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...
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.
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.
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...
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...
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.
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.
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...
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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