Étude du rôle des domaines structuraux et du motif de ciblage YXXO dans le transport intracellulaire et de l'activité fusogénique de la gp41 du VIH-1

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Tissue-specificity of antibodies raised against TrkB and p75NTR receptors ; implications for platelets as models of neurodegenerative diseases

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF

Platelets selectively regulate the release of BDNF, but not that of its precursor protein, proBDNF

Background: Brain-derived neurotrophic factor (BDNF) plays a role in synaptic plasticity and neuroprotection. BDNF has well-established pro-survival effects, whereas its precursor protein, proBDNF, induces apoptosis. Thus, it has been suggested that the proBDNF/BDNF ratio could be an indicator of neuronal health. Access to neurons is, understandably, limited. Because of their similarities, platelets have been put forward as a non-invasive biomarker of neuronal health; indeed, they store large quantities of BDNF and can release it into circulation upon activation, similarly to neurons. However, whether platelets also express the precursor proBDNF protein remains unknown. We therefore sought to characterize proBDNF levels in human platelets and plasma. Methods: The presence of proBDNF was assessed by immunoblotting, cell fractionation, flow cytometry, and confocal microscopy in washed platelets from 10 healthy volunteers. Platelets from 20 independent healthy volunteers were activated with several classical agonists and the release of BDNF and proBDNF into plasma was quantified by ELISA. Results: Platelets expressed detectable levels of proBDNF (21 ± 13 fmol/250 x 106 platelets). ProBDNF expression was mainly localized in the intracellular compartment. The proBDNF to BDNF molar ratio was ~1:5 in platelets and 10:1 in plasma. In stark contrast to the release of BDNF during platelet activation, intraplatelet and plasma concentrations of proBDNF remained stable following stimulation with classical platelet agonists, consistent with non-granular expression. Conclusions: Platelets express both the mature and the precursor form of BDNF. Whether the intraplatelet proBDNF to BDNF ratio could be used as a non-invasive biomarker of cognitive health warrants further investigation

Head‐to‐Head comparison of consensus‐recommended platelet function tests to assess P2Y12 Inhibition : insights for multi‐center trials

The vasodilator‐associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole‐blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR‐C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA‐ and flow cytometry‐ based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry‐based VASP assay (r = 0.79, p < 0.0001) than for the ELISA‐based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus‐recommended assays with their standardized cut‐offs should not be used interchangeably in multi‐center clinical studies but, rather, they should be standardized throughout sites

Clinical correlates identify ProBDNF and thrombo-inflammatory markers as key predictors of circulating p75NTR extracellular domain levels in older adults

The p75NTR receptor binds all neurotrophins and is mostly known for its role in neuronal survival and apoptosis. Recently, the extracellular domain (ECD) of p75NTR has been reported in plasma, its levels being dysregulated in numerous neurological diseases. However, the factors associated with p75NTR ECD levels remain unknown. We investigated clinical correlates of plasma p75NTR ECD levels in older adults without clinically manifested neurological disorders. Circulating p75NTR levels were measured by enzyme-linked immunosorbent assay in plasma obtained from participants in the BEL-AGE cohort (n = 1,280). Determinants of plasma p75NTR ECD levels were explored using linear and non-linear statistical models. Plasma p75NTR ECD levels were higher in male participants; were positively correlated with circulating concentrations of pro-brain-derived neurotrophic factor, and inflammatory markers interleukin-6 and CD40 Ligand; and were negatively correlated with the platelet activation marker P-selectin. While most individuals had p75NTR levels ranging from 43 to 358 pg/ml, high p75NTR levels reaching up to 9,000 pg/ml were detectable in a subgroup representing 15% of the individuals studied. In this cohort of older adults without clinically manifested neurological disorders, there was no association between plasma p75NTR ECD levels and cognitive performance, as assessed by the Montreal Cognitive Assessment score. The physiological relevance of high p75NTR ECD levels in plasma warrants further investigation. Further research assessing the source of circulating p75NTR is needed for a deeper understanding of the direction of effect, and to investigate whether high p75NTR ECD levels are predictive biomarkers or consequences of neuropathology

Perturbation of adhesion molecule-mediated chondrocyte-matrix interactions by 4-hydroxynonenal binding: implication in osteoarthritis pathogenesis

ABSTRACT: INTRODUCTION: Objectives were to investigate whether interactions between human osteoarthritic chondrocytes and 4-hydroxynonenal (HNE)-modified type II collagen (Col II) affect cell phenotype and functions and to determine the protective role of carnosine (CAR) treatment in preventing these effects. METHODS: Human Col II was treated with HNE at different molar ratios (MR) (1:20 to 1:200; Col II:HNE). Articular chondrocytes were seeded in HNE/Col II adduct-coated plates and incubated for 48 hours. Cell morphology was studied by phase-contrast and confocal microscopy. Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and alpha1beta1 integrin at protein and mRNA levels were quantified by Western blotting, flow cytometry and real-time reverse transcription-polymerase chain reaction. Cell death, caspases activity, prostaglandin E2 (PGE2), metalloproteinase-13 (MMP-13), mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) were assessed by commercial kits. Col II, cyclooxygenase-2 (COX-2), MAPK, NF-kappaB-p65 levels were analyzed by Western blotting. The formation of alpha1beta1 integrin-focal adhesion kinase (FAK) complex was revealed by immunoprecipitation. RESULTS: Col II modification by HNE at MR approximately 1:20, strongly induced ICAM-1, alpha1beta1 integrin and MMP-13 expression as well as extracellular signal-regulated kinases 1 and 2 (ERK1/2) and NF-kappaB-p65 phosphorylation without impacting cell adhesion and viability or Col II expression. However, Col II modification with HNE at MR approximately 1:200, altered chondrocyte adhesion by evoking cell death and caspase-3 activity. It inhibited alpha1beta1 integrin and Col II expression as well as ERK1/2 and NF-kappaB-p65 phosphorylation, but, in contrast, markedly elicited PGE2 release, COX-2 expression and p38 MAPK phosphorylation. Immunoprecipitation assay revealed the involvement of FAK in cell-matrix interactions through the formation of alpha1beta1 integrin-FAK complex. Moreover, the modification of Col II by HNE at a 1:20 or approximately 1:200 MR affects parameters of the cell shape. All these effects were prevented by CAR, an HNE-trapping drug. CONCLUSIONS: Our novel findings indicate that HNE-binding to Col II results in multiple abnormalities of chondrocyte phenotype and function, suggesting its contribution in osteoarthritis development. CAR was shown to be an efficient HNE-snaring agent capable of counteracting these outcomes

Outcome of Occupational Asthma after Removal from Exposure: A Follow-Up Study

BACKGROUND: Despite being removed from their workplace, the majority of workers with occupational asthma (OA) remain afflicted with asthma

Outcome of occupational asthma after removal from exposure: A follow-up study

BACKGROUND: Despite being removed from their workplace, the majority of workers with occupational asthma (OA) remain afflicted with asthma

Protective role against hydrogen peroxide and fibroblast stimulation via Ce-doped TiO2 nanostructured materials

Background: Cerium oxide (CeO2) and Ce-doped nanostructured materials (NMs) are being seen as innovative therapeutic tools due to their exceptional antioxidant effects; nevertheless their bio-applications are still in their infancy. Methods: TiO2, Ce–TiO2 and CeO2–TiO2 NMs were synthesized by a bottom-up microemulsion-mediated strategy and calcined during 7 h at 650 °C under air flux. The samples were compared to elucidate the physicochemical characteristics that determine cellular uptake, toxicity and the influence of redox balance between the Ce3 +/Ce4 + on the cytoprotective role against an exogenous ROS source: H2O2. Fibroblasts were selected as a cell model because of their participation in wound healing and fibrotic diseases. Results: Ce–TiO2 NM obtained via sol–gel reaction chemistry of metallic organic precursors exerts a real cytoprotective effect against H2O2 over fibroblast proliferation, while CeO2 pre-formed nanoparticles incorporated to TiO2 crystalline matrix lead to a harmful CeO2–TiO2 material. TiO2 was processed by the same pathways as Ce–TiO2 and CeO2–TiO2 NM but did not elicit any adverse or protective influence compared to controls. Conclusions: It was found that the Ce atoms source and its concentration have a clear effect on material's physicochemical properties and its subsequent influence in the cellular response. It can induce a range of biological reactions that vary from cytotoxic to cytoprotective. General significance: Even though there are still some unresolved issues and challenges, the unique physical and chemical properties of Ce-based NMs are fascinating and versatile resources for different biomedical applications.Fil: Gravina, Noel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; ArgentinaFil: Maghni, Karim. Hôpital du Sacré-Coeur-de-Montréal; CanadáFil: Welman, Mélanie. Hôpital du Sacré-Coeur-de-Montréal; CanadáFil: Yahia, L'Hocine. École Polytechnique de Montréal; CanadáFil: Mbeh, Doris Antoinette. École Polytechnique de Montréal; CanadáFil: Messina, Paula Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentin

The brain-derived neurotrophic factor prompts platelet aggregation and secretion

Brain-derived neurotrophic factor (BDNF) has both autocrine and paracrine roles in neurons, and its release and signaling mechanisms have been extensively studied in the central nervous system. Large quantities of BDNF have been reported in circulation, essentially stored in platelets with concentrations reaching 100- to 1000-fold those of neurons. Despite this abundance, the function of BDNF in platelet biology has not been explored. At low concentrations, BDNF primed platelets, acting synergistically with classical agonists. At high concentrations, BDNF induced complete biphasic platelet aggregation that in part relied on amplification from secondary mediators. Neurotrophin-4, but not nerve growth factor, and an activating antibody against the canonical BDNF receptor tropomyosin-related kinase B (TrkB) induced similar platelet responses to BDNF, suggesting TrkB could be the mediator. Platelets expressed, both at their surface and in their intracellular compartment, a truncated form of TrkB lacking its tyrosine kinase domain. BDNFinduced platelet aggregation was prevented by inhibitors of Ras-related C3 botulinum toxin substrate 1 (Rac1), protein kinase C, and phosphoinositide 3-kinase. BDNF-stimulated platelets secreted a panel of angiogenic and inflammatory cytokines, which may play a role in maintaining vascular homeostasis. Two families with autism spectrum disorder were found to carry rare missense variants in the BDNF gene. Platelet studies revealed defects in platelet aggregation to low concentrations of collagen, as well as reduced adenosine triphosphate secretion in response to adenosine diphosphate. In summary, circulating BDNF levels appear to regulate platelet activation, aggregation, and secretion through activation of a truncated TrkB receptor and downstream kinase-dependent signaling