A031 Développement d’un peptido-mimétique de la glycorpotein VI plaquettaire comme outil d’imagerie de la fibrose

ObjectifLa glycoprotéine VI est le récepteur d’activation des plaquettes par les collagènes de type I et de type III. Nous avons émis l’hypothèse que nous pourrions développer une sonde spécifique du collagène basée sur la spécificité de GPVI et que cette sonde permettrait de visualiser la fibrose in vivo par une méthode non invasive.MéthodesUn anticorps bloquant la liaison de GPVI au collagène a été utilisé pour cribler une banque peptidique permettant d’identifier un motif peptidique cyclique. La capacité du peptide à mimer la GPVI a été analysée par des études de liaison et de compétition en phase solide. La liaison au collagène tissulaire a été analysée par histochimie. L’imagerie in vivo a été réalisée par injection du peptide-marqué au Tc-99m dans un modèle de fibrose cicatricielle sur infarctus du myocarde chez le rat, scintigraphie et autoradiographieRésultatsLe peptide, nommé collagelin, se lie de manière spécifique à l’anticorps anti GPVI 9O12.2 et aux collagènes I et III in vitro et la liaison est inhibée par GPVI indiquant que le peptide mime GPVI. Cependant le collagelin n’inhibe pas l’agrégation des plaquettes induite par le collagène. Les études d’histochimie montrent que le collagelin se lie au collagène tissulaire sur coupe d’aorte et de queue de rat indiquant que le collagelin se comporte comme un traceur du collagène. Dans le modèle d’infarctus cicatriciel, une accumulation du collagelin radiomarqué est observée dans la zone cardiaque par scintigraphie planaire et tomographie chez les animaux avec MI mais pas chez les animaux contrôles ni avec un peptide contrôle. L’accumulation du traceur dans les zones de fibrose a été mise en évidence ex vivo par superposition des images d’autoradiographies et d’histologie sur coupes congelées.ConclusionNous avons produit un peptide qui mime en partie le site de liaison de GPVI au collagène. Ce peptide se comporte comme un traceur spécifique du collagène in vitro et in vivo. Nous proposons que ce traceur pourrait être utile pour le diagnostic et le suivi évolutif de la fibrose dans un grand nombre de pathologies

Non-Invasive Molecular Imaging of Fibrosis Using a Collagen-Targeted Peptidomimetic of the Platelet Collagen Receptor Glycoprotein VI

Background: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III. Methodology/Principal: Findings An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10−7 M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis. Conclusion/Significance: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.Psycholog

Discovery of novel GPVI receptor antagonists by structure-based repurposing.

Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug 'resistance', underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing

Specific Binding of the Pathogenic Prion Isoform: Development and Characterization of a Humanized Single-Chain Variable Antibody Fragment

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

A comparative study of ion beam nitriding of single-crystalline and polycrystalline 316L austenitic stainless steel

Surface and coatings technology: 13th international conference on surface modification of materials by ion beams, San Antonio, Texas, 21-26 September, 2003Nitriding of an austenitic stainless steel (ASS) at moderate temperature (400 °C) increases significantly its hardness without loss of corrosion resistance. This study presents the first comparison between single crystalline ([001], [011] and [013] orientations) and polycrystalline 316L ASS ion beam nitriding. The current density, ion energy and temperature were 0.5 mA·cm−2, 1.2 keV and 400 °C, respectively. The nitrogen distribution profiles were measured by nuclear reaction analysis (NRA). A notable difference of nitrogen penetration between polycrystalline and single-crystalline samples is observed, depending on the orientation of the single crystals. The nitrogen penetration is higher for [100] and [013] oriented single crystals. X-ray diffraction (XRD) reveals the presence of the γN phase in both types of samples. Moreover, it shows that the nitrided layer in single crystals exhibits disorientations in relation to the matrixVytauto Didžiojo universiteta