43 research outputs found

    A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector

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    AbstractMonoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning. A single-chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations

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

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    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

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    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

    Anticorps recombinants : Vers une nouvelle génération d’antivenins?

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    Conception de fragments d'anticorps recombinants (application à la neutralisation de toxines animales et bactériennes)

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    The work undertaken during this PhD thesis consisted in the engeneering of therapeutic recombinant antibody fragments restricted to antibody V-domains. These molecules harbour numerous potential advantages for diagnostic and therapeutic applications in cancerology and infectiology. As an alternative to serotherapy, the first goal was to design a bivalent and bispecific recombinant antibody fragment that protects from the toxicity of a scorpion venom. The second goal consisted in designing recombinant scFvs and diabodies directed to the anthrax toxin and suitable for neutralization. These antibody fragments were also used to identify neutralizing mimotopes.Des fragments d anticorps recombinants à potentiel thérapeutique ont été conçus. Il s agit de petites molécules hautement diffusibles dans les tissus, adaptées à la neutralisation de toxines d origines animale (neurotoxines de scorpion) ou bactérienne (toxine charbonneuse).Les toxines de scorpions sont des polypeptides neurotoxiques de 30 à 70 résidus d acides aminés, agissant avec une spécificité élevée sur différents canaux ioniques des membranes des cellules excitables. La quasi-totalité du pouvoir toxique du venin de scorpion Androctonus australis, originaire du pourtour méditerranéen, lui est conféré par trois toxines, parmi les plus puissantes neurotoxines d origine animale (AahI, AahII et AahIII). Le seul traitement spécifique efficace est la sérothérapie basée sur l utilisation de fragments d anticorps polyclonaux hétérologues. Ce traitement n a pas connu d évolution significative depuis sa découverte et reste associé à de nombreux inconvénients : variabilité des lots, effets secondaires grâves et risques de transmission d agents pathogènes. Un fragment d anticorps bispécifique de type scFv en tandem (50 kDa) qui possède un site de reconnaissance antigénique dirigé contre la toxine AahI (ou AahIII) et un autre site dirigé contre la toxine AahII a été développé. Protéine recombinante produite en système procaryotique, cette molécule bispécifique conserve une haute affinité pour les toxines et présente la capacité de neutraliser à elle seule les effets toxiques du venin entier in vivo, dans des conditions expérimentales proches de celle d une envenimation naturelle. Les fragments d anticorps recombinants peuvent également avoir un intérêt pour la neutralisation de toxines bactériennes, celle du charbon en est un exemple. La maladie est une zoonose de distribution mondiale ayant pour agent étiologique Bacillus anthracis. Lorsqu elle est diagnostiquée suffisamment tôt, elle peut être traitée par antibiothérapie. Le plus souvent, les premiers signes cliniques échappent à la vigilance des personnes infectées et l antibiothérapie devient alors inefficace en raison de la trop grande quantité de toxine présente dans l organisme. L immunothérapie passive basée sur l utilisation d anticorps neutralisants la toxine pourrait être un traitement adjuvant efficace et complémentaire à l antibiothérapie. Des fragments d anticorps de primates non-humains (monovalent et bivalent), dirigés contre la toxine de Bacillus anthracis ont été conçus et caractérisés sur le plan structural et fonctionnel. Le rendement de production dans des bactéries recombinantes est élevé. Ils conservent leur activité de liaison et une affinité élevée pour l antigène. Un test de cytotoxicité in vitro a également montré que le scFv35PA83 est neutralisant. Une séquence mimotope de l épitope reconnu par ce scFv a été identifiée et pourrait avoir un potentiel vaccinal. Des souris immunisées avec le peptide d intérêt développent des anticorps anti-PA, neutralisants partiellement les effet létaux de la toxine dans un test de neutralisation in vitro. En parallèle, une méthode rapide, simple et générale a été développée permettant de cloner plus facilement l ADNc d un domaine variable fonctionnel de la chaîne légère (Analytical Biochemistry 349 (2006) : 159-161). En effet, la présence d un ARNm aberrant codant une région Kappa non fonctionnelle, exprimé par les cellules de myélome utilisées pour produire les hybridomes, rend souvent délicat le clonage de l ADNc correspondant au VL d un anticorps monoclonal.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

    La salive de la sangsue Hirudo medicinalis (effets pharmacologiques et applications thérapeutiques)

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    CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF
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