Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes

International audienceTherapeutic failure limits prophylaxis of nematode diseases and has been mainly attributed to mutations in cellular targets of anthelmintics. Besides these specific mechanisms, alterations of drug transport also occur in parasites resistant to anthelmintics and depend on both the presence of membrane pumps such as P-glycoprotein (Pgp) and on the lipid composition of membranes. We recently showed in the nematode Haemonchus contortus, using eggs as a model, that the total cholesterol (TC) concentration alters the transport of lipophilic molecules due to membrane pumps such as P-glycoprotein and the resistance to anthelmintics. The effect of TC may depend on the presence of other lipids interacting with TC. Therefore, we analysed the lipid composition and its relationship with Pgp and resistance to anthelmintics. Better correlations were found between Pgp and free cholesterol (FC) than with TC. We also showed that the relationships between lipid composition and resistance to anthelmintics or Pgp depended on the equilibrium between FC and phospholipids (PLs), mainly PLs known to be present primarily in either the external leaflets of cell membranes or the internal leaflets. The PLs phosphatidylcholine and phosphatidylethanolamine played the most significant role, but phosphatidic acid also influenced drug resistance

Efflux pump inhibitors: a progress in parasitic nematode control

Animal and human nematode infestations are controlled primarily with anthelmintics. However, their continuous administration during outbreaks represents a significant expense for livestock farms. In humans also, their high cost limits their use in poor areas where parasitic worms are most prevalent and most pathogenic. Furthermore, nematodes have developed drug resistance mechanisms, specific or not, which reduce the efficiency of treatments. Among these mechanisms, the accelerated removal of anthelmintics by efflux pumps present in cell membranes, eggshells and cuticles is a major limiting factor. This accelerated efflux is very similar to the mechanism of multidrug resistance (MDR) observed in cancer cells and protozoa. This phenomenon is all the more worrying that it applies simultaneously to several chemical families of drugs. One solution is to block the efflux pumps in parasites with inhibitors. These pumps belong to the large family of ABC transporters, which have many characteristics in common. Some have major physiological functions or protect organs from toxic agents. As much as possible, inhibitors should not have any effect on the pumps of the host and target the parasite exclusively. The diversity of these pumps is greater in nematodes than in vertebrates, and there are differences in their protein structures. Some parts of these proteins are relatively well-conserved in the animal kingdom, while other parts show little homology from one transporter to another or from one species to another. The affinity of these pumps for the substrates can vary with the mutation of a single amino acid. These differences could be used to develop inhibitors specific of nematode pumps, which could then be combined with anthelmintics.Les anthelminthiques constituent le moyen majeur de lutte contre les nématodoses animales et humaines. Administrés de façon continue en période d'infestation, ils représentent un coût considérable pour les élevages. Chez l'homme, ce coût limite leur distribution dans les zones géographiques défavorisées où pourtant les vers parasites sont les plus nombreux et les plus pathogènes. De plus, les nématodes ont développé des mécanismes de chimiorésistance, spécifiques ou non, qui réduisent l'efficacité des anthelminthiques. Parmi ces mécanismes, le rejet accéléré des molécules thérapeutiques par des pompes d'efflux est semblable au mécanisme de multi-résistance aux médicaments (MDR) des cellules cancéreuses et des protozoaires. Ces pompes sont présentes dans les membranes cellulaires, la coque des œufs et les cuticules des nématodes. Ce rejet accéléré est d'autant plus préoccupant qu'il s'applique simultanément à plusieurs familles chimiques d'antiparasitaires. Une des solutions consiste à bloquer les pompes d'efflux des parasites à l'aide d'inhibiteurs. Ces pompes appartiennent à la grande famille des transporteurs ABC dotés de nombreuses caractéristiques communes. Certains de ces transporteurs jouent des rôles physiologiques déterminants ou protègent les organes des molécules toxiques. Les inhibiteurs doivent donc être autant que possible dépourvus d'action sur les pompes de l'hôte. La variabilité des pompes est plus importante chez les nématodes que chez les vertébrés, et il existe des différences dans leur structure protéique. Certaines parties de ces protéines sont bien conservées dans le règne animal, tandis que d'autres présentent peu d'homologie d'un transporteur à l'autre ou, pour un même transporteur, d'une espèce à l'autre. L'affinité de ces pompes pour les substrats peut varier en fonction de la mutation d'un seul acide aminé. Ces différences pourraient être mises à profit pour développer des inhibiteurs spécifiques des pompes des nématodes et les utiliser en association avec les anthelminthiques

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

AbstractBluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)–matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes

La Plateforme d'infectiologie expérimentale du centre INRA Val de Loire : Une animalerie protégée pour«Modèles animaux en Infectiologie» de dimension européenne

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Les modèles précliniques (petits et gros animaux) proposés à l’INRA

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INRA's infectiology platform (PFIE)

Session : Animal Health R&D Service PresentationsNational audienc

Rôles et régulation des P-glycoprotéines de membrane impliquées dans les mécanismes de résistance multiple des nématodes parasites des petits ruminants aux anthelminthiques

Les P-glycoprotéines (P-gp) jouent un rôle primordial dans la résistance multiple aux anthelminthiques des nématodes parasites des petits ruminants. Ce travail de thèse a permis de décrire le premier schéma du transport des anthelminthiques pour un modèle œuf de nématode impliquant les P-gp et de montrer le rôle de l environnement membranaire dans leur activité. Les anthelminthiques pénétreraient dans les œufs soit par diffusion passive soit par capture directe par les P-gp (influx). Les anthelminthiques activeraient les P-gp et changeraient leur conformation. Les P-gp activées éliminent les anthelminthiques (efflux) et préserveraient ainsi l œuf de leurs effets toxiques. Ce transport des anthelminthiques est altéré par la modification de la teneur en cholestérol des œufs. Le cholestérol agirait soit indirectement sur la solubilisation des anthelminthiques dans les membranes soit directement sur l activité des P-gp.P-glycoproteins (Pgp) play an important role in the multiresistance to anthelmintics in the parasitic nematodes of small domestic ruminants. This work allowed to describe the first scheme for the transport of anthelmintics in nematode egg implying Pgp and to show the role of membrane environment in their activity. The anthelmintics could penetrate into eggs either by passive diffusion or by direct capture by Pgp (influx). The presence of anthelmintics would activate Pgp by changing their conformation. Once activated, Pgp eliminate the anthelmintics (efflux) and protect eggs from their toxic effects. The transport of anthelmintics is altered by changes in cholesterol content of eggs. Cholesterol could act either indirectly on the solubilisation of anthelmintics in membranes or directly on Pgp activity.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

L'accès aux dispositifs d'expérimentation animale toutes espèces : accompagner les entreprises vers les acteurs de la recherche

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Salivary Veterinary ELISA Diagnosis for Apicomplexa Detection combined with a ethical saliva collection tool

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Cryptic Color Change in a Crab Spider (Misumena vatia): Identification and Quantification of Precursors and Ommochrome Pigments by HPLC

International audienceMimicry is used widely by arthropods to survive in a hostile environment. Often mimicry is associated with the production of chemical compounds such as pigments. In crab spiders, the change of color is based on a complex physiological process that still is not understood. The aim of this study was to identify and quantify the ommochrome pigments and precursors responsible for the color change in the mimetic crab spider Misumena vatia (Thomisidae). A modified high performance reverse phase ion-pair chromatography technique enabled us to separate and quantify the ommochrome pigments, their precursors, and related metabolites in individual spiders. Compounds such as tryptophan, kynurenine, and kynurenic acid occurred only or mainly in white crab spiders. In contrast, compounds such as 3-hydroxy-kynurenine, xanthommatin, and ommatin D occurred only or mainly in yellow crab spiders. Factor analysis ranked the different color forms in accordance with their metabolites. The biochemical results enabled us to associate the different phases of formation of pigment granules with specific metabolites. Yellow crab spiders contain many unknown ommochrome-like compounds not present in white crab spiders. We also found large quantities of decarboxylated xanthommatin, whose role as precursor of new pathways in ommochrome synthesis needs to be assessed. The catabolism of ommochromes, a process occurring when spiders revert from yellow to white, warrants further study