Modulation de la réponse autoimmune par vaccination adn (gene gun) codant pour la décarboxylase de l'acide glutamique, chez le chien et la souris nod

Le diabète insulinodépendant de type 1 est une maladie autoimmune qui résulte de la destruction des cellules B insulinosécrétrices du pancréas. Cette destruction impliquerait surtout les lymphocytes T CD4+ de sous-type th1 et les lymphocytes T CD8+. A l'inverse, certains lymphocytes T régulateurs, notamment les lymphocytes T de sous-type Th2/Th3 semblent capables de prévenir l'apparition de la maladie.NANTES-Ecole Nat.Vétérinaire (441092302) / SudocSudocFranceF

Apport des modèles animaux dans l'étude de la pathogénie du diabète de type 1 (mise au point d'un modèle canin de diabète insulino-dépendant ; étude de l'implication du mimétisme moléculaire par immunisation ADN chez la souris NOD et le chien)

Le diabète de type 1 (DT1) résulte de la destruction autoimmune des cellules insulinosécrétrices du pancréas. Cette destruction impliquerait surtout les lymphocytes T CD4+ de sous-type Th1 et les lymphocytes T CD8+ de sous-type Tc1 autoagressifs et serait influencée par des facteurs environnementaux qui interviendraient en initiant ou en modulant la réponse autoimmune. Le mécanisme de mimétisme moléculaire entre un peptide microbien et un autoantigène pourrait expliquer, dans certains cas, l'action des facteurs environnementaux sur le déclenchement ou la modulation de la maladie. L'objectif de notre étude a été de déterminer l'influence d'une protéine mimétique d'un autoantigène du DT1, la décarboxylase de l'acide glutamique de 65 kDa (GAD65), présentée par les cellules dendritiques cutanées sur la modulation du processus autoimmun, chez la souris NOD et le chien. Afin de cibler préférentiellement les cellules dendritiques de la peau, nous avons utilisé la technique du gene-gun qui correspond à l'administration intracutanée de microparticules d'or coatées avec de l'ADN plasmidique. Parallèlement, nous avons développé un modèle de diabète insulino-dépendant induit par de faibles doses de streptozotocine (STZ) chez le chien et testé l'effet d'une vaccination ADN par gene-gun codant pour la GAD65 humaine sur l'apparition de ce diabète expérimental. Chez la souris NOD, nous avons identifié un déficit fonctionnel des cellules dendritiques cutanées qui pourrait être impliqué dans l'altération des mécanismes immuno-régulateurs conduisant à l'initiation du processus autoimmun. Chez le chien sain, nos résultats suggèrent que l'expression cutanée de la GAD65 humaine conduit à l'activation d'un mécanisme de tolérance périphérique vis-à-vis de l'autoantigène. L'activation d'une réponse T régulatrice de sous-type Th2 chez la souris BALB/c et la protection de 2 chiens/ 3 du diabète induit par de faibles doses de STZ, après l'immunisation ADN, suggèrent un impact immunorégulateur des peptides mimétiques d'autoantigènes comme la GAD, présentés par les cellules dendritiques cutanées, sur l'initiation de la réponse autoimmune destructrice.NANTES-BU Sciences (441092104) / SudocSudocFranceF

Diverse profiles of N‐acyl‐homoserine lactone molecules found in cnidarians

Many marine habitats, such as the surface and tissues of marine invertebrates, including corals, harbour diverse populations of microorganisms, which are thought to play a role in the health of their hosts and influence mutualistic and competitive interactions. Investigating the presence and stability of quorum sensing (QS) in these ecosystems may shed light on the roles and control of these bacterial communities. Samples of 13 cnidarian species were screened for the presence and diversity of N-acyl-homoserine lactones (AHLs; a prevalent type of QS molecule) using thin-layer chromatography and an Agrobacterium tumefaciens NTL4 biosensor. Ten of 13 were found to harbour species-specific, conserved AHL profiles. AHLs were confirmed in Anemonia viridis using liquid chromatography tandem mass spectrometry. To assess temporal role and stability, AHLs were investigated in A. viridis from intertidal pools over 16 h. Patterns of AHLs showed conserved profiles except for two mid-chain length AHLs, which increased significantly over the day, peaking at 20:00, but had no correlation with pool chemistry. Denaturing gel electrophoresis of RT-PCR-amplified bacterial 16S rRNA showed the presence of an active bacterial community that changed in composition alongside AHL profiles and contained a number of bands that affiliate with known AHL-producing bacteria. Investigations into the quorum sensing-controlled, species-specific roles of these bacterial communities and how these regulatory circuits are influenced by the coral host and members of the bacterial community are imperative to expand our knowledge of these interactions with respect to the maintenance of coral health

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain

International audienceSevere deficiency in lysosomal β-glucuronidase (β-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in β-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the \textgreater200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, β-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases

Gene therapy of the brain in the dog model of Hurler's syndrome

International audienceObjective A defect of the lysosomal enzyme α-L-iduronidase (IDUA) interrupts the degradation of glycosaminoglycans in mucopolysaccharidosis type I, causing severe neurological manifestations in children with Hurler's syndrome. Delivery of the missing enzyme through stereotactic injection of adeno-associated virus vectors coding for IDUA prevents neuropathology in affected mice. We examined the efficacy and the safety of this approach in enzyme-deficient dogs. Methods Because deficient dogs raise antibodies against IDUA in response to infusion, intracerebral vector injections were combined with an immunosuppressive regimen. Results Treatment was tolerated well. We observed broad dispersion of vector genomes in the brain of efficiently immunosuppressed dogs. The delivery of IDUA to large areas, which could encompass the entire brain, prevented glycosaminoglycan and secondary ganglioside accumulations. This condition was associated with drastic reduction of neuropathology throughout the encephalon. In contrast, vector injection combined with partial immunosuppression was associated with subacute encephalitis, production of antibodies against IDUA in brain tissues, and elimination of genetically modified cells. Interpretation Gene therapy directed to the entire brain is feasible and may be beneficial to children with Hurler's syndrome. The possibility of subacute encephalitis emphasizes the importance of preventing immune response against IDUA, a problem that needs to be considered in similar therapies for other genetic defects. Ann Neurol 2006

Intra-CSF AAV9 and AAVrh10 Administration in Nonhuman Primates: Promising Routes and Vectors for Which Neurological Diseases?

International audienc

Intravenous Administration of Self-complementary AAV9 Enables Transgene Delivery to Adult Motor Neurons

Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood–brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients

Safe, Efficient, and Reproducible Gene Therapy of the Brain in the Dog Models of Sanfilippo and Hurler Syndromes

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome