140 research outputs found
A preliminary study of engineered nanoparticles effects on barrier function of airway epithelial monolayers
The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARβ induction
AbstractThe mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all-trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARβ protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARβ, suggesting that the receptor is involved in the regulation of the transporter gene
Oligodendroglioma cells lack glutamine synthetase and are auxotrophic for glutamine, but do not depend on glutamine anaplerosis for growth
In cells derived from several types of cancer, a transcriptional program drives high consumption of glutamine (Gln), which is used for anaplerosis, leading to a metabolic addiction for the amino acid. Low or absent expression of Glutamine Synthetase (GS), the only enzyme that catalyzes de novo Gln synthesis, has been considered a marker of Gln-addicted cancers. In this study, two human cell lines derived from brain tumors with oligodendroglioma features, HOG and Hs683, have been shown to be GS-negative. Viability of both lines depends from extracellular Gln with EC of 0.175 ± 0.056 mM (Hs683) and 0.086 ± 0.043 mM (HOG), thus suggesting that small amounts of extracellular Gln are sufficient for OD cell growth. Gln starvation does not significantly affect the cell content of anaplerotic substrates, which, consistently, are not able to rescue cell growth, but causes hindrance of the Wnt/β-catenin pathway and protein synthesis attenuation, which is mitigated by transient GS expression. Gln transport inhibitors cause partial depletion of intracellular Gln and cell growth inhibition, but do not lower cell viability. Therefore, GS-negative human oligodendroglioma cells are Gln-auxotrophic but do not use the amino acid for anaplerosis and, hence, are not Gln addicted, exhibiting only limited Gln requirements for survival and growth
Cerium oxide nanoparticles rescue α-synuclein-induced toxicity in a yeast model of Parkinson’s disease
Over the last decades, cerium oxide nanoparticles (CeO2 NPs) have gained great interest due to their potential applications, mainly in the fields of agriculture and biomedicine. Promising effects of CeO2 NPs are recently shown in some neurodegenerative diseases, but the mechanism of action of these NPs in Parkinson’s disease (PD) remains to be investigated. This issue is addressed in the present study by using a yeast model based on the heterologous expression of the human α-synuclein (α-syn), the major component of Lewy bodies, which represent a neuropathological hallmark of PD. We observed that CeO2 NPs strongly reduce α-syn-induced toxicity in a dose-dependent manner. This effect is associated with the inhibition of cytoplasmic α-syn foci accumulation, resulting in plasma membrane localization of α-syn after NP treatment. Moreover, CeO2 NPs counteract the α-syn-induced mitochondrial dysfunction and decrease reactive oxygen species (ROS) production in yeast cells. In vitro binding assay using cell lysates showed that α-syn is adsorbed on the surface of CeO2 NPs, suggesting that these NPs may act as a strong inhibitor of α-syn toxicity not only acting as a radical scavenger, but through a direct interaction with α-syn in vivo
Pathways of L-glutamic acid transport in cultured human fibroblasts.
The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax
Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro.
BACKGROUND/PURPOSE
Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype.
MATERIALS AND METHODS
We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates.
RESULTS
Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages.
CONCLUSION
These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application
The TLR4/NFÎşB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles
Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans
Lipopolysaccharide adsorbed to the bio-corona of TiO2 nanoparticles powerfully activates selected pro-inflammatory transduction pathways
An in vitro strategy to assess mitigation of hazardous properties of engineered metal nanoparticles
The huge progress in the nanotechnology field has requested the production of increasingly
advanced engineered nanoparticles (NPs). In particular, metal-based advanced NPs are
widely used in several industrial applications. However, their potential effects on human
health during occupational exposure are still incompletely characterized thus far and
possible strategies to decrease their hazardous properties are not yet clearly defined. In this
project we are developing an in vitro approach to test the cytotoxic effects of metal-based
NPs, as derived from production lines or modified through coating with organic or inorganic
moieties. We have used two cell models widely employed in toxicological studies, the human
alveolar cell line A549 and the murine macrophage cell line RAW264.7, to avoid possible
limitations due to cell specific effects. Moreover, in order to evaluate the effectiveness of
mitigation approaches for NPs endowed with little acute cytotoxicity, additional endpoints,
alternative to viability, have also been assessed. Colloidal suspensions of Ag, TiO2 and ZrO2
NPs were tested as provided by industries or modified with SiO2 NPs or citrate used as
coating remediation agents. Heterocoagulation of opposite charged phases was applied in
order to promote the coating of pristine surfaces by modifying agents. Heterocoagulated sols
were obtained by ball milling sols of positive charged Ag, TiO2 and ZrO2 NPs with negative
charged SiO2 NPs or citrate ions. Modified samples, obtained by spray-drying and re-
dispersing in water the corresponding sols, were also obtained in order to compare
reactivity. Original and modified NPs were added to culture media starting from water
colloidal suspensions. Viability was determined with the resazurin method in a range of
doses from 2.5 to 80 nfg/cm2 (0.3125 to 20 g/cm2 for Ag NPs) of monolayer surface at three
experimental times (24, 48 and 72h). The expression of the inducible form of nitric oxide
synthase (Nos2), an indicator of macrophage activation and, hence, of pro-inflammatory
activity, was assessed with RT-PCR as an end-point alternative to viability.
Among the NPs tested, only Ag NP caused a significant loss of viability, with an IC50 of about
0.8 g/cm2 for Raw264.7 cells and 2.4 g/cm2 for A549 cells at the 24h-experimental time. In
a preliminary experiment, SiO2 NPs were demonstrated to have no significant effect on cell
viability. The comparison between original and SiO2-coated Ag NPs, performed in the same
experiment, suggested a coating-independent mitigation effect of bioreactivity exerted by the
spray drying procedure. However, once corrected for the actual Ag content of the spray-
dried powder, no significant difference was found in the IC50 values, indicating that neither
silica coating nor spray drying mitigate cytotoxicity. The effects on viability of original TiO2
and ZrO2 NPs were assessed using P25 Aeroxide TiO2 NPs as a reference material. These
materials did not affect significantly cell viability at any time point tested, so that it was not
possible to estimate IC50 values for either cell line. However, titania produced a clear-cut
induction of Nos2 expression in Raw264.7 cells, thus indicating their potential pro-
inflammatory activity. Citrate coating did not produce any significant attenuation of the
biological effect. In summary, these preliminary results showed no mitigating effect of the
surface modifications tested on the biological effects of the engineered NPs investigated.
However, the exploitation of this in vitro experimental strategy can be useful for the
preliminary assessment of the mitigation potential of surface modifications of both low-toxic
and high-toxic engineered NPs.
Supported by EU Grant NMP4-SL-2012-280716 (Sanowork Project
Identifying contact mediated, localized toxic effects of MWCNT aggregates on epithelial monolayers: a single-cell monitoring toxicity assay
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