33 results on '"Marescotti D"'
Search Results
2. P01-02 AOP-based in vitro assay development for assessment of inhalational toxicants — oxidative stress leading to decreased lung function
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Goralczyk, A., primary, Pereira, J., additional, Torres, L. Ortega, additional, Iskandar, A., additional, van der Toor, M., additional, Talikka, M., additional, Luettich, K., additional, and Marescotti, D., additional
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- 2022
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3. P006 3D Multicellular intestine-on-a-chip model for disease modelling and drug discovery
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Lo Sasso, G, primary, Gijzen, L, additional, Marescotti, D, additional, Naumovska, E, additional, Raineri, E, additional, Nicolas, A, additional, Lanz, H, additional, Guerrera, D, additional, van Vught, R, additional, Joore, J, additional, Vulto, P, additional, Peitsch, M C, additional, Hoeng, J, additional, and Kurek, D, additional
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- 2020
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4. Inhibition of Serine-Peptidase Activity Enhances the Generation of a Survivin-Derived HLA-A2-Presented CTL Epitope in Colon-Carcinoma Cells
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Preta, G., Marescotti, D., Fortini, C., Carcoforo, P., Castelli, C., Masucci, M., and Gavioli, R.
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- 2008
5. Characterization of a lung/liver organ-on-a-chip model
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Bovard, D., primary, Sandoz, A., additional, Morelli, M., additional, Trivedi, K., additional, Marescotti, D., additional, Frentzel, S., additional, Luettich, K., additional, and Hoeng, J., additional
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- 2018
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6. A comparative high-content screening-based assessment of e-cigarette liquids in primary bronchial epithelial cells
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Marescotti, D., primary, Taylor, M., additional, Gonzalez-Suarez, I., additional, Acali, S., additional, Walker, P., additional, Belcastro, V., additional, Martin, F., additional, Frentzel, S., additional, Thorne, D., additional, Peitsch, M.C., additional, Proctor, C., additional, Gaca, M., additional, and Hoeng, J., additional
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- 2018
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7. High Content Screening (HCS) approach to characterize phenotypic changes occurring during long-term treatment of human bronchial epithelial cells with cigarette smoke total particulate matter
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Marescotti, D., primary, Van Det Toorn, M., additional, Laurent, A., additional, Luettich, K., additional, Frentzel, S., additional, Peitsch, M., additional, and Hoeng, J., additional
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- 2016
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8. Establishment and characterization of a lung/liver organ-on-a-chip model. Phase 1: Model characterization
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Bovard, D., primary, Luettich, K., additional, Frentzel, S., additional, Marescotti, D., additional, Acali, S., additional, Trivedi, K., additional, and Hoeng, J., additional
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- 2016
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9. AI-driven laboratory workflows enable operation in the age of social distancing.
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Marescotti D, Narayanamoorthy C, Bonjour F, Kuwae K, Graber L, Calvino-Martin F, Ghosh S, and Hoeng J
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- Artificial Intelligence, Humans, Laboratories, Workflow, COVID-19 diagnosis, Physical Distancing
- Abstract
The COVID-19 (Coronavirus disease 2019) global pandemic has upended the normal pace of society at multiple levels-from daily activities in personal and professional lives to the way the sciences operate. Many laboratories have reported shortage in vital supplies, change in standard operating protocols, suspension of operations because of social distancing and stay-at-home guidelines during the pandemic. This global crisis has opened opportunities to leverage internet of things, connectivity, and artificial intelligence (AI) to build a connected laboratory automation platform. However, laboratory operations involve complex, multicomponent systems. It is unrealistic to completely automate the entire diversity of laboratories and processes. Recently, AI technology, particularly, game simulation has made significant strides in modeling and learning complex, multicomponent systems. Here, we present a cloud-based laboratory management and automation platform which combines multilayer information on a simulation-driven inference engine to plan and optimize laboratory operations under various constraints of COVID-19 and risk scenarios. The platform was used to assess the execution of two cell-based assays with distinct parameters in a real-life high-content screening laboratory scenario. The results show that the platform can provide a systematic framework for assessing laboratory operation scenarios under different conditions, quantifying tradeoffs, and determining the performance impact of specific resources or constraints, thereby enabling decision-making in a cost-effective manner. We envisage the laboratory management and automation platform to be further expanded by connecting it with sensors, robotic equipment, and other components of scientific operations to provide an integrated, end-to-end platform for scientific laboratory automation., Competing Interests: Declaration of Competing Interest Diego Marescotti, Filipe Bonjour, Luc Graber, Florian Calvino-Martin, and Julia Hoeng are employees of Philip Morris International or had worked for Philip Morris International under contractual agreements. Chandrasekaran Narayanamoorthy, Ken Kuwae, and Samik Ghosh (SBX Corporation, Japan) were part of the team responsible for customizing and developing the platform based on a proprietary software licensed by SBX Corporation, Japan., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2022
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10. Toxicological Assessment of Flavor Ingredients in E-Vapor Products.
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Sciuscio D, Calvino-Martin F, Kumar A, Langston TB, Martin E, Marescotti D, Mathis C, Hoeng J, Peitsch MC, Smith DC, Gogova M, Vanscheeuwijck P, and Lee KM
- Abstract
Many flavor ingredients are often used in potentially reduced-risk tobacco products (such as e-vapor products). Although most are "generally recognized as safe (GRAS)" when used in food, there is limited information available on their long-term health effects when delivered by inhalation. While obtaining route-of-exposure-specific toxicological data on flavor ingredients is critical to product evaluation, the large number of individual flavor ingredients available and their potential combinations render classical toxicological assessment approaches impractical, as they may require years of preclinical investigations and thousands of laboratory animals. Therefore, we propose a pragmatic approach in which flavor ingredients are initially assigned to groups of structurally related compounds (Flavor Groups), from which flavor group representatives (FGR) are then selected and tested individually and as a mixture in vitro and in vivo . The premise is that structurally related compounds would have comparable metabolic and biological activity and that the data generated using FGRs could support the toxicological assessment of other structurally related flavor ingredients of their respective Flavor Groups. This approach is explained in a step-wise manner and exemplified by a case study, along with its strengths, limitations as well as recommendations for further confirmatory testing. Once completed, this FGR approach could significantly reduce the time and resources required for filling the data gap in understanding the health risks of many flavor ingredients while also minimizing the need for laboratory animals., Competing Interests: Authors DS, FC-M, EM, DM, CM, JH, MCP, and PV were employed by the company Philip Morris Products S.A. Authors AK, TBL, DCS, MG, and KML were employed by the company Altria Client Services LLC., (Copyright © 2022 Sciuscio, Calvino-Martin, Kumar, Langston, Martin, Marescotti, Mathis, Hoeng, Peitsch, Smith, Gogova, Vanscheeuwijck and Lee.)
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- 2022
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11. Impact of aerosols on liver xenobiotic metabolism: A comparison of two methods of exposure.
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Bovard D, Renggli K, Marescotti D, Sandoz A, Majeed S, Pinard L, Ferreira S, Pak C, Barbier A, Beguin A, Iskandar A, Frentzel S, Hoeng J, and Peitsch MC
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- Cells, Cultured, Cytochrome P-450 Enzyme System drug effects, Humans, Liver enzymology, Liver metabolism, Smoke adverse effects, Spheroids, Cellular drug effects, Tissue Array Analysis methods, Tobacco Products adverse effects, Toxicity Tests methods, Aerosols toxicity, Cytochrome P-450 Enzyme System metabolism, Liver drug effects, Lung drug effects
- Abstract
Assessment of aerosols effects on liver CYP function generally involves aqueous fractions (AF). Although easy and efficient, this method has not been optimized recently or comparatively assessed against other aerosol exposure methods. Here, we comparatively evaluated the effects of the AFs of cigarette smoke (CS) and Tobacco Heating System (THS) aerosols on CYP activity in liver spheroids. We then used these data to develop a physiological aerosol exposure system combining a multi-organs-on-a-chip, 3D lung tissues, liver spheroids, and a direct aerosol exposure system. Liver spheroids incubated with CS AF showed a dose-dependent increase in CYP1A1/1B1, CYP1A2, and CYP2B6 activity and a dose-dependent decrease in CYP2C9, CYP2D6, and CYP3A4 activity relative to untreated tissues. In our physiological exposure system, repeated CS exposure of the bronchial tissues also caused CYP1A1/1B1 and CYP1A2 induction in the bronchial tissues and liver spheroids; but the spheroids showed an increase in CYP3A4 activity and no effect on CYP2C9 or CYP2D6 activity relative to air-exposed tissues, which resembles the results reported in smokers. THS aerosol did not affect CYP activity in bronchial or liver tissues, even at 4 times higher concentrations than CS. In conclusion, our system allows us to physiologically test the effects of CS or other aerosols on lung and liver tissues cultured in the same chip circuit, thus delivering more in vivo like data., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2022
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12. Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds.
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Marescotti D, Lo Sasso G, Guerrera D, Renggli K, Ruiz Castro PA, Piault R, Jaquet V, Moine F, Luettich K, Frentzel S, Peitsch MC, and Hoeng J
- Abstract
Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine ( R / S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation., Competing Interests: All authors are employees of Philip Morris International R&D or had worked for Philip Morris International R&D under contractual agreements., (Copyright © 2021 Marescotti, Lo Sasso, Guerrera, Renggli, Ruiz Castro, Piault, Jaquet, Moine, Luettich, Frentzel, Peitsch and Hoeng.)
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- 2021
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13. Comparison of the biological impact of aerosol of e-vapor device with MESH® technology and cigarette smoke on human bronchial and alveolar cultures.
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Giralt A, Iskandar AR, Martin F, Moschini E, Serchi T, Kondylis A, Marescotti D, Leroy P, Ortega-Torres L, Majeed S, Merg C, Trivedi K, Guedj E, Frentzel S, Ivanov NV, Peitsch MC, Gutleb AC, and Hoeng J
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- Adenylate Kinase metabolism, Adult, Aerosols, Cell Survival drug effects, Cilia drug effects, Humans, Male, Nicotine chemistry, Organ Culture Techniques, RNA, Messenger biosynthesis, Nicotiana, Transcription, Genetic drug effects, Bronchi drug effects, Electronic Nicotine Delivery Systems, Pulmonary Alveoli drug effects, Smoke adverse effects
- Abstract
Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)
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- 2021
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14. An Intestine-on-a-Chip Model of Plug-and-Play Modularity to Study Inflammatory Processes.
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Gijzen L, Marescotti D, Raineri E, Nicolas A, Lanz HL, Guerrera D, van Vught R, Joore J, Vulto P, Peitsch MC, Hoeng J, Lo Sasso G, and Kurek D
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- Caco-2 Cells, Humans, Intestines, Intestinal Mucosa, Lab-On-A-Chip Devices
- Abstract
Development of efficient drugs and therapies for the treatment of inflammatory conditions in the intestine is often hampered by the lack of reliable, robust, and high-throughput in vitro and in vivo models. Current models generally fail to recapitulate key aspects of the intestine, resulting in low translatability to the human situation. Here, an immunocompetent 3D perfused intestine-on-a-chip platform was developed and characterized for studying intestinal inflammation. Forty independent polarized 3D perfused epithelial tubular structures were grown from cells of mixed epithelial origin, including enterocytes (Caco-2) and goblet cells (HT29-MTX-E12). Immune cells THP-1 and MUTZ-3, which can be activated, were added to the system and assessed for cytokine release. Intestinal inflammation was mimicked through exposure to tumor necrosis factor-α (TNFα) and interleukin (IL)-1β. The effects were quantified by measuring transepithelial electrical resistance (TEER) and proinflammatory cytokine secretion on the apical and basal sides. Cytokines induced an inflammatory state in the culture, as demonstrated by the impaired barrier function and increased IL-8 secretion. Exposure to the known anti-inflammatory drug TPCA-1 prevented the inflammatory state. The model provides biological modularity for key aspects of intestinal inflammation, making use of well-established cell lines. This allows robust assays that can be tailored in complexity to serve all preclinical stages in the drug discovery and development process.
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- 2020
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15. In Vitro High-Content Imaging-Based Phenotypic Analysis of Bronchial 3D Organotypic Air-Liquid Interface Cultures.
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Marescotti D, Bovard D, Morelli M, Sandoz A, Luettich K, Frentzel S, Peitsch M, and Hoeng J
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- Humans, Mucin 5AC metabolism, Phenotype, Tubulin metabolism, Air, Bronchi cytology, Cell Culture Techniques methods, Imaging, Three-Dimensional
- Abstract
High-content imaging (HCI) is a powerful method for quantifying biological effects in vitro. Historically, HCI has been applied to adherent cells growing in monolayers. With the advent of confocal versions of HCI devices, researchers now have the option of performing analyses on 3D cell cultures. However, some obstacles remain in integrating the third dimension, such as limited light penetration and less sophisticated image analysis. Here, we report the development of an HCI technique for imaging human bronchial 3D organotypic air-liquid interface (ALI) cultures (hBR-ALI). In this method, we monitored differentiation status through HCI evaluation markers representative of ciliated epithelial cells and goblet cells (Muc5AC [mucin 5AC]). As a second use case for demonstrating the utility of this technique, we induced goblet cell hyperplasia in hBR-ALI by using interleukin (IL)-13. Our results demonstrate the utility of the HCI technique for imaging hBR-ALI grown on Transwell inserts. This technique may be expanded to other cell culture systems, such as skin epithelia and 3D intestinal systems.
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- 2020
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16. Systems toxicology assessment of a representative e-liquid formulation using human primary bronchial epithelial cells.
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Marescotti D, Mathis C, Belcastro V, Leroy P, Acali S, Martin F, Dulize R, Bornand D, Peric D, Guedj E, Ortega Torres L, Biasioli M, Fuhrimann M, Fernandes E, Frauendorfer F, Gonzalez Suarez I, Sciuscio D, Ivanov NV, Peitsch MC, and Hoeng J
- Abstract
The development of reduced-risk products aims to provide alternatives to cigarettes that present less risk of harm for adult smokers. Responsible use of flavoring substances in these products may fulfill an important role in product acceptance. While most flavoring substances used in such products are also used by the food industry and are considered safe when ingested, their impact when inhaled may require further assessment. To aid in such an assessment, a three-step approach combining real-time cellular analysis, phenotypic high-content screening assays, and gene expression analysis was developed and tested in normal human bronchial epithelial cells with 28 flavoring substances commonly used in e-liquid formulations, dissolved individually or as a mixture in a base solution composed of propylene glycol, vegetable glycerin, and 0.6% nicotine. By employing this approach, we identified individual flavoring substances that potentially contribute greatly to the overall mixture effect (citronellol and alpha-pinene). By assessing modified mixtures, we showed that, although cytotoxic effects were found when assessed individually, alpha-pinene did not contribute to the overall mixture cytotoxicity. Most of the cytotoxic effect appeared to be attributable to citronellol, with the remaining substances contributing due to synergistic effects. We developed and used different scoring methods (Tox-Score, Phenotypic Score, and Biological Impact Factor/Network Perturbation Amplitude), ultimately enabling a ranking based on cytotoxicity, phenotypic outcome, and molecular network perturbations. This case study highlights the benefits of testing both individual flavoring substances and mixtures for e-liquid flavor assessment and emphasized the importance of data sharing for the benefit of consumer safety., (© 2019 The Authors.)
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- 2019
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17. Application of a multi-layer systems toxicology framework for in vitro assessment of the biological effects of Classic Tobacco e-liquid and its corresponding aerosol using an e-cigarette device with MESH™ technology.
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Iskandar AR, Zanetti F, Marescotti D, Titz B, Sewer A, Kondylis A, Leroy P, Belcastro V, Torres LO, Acali S, Majeed S, Steiner S, Trivedi K, Guedj E, Merg C, Schneider T, Frentzel S, Martin F, Ivanov NV, Peitsch MC, and Hoeng J
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- Adenylate Kinase metabolism, Bronchi metabolism, Bronchi pathology, Cell Line, Cell Survival drug effects, Epithelial Cells metabolism, Epithelial Cells pathology, Humans, Male, Middle Aged, Primary Cell Culture, Proteome metabolism, Toxicity Tests, Transcriptome drug effects, Aerosols toxicity, Bronchi drug effects, Electronic Nicotine Delivery Systems, Epithelial Cells drug effects, Tobacco Products toxicity
- Abstract
We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.
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- 2019
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18. GladiaTOX: GLobal Assessment of Dose-IndicAtor in TOXicology.
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Belcastro V, Cano S, Marescotti D, Acali S, Poussin C, Gonzalez-Suarez I, Martin F, Bonjour F, Ivanov NV, Peitsch MC, and Hoeng J
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- Quality Control, Toxicology, Biomedical Research, Software
- Abstract
Summary: GladiaTOX R package is an open-source, flexible solution to high-content screening data processing and reporting in biomedical research. GladiaTOX takes advantage of the 'tcpl' core functionalities and provides a number of extensions: it provides a web-service solution to fetch raw data; it computes severity scores and exports ToxPi formatted files; furthermore it contains a suite of functionalities to generate PDF reports for quality control and data processing., Availability and Implementation: GladiaTOX R package (bioconductor). Also available via: git clone https://github.com/philipmorrisintl/GladiaTOX.git., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press.)
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- 2019
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19. Corrigendum to "The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product" published in Toxicology in Vitro, Volume 50, August 2018, Pages 95-108.
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van der Toorn M, Sewer A, Marescotti D, Johne S, Baumer K, Bornand D, Dulize R, Merg C, Corciulo M, Scotti E, Pak C, Leroy P, Guedj E, Ivanov N, Martin F, Peitsch M, Hoeng J, and Luettich K
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- 2019
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20. How complex should an in vitro model be? Evaluation of complex 3D alveolar model with transcriptomic data and computational biological network models.
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Marescotti D, Serchi T, Luettich K, Xiang Y, Moschini E, Talikka M, Martin F, Baumer K, Dulize R, Peric D, Bornand D, Guedj E, Sewer A, Cambier S, Contal S, Chary A, Gutleb AC, Frentzel S, Ivanov NV, Peitsch MC, and Hoeng J
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- Alveolar Epithelial Cells cytology, Gene Expression, Humans, Lung cytology, Lung physiology, Macrophages cytology, Coculture Techniques, Computational Biology, In Vitro Techniques, Models, Biological, Transcriptome
- Abstract
To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This will allow us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-and-effect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the assembled tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages; the addition of rested macrophages decreased the basal inflammatory and cell stress status of the co-culture. Finally, we compared transcriptional profiles from publicly available datasets of conventional in vitro models representative of the airways and of healthy human lung tissues to assess similarities between our model and other in vitro models and the human lung. On the transcriptional level, we found an increasing correlation between airway models and normal human lung tissue, particularly as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart.
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- 2019
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21. A lung/liver-on-a-chip platform for acute and chronic toxicity studies.
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Bovard D, Sandoz A, Luettich K, Frentzel S, Iskandar A, Marescotti D, Trivedi K, Guedj E, Dutertre Q, Peitsch MC, and Hoeng J
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- Aflatoxin B1 toxicity, Cells, Cultured, Coculture Techniques instrumentation, Equipment Design, Humans, Inhalation Exposure analysis, Liver drug effects, Lung drug effects, Spheroids, Cellular cytology, Spheroids, Cellular drug effects, Liver cytology, Lung cytology, Models, Biological, Tissue Array Analysis instrumentation, Tissue Array Analysis methods, Toxicity Tests instrumentation, Toxicity Tests methods
- Abstract
The merging of three-dimensional in vitro models with multi-organ-on-a-chip (MOC) technology has taken in vitro assessment of chemicals to an unprecedented level. By connecting multiple organotypic models, MOC allows for the crosstalk between different organs to be studied to evaluate a compound's safety and efficacy better than with single cultures. The technology could also improve the toxicological assessment of aerosols that have been implicated in the development of chronic obstructive pulmonary disease, asthma, or lung cancer. Here we report the development of a lung/liver-on-a-chip, connecting in a single circuit, normal human bronchial epithelial (NHBE) cells cultured at the air-liquid interface (ALI), and HepaRG™ liver spheroids. Maintenance of the individual tissues in the chip increased NHBE ALI tissue transepithelial electrical resistance and decreased HepaRG™ spheroid adenosine triphosphate content as well as cytochrome P450 (CYP) 1A1/1B1 inducibility. CYP inducibility was partly restored when HepaRG™ spheroids were cocultured with NHBE ALI tissues. Both tissues remained viable and functional for 28 days when cocultured in the chip. The capacity of the HepaRG™ spheroids to metabolize compounds present in the medium and to modulate their toxicity was proven using aflatoxin B1 (AFB1). AFB1 toxicity in NHBE ALI tissues decreased when HepaRG™ spheroids were present in the same chip circuit, proving that the HepaRG™-mediated detoxification is protecting/decreasing from AFB1-mediated cytotoxicity. The lung/liver-on-a-chip platform presented here offers new opportunities to study the toxicity of inhaled aerosols or to demonstrate the safety and efficacy of new drug candidates targeting the human lung.
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- 2018
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22. In vitro systems toxicology-based assessment of the potential modified risk tobacco product CHTP 1.2 for vascular inflammation- and cytotoxicity-associated mechanisms promoting adhesion of monocytic cells to human coronary arterial endothelial cells.
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Poussin C, Laurent A, Kondylis A, Marescotti D, van der Toorn M, Guedj E, Goedertier D, Acali S, Pak C, Dulize R, Baumer K, Peric D, Maluenda E, Bornand D, Suarez IG, Schlage WK, Ivanov NV, Peitsch MC, and Hoeng J
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- Cells, Cultured, Coronary Vessels cytology, Endothelium, Vascular cytology, Humans, Monocytes cytology, Smoke adverse effects, Toxicity Tests, Cell Adhesion drug effects, Coronary Vessels drug effects, Endothelium, Vascular drug effects, Monocytes drug effects, Plant Extracts toxicity, Nicotiana chemistry, Vasculitis chemically induced
- Abstract
Cigarette smoking causes cardiovascular diseases. Heating tobacco instead of burning it reduces the amount of toxic compounds in the aerosol and may exert a reduced impact on health compared with cigarette smoke. Aqueous extract from the aerosol of a potential modified risk tobacco product, the Carbon Heated Tobacco Product (CHTP) 1.2, was compared in vitro with aqueous extract from the smoke of a 3R4F reference cigarette for its impact on the adhesion of monocytic cells to artery endothelial cells. Human coronary artery endothelial cells (HCAEC) were treated for 4 h with conditioned media from human monocytic Mono Mac 6 (MM6) cells exposed to CHTP1.2 or 3R4F extracts for 2 h or directly with those extracts freshly generated. In vitro monocyte-endothelial cell adhesion was measured concomitantly with inflammatory, oxidative stress, cytotoxicity, and death markers. Furthermore, transcriptomics analyses enabled to quantify the level of perturbation in HCAECs, and provide biological interpretation for the underlying molecular changes following exposure to 3R4F or CHTP1.2 extract. Our systems toxicology study demonstrated that approximately 10-15-fold higher concentrations of the CHTP 1.2 aerosol extract were needed to elicit similar effects as the 3R4F smoke extract on cardiovascular disease-relevant inflammation and cytotoxicity-related mechanisms and markers investigated in vitro., (Copyright © 2018 PMI R&D, Philip Morris Products S.A. Published by Elsevier Ltd.. All rights reserved.)
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- 2018
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23. The biological effects of long-term exposure of human bronchial epithelial cells to total particulate matter from a candidate modified-risk tobacco product.
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van der Toorn M, Sewer A, Marescotti D, Johne S, Baumer K, Bornand D, Dulize R, Merg C, Corciulo M, Scotti E, Pak C, Leroy P, Guedj E, Ivanov N, Martin F, Peitsch M, Hoeng J, and Luettich K
- Subjects
- Antigens, CD, Bronchi cytology, Cadherins metabolism, Cell Adhesion drug effects, Cell Line, Epithelial Cells metabolism, Epithelial Cells physiology, Epithelial-Mesenchymal Transition drug effects, Hot Temperature, Humans, Transcriptome drug effects, Epithelial Cells drug effects, Particulate Matter toxicity, Tobacco Products toxicity
- Abstract
Cigarette smoking is the leading cause of preventable lung cancer (LC). Reduction of harmful constituents by heating rather than combusting tobacco may have the potential to reduce the risk of LC. We evaluated functional and molecular changes in human bronchial epithelial BEAS-2B cells following a 12-week exposure to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product (cMRTP) in comparison with those following exposure to TPM from the 3R4F reference cigarette. Endpoints linked to lung carcinogenesis were assessed. Four-week 3R4F TPM exposure resulted in crisis and epithelial to mesenchymal transition (EMT) accompanied by decreased barrier function and disrupted cell-to-cell contacts. By week eight, cells regained E-cadherin expression, suggesting that EMT was reversible. Increased levels of inflammatory mediators were noted in cells treated to 3R4F TPM but not in cells treated to the same or a five-fold higher concentration of cMRTP TPM. A 20-fold higher concentration of cMRTP TPM increased oxidative stress and DNA damage and caused reversible EMT. Anchorage-independent growth was observed in cells treated to 3R4F or a high concentration of cMRTP TPM. 3R4F TPM-derived clones were invasive, while cMRTP TPM-derived clones were not. Long-term exposure to TPM from the cMRTP had a lower biological impact on BEAS-2B cells compared with that of exposure to TPM from 3R4F., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2018
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24. A framework for in vitro systems toxicology assessment of e-liquids.
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Iskandar AR, Gonzalez-Suarez I, Majeed S, Marescotti D, Sewer A, Xiang Y, Leroy P, Guedj E, Mathis C, Schaller JP, Vanscheeuwijck P, Frentzel S, Martin F, Ivanov NV, Peitsch MC, and Hoeng J
- Subjects
- Aerosols, Cell Culture Techniques, Cell Survival drug effects, Cells, Cultured, Equipment Design, High-Throughput Screening Assays, Humans, Systems Biology, Volatilization, Air Pollutants toxicity, Electronic Nicotine Delivery Systems adverse effects, Toxicity Tests instrumentation, Toxicity Tests methods
- Abstract
Various electronic nicotine delivery systems (ENDS), of which electronic cigarettes (e-cigs) are the most recognized prototype, have been quickly gaining ground on conventional cigarettes because they are perceived as less harmful. Research assessing the potential effects of ENDS exposure in humans is currently limited and inconclusive. New products are emerging with numerous variations in designs and performance parameters within and across brands. Acknowledging these challenges, we present here a proposed framework for an in vitro systems toxicology assessment of e-liquids and their aerosols, intended to complement the battery of assays for standard toxicity assessments. The proposed framework utilizes high-throughput toxicity assessments of e-liquids and their aerosols, in which the device-to-device variability is minimized, and a systems-level investigation of the cellular mechanisms of toxicity is an integral part. An analytical chemistry investigation is also included as a part of the framework to provide accurate and reliable chemistry data solidifying the toxicological assessment. In its simplest form, the framework comprises of three main layers: (1) high-throughput toxicity screening of e-liquids using primary human cell culture systems; (2) toxicity-related mechanistic assessment of selected e-liquids, and (3) toxicity-related mechanistic assessment of their aerosols using organotypic air-liquid interface airway culture systems. A systems toxicology assessment approach is leveraged to enable in-depth analyses of the toxicity-related cellular mechanisms of e-liquids and their aerosols. We present example use cases to demonstrate the suitability of the framework for a robust in vitro assessment of e-liquids and their aerosols.
- Published
- 2016
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25. High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC).
- Author
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Marescotti D, Gonzalez Suarez I, Acali S, Johne S, Laurent A, Frentzel S, Hoeng J, and Peitsch MC
- Subjects
- Aerosols, Risk Factors, Smoke, Nicotiana, Toxicology methods
- Abstract
Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA). For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound's toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC. Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (< 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator. This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.
- Published
- 2016
- Full Text
- View/download PDF
26. In Vitro Systems Toxicology Assessment of a Candidate Modified Risk Tobacco Product Shows Reduced Toxicity Compared to That of a Conventional Cigarette.
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Gonzalez-Suarez I, Martin F, Marescotti D, Guedj E, Acali S, Johne S, Dulize R, Baumer K, Peric D, Goedertier D, Frentzel S, Ivanov NV, Mathis C, Hoeng J, and Peitsch MC
- Subjects
- Aerosols chemistry, Bronchi cytology, Bronchi drug effects, Dose-Response Relationship, Drug, Epithelial Cells drug effects, Epithelial Cells metabolism, Humans, Risk Factors, Smoke adverse effects, Tobacco Products toxicity
- Abstract
Cigarette smoke increases the risk for respiratory and other diseases. Although smoking prevalence has declined over the years, millions of adults choose to continue to smoke. Modified risk tobacco products (MRTPs) are potentially valuable tools for adult smokers that are unwilling to quit their habit. Here, we investigated the biological impact of a candidate MRTP, the tobacco-heating system (THS) 2.2, compared to that of the 3R4F reference cigarette in normal primary human bronchial epithelial cells. Chemical characterization of the THS 2.2 aerosol showed reduced levels of harmful constituents compared to those of a combustible cigarette. Multiparametric indicators of cellular toxicity were measured via real-time cellular analysis and high-content screening. The study was complemented by a whole transcriptome analysis, followed by computational approaches to identify and quantify perturbed molecular pathways. Exposure of cells to 3R4F cigarette smoke resulted in a dose-dependent response in most toxicity end points. Moreover, we found a significant level of perturbation in multiple biological pathways, particularly in those related to cellular stress. By contrast, exposure to THS 2.2 resulted in an overall lower biological impact. At 3R4F doses, no toxic effects were observed. A toxic response was observed for THS 2.2 in some functional end points, but the responses occurred at doses between 3 and 15 times higher than those of 3R4F. The level of biological network perturbation was also significantly reduced following THS 2.2 aerosol exposure compared to that of 3R4F cigarette smoke. Taken together, the data suggest that THS 2.2 aerosol is less toxic than combustible cigarette smoke and thus may have the potential to reduce the risk for smoke-related diseases.
- Published
- 2016
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27. Systems biology approach for evaluating the biological impact of environmental toxicants in vitro.
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Gonzalez-Suarez I, Sewer A, Walker P, Mathis C, Ellis S, Woodhouse H, Guedj E, Dulize R, Marescotti D, Acali S, Martin F, Ivanov NV, Hoeng J, and Peitsch MC
- Subjects
- Acrolein chemistry, Acrolein toxicity, Apoptosis drug effects, Catechols chemistry, Catechols toxicity, Cell Proliferation drug effects, Cells, Cultured, DNA Damage drug effects, Epithelial Cells cytology, Epithelial Cells drug effects, Formaldehyde chemistry, Formaldehyde toxicity, Gene Expression Profiling, Humans, Membrane Potential, Mitochondrial drug effects, Mitochondria drug effects, Mitochondria metabolism, Mitogen-Activated Protein Kinase Kinases metabolism, Reactive Oxygen Species metabolism, Smoke
- Abstract
Exposure to cigarette smoke is a leading cause of lung diseases including chronic obstructive pulmonary disease and cancer. Cigarette smoke is a complex aerosol containing over 6000 chemicals and thus it is difficult to determine individual contributions to overall toxicity as well as the molecular mechanisms by which smoke constituents exert their effects. We selected three well-known harmful and potentially harmful constituents (HPHCs) in tobacco smoke, acrolein, formaldehyde and catechol, and established a high-content screening method using normal human bronchial epithelial cells, which are the first bronchial cells in contact with cigarette smoke. The impact of each HPHC was investigated using 13 indicators of cellular toxicity complemented with a microarray-based whole-transcriptome analysis followed by a computational approach leveraging mechanistic network models to identify and quantify perturbed molecular pathways. HPHCs were evaluated over a wide range of concentrations and at different exposure time points (4, 8, and 24 h). By high-content screening, the toxic effects of the three HPHCs could be observed only at the highest doses. Whole-genome transcriptomics unraveled toxicity mechanisms at lower doses and earlier time points. The most prevalent toxicity mechanisms observed were DNA damage/growth arrest, oxidative stress, mitochondrial stress, and apoptosis/necrosis. A combination of multiple toxicological end points with a systems-based impact assessment allows for a more robust scientific basis for the toxicological assessment of HPHCs, allowing insight into time- and dose-dependent molecular perturbations of specific biological pathways. This approach allowed us to establish an in vitro systems toxicology platform that can be applied to a broader selection of HPHCs and their mixtures and can serve more generally as the basis for testing the impact of other environmental toxicants on normal bronchial epithelial cells.
- Published
- 2014
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28. High avidity binding to DNA protects ubiquitylated substrates from proteasomal degradation.
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Coppotelli G, Mughal N, Marescotti D, and Masucci MG
- Subjects
- Cell Nucleus genetics, DNA genetics, Epstein-Barr Virus Nuclear Antigens genetics, HeLa Cells, Herpesvirus 4, Human genetics, Humans, Proteasome Endopeptidase Complex genetics, Protein Sorting Signals genetics, Ubiquitinated Proteins genetics, Cell Nucleus metabolism, DNA metabolism, Epstein-Barr Virus Nuclear Antigens metabolism, Herpesvirus 4, Human metabolism, Proteasome Endopeptidase Complex metabolism, Ubiquitinated Proteins metabolism
- Abstract
Protein domains that act as degradation and stabilization signals regulate the rate of turnover of proteasomal substrates. Here we report that the bipartite Gly-Arg repeat of the Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 acts as a stabilization signal that inhibits proteasomal degradation in the nucleus by promoting binding to cellular DNA. Protection can be transferred by grafting the domain to unrelated proteasomal substrates and does not involve changes of ubiquitylation. Protection is also afforded by other protein domains that, similar to the Gly-Arg repeat, mediate high avidity binding to DNA, as exemplified by resistance to detergent extraction. Our findings identify high avidity binding to DNA as a portable inhibitory signal that counteracts proteasomal degradation.
- Published
- 2011
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29. IFN-α boosts epitope cross-presentation by dendritic cells via modulation of proteasome activity.
- Author
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Lattanzi L, Rozera C, Marescotti D, D'Agostino G, Santodonato L, Cellini S, Belardelli F, Gavioli R, and Ferrantini M
- Subjects
- CD8-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes immunology, Cell Line, Tumor, Cell Proliferation, Cross-Priming, Cytotoxicity, Immunologic, Dendritic Cells cytology, Dendritic Cells immunology, Epitopes metabolism, Epitopes, T-Lymphocyte metabolism, HLA-A Antigens metabolism, HLA-A2 Antigen, Humans, Immunologic Memory, Interferon-alpha immunology, Lymphocyte Activation, Neoplasm Proteins metabolism, Proteasome Endopeptidase Complex immunology, Protein Binding, Viral Matrix Proteins metabolism, CD8-Positive T-Lymphocytes metabolism, Dendritic Cells metabolism, Immunotherapy, Adoptive, Interferon-alpha metabolism, Proteasome Endopeptidase Complex metabolism
- Abstract
We have investigated the molecular mechanisms underlying the peculiar cross-presentation efficiency of human dendritic cells (DCs) differentiated from monocytes in the presence of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Interferon (IFN)-α (IFN-DCs). To this end, we evaluated the capability of IFN-DCs to present and cross-present epitopes derived from Epstein-Barr Virus (EBV) or human melanoma-associated antigens after exposure to cell lysates or apoptotic cells. In an autologous setting, IFN-DCs loaded with Lymphoblastoid Cell Lines (LCL) lysates or apoptotic LCL were highly efficient in expanding, respectively, EBV-specific class II- or class I-restricted memory T cell responses. Of note, IFN-DCs loaded with apoptotic LCL were more potent than fully mature DCs in triggering the cytotoxicity of CD8(+) T lymphocytes recognizing a subdominant HLA-A*0201-restricted epitope derived from EBV latent membrane protein 2 (LMP2). In addition, IFN-DCs loaded with apoptotic human melanoma cells were highly efficient in cross-presenting the MART-1(27-35) epitope to a specific CD8(+) cytotoxic T cell clone, and this functional activity was proteasome-dependent. These IFN-DC properties were associated with an enhanced expression of all the immunoproteasome subunits as well as of TAP-1, TAP-2 and tapasin, and, interestingly, to a higher proteasome proteolytic activity as compared to immature or mature DCs. Altogether, these results highlight new mechanisms by which IFN-α influences antigen processing and cross-presentation ability of monocyte-derived DCs, with potentially important implications for the development of DC-based therapeutic vaccines., (Copyright © 2010 Elsevier GmbH. All rights reserved.)
- Published
- 2011
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30. Proteasome inhibitors induce the presentation of an Epstein-Barr virus nuclear antigen 1-derived cytotoxic T lymphocyte epitope in Burkitt's lymphoma cells.
- Author
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Destro F, Sforza F, Sicurella M, Marescotti D, Gallerani E, Baldisserotto A, Marastoni M, and Gavioli R
- Subjects
- Antigen Presentation immunology, Blotting, Western, Boronic Acids pharmacology, Bortezomib, Burkitt Lymphoma metabolism, Cell Line, Epitopes, T-Lymphocyte metabolism, Epstein-Barr Virus Nuclear Antigens metabolism, Fluorescent Antibody Technique, Humans, Leupeptins pharmacology, Oligopeptides pharmacology, Proteasome Endopeptidase Complex drug effects, Proteasome Endopeptidase Complex immunology, Proteasome Endopeptidase Complex metabolism, Pyrazines pharmacology, T-Lymphocytes, Cytotoxic metabolism, Antigen Presentation drug effects, Burkitt Lymphoma immunology, Epitopes, T-Lymphocyte immunology, Epstein-Barr Virus Nuclear Antigens immunology, Protease Inhibitors pharmacology, T-Lymphocytes, Cytotoxic immunology
- Abstract
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is generally expressed in all EBV-associated tumours and is therefore an interesting target for immunotherapy. However, evidence for the recognition and elimination of EBV-transformed and Burkitt's lymphoma (BL) cells by cytotoxic T lymphocytes (CTLs) specific for endogenously presented EBNA1-derived epitopes remains elusive. We confirm here that CTLs specific for the HLA-B35/B53-presented EBNA1-derived HPVGEADYFEY (HPV) epitope are detectable in the majority of HLA-B35 individuals, and recognize EBV-transformed B lymphocytes, thereby demonstrating that the GAr domain does not fully inhibit the class I presentation of the HPV epitope. In contrast, BL cells are not recognized by HPV-specific CTLs, suggesting that other mechanisms contribute to providing a full protection from EBNA1-specific CTL-mediated lysis. One of the major differences between BL cells and lymphoplastoid cell lines (LCLs) is the proteasome; indeed, proteasomes from BL cells demonstrate far lower chymotryptic and tryptic-like activities compared with proteasomes from LCLs. Hence, inefficient proteasomal processing is likely to be the main reason for the poor presentation of this epitope in BL cells. Interestingly, we show that treatments with proteasome inhibitors partially restore the capacity of BL cells to present the HPV epitope. This indicates that proteasomes from BL cells, although less efficient in degrading reference substrates than proteasomes from LCLs, are able to destroy the HPV epitope, which can, however, be generated and presented after partial inhibition of the proteasome. These findings suggest the use of proteasome inhibitors, alone or in combination with other drugs, as a strategy for the treatment of EBNA1-carrying tumours., (© 2011 The Authors. Immunology © 2011 Blackwell Publishing Ltd.)
- Published
- 2011
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31. Characterization of an human leucocyte antigen A2-restricted Epstein-Barr virus nuclear antigen-1-derived cytotoxic T-lymphocyte epitope.
- Author
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Marescotti D, Destro F, Baldisserotto A, Marastoni M, Coppotelli G, Masucci M, and Gavioli R
- Subjects
- Amino Acid Sequence, Antigen Presentation drug effects, Antigen Presentation immunology, Cell Line, Cytotoxicity Tests, Immunologic, Endoplasmic Reticulum metabolism, Epitopes, T-Lymphocyte genetics, Epstein-Barr Virus Nuclear Antigens genetics, HLA-A Antigens immunology, HLA-A Antigens metabolism, HLA-A2 Antigen metabolism, HLA-A24 Antigen, Humans, Interferon-gamma metabolism, Leukocytes, Mononuclear immunology, Peptides genetics, Peptides immunology, Peptides metabolism, Protease Inhibitors pharmacology, Proteasome Endopeptidase Complex metabolism, Proteasome Inhibitors, Protein Binding immunology, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, T-Lymphocytes, Cytotoxic metabolism, Transfection, tat Gene Products, Human Immunodeficiency Virus genetics, Epitopes, T-Lymphocyte immunology, Epstein-Barr Virus Nuclear Antigens immunology, HLA-A2 Antigen immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus-infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) -A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1-specific cytotoxic T-lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT-specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA-A2-restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT-specific CTL clones did not lyse EBV-carrying lymphoblastoid cell lines and Burkitt's lymphoma cell lines nor EBNA1-transfected Burkitt's lymphoma cells but specifically released interferon-gamma upon stimulation with HLA-matched EBNA1-expressing cells and this response was enhanced by deletion of the Gly-Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.
- Published
- 2010
- Full Text
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32. Membrane binding and anticoagulant properties of protein S natural variants.
- Author
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Baroni M, Pavani G, Marescotti D, Kaabache T, Borgel D, Gandrille S, Marchetti G, Legnani C, D'Angelo A, Pinotti M, and Bernardi F
- Subjects
- Animals, Binding Sites genetics, Blood Coagulation genetics, Cell Line, Cell Line, Transformed, Cell Transformation, Viral, Complement C4b-Binding Protein, Cricetinae, Fibroblasts cytology, Herpesvirus 4, Human physiology, Histocompatibility Antigens metabolism, Humans, Kidney cytology, Membranes metabolism, Phospholipids metabolism, Protein Binding genetics, Protein C metabolism, Protein Conformation, Protein S Deficiency genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Simian virus 40 physiology, Anticoagulants metabolism, Cell Membrane metabolism, Liposomes metabolism, Protein S genetics, Protein S metabolism
- Abstract
Introduction: Protein S (PS) is a vitamin K-dependent plasma glycoprotein with a key role in the control of coagulation pathway on phospholipid membranes. We compared anticoagulant and membrane binding properties of PS altered by natural mutations (N217S, DelI203D204) affecting the epidermal growth factor like-domain 4 (EGF4) and causing PS deficiency., Materials and Methods: Binding of recombinant, immunopurified PS (rPS) to several conformation-specific antibodies, to C4BP and to phospholipid liposomes was investigated by ELISA. PS binding to cells was analysed by flow cytometry. PS inhibitory activities were studied in plasma and purified systems., Results and Conclusions: Conformational changes produced by mutations were revealed by mapping with calcium-dependent antibodies. The immunopurified recombinant mutants (rPS) showed at 200-800 nM concentration reduced inhibition of coagulation (rPS217S, 10.2-17.3%; rPSDelI203D204, 5.8-8.9% of rPSwt) in FXa 1-stage clotting assay with APC. In thrombin generation assays the inhibition of ETP was reduced to 51.6% (rPS217S) and 24.1% (rPSDelI203D204) of rPSwt. A slightly shortened lag time (minutes) was also observed (rPS217S, 2.58; rPSDelI203D204, 2.33; rPSwt, 3.17; PS deficient plasma, 2.17). In flow cytometry analysis both mutants efficiently bound apoptotic cells in adhesion or in suspension. The affinity for phosphatidylserine-rich vesicles (apparent Kd: rPSwt 27.7+/-1.6 nM, rPS217S 146.0+/-16.1 nM and rPSDelI203D204 234.1+/-28.1 nM) was substantially increased by membrane oxidation (10.9+/-0.6, 38.2+/-3.5 and 81.4+/-6.0 nM), which resulted in a virtually normal binding capacity of mutants at physiological PS concentration. These properties help to define the molecular bases of PS deficiency, and provide further elements for PS-mediated bridging of coagulation and inflammation., (Copyright 2009 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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- View/download PDF
33. The Epstein-Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species.
- Author
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Gruhne B, Sompallae R, Marescotti D, Kamranvar SA, Gastaldello S, and Masucci MG
- Subjects
- Antigens, Viral chemistry, Antioxidants, Catalytic Domain, Cell Transformation, Neoplastic, DNA Damage, Disease Progression, Genomic Instability, Humans, Membrane Glycoproteins metabolism, Models, Biological, NADP chemistry, NADPH Oxidase 2, NADPH Oxidases metabolism, Neoplasms pathology, Transcriptional Activation, Epstein-Barr Virus Nuclear Antigens metabolism, Reactive Oxygen Species
- Abstract
The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is the only viral protein expressed in all EBV-carrying malignancies, but its contribution to oncogenesis has remained enigmatic. We show that EBNA-1 induces chromosomal aberrations, DNA double-strand breaks, and engagement of the DNA damage response (DDR). These signs of genomic instability are associated with the production of reactive oxygen species (ROS) and are reversed by antioxidants. The catalytic subunit of the leukocyte NADPH oxidase, NOX2/gp91(phox), is transcriptionally activated in EBNA-1-expressing cells, whereas inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 and a possible mechanism by which expression of this viral protein could contribute to malignant transformation and tumor progression.
- Published
- 2009
- Full Text
- View/download PDF
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