Nano-graphene oxide (GO) has attracted great interest in nanomedicine due to its own intrinsic properties and its possible biomedical applications such as drug delivery, tissue engineering and ...hyperthermia cancer therapy. However, the toxicity of GO nanosheets is not yet well-known and it is necessary to understand its entry mechanisms into mammalian cells in order to avoid cell damage and human toxicity. In the present study, the cellular uptake of pegylated GO nanosheets of ca. 100 nm labeled with fluorescein isothiocyanate (FITC-PEG-GOs) has been evaluated in the presence of eight inhibitors (colchicine, wortmannin, amiloride, cytochalasin B, cytochalasin D, genistein, phenylarsine oxide and chlorpromazine) that specifically affect different endocytosis mechanisms. Three cell types were chosen for this study: human Saos-2 osteoblasts, human HepG2 hepatocytes and murine RAW-264.7 macrophages. The results show that different mechanisms take part in FITC-PEG-GOs uptake, depending on the characteristics of each cell type. However, macropinocytosis seems to be a general internalization process in the three cell lines analyzed. Besides macropinocytosis, FITC-PEG-GOs can enter through pathways dependent on microtubules in Saos-2 osteoblasts, and through clathrin-dependent mechanisms in HepG2 hepatocytes and RAW-264.7 macrophages. HepG2 cells can also phagocytize FITC-PEG-GOs. These findings help to understand the interactions at the interface of GO nanosheets and mammalian cells and must be considered in further studies focused on their use for biomedical applications.
Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to be addressed. In this study, ...the biocompatibility of SiO2-CaO NanoMBGs was evaluated on different mouse immune cells, including spleen cells subsets, bone marrow-derived dendritic cells (BMDCs), or cell lines like SR.D10 Th2 CD4+ lymphocytes and DC2.4 dendritic cells. Flow cytometry and confocal microscopy show that the nanoparticles were rapidly and efficiently taken up in vitro by T and B lymphocytes or by specialized antigen-presenting cells (APCs) like dendritic cells (DCs). Nanoparticles were not cytotoxic and had no effect on cell viability or proliferation under T-cell (anti-CD3) or B cell (LPS) stimuli. Besides, NanoMBGs did not affect the balance of spleen cell subsets, or the production of intracellular or secreted pro- and anti-inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-6, IL-10) by activated T, B, and dendritic cells (DC), as determined by flow cytometry and ELISA. T cell activation surface markers (CD25, CD69 and Induced Costimulator, ICOS) were not altered by NanoMBGs. Maturation of BMDCs or DC2.4 cells in vitro was not altered by NanoMBGs, as shown by expression of Major Histocompatibility Complex (MHC) and costimulatory molecules (CD40, CD80, CD86), or IL-6 secretion. The effect of wortmannin and chlorpromazine indicate a role for phosphoinositide 3-kinase (PI3K), actin and clathrin-dependent pathways in NanoMBG internalization. We thus demonstrate that these NanoMBGs are both non-toxic and non-inflammagenic for murine lymphoid cells and myeloid DCs despite their efficient intake by the cells.
Key message
Several members of
WOX
and
KNOX
gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer
Pinus ...pinea
.
Similar to
Arabidopsis thaliana
,
Pinus pinea
shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in
P. pinea
cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in
P. pinea
. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2–6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes
PpWUS
,
PpWOX5
,
PpKN2
,
PpKN3
and
PipiRR1
.
WUS
is functional in pines and has an important role in caulogenesis. Interestingly,
WOX5
also seems to participate in the process, although its specific role has not been determined.
A newborn boy presented with a progressively infiltrating and painful congenital ulcerated plaque on the back of his left foot. A partial excision was performed and histopathologic examination ...confirmed a diagnosis of a plexiform fibrohistiocytic tumor. This rare tumor usually appears in children and adolescents, with congenital presentations even more uncommon. This case details the exceptional presentation of a congenital ulcerated plexiform fibrohistiocytic tumor with a review of the current literature.
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Dental bleaching with H2O2 is a common daily practice in dentistry to correct discoloration of anterior teeth. The aim of this study has been to determine whether this treatment of ...human teeth affects growth, differentiation and activity of osteoclast-like cells, as well as the putative modulatory action of osteostatin and fibroblast growth factor 2 (FGF-2).
Previously to the in vitro assays, structural, physical–chemical and morphological features of teeth after bleaching were studied. Osteoclast-like cells were cultured on human dentin disks, pre-treated or not with 38% H2O2 bleaching gel, in the presence or absence of osteostatin (100nM) or FGF-2 (1ng/ml). Cell proliferation and viability, intracellular content of reactive oxygen species (ROS), pro-inflammatory cytokine (IL-6 and TNFα) secretion and resorption activity were evaluated.
Bleaching treatment failed to affect either the structural or the chemical features of both enamel and dentin, except for slight morphological changes, increased porosity in the most superficial parts (enamel), and a moderate increase in the wettability degree. In this scenario, bleaching produced an increased osteoclast-like cell proliferation but decreased cell viability and cytokine secretion, while it augmented resorption activity on dentin. The presence of either osteostatin or FGF-2 reduced the osteoclast-like cell proliferation induced by bleaching. FGF-2 enhanced ROS content, whereas osteostatin decreased ROS but increased TNFα secretion. The bleaching effect on resorption activity was increased by osteostatin, but this effect was less evident with FGF-2.
These findings further confirm the deleterious effects of tooth bleaching by affecting osteoclast growth and function as well as different modulatory actions of osteostatin and FGF-2.
The role of membrane cofactor protein (MCP, CD46) on human T cell activation has been analyzed. Coligation of CD3 and CD46 in the presence of PMA or CD28 costimuli enhanced IL‐2, IFN‐γ, or IL‐10 ...secretion by CD4+ T lymphocytes. The effect of CD46 on IL‐10 secretion did not require additional costimuli like anti‐CD28 antibodies or phorbol esters. CD46 also enhanced IL‐2 or IFN‐γ secretion by CD4+ blasts. In contrast, IL‐5 secretion was inhibited upon CD46‐CD3 coligation, in all the cells analyzed. These effects were independent of IL‐12 and suggest that CD46 costimulation promotes a Th1‐biased response in human CD4+ T lymphocytes. CD46 enhanced TCR/CD3‐induced tyrosine phosphorylation of CD3ζ and ZAP‐70, as well as the activation of the ERK, JNK, and p38, but did not modify intracellular calcium. The effect of specific inhibitors shows that enhanced ERK activation contributes to augmented IFN‐γ and lower IL‐5 secretion and, consequently, to the Th1 bias. Cross‐linking CD46 alone induced weak tyrosine phosphorylation of CD3ζ and ZAP‐70. However, CD46 cross‐linking by itself did not induce cell proliferation or lymphokine secretion, and pretreatment of CD4+ T lymphocytes with anti‐CD46 antibodies did not significantly alter TCR/CD3 activation.
Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF–Cys2 mutant was ...recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group’s side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF–Cys2–heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF–Cys2–heparin complex might represent a biologically relevant complex in physiological media.
Calcium phosphate (CaP) based hybrid materials have been synthesized through a precipitation method in aqueous medium in the presence of the anionic surfactant sodium dodecylbenzene sulfonate (SDBS) ...as structure directing agent. Parameters of synthesis, such as Ca/P molar ratio, surfactant concentration and initial pH, have been investigated trying to get mesostructured CaP phases. A lamellar‐like hybrid mesophase consisting in several wavy layers of apatite or apatite‐brushite nanoplates was successfully obtained. Materials have been characterized by several physico‐chemical techniques. A potential application of these lamellar calcium phosphate based hybrids can be as matrixes in drug delivery systems with the possibility of loading hydrophobic drugs in the organic interlayers. To evaluate their potential as biomaterials, the biocompatibility of two hybrids has been studied in vitro with human Saos‐2 osteoblasts. Cell assays showed that, upon the appropriate synthesis and stabilization conditions, a biocompatible CaP and SDBS mesolamellar hybrid can be prepared.
Mesostructured lamellar calcium phosphates have been prepared via surfactant templating with sodium dodecylbenzenesulfonate. Synthesis parameters were optimized leading to a novel hybrid material consisting of mesolamellar apatite. The hybrids biocompatibility has been evaluated in vitro with human osteoblasts showing that biocompatible hybrids can be prepared, therefore suggesting their possible application as matrixes in drug delivery systems.
Mesoporous bioactive glass nanospheres (NanoMBGs) have high potential for clinical applications. However, the impact of these nanoparticles on the immune system needs to be addressed. In this study, ...the biocompatibility of SiO
-CaO NanoMBGs was evaluated on different mouse immune cells, including spleen cells subsets, bone marrow-derived dendritic cells (BMDCs), or cell lines like SR.D10 Th2 CD4
lymphocytes and DC2.4 dendritic cells. Flow cytometry and confocal microscopy show that the nanoparticles were rapidly and efficiently taken up in vitro by T and B lymphocytes or by specialized antigen-presenting cells (APCs) like dendritic cells (DCs). Nanoparticles were not cytotoxic and had no effect on cell viability or proliferation under T-cell (anti-CD3) or B cell (LPS) stimuli. Besides, NanoMBGs did not affect the balance of spleen cell subsets, or the production of intracellular or secreted pro- and anti-inflammatory cytokines (TNF-α, IFN-γ, IL-2, IL-6, IL-10) by activated T, B, and dendritic cells (DC), as determined by flow cytometry and ELISA. T cell activation surface markers (CD25, CD69 and Induced Costimulator, ICOS) were not altered by NanoMBGs. Maturation of BMDCs or DC2.4 cells in vitro was not altered by NanoMBGs, as shown by expression of Major Histocompatibility Complex (MHC) and costimulatory molecules (CD40, CD80, CD86), or IL-6 secretion. The effect of wortmannin and chlorpromazine indicate a role for phosphoinositide 3-kinase (PI3K), actin and clathrin-dependent pathways in NanoMBG internalization. We thus demonstrate that these NanoMBGs are both non-toxic and non-inflammagenic for murine lymphoid cells and myeloid DCs despite their efficient intake by the cells.
The antigen T cell receptor (TCR)-CD3 complexes present on the cell surface of CD4(+) T lymphocytes and T cell lines express CD3 epsilon chain isoforms with different isoelectric points (pI), with ...important structural and functional consequences. The pI values of the isoforms fit the predicted pI values of CD3 epsilon chains lacking one, two, and three negatively charged amino acid residues present in the N-terminal region. Different T cells have different ratios of CD3 epsilon chain isoforms. At a high pI, degraded CD3 epsilon isoforms can be better recognized by certain anti-CD3 monoclonal antibodies such as YCD3-1, the ability of which to bind to the TCR-CD3 complex is directly correlated with the pI of CD3 epsilon. The abundance of CD3 epsilon isoforms can be modified by treatment of T cells with the proteinase inhibitor phenanthroline. In addition, these CD3 epsilon isoforms have functional importance. This is shown, first, by the different structure of TCR-CD3 complexes in cells possessing different amounts of isoforms (as observed in surface biotinylation experiments), by their different antigen responses, and by the stronger interaction between low pI CD3 epsilon isoforms and the TCR. Second, incubation of cells with phenanthroline diminished the proportion of degraded high pI CD3 epsilon isoforms, but also the ability of the cells to deliver early TCR activation signals. Third, cells expressing mutant CD3 epsilon chains lacking N-terminal acid residues showed facilitated recognition by antibody YCD3-1 and enhanced TCR-mediated activation. Furthermore, the binding avidity of antibody YCD3-1 was different in distinct thymus populations. These results suggest that changes in CD3 epsilon N-terminal chains might help to fine-tune the response of the TCR to its ligands in distinct activation situations or in thymus selection.
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