"In vitro methods to study the cytotoxicity, cell transformation capacity and genotoxicity of nanoparticles - application to cobalt ferrite and silver nanoparticles"

The objective of this thesis was to investigate the in vitro cytotoxic, genotoxic and transforming effects induced by nanoparticles (NPs) of industrial interest on a range of cell cultures. The cytotoxicity of two sizes of CoFe2O4 NPs, paramagnetic particles of interest in different biomedical applications, was investigated using the Neutral Red uptake (NR) and Colony Forming Efficiency (CFE) assays using six mammalian cell lines at concentrations between 10 and 120 μM. More specifically, cytotoxicity was evaluated after 72-hour treatment of five cell lines (A549, CaCo2, HaCaT, HepG2, MDCK) with 10, 20, 40, 60, 80, 100 and 120 μM concentrations of NPs using NR, and 10, 20, 30, 40, 50, 60, 80, 100 and 120 μM concentrations of NPs using CFE. In parallel with these tests, cytotoxicity was also evaluated in mouse Balb3T3 fibroblasts using (i) NR after 72-hour treatment with 10, 20, 40, 60, 80, 100 and 120 μM concentrations of NPs, and (ii) CFE after both 72-hour treatment with 1, 5, 10, 20, 30, 40, 50, 60, 80, 100 and 120 μM concentrations of NPs, and 24-hour treatment with 1, 10, 60 and 120 μM concentrations of NPs. The cytotoxic effect exhibited a dose-effect relationship for Balb3T3 cells as assessed using the CFE assay. The testing of a more extensive concentration range of NPs in Balb3T3 cells (i.e., 1 and 5 μM in addition to the concentrations tested in the other five cell lines) over a 72-hour exposure time using CFE, together with the additional test using a 24-hour exposure time allowed appropriate concentration ranges to be determined for use in subsequent experiments using the Cell Transformation (CTA) and Cytokinesis-Block Micronucleus (CBMN) assays. The cell transformation capacity and genotoxicity of CoFe2O4 NPs were investigated using the Balb3T3 model, and assessed using the CTA (specifically at concentrations of 1, 5, 20 and 60 μM for 72 hours of treatment) and CBMN (specifically at concentrations of 1, 10 and 60 μM for 24 hours of treatment). The CoFe2O4 NPs induced neither effect at the doses and time points investigated. Four sizes of Ag NPs, chosen for their antimicrobial properties, were assessed for cytotoxicity (using CFE at concentrations of 0.1, 0.5, 1, 5 and 10 μM for 24 hours of treatment and of 0.01, 0.1, 0.5, 1, 2.5, 5 and 10 μM for 72 hours of treatment), cell transformation capacity (using CTA at concentrations of 0.5, 2.5 and 5 μM for 72 hours of treatment) and genotoxicity to Balb3T3 mouse fibroblasts (using CBMN at concentrations of 1, 5 and 10 μM for 24 hours of treatment). The Ag NPs had a significant cytotoxic effect, but no cell transformation or genotoxic effects at the doses and time points investigated. Physicochemical characterization of the chosen NPs was performed; size distribution and surface charge were measured by Dynamic Light Scattering (DLS), imaging by Scanning Electron Microscopy (SEM), the purity and ion leakage by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and the sedimentation by UV-Visible spectrometry

Sedimentation of Nanoparticles in in vitro Toxicity Assays

Attached extended abstract published in the conference proceedingsJRC.DG.I.5-Nanobioscience

Atypical teratoid/rhabdoid tumor in adults: a systematic review of the literature with meta‑analysis and additional reports of 4 cases

Introduction Atypical teratoid/rhabdoid tumor (AT/RT) is a highly aggressive embryonal CNS neoplasm, characterized by inactivation of SMARCB1 (INI1) or rarely of SMARCA4 (BRG1). While it is predominantly a childhood tumor, AT/RT is rare in adults. Methods We provide a comprehensive systematic review of literature with meta-analysis; 92 adult cases were found from 74 articles. We additionally present 4 cases of adult AT/RTs (age ranging from 19 to 29 years), located to cerebellum in 2 cases, to ponto-cerebellar angle in 1 case and to spinal cord in the remaining case. Results Microscopic features of our 4 cases showed a highly cellular tumor with rhabdoid morphology and high mitotic activity. All tumor cells lacked nuclear SMARCB1/INI1 protein expression. In case no. 3 we also performed methylation profiling which clustered the tumor with pediatric AT/RT-MYC subgroup. Prognosis remains poor in both pediatric and adult population with a median overall survival of 11 months. Our review demonstrated median overall survival of 15 months among the adult populations. However, consistent with a recent review, adult AT/RT seems to have highly variable prognosis and some patients reach long term survival with 22.9% of 5-year survival without evidence of disease and mean follow up time of 35.9 months (SD = 36.5). 27.1% of dissemination was also reported among the adult population. Conclusions Adult AT/RTs predominantly arise in female patients and in supratentorial location. Midline structures, including the sellar region, are the most affected sites, especially among females aged > 40 years. Male gender is more prevalent between the age of 18 and 40 years and more frequently associated with non-midline tumors. Factors significantly associated with better prognosis are patient’s age (< 40 years), combined radio-chemotherapy adjuvant approach and Ki-67 score < 40%

Type III interferons disrupt the lung epithelial barrier upon viral recognition

Viral infections of the lower respiratory tract are a leading cause of mortality. Mounting evidence indicates that most severe cases are characterized by aberrant immune responses and do not depend on viral burden. In this study, we assessed how type III interferons (IFN-λ) contribute to the pathogenesis induced by RNA viruses. We report that IFN-λ is present in the lower, but not upper, airways of patients with coronavirus disease 2019 (COVID-19). In mice, we demonstrate that IFN-λ produced by lung dendritic cells in response to a synthetic viral RNA induces barrier damage, causing susceptibility to lethal bacterial superinfections. These findings provide a strong rationale for rethinking the pathophysiological role of IFN-λ and its possible use in clinical practice against endemic viruses, such as influenza virus as well as the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection

Mindful approach to University education

A mindful approach to education and training could improve students' reflective capacities and have positive effects on clinical practice because it facilitates a helping relationship. The main aims of this study were to investigate whether participation in a mindful-based University training was associated with increases in mindfulness skills as measured by the 5-Facet M Questionnaire, and to present the Italian validation of the questionnaire. Sixty-seven students from the course Neuro and Psychomotor Therapy were enrolled. They filled in the self-administered 5-Facet M Questionnaire before and 1 month after a mindfulness-based training, focused on role-playing and followed by a feedback group discussion. The Italian version of the 5-Facet M Questionnaire had good psychometric properties. The pre- and post-training analysis showed a significant increase in the subscale 'Observing'. Findings suggest that role-playing and feedback group sessions are valid tools to improve students' mindfulness skills

Interferon (IFN)-λ Takes the Helm: Immunomodulatory Roles of Type III IFNs

Type III interferons (IFNs) (or IFN-λ) are the latest addition to the IFN family. Even though they share little protein homology with type I IFN, both exhibit remarkable functional similarities: each can be induced in response to viral infections, and both lead to Janus kinases (JAK) and signal transducer and activator of transcription (STAT) activation. The JAK/STAT pathway induces antiviral responses and IFN-stimulated gene transcription. However, despite the similarities in their effector functions with type I IFNs, IFN-λ also has a non-redundant role in protecting barrier organs: epithelial cells preferentially produce IFN-λ rather than type I IFNs; and interferon lambda receptor 1 (IFNLR1), the specific receptor for IFN-λ, is highly expressed on cells of epithelial lineage. Thus far, IFN-λ has been considered mainly as an epithelial cytokine, which restricts viral replication in epithelial cells and constitutes an added layer of protection at mucosal sites. However, it is now increasingly recognized that IFNLR1 is expressed broadly, and that immune cells such as neutrophils and dendritic cells also respond to IFN-λ. Moreover, in many in vivo models, IFN-λ modulates immune cell functions and thereby configures itself less as a cytokine that is only specific to the epithelium, and more as a cytokine that directly controls the inflammatory response at mucosal sites. Here, we critically review the recent literature on immune modulatory roles for IFN-λ, and distinguish between the direct and indirect effects of this IFN on immune cell functions in different inflammatory settings

Interferon (IFN)-λ Takes the Helm: Immunomodulatory Roles of Type III IFNs

Type III interferons (IFNs) (or IFN-λ) are the latest addition to the IFN family. Even though they share little protein homology with type I IFN, both exhibit remarkable functional similarities: each can be induced in response to viral infections, and both lead to Janus kinases (JAK) and signal transducer and activator of transcription (STAT) activation. The JAK/STAT pathway induces antiviral responses and IFN-stimulated gene transcription. However, despite the similarities in their effector functions with type I IFNs, IFN-λ also has a non-redundant role in protecting barrier organs: epithelial cells preferentially produce IFN-λ rather than type I IFNs; and interferon lambda receptor 1 (IFNLR1), the specific receptor for IFN-λ, is highly expressed on cells of epithelial lineage. Thus far, IFN-λ has been considered mainly as an epithelial cytokine, which restricts viral replication in epithelial cells and constitutes an added layer of protection at mucosal sites. However, it is now increasingly recognized that IFNLR1 is expressed broadly, and that immune cells such as neutrophils and dendritic cells also respond to IFN-λ. Moreover, in many in vivo models, IFN-λ modulates immune cell functions and thereby configures itself less as a cytokine that is only specific to the epithelium, and more as a cytokine that directly controls the inflammatory response at mucosal sites. Here, we critically review the recent literature on immune modulatory roles for IFN-λ, and distinguish between the direct and indirect effects of this IFN on immune cell functions in different inflammatory settings

Migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of iTreg cells

International audienc

Amorphous silica nanoparticles do not induce cytotoxicity, cell transformation or genotoxicity in Balb/3T3 mouse fibroblasts

Although amorphous silica nanoparticles (aSiO2NPs) are believed to be non-toxic and are currently used in several industrial and biomedical applications including cosmetics, food additives and drug delivery systems, there is still no conclusive information on their cytotoxic, genotoxic and carcinogenic potential. For this reason, this work has investigated the effects of aSiO2NPs on Balb/3T3 mouse fibroblasts, focusing on cytotoxicity, cell transformation and genotoxicity. Results obtained using aSiO2NPs, with diameters between 15 nm and 300 nm and exposure times up to 72 h, have not shown any cytotoxic effect on Balb/3T3 cells as measured by the MTT test and the Colony Forming Efficiency (CFE) assay. Furthermore, aSiO2NPs have induced no morphological transformation in Balb/3T3 cells and have not resulted in genotoxicity, as shown by Cell Transformation Assay (CTA) and Micronucleus (MN) assay, respectively. To understand whether the absence of any toxic effect could result from a lack of internalization of the aSiO2NPs by Balb/3T3 cells, we have investigated the uptake and the intracellular distribution following exposure to 85 nm fluorescently-labelled aSiO2NPs. Using fluorescence microscopy, it was observed that fluorescent aSiO2NPs are internalized and located exclusively in the cytoplasmic region. In conclusion, we have demonstrated that although aSiO2NPs are internalized in vitro by Balb/3T3 mouse fibroblasts, they do not trigger any cytotoxic or genotoxic effect and do not induce morphological transformation, suggesting that they might be a useful component in industrial applications.JRC.I.4-Nanobioscience