Isabelle Cloutier - Academia.edu (original) (raw)

Papers by Isabelle Cloutier

Research paper thumbnail of Abstract 4933: A 17B-estradiol Conjugated Chitosan-phosphorylcholine Polymer Supports Survival and Amplification of Endothelial Progenitor Cells (EPCs)

Research paper thumbnail of Selective Binding of Bacterial Toxins to Major Histocompatibility Complex Class II-Expressing Cells is Controlled by Invariant Chain and HLA-DM

Proceedings of the National Academy of Sciences of the United States of America, Jun 24, 1997

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules ... more Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a Vbeta -restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the micro-heterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Research paper thumbnail of The processing routes determined by negatively charged residues in DR1-restricted T cell determinants

The Journal of Immunology

The presentation pathways followed by DR1-restricted determinants from the fusion protein of meas... more The presentation pathways followed by DR1-restricted determinants from the fusion protein of measles virus were studied. By assessing the capacity of various APC preparations to stimulate fusion protein-specific T cells, it was shown that the determinant contained within the fusion protein sequence 254-268 (F254) relies on newly synthesized DR1 protein for its presentation. By contrast, the determinant contained within the fusion protein sequence 314-328 (F314) is captured by DR1 protein recycled from the plasma membrane. In vitro binding analyses showed that the F254 and F314 peptides optimally bind to DR1 at pH 4 and pH 5, respectively. In addition, it was found that binding of the F254 peptide to DR1 is much poorer at pH 7 than at pH 4, while binding of the F314 peptide was decreased only moderately at pH 7 as compared with pH 5. Substitution of the glutamic acid 261 for an alanine in the F254 peptide resulted in an analogue with an improved capacity of binding to DR1 at neutral ...

Research paper thumbnail of Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific... more Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosin...

Research paper thumbnail of Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum

The EMBO journal, Jan 15, 1996

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules f... more Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50...

Research paper thumbnail of Vascular Smooth Muscle Cell Alterations Triggered by Mice Adipocytes: Role of High-Fat Diet

Journal of Atherosclerosis and Thrombosis, 2012

Aim: Inherent mechanisms leading to vascular smooth muscle cells (VSMC) alterations in obesitylin... more Aim: Inherent mechanisms leading to vascular smooth muscle cells (VSMC) alterations in obesitylinked type 2 diabetes (T2D) situation remain to be clarified. This study evaluates the impact of supernatant of adipocytes extracted from mice fed high-fat-diets (HFD) on the proliferation and apoptosis of VSMC. Methods: Adipocytes were extracted from visceral white fat pads of male and female C57Bl6 mice showing different stages of metabolic alterations after 20 weeks of vegetal or animal HFD feeding. These cells were stimulated or not with insulin or glucose to condition VSMC media. After 24h of stimulation with adipocyte supernatants (AdS), VSMC proliferation and sustainability were assessed in the absence and presence of AdS. CD36 and insulin receptor mRNA levels were also evaluated.

Research paper thumbnail of High-Fat Diets-Induced Metabolic Alterations Alter the Differentiation Potential of Adipose Tissue-Derived Stem Cells

Open Journal of Endocrine and Metabolic Diseases, 2013

Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adi... more Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adipocytes or endothelial cells to help in the adipogenesis, vasculogenesis and vascular repair. This study aims at determining the impact of highfat diets (HFD)-induced type 2 diabetes (T2D) on the differentiation potential of ASC. Results: C57BL/6J male mice were fed a vegetal (VD) or an animal (AD) HFD. Isolation of ACS from mice showing different levels of metabolic alterations reveals that advanced T2D did not affect the number of cells per gram of tissue. Rather, a higher proportion of inflammatory CD36 + cells was identified in HFD fed mice. Despite a marked decreased expression of adipogenic genes (aP2, C/EBPα and PPARγ2), ASC from HFD groups had a higher adipogenic potential and a lower endothelial differentiation potential in vitro compared to control. ASC from the VD group had enhanced cyclin B1 expression and had more adipogenic potential compared to AD group. Conclusion: Our results demonstrate that the metabolic modifications, linked to the nature of fatty acids in diets, modulate the differentiation potential of ASC with increased adipogenesis to the detriment of the endothelial pathway. Results highlight the importance of evaluating the ASC differentiation behavior in a context of autologous cell-based therapy for the repair of vascular tissues in diabetic patients.

Research paper thumbnail of Subsets of HLA-DR1 molecules defined by SEB and TSST-1 binding

Science, 1994

Superantigens bind to major histocompatibility complex class II molecules on antigen-presenting c... more Superantigens bind to major histocompatibility complex class II molecules on antigen-presenting cells and stimulate T cells. Staphylococcus aureus enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) bind to the same region of human lymphocyte antigen (HLA)-DR1 but do not compete with each other, which indicates that they bind to different subsets of DR1 molecules. Here, a mutation in the peptide-binding groove disrupted the SEB and TSST-1 binding sites, which suggests that peptides can influence the interaction with bacterial toxins. In support of this, the expression of the DR1 molecule in various cell types differentially affected the binding of these toxins.

Research paper thumbnail of Selective binding of bacterial toxins to major histocompatibility complex class II-expressing cells is controlled by invariant chain and HLA-DM

Proceedings of the National Academy of Sciences, 1997

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules ... more Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a V␤-restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the microheterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Research paper thumbnail of Key Fatty Acid Combinations Define Vascular Smooth Muscle Cell Proliferation and Viability

Lipids, 2012

High plasma concentrations of free fatty acids (FFA) and insulin are common features in atheroscl... more High plasma concentrations of free fatty acids (FFA) and insulin are common features in atherosclerotic patients with type 2 diabetes. FFA, according to their nature, can have various effects on vascular smooth muscle cells (VSMC). These cells play important roles throughout atherosclerosis pathogenesis, from plaque development to plaque instability. Thus, this study aims to assess the impact of two FFA combinations and insulin on murine VSMC viability. The two combinations contain the same FFA but at different ratios, one being richer in saturated fatty acids (SFA) and the other having a higher proportion of monounsaturated fatty acids (MUFA). Both combinations inhibited VSMC proliferation due to their pro-apoptotic potential, with SFA being the major inducers of apoptosis. However, the presence of oleic acid (OLA) attenuated this impact in a dose-dependent manner. OLA had also the capacity to reduce apoptosis rates more strongly when combined with a SFA than when used alone in VSMC treatments. This effect was significant only for specific proportions of these FFA and was even more effective in presence of insulin. These results highlight the presence of a competition between pro-apoptotic and anti-apoptotic mechanisms in VSMC that is dependent on FFA ratios (saturated vs. monounsaturated) and on insulinemia. They also underline the importance of the presence of MUFA such as OLA in diets containing high proportions of SFA.

Research paper thumbnail of Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage φKZ gp144 lytic transglycosylase

FEMS Microbiology Letters, 2007

The gp144 endolysin gene from the Pseudomonas aeruginosa phage fKZ was cloned and studies of gp14... more The gp144 endolysin gene from the Pseudomonas aeruginosa phage fKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN 3 ), an inhibitor of the protein export machinery, and into an E. coli MM52 secA ts mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in a-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from fKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.

Research paper thumbnail of Vα domain modulates the multiple topologies of mouse T cell receptor Vβ20/staphylococcal enterotoxins A and E complexes

European Journal of Immunology, 1997

ABSTRACT The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major hi... more ABSTRACT The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the alpha and beta chains. We have investigated the role of the T cell receptor (TCR) alpha chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse V beta20 T cell lines expressing different V alpha domains and two T cell hybridomas expressing mouse V beta1 or V beta11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position alpha39K and beta81H) to which the superantigens can still bind but with an altered conformation. Although V beta20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR V alpha domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the V alpha domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given V beta20 T cell line.

Research paper thumbnail of 072 The impact of fatty acid combinations and insulin on vascular smooth muscle cell functionality

Canadian Journal of Cardiology, 2011

Research paper thumbnail of Tuning the Properties and Functions of 17β-Estradiol-polysaccharide Conjugates in Thin Films: Impact of Sample History

Biomacromolecules, 2012

In addition to its role in the regulation of sexrelated processes, 17β-estradiol (E2) participate... more In addition to its role in the regulation of sexrelated processes, 17β-estradiol (E2) participates in the prevention and treatment of cardiovascular diseases via nongenomic pathways mediated by estrogen receptors (ERα) located in the cell membrane. To achieve specific nongenomic activity of E2, we linked E2 (4.4 mol %) to chitosan-phosphorylcholine (CH-PC) (20 mol % PC). Injections of ER-α solutions (5 to 100 nmol L −1 ) over rehydrated CH-PC-E2 thin films led to permanent adsorption of ER-α to the film surface, as detected by quartz crystal microbalance with dissipation (QCM-D). However, ER-α did not bind onto CH-PC-E2 films formed in situ and never dried. Xray photoelectron spectroscopy (XPS) analysis of spin-cast CH-PC-E2 films revealed significant E2 enrichment of the topmost section of the film, attributed to the preferential migration of E2 toward the film/air interface upon drying. Mechanical analysis of CH-PC-E2 films in the frequency domain probed by QCM-D indicated that rehydrated films behave as an entangled network with junction points formed by self-assembly of hydrophobic E2 moieties and by ion pairing among PC groups, whereas films formed in situ are entangled polymer solutions with temporary junctions. The structural analysis presented offers useful guidelines for the study of amphiphilic biomacromolecules designed for therapeutic use as thin films.

Research paper thumbnail of A phosphorylcholine-modified chitosan polymer as an endothelial progenitor cell supporting matrix

Biomaterials, 2011

The aim of the present study was to develop a new biopolymer to increase endothelial progenitor c... more The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosanephosphorylcholine (CHePC). On CHePC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CHePC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH ePC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CHePC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CHePC) increased the percentage of progenitor cells (CD117 þ CD34 þ ) (7.1 AE 0.8%, FN: 4.1 AE 0.8%) and EPC (CD117 þ CD34 þ VEGFR-2 þ CD31 þ ) (2.33 AE 0.6%, FN: 0.25 AE 0.1%), while the mesenchymal stem cell fraction (CD44 þ CD106 þ CD90 þ ) was decreased (0.07 AE 0.01%, FN: 0.55 AE 0.22%). Polymeric substrate CHePC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.

Research paper thumbnail of Stent implantation in coronary porcine arteries is associated with early activation of TNFα and TNFα receptor II expression

Atherosclerosis, 2007

Inflammation present in restenosis after angioplasty is associated with production of cytokines s... more Inflammation present in restenosis after angioplasty is associated with production of cytokines such as tumor necrosis factor (TNFα). However, limited data exist on the possible increase in TNFα and TNFα receptor expression induced during the chronic phase after stenting. To this end, swine underwent balloon denudation (PTCA) and stent implantation in coronary arteries. At day 1, 7 or 28 post-procedure,

Research paper thumbnail of Estrogen-Induced Gene Expression in Bone Marrow c-kit+ Stem Cells and Stromal Cells: Identification of Specific Biological Processes Involved in the Functional Organization of the Stem Cell Niche

Stem Cells and Development, 2008

The recent interest in the role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) a... more The recent interest in the role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) and the benefits of estrogen on cardiovascular health brought us to evaluate if estrogen could affect cardiac repair more broadly by regulating biological processes involved in the functional organization of the BM stem cell (SC) niche. To assess such possibility, we evaluated gene expression profiles of BM c-kit+ SCs and CD44+ stromal cells (StroCs) after exposure to a physiological concentration of 17beta-estradiol (17betaE). Data analysis showed that 17betaE altered the expression (>1.5 fold) of 509 and 682 gene probes in c-kit+ SCs and CD44+ StroCs, respectively. Among them, 199 genes in c-kit+ SCs and 283 in CD44+ StroCs were associated to biological process categories of the Gene Ontology classification. Within processes highly regulated by 17betaE, we identified key factors involved in adhesion, migration, proteolysis, and signaling by which 17betaE influences physiological regulation of the functional organization of the SC niche. Together, our results demonstrate that estrogen benefits on cardiovascular health could involve other BM-derived cells than EPCs and that this capacity of estrogen to influence the physiology of the BM SC niche deserves to be investigated clinically.

Research paper thumbnail of Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: An ERα-mediated pathway

Cardiovascular Research, 2006

Objective: Interferon gamma (IFN-γ) was shown to induce CD40 and CD40L expression on endothelial ... more Objective: Interferon gamma (IFN-γ) was shown to induce CD40 and CD40L expression on endothelial cells (ECs) and consequently to promote neutrophil adhesion. The pro-and anti-inflammatory effects of estrogens are well recognized but their role on the regulation of CD40 and CD40L expression on ECs remains undefined. Methods and results: Treatment of porcine aortic endothelial cells (PAEC) with IFN-γ for 24 h enhanced CD40 and CD40L expression by 97% and 78%, respectively. Pretreatment of PAEC with 17-beta-estradiol (17βE) for 24 h prevented the latter expression of CD40/CD40L. Treatment of PAEC with antisense oligomers targeting ERα mRNA attenuated the ability of 17βE to inhibit the IFN-γ-induced CD40 and CD40L protein expression. The IFN-γ activation pathway of CD40 is known to involve the phosphorylation of the Janus activated kinase (JAK) and the signal transducer and activator of transcription 1 (Stat1). 17βE, acting via the estrogen receptor α (ERα), abrogated IFN-γmediated effects on Stat1 but failed to inhibit Jak1 and Jak2 phosphorylation. Furthermore, 17βE prevented neutrophil adhesion induced by IFN-γ. Conlusion: In summary, 17βE binding to ERα blocked IFN-γ-induced Stat1 phosphorylation, CD40 and CD40L protein expression, and neutrophil adhesion onto ECs.

Research paper thumbnail of Menstrual cycle influences endothelial progenitor cell regulation: A link to gender differences in vascular protection?

International Journal of Cardiology, 2009

BackgroundThe potential role of endothelial progenitor cells (EPCs) in the beneficial effects of ... more BackgroundThe potential role of endothelial progenitor cells (EPCs) in the beneficial effects of estrogen on women's cardiovascular health is of great interest. We thus evaluated if menstrual cycle influences circulating levels of EPC subpopulations in normally menstruating women and if this could underline gender differences.

Research paper thumbnail of Biophysical studies of the interactions between the phage varphiKZ gp144 lytic transglycosylase and model membranes

European biophysics journal : EBJ, 2010

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a... more The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was ob...

Research paper thumbnail of Abstract 4933: A 17B-estradiol Conjugated Chitosan-phosphorylcholine Polymer Supports Survival and Amplification of Endothelial Progenitor Cells (EPCs)

Research paper thumbnail of Selective Binding of Bacterial Toxins to Major Histocompatibility Complex Class II-Expressing Cells is Controlled by Invariant Chain and HLA-DM

Proceedings of the National Academy of Sciences of the United States of America, Jun 24, 1997

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules ... more Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a Vbeta -restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the micro-heterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Research paper thumbnail of The processing routes determined by negatively charged residues in DR1-restricted T cell determinants

The Journal of Immunology

The presentation pathways followed by DR1-restricted determinants from the fusion protein of meas... more The presentation pathways followed by DR1-restricted determinants from the fusion protein of measles virus were studied. By assessing the capacity of various APC preparations to stimulate fusion protein-specific T cells, it was shown that the determinant contained within the fusion protein sequence 254-268 (F254) relies on newly synthesized DR1 protein for its presentation. By contrast, the determinant contained within the fusion protein sequence 314-328 (F314) is captured by DR1 protein recycled from the plasma membrane. In vitro binding analyses showed that the F254 and F314 peptides optimally bind to DR1 at pH 4 and pH 5, respectively. In addition, it was found that binding of the F254 peptide to DR1 is much poorer at pH 7 than at pH 4, while binding of the F314 peptide was decreased only moderately at pH 7 as compared with pH 5. Substitution of the glutamic acid 261 for an alanine in the F254 peptide resulted in an analogue with an improved capacity of binding to DR1 at neutral ...

Research paper thumbnail of Molecular characterization and role in T cell activation of staphylococcal enterotoxin A binding to the HLA-DR alpha-chain

Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1997

Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific... more Superantigens bind to MHC class II-positive cells and stimulate T lymphocytes expressing specific V beta regions of the TCR. Two distinct regions of staphylococcal enterotoxin A superantigen (SEA) have been shown to affect the binding to MHC class II molecules. Results presented here demonstrate for the first time that the SEA-DR interaction can be affected by mutations on the class II alpha-chain. Furthermore, we have precisely mapped the interaction of the SEA N-terminal domain with the alpha1 domain of HLA-DR. Scatchard analysis using DAP cells transfected with mutant class II molecules showed a role for residue DR alpha K39 in the binding of SEA. Also, complementation experiments using mutant SEA molecules revealed an interaction between SEA residue F47 and position alphaQ18 on an outer loop of HLA-DR. These interactions between SEAF47 and the DR alpha-chain are critical, as they allow the recognition by an otherwise nonreactive V beta1+ T cell hybridoma and induction of tyrosin...

Research paper thumbnail of Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum

The EMBO journal, Jan 15, 1996

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules f... more Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50...

Research paper thumbnail of Vascular Smooth Muscle Cell Alterations Triggered by Mice Adipocytes: Role of High-Fat Diet

Journal of Atherosclerosis and Thrombosis, 2012

Aim: Inherent mechanisms leading to vascular smooth muscle cells (VSMC) alterations in obesitylin... more Aim: Inherent mechanisms leading to vascular smooth muscle cells (VSMC) alterations in obesitylinked type 2 diabetes (T2D) situation remain to be clarified. This study evaluates the impact of supernatant of adipocytes extracted from mice fed high-fat-diets (HFD) on the proliferation and apoptosis of VSMC. Methods: Adipocytes were extracted from visceral white fat pads of male and female C57Bl6 mice showing different stages of metabolic alterations after 20 weeks of vegetal or animal HFD feeding. These cells were stimulated or not with insulin or glucose to condition VSMC media. After 24h of stimulation with adipocyte supernatants (AdS), VSMC proliferation and sustainability were assessed in the absence and presence of AdS. CD36 and insulin receptor mRNA levels were also evaluated.

Research paper thumbnail of High-Fat Diets-Induced Metabolic Alterations Alter the Differentiation Potential of Adipose Tissue-Derived Stem Cells

Open Journal of Endocrine and Metabolic Diseases, 2013

Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adi... more Background: Adipose tissue-derived stem cells (ASC) possess the ability to differentiate into adipocytes or endothelial cells to help in the adipogenesis, vasculogenesis and vascular repair. This study aims at determining the impact of highfat diets (HFD)-induced type 2 diabetes (T2D) on the differentiation potential of ASC. Results: C57BL/6J male mice were fed a vegetal (VD) or an animal (AD) HFD. Isolation of ACS from mice showing different levels of metabolic alterations reveals that advanced T2D did not affect the number of cells per gram of tissue. Rather, a higher proportion of inflammatory CD36 + cells was identified in HFD fed mice. Despite a marked decreased expression of adipogenic genes (aP2, C/EBPα and PPARγ2), ASC from HFD groups had a higher adipogenic potential and a lower endothelial differentiation potential in vitro compared to control. ASC from the VD group had enhanced cyclin B1 expression and had more adipogenic potential compared to AD group. Conclusion: Our results demonstrate that the metabolic modifications, linked to the nature of fatty acids in diets, modulate the differentiation potential of ASC with increased adipogenesis to the detriment of the endothelial pathway. Results highlight the importance of evaluating the ASC differentiation behavior in a context of autologous cell-based therapy for the repair of vascular tissues in diabetic patients.

Research paper thumbnail of Subsets of HLA-DR1 molecules defined by SEB and TSST-1 binding

Science, 1994

Superantigens bind to major histocompatibility complex class II molecules on antigen-presenting c... more Superantigens bind to major histocompatibility complex class II molecules on antigen-presenting cells and stimulate T cells. Staphylococcus aureus enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) bind to the same region of human lymphocyte antigen (HLA)-DR1 but do not compete with each other, which indicates that they bind to different subsets of DR1 molecules. Here, a mutation in the peptide-binding groove disrupted the SEB and TSST-1 binding sites, which suggests that peptides can influence the interaction with bacterial toxins. In support of this, the expression of the DR1 molecule in various cell types differentially affected the binding of these toxins.

Research paper thumbnail of Selective binding of bacterial toxins to major histocompatibility complex class II-expressing cells is controlled by invariant chain and HLA-DM

Proceedings of the National Academy of Sciences, 1997

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules ... more Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a V␤-restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate that Staphylococcus aureus have retained the ability to express numerous SAgs in adaptation to the microheterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.

Research paper thumbnail of Key Fatty Acid Combinations Define Vascular Smooth Muscle Cell Proliferation and Viability

Lipids, 2012

High plasma concentrations of free fatty acids (FFA) and insulin are common features in atheroscl... more High plasma concentrations of free fatty acids (FFA) and insulin are common features in atherosclerotic patients with type 2 diabetes. FFA, according to their nature, can have various effects on vascular smooth muscle cells (VSMC). These cells play important roles throughout atherosclerosis pathogenesis, from plaque development to plaque instability. Thus, this study aims to assess the impact of two FFA combinations and insulin on murine VSMC viability. The two combinations contain the same FFA but at different ratios, one being richer in saturated fatty acids (SFA) and the other having a higher proportion of monounsaturated fatty acids (MUFA). Both combinations inhibited VSMC proliferation due to their pro-apoptotic potential, with SFA being the major inducers of apoptosis. However, the presence of oleic acid (OLA) attenuated this impact in a dose-dependent manner. OLA had also the capacity to reduce apoptosis rates more strongly when combined with a SFA than when used alone in VSMC treatments. This effect was significant only for specific proportions of these FFA and was even more effective in presence of insulin. These results highlight the presence of a competition between pro-apoptotic and anti-apoptotic mechanisms in VSMC that is dependent on FFA ratios (saturated vs. monounsaturated) and on insulinemia. They also underline the importance of the presence of MUFA such as OLA in diets containing high proportions of SFA.

Research paper thumbnail of Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage φKZ gp144 lytic transglycosylase

FEMS Microbiology Letters, 2007

The gp144 endolysin gene from the Pseudomonas aeruginosa phage fKZ was cloned and studies of gp14... more The gp144 endolysin gene from the Pseudomonas aeruginosa phage fKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN 3 ), an inhibitor of the protein export machinery, and into an E. coli MM52 secA ts mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in a-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from fKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.

Research paper thumbnail of Vα domain modulates the multiple topologies of mouse T cell receptor Vβ20/staphylococcal enterotoxins A and E complexes

European Journal of Immunology, 1997

ABSTRACT The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major hi... more ABSTRACT The superantigens staphylococcal enterotoxin A and E (SEA and SEE) both contact major histocompatibility complex (MHC) class II molecules on two sites located on the alpha and beta chains. We have investigated the role of the T cell receptor (TCR) alpha chain in the modulation of the various topologies of TCR/SEA (or SEE)/class II complexes. For this purpose, we have used three mouse V beta20 T cell lines expressing different V alpha domains and two T cell hybridomas expressing mouse V beta1 or V beta11 segments. The response of these T cells to SEA and SEE was studied in the context of presentation by wild-type human MHC class II molecules; or by mutants on MHC, in each of the two superantigen binding sites (position alpha39K and beta81H) to which the superantigens can still bind but with an altered conformation. Although V beta20 T cell lines are efficiently stimulated using SEA and SEE presented by wild-type HLA-DR1 molecules, our results show that the nature of the TCR V alpha domain can affect differently the recognition of the toxins bound to mutant class II molecules. This suggests that various functional topologies exist for both SEA and SEE/class II complexes and that the T cell response to each of these complexes can be modulated by the V alpha domain of the TCR. Interestingly, the recognition of SEA and SEE is achieved in different fashions by a given V beta20 T cell line.

Research paper thumbnail of 072 The impact of fatty acid combinations and insulin on vascular smooth muscle cell functionality

Canadian Journal of Cardiology, 2011

Research paper thumbnail of Tuning the Properties and Functions of 17β-Estradiol-polysaccharide Conjugates in Thin Films: Impact of Sample History

Biomacromolecules, 2012

In addition to its role in the regulation of sexrelated processes, 17β-estradiol (E2) participate... more In addition to its role in the regulation of sexrelated processes, 17β-estradiol (E2) participates in the prevention and treatment of cardiovascular diseases via nongenomic pathways mediated by estrogen receptors (ERα) located in the cell membrane. To achieve specific nongenomic activity of E2, we linked E2 (4.4 mol %) to chitosan-phosphorylcholine (CH-PC) (20 mol % PC). Injections of ER-α solutions (5 to 100 nmol L −1 ) over rehydrated CH-PC-E2 thin films led to permanent adsorption of ER-α to the film surface, as detected by quartz crystal microbalance with dissipation (QCM-D). However, ER-α did not bind onto CH-PC-E2 films formed in situ and never dried. Xray photoelectron spectroscopy (XPS) analysis of spin-cast CH-PC-E2 films revealed significant E2 enrichment of the topmost section of the film, attributed to the preferential migration of E2 toward the film/air interface upon drying. Mechanical analysis of CH-PC-E2 films in the frequency domain probed by QCM-D indicated that rehydrated films behave as an entangled network with junction points formed by self-assembly of hydrophobic E2 moieties and by ion pairing among PC groups, whereas films formed in situ are entangled polymer solutions with temporary junctions. The structural analysis presented offers useful guidelines for the study of amphiphilic biomacromolecules designed for therapeutic use as thin films.

Research paper thumbnail of A phosphorylcholine-modified chitosan polymer as an endothelial progenitor cell supporting matrix

Biomaterials, 2011

The aim of the present study was to develop a new biopolymer to increase endothelial progenitor c... more The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosanephosphorylcholine (CHePC). On CHePC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CHePC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH ePC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CHePC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CHePC) increased the percentage of progenitor cells (CD117 þ CD34 þ ) (7.1 AE 0.8%, FN: 4.1 AE 0.8%) and EPC (CD117 þ CD34 þ VEGFR-2 þ CD31 þ ) (2.33 AE 0.6%, FN: 0.25 AE 0.1%), while the mesenchymal stem cell fraction (CD44 þ CD106 þ CD90 þ ) was decreased (0.07 AE 0.01%, FN: 0.55 AE 0.22%). Polymeric substrate CHePC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.

Research paper thumbnail of Stent implantation in coronary porcine arteries is associated with early activation of TNFα and TNFα receptor II expression

Atherosclerosis, 2007

Inflammation present in restenosis after angioplasty is associated with production of cytokines s... more Inflammation present in restenosis after angioplasty is associated with production of cytokines such as tumor necrosis factor (TNFα). However, limited data exist on the possible increase in TNFα and TNFα receptor expression induced during the chronic phase after stenting. To this end, swine underwent balloon denudation (PTCA) and stent implantation in coronary arteries. At day 1, 7 or 28 post-procedure,

Research paper thumbnail of Estrogen-Induced Gene Expression in Bone Marrow c-kit+ Stem Cells and Stromal Cells: Identification of Specific Biological Processes Involved in the Functional Organization of the Stem Cell Niche

Stem Cells and Development, 2008

The recent interest in the role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) a... more The recent interest in the role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) and the benefits of estrogen on cardiovascular health brought us to evaluate if estrogen could affect cardiac repair more broadly by regulating biological processes involved in the functional organization of the BM stem cell (SC) niche. To assess such possibility, we evaluated gene expression profiles of BM c-kit+ SCs and CD44+ stromal cells (StroCs) after exposure to a physiological concentration of 17beta-estradiol (17betaE). Data analysis showed that 17betaE altered the expression (>1.5 fold) of 509 and 682 gene probes in c-kit+ SCs and CD44+ StroCs, respectively. Among them, 199 genes in c-kit+ SCs and 283 in CD44+ StroCs were associated to biological process categories of the Gene Ontology classification. Within processes highly regulated by 17betaE, we identified key factors involved in adhesion, migration, proteolysis, and signaling by which 17betaE influences physiological regulation of the functional organization of the SC niche. Together, our results demonstrate that estrogen benefits on cardiovascular health could involve other BM-derived cells than EPCs and that this capacity of estrogen to influence the physiology of the BM SC niche deserves to be investigated clinically.

Research paper thumbnail of Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: An ERα-mediated pathway

Cardiovascular Research, 2006

Objective: Interferon gamma (IFN-γ) was shown to induce CD40 and CD40L expression on endothelial ... more Objective: Interferon gamma (IFN-γ) was shown to induce CD40 and CD40L expression on endothelial cells (ECs) and consequently to promote neutrophil adhesion. The pro-and anti-inflammatory effects of estrogens are well recognized but their role on the regulation of CD40 and CD40L expression on ECs remains undefined. Methods and results: Treatment of porcine aortic endothelial cells (PAEC) with IFN-γ for 24 h enhanced CD40 and CD40L expression by 97% and 78%, respectively. Pretreatment of PAEC with 17-beta-estradiol (17βE) for 24 h prevented the latter expression of CD40/CD40L. Treatment of PAEC with antisense oligomers targeting ERα mRNA attenuated the ability of 17βE to inhibit the IFN-γ-induced CD40 and CD40L protein expression. The IFN-γ activation pathway of CD40 is known to involve the phosphorylation of the Janus activated kinase (JAK) and the signal transducer and activator of transcription 1 (Stat1). 17βE, acting via the estrogen receptor α (ERα), abrogated IFN-γmediated effects on Stat1 but failed to inhibit Jak1 and Jak2 phosphorylation. Furthermore, 17βE prevented neutrophil adhesion induced by IFN-γ. Conlusion: In summary, 17βE binding to ERα blocked IFN-γ-induced Stat1 phosphorylation, CD40 and CD40L protein expression, and neutrophil adhesion onto ECs.

Research paper thumbnail of Menstrual cycle influences endothelial progenitor cell regulation: A link to gender differences in vascular protection?

International Journal of Cardiology, 2009

BackgroundThe potential role of endothelial progenitor cells (EPCs) in the beneficial effects of ... more BackgroundThe potential role of endothelial progenitor cells (EPCs) in the beneficial effects of estrogen on women's cardiovascular health is of great interest. We thus evaluated if menstrual cycle influences circulating levels of EPC subpopulations in normally menstruating women and if this could underline gender differences.

Research paper thumbnail of Biophysical studies of the interactions between the phage varphiKZ gp144 lytic transglycosylase and model membranes

European biophysics journal : EBJ, 2010

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a... more The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was ob...