The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection wa... more The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (...
Cardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis ... more Cardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis patients. This morbidity is due to the accelerated atherosclerosis observed in these patients. Atherosclerosis is a chronic inflammatory disease characterized by key players such as monocytes, macrophages or oxidized LDLs. Monocytes-macrophages are classified into subsets of polarized cells, with M1 and M2 macrophages considered respectively as pro- and anti-inflammatory.The monocyte subsets and phenotypes were analyzed by flow cytometry. These data was completed by the quantification of plasma M-CSF, IL-8, CRP, Mox-LDLs, Apo-B and Apo-AI, chloro-tyrosine and homocitrulline concentrations.The statistical differences and associations between two continuous variables were assessed using the Mann-Whitney U test and Spearman’s correlation coefficient, respectively.Hemodialyzed patients showed a significant increase in the concentrations of CRP, M-CSF and IL-8 (inflammation biomarkers) as wel...
Animal by-products are valuable protein sources in animal nutrition. Among them are blood product... more Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milkderived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method.
Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been n... more Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been neglected up to now. Macrophages are prone to polarization and subsets of polarized macrophages have been described in atheromas. LDLs can be oxidized not only chemically by copper (Ox-LDLs) but also enzymatically by myeloperoxidase (MpOx-LDLs) resulting in oxidized LDLs poor in lipid peroxides. The effects of physiologically relevant myeloperoxidase-oxidized LDLs on macrophage polarization or on polarized macrophages remain largely unknown. In this study, the effects of LDLs on macrophage polarization were investigated by monitoring the expression of M1 and M2 genes following stimulation with native LDLs, Ox-LDLs, or MpOx-LDLs in RAW 264.7 cells. Except forMRC1, which is induced only by Ox-LDLs, MpOx-LDLs induced an overexpression of most of the selected marker genes at the mRNA level. MpOx-LDLs also modulate marker gene expression in polarized macrophages favoring notably anti-inflammat...
The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with proces... more The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs.
Background: Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs... more Background: Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. Methods: We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. Results: mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. Conclusions: These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis.
Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necr... more Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia.
Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into a... more Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 pr...
The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human hi... more The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite.
ABSTRACT A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and o... more ABSTRACT A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and one phenanthroline (phen) ligands was synthesized and then tethered to (antisense) oligonucleotides (Ru–ASO) to target a destabilized GFP (dGFP). The specificity of the photoreaction of this Ru–ASO conjugate was studied in vitro by polyacrylamide gel electrophoresis (PAGE) experiments in denaturing conditions. Other nonspecific Ru–ASO conjugates were also prepared and evaluated with human keratinocytes that expressed dGFP. An illumination-dependent cytotoxicity was observed for most Ru–ASO conjugates that varied from 10 to almost 40 %, but only the specific Ru–ASO conjugate was able to significantly reduce GFP expression in illuminated cells.
Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 INK-4a and ab... more Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. Methodology/Principal Findings: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29). Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCd. Conclusions/Significance: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.
Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, lead... more Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, leads to temporal fluctuations in tissue oxygen tension exposing tumor and stromal cells to cycling hypoxia. Inflammation is another feature of tumor microenvironment and is considered as a new enabling characteristic of tumor progression. As cycling hypoxia is known to participate in tumor aggressiveness, the purpose of this study was to evaluate its role in tumor-promoting inflammation. Firstly, we assessed the impact of cycling hypoxia in vitro on endothelial inflammatory response induced by tumor necrosis factor α. Results showed that endothelial cells exposed to cycling hypoxia displayed an amplified proinflammatory phenotype, characterized by an increased expression of inflammatory cytokines, namely, interleukin (IL)-6 and IL-8; by an increased expression of adhesion molecules, in particular intercellular adhesion molecule-1 (ICAM-1); and consequently by an increase in THP-1 monocyte a...
Two-dimensional remains one of the main experimental approaches in proteome analysis. However, co... more Two-dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification, and PTM detection. We have optimized a third additional step of in-gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described.
Background: Blood fluidity is maintained by a delicate balance between coagulation and fibrinolys... more Background: Blood fluidity is maintained by a delicate balance between coagulation and fibrinolysis. The endothelial cell surface is a key player in this equilibrium and cell surface disruptions can upset the balance. We investigated the role of pericellular myeloperoxidase oxidized LDLs (Mox-LDLs) in this balance. Methods and Results: We designed a technical device that enabled us to monitor fibrinolysis in real-time at the surface of an endothelial cell line (EA.hy926), and showed that Mox-LDL decreased pericellular fibrinolysis. There were no changes in fibrinolysis when EA.hy926 endothelial cells were exposed to native LDL (24 hours) at doses of 10, 50, 100 and up to 1250 mg/ml. However, treatment of EA.hy926 endothelial cells with 10 and 50 mg/ml of Mox-LDL (physiological serum concentrations) increased the lysis time by 15 and 13%, respectively (p,0.001), although this effect was not present at higher concentrations of 100 mg/ml. This effect was not correlated with any changes in PAI-1 or t-PA or PA Receptor (PAR) expression. No effect was observed at the surface of smooth muscle cells used as controls. Conclusion: Our data link the current favorite hypothesis that modified LDL has a causal role in atheroma plaque formation with an old suggestion that fibrin may also play a causal role. Our data help complete the paradigm of atherosclerosis: Modified LDL locally enhances fibrin deposition (present work); fibrin deposits enhance endothelial permeability; this effect allows subendothelial accumulation of lipid and foam cells.
Journal of Biomaterials Science, Polymer Edition, 2012
Taylor & Francis. The accuracy of the Content should not be relied upon and should be independent... more Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of... more The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro-and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the threedimensional structure of MPO.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2010
The intensification of shrimp farming has been related to the increasing use of chemotherapeutics... more The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg −1) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were underexpressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.
Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we inve... more Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin.
The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection wa... more The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (...
Cardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis ... more Cardiovascular diseases represent a major issue in terms of morbidity and mortality for dialysis patients. This morbidity is due to the accelerated atherosclerosis observed in these patients. Atherosclerosis is a chronic inflammatory disease characterized by key players such as monocytes, macrophages or oxidized LDLs. Monocytes-macrophages are classified into subsets of polarized cells, with M1 and M2 macrophages considered respectively as pro- and anti-inflammatory.The monocyte subsets and phenotypes were analyzed by flow cytometry. These data was completed by the quantification of plasma M-CSF, IL-8, CRP, Mox-LDLs, Apo-B and Apo-AI, chloro-tyrosine and homocitrulline concentrations.The statistical differences and associations between two continuous variables were assessed using the Mann-Whitney U test and Spearman’s correlation coefficient, respectively.Hemodialyzed patients showed a significant increase in the concentrations of CRP, M-CSF and IL-8 (inflammation biomarkers) as wel...
Animal by-products are valuable protein sources in animal nutrition. Among them are blood product... more Animal by-products are valuable protein sources in animal nutrition. Among them are blood products and blood meal, which are used as high-quality material for their beneficial effects on growth and health. Within the framework of the feed ban relaxation, the development of complementary methods in order to refine the identification of processed animal proteins remains challenging. The aim of this study was to identify specific biomarkers that would allow the detection of bovine blood products and processed animal proteins using tandem mass spectrometry. Seventeen biomarkers were identified: nine peptides for bovine plasma powder; seven peptides for bovine haemoglobin powder, including six peptides for bovine blood meal; and one peptide for porcine blood. They were not detected in several commercial compound feed or feed materials, such as blood by-products of other animal origins, milkderived products and fish meal. These biomarkers could be used for developing a species-specific and blood-specific detection method.
Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been n... more Macrophages and oxidized LDLs play a key role in atherogenesis but their heterogeneity has been neglected up to now. Macrophages are prone to polarization and subsets of polarized macrophages have been described in atheromas. LDLs can be oxidized not only chemically by copper (Ox-LDLs) but also enzymatically by myeloperoxidase (MpOx-LDLs) resulting in oxidized LDLs poor in lipid peroxides. The effects of physiologically relevant myeloperoxidase-oxidized LDLs on macrophage polarization or on polarized macrophages remain largely unknown. In this study, the effects of LDLs on macrophage polarization were investigated by monitoring the expression of M1 and M2 genes following stimulation with native LDLs, Ox-LDLs, or MpOx-LDLs in RAW 264.7 cells. Except forMRC1, which is induced only by Ox-LDLs, MpOx-LDLs induced an overexpression of most of the selected marker genes at the mRNA level. MpOx-LDLs also modulate marker gene expression in polarized macrophages favoring notably anti-inflammat...
The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with proces... more The outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986, with processed animal proteins (PAPs) as the main vector of the disease, has led to their prohibition in feed. The progressive release of the feed ban required the development of new analytical methods to determine the exact origin of PAPs from meat and bone meal. We set up a promising MS-based method to determine the species and the source (legal or not) present in PAPs: a TCA-acetone protein extraction followed by a cleanup step, an in-solution tryptic digestion of 5 h (with a 1:20 protein/trypsin ratio), and mass spectrometry analyses, first without any a priori, with a Q-TOF, followed by a targeted triple-quadrupole analysis. Using this procedure, we were able to overcome some of the major limitations of the official methods to analyze PAPs, detecting and identifying prohibited animal products in feedstuffs by the monitoring of peptides specific for cows, pigs, and sheep in PAPs.
Background: Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs... more Background: Tumor associated macrophages (TAMs) are present in high density in solid tumors. TAMs share many characteristics with alternatively activated macrophages, also called M2. They have been shown to favor tumor development and a role in chemoresistance has also been suggested. Here, we investigated the effects of M2 in comparison to M1 macrophages on cancer cell sensitivity to etoposide. Methods: We set up a model of macrophage polarization, starting from THP-1 monocytes differentiated into macrophages using PMA (Phorbol 12-myristate 13-acetate). Once differentiated (M0 macrophages), they were incubated with IL-4 and IL-13 in order to obtain M2 polarized macrophages or with IFN-gamma and LPS for classical macrophage activation (M1). To mimic the communication between cancer cells and TAMs, M0, M1 or M2 macrophages and HepG2 or A549 cancer cells were co-cultured during respectively 16 (HepG2) or 24 (A549) hours, before etoposide exposure for 24 (HepG2) or 16 (A549) hours. After the incubation, the impact of etoposide on macrophage polarization was studied and cancer cell apoptosis was assessed by western-blot for cleaved caspase-3 and cleaved PARP-1 protein, caspase activity assay and FACS analysis of Annexin V and PI staining. Results: mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1-derived macrophages, which provide a new, easy and well-characterized model of polarized human macrophages. Etoposide-induced cancer cell apoptosis was markedly reduced in the presence of THP-1 M2 macrophages, while apoptosis was increased in cells co-cultured with M1 macrophages. On the other hand, etoposide did not influence M1 or M2 polarization. Conclusions: These results evidence for the first time a clear protective effect of M2 on the contrary to M1 macrophages on etoposide-induced cancer cell apoptosis.
Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necr... more Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL) both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia.
Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into a... more Exposure to environmental pollutants such as polychlorinated biphenyls (PCBs) is now taken into account to partly explain the worldwide decline of amphibians. PCBs induce deleterious effects on developing amphibians including deformities and delays in metamorphosis. However, the molecular mechanisms by which they express their toxicity during the development of tadpoles are still largely unknown. A proteomics analysis was performed on developing Xenopus laevis tadpoles exposed from 2 to 5 days postfertilization to either 0.1 or 1 ppm Aroclor 1254, a PCB mixture. Two-dimensional DIGE with a minimal labeling method coupled to nanoflow liquid chromatography-tandem mass spectrometry was used to detect and identify proteins differentially expressed under PCBs conditions. Results showed that 59 spots from the 0.1 ppm Aroclor 1254 condition and 57 spots from the 1 ppm Aroclor 1254 condition displayed a significant increase or decrease of abundance compared with the control. In total, 28 pr...
The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human hi... more The protozoan parasite Trypanosoma brucei is lysed by apolipoprotein L-I, a component of human high-density lipoprotein (HDL) particles that are also characterized by the presence of haptoglobin-related protein. We report that this process is mediated by a parasite glycoprotein receptor, which binds the haptoglobin-hemoglobin complex with high affinity for the uptake and incorporation of heme into intracellular hemoproteins. In mice, this receptor was required for optimal parasite growth and the resistance of parasites to the oxidative burst by host macrophages. In humans, the trypanosome receptor also recognized the complex between hemoglobin and haptoglobin-related protein, which explains its ability to capture trypanolytic HDLs. Thus, in humans the presence of haptoglobin-related protein has diverted the function of the trypanosome haptoglobin-hemoglobin receptor to elicit innate host immunity against the parasite.
ABSTRACT A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and o... more ABSTRACT A photoreactive ruthenium(II) complex that contains two tetraazaphenanthrene (TAP) and one phenanthroline (phen) ligands was synthesized and then tethered to (antisense) oligonucleotides (Ru–ASO) to target a destabilized GFP (dGFP). The specificity of the photoreaction of this Ru–ASO conjugate was studied in vitro by polyacrylamide gel electrophoresis (PAGE) experiments in denaturing conditions. Other nonspecific Ru–ASO conjugates were also prepared and evaluated with human keratinocytes that expressed dGFP. An illumination-dependent cytotoxicity was observed for most Ru–ASO conjugates that varied from 10 to almost 40 %, but only the specific Ru–ASO conjugate was able to significantly reduce GFP expression in illuminated cells.
Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 INK-4a and ab... more Background: Repeated exposures to UVB of human keratinocytes lacking functional p16 INK-4a and able to differentiate induce an alternative state of differentiation rather than stress-induced premature senescence. Methodology/Principal Findings: A 2D-DIGE proteomic profiling of this alternative state of differentiation was performed herein at various times after the exposures to UVB. Sixty-nine differentially abundant protein species were identified by mass spectrometry, many of which are involved in keratinocyte differentiation and survival. Among these protein species was TRIpartite Motif Protein 29 (TRIM29). Increased abundance of TRIM29 following UVB exposures was validated by Western blot using specific antibody and was also further analysed by immunochemistry and by RT-PCR. TRIM29 was found very abundant in keratinocytes and reconstructed epidermis. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCd. Conclusions/Significance: These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress.
Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, lead... more Abnormal architecture of the tumor blood network, as well as heterogeneous erythrocyte flow, leads to temporal fluctuations in tissue oxygen tension exposing tumor and stromal cells to cycling hypoxia. Inflammation is another feature of tumor microenvironment and is considered as a new enabling characteristic of tumor progression. As cycling hypoxia is known to participate in tumor aggressiveness, the purpose of this study was to evaluate its role in tumor-promoting inflammation. Firstly, we assessed the impact of cycling hypoxia in vitro on endothelial inflammatory response induced by tumor necrosis factor α. Results showed that endothelial cells exposed to cycling hypoxia displayed an amplified proinflammatory phenotype, characterized by an increased expression of inflammatory cytokines, namely, interleukin (IL)-6 and IL-8; by an increased expression of adhesion molecules, in particular intercellular adhesion molecule-1 (ICAM-1); and consequently by an increase in THP-1 monocyte a...
Two-dimensional remains one of the main experimental approaches in proteome analysis. However, co... more Two-dimensional remains one of the main experimental approaches in proteome analysis. However, comigration of protein leads to several limitations: lack of accuracy in protein identification, impaired comparative quantification, and PTM detection. We have optimized a third additional step of in-gel separation to alleviate comigration associated drawbacks. Spot resolution is strikingly improved following this simple and rapid method and the positive impact on protein and peptide identification from MS/MS data, on the analysis of relative changes in protein abundance, and on the detection of PTM is described.
Background: Blood fluidity is maintained by a delicate balance between coagulation and fibrinolys... more Background: Blood fluidity is maintained by a delicate balance between coagulation and fibrinolysis. The endothelial cell surface is a key player in this equilibrium and cell surface disruptions can upset the balance. We investigated the role of pericellular myeloperoxidase oxidized LDLs (Mox-LDLs) in this balance. Methods and Results: We designed a technical device that enabled us to monitor fibrinolysis in real-time at the surface of an endothelial cell line (EA.hy926), and showed that Mox-LDL decreased pericellular fibrinolysis. There were no changes in fibrinolysis when EA.hy926 endothelial cells were exposed to native LDL (24 hours) at doses of 10, 50, 100 and up to 1250 mg/ml. However, treatment of EA.hy926 endothelial cells with 10 and 50 mg/ml of Mox-LDL (physiological serum concentrations) increased the lysis time by 15 and 13%, respectively (p,0.001), although this effect was not present at higher concentrations of 100 mg/ml. This effect was not correlated with any changes in PAI-1 or t-PA or PA Receptor (PAR) expression. No effect was observed at the surface of smooth muscle cells used as controls. Conclusion: Our data link the current favorite hypothesis that modified LDL has a causal role in atheroma plaque formation with an old suggestion that fibrin may also play a causal role. Our data help complete the paradigm of atherosclerosis: Modified LDL locally enhances fibrin deposition (present work); fibrin deposits enhance endothelial permeability; this effect allows subendothelial accumulation of lipid and foam cells.
Journal of Biomaterials Science, Polymer Edition, 2012
Taylor & Francis. The accuracy of the Content should not be relied upon and should be independent... more Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of... more The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro-and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and the threedimensional structure of MPO.
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2010
The intensification of shrimp farming has been related to the increasing use of chemotherapeutics... more The intensification of shrimp farming has been related to the increasing use of chemotherapeutics and potentially suboptimal rearing conditions. For the purpose of assessing the stress level of cultured giant tiger shrimp Penaeus monodon, a proteomic analysis (2D-DIGE) was performed on hemolymph. On the one hand, shrimp were exposed for 7 days to the antibiotics enrofloxacin or furazolidone via feed (4 g kg −1) under laboratory conditions. On the other hand, shrimp were submitted to enrofloxacin directly in field conditions in Vietnam, for which two different culture systems were distinguished (intensive and improved extensive). No significant different protein abundance pattern was induced by antibiotics under laboratory conditions, while only one protein spot displayed a 1.53-fold reduction in intensity after exposure to enrofloxacin in improved extensive ponds. When we compared the proteome of shrimp bred either in intensive or in improved extensive system, we observed 9 protein spots displaying significant difference in abundance. Among them, 3 spots of hemocyanin were underexpressed in shrimp from improved extensive ponds. At the opposite 2 spots corresponding to Sarcoplasmic Calcium-binding Protein (SCP) were less abundant in hemolymph of shrimp from intensive ponds. These results demonstrate that the very subtle effects of tested antibiotics on patterns of hemolymph protein expression are overwhelmed by the effects of conditions encountered in different production management systems, such as different oxygen and nitric concentrations.
Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we inve... more Skin carcinomas are among the most commonly diagnosed tumors in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using the antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based copolymers. In order to improve the transfection efficiency of the classic PDMAEMA polymers, copolymers were synthesized including a poly(N-morpholino)ethylmethacrylate) (PMEMA) moiety for an improved proton-sponge effect, intended to favour the release of the oligonucleotide from the acidic endosome. These copolymers were synthesized either statistically (with alternating PDMAEMA and PMEMA fragments) or in blocks (one PDMAEMA block followed by one PMEMA block). MTT assays were performed using the PDMAEMA-PMEMA copolymers and revealed no significant cytotoxicity of these polymers at an N/P ratio of 7.3. Using fluorescent oligonucleotides and analyzing transfection efficiency by flow cytometry, we noticed no significant differences between the two kinds of copolymers. However copolymers with a higher DMAEMA content and a higher Mn were also those displaying the highest vectorization efficiency. Confocal microscopy showed that these copolymers induced a fine granular distribution of the transfected antisense oligonucleotides inside the cells. We also assessed the functionality of the transfected antisense oligonucleotide by transfecting immortalized GFP expressing keratinocytes with a GFP antisense oligonucleotide using these copolymers. A significant silencing was achieved with a PDMAEMA-PMEMA in block copolymer (Mn=41,000, 89 % PDMAEMA). Together, these results suggest that PDMAEMA-PMEMA copolymers combining low toxicity, vectorization and proton sponge properties, can be efficiently used to transfect immortalized keratinocytes and so open new perspectives in the therapy of skin carcinomas as well as of other skin diseases of genetic or immunological origin.
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Papers by Martine Raes