Manganese-induced integrin affinity maturation promotes recruitment of alpha V beta 3 integrin to focal adhesions in endothelial cells: evidence for a role of phosphatidylinositol 3-kinase and Src.

Integrin activity is controlled by changes in affinity (i.e. ligand binding) and avidity (i.e. receptor clustering). Little is known, however, about the effect of affinity maturation on integrin avidity and on the associated signaling pathways. To study the effect of affinity maturation on integrin avidity, we stimulated human umbilical vein endothelial cells (HUVEC) with MnCl(2) to increase integrin affinity and monitored clustering of beta 1 and beta 3 integrins. In unstimulated HUVEC, beta 1 integrins were present in fibrillar adhesions, while alpha V beta 3 was detected in peripheral focal adhesions. Clustered beta 1 and beta 3 integrins expressed high affinity/ligand-induced binding site (LIBS) epitopes. MnCl(2)-stimulation promoted focal adhesion and actin stress fiber formation at the basal surface of the cells, and strongly enhanced mAb LM609 staining and expression of beta 3 high affinity/LIBS epitopes at focal adhesions. MnCl(2)-induced alpha V beta 3 clustering was blocked by a soluble RGD peptide, by wortmannin and LY294002, two pharmacological inhibitors of phosphatidylinositol 3-kinase (PI 3-K), and by over-expressing a dominant negative PI 3-K mutant protein. Conversely, over-expression of active PI 3-K and pharmacological inhibiton of Src with PP2 and CGP77675, enhanced basal and manganese-induced alpha V beta 3 clustering. Transient increased phosphorylation of protein kinase B/Akt, a direct target of PI 3K, occurred upon manganese stimulation. MnCl(2) did not alter beta 1 integrin distribution or beta1 high-affinity/LIBS epitope expression. Based on these results, we conclude that MnCl(2)-induced alpha V beta 3 integrin affinity maturation stimulates focal adhesion and actin stress fiber formation, and promotes recruitment of high affinity alpha V beta 3 to focal adhesions. Affinity-modulated alpha V beta 3 clustering requires PI3-K signaling and is negatively regulate by Src.

Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation.

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.

Dramatic influence of V beta gene polymorphism on an antigen-specific CD8+ T cell response in vivo.

According to recent crystallographic studies, the TCR-alpha beta contacts MHC class I-bound antigenic peptides via the polymorphic V gene-encoded complementarity-determining region 1 beta (CDR1 beta) and the hypervariable (D)J-encoded CDR3 beta and CDR3 alpha domains. To evaluate directly the relative importance of CDR1 beta polymorphism on the fine specificity of T cell responses in vivo, we have taken advantage of congenic V beta a and V beta b mouse strains that differ by a CDR1 polymorphism in the V beta 10 gene segment. The V beta 10-restricted CD8+ T cell response to a defined immunodominant epitope was dramatically reduced in V beta a compared with V beta b mice, as measured either by the expansion of V beta 10+ cells or by the binding of MHC-peptide tetramers. These data indicate that V beta polymorphism has an important impact on TCR-ligand binding in vivo, presumably by modifying the affinity of CDR1 beta-peptide interactions.

"V.N. Volochinov en contexte: essai d'épistémologie historique"

Cette thèse de doctorat porte sur la pensée de Valentin Volochinov (1895-1936) et le contexte intellectuel russe du début du XXe siècle dans lequel elle a été élaborée. Le cadre spatial de l'étude est la Russie. Le cadre temporel correspond aux années 1890-1920. Fait dans le cadre des « études bakhtiniennes », ce travail propose une démarche inverse de celle utilisée jusqu'à présent dans ce domaine. Son objectif est d'explorer les conséquences pratiques de trois hypothèses : 1) Valentin Volochinov (et non pas Mikhaïl Bakhtine) est l'auteur des textes parus sous son nom dans les années 1920 ; 2) Valentin Volochinov est un chercheur indépendant de Mikhaïl Bakhtine et de Pavel Medvedev (et non pas seulement un des membres du « Cercle de Bakhtine » ou du « groupe B.M.V. » dont le projet scientifique serait incompréhensible sans le recours aux textes bakhtiniens et/ou de Medvedev) ; 3) la connaissance du contexte intellectuel général dans lequel a travaillé Valentin Volochinov (c'est-à-dire l'ensemble des textes et des discussions scientifiques qui se sont déroulées dans les revues et les institutions scientifiques autour des thèmes abordés dans les textes signés dans les années 1920 par Volochinov) joue un rôle de premier plan dans l'interprétation de sa conception. L'analyse de la terminologie utilisée dans les travaux de Volochinov (en particulier de la notion d'« idéologie » et de méthode « marxiste »), ainsi que l'examen des idées ayant trait à la philosophie du langage (la réception des idées de Saussure, la polémique avec Rozalija Chor sur des principes de la science du langage « marxiste », la critique de la conception du «mot» de Gustav Chpet), psychologiques (la critique de la théorie psychanalytique de Sigmund Freud, l'élaboration de la notion de conscience, la recherche des bases de la psychologie « marxiste ») et sociologiques (l'analyse de l'interaction socio-verbale et l'élaboration de la théorie de l'énoncé) amènent à la conclusion que les hypothèses avancées sont justes et le mode de lecture adopté dans le travail est rentable : il suscite des interprétations différentes de celles proposées jusqu'à présent. Il permet, par exemple, de mettre en évidence les particularités du marxisme russe des années 1920 considéré par les intellectuels de l'époque, d'une part, comme une méthode des recherches dont les principes fondamentaux sont le matérialisme, le monisme, le déterminisme (y compris social), la dialectique et, d'autre part, comme une doctrine sociologique. Compris en ce sens, le marxisme fait partie de l'histoire de la sociologie russe qui comprend également des conceptions dites « bourgeoises » ou « positivistes » comme, par exemple, celle d'Eugène de Roberty, qui insiste dans ses travaux sur le primat du social sur l'individuel, autrement dit qui met en avant le principe du déterminisme social souvent associé uniquement au «sociologisme marxisant». L'oeuvre de Volochinov ne contient pas, par conséquent, d'éléments de «sociologisme vulgaire», elle s'inscrit dans l'histoire des idées sociologiques russes où il n'y a pas de rupture nette entre les conceptions dites «bourgeoises» et marxistes. Le marxisme de Volochinov n'est pas révolutionnaire. Il ne peut pas non plus être associé aux idées psychanalytiques. La preuve est le refus radical de l'existence de l'inconscient (ou plutôt d'une force inconsciente) qui déterminerait le comportement des individus sans qu'ils s'en rendent compte. Le projet scientifique de Volochinov consiste à analyser la conscience (les faits psychiques), ainsi que le langage, l'énoncé et les structures syntaxiques dans lesquelles l'énoncé se réalise en tant qu'éléments constitutifs de l'échange social et/ou verbal, qui se trouve au centre de vives discussions menées à la charnière des XIXe-XXe siècles par les chercheurs russes d'orientation marxiste et non marxiste. Pour comprendre ce projet il n'est pas nécessaire de faire appel aux idées de Bakhtine et de Medvedev : la lecture en contexte des textes signés dans les années 1920 par Volochinov met ainsi en doute l'idée qui domine actuellement dans le monde francophone que Bakhtine serait leur véritable auteur.

Clonal deletion of V beta 14-bearing T cells in mice transgenic for mammary tumour virus.

Autoreactive T lymphocytes are clonally deleted during maturation in the thymus. Deletion of T cells expressing particular receptor V beta elements is controlled by poorly defined autosomal dominant genes. A gene has now been identified by expression of transgenes in mice which causes deletion of V beta 14+ T cells. The gene lies in the open reading frame of the long terminal repeat of the mouse mammary tumour virus.

Immunotherapy with Bet v 1 derived contiguous overlapping peptides leads to long-term immunoregulatory responses

Background: Allergen-specific immunotherapy with whole pollen extract may induce anaphylaxis, is poorly standardized and of long duration.We thus designed a randomized, placebo-controlled phase I/II clinical trial in volunteers with birch pollen allergic rhinitis and asthma to evaluate the safety and immunogenicity of a novel immunotherapy based on contiguous overlapping peptides (COPs) derived from Bet v 1, the major birch pollen allergen. Methods: A mixture of three COPs (AllerT™, Anergis SA, Switzerland) spanning the whole Bet v 1 molecule was selected for its inability to bind IgE. Prior to the pollen season, AllerT (in Alum) was injected subcutaneously to 15 adult volunteers at D0 (57 g), D7, D14, D21 and D51 (95 g each). Control volunteers (n = 5) only received the adjuvant. Results: Overall AllerT was safe. No serious adverse events and no immediate allergic reactions were reported. AllerT induced a vigorous early Bet v 1 specific immune response marked by vaccine associated INF- and IL- 10 secretion. This contributed to a strong anti-Bet v 1-specific IgG4 enhancement. Moreover, 2 months after the second season post treatment (July 2010), serum Bet v 1 specific IgG4 response was still markedly increased as compared to pre-treatment values and to placebo whereas post seasonal Bet v 1 specific IgE titers were similar to baseline values. Conclusion: Our data indicate that immunotherapy with a mixture of three COPs derived from Bet v 1 (AllerT) was safe and immunogenic, and led to long-term immunological memory.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas.

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.