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

Preparatory planning framework for Created Out of Mind: Shaping perceptions of dementia through art and science [version 1; referees: 2 approved]

Created Out of Mind is an interdisciplinary project, comprised of individuals from arts, social sciences, music, biomedical sciences, humanities and operational disciplines. Collaboratively we are working to shape perceptions of dementias through the arts and sciences, from a position within the Wellcome Collection. The Collection is a public building, above objects and archives, with a porous relationship between research, museum artefacts, and the public. This pre-planning framework will act as an introduction to Created Out of Mind. The framework explains the rationale and aims of the project, outlines our focus for the project, and explores a number of challenges we have encountered by virtue of working in this way

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

Cross-recognition of a pit viper (Crotalinae) polyspecific antivenom explored through high-density peptide microarray epitope mapping

Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.Novo Nordisk Foundation/[NNF13OC0005613]/NNF/DinamarcaNovo Nordisk Foundation/[NNF16OC0019248]/NNF/DinamarcaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Production of a functional anti-scorpion hemocyanin scFv in Escherichia coli.

A58 is a murine monoclonal antibody raised against the internal dimeric subunit A alpha 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH--VL structure-function relationships of antibodies

Production of a functional anti-scorpion hemocyanin scFv in Escherichia coli.

A58 is a murine monoclonal antibody raised against the internal dimeric subunit A alpha 1 of the hemocyanin of the scorpion Androctonus australis. When A58 is tested against a range of related hemocyanins, it exhibits cross-reactivity with a limited number of scorpion species. All of them belong to a single Scorpion Family: the Buthidae. We have utilized a bacterial expression-export system in which the variable regions of A58 are expressed as a single-chain variable fragment (scFv). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions which were assembled into a scFv. After insertion into a vector which contains the pectate lyase signal sequence from Erwinia caratovora for periplasmic expression, a scFv protein was produced in high yield as a soluble and functional protein. The bacterial produced A58 scFv has binding properties similar to those of the original murine antibody. It binds specifically the heterodimeric subunit of Androctonus australis hemocyanin, cross-reacts with only one subunit of each Buthidae investigated, and blocks the recognition of the hemocyanin by antibody A58 in competitive enzyme-linked immunosorbant assay. Previous reports have demonstrated the value of monoclonal antibodies in taxonomical and molecular evolution studies. A58scFv is the first of a new generation of antigen-binding proteins which should prove useful both in taxonomical studies and in the analysis of VH--VL structure-function relationships of antibodies

scFv single chain antibody variable fragment as inverse agonist of the β(2) -adrenergic receptor

Antibodies directed against the second extracellular loop of G protein-coupled receptors were shown to possess functional activities. Using a functional monoclonal antibody against the human {beta}2-adrenergic receptor, a scFv fragment with high affinity for the target epitope was constructed and produced. The fragment recognized the {beta} 2-adrenergic receptors on A431 cells, blocked cAMP accumulation induced by the {beta}2-agonist salbutamol, and decreased basal cAMP accumulation in the same cells. Their in vitro activity was tested on neonatal rat cardiomyocytes. The antibody fragments blocked the chronotropic activity induced by the {beta}2-agonist clenbuterol. They also decreased the in vivo heart beating frequency of mice pretreated with bisoprolol (a {beta}1-adrenergic receptor antagonist) for 4 min after injection. The immunological approach presented here may serve as a strategy for the synthesis of a new class of allosteric modulators for G protein-coupled receptors

scFv single chain antibody variable fragment as inverse agonist of the beta(2)-adrenergic receptor

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