Sélections d'aptamères pour la recherche, la thérapie et l'imagerie des cancers

L'un des objectifs les plus importants de la recherche contre le cancer concerne le développement de molécules ciblant spécifiquement des marqueurs de tumeurs et permettant une discrimination élevée avec les tissus normaux. Dans ce contexte, les aptamères représentent une nouvelle classe d'agents de ciblage aux propriétés prometteuses. L'objectif de ces travaux de thèse était de sélectionner de nouveaux aptamères, dirigés contre des cibles exprimées à la surface des cellules endothéliales lors de l'angiogenèse tumorale. Dans ce but, nous avons utilisé une stratégie de sélection sur cellules (cell-SELEX) dirigée d'une part contre VEGR2 (vascular endothelial growth factor receptor 2) et d'autre part contre ETBR (endothelin type B receptor). Ces sélections n'ont pu aboutir à l'obtention d'aptamères dirigés contre ces cibles. Néanmoins, notre travail a permis d'identifier deux aptamères (E8 et V8) ayant une très forte affinité pour des marqueurs surexprimés à la surface de la lignée cellulaire de cancer du sein MCF -7. Les cibles des aptamères E8 et V8 ont par la suite été identifiées comme étant respectivement l'annexine A2 et la protéine LAR (leucocyte common antigen related). Ces aptamères ont été validés comme outils de détection in vitro par des expériences de cytométrie en flux et de microscopie. De plus, en utilisant des techniques d'imagerie in vivo par fluorescence, nous avons démontré que E8 et V8 ont un ciblage tumoral plus élevé par rapport à une séquence contrôle. Enfin, ils ont montré un effet inhibiteur dans un modèle d'angiogenèse in vitro, mettant en évidence une utilisation potentielle de ces aptamères comme outils thérapeutiques.One of the most important goals in cancer research is the development of binding molecules that are specific to tumour-associated markers with high discrimination between tumour and normal tissue. ln this context, aptamers represent a new class of targeting agents with promising properties. The objective of this thesis was to select new aptamers directed against highly accessible targets expressed on the surface of endothelial cells during tumor angiogenesis. For this purpose, we used a cell-SELEX strategy against cells that overexpress VEGFR2 (vascular endothelial growth factor receptor 2) and ETBR (endothelin type B receptor). Although these selections did not result in the identification of suitable aptamers directed against these initial targets, two other aptamers (E8 and V8) with very high affinity for markers overexpressed on the surface of the breast cancer cellline MCF-7 were successfully developed. The targets of aptamers E8 and V8 have subsequently been identified as Annexin A2 and leukocyte common antigen related (LAR) protein, respectively. We have subsequently validated the use of these aptamers as tools for in vitro experiments using flow cytometry and microscopy. ln addition, using in vivo fluorescence imaging techniques, we demonstrated that E8 and V8 achieved significantly higher tumour uptake compared to a control sequence. Finally, these two aptamers also showed an inhibitory effect in a model of angiogenesis in vitro, highlighting the potential use of these aptamers for therapeutic applications.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Mitogenic functions of endocrine gland-derived vascular endothelial growth factor and Bombina variegata 8 on steroidogenic adrenocortical cells.

International audienceEndocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its homolog Bombina variegata (Bv8), also termed prokineticin-1 and -2 (PK1 and PK2) respectively, are newly identified peptides with specific mitogenic activity on endocrine gland-derived endothelial cells. In the present study, we analyzed the sites of expression of EG-VEGF/PK1, Bv8/PK2, and their receptors (PKR1 and PKR2) in the adrenal cortex and checked for new biological functions of these factors on the endocrine cell compartment. RT-PCR and immunostaining analyses revealed that glomerulosa and fasciculata cells express both factors and both receptors. EG-VEGF/PK1 had no effect on the steroidogenic activity of both bovine glomerulosa and fasciculata cells but appeared to be mitogenic for both cell types. Binding of EG-VEGF/PK1 to fasciculata cells stimulated the phosphorylation of ERK1/2. Pretreatment with pertussis toxin suppressed this effect, indicating that it was Gi mediated. EG-VEGF/PK1 also increased the phosphorylation of Akt in endocrine cells of the adrenal cortex. EG-VEGF/PK1 and Bv8/PK2 thus represent new regulatory peptides acting as autocrine mitogens for endocrine cells

Aptamers based photodiagnostic

Nucleic acid aptamers are molecules that are being used in a large number of biomedical applications. Aptamers have the properties to bind to a wide range of molecules with high specificity and affinity for their target. These properties together with their small size and their ease of synthesis make them very attractive and promising for targeting diseases. Aptamers can serve as cancer diagnostic tools by detecting specific biomarkers, circulating cancer cells or imaging diseased tissue. On the other hand, aptamers can be used as therapeutic agents due to their potential antagonist activity, or as targeting agents. The objective of this work is to use a fluorescent labelled aptamer to detect cancer cells. A nuclease resistant 2'Fluoro-Pyrimidine RNA aptamer has been selected using the cell-SELEX strategy and binds a cell surface biomarker that seems to be overexpressed in several cancers. Identification of its target is ongoing. A fluorescent moiety (Alexa Fluor probes) is added on the aptamer for labelling. We have characterised the affinity of this aptamer for several cancer cell lines using flow cytometry and binding experiments. An efficient binding has been obtained with the breast adenocarcinoma MCF-7 and the pharynx squamous cell carcinoma FaDu cell lines (92% and 82% labelling respectively). Localisation and internalisation studies of the fluorescent aptamer are also realised on the selected cell lines using fluorescent microscope. Finally, our ongoing studies aim the coupling of this aptamer to a chlorin loaded nanoparticle for aptamer-targeted photodynamic treatment

Differential impacts of trail and ultra-trail running on cytokine profiles: An observational study

BACKGROUND: Endurance running events are known to cause inflammation and result in increased cytokine production. However, the effects of ultramarathons on cytokine profiles are not well characterized. OBJECTIVE: The aim of this study was to describe and compare the effects of a trail (40 km) race and an ultra-trail (171 km) race on leukocyte concentrations and cytokine profiles. METHODS: The study was conducted during the Ultra-Trail du Mont Blanc® ultra-marathon running event, and included 11 runners who completed the 40 km trail run and 12 runners who completed the 171 km ultra-trail. Blood samples were taken before and after the races. RESULTS: Leukocyte concentrations significantly increased after both races. Circulating levels of IL-6, IL-1β, MCP-1, and IFN-γ were significantly higher after the longer race compared to the shorter race. Furthermore, while both races resulted in significant increases in IL-6 and IL-8, only the longer race resulted in significant increases in MIP-1β, IL-7, IL-17a, and IL-4. CONCLUSIONS: These results illustrate that a 171 km ultra-trail race results in greater modulations in cytokine profiles than a traditional trail race.</jats:p

Impact of a 10 km running trial on eryptosis, red blood cell rheology, and electrophysiology in endurance trained athletes: a pilot study

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Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

International audienceRed blood cells (RBCs) can act as carriers for therapeutic agents and can substantially improve the safety, pharmacokinetics, and pharmacodynamics of many drugs. Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier. We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity. Several parameters were compared between processed RBCs loaded with l-asparaginase (“eryaspase”), processed RBCs without drug and non-processed RBCs. Processed RBCs were less hydrated and displayed a reduction of intracellular content. We observed a change in the metabolomic but not in the proteomic profile of processed RBCs. Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release. Despite a decrease in deformability, processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance. Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms. Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function