A fiber-modified adenoviral vector interacts with immunoevasion molecules of the B7 family at the surface of murine leukemia cells derived from dormant tumors

Tumor cells can escape the immune system by overexpressing molecules of the B7 family, e.g. B7-H1 (PD-L1 or CD86), which suppresses the anti-tumor T-cell responses through binding to the PD-1 receptor, and similarly for B7.1 (CD80), through binding to CTLA-4. Moreover, direct interactions between B7-H1 and B7.1 molecules are also likely to participate in the immunoevasion mechanism. In this study, we used a mouse model of tumor dormancy, DA1-3b leukemia cells. We previously showed that a minor population of DA1-3b cells persists in equilibrium with the immune system for long periods of time, and that the levels of surface expression of B7-H1 and B7.1 molecules correlates with the dormancy time. We found that leukemia cells DA1-3b/d365 cells, which derived from long-term dormant tumors and overexpressed B7-H1 and B7.1 molecules, were highly permissive to Ad5FB4, a human adenovirus serotype 5 (Ad5) vector pseudotyped with chimeric human-bovine fibers. Both B7-H1 and B7.1 were required for Ad5FB4-cell binding and entry, since (i) siRNA silencing of one or the other B7 gene transcript resulted in a net decrease in the cell binding and Ad5FB4-mediated transduction of DA1-3b/d365; and (ii) plasmid-directed expression of B7.1 and B7-H1 proteins conferred to Ad5FB4-refractory human cells a full permissiveness to this vector. Binding data and flow cytometry analysis suggested that B7.1 and B7-H1 molecules played different roles in Ad5FB4-mediated transduction of DA1-3b/d365, with B7.1 involved in cell attachment of Ad5FB4, and B7-H1 in Ad5FB4 internalization. BRET analysis showed that B7.1 and B7-H1 formed heterodimeric complexes at the cell surface, and that Ad5FB4 penton, the viral capsomere carrying the fiber projection, could negatively interfere with the formation of B7.1/B7-H1 heterodimers, or modify their conformation. As interactors of B7-H1/B7.1 molecules, Ad5FB4 particles and/or their penton capsomeres represent potential therapeutic agents targeting cancer cells that had developed immunoevasion mechanisms

Encephalopathy induced by Alzheimer brain inoculation in a non-human primate.

Alzheimer's disease is characterized by cognitive alterations, cerebral atrophy and neuropathological lesions including neuronal loss, accumulation of misfolded and aggregated β-amyloid peptides (Aβ) and tau proteins. Iatrogenic induction of Aβ is suspected in patients exposed to pituitary-derived hormones, dural grafts, or surgical instruments, presumably contaminated with Aβ. Induction of Aβ and tau lesions has been demonstrated in transgenic mice after contamination with Alzheimer's disease brain homogenates, with very limited functional consequences. Unlike rodents, primates naturally express Aβ or tau under normal conditions and attempts to transmit Alzheimer pathology to primates have been made for decades. However, none of earlier studies performed any detailed functional assessments. For the first time we demonstrate long term memory and learning impairments in a non-human primate (Microcebus murinus) following intracerebral injections with Alzheimer human brain extracts. Animals inoculated with Alzheimer brain homogenates displayed progressive cognitive impairments (clinical tests assessing cognitive and motor functions), modifications of neuronal activity (detected by electroencephalography), widespread and progressive cerebral atrophy (in vivo MRI assessing cerebral volume loss using automated voxel-based analysis), neuronal loss in the hippocampus and entorhinal cortex (post mortem stereology). They displayed parenchymal and vascular Aβ depositions and tau lesions for some of them, in regions close to the inoculation sites. Although these lesions were sparse, they were never detected in control animals. Tau-positive animals had the lowest performances in a memory task and displayed the greatest neuronal loss. Our study is timely and important as it is the first one to highlight neuronal and clinical dysfunction following inoculation of Alzheimer's disease brain homogenates in a primate. Clinical signs in a chronic disease such as Alzheimer take a long time to be detectable. Documentation of clinical deterioration and/or dysfunction following intracerebral inoculations with Alzheimer human brain extracts could lead to important new insights about Alzheimer initiation processes

TRANSFERT DE GENE PAR UN VECTEUR NON VIRAL COMPOSE D'UNE SEQUENCE DE CIBLAGE DES INTEGRINES ET D'UN LIPOSOME CATIONIQUE (EFFICACITE ET DEVENIR INTRACELLULAIRE)

UN DES BUTS DE LA THERAPIE GENIQUE EST D'INTRODUIRE DANS UNE CELLULE UN GENE-MEDICAMENT AFIN DE CORRIGER UN DYSFONCTIONNEMENT DE SON PROGRAMME GENETIQUE. CETTE NOUVELLE THERAPEUTIQUE NECESSITE, LA PLUPART DU TEMPS, LA MISE AU POINT D'UNE VECTORISATION AFIN DE TRANSPORTER CE GENE-MEDICAMENT DANS LA CELLULE. NOUS AVONS AINSI MONTRE, DANS UN MODELE DE CELLULES TRACHEALES HUMAINES, UNE AUGMENTATION SPECIFIQUE D'UN FACTEUR 10 DE L'EXPRESSION D'UN PLASMIDE (ADNP) CODANT POUR LE GENE RAPPORTEUR LUCIFERASE LORSQUE CELUI-CI EST CONDENSE PAR UN PEPTIDE CYCLIQUE SPECIFIQUE DES INTEGRINES, COMPRENANT UNE SEQUENCE POLYLYSINE-ARG-GLY-ASP (K 1 6RGD). L'ADDITION D'UN LIPIDE CATIONIQUE AU COMPLEXE ADNP:K 1 6RGD PERMET D'OBTENIR DES NIVEAUX D'EXPRESSION COMPARABLES A CEUX OBTENUS AVEC UN ADENOVIRUS, EN AMELIORANT L'EFFICACITE DU VECTEUR D'UN FACTEUR 30. CE LIPOPOLYPLEXE (ADNP:K 1 6RGD : LIPIDE) PENETRE DANS LA CELLULE PAR ENDOCYTOSE MEDIEE PAR LES INTEGRINES, IMPLIQUANT LES PUITS RECOUVERTS DE CLATHRINE. L'AUGMENTATION DE L'EXPRESSION DU GENE RAPPORTEUR LUCIFERASE, EN PRESENCE DU LIPIDE, EST CORRELEE AVEC UNE AUGMENTATION DU PASSAGE DE L'ADNP DANS LES ENDOSOMES/LYSOSOMES, UNE DIMINUTION DE L'EXOCYTOSE DE L'ADNP ET UN MEILLEUR TRANSFERT NUCLEAIRE. LE TRAFIC INTRACELLULAIRE NECESSITE UNE FUSION ENDOSOMALE ET UN ENVIRONNEMENT VESICULAIRE ACIDE EST BENEFIQUE A L'EFFICACITE DU VECTEUR LIPIDIQUE. LE PASSAGE DE L'ADNP A TRAVERS L'ENVELOPPE NUCLEAIRE SERAIT UN MECANISME ACTIF IMPLIQUANT LES PORES NUCLEAIRES ET NE NECESSITANT PAS L'APPORT D'ELEMENTS CYTOSOLIQUES. LE LIPOPOLYPLEXE PEUT TRANSFECTER DES CELLULES QUIESCENTES MAIS SON ACTIVITE EST MULTIPLIEE PAR QUATRE SUR DES CELLULES EN MITOSE. DES ETUDES IN VIVO CHEZ LA SOURIS NOUS ONT PERMIS D'ETABLIR QUE L'HEMOGLOBINE MASQUAIT LA DETECTION DE L'ACTIVITE LUCIFERASE. UNE PERFUSION APPROPRIEE DES ORGANES PERMETTANT D'EXTRAIRE L'HEMOGLOBINE S'IMPOSE ALORS, AFIN DE DETERMINER L'EXPRESSION IN VIVO DU GENE RAPPORTEUR LUCIFERASE. CES TRAVAUX PARTICIPENT A L'AMELIORATION DES CONNAISSANCES DEVANT PERMETTRE A TERME, UNE OPTIMISATION DES VECTEURS NON VIRAUX NECESSAIRE A L'OBTENTION D'ESSAIS CLINIQUES CONCLUANTS.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Factors involved in the sensitivity of different hematopoietic cell lines to infection by subgroup C adenovirus: implication for gene therapy of human lymphocytic malignancies

AbstractGene transfer approaches using viruses such human adenovirus (HAdV) may provide an alternative treatment for diseases involving hematopoietic cells.Better understanding of the cellular mechanisms by which the HAdV introduces DNA into these cells should help in vector design. We examined HAdV intracellular delivery in several cell lines including B and T lymphocytes. We demonstrated that HAdV resistance in most B lymphocytes is the result of moderate HAdV uptake. In contrast, high levels of coxsackie and HAdV receptor (hCAR) are expressed on the surface of HSB2 (T cells), allowing efficient binding and uptake but no transgene expression, probably because of deficient endosomolysis and subsequent exocytose. This work demonstrates the existence of hCAR-dependent and -independent endocytic route in hematopoietic cells. Moreover, it precises the intracellular barriers to be overcome by HAdV in such cells to be infectious and gives previous information's to design new vectors for gene transfer

Les vésicules extracellulaires

Les vésicules extracellulaires (VE) sont libérées par une grande variété de cellules et contiennent des protéines, des ARN et des lipides, qui sont ainsi échangés entre ces cellules. Elles représentent donc un mode de communication intercellulaire majeur aussi bien en conditions physiologiques que pathologiques. C’est notamment le cas dans le système nerveux (SN) où les neurones et les cellules gliales forment un réseau très dense et où des milliards de connexions s’établissent. Cette revue fournit un aperçu des différents rôles joués par les VE dans un cerveau sain lors du renforcement des réseaux par exemple, mais également dans un cerveau malade où les VE participent, entre autres, à la progression des maladies neurodégénératives et tumorales.</jats:p

Les vésicules extracellulaires

Les vésicules extracellulaires (VE) sont libérées par une grande variété de cellules et contiennent des protéines, des ARN et des lipides, qui sont ainsi échangés entre ces cellules. Elles représentent donc un mode de communication intercellulaire majeur aussi bien en conditions physiologiques que pathologiques. C’est notamment le cas dans le système nerveux (SN) où les neurones et les cellules gliales forment un réseau très dense et où des milliards de connexions s’établissent. Cette revue fournit un aperçu des différents rôles joués par les VE dans un cerveau sain lors du renforcement des réseaux par exemple, mais également dans un cerveau malade où les VE participent, entre autres, à la progression des maladies neurodégénératives et tumorales

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes

AbstractMost adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 (Aviadenovirus), DAdV-1, and BAdV-4 (Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy

From the prion-like propagation hypothesis to therapeutic strategies of anti-tau immunotherapy

Abstract The term “propagon” is used to define proteins that may transmit misfolding in vitro, in tissues or in organisms. Among propagons, misfolded tau is thought to be involved in the pathogenic mechanisms of various “tauopathies” that include Alzheimer's disease, progressive supranuclear palsy, and argyrophilic grain disease. Here, we review the available data in the literature and point out how the prion-like tau propagation has been extended from Alzheimer's disease to tauopathies. First, in Alzheimer’s disease, the progression of tau aggregation follows stereotypical anatomical stages which may be considered as spreading. The mechanisms of the propagation are now subject to intensive and controversial research. It has been shown that tau may be secreted in the interstitial fluid in an active manner as reflected by high and constant concentration of extracellular tau during Alzheimer’s pathology. Animal and cell models have been devised to mimic tau seeding and propagation, and despite their limitations, they have further supported to the prion-like propagation hypothesis. Finally, such new ways of thinking have led to different therapeutic strategies in anti-tau immunotherapy among tauopathies and have stimulated new clinical trials. However, it appears that the prion-like propagation hypothesis mainly relies on data obtained in Alzheimer’s disease. From this review, it appears that further studies are needed (1) to characterize extracellular tau species, (2) to find the right pathological tau species to target, (3) to follow in vivo tau pathology by brain imaging and biomarkers and (4) to interpret current clinical trial results aimed at reducing the progression of these pathologies. Such inputs will be essential to have a comprehensive view of these promising therapeutic strategies in tauopathies.</jats:p

Elaboration of nanoneedle arrays via nanosphere lithography and single step continuous ICP etching. Application for detections of neurodegenerative diseases' biomarkers by mass spectrometry

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