Publications by authors named "Stéphane Robert"

50 Publications

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock.

Blood 2022 Jan 13. Epub 2022 Jan 13.

AP-HM, France.

Microvesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We therefore hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using a MV-PGC kinetic assay, ELISA and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that high PGC level displayed by Gran-MVs reduce thrombus formation and improve survival conferring to Gran-MVs a protective role in a murine model of sepsis.
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http://dx.doi.org/10.1182/blood.2021013328DOI Listing
January 2022

Experimental identification of a grating profile using neural network classifiers in optical scatterometry.

Appl Opt 2021 Sep;60(26):7929-7936

In this paper, we develop a new technique, to the best of our knowledge, of grating characterization based on two separate steps. First, an artificial neural network (ANN) is implemented in a classifier mode to identify the shape of the geometrical profile from a measured optical signature. Then, a second ANN is used in a regression mode to determine the geometrical parameters corresponding to the selected geometrical model. The advantage of this approach is highlighted by discussions and studies involving the error criterion that is used widely in scatterometry. In addition, experimental tests are provided on diffraction grating structures with a period of 500 and 750 nm.
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http://dx.doi.org/10.1364/AO.432987DOI Listing
September 2021

Systemic Administration of G-CSF Accelerates Bone Regeneration and Modulates Mobilization of Progenitor Cells in a Rat Model of Distraction Osteogenesis.

Int J Mol Sci 2021 Mar 28;22(7). Epub 2021 Mar 28.

ISM, CNRS, Aix Marseille University, 13009 Marseille, France.

Granulocyte colony-stimulating factor (G-CSF) was shown to promote bone regeneration and mobilization of vascular and osteogenic progenitor cells. In this study, we investigated the effects of a systemic low dose of G-CSF on both bone consolidation and mobilization of hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs) and mesenchymal stromal cells (MSCs) in a rat model of distraction osteogenesis (DO). Neovascularization and mineralization were longitudinally monitored using positron emission tomography and planar scintigraphy. Histological analysis was performed and the number of circulating HSPCs, EPCs and MSCs was studied by flow cytometry. Contrary to control group, in the early phase of consolidation, a bony bridge with lower osteoclast activity and a trend of an increase in osteoblast activity were observed in the distracted callus in the G-CSF group, whereas, at the late phase of consolidation, a significantly lower neovascularization was observed. While no difference was observed in the number of circulating EPCs between control and G-CSF groups, the number of MSCs was significantly lower at the end of the latency phase and that of HSPCs was significantly higher 4 days after the bone lengthening. Our results indicate that G-CSF accelerates bone regeneration and modulates mobilization of progenitor cells during DO.
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http://dx.doi.org/10.3390/ijms22073505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037338PMC
March 2021

CeO Nanomaterials from Diesel Engine Exhaust Induce DNA Damage and Oxidative Stress in Human and Rat Sperm In Vitro.

Nanomaterials (Basel) 2020 Nov 24;10(12). Epub 2020 Nov 24.

IMBE, CNRS, IRD, Avignon Université, Aix Marseille University, 13005 Marseille, France.

Cerium dioxide nanomaterials (CeO NMs) are widely used in nano-based diesel additives to decrease the emission of toxic compounds, but they have been shown to increase the emission of ultrafine particles as well as the amount of released Ce. The Organization for Economic Cooperation and Development included CeO NMs in the priority list of nanomaterials that require urgent evaluation, and the potential hazard of aged CeO NM exposure remains unexplored. Herein, human and rat sperm cells were exposed in vitro to a CeO NM-based diesel additive (called Envirox), burned at 850 °C to mimic its release after combustion in a diesel engine. We demonstrated significant DNA damage after in vitro exposure to the lowest tested concentration (1 µg·L) using the alkaline comet assay (ACA). We also showed a significant increase in oxidative stress in human sperm after in vitro exposure to 1 µg·L aged CeO NMs evaluated by the HDCF-DA probe. Electron microscopy showed no internalization of aged CeO NMs in human sperm but an affinity for the head plasma membrane. The results obtained in this study provide some insight on the complex cellular mechanisms by which aged CeO NMs could exert in vitro biological effects on human spermatozoa and generate ROS.
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http://dx.doi.org/10.3390/nano10122327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760532PMC
November 2020

Evaluation of PIG-A-mutated granulocytes and ex-vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans.

Environ Mol Mutagen 2021 01 20;62(1):18-28. Epub 2020 Nov 20.

Aix Marseille University, Avignon Université, CNRS, IRD, IMBE, Marseille, France.

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.
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http://dx.doi.org/10.1002/em.22413DOI Listing
January 2021

Replicated anthropogenic hybridisations reveal parallel patterns of admixture in marine mussels.

Evol Appl 2020 Mar 22;13(3):575-599. Epub 2019 Nov 22.

ISEM Univ Montpellier CNRS EPHE IRD Montpellier France.

Human-mediated transport creates secondary contacts between genetically differentiated lineages, bringing new opportunities for gene exchange. When similar introductions occur in different places, they provide informally replicated experiments for studying hybridisation. We here examined 4,279 mussels, sampled in Europe and genotyped with 77 ancestry-informative markers. We identified a type of introduced mussels, called "dock mussels," associated with port habitats and displaying a particular genetic signal of admixture between and the Mediterranean lineage of . These mussels exhibit similarities in their ancestry compositions, regardless of the local native genetic backgrounds and the distance separating colonised ports. We observed fine-scale genetic shifts at the port entrance, at scales below natural dispersal distance. Such sharp clines do not fit with migration-selection tension zone models, and instead suggest habitat choice and early-stage adaptation to the port environment, possibly coupled with connectivity barriers. Variations in the spread and admixture patterns of dock mussels seem to be influenced by the local native genetic backgrounds encountered. We next examined departures from the average admixture rate at different loci, and compared human-mediated admixture events, to naturally admixed populations and experimental crosses. When the same background was involved, positive correlations in the departures of loci across locations were found; but when different backgrounds were involved, no or negative correlations were observed. While some observed positive correlations might be best explained by a shared history and saltatory colonisation, others are likely produced by parallel selective events. Altogether, genome-wide effect of admixture seems repeatable and more dependent on genetic background than environmental context. Our results pave the way towards further genomic analyses of admixture, and monitoring of the spread of dock mussels both at large and at fine spacial scales.
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http://dx.doi.org/10.1111/eva.12879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045717PMC
March 2020

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

miR-9 Does Not Regulate Lamin A Expression in Metastatic Cells from Lung Adenocarcinoma.

Int J Mol Sci 2020 Feb 26;21(5). Epub 2020 Feb 26.

Aix Marseille Univ, APHM, INSERM, MMG, Hôpital la Timone, Service de Biologie Cellulaire, 13005 Marseille, France.

In lung adenocarcinoma, low lamin A expression in pleural metastatic cells has been proposed as a pejorative factor. miR-9 physiologically inhibits the expression of lamin A in neural cells and seems to be a central actor in the carcinogenesis and the metastatic process in lung cancer. Thus, it could be a good candidate to explain the reduction of lamin A expression in lung adenocarcinoma cells. miR-9 expression was analyzed in 16 pleural effusions containing metastatic cells from lung adenocarcinoma and was significantly reduced in patients from the 'Low lamin A expression' group compared to patients from the 'High lamin A expression' group. Then, carcinoma cells selection by fluorescence-activated cell sorting (FACS) was performed according to epithelial membrane antigen (EMA) expression, reflecting lamin A expression. miR-9 was underexpressed in lamin A- carcinoma cells compared to lamin A+ carcinoma cells in patients from the 'Low lamin A expression' group, whereas there was no difference of miR-9 expression between lamin A+ and lamin A- carcinoma cells in patients from the 'High lamin A expression' group. These results suggest that miR-9 does not regulate lamin A expression in metastatic cells from lung adenocarcinoma. On the contrary, miR-9 expression was shown to be reduced in lamin A-negative carcinoma cells.
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http://dx.doi.org/10.3390/ijms21051599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7084260PMC
February 2020

Deciphering the Crosstalk Between Myeloid-Derived Suppressor Cells and Regulatory T Cells in Pancreatic Ductal Adenocarcinoma.

Front Immunol 2019 22;10:3070. Epub 2020 Jan 22.

Aix Marseille Univ, INSERM, CRO2, Centre de Recherche en Oncologie biologique et Oncopharmacologie, Marseille, France.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with rising incidence and a remarkable resistance to current therapies. The reasons for this therapeutic failure include the tumor's extensive infiltration by immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). By using light sheet fluorescent microscopy, we identified here direct interactions between these major immunoregulatory cells in PDAC. The depletion of MDSCs led to a significant reduction in Tregs in the pancreatic tumors. Through videomicroscopy and functional assays we have shown that (i) MDSCs are able to induce Treg cells in a cell-cell dependent manner; (ii) Treg cells affect the survival and/or the proliferation of MDSCs. Furthermore, we have observed contacts between MDSCs and Treg cells at different stages of human cancer. Overall our findings suggest that interactions between MDSCs and Treg cells contribute to PDAC immunosuppressive environment.
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http://dx.doi.org/10.3389/fimmu.2019.03070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6987391PMC
November 2020

A squalamine derivative, NV669, as a novel PTP1B inhibitor: and effects on pancreatic and hepatic tumor growth.

Oncotarget 2019 Nov 19;10(62):6651-6667. Epub 2019 Nov 19.

Aix Marseille Univ, INSERM, CRO2, Centre de Recherche en Oncologie biologique et Oncopharmacologie, Faculté de médecine, Marseille, France.

NV669 is an aminosterol derived from squalamine found to possess strong anticancer effects. The aim of this study was to investigate NV669's beneficial effects on human pancreatic and hepatic cancer models and to decipher the cellular and molecular mechanisms involved in tumor growth decrease upon treatment with NV669. Pancreatic (BxPC3, MiaPaCa-2) and hepatic (HepG2, Huh7) cancer cells were treated with NV669, and the effects recorded on proliferation, cell cycle and death. Results showed that NV669 inhibited the viability of cancer cells, induced cell cycle arrest and subsequently promoted apoptosis. This was accompanied by a decrease in the expression of cyclin B1 and phosphorylated Cdk1 and by a cleavage of pro-apoptotic caspase-8 and PARP-1. Taken together, our studies showed that NV669 inhibits the proliferation of pancreatic and hepatic cancer cells through the regulation of G2/M phase transition the cyclin B1-Cdk1 complex. NV669 inhibits PTP1B activity and FAK expression. NV669 impacts on the expression of adhesion molecules CDH-1, -2 and -3 in BxPC3 and Huh7 lines that form cell monolayers. Consecutively NV669 induces cell detachment. This suggests that NV669 by inhibiting PTP1B induces cell detachment and apoptosis. Subsequently, our results showed that NV669 inhibited the growth of pancreatic and hepatic tumor xenografts with a significant cell cycle arrest in pre-mitotic phase and an increase of tumor cell apoptosis. Therefore, NV669 may serve as an alternative anticancer agent, used alone or in association with other medications, for the treatment of pancreatic adenocarcinoma and hepatocellular carcinoma.
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http://dx.doi.org/10.18632/oncotarget.27286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877102PMC
November 2019

The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis.

Cancer Res 2020 01 14;80(2):291-303. Epub 2019 Nov 14.

Aix Marseille Univ, INSERM, INRA, Marseille, France.

Platelets promote metastasis, however, their role in tumor growth remains controversial. Here, we investigated the effect of platelet interactions with colorectal tumor cells. Platelets extravasated into the tumor microenvironment and interacted with tumor cells in a cadherin-6-dependent manner. The interaction induced platelet spreading, release of their granule content, and the generation of three types of microparticles (iMP) that expressed platelet markers, tumor markers, or both. The presence of iMPs was confirmed in colorectal cancer tissue specimens. Platelets significantly reduced tumor growth and increased intratumoral macrophages. This was mediated by iMP recruitment of macrophages via the chemoattractants RANTES, MIF, CCL2, and CXCL12 and activation of their tumor cell killing capacity through IFNγ and IL4, which led to cell-cycle arrest of tumor cells in a p21-dependent manner. In contrast, in the bloodstream, iMPs activated endothelial cells and platelets and induced epithelial-to-mesenchymal transition of tumor cells, promoting metastasis. Altogether, these results indicate that depending on the environment, local or bloodstream, the consequences of the interactions between platelets and a tumor may promote or prevent cancer progression. SIGNIFICANCE: Tumor cell interaction with platelets produces chimeric extracellular vesicles that suppress primary tumor growth by activating tumor-eliminating macrophages, while promoting metastasis through EMT and endothelial activation.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1181DOI Listing
January 2020

Reconstruction of a complex profile shape by weighting basic characterization results for nanometrology.

Appl Opt 2019 Aug;58(22):6118-6125

Scatterometry has become an essential method of characterization during the fabrication process of nanostructures. This nondestructive technique based on diffracted light analysis is composed of two steps: the measurement of the optical signatures and the treatment to reconstruct the profile of the periodic structure. The artificial neural network has proved its effectiveness in solving the inverse scattering problem. Usually the scatterometry characterization process requires a previous geometrical profile shape to be defined. This study proposes a method for the profile reconstruction of any geometrical shape, not necessarily one that is included in the initial model. For example, this could include the detection or the identification of an incorrect profile shape in a lithography or etching process. This so-called weighting profile approach consists in combining several results of basic characterizations for the reconstruction of the real profile shape. In this study, the feasibility of this method is demonstrated on theoretical samples to satisfy the requirements of scatterometry. Finally, the weighting profile method is compared with a classical scatterometry method involving a generic profile shape.
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http://dx.doi.org/10.1364/AO.58.006118DOI Listing
August 2019

Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation.

Sci Rep 2019 07 16;9(1):10299. Epub 2019 Jul 16.

Aix Marseille Univ, INSERM, C2VN, Marseille, France.

Human immunodeficiency virus type 1 (HIV-1) infection promotes a generalized activation of host responses that involves not only CD4 T cells, but also cells of the microenvironment, which are not directly infected, such as endothelial cells. The mechanisms triggering HIV-1-associated vascular alterations remain poorly understood. Extracellular vesicles (EVs), implicated in cell-to-cell communication, have been recently described as carriers of microRNAs (miRNAs). Here, we show that miR-146b-5p is upregulated in both CD4 T cells, CD4 T cell-derived EVs and circulating EVs obtained from antiretroviral therapy-naive HIV-1-infected patients. We further demonstrate that EVs from T cell line overexpressing miR-146b-5p mimics (miR-146b-EVs): 1) protect their miRNA cargo from RNase degradation, 2) transfer miR-146b-5p mimics into endothelial cells and 3) reduce endothelial inflammatory responses in vitro and in vivo in the lungs of mice through the downregulation of nuclear factor-κB-responsive molecules. These data advance our understanding on chronic inflammatory responses affecting endothelial homeostasis, in infectious and non-infectious diseases and pave the way for potential new anti-inflammatory strategies.
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http://dx.doi.org/10.1038/s41598-019-44743-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635508PMC
July 2019

Neutrophil extracellular traps are associated with the pathogenesis of diffuse alveolar hemorrhage in murine lupus.

J Autoimmun 2019 06 28;100:120-130. Epub 2019 Mar 28.

Aix-Marseille Univ, INSERM, INRA, C2VN, Marseille, France; Department of Internal Medicine and Clinical Immunology CHU Conception, Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Diffuse alveolar hemorrhage (DAH) is a life-threatening complication of systemic lupus erythematosus (SLE) and systemic vasculitis. Although initially described to have antibacterial properties, increasing evidence suggests that neutrophil extracellular traps (NETs) have a detrimental role in both autoimmune diseases and acute lung injury. We investigated whether NETs could be detected in a murine model of pristane-induced lupus DAH and contribute to lung injury. Such NETs might constitute a therapeutic target. NETs were characterized by immunofluorescence staining of DNA, neutrophil elastase and citrullinated histones. Evaluation of lung injury was performed by haematoxylin-eosin staining and a quantification program. Clinical status of the mice was assessed by measurement of arterial oxygen saturation and survival curves after recombinant human deoxyribonuclease-1 (Rh-DNase-1) inhalations or polymorphonuclear neutrophil (PMN) depletion. Pristane was found to promote NETs formation in vitro and in vivo. Treatment of mice with Rh-DNase-1 inhalations cleared NETs and reduced lung injury. Clinical status improved significantly, with increased arterial oxygenation and survival. Following PMN depletion, NETs were absent with a subsequent reduction of lung injury and improved arterial oxygenation. These results support a pathogenic role of PMNs and NETs in lung injury during pristane-induced DAH. Targeting NETs with Rh-DNase-1 inhalations could constitute an interesting adjuvant therapy in human DAH.
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http://dx.doi.org/10.1016/j.jaut.2019.03.009DOI Listing
June 2019

CD146 deficiency promotes plaque formation in a mouse model of atherosclerosis by enhancing RANTES secretion and leukocyte recruitment.

J Mol Cell Cardiol 2019 05 27;130:76-87. Epub 2019 Mar 27.

Aix-Marseille Univ., INSERM 1263, INRA 1260, C2VN, Marseille, France. Electronic address:

Aims: The progression of atherosclerosis is based on the continued recruitment of leukocytes in the vessel wall. The previously described role of CD146 in leukocyte infiltration suggests an involvement for this adhesion molecule in the inflammatory response. In this study, we investigated the role of CD146 in leukocyte recruitment by using an experimental model of atherogenesis.

Methods And Results: The role of CD146 was explored in atherosclerosis by crossing CD146-/- mice with ApoE-/- mice. CD146 -/-/ApoE -/- and ApoE -/- mice were fed a Western diet for 24 weeks and were monitored for aortic wall thickness using high frequency ultrasound. The arterial wall was significantly thicker in CD146-deficient mice. After 24 weeks of Western diet, a significant increase of atheroma in both total aortic lesion and aortic sinus of CD146-null mice was observed. In addition, atherosclerotic lesions were more inflammatory since plaques from CD146-deficient mice contained more neutrophils and macrophages. This was due to up-regulation of RANTES secretion by macrophages in CD146-deficient atherosclerotic arteries. This prompted us to further address the function of CD146 in leukocyte recruitment during acute inflammation by using a second experimental model of peritonitis induced by thioglycollate. Neutrophil recruitment was significantly increased in CD146-deficient mice 12 h after peritonitis induction and associated with higher RANTES levels in the peritoneal cavity. In CD146-null macrophages, we also showed that increased RANTES production was dependent on constitutive inhibition of the p38-MAPK signaling pathway. Finally, Maraviroc, a RANTES receptor antagonist, was able to reduce atherosclerotic lesions and neutrophilia in CD146-deficient mice to the same level as that found in ApoE -/- mice.

Conclusions: Our data indicate that CD146 deficiency is associated with the upregulation of RANTES production and increased inflammation of atheroma, which could influence the atherosclerotic plaque fate. Thus, these data identify CD146 agonists as potential new therapeutic candidates for atherosclerosis treatment.
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http://dx.doi.org/10.1016/j.yjmcc.2019.03.017DOI Listing
May 2019

Dendritic cell-based vaccination: powerful resources of immature dendritic cells against pancreatic adenocarcinoma.

Oncoimmunology 2018;7(12):e1504727. Epub 2018 Sep 25.

Aix Marseille Univ, INSERM, CRO2, Centre de Recherche en Oncologie biologique et Oncopharmacologie, Marseille, France.

Pancreatic adenocarcinoma (PAC) has a poor prognosis. One treatment approach, investigated here, is to reinforce antitumor immunity. Dendritic cells (DCs) are essential for the development and regulation of adaptive host immune responses against tumors. A major role for DCs may be as innate tumoricidal effector cells. We explored the efficacy of vaccination with immature (i)DCs, after selecting optimal conditions for generating immunostimulatory iDCs. We used two models, C57BL/6Jrj mice with ectopic tumors induced by the PAC cell line, Panc02, and genetically engineered (KIC) mice developing PAC. Therapeutic iDC-vaccination resulted in a significant reduction in tumor growth in C57BL/6Jrj mice and prolonged survival in KIC mice. Prophylactic iDC-vaccination prevented subcutaneous tumor development. These protective effects were long-lasting in Panc02-induced tumor development, but not in melanoma. iDC-vaccination impacted the immune status of the hosts by greatly increasing the percentage of CD8 T-cells, and natural killer (NK)1.1 cells, that express granzyme B associated with Lamp-1 and IFN-γ. Efficacy of iDC-vaccination was CD8 T-cell-dependent but NK1.1 cell-independent. We demonstrated the ability of DCs to produce peroxynitrites and to kill tumor cells; this killing activity involved peroxynitrites. Altogether, these findings make killer DCs the pivotal actors in the beneficial clinical outcome that accompanies antitumor immune responses. We asked whether efficacy can be improved by combining DC-vaccination with the FOLFIRINOX regimen. Combined treatment significantly increased the lifespan of KIC mice with PAC. Prolonged treatment with FOLFIRINOX clearly augmented this beneficial effect. Combining iDC-vaccination with FOLFIRINOX may therefore represent a promising therapeutic option for patients with PAC.
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http://dx.doi.org/10.1080/2162402X.2018.1504727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279335PMC
September 2018

Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release.

Front Immunol 2018 18;9:1665. Epub 2018 Jul 18.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France.

The pathophysiology of systemic sclerosis (SSc) involves early endothelial and immune activation, both preceding the onset of fibrosis. We previously identified soluble fractalkine and circulating endothelial microparticles (EMPs) as biomarkers of endothelial inflammatory activation in SSc. Fractalkine plays a dual role as a membrane-bound adhesion molecule expressed in inflamed endothelial cells (ECs) and as a chemokine involved in the recruitment, transmigration, and cytotoxic activation of immune cells that express CX3CR1, the receptor of fractalkine, namely CD8 and γδ T cells and natural killer (NK) cells. We aimed to quantify circulating cytotoxic immune cells and their expression of CX3CR1. We further investigated the expression profile of NK cells chemokine receptors and activation markers and the potential of NK cells to induce EC activation in SSc. We performed a monocentric study (NCT 02636127) enrolling 15 SSc patients [15 females, median age of 55 years (39-63), 11 limited cutaneous form and 4 diffuse] and 15 healthy controls. Serum fractalkine levels were significantly increased in SSc patients. Circulating CD8 T cells numbers were decreased in SSc patients with no difference in their CX3CR1 expression. Circulating γδ T cells and NK cells numbers were preserved. CX3CR1 expression in CD8 and γδ T cells did not differ between SSc patients and controls. The percentage and level of CX3CR1 expression in NK cells were significantly lowered in SSc patients. Percentages of CXCR4, NKG2D, CD69-expressing NK cells, and their expression levels were decreased in NK cells. Conversely, CD16 level expression and percentages of CD16 NK cells were preserved. The exposure of human microvascular dermic EC line (HMVEC-d) to peripheral blood mononuclear cells resulted in similar NK cells degranulation activity in SSc patients and controls. We further showed that NK cells purified from the blood of SSc patients induced enhanced release of EMPs than NK cells from controls. This study evidenced a peculiar NK cells phenotype in SSc characterized by decreased chemokine and activation receptors expression, that might reflect NK cells involvement in the pathogenic process. It also highlighted the role of NK cells as a potent mechanism inducing endothelial activation through enhanced EMPs release.
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http://dx.doi.org/10.3389/fimmu.2018.01665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058015PMC
July 2018

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance.

J Extracell Vesicles 2018 16;7(1):1494482. Epub 2018 Jul 16.

Aix-Marseille Université, C2VN, UMR-1263, INSERM, INRA 1260, UFR de Pharmacie, Marseille, France.

Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8-3.0] 3.1 [1.7-18]  × 10/min,  = 0.02; 1.4 [1-1.6] 5.2 [2.2-16]  × 10/min,  = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance.
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http://dx.doi.org/10.1080/20013078.2018.1494482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052415PMC
July 2018

A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule.

Oncotarget 2017 Dec 28;8(68):112283-112296. Epub 2017 Nov 28.

INSERM UMR-S 1076, Aix-Marseille University, UFR Pharmacy, Marseille, France.

CD146 is an adhesion molecule present on many tumors (melanoma, kidney, pancreas, breast, ...). In addition, it has been shown to be expressed on vascular endothelial and smooth muscle cells. Generating an antibody able to specifically recognize CD146 in cancer cells (designated as tumor CD146), but not in normal cells, would thus be of major interest for targeting tumor CD146 without affecting the vascular system. We thus generated antibodies against the extracellular domain of the molecule produced in cancer cells and selected an antibody that specifically recognizes tumor CD146. This antibody (TsCD146 mAb) was able to detect CD146-positive tumors in human biopsies and , by PET imaging, in a murine xenograft model. In addition, TsCD146 mAb antibody was able to specifically detect CD146-positive cancer microparticles in the plasma of patients. TsCD146 mAb displayed also therapeutic effects since it was able to reduce the growth of human CD146-positive cancer cells xenografted in nude mice. This effect was due to a decrease in the proliferation and an increase in the apoptosis of CD146-positive cancer cells after TsCD146-mediated internalization of the cell surface CD146. Thus, TsCD146 mAb could be of major interest for diagnostic and therapeutic strategies against CD146-positive tumors in a context of personalized medicine.
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http://dx.doi.org/10.18632/oncotarget.22736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762510PMC
December 2017

Biogenesis of Pro-senescent Microparticles by Endothelial Colony Forming Cells from Premature Neonates is driven by SIRT1-Dependent Epigenetic Regulation of MKK6.

Sci Rep 2017 08 15;7(1):8277. Epub 2017 Aug 15.

Aix Marseille Univ, INSERM, VRCM, Marseille, France.

Senescent cells may exert detrimental effect on microenvironment through the secretion of soluble factors and the release of extracellular vesicles, such as microparticles, key actors in ageing and cardiovascular diseases. We previously reported that sirtuin-1 (SIRT1) deficiency drives accelerated senescence and dysfunction of endothelial colony-forming cells (ECFC) in PT neonates. Because preterm birth (PT) increases the risk for cardiovascular diseases during neonatal period as well as at adulthood, we hypothesized that SIRT1 deficiency could control the biogenesis of microparticles as part of a senescence-associated secretory phenotype (SASP) of PT-ECFC and investigated the related molecular mechanisms. Compared to control ECFC, PT-ECFC displayed a SASP associated with increased release of endothelial microparticles (EMP), mediating a paracrine induction of senescence in naïve endothelial cells. SIRT1 level inversely correlated with EMP release and drives PT-ECFC vesiculation. Global transcriptomic analysis revealed changes in stress response pathways, specifically the MAPK pathway. We delineate a new epigenetic mechanism by which SIRT1 deficiency regulates MKK6/p38/Hsp27 pathway to promote EMP biogenesis in senescent ECFC. These findings deepen our understanding of the role of ECFC senescence in the disruption of endothelial homeostasis and provide potential new targets towards the control of cardiovascular risk in individuals born preterm.
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http://dx.doi.org/10.1038/s41598-017-08883-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557933PMC
August 2017

Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains.

Sci Rep 2017 07 17;7(1):5610. Epub 2017 Jul 17.

EA3064-GIMAP, Université de Lyon, 42023, Saint-Etienne, France.

Beyond their hemostatic functions, platelets alter their inflammatory response according to the bacterial stimulus. Staphylococcus aureus is associated with exacerbated inflammation and thrombocytopenia, which is associated with poor prognosis during sepsis. Acetylsalicylic acid and statins prevent platelet aggregation and decrease the mortality rate during sepsis. Therefore, we assessed whether these two molecules could reduce in vitro platelet activation and the inflammatory response to S. aureus. Platelets were exposed to clinical strains of S. aureus in the presence or absence of acetylsalicylic acid or fluvastatin. Platelet activation, aggregation, and release of soluble sCD62P, sCD40 Ligand, RANTES and GROα were assessed. Platelet cell death was evaluated by analyzing the mitochondrial membrane potential, phosphatidylserine exposure, platelet microparticle release and caspase-3 activation. All S. aureus strains induced platelet activation but not aggregation and decreased the platelet count, the expression of cell death markers and the release of RANTES and GROα. Acetylsalicylic acid but not fluvastatin limited platelet activation and inflammatory factor release and restored the platelet count by protecting platelets from Staphylococcus-induced expression of cell death markers. This study demonstrates that acetylsalicylic acid limits S. aureus-induced effects on platelets by reducing cell death, revealing new strategies to reduce the platelet contribution to bacteremia-associated inflammation.
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http://dx.doi.org/10.1038/s41598-017-06024-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514152PMC
July 2017

[The PIG-A gene as a new biomarker of mutagenesis: proof of concept and technical specifications].

Med Sci (Paris) 2017 Apr 12;33(4):432-439. Epub 2017 May 12.

Institut Méditerranéen de Biodiversité et d'Écologie (IMBE), équipe Biogénotoxicologie, Santé Humaine et Environnement, Aix-Marseille Université (AMU), CNRS, IRD, Avignon Université, Faculté de Médecine de Marseille, 27, boulevard Jean Moulin, 13005 Marseille, France.

Gene mutations are not directly detected by current genotoxicity assays and most of them need a cell culture step. The whole blood PIG-A assay consists in the detection of the mutation frequency within the PIG-A sentinel gene by identification of glycosyl-phosphatidyl-inositol (GPI-) deficient cells. PIG-A mutated/GPI-deficient cells can be detected by flow cytometry as they no longer express surface fluorescence for GPI-linked markers. The last researches have focused on cell enrichment techniques leading to increased throughput and sensitivity. The results of this new and promising biomarker of mutagenesis, performed in humans or rodents, are now available within 2 hours after blood collection.
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http://dx.doi.org/10.1051/medsci/20173304014DOI Listing
April 2017

Soluble CD146, an innovative and non-invasive biomarker of embryo selection for in vitro fertilization.

PLoS One 2017 14;12(3):e0173724. Epub 2017 Mar 14.

Aix Marseille Univ, Inserm U1076, Marseille, France.

Although progress was made in in vitro fertilization (IVF) techniques, the majority of embryos transferred fail to implant. Morphology embryo scoring is the standard procedure for most of IVF centres for choosing the best embryo, but remains limited since even the embryos classified as "top quality" may not implant. As it has been shown that i) CD146 is involved in embryo implantation and ii) membrane form is shed to generate soluble CD146 (sCD146), we propose that sCD146 in embryo supernatants may constitute a new biomarker of embryo selection. Immunocytochemical staining showed expression of CD146 in early embryo stages and sCD146 was detected by ELISA and Western-blot in embryo supernatants from D2. We retrospectively studied 126 couples who underwent IVF attempt. The embryo culture medium from each transferred embryo (n = 222) was collected for measurement of sCD146 by ELISA. Significantly higher sCD146 concentrations were present in embryo supernatants that did not implant (n = 185) as compared to those that successfully implanted (n = 37) (1310 +/- 1152 pg.mL-1 vs. 845+/- 1173 pg.mL-1, p = 0.024). Sensitivity analysis performed on single embryo transfers (n = 71) confirmed this association (p = 0.0054). The computed ROC curve established that the optimal sCD146 concentration for embryo implantation is under 1164 pg.mL-1 (sensitivity: 76%, specificity: 48%, PPV: 25% and NPV: 92%). Over this sCD146 threshold, the implantation rate was significantly lower (9% with sCD146 levels >1164 pg.ml-1 vs. 22% with sCD146 levels ≤ 1164 pg.mL-1, p = 0.01). Among the embryos preselected by morphologic scoring, sCD146 determination could allow a better selection of the embryo(s), thus improving the success of elective single embryo transfer. This study establishes the proof of concept for the use of sCD146 as a biomarker for IVF by excluding the embryo with the highest sCD146 level. A multicentre prospective study will now be necessary to further establish its use in clinical practice.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173724PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349662PMC
September 2017

High-Throughput Isolation of Giant Viruses in Liquid Medium Using Automated Flow Cytometry and Fluorescence Staining.

Front Microbiol 2016 29;7:26. Epub 2016 Jan 29.

Centre National de la Recherche Scientifique 7278, Institut National de la Santé et de la Recherche Médicale U1095, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes, UM 63, IRD 198, Facultés de Médecine et de Pharmacie, Aix Marseille UniversitéMarseille, France; Institut Hospitalo-Universitaire, Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique - Hôpitaux de Marseille and Aix Marseille UniversitéMarseille, France.

The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than 10 strains of previously known species of giant viruses and seven new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.
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http://dx.doi.org/10.3389/fmicb.2016.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731542PMC
February 2016

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions.

Oncotarget 2016 Jan;7(3):3357-66

VRCM, UMR-S1076, Aix-Marseille Université, INSERM, Faculté de Pharmacie, Marseille, France.

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.
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http://dx.doi.org/10.18632/oncotarget.6581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823111PMC
January 2016

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

Transfus Apher Sci 2015 Oct 27;53(2):110-26. Epub 2015 Oct 27.

Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), CHU UCL Namur, Université catholique de Louvain, Yvoir, Belgium.

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.
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http://dx.doi.org/10.1016/j.transci.2015.10.008DOI Listing
October 2015

A pancreatic tumor-specific biomarker characterized in humans and mice as an immunogenic onco-glycoprotein is efficient in dendritic cell vaccination.

Oncotarget 2015 Sep;6(27):23462-79

Aix-Marseille Université, CRO2, Centre de Recherche en Oncologie Biologique et Oncopharmacologie, Marseille, France.

Oncofetal fucose-rich glycovariants of the pathological bile salt-dependent lipase (pBSDL) appear during human pancreatic oncogenesis and are detected by themonoclonal antibody J28 (mAbJ28). We aimed to identify murine counterparts onpancreatic ductal adenocarcinoma (PDAC) cells and tissue and investigate the potential of dendritic cells (DC) loaded with this unique pancreatic tumor antigen to promote immunotherapy in preclinical trials. Pathological BSDLs purified from pancreatic juices of patients with PDAC were cleaved to generate glycosylated C-terminal moieties (C-ter) containing mAbJ28-reactive glycoepitopes. Immunoreactivity of the murine PDAC line Panc02 and tumor tissue to mAbJ28 was detected by immunohistochemistry and flow cytometry. C-ter-J28+ immunization promoted Th1-dominated immune responses. In vitro C-ter-J28+-loaded DCskewed CD3+ T-cells toward Th1 polarization. C-ter-J28+-DC-vaccinations selectively enhanced cell immunoreactivity to Panc02, as demonstrated by CD4+- and CD8+-T-cell activation, increased percentages of CD4+- and CD8+-T-cells and NK1.1+ cells expressing granzyme B, and T-cell cytotoxicity. Prophylactic and therapeutic C-ter-J28+-DC-vaccinations reduced ectopic Panc02-tumor growth, provided long-lasting protection from Panc02-tumor development in 100% of micebut not from melanoma, and attenuated progression of orthotopic tumors as revealed by MRI. Thusmurine DC loaded with pancreatic tumor-specific glycoepitope C-ter-J28+ induce efficient anticancer adaptive immunity and represent a potential adjuvant therapy for patients afflicted with PDAC.
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http://dx.doi.org/10.18632/oncotarget.4359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4695130PMC
September 2015

The origin and concentration of circulating microparticles differ according to cancer type and evolution: A prospective single-center study.

Int J Cancer 2016 Feb 1;138(4):939-48. Epub 2015 Oct 1.

Aix Marseille Université, INSERM UMR-S1076, VRCM, Marseille, France.

Microparticles are plasma membrane vesicles produced by apoptotic or activated cells and resting cancer cells. The concentration, origin and procoagulant properties of circulating microparticles are reported to differ according to pathological settings (inflammation, cancer and cardiovascular diseases). In case of cancer, different studies have reported a variation in the concentration of circulating microparticles, with an increase in procoagulant and tumor-associated antigen-bearing microparticles. However, the cancer specificity of these results remains unknown. The objective was to establish a specific signature of colorectal and pancreatic cancers (CRC, PC) by characterizing circulating microparticles. Patients presenting with CRC, PC, inflammatory bowel or pancreatic diseases, and healthy subjects, were prospectively included. Circulating microparticles were analyzed by flow cytometry, combining the analysis of Annexin V-positive with characterization of their origin and determination of their procoagulant activities. We included 85, 36, 15, 18 and 20 patients presenting with CRC, PC, inflammatory bowel or pancreatic diseases, and healthy subjects, respectively. Here, we depict a specific signature, which differed between CRC, PC, associated inflammatory bowel and pancreatic diseases and healthy subjects. Furthermore, in patients with remission, this signature returned to the levels observed in associated inflammatory or healthy patients. Our results indicate that circulating microparticles differ depending on the evolution of a cancer. The analysis of the circulating microparticles reveals the specificity of the signature and can be used as a new complex biomarker reflecting the evolution of the disease.
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http://dx.doi.org/10.1002/ijc.29837DOI Listing
February 2016

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

Br J Haematol 2015 Nov 24;171(4):615-24. Epub 2015 Jul 24.

Centre de Référence Maladies Rares Thalassémies, Marseille-Lyon, Service d'Hémato-Oncologie Pédiatrique, Hôpital de la Timone, APHM, Marseille, France.

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients.
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http://dx.doi.org/10.1111/bjh.13609DOI Listing
November 2015

Prelamin A accumulation in endothelial cells induces premature senescence and functional impairment.

Atherosclerosis 2014 Nov 28;237(1):45-52. Epub 2014 Aug 28.

APHM, CHU de la Timone, Laboratoire de Génétique Moléculaire, Marseille, France; Aix-Marseille Université, Inserm UMR_S U910, Faculté de Médecine, Marseille, France. Electronic address:

Background: Defects in lamin A maturation result in premature aging syndromes and severe atherosclerosis as observed in the Hutchinson-Gilford Progeria Syndrome. In age-related atherosclerosis, several features of cellular senescence have been characterized in endothelial cells including telomere shortening and increased oxidative stress. However, to date, very little is known about lamin A alterations in these cells.

Objectives: To study lamin A-related senescence and its consequences in the activation status of primary endothelial cells.

Methods: Healthy primary endothelial cells and progenitors issued from human umbilical vein or cord blood were used. Lamin A defects were induced by protease inhibitor (Atazanavir) treatment for 48 h.

Results: We show that protease inhibitor treatment leads to the accumulation of farnesylated prelamin A, inducing nuclear shape abnormalities and premature senescence in both differentiated and progenitor endothelial cells. ICAM-1-dependent activation and monocytes adhesion was increased in mature endothelial cells. In parallel, the ability to generate microvascular networks in matrigel was decreased for endothelial progenitors. The effects of protease inhibitor treatment on nuclear shapes were reversed when cells were treated in combination with Pravastatin and Zoledronate in both mature and progenitor endothelial cells. Reversion was also demonstrated with a morpholino antisense-oligonucleotide targeting lamin A-specific splice site.

Discussion: This study shows that protease inhibitor treatment reproduces premature senescence due to lamin A defects in primary endothelial cells and progenitors after 48 h exposure. The cells used were non-aged as extracted from cord blood or umbilical vein, allowing one to consider that other senescence pathways were not activated and that the observed alterations were specific of prelamin A accumulation. Both mature endothelial cells and precursors were sensitive to prelamin accumulation and thus, could be used in the future as a valuable model to test different approaches aimed at specifically reversing lamin A-related cells senescence.
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http://dx.doi.org/10.1016/j.atherosclerosis.2014.08.036DOI Listing
November 2014
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