Papers by Cédric Broussard
Stem Cells, Apr 9, 2012
Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor in... more Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem-like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio-chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two-dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma-derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC-conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC-conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti-HDGF antibodies. In vivo, GSC-conditioned medium induced neoangiogenesis, whereas HDGF-targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high-grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties.
Revue Francaise D Allergologie, Nov 1, 2022
The European respiratory journal, Oct 17, 2011
Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis... more Anti-endothelial cell antibodies (AECAs) have been identified in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and in patients with idiopathic pulmonary arterial hypertension (iPAH). However, their target antigens remain poorly identified. Sera from 24 patients with SSc without PAH, 20 patients with SSc with PAH, 30 with iPAH and 12 healthy controls were collected. Target antigens were identified by two-dimensional electrophoresis and immunoblotting in protein extracts of human umbilical vein endothelial cells. Targeted antigens were identified by mass spectrometry. Serum immunoglobulin G from patients with SSc with or without PAH and patients with iPAH specifically recognised 110, 82 and 37 protein spots, respectively. Among others, target antigens of AECAs included lamin A/C, tubulin b-chain and vinculin. One-dimension immunoblotting experiments confirmed the identification of lamin A/C and tubulin b-chain. In conclusion, our results confirm the presence of AECA in patients with systemic sclerosis with and without pulmonary arterial hypertension and in those with idiopathic pulmonary arterial hypertension, and provide evidence for the identification of target antigens of these autoantibodies including lamin A/C and tubulin b-chain.
Annals of the Rheumatic Diseases, Jun 1, 2013
Background Anti-endothelial cell antibodies (AECA) are frequently detected in anti-neutrophil cyt... more Background Anti-endothelial cell antibodies (AECA) are frequently detected in anti-neutrophil cytoplasm antibodies (ANCA)-associated systemic vasculitis (AAV) and are considered to play pathological roles but their antigenic specificities are still unknown. Objectives We used a proteomic approach combining two-dimensional electrophoresis and immunoblotting to identify the target antigens of AECA in patients with ANCA-associated vasculitis. Methods Sera from 30 ANCA-associated vasculitis patients (12 with Granulomatosis with polyangeitis (GPA), 9 with microscopic polyangeitis (MPA), 9 with Churg Strauss syndrome (CSS)), tested in pools of 3 sera, were compared to a sera pool from 12 healthy controls (HC). Serum IgG reactivity was analyzed by use of a 2-D electrophoresis and immunoblotting technique with normal human umbilical vein endothelial cell (HUVEC) antigens. Results Serum IgG in the HC sera pool recognized 85 protein spots and serum IgG from patients with AAV recognized 134±65 different protein spots. We focused on protein recognized by at least 3/4 pools of patients with GPA and 2/3 pools of patients with MPA and CSS and not by HC and identified 20, 7 and 8 proteins, respectively. In addition, among the 330 spots recognized by at least one pool of patients with AAV, ten different spots were recognized by at least 6/10 pools. Among identified proteins, IgG reactivity was detected against alpha-enolase, lamin A/C and protein disulfide-isomerase A3. Interestingly, Ingenuity Pathway Analysis revealed that most of these antigens interact with TGF-β and signalling pathways such as Jun and MAPK. Conclusions AECA detected in patients with AAV recognize cellular targets playing key roles in cell biology. Target antigens interact with protein and complexes known to play a crucial role in AAV pathophysiology. Disclosure of Interest: None Declared
Revue de Médecine Interne, Jun 1, 2010
Presse Medicale, Apr 1, 2013
Introduction.-Calprotectin (MRP8/14) is an endogenous TLR4 agonist, expressed in neutrophils, mon... more Introduction.-Calprotectin (MRP8/14) is an endogenous TLR4 agonist, expressed in neutrophils, monocytes and infiltrating macrophages, promoting endothelial activation and transcription of proinflammatory cytokines. We have shown patients with active ANCA-associated vasculitis (AAV) have elevated cell surface and serum levels, and relapsers from the NORAM trial have higher early serum levels than non-relapsers. Calprotectin (+) macrophages are found in crescentic renal lesions but not sclerotic lesions, and calprotectin deficient mice (cal-/-) are protected in a nephrotoxic nephritis (NTN) animal model. We investigate the proinflammatory mechanisms of calprotectin on bone marrow derived macrophages (BMDMs), endothelial cells (EC) and mesangial cells (MC). Methods.-EC isolated from wild-type (WT) mice; BMDMs from WT, TLR4-/-and cal-/-mice; MC from WT and TLR4-/-mice-were stimulated with calprotectin. WT EC were co-cultured with WT BMDMs or cal-/-BMDMs. Cytokines measured in supernatants by ELISA. The phagocytosis ability of WT and Cal-/-BMDMs were compared using opsonised beads. Serum calprotectin levels were measured in WT mice during NTN. Results.-The calprotectin induced increase in IL-8, TNF-a, MCP-1 in BMDMs was abrogated in TLR4-/-BMDM (P < 0.001), but no differences seen in MC. Cal-/-vs WT BMDM stimulated with exogenous calprotectin demonstrate little pro-inflammatory activity and less TNFa, IL-6, IL-8 (P < 0.005). The increase in IL-6, IL-8 and MCP-1 following co-culture of EC and WT BMDM was absent with cal-/-BMDM. Cal-/-BMDMs demonstrate decreased phagocytosis (P < 0.005). WT mice have increased serum calprotectin (correlates with thrombosis). Conclusion.-Calprotectin has inflammatory effects mediated by TLR4 on BMDMs and a TLR4 independent effect on MC possibly through mesangial RAGE receptors, known to bind calprotectin. Cal-/-BMDMs lack a pro-inflammatory effect, suggesting a role for calprotectin in amplifying endothelial and glomerular damage in AAV, and may be a potential therapeutic target.
bioRxiv (Cold Spring Harbor Laboratory), Mar 27, 2018
In contrast to mammalian erythroid cells that lost their nucleus at the end of the differentiatio... more In contrast to mammalian erythroid cells that lost their nucleus at the end of the differentiation process, circulating chicken erythrocytes, like erythrocytes of most other nonmammalian vertebrates, are nucleated although their nucleus is believed to be transcriptionally silent. This major difference suggests that the erythroid differentiation process is likely to present both similarities and differences in mammals compared to other vertebrates. Since proteins are the major cellular effectors, analysis of the proteome is more prone to reflect true differences than analysis of the pattern of mRNA expression. We have previously reported the evolution of the proteome of human erythroid cells throughout their differentiation process. Here we report the analysis of the proteome of chicken erythroblasts during their terminal differentiation. We used the T2EC cellular model that allows to obtain homogenous populations of immature erythroblasts. Induction of their terminal
Key Engineering Materials, 2016
Mollusc shells are organic-inorganic composites that are often preserved in the fossil record. Ho... more Mollusc shells are organic-inorganic composites that are often preserved in the fossil record. However, the way the organic fraction, also called shell matrix, gets fossilized remains an unsolved question, in spite of several old and more recent studies. In the present paper, we have tried to mimic a diagenetic process by constantly heating for ten days at 100°C fresh nacre powder samples of the Polynesian pearl oyster Pinctadamargaritifera. Each day, aliquots of nacre powder were sampled and the matrix was subsequently extracted. It was further analysed by direct weigh quantification, by immunological techniques and by proteomics. Our preliminary data suggest that nacre proteins, when heated at 100°C in dry condition, degrade rather slowly. We evidenced a differential degradation pattern of the soluble and insoluble fractions, and showed that some nacre proteins of the insoluble fraction are stable after ten days of heating. Factors that influence the diagenetic stability of some shell proteins are discussed.
PLOS ONE, Nov 9, 2017
Pregnancy-associated malaria (PAM) is one of the severe forms of Plasmodium falciparum infection.... more Pregnancy-associated malaria (PAM) is one of the severe forms of Plasmodium falciparum infection. The main antigen VAR2CSA is the target of vaccine development. However, the large size of VAR2CSA protein and its high degree of variability limit to the efficiency of the vaccination. Using quantitative mass spectrometry method, we detected and quantified proteotypic peptides from 5 predicted PAM associated proteins. Our results confirmed that PFI1785w is over-expressed in PAM samples. Then, we investigated PFI1785w variability among a set of parasite samples from various endemic areas. PFI1785w appear to be more conserved than VAR2CSA. PFB0115w, another PAM associated protein, seems also associated with the pathology. Further vaccination strategies could integrate other proteins in addition to the major VAR2CSA antigen to improve immune response to vaccination.
Molecular Neurobiology, Oct 5, 2017
Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involve... more Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble Nterminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPαinduced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.
PLOS ONE, Jul 1, 2015
Serological proteome analysis (SERPA) combines classical proteomic technology with effective sepa... more Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest.
Autoimmunity Reviews, Apr 1, 2017
Clinical Immunology, Jul 1, 2014
Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antig... more Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens. Methods: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry. Results: Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2. Conclusions: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.
Annals of the Rheumatic Diseases, Nov 14, 2011
Pulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with ... more Pulmonary arterial hypertension (PAH) is characterised by remodelling of pulmonary arteries with enhanced vascular smooth muscle cell (VSMC) contraction, migration and proliferation. The authors investigated the presence of antibodies to human VSMCs in the serum of patients with systemic sclerosis with or without PAH and idiopathic PAH (iPAH). Antibodies to VSMCs were detected by immunofluorescence in sera from healthy controls and patients with scleroderma without PAH, scleroderma-associated PAH and iPAH. Serum IgG from these patients induced contraction of VSMCs in a collagen matrix in contrast with IgG from healthy controls. Several protein spots of interest and target antigens were identified by two-dimensional immunoblotting and MS, including stress-induced phosphoprotein 1 and α-enolase. Finally, antibodies to stress-induced phosphoprotein 1 were detected by ELISA in sera from 84%, 76% and 24% of patients with scleroderma without PAH, scleroderma-associated PAH and iPAH, respectively, compared with only 3% of healthy controls. The authors have identified IgG that binds to VSMCs in the serum of patients with scleroderma and iPAH. These antibodies may be pathogenic by modulating vascular contraction. The target antigens of these antibodies are stress-induced phosphoprotein 1 and α-enolase.
Proteomics, Mar 1, 2008
Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antig... more Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens. Methods: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry. Results: Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2. Conclusions: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.
Chemistry – A European Journal
Cell Death & Differentiation
Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E... more Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E/pro-erythroblast differentiation, depending on the balance between erythropoietin (EPO) levels and activation of the Fas/FasL pathway. At this stage, activation of transient caspases through depolarization via mitochondrial outer membrane permeabilization (MOMP) is also required for terminal erythroid differentiation. Molecular mechanisms regulating the differential levels of MOMP during differentiation and apoptosis, however, remain poorly understood. Here we show a novel and essential role for the caspase-10-P13-tBID axis in erythroid terminal differentiation. Caspase-10 (but not caspase-8, which is activated during apoptosis) is activated at the early stages of erythroid terminal differentiation leading to the cleavage of P22-BID into P18-tBID, and later into P13-tBID. Erythropoietin (EPO) by inducing casein kinase I alpha (CKIα) expression, which in turn phosphorylates P18-tBID, pre...
HAL (Le Centre pour la Communication Scientifique Directe), Nov 1, 2021
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Papers by Cédric Broussard