Cellular crosstalk between airway epithelial and endothelial cells regulates barrier functions during exposure to double-stranded RNA

Introduction: The epithelial and endothelial barriers of the airway mucosa are critical for regulation of tissue homeostasis and protection against pathogens or other tissue damaging agents. In response to a viral infection, epithelial cells must signal to the endothelium to initiate immune cell recruitment. This is a highly temporal regulated process; however, the mechanisms of this cross-talk are not fully understood. Methods: In a close-contact co-culture model of human airway epithelial and endothelial cells cellular crosstalk was analysed using transepithelial electrical resistance (TER) measurements, immunofluorescence, electron microscopy and ELISA. Viral infections were simulated by exposing airway epithelial cells apically to double-stranded RNA (Poly(I:C)). Using a microfluidic culture system the temporal release of mediators was analysed in the co-culture model. Results: Within 4h of challenge, double-stranded RNA induced the release of TNF-a by epithelial cells. This activated endothelial cells by triggering the release of the chemoattractant CX3CL1 (fractalkine) by 8h post-challenge and expression of adhesion molecules E-selectin and ICAM-1. These responses were significantly reduced by neutralising TNF-a. Conclusion: By facilitating kinetic profiling, the microfluidic co-culture system has enabled identification of a key signalling mechanism between the epithelial and endothelial barriers. Better understanding of cell-cell cross-talk and its regulatory mechanisms has the potential to identify new therapeutic strategies to control airway inflammation

L'open innovation applicata all'ideazione di prodotti basati sulle leghe a memoria di forma

LAUREA MAGISTRALELo scopo di questo lavoro di tesi è di incentivare il metodo dell'Open Innovation, ovvero la collaborazione tra aziende e centri di ricerca, mirata a migliorare il processo di innovazione. In quest'ottica la nostra tesi si propone come un esempio applicativo del metodo prima citato. In particolare, il nostro lavoro prevede l'utilizzo di Leghe a Memoria di Forma (Shape Memory Alloys – SMA) per lo sviluppo di applicazioni innovative. Come punto di partenza per la generazione delle idee si è scelto di utilizzare la conoscenza presente nei brevetti. Questo perché si tratta di informazioni ad accesso libero e di elevato contenuto tecnico. Con un'attenta ricerca brevettuale si sono individuati i settori tecnologici in cui gli SMA non si sono ancora particolarmente diffusi, e che quindi rappresentano un terreno fertile per la ricerca di nuove soluzioni. Si è inoltre cercato di elaborare un metodo di ricerca brevettuale che, al di là del nostro specifico caso, potesse trovare un impiego generale. Coerentemente con il percorso di Open Innovation, le idee innovative generate sono state selezionate anche con l'aiuto di esperti esterni. A partire dai risultati ottenuti, è stato realizzato un prototipo fisico ed uno virtuale delle proposte più promettenti. Il lavoro si conclude con un secondo confronto con esperti esterni, che hanno giudicato non più proposte astratte, ma modelli concreti. In questo modo, da un lato si chiariscono le possibili prospettive di sviluppo futuro dei prototipi, dall'altro si nota come variano i giudizi presentando non più delle idee ma dei prototipi concreti.The theme of this thesis is the implementation of Open Innovation: more specifically, collaboration between companies and research centres with the final aim of promoting and improving the process of innovation. Towards this end, our thesis proposes an applicable example of a method for such implementation. In detail, our work employs the use of Shape Memory Alloys (SMA) for the development of innovative applications. As a basis for the generation of ideas, we have accessed existing available patented material because this information is free and has a high level of technical content. From a careful patent research, we have identified the technological areas in which SMA are not yet particularly widespread and concluded that the same represent fertile pitches for the identification of new applicable solutions. Furthermore, we have sought to develop a patent research method that, beyond our specific context, can be generically applied. Coherent with the aim towards Open Innovation, our newly generated ideas have been selected also with the input of external experts. From the results obtained, we have made a physical and a virtual prototype of the most promising proposals. The work concludes with the report of a second comparison with external experts, this time no longer called upon to evaluate abstract proposals but rather concrete models. In this manner, firstly we are able to better clarify the possible prospects for the future development of said prototypes, and secondly, we can observe how much the evaluations change if presented not only in an abstract form in terms of ideas but in a more concrete form in terms of tangible prototypes

Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles

Background: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles.Results: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFa signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust.Conclusions: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health

A roadmap towards safe and sustainable by design nanotechnology: titanium dioxide coated photocatalytic depolluting surfaces production by the ASINA know-how

This report, the second of its kind from ASINA project, aims at providing a roadmap with quantitative metrics for Safe(r) and (more) Sustainable by Design (SSbD) solutions for titanium dioxide (TiO2) nanomaterials (NMs). We begin with a brief description of ASINA’s methodology across the product lifecycle, highlighting the quantitative elements, such as the Key Performance Indicators (KPIs). We then propose a decision support tool for implementing SSbD objectives across various dimensions—functionality, cost, environment, and human health safety. This is followed by the main innovative findings, a consolidation of the technical processes involved, design rationales, experimental procedures, tools and models, used and developed, to deliver photocatalytic depolluting surfaces by spray- finishing techniques based on TiO2 NMs formulations. The roadmap is thoroughly described to inform similar projects through the integration of KPIs into SSbD methodologies, fostering data-driven decision-making. While specific results are beyond this report's scope, its primary aim is to demonstrate the roadmap (SSbD know-how) and promote SSbD-oriented innovation in nanotechnology. Finally, we provide a comparison of the approaches followed in two case studies that target different industrial sectors. This case-specific SSbD assessments provide a concrete exemplification of the addressed methodology that contributes to the efforts towards attaining a common roadmap for implementing SSbD solutions aligned with the EU’s Green Deal objectives

Lung Toxicity of Condensed Aerosol from E-CIG Liquids: Influence of the Flavor and the In Vitro Model Used

The diffusion of e-cigarette (e-CIG) opens a great scientific and regulatory debate about its safety. The huge number of commercialized devices, e-liquids with almost infinite chemical formulations and the growing market demand for a rapid and efficient toxicity screen system that is able to test all of these references and related aerosols. A consensus on the best protocols for the e-CIG safety assessment is still far to be achieved, since the huge number of variables characterizing these products (e.g., flavoring type and concentration, nicotine concentration, type of the device, including the battery and the atomizer). This suggests that more experimental evidences are needed to support the regulatory frameworks. The present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from three main e-liquid categories (tobacco, mint, and cinnamon as food-related flavor), with (18 mg/mL) or without nicotine. Two in vitro models, represented by a monoculture of human epithelial alveolar cells and a three-dimensional (3D) co-culture of alveolar and lung microvascular endothelial cells were used. Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity were investigated as inhalation toxicity endpoints. Results showed that nicotine itself had almost no influence on the modulation of the toxicity response, while flavor composition did have. The cell viability was significantly decreased in monoculture and ABB after exposure to the mints and cinnamon CAs. The barrier integrity was significantly affected in the ABB after exposure to cytotoxic CAs. With the exception of the significant IL-8 release in the monoculture after Cinnamon exposure, no increase of inflammatory cytokines (IL-8 and MCP-1) release was observed. These findings point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from liquids with different flavors, providing the companies and regulatory bodies with useful tools for the preliminary screening of marketable products

Lung Toxicity of Condensed Aerosol from E-CIG Liquids: Influence of the Flavor and the In Vitro Model Used

The diffusion of e-cigarette (e-CIG) opens a great scientific and regulatory debate about its safety. The huge number of commercialized devices, e-liquids with almost infinite chemical formulations and the growing market demand for a rapid and efficient toxicity screen system that is able to test all of these references and related aerosols. A consensus on the best protocols for the e-CIG safety assessment is still far to be achieved, since the huge number of variables characterizing these products (e.g., flavoring type and concentration, nicotine concentration, type of the device, including the battery and the atomizer). This suggests that more experimental evidences are needed to support the regulatory frameworks. The present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from three main e-liquid categories (tobacco, mint, and cinnamon as food-related flavor), with (18 mg/mL) or without nicotine. Two in vitro models, represented by a monoculture of human epithelial alveolar cells and a three-dimensional (3D) co-culture of alveolar and lung microvascular endothelial cells were used. Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity were investigated as inhalation toxicity endpoints. Results showed that nicotine itself had almost no influence on the modulation of the toxicity response, while flavor composition did have. The cell viability was significantly decreased in monoculture and ABB after exposure to the mints and cinnamon CAs. The barrier integrity was significantly affected in the ABB after exposure to cytotoxic CAs. With the exception of the significant IL-8 release in the monoculture after Cinnamon exposure, no increase of inflammatory cytokines (IL-8 and MCP-1) release was observed. These findings point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from liquids with different flavors, providing the companies and regulatory bodies with useful tools for the preliminary screening of marketable products

Cellular Mechanisms Involved in the Combined Toxic Effects of Diesel Exhaust and Metal Oxide Nanoparticles

Diesel exhaust particles (DEPs) and non-exhaust particles from abrasion are two main representative sources of air pollution to which humans are exposed daily, together with emerging nanomaterials, whose emission is increasing considerably. In the present work, we aimed to investigate whether DEPs, metal oxide nanoparticles (MeO-NPs), and their mixtures could affect alveolar cells. The research was focused on whether NPs induced different types of death in cells, and on their effects on cell motility and migration. Autophagy and cell cycles were investigated via cytofluorimetric analyses, through the quantification of the autophagic biomarker LC3B and PI staining, respectively. Cellular ultrastructures were then observed via TEM. Changes in cell motility and migration were assessed via transwell migration assay, and by the cytofluorimetric analysis of E-cadherin expression. A colony-forming efficiency (CFE) assay was performed in order to investigate the interactions between cells inside the colonies, and to see how these interactions change after exposure to the single particles or their mixtures. The results obtained suggest that NPs can either reduce the toxicity of DEPs (CuO) or enhance it (ZnO), through a mechanism that may involve autophagy as cells’ response to stressors and as a consequence of particles’ cellular uptake. Moreover, NPs can induce modification of E-cadherin expression and, consequentially, of colonies’ phenotypes.</jats:p

Effect of Nanoparticles and Environmental Particles on a Cocultures Model of the Air-Blood Barrier

Exposure to engineered nanoparticles (NPs) and to ambient particles (PM) has increased significantly. During the last decades the application of nano-objects to daily-life goods and the emissions produced in highly urbanized cities have considerably augmented. As a consequence, the understanding of the possible effects of NPs and PM on human respiratory system and particularly on the air-blood barrier (ABB) has become of primary interest. The crosstalk between lung epithelial cells and underlying endothelial cells is indeed essential in determining the effects of inhaled particles. Here we report the effects of metal oxides NPs (CuO and TiO2) and of PM on an in vitro model of the ABB constituted by the type II epithelial cell line (NCI-H441) and the endothelial one (HPMEC-ST1.6R). The results demonstrate that apical exposure of alveolar cells induces significant modulation of proinflammatory proteins also in endothelial cells