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1.
J Extracell Vesicles ; 7(1): 1446660, 2018.
Article in English | MEDLINE | ID: mdl-29696074

ABSTRACT

Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

2.
Vascul Pharmacol ; 75: 7-18, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26254104

ABSTRACT

A defect in neo-vascularization process involving circulating angiogenic mononuclear cells (CACs) dysfunction is associated with diabetes. We showed that oxidative stress was elevated in CACs cultured from blood of individuals with metabolic syndrome (MetS) and diabetes. We then assessed the action of palmitic acid (PA), a deregulated and increased NEFA in metabolic disorders, focusing on its oxidant potential. We observed that the phyto-polyphenol resveratrol normalized oxidative stress both in CACs isolated from MetS patients or treated with PA. Resveratrol further decreased the deleterious action of PA on gene expression of vascularization factors (TNFα, VEGF-A, SDF1α, PECAM-1, VEGFR2, Tie2 and CXCR4) and improved CAC motility. Particularly, resveratrol abolished the PA-induced over-expression of the pro-oxidant protein p66Shc. Neither KLF2 nor SIRT1, previously shown in resveratrol and p66Shc action, was directly involved. Silencing p66Shc normalized PA action on VEGF-A and TNFα specifically, without abolishing the PA-induced oxidative stress, which suggests a deleterious role of p66Shc independently of any major modulation of the cellular oxidative status in a high NEFA levels context. Besides showing that resveratrol reverses PA-induced harmful effects on human CAC function, certainly through profound cellular modifications, we establish p66Shc as a major therapeutic target in metabolic disorders, independent from glycemic control.


Subject(s)
Oxidative Stress/drug effects , Palmitic Acid/metabolism , Shc Signaling Adaptor Proteins/genetics , Stilbenes/pharmacology , Antioxidants/pharmacology , Case-Control Studies , Cell Movement/drug effects , Cells, Cultured , Diabetes Mellitus, Type 2/physiopathology , Gene Expression Regulation/drug effects , Gene Silencing , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Metabolic Syndrome/physiopathology , Middle Aged , Neovascularization, Physiologic/drug effects , Resveratrol , Src Homology 2 Domain-Containing, Transforming Protein 1
3.
Mol Immunol ; 46(6): 1240-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19162326

ABSTRACT

Antigen presenting cells (APC) express a variety of pattern recognition receptors, including the C-type lectin receptors (CLR) that specifically recognize carbohydrate structures expressed on self-tissue and pathogens. The CLR play an important role in antigen uptake and presentation and have been shown to mediate cellular interactions. The ligand specificity of the human macrophage galactose-type lectin (MGL) has been characterized extensively. Here, we set out to determine the glycan specificity of the murine homologues, MGL1 and MGL2, using a glycan array. Murine MGL1 was found to be highly specific for Lewis X and Lewis A structures, whereas mMGL2, more similar to the human MGL, recognized N-acetylgalactosamine (GalNAc) and galactose, including the O-linked Tn-antigen, TF-antigen and core 2. The generation of MGL1 and MGL2-Fc proteins allowed us to identify ligands in lymph nodes, and MGL1-Fc additionally recognized high endothelial venules. Strikingly, MGL2 interacted strongly to adenocarcinoma cells, suggesting a potential role in tumor immunity.


Subject(s)
Asialoglycoproteins/metabolism , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Polysaccharides/metabolism , Adenocarcinoma/metabolism , Animals , Antigen-Presenting Cells/immunology , Asialoglycoproteins/genetics , Asialoglycoproteins/immunology , Bone Marrow Cells/metabolism , Cell Line, Tumor , Cells, Cultured , Endothelium, Vascular/metabolism , Extracellular Matrix/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/immunology , Ligands , Lymph Nodes/metabolism , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred C57BL , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Skin/metabolism
4.
Arterioscler Thromb Vasc Biol ; 27(5): 1023-9, 2007 May.
Article in English | MEDLINE | ID: mdl-17322099

ABSTRACT

OBJECTIVE: The purpose of this study was to investigate the presence and functionality of P-selectin glycoprotein ligand-1 (PSGL-1) on activated endothelial cells (ECs). METHODS AND RESULTS: We show here that PSGL-1 is expressed at the mRNA and protein levels in umbilical vein and microvascular ECs. Furthermore, this endothelial PSGL-1 (ePSGL-1) is functional and mediates adhesion of monocytes or platelet-monocyte complexes (PMCs) to the activated endothelium in a flow model. ePSGL-1 expression was not affected by treating ECs with inflammatory stimuli (tumor necrosis factor alpha, interleukin-1beta, thrombin, or histamine). However, the functional binding capacity of ePSGL-1 to monocytes or P-selectin/Fc chimera significantly increased by stimulation of the ECs with TNFalpha. By means of a siRNA approach to specifically knock-down the genes involved in the glycosylation of PSGL-1 we could show that tumor necrosis factor alpha-induced glycosylation of ePSGL-1 is critical for its binding capacity. CONCLUSION: Our results show that ECs express functional PSGL-1 which mediates tethering and firm adhesion of monocytes and platelets to inflamed endothelium.


Subject(s)
Cell Adhesion/physiology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Gene Expression , Membrane Glycoproteins/genetics , RNA/genetics , Blood Platelets/metabolism , Blood Platelets/pathology , Blotting, Western , Cells, Cultured , Coronary Artery Disease/metabolism , Coronary Artery Disease/pathology , Endothelial Cells/cytology , Endothelium, Vascular/metabolism , Flow Cytometry , Humans , Membrane Glycoproteins/biosynthesis , Microscopy, Confocal , Monocytes/metabolism , Monocytes/pathology , RNA/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Umbilical Veins/cytology
5.
Mol Immunol ; 44(10): 2605-15, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17241663

ABSTRACT

In schistosomiasis, a parasitic disease caused by helminths, the parasite eggs induce a T helper 2 cell (T(H)2) response in the host. Here, the specific role of human monocyte-derived dendritic cells (DCs) in initiation and polarization of the egg-specific T cell responses was examined. We demonstrate that immature DCs (iDCs) pulsed with schistosome soluble egg antigens (SEA) do not show an increase in expression of co-stimulatory molecules or cytokines, indicating that no conventional maturation was induced. The ability of SEA to affect the Toll-like receptor (TLR) induced maturation of iDCs was examined by copulsing the DCs with SEA and TLR-ligands. SEA suppressed both the maturation of iDCs induced by poly-I:C and LPS, as indicated by a decrease in co-stimulatory molecule expression and production of IL-12, IL-6 and TNF-alpha. In addition, SEA suppressed T(H)1 responses induced by the poly-I:C-pulsed DCs, and skewed the LPS-induced mixed response towards a T(H)2 response. Immature DCs rapidly internalized SEA through the C-type lectins DC-SIGN, MGL and the mannose receptor and the antigens were targeted to MHC class II-positive lysosomal compartments. The internalization of SEA by multiple C-type lectins may be important to regulate the response of the iDCs to TLR-induced signals.


Subject(s)
Antigens, Helminth/immunology , Dendritic Cells/immunology , Lectins, C-Type/immunology , Schistosoma mansoni/immunology , Toll-Like Receptors/immunology , Animals , Antigen Presentation , Antigens, Helminth/pharmacology , Cytokines/metabolism , Dendritic Cells/drug effects , Histocompatibility Antigens Class II/immunology , Humans , Ligands , Lipopolysaccharides/pharmacology , Lysosomal-Associated Membrane Protein 1/metabolism , Ovum/immunology , Poly I-C/pharmacology , T-Lymphocytes/immunology
6.
FEBS Lett ; 580(26): 6123-31, 2006 Nov 13.
Article in English | MEDLINE | ID: mdl-17055489

ABSTRACT

The dendritic cell specific C-type lectin dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) binds to "self" glycan ligands found on human cells and to "foreign" glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC-SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC-SIGN binds with K(d)<2muM to a neoglycoconjugate in which Galbeta1-4(Fucalpha1-3)GlcNAc (Le(x)) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le(x) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing core-fucose linked either alpha1-6 or alpha1-3 to the Asn-linked GlcNAc of N-glycans. These results demonstrate that DC-SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose- and complex-type N-glycans.


Subject(s)
Cell Adhesion Molecules/metabolism , Fucose , Lectins, C-Type/metabolism , Mannose , Polysaccharides/metabolism , Receptors, Cell Surface/metabolism , Humans , Ligands , Polysaccharides/chemistry , Protein Array Analysis , Protein Binding
7.
Mol Cell Biol ; 25(15): 6454-63, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16024783

ABSTRACT

Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The beta1,4-galactosyltransferase 1 (beta4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3beta is required to bind beta4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Ikappabeta kinase and protein kinase Cdelta is involved in the phosphorylation of 14-3-3beta on two serine residues, paralleled by release of binding of TTP and 14-3-3beta from beta4GalT1 mRNA, nuclear sequestration of TTP, and beta4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3beta.


Subject(s)
14-3-3 Proteins/metabolism , DNA-Binding Proteins/metabolism , Immediate-Early Proteins/metabolism , Protein Kinase C/physiology , Protein Serine-Threonine Kinases/physiology , RNA Stability , RNA, Messenger/metabolism , Cells, Cultured , Endothelial Cells/enzymology , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Humans , I-kappa B Kinase , Mutagenesis, Site-Directed , Phosphorylation , Protein Kinase C-delta , Serine/metabolism , Signal Transduction/physiology , Tristetraprolin , Tumor Necrosis Factor-alpha/metabolism , Umbilical Veins/enzymology
8.
J Biol Chem ; 280(13): 12676-82, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15668241

ABSTRACT

During the course of an inflammatory response, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFalpha) triggers endothelial cells to increase the expression levels of adhesion molecules that are pivotal for the rolling, adhesion, and transmigration of leukocytes over the endothelial cell wall. Here we show that TNFalpha, in addition, has a regulatory function in the biosynthesis of proper carbohydrate molecules on endothelial cells that constitute ligands for adhesion molecules on leukocytes. Our data show that TNFalpha induced an increase in the expression of beta1,4-galactosyltransferase-1 (beta4GalT-1) in primary human umbilical vein endothelial cells in a time- and concentration-dependent manner. The beta4GalT-1 mRNA up-regulation correlated with an increase in the Golgi expression and catalytic activity of the enzyme. Furthermore, an enhanced incorporation of galactose was observed in newly synthesized glycoproteins. Analysis of the molecular mechanism behind the up-regulation of beta4GalT-1 showed that the increase in mRNA levels is due to an enhanced stability of the transcripts. These data strongly demonstrate that TNFalpha modulates the glycosylation of endothelial cells by a mechanism that directly enhances the stability of beta4GalT-1 mRNA transcripts.


Subject(s)
Endothelium, Vascular/cytology , N-Acetyllactosamine Synthase/metabolism , Tumor Necrosis Factor-alpha/metabolism , Up-Regulation , 3' Untranslated Regions , Carbohydrates/chemistry , Cell Adhesion , Cells, Cultured , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Galactose/metabolism , Galactosyltransferases/metabolism , Glycoproteins/chemistry , Glycoproteins/metabolism , Glycosylation , Golgi Apparatus/metabolism , HeLa Cells , Humans , Inflammation , Leukocytes/cytology , Ligands , Microscopy, Fluorescence , Molecular Sequence Data , Oligonucleotides/chemistry , Plasmids/metabolism , Protein Structure, Tertiary , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
9.
J Leukoc Biol ; 78(2): 453-61, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15647324

ABSTRACT

Alpha1-acid glycoprotein (AGP) is a major acute-phase protein present in human plasma as well as in polymorphonuclear leukocytes (PMN). In this report, we show that PMN synthesize a specific glycoform of AGP, which is stored in the specific and azurophilic granules. Activation of PMN results in the rapid release of soluble AGP. PMN AGP exhibits a substantially higher apparent molecular weight than plasma AGP (50-60 kD vs. 40-43 kD), owing to the presence of strongly fucosylated and sialylated polylactosamine units on its five N-linked glycans. PMN AGP is also released in vivo from activated PMN, as appeared from studies using well-characterized myocard slices of patients that had died within 2 weeks after an acute myocardial infarction. AGP was found deposited transiently on damaged cardiomyocytes in areas with infiltrating PMN only. It is interesting that this was inversely related to the deposition of activated complement C3. Strongly fucosylated and sialylated AGP glycoforms have the ability to bind to E-selectin and to inhibit complement activation. We suggest that AGP glycoforms in PMN provide an endogenous feedback-inhibitory response to excessive inflammation.


Subject(s)
Cytoplasmic Granules/metabolism , Myocardial Infarction/metabolism , Neutrophils/metabolism , Orosomucoid/biosynthesis , Protein Processing, Post-Translational , Complement Activation , Complement C3a/metabolism , E-Selectin/metabolism , Female , Fucose/biosynthesis , Glycosylation , Humans , Inflammation/metabolism , Male , Myocardial Infarction/pathology , Myocardium/metabolism , Myocardium/pathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Neutrophils/pathology , Protein Binding
10.
Transfusion ; 42(1): 66-75, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11896315

ABSTRACT

BACKGROUND: Although transfusion or return of salvaged shed blood has become popular in major orthopedic procedures, this blood-saving method is still controversial because shed blood may be contaminated with chemical and tissular debris, such as fat particles, which may increase the risk of fat embolism after bone surgery. STUDY DESIGN AND METHODS: In an effort to find an easy, reliable method for determination of both fat particle content and removal from shed blood, analyses of perioperative blood samples were performed with a cell counter (Technicon H3 [H3]) in orthopedic patients undergoing spinal fusion in which postoperative shed blood was collected and returned with a blood collection canister. A screen or surface filter was intercalated in the return line to eliminate microaggregates, fat particles, and/or WBCs. RESULTS: Fat particles in shed blood are clearly detected as a condensed, sigmoidal-shaped area at the right-hand side of the PMN zone in the channel in which the H3 measures particles according to their degree of lobularity. This signal can be reproduced by the addition of animal or vegetable fat to venous blood, but not by the addition of activated platelets or RBC membranes. Fat particles, together with WBCs and microaggregates, in shed blood were effectively removed by surface filters, whereas screen filters were not effective. CONCLUSION: The use of the TH3 seems to be an easy, reliable, and low-cost approach for monitoring fat particle content and removal from postoperative salvaged shed blood in orthopedic procedures.


Subject(s)
Blood Transfusion, Autologous/methods , Blood , Embolism, Fat/prevention & control , Fats , Orthopedic Procedures , Blood Cell Count/instrumentation , Elective Surgical Procedures , Filtration , Humans , Particle Size , Postoperative Period , Safety
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