical properties of the acellular lungs were measured before and after subjecting them to three consecutive cycles of freezing/thawing. The resistance (RL) and elastance (EL) of the decellularized lungs were computed by linear regression fitting of the recorded signals (tracheal pressure, flow, and volume) during mechanical ventilation. RL was not significantly modified by freezing-thawing: from 0.88 ± 0.37 to 0.90 ± 0.38 cmH2O·s·mL-1 (mean ± SE). EL slightly increased from 64.4 ± 11.1 to 73.0 ± 16.3 cmH2O·mL-1 after the three freeze-thaw cycles (p = 0.0013). In conclusion, the freezing/thawing process that is commonly used for both organ decellularization and storage induces only minor changes in the ventilation mechanical properties of the organ scaffold.

JTD Keywords: Elastance, Freezing/thawing, Lung bioengineering, Lung decellularization, Mechanical ventilation, Organ scaffold

Lung decellularization is based on the use of physical, chemical, or enzymatic methods to break down the integrity of the cells followed by a treatment to extract the cellular material from the lung scaffold. The aim of this study was to characterize the mechanical changes throughout the different steps of lung decellularization process. Four lungs from mice (C57BL/6) were decellularized by using a conventional protocol based on sodium dodecyl sulfate. Lungs resistance (RL) and elastance (EL) were measured along decellularization steps and were computed by linear regression fitting of tracheal pressure, flow, and volume during mechanical ventilation. Transients differences found were more distinct in an intermediate step after the lungs were rinsed with deionized water and treated with 1% SDS, whereupon the percentage of variation reached approximately 80% for resistance values and 30% for elastance values. In conclusion, although a variation in extracellular matrix stiffness was observed during the decellularization process, this variation can be considered negligible overall because the resistance and elastance returned to basal values at the final decellularization step.

JTD Keywords: Lung bioengineering, Lung decellularization, Organ scaffold, dodecyl sulfate sodium, animal tissue, article, artificial ventilation, compliance (physical), controlled study, enzyme chemistry, extracellular matrix, female, flow, lung, lung decellularization, lung pressure, lung resistance, mouse, nonhuman, positive end expiratory pressure, priority journal, rigidity, tissue engineering, trachea pressure

Introduction: Alveolar epithelial cells undergo stretching during mechanical ventilation. Stretch can modify the oxidative balance in the alveolar epithelium. The aim of the present study was to evaluate the antioxidant role of human adult adipose tissue-derived stromal cells (hADSCs) when human alveolar epithelial cells were subjected to injurious cyclic overstretching. Methods: A549 cells were subjected to biaxial stretch (0-15% change in surface area for 24. h, 0.2. Hz) with and without hADSCs. At the end of the experiments, oxidative stress was measured as superoxide generation using positive nuclear dihydroethidium (DHE) staining, superoxide dismutase (SOD) activity in cell lysates, 8-isoprostane concentrations in supernatant, and 3-nitrotyrosine by indirect immunofluorescence in fixed cells. Results: Cyclically stretching of AECs induced a significant decrease in SOD activity, and an increase in 8-isoprostane concentrations, DHE staining and 3-nitrotyrosine staining compared with non-stretched cells. Treatment with hADSCs significantly attenuated stretch-induced changes in SOD activity, 8-isoprostane concentrations, DHE and 3-nitrotyrosine staining. Conclusion: These data suggest that hADSCs have an anti-oxidative effect in human alveolar epithelial cells undergoing cyclic stretch.

JTD Keywords: Acute lung injury, Cyclic stretch, Human adipose-derived stromal stem cells, Oxidative stress

Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). This study investigated whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 mL.kg(-1)). MSCs (5 x 10(6)) were intravenously or intratracheally administered (n=8 each) 30 min before starting over-ventilation and eight rats were MSC-untreated. Spontaneously breathing anesthetised rats (n=8) served as controls. After 3 h of over-ventilation or control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI features. Lung oedema, histological lung injury index, concentrations of total protein, interleukin-1 beta, macrophage inflammatory protein-2 and number of neutrophils in BALF and vascular cell adhesion protein-1 in lung tissue significantly increased in over-ventilated rats. All these indices of VILI moved significantly towards normalisation in the rats treated with MSCs, whether intravenously or intratracheally. Both local and systemic pre-treatment with MSCs reduced VILI in a rat model.

JTD Keywords: Acute lung injury, Cell therapy, Injurious ventilation, Lung inflammation, Lung oedema, Mechanical ventilation

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +/- 0.58 (m +/- SE) and 1.00 +/- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +/- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +/- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

JTD Keywords: Induced acute lung, Sleep-apnea, Intermitent hypoxia, Cardiovascular-disease, Progenito Cells, Rat model, Inflammation, Mechanisms, Repair, Blood

The importance of quality control in both general and respiratory medicine has increased in parallel with the complexity of healthcare provision. Only a few decades ago, the respiratory physician and/or scientist had a very limited number of diagnostic and therapeutic tools available and, moreover, medical practice was based almost exclusively on the personal interaction between doctor and patient. Consequently, at that time the quality of the respiratory healthcare depended entirely on the professional competence of the doctor. Although nowadays the relationship between physician and patient undoubtedly still lies at the heart of respiratory medical practice, the quality of the medical service received by the patient also depends on many other participants in a complex healthcare network: various medical specialists, lung function technicians, nurses, respiratory therapists, social workers and administrative staff. Accordingly, several quality control programmes are applied in order to avoid, or at least to reduce, errors in diagnosis, improper performance of procedures, errors in medication, and failure to supervise or monitor care or recognise complications associated with treatment

JTD Keywords: Airway pressure devices, Clinical-trial, Standardization, Spirometry, Lung, Home, Ventilators, Publication, Performance, Technology

The aim was to assess whether induction of ventilator-induced lung injury (VILI) in one lung triggers a concomitant inflammatory response in the normally ventilated contralateral lung. To this end, a differential ventilator was used in 6 rats. One lung was normally ventilated (3.5 ml/kg b.w.) and the contralateral lung was overstretched (15 ml/kg b.w.). Six control rats were normally ventilated (3.5 ml/kg b.w. each lung). After 3h, edema and gene expression of MIP-2 in the lung, and plasma and liver TNF-alpha were assessed. Overexpression of MIP-2 and edema were found in the overventilated lung but not in the normally ventilated contralateral lung. No detectable levels of circulating and liver TNF-alpha were detected. These data do not support the hypothesis of an early positive feedback in the lung inflammation during the mechanical ventilation.

JTD Keywords: Mechanical ventilation, Lung injury, Lung edema, Lung over stretch, High volume ventilation, Differential ventilation