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      Air–Liquid Interface In Vitro Models for Respiratory Toxicology Research: Consensus Workshop and Recommendations

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          Abstract

          In vitro air–liquid interface (ALI) cell culture models can potentially be used to assess inhalation toxicology endpoints and are usually considered, in terms of relevancy, between classic (i.e., submerged) in vitro models and animal-based models. In some situations that need to be clearly defined, ALI methods may represent a complement or an alternative option to in vivo experimentations or classic in vitro methods. However, it is clear that many different approaches exist and that only very limited validation studies have been carried out to date. This means comparison of data from different methods is difficult and available methods are currently not suitable for use in regulatory assessments. This is despite inhalation toxicology being a priority area for many governmental organizations. In this setting, a 1-day workshop on ALI in vitro models for respiratory toxicology research was organized in Paris in March 2016 to assess the situation and to discuss what might be possible in terms of validation studies. The workshop was attended by major parties in Europe and brought together more than 60 representatives from various academic, commercial, and regulatory organizations. Following plenary, oral, and poster presentations, an expert panel was convened to lead a discussion on possible approaches to validation studies for ALI inhalation models. A series of recommendations were made and the outcomes of the workshop are reported.

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          Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling.

          The small airways of the human lung undergo pathological changes in pulmonary disorders, such as chronic obstructive pulmonary disease (COPD), asthma, bronchiolitis obliterans and cystic fibrosis. These clinical problems impose huge personal and societal healthcare burdens. The changes, termed 'pathological airway remodeling', affect the epithelium, the underlying mesenchyme and the reciprocal trophic interactions that occur between these tissues. Most of the normal human airway is lined by a pseudostratified epithelium of ciliated cells, secretory cells and 6-30% basal cells, the proportion of which varies along the proximal-distal axis. Epithelial abnormalities range from hypoplasia (failure to differentiate) to basal- and goblet-cell hyperplasia, squamous- and goblet-cell metaplasia, dysplasia and malignant transformation. Mesenchymal alterations include thickening of the basal lamina, smooth muscle hyperplasia, fibrosis and inflammatory cell accumulation. Paradoxically, given the prevalence and importance of airway remodeling in lung disease, its etiology is poorly understood. This is due, in part, to a lack of basic knowledge of the mechanisms that regulate the differentiation, maintenance and repair of the airway epithelium. Specifically, little is known about the proliferation and differentiation of basal cells, a multipotent stem cell population of the pseudostratified airway epithelium. This Perspective summarizes what we know, and what we need to know, about airway basal cells to evaluate their contributions to normal and abnormal airway remodeling. We contend that exploiting well-described model systems using both human airway epithelial cells and the pseudostratified epithelium of the genetically tractable mouse trachea will enable crucial discoveries regarding the pathogenesis of airway disease.
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            The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century.

            The major aim of our current work is to develop a deep understanding of biological effects of nanoparticles and how these effects are mediated by proteins that are adsorbed on the nanoparticles under different biological circumstances. Due to their small size, nanoparticles have distinct properties compared to the bulk form of the same materials, and these properties are rapidly revolutionizing many areas of medicine and technology. However, relatively little is known about the interaction of nanoscale objects with biological systems, as this requires quite different concepts from more established nanoscience. Thus, we have argued that in a biological fluid, proteins associate with nanoparticles, and it is the amount and presentation of the proteins on the surface rather than the particles themselves that are the cause of numerous biological responses. It is this outer layer of proteins that is seen by the biological cells, and leads to their responses. We are developing novel techniques to identify and quantify the proteins that are consistently associated with nanoparticles, as a function of the nanoparticle size, shape, and surface properties, and to correlate the adsorbed protein identities with their association and dissociation rates to and from the nanoparticles. We also seek to understand the degree of conformational change that they undergo upon adsorption to the nanoparticles. In essence, we wish to create "epitope maps" of the protein corona that surrounds nanoparticles in biological solutions, as it is the particle-protein complex that is the biologically active entity.
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              Surface charge of gold nanoparticles mediates mechanism of toxicity.

              Recently gold nanoparticles (Au NPs) have shown promising biological and military applications due to their unique electronic and optical properties. However, little is known about their biocompatibility in the event that they come into contact with a biological system. In the present study, we have investigated whether modulating the surface charge of 1.5 nm Au NPs induced changes in cellular morphology, mitochondrial function, mitochondrial membrane potential (MMP), intracellular calcium levels, DNA damage-related gene expression, and of p53 and caspase-3 expression levels after exposure in a human keratinocyte cell line (HaCaT). The evaluation of three different Au NPs (positively charged, neutral, and negatively charged) showed that cell morphology was disrupted by all three NPs and that they demonstrated a dose-dependent toxicity; the charged Au NPs displayed toxicity as low as 10 µg ml(-1) and the neutral at 25 µg ml(-1). Furthermore, there was significant mitochondrial stress (decreases in MMP and intracellular Ca2+ levels) following exposure to the charged Au NPs, but not the neutral Au NPs. In addition to the differences observed in the MMP and Ca2+ levels, up or down regulation of DNA damage related gene expression suggested a differential cell death mechanism based on whether or not the Au NPs were charged or neutral. Additionally, increased nuclear localization of p53 and caspase-3 expression was observed in cells exposed to the charged Au NPs, while the neutral Au NPs caused an increase in both nuclear and cytoplasmic p53 expression. In conclusion, these results indicate that surface charge is a major determinant of how Au NPs impact cellular processes, with the charged NPs inducing cell death through apoptosis and neutral NPs leading to necrosis.
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                Author and article information

                Journal
                Appl In Vitro Toxicol
                Appl In Vitro Toxicol
                aivt
                Applied in Vitro Toxicology
                Mary Ann Liebert, Inc. (140 Huguenot Street, 3rd FloorNew Rochelle, NY 10801USA )
                2332-1512
                2332-1539
                01 June 2018
                01 June 2018
                01 June 2018
                : 4
                : 2
                : 91-106
                Affiliations
                [ 1 ]Chronic Risks Division, Institut National de l'Environnement Industriel et des RISques , Verneuil-en-Halatte, France.
                [ 2 ]In Vitro und Mechanistische Toxikologie, Fraunhofer ITEM, Hannover, Germany.
                [ 3 ]Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology, Esch-sur-Alzette, Luxembourg.
                [ 4 ]Inhalation Toxicology Group, National Research Centre for the Working Environment, Copenhagen, Denmark.
                [ 5 ]Systems Toxicology Department, Philip Morris International R&D, Neuchâtel, Switzerland.
                [ 6 ]Epithelix Sarl, Plan-les-Outes, Switzerland.
                [ 7 ]BioNanomaterials, Adolphe Merkle Institute, University of Fribourg , Fribourg, Switzerland.
                [ 8 ]Laboratory for Materials-Biology Interactions, EMPA, Swiss Federal Laboratories for Materials , Science and Technology, St Gallen, Switzerland.
                [ 9 ]Department of Pulmonology, Leiden University Medical Center, Leiden, The Netherlands.
                [ 10 ]Directorate F-Health, Consumers and Reference Materials Chemicals Safety and Alternative Methods Unit (F.3), EURL ECVAM, JRC, Ispra, Italy.
                [ 11 ]Environment, Health and Safety Division, OECD, Paris, France.
                [ 12 ]European Affairs and Standardization, Laboratoire National de Métrologie et d'Essais, Paris, France.
                [ 13 ]Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany.
                [ 14 ]Toxicological and Environmental Risk Assessment (TERA) Department, Solvay, Brussels, Belgium.
                [ 15 ]Department of Circular Environment and Environment (CEE), TNO, Utrecht, The Netherlands.
                Author notes
                Address correspondence to: Dr. Ghislaine Lacroix, Institut National de l'Environnement industriel et des RISques (INERIS) DRC/VIVA/TOXI, Parc Technologique ALATA, BP2, F-60550 Verneuil-en-Halatte, France

                E-mail: ghislaine.lacroix@ 123456ineris.fr
                Article
                10.1089/aivt.2017.0034
                10.1089/aivt.2017.0034
                7500038
                32953944
                e439e7ea-e88b-48c1-ada7-13e653fb1aa3
                © Ghislaine Lacroix et al., 2018; Published by Mary Ann Liebert, Inc.

                This Open Access article is distributed under the terms of the Creative Commons License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Page count
                Figures: 4, References: 124, Pages: 16
                Categories
                Reviews

                air–liquid interface,in vitro,inhalation,lung cell models,toxicology,validation

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