18 research outputs found
Cellular Uptake of Gold Nanoparticles Bearing HIV gp120 Oligomannosides
Dendritic cells are the most potent of the professional
antigen-presenting
cells which display a pivotal role in the generation and regulation
of adaptive immune responses against HIV-1. The migratory nature of
dendritic cells is subverted by HIV-1 to gain access to lymph nodes
where viral replication occurs. Dendritic cells express several calcium-dependent
C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing
non-integrin (DC-SIGN), which constitute a major receptor for HIV-1.
DC-SIGN recognizes <i>N</i>-linked high-mannose glycan clusters
on HIV gp120 through multivalent and Ca<sup>2+</sup>-dependent proteinācarbohydrate
interactions. Therefore, mimicking the cluster presentation of oligomannosides
from the virus surface is a strategic approach for carbohydrate-based
microbicides. We have shown that gold nanoparticles (<i>manno</i>GNPs) displaying multiple copies of structural motifs (di-, tri-,
tetra-, penta-, or heptaoligomanosides) of the <i>N</i>-linked
high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated <i>trans-</i>infection of human T cells. We have now prepared the
corresponding fluorescent-labeled glyconanoparticles (FITC-<i>manno</i>GNPs) and studied their uptake by DC-SIGN expressing
Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocyte-derived
immature dendritic cells (iDCs) by flow cytometry and confocal laser
scanning microscopy. We demonstrate that the 1.8 nm oligomannoside
coated nanoparticles are endocytosed following both DC-SIGN-dependent
and -independent pathways and part of them colocalize with DC-SIGN
in early endosomes. The blocking and sequestration of DC-SIGN receptors
by <i>manno</i>GNPs could explain their ability to inhibit
HIV-1 <i>trans</i>-infection of human T cells <i>in
vitro</i>
Transcriptome of Extracellular Vesicles Released by Hepatocytes
<div><p>The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide <i>in vitro</i> and <i>in vivo</i> evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.</p></div
Comprehensive Pathway analysis of EVs transcriptome.
<p>Ingenuity Pathway Analysis of transcripts detected in EVs released by non-tumoral hepatic cellular models.</p
Comparison between cellular and EVs transcriptomes in MLP29 cellular model.
<p>(A) Plot of fluorescent intensities for MLP29 EVs and cells reveals a group of genes enriched or underrepresented in EVs. (B) Plot shows the correlation of the fold changes estimated by microarray analysis and the fold change calculated by qPCR for a set of genes.</p
Hepatic stellate cells capture hepatocyte-released EVs and become activated.
<p>HSC 8B cells capture EVs from RH and respond to these stimuli by increasing <i>Nos2</i> transcription. (A) The capture can be followed by detecting the presence of <i>Alb</i>, an RH-specific transcript, for up to 24 hours after capture. When EVs were treated with RNase in the presence of detergent before incubation with the cells, <i>Alb</i> transcript was not detected (6 h incubation time), indicating that the transcript must have been transferred from the hepatocyte-released EVs. (B) The activation of HSCs can be followed by the expression of the protein nitric oxide synthase 2 (Nos2). <i>Nos2</i> is expressed at a very low level in HSC 8B (lanes 1 and 2), and clearly expressed if the cells are treated with EVs (lane 3 and 4), as previously described (31). After incubation with EVs pre-treated with RNase in the presence of detergent, we do not detect <i>Nos2</i> transcription (lane 5). HSC 8B cells incubated with EVs pre-treated only with detergent increase the transcription of <i>Nos</i>2 (lane 6), although to lower levels than the cells treated with intact EVs (lanes 3 and 4). In the graph, error bars represent SD (nā=ā2),* denotes p<0.05 respect to control, lane 1.</p
Characterization of EVs.
<p>Characterization of EVs from MLP29 (A, C) and rat primary hepatocytes (RH) (B, D). The NTA analyses of two independent samples for each cell type show more heterogeneous vesicle populations released by primary culture of hepatocytes. Cryo-TEM pictures show membrane vesicles of different sizes (insets AāB). Bioanalyzer profiles of total RNA extracted by RNeasy from both cell types are similar with a wide distribution on size and also the presence of a reduced amount of ribosomal RNAs (CāD).</p
Human Mammospheres Secrete Hormone-Regulated Active Extracellular Vesicles
<div><p>Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression.</p></div
Effect of EVs secreted by MDA-MB-468 mammospheres on MCF7 cells.
<p>(A) MCF-7 cells were incubated with 0, 25 and 50 Āµg/mL of MDA-MB-468 EVs and the number of mammospheres formed after 7-days was counted [data are mean Ā±SD; <i>n</i>ā=ā3, *<i>p</i><0.05, **<i>p</i><0.01, relative to the values in the control]. (B) Quantitative polymerase chain reaction analysis was conducted to examine the expression of the factors <i>Zeb1</i> and <i>Snaill</i> [data are mean Ā±SD; <i>n</i>ā=ā3, <i>p</i><0.05 and 0.05 respectively, relative to the values in the control].</p
Effect of EVs from MDA-MB-468 on stem cell and EMT markers in U2OS cells.
<p>U2OS cells were incubated with 0 or 50 Āµg/mL of MDA-MB-468-derived EVs and quantitative polymerase chain reaction analysis was conducted to examine the expression of the transcription factors <i>Nanog</i>, <i>Oct-4</i>, <i>Sox2</i>, <i>Zeb1</i> and <i>Snaill</i> involved in development and maintenance of stem cells [data are mean Ā±SD; <i>n</i>ā=ā3, <i>p</i><0.05, 0.05, 0.01, 0.05 and 0.01, respectively, relative to the values in the control].</p
Comparison between the transcriptomes from MLP29 and RHās EVs, and other published transcriptomes.
<p>Published transcriptomes from endothelial, mesenchymal <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068693#pone.0068693-Valadi1" target="_blank">[13]</a> and cardiomyocyte <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068693#pone.0068693-Waldenstrom1" target="_blank">[33]</a> EVs. The common genes among the five cell types (A) are enriched in translation and ribosomal machinery categories. For the transcripts only detected MLP29 and RH (B), we found enrichment in pathways related to fatty acid metabolism and ethanol degradation.</p