53 research outputs found
Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake
Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.JRC.I.4-Nanobioscience
ECVAM Technical Report on the Status of Alternative Methods for Cosmetics Testing (2008-2009)
The ECVAM technical report presents the progress made in the development and validation of alternative methods for the human health effects relevant to the Cosmetics Directive. It provides an update on the activities described by ECVAM in 2005 , 2006 and 2007 .
The report intends to present the latest scientific and technical developments in the field during 2008-2009. As required by Directive 2003/15/EC, the seventh amendment to Directive 76/768/EEC, developments in refinement and reduction methods are also described (EU, 2003).
Most successes in the development of alternative methods are in acute local toxicity and short-term testing, such as e.g. skin and eye irritation/corrosion, phototoxicity and skin penetration The test methods consuming a high number of animals, however, are in long-term testing and systemic toxicity, such as e.g. reproductive toxicity and repeated dose toxicity. In these complex fields, several research initiatives are ongoing. However full replacement approaches are still lacking.JRC.DG.I.3-In-vitro method
Phospholipid Scramblase-1-Induced Lipid Reorganization Regulates Compensatory Endocytosis in Neuroendocrine Cells
Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1â/âmice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis
Interactions of Epstein-Barr virus origins of replication with nuclear matrix in the latent and in the lytic phases of viral infection
Eukaryotic DNA is organized into domains or loops generated by the attachment of chromatin fibers to the nuclear matrix via specific regions called scaffold or matrix attachment regions. The role of these regions in DNA replication is currently under investigation since they have been found in close association with origins of replication. Also, viral DNA sequences, containing the origins of replication, have been found attached to the nuclear matrix. To investigate the functional role of this binding we have studied, in Raji cells, the interaction between Epstein-Barr virus (EBV) origins of replication and the nuclear matrix in relation to the viral cycle of infection. We report here that both the latent (ori P) and the lytic (ori Lyt) EBV origins of replication are attached to the nuclear matrix, the first during the latent cycle of infection and the second after induction of the lytic cycle. These findings suggest that the binding of the origins of replication with the nuclear matrix modulates viral replication and expression in the two different phases of infection
Differentation-specific nuclear matrix proteins cross-linked to DNA by cis-diamminedichloroplatinum.
DNA-protein cross-linkages were performed in intact undifferentiated and differentiated-HL60 cells by the action of cis-diammine dichloroplatinum. Total nuclear matrix proteins and DNA cross-linked nuclear matrix proteins were resolved by two-dimensional gel electrophoresis. The comparison of the electrophoretic patterns allowed the identification of a set of differentiation-induced nuclear matrix proteins cross-linked to DNA. One of these proteins binds cloned histone SAR sequences. Our results outline an experimental strategy for isolating and characterizing nuclear matrix components that may play a fundamental role in the overall control and coordination of gene expression during differentiation
Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis
Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding. KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCgamma binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphor-phosphorylation of PLCgamma as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR. (C) 2004 Elsevier Inc. All rights reserved
ECVAM Bottom-Up/Top-Down Testing Approach: Testing Strategy to Reduce/Replace the Draize Eye Test and Validation/Regulatory Acceptance of In Vitro Assays: Current Status
To reduce and/or replace the Draize test, testing schemes combining strengths of particular in vitro assays were proposed during a 2005 ECVAM expert meeting. The testing scheme proposes, based on expected irritancy of the test substance, a Bottom-Up approach, beginning with test methods that accurately identify non-irritants, or a Top-Down approach, beginning with test methods that accurately identify severe irritants before progression of further in vitro testing. Furthermore as its core activity, ECVAM participated in the retrospective validation of and has peer reviewed scientific validity of four organotypic assays, and undertook retrospective validation of four cell function/cytotoxicity assays. The BCOP and ICE organotypic assays were ICCVAM and ESAC endorsed as scientifically valid for identifying severe irritants, and OECD Test Guidelines are under adoption. NRR, FL and CM cell function/cytotoxicity assays were recommended by an ECVAM Validation Management Group for identification of non-irritants or severe eye irritants in the Bottom-Up/Top-Down approaches. These assays were peer reviewed by ESAC during 2009. Finally, a joint ECVAM-COLIPA prospective validation study was initiated in 2008 to evaluate two Reconstructed human Tissue assays to discriminate non-classified materials from eye irritants, based on the proposed test strategies. The ultimate goal is to combine validated in vitro assays, based on their performances and applicability domains, to define the most suitable testing strategy to classify substances for eye irritation potential and ultimately replace the Draize test. This manuscript presents the proposed testing scheme and provides details on the validation/regulatory status of in vitro assays for use in this scheme.JRC.I.2-Validation of Alternative Method
The Rho Guanine Nucleotide Exchange Factors Intersectin 1L and b-Pix Control Calcium-Regulated Exocytosis in Neuroendocrine PC12 Cells
International audienceGTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmis-sion and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and b-Pix, play essential roles in neuroen-docrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells
The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation
Background: Significant progress of nanotechnology, including in particular biomedical and pharmaceutical
applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover,
a determination of so-called âcritical quality attributesâ, that is specific physicochemical properties of nanomaterials
triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel
safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of
nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following
this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the
agglomeration state can influence the level and the mechanism of NP cellular internalization.
Results: We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated
form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene
silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed
differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis
was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration
state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted.
Conclusions: We demonstrated that the agglomeration state of NPs is an important factor influencing the level of
cell uptake and the mechanism of endocytosis of silica NPs.JRC.F.2-Consumer Products Safet
The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation
Abstract Background Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called âcritical quality attributesâ, that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. Results We employed fluorescently-labelled 30 and 80Â nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. Conclusions We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs
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