Cytotoxicity, mutagenicity and genotoxicity of electronic cigarettes emission aerosols compared to cigarette smoke: the REPLICA project.

Concerns have recently increased that the integrity of some scientific research is questionable due to the inability to reproduce the claimed results of some experiments and thereby confirm that the original researcher's conclusions were justified. This phenomenon has been described as 'reproducibility crisis' and affects various fields from medicine to basic applied sciences. In this context, the REPLICA project aims to replicate previously conducted in vitro studies on the toxicity of cigarette smoke and e-cigarette aerosol, sometimes adding experiments or conditions where necessary, in order to verify the robustness and replicability of the data. In this work the REPLICA Team replicated biological and toxicological assessment published by Rudd and colleagues in 2020. As in the original paper, we performed Neutral Red Uptake (NRU) assay for the evaluation of cytotoxicity, Ames test for the evaluation of mutagenesis and In Vitro Micronuclei (IVMN) assay for the evaluation of genotoxicity on cells treated with cigarette smoke or e-cigarette aerosol. The results showed high cytotoxicity, mutagenicity and genotoxicity induced by cigarette smoke, but slight or no cytotoxic, mutagenic and genotoxic effects induced by the e-cigarette aerosol. Although the two studies presented some methodological differences, the findings supported those previously presented by Rudd and colleagues.

Low levels of endogenous anabolic androgenic steroids in females with severe asthma taking corticosteroids.

Rationale: Patients with severe asthma are dependent upon treatment with high doses of inhaled corticosteroids (ICS) and often also oral corticosteroids (OCS). The extent of endogenous androgenic anabolic steroid (EAAS) suppression in asthma has not previously been described in detail. The objective of the present study was to measure urinary concentrations of EAAS in relation to exogenous corticosteroid exposure. Methods: Urine collected at baseline in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease outcomes) study of severe adult asthmatics (SA, n=408) was analysed by quantitative mass spectrometry. Data were compared to that of mild-to-moderate asthmatics (MMA, n=70) and healthy subjects (HC, n=98) from the same study. Measurements and main results: The concentrations of urinary endogenous steroid metabolites were substantially lower in SA than in MMA or HC. These differences were more pronounced in SA patients with detectable urinary OCS metabolites. Their dehydroepiandrosterone sulfate (DHEA-S) concentrations were <5% of those in HC, and cortisol concentrations were below the detection limit in 75% of females and 82% of males. The concentrations of EAAS in OCS-positive patients, as well as patients on high-dose ICS only, were more suppressed in females than males (p<0.05). Low levels of DHEA were associated with features of more severe disease and were more prevalent in females (p<0.05). The association between low EAAS and corticosteroid treatment was replicated in 289 of the SA patients at follow-up after 12-18 months. Conclusion: The pronounced suppression of endogenous anabolic androgens in females might contribute to sex differences regarding the prevalence of severe asthma.

Cigarette smoke attenuates mesenchymal stem cell-based suppression of immune cell-driven acute liver failure.

Detrimental effects of smoking on mesenchymal stem cell (MSC)-dependent immunosuppression and hepatoprotection are unknown. Herewith, by using α-galactosylceramide (α-GalCer)-induced liver injury, a well-established murine model of fulminant hepatitis, we examined molecular mechanisms which were responsible for negative effects of cigarette smoke on MSC-dependent immunomodulation. MSC which were grown in cigarette smoke-exposed medium (MSCWS-CM) obtained pro-inflammatory phenotype, were not able to optimally produce hepatoprotective and immunosuppressive cytokines (TGF-ß, HGF, IL-10, NO, KYN), and secreted significantly higher amounts of inflammatory cytokines (IFN-γ, TNF-α, IL-17, IL-6) than MSC that were cultured in standard medium never exposed to cigarette smoke (MSCCM). In contrast to MSCCM, which efficiently attenuated α-GalCer-induced hepatitis, MSCWS-CM were not able to prevent hepatocyte injury and liver inflammation. MSCWS-CM had reduced capacity for the suppression of liver-infiltrated inflammatory macrophages, dendritic cells (DCs) and lymphocytes. Although significantly lower number of IL-12-producing macrophages and DCs, TNF-α, IFN-γ or IL-17-producing CD4 + and CD8 +T lymphocytes, NK and NKT cells were noticed in the livers of α-GalCer+MSCCM-treated mice compared to α-GalCer+saline-treated animals, this phenomenon was not observed in α-GalCer-injured mice that received MSCWS-CM. MSCWS-CM could not induce expansion of anti-inflammatory IL-10-producing FoxP3 +CD4 + and CD8 + T regulatory cells and were not able to create immunosuppressive microenvironment in the liver as MSCCM. Similarly as it was observed in mice, MSCWS-CM were not able to optimally inhibit production of inflammatory and hepatototoxic cytokines in activated human Th1/Th17 and NKT1/NKT17 cells, confirming the hypothesis that cigarette smoke significantly attenuates therapeutic potential of MSC in cell-based immunotherapy of inflammatory liver diseases.

The role of inflammation in anxiety and depression in the European U-BIOPRED asthma cohorts.

BACKGROUND: Growing evidence indicates high comorbid anxiety and depression in patients with asthma. However, the mechanisms underlying this comorbid condition remain unclear. The aim of this study was to investigate the role of inflammation in comorbid anxiety and depression in three asthma patient cohorts of the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) project. METHODS: U-BIOPRED was conducted by a European Union consortium of 16 academic institutions in 11 European countries. A subset dataset from subjects with valid anxiety and depression measures and a large blood biomarker dataset were analysed, including 198 non-smoking patients with severe asthma (SAn), 65 smoking patients with severe asthma (SAs), 61 non-smoking patients with mild-to-moderate asthma (MMA), and 20 healthy non-smokers (HC). The Hospital Anxiety and Depression Scale was used to measure anxiety and depression and a series of inflammatory markers were analysed by the SomaScan v3 platform (SomaLogic, Boulder, Colo). ANOVA and the Kruskal-Wallis test were used for multiple-group comparisons as appropriate. RESULTS: There were significant group effects on anxiety and depression among the four cohort groups (p < 0.05). Anxiety and depression of SAn and SAs groups were significantly higher than that of MMA and HC groups (p < 0.05. There were significant differences in serum IL6, MCP1, CCL18, CCL17, IL8, and Eotaxin among the four groups (p < 0.05). Depression was significantly associated with IL6, MCP1, CCL18 level, and CCL17; whereas anxiety was associated with CCL17 only (p < 0.05). CONCLUSIONS: The current study suggests that severe asthma patients are associated with higher levels of anxiety and depression, and inflammatory responses may underlie this comorbid condition.

Comparing the Effectiveness, Tolerability, and Acceptability of Heated Tobacco Products and Refillable Electronic Cigarettes for Cigarette Substitution (CEASEFIRE): Randomized Controlled Trial.

BACKGROUND: People who smoke and who face challenges trying to quit or wish to continue to smoke may benefit by switching from traditional cigarettes to noncombustible nicotine delivery alternatives, such as heated tobacco products (HTPs) and electronic cigarettes (ECs). HTPs and ECs are being increasingly used to quit smoking, but there are limited data about their effectiveness. OBJECTIVE: We conducted the first randomized controlled trial comparing quit rates between HTPs and ECs among people who smoke and do not intend to quit. METHODS: We conducted a 12-week randomized noninferiority switching trial to compare effectiveness, tolerability, and product satisfaction between HTPs (IQOS 2.4 Plus) and refillable ECs (JustFog Q16) among people who do not intend to quit. The cessation intervention included motivational counseling. The primary endpoint of the study was the carbon monoxide-confirmed continuous abstinence rate from week 4 to week 12 (CAR weeks 4-12). The secondary endpoints included the continuous self-reported ≥50% reduction in cigarette consumption rate (continuous reduction rate) from week 4 to week 12 (CRR weeks 4-12) and 7-day point prevalence of smoking abstinence. RESULTS: A total of 211 participants completed the study. High quit rates (CAR weeks 4-12) of 39.1% (43/110) and 30.8% (33/107) were observed for IQOS-HTP and JustFog-EC, respectively. The between-group difference for the CAR weeks 4-12 was not significant (P=.20). The CRR weeks 4-12 values for IQOS-HTP and JustFog-EC were 46.4% (51/110) and 39.3% (42/107), respectively, and the between-group difference was not significant (P=.24). At week 12, the 7-day point prevalence of smoking abstinence values for IQOS-HTP and JustFog-EC were 54.5% (60/110) and 41.1% (44/107), respectively. The most frequent adverse events were cough and reduced physical fitness. Both study products elicited a moderately pleasant user experience, and the between-group difference was not significant. A clinically relevant improvement in exercise tolerance was observed after switching to the combustion-free products under investigation. Risk perception for conventional cigarettes was consistently higher than that for the combustion-free study products under investigation. CONCLUSIONS: Switching to HTPs elicited a marked reduction in cigarette consumption among people who smoke and do not intend to quit, which was comparable to refillable ECs. User experience and risk perception were similar between the HTPs and ECs under investigation. HTPs may be a useful addition to the arsenal of reduced-risk alternatives for tobacco cigarettes and may contribute to smoking cessation. However, longer follow-up studies are required to confirm significant and prolonged abstinence from smoking and to determine whether our results can be generalized outside smoking cessation services offering high levels of support. TRIAL REGISTRATION: ClinicalTrials.gov NCT03569748; https://clinicaltrials.gov/ct2/show/NCT03569748.

Heparan Sulfate and Enoxaparin Interact at the Interface of the Spike Protein of HCoV-229E but Not with HCoV-OC43.

It is known that the spike protein of human coronaviruses can bind to a secondary receptor, or coreceptor, to facilitate the virus entry. While HCoV-229E uses human aminopeptidase N (hAPN) as a receptor, HCoV-OC43 binds to 9-O-acetyl-sialic acid (9-O-Ac-Sia), which is linked in a terminal way to the oligosaccharides that decorate glycoproteins and gangliosides on the surface of the host cell. Thus, evaluating the possible inhibitory activity of heparan sulfate, a linear polysaccharide found in animal tissues, and enoxaparin sodium on these viral strains can be considered attractive. Therefore, our study also aims to evaluate these molecules' antiviral activity as possible adsorption inhibitors against non-SARS-CoV. Once the molecules' activity was verified in in vitro experiments, the binding was studied by molecular docking and molecular dynamic simulations confirming the interactions at the interface of the spike proteins.

Stratification of asthma by lipidomic profiling of induced sputum supernatant.

BACKGROUND: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features. OBJECTIVE: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls. METHODS: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes. RESULTS: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts. CONCLUSION: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator.

Comparative assessment of electronic nicotine delivery systems aerosol and cigarette smoke on endothelial cell migration: The Replica Project.

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations evaluated in vitro the effect of electronic cigarette (EC) compared with cigarette smoke demonstrating a significant reduction in human umbilical vein endothelial cells (HUVECs) migration inhibition following EC aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared with aerosol from EC and heated tobacco products (HTPs). We performed an in vitro scratch wound assay on endothelial cells with a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study, also testing the effect of aerosol from two HTPs on endothelial cells. Consistently with the original study, we observed a substantial reduction of the effects of aerosol from EC and HTPs on endothelial cell migration compared with cigarette smoke. While cigarette smoke reduced endothelial wound healing ability already at low concentrations (12.5%) and in a concentration-dependent manner, EC and HTPs aerosol showed no effect on endothelial cells until 80%-100% concentrations. In conclusion, our study further confirms the importance of EC and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

In vitro cytoxicity profile of e-cigarette liquid samples on primary human bronchial epithelial cells.

Cigarette smoke is associated to severe chronic diseases. The most harmful components of cigarette smoke derive from the combustion process, which are significantly reduced in the electronic cigarette aerosol, thus providing a valid option in harm reduction strategies. To develop safer products, it is therefore necessary to screen electronic cigarette liquids (e-liquids) to meet high safety standards defined by government regulations. The aim of the present study was to evaluate the presence of metal- and plastic-derived contaminants in four different commercial e-liquids with high concentration of nicotine and their cytotoxic effect in normal human bronchial epithelial cells by a number of in vitro assays, in comparison with the 1R6F reference cigarette, using an air-liquid interface (ALI) exposure system. Moreover, we evaluated the effect of aerosol exposure on oxidative stress by measuring the production of reactive oxygen species and mitochondrial potential. Our results showed no contaminants in all e-liquids and a significantly reduced cytotoxic effect of e-liquid aerosol compared to cigarette smoke as well as a maintained mitochondria integrity. Moreover, no production of reactive oxygen species was detected with e-cigarette aerosol. In conclusion, these results support the reduced toxicity potential of e-cigs compared to tobacco cigarettes in an in vitro model resembling real life smoke exposure.

Lactate modulates microglia polarization via IGFBP6 expression and remodels tumor microenvironment in glioblastoma.

Lactic acidosis has been reported in solid tumor microenvironment (TME) including glioblastoma (GBM). In TME, several signaling molecules, growth factors and metabolites have been identified to induce resistance to chemotherapy and to sustain immune escape. In the early phases of the disease, microglia infiltrates TME, contributing to tumorigenesis rather than counteracting its growth. Insulin-like Growth Factor Binding Protein 6 (IGFBP6) is expressed during tumor development, and it is involved in migration, immune-escape and inflammation, thus providing an attractive target for GBM therapy. Here, we aimed at investigating the crosstalk between lactate metabolism and IGFBP6 in TME and GBM progression. Our results show that microglia exposed to lactate or IGFBP6 significantly increased the Monocarboxylate transporter 1 (MCT1) expression together with genes involved in mitochondrial metabolism. We, also, observed an increase in the M2 markers and a reduction of inducible nitric oxide synthase (iNOS) levels, suggesting a role of lactate/IGFBP6 metabolism in immune-escape activation. GBM cells exposed to lactate also showed increased levels of IGFBP6 and vice-versa. Such a phenomenon was coupled with a IGFBP6-mediated sonic hedgehog (SHH) ignaling increase. We, finally, tested our hypothesis in a GBM zebrafish animal model, where we observed an increase in microglia cells and igfbp6 gene expression after lactate exposure. Our results were confirmed by the analysis of human transcriptomes datasets and immunohistochemical assay from human GBM biopsies, suggesting the existence of a lactate/IGFBP6 crosstalk in microglial cells, so that IGFBP6 expression is regulated by lactate production in GBM cells and in turn modulates microglia polarization.

The Impact of Tobacco Cigarettes, Vaping Products and Tobacco Heating Products on Oxidative Stress.

Cells constantly produce oxidizing species because of their metabolic activity, which is counteracted by the continuous production of antioxidant species to maintain the homeostasis of the redox balance. A deviation from the metabolic steady state leads to a condition of oxidative stress. The source of oxidative species can be endogenous or exogenous. A major exogenous source of these species is tobacco smoking. Oxidative damage can be induced in cells by chemical species contained in smoke through the generation of pro-inflammatory compounds and the modulation of intracellular pro-inflammatory pathways, resulting in a pathological condition. Cessation of smoking reduces the morbidity and mortality associated with cigarette use. Next-generation products (NGPs), as alternatives to combustible cigarettes, such as electronic cigarettes (e-cig) and tobacco heating products (THPs), have been proposed as a harm reduction strategy to reduce the deleterious impacts of cigarette smoking. In this review, we examine the impact of tobacco smoke and MRPs on oxidative stress in different pathologies, including respiratory and cardiovascular diseases and tumors. The impact of tobacco cigarette smoke on oxidative stress signaling in human health is well established, whereas the safety profile of MRPs seems to be higher than tobacco cigarettes, but further, well-conceived, studies are needed to better understand the oxidative effects of these products with long-term exposure.

Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle.

Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell-substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.

Clinical and transcriptomic features of persistent exacerbation-prone severe asthma in U-BIOPRED cohort.

BACKGROUND: Exacerbation-prone asthma is a feature of severe disease. However, the basis for its persistency remains unclear. OBJECTIVES: To determine the clinical and transcriptomic features of frequent exacerbators (FEs) and persistent FEs (PFEs) in the U-BIOPRED cohort. METHODS: We compared features of FE (≥2 exacerbations in past year) to infrequent exacerbators (IE, <2 exacerbations) and of PFE with repeat ≥2 exacerbations during the following year to persistent IE (PIE). Transcriptomic data in blood, bronchial and nasal epithelial brushings, bronchial biopsies and sputum cells were analysed by gene set variation analysis for 103 gene signatures. RESULTS: Of 317 patients, 62.4% had FE, of whom 63.6% had PFE, while 37.6% had IE, of whom 61.3% had PIE. Using multivariate analysis, FE was associated with short-acting beta-agonist use, sinusitis and daily oral corticosteroid use, while PFE was associated with eczema, short-acting beta-agonist use and asthma control index. CEA cell adhesion molecule 5 (CEACAM5) was the only differentially expressed transcript in bronchial biopsies between PE and IE. There were no differentially expressed genes in the other four compartments. There were higher expression scores for type 2, T-helper type-17 and type 1 pathway signatures together with those associated with viral infections in bronchial biopsies from FE compared to IE, while there were higher expression scores of type 2, type 1 and steroid insensitivity pathway signatures in bronchial biopsies of PFE compared to PIE. CONCLUSION: The FE group and its PFE subgroup are associated with poor asthma control while expressing higher type 1 and type 2 activation pathways compared to IE and PIE, respectively.

Plasma proteins elevated in severe asthma despite oral steroid use and unrelated to Type-2 inflammation.

RATIONALE: Asthma phenotyping requires novel biomarker discovery. OBJECTIVES: To identify plasma biomarkers associated with asthma phenotypes by application of a new proteomic panel to samples from two well-characterised cohorts of severe (SA) and mild-to-moderate (MMA) asthmatics, COPD subjects and healthy controls (HCs). METHODS: An antibody-based array targeting 177 proteins predominantly involved in pathways relevant to inflammation, lipid metabolism, signal transduction and extracellular matrix was applied to plasma from 525 asthmatics and HCs in the U-BIOPRED cohort, and 142 subjects with asthma and COPD from the validation cohort BIOAIR. Effects of oral corticosteroids (OCS) were determined by a 2-week, placebo-controlled OCS trial in BIOAIR, and confirmed by relation to objective OCS measures in U-BIOPRED. RESULTS: In U-BIOPRED, 110 proteins were significantly different, mostly elevated, in SA compared to MMA and HCs. 10 proteins were elevated in SA versus MMA in both U-BIOPRED and BIOAIR (alpha-1-antichymotrypsin, apolipoprotein-E, complement component 9, complement factor I, macrophage inflammatory protein-3, interleukin-6, sphingomyelin phosphodiesterase 3, TNF receptor superfamily member 11a, transforming growth factor-ß and glutathione S-transferase). OCS treatment decreased most proteins, yet differences between SA and MMA remained following correction for OCS use. Consensus clustering of U-BIOPRED protein data yielded six clusters associated with asthma control, quality of life, blood neutrophils, high-sensitivity C-reactive protein and body mass index, but not Type-2 inflammatory biomarkers. The mast cell specific enzyme carboxypeptidase A3 was one major contributor to cluster differentiation. CONCLUSIONS: The plasma proteomic panel revealed previously unexplored yet potentially useful Type-2-independent biomarkers and validated several proteins with established involvement in the pathophysiology of SA.

Mapping atopic dermatitis and anti-IL-22 response signatures to type 2-low severe neutrophilic asthma.

BACKGROUND: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. OBJECTIVE: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti-IL-22 (fezakinumab [FZ]) is enriched in severe asthma. METHODS: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. RESULTS: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, TH2, and TH17/TH22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P < .05). The FZ-response signature (296 downregulated genes) was enriched in asthmatic blood (P < .05) and particularly in neutrophilic and mixed granulocytic sputum (P < .05). These data were confirmed in sputum of the Airway Disease Endotyping for Personalized Therapeutics cohort. IL-22 mRNA across tissues did not correlate with FZ-response enrichment scores, but this response signature correlated with TH22/IL-22 pathways. CONCLUSIONS: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

Electronic nicotine delivery systems exhibit reduced bronchial epithelial cells toxicity compared to cigarette: the Replica Project.

Electronic nicotine delivery systems (ENDS) may reduce health risks associated with chronic exposure to smoke and their potential benefits have been the matter of intense scientific debate. We aimed to replicate three published studies on cytotoxic and inflammatory effects of cigarette smoke and ENDS aerosol in an independent multi-center ring study. We aimed to establish the reliability of results and the robustness of conclusions by replicating the authors' experimental protocols and further validating them with different techniques. Human bronchial epithelial cells (NCI-H292) were exposed to cigarette whole smoke and vapor phase and to aerosol from ENDS. We also assessed the inflammatory cytokines interleukin-6 and interleukin-8 and the remodeling mediator matrix metalloproteinase-1. We replicated cell viability results and confirmed that almost 80% of cytotoxic effects are due to volatile compounds in the vapor phase of smoke. Our findings substantiated the reduced cytotoxic effects of ENDS aerosol. However, our data on inflammatory and remodeling activity triggered by smoke differed significantly from those in the original reports. Taken together, independent data from multiple laboratories clearly demonstrated the reduced toxicity of ENDS products compared to cigarettes.