Initiation de l'adhésion leucocytaire : rôle des fucosyltransférases et des ligands des sélectines

RESUME La dissémination extramédullaire des cellules blastiques est une complication majeure des leucémies myéloïdes (LMA) ou lymphoïdes aiguës (LLA). La migration des cellules blastiques dépend de mécanismes semblables à ceux qui régulent la migration des leucocytes dans un site d'inflammation. Parmi ceux-ci, les oligosaccharides fucosylés décorant les ligands des sélectines jouent un rôle clé en interagissant avec les sélectines. PSGL-1 (P-Selectin Glycoprotein Ligand-1) est une protéine de 240 kD, exprimée à la surface des leucocytes, permettant de soutenir le roulement leucocytaire sur les sélectines, le long de la paroi vasculaire. L'interaction de PSGL-1 avec les sélectines nécessite des modifications post-traductionnelles de type sialylation, sulfatation , N et 0-glycosylation. Parmi les enzymes impliqués, les α1,3-fucosyltransférases jouent un rôle important dans la biosynthèse d'oligosaccharides fucosylés, ligands des sélectines (sLex, Lex, VIM-2, CLA). Comme l'expression des α1,3-fucosyltransférases par les cellules blastiques leucémiques n'a pas été étudiée précédemment, nous l'avons recherchée dans 120 cas de leucémies aiguës. Les ARNm des FucT-IV et -VII ont été détectés, par RT-PCR, dans tous les cas testés. L'ARNm de la FucT-IX n'a été observé que dans 40% des leucémies aiguës (48/120). L'ARNm de la FucT-IX est détecté dans 65% des LMA (47/72) et, moins fréquemment, dans 26% des LLA (11/42). A noter que les cas de LLA exprimant la FucT-IX correspondent essentiellement à des LLA secondaires à la transformation d'une leucémie myéloïde chronique ou des LLA de la lignée B de type leucémie/lymphome de Burkitt. L'expression de PSGL-1 et des oligosaccharides fucosylés par les blastes varie significativement parmi les LMA et les LLA : Lex, VIM-2 et sLex étant exprimés plus fréquemment par les myéloblastes que par les lymphoblastes. Le rôle des FucT-IV, -VII et -IX dans la synthèse des Lex, VIM-2, CLA et sLex a été examiné en exprimant l'ADNc de chaque FucT dans des cellules CHO. L'immunophénotypisation des transfectants indique que la FucT-VII synthétise sLex et CLA, mais pas Lex et VIM-2. Lex et VIM-2 sont générés par la FucT-IV. La FucT-IX ne participe qu'à la synthèse de Lex, sa capacité de synthèse de VIM-2 dans les cellules CHO est très faible. Le rôle de la FucT-IX dans la régulation du roulement cellulaire dépendant des sélectines a été testé dans des conditions de flux. Les vitesses de roulement des cellules CHO co-exprimant la FucT-LX, la core-2 01,6-N-acetylglucosaminyltransferase et PSGL-1 sont très élevées sur la P-sélectine (médiane : 497.95 µm/s, n=96) alors qu'elles sont beaucoup plus lentes sur la E-sélectine (médiane 7 µm/s, n=64). Les recrutements sur la E-sélectine des cellules CHO-C2F9PSGL¬1 et des CHO-C2F7PSGL-1 sont similaires (moyenne ± SEM : 127.44 ± 4.38 vs. 151.16 ± 3.16 cellules/min/mm2, n=5). Celui des cellules CHO-C2F4PSGL-1 est par contre plus faible (54.20 ± 2.13 cellules/min/mm2, n=5). Ces résultats indiquent que la FucT-IX est impliquée dans la biosynthèse de Lex, VIM-2 et CLA et qu'elle régule l'interaction des cellules CHO avec la E-sélectine. Contrairement aux FucT-IV et -VII, la FucT-IX ne joue qu'un rôle mineur dans la régulation du roulement cellulaire sur la L- et la P-sélectine. L'expression fréquente de la FucT-IX par les myéloblastes suggère qu'elle pourrait participer avec les FucT-IV et -VII à la régulation de la migration cellulaire dépendant de la E-sélectine. Finalement, ce travail de thèse a été étendu à l'identification des protéines cytoplasmiques qui interagissent avec le domaine cytoplasmique de PSGL-1 et qui pourraient être impliquées dans la transmission de signaux intracellulaires. Les ligands intracellulaires de PSGL-1 seront identifiés par la technique du double hybride qui nous a déjà permis de confirmer que syk et la N-moésine se lient au domaine cytoplasmique de PSGL-1. Des ligands supplémentaires seront identifiés employant une librairie provenant des cellules souches hématopoïétiques comme proie. ABSTRACT Blast cell dissemination is a major complication of acute myeloblastic (AML) and lymphoblastic leukemia (ALL). Blast cell migration is dependent on mechanisms that are similar to those which regulate leukocyte migration into inflammatory lesions. Among them, fticosylated oligosaccharides that decorate selectin ligands play a key role by interacting with selectins. PSGL-1 (P-Selectin Glycoprotein Ligand-1) is a 240 kD glycoprotein constitutively expressed on leucocytes and which supports leukocyte rolling on selectins. PSGL-1 interaction with selectins is dependent on post-translational modifications such as sialylation, sulfation, N- and 0-glycosylation. Among the involved enzymes, the α1,3-fucosyltransferases (FucT) play a major role in generating cell surface glycoconjugates carrying fucosylated oligosaccharides which interact with selectins (sLex, Lex, VIM-2, CLA). Since no information is available on the expression of α1,3-fucosyltransferases by leukemic blast cells, we examined it in 120 cases of acute leukemia. FucT-IV and -VII mRNAs were detected, by RT-PCR, in all tested cases. In contrast, the presence of FucT-IX mRNA was shown in only 40% of patients with acute leukemia (48/120). FucT-IX mRNA was detected in 65% of AML (47/72) and, less frequently, in 26% of ALL (11/42). Importantly, all ALL cases expressing FucT-IX were either secondary leukemia resulting from the transformation of chronic myelocytic leukemia in acute lymphoblastic leukemia or mature B-ALL (FAB L3 subtype or Burkitt lymphoma/leukemia according to WHO classification). FucT-IX was not detected in precursor B or T-ALL. The expression of PSGL-1 and fucosylated epitopes was significantly different among AML and ALL, Lex, VIM-2 and sLex being more frequently expressed by myeloblasts than by lymphoblasts. The role of FucT-IV, -VII and -IX in the biosynthesis of Lex, VIM-2, CLA and sLex was examined by expressing the cDNA of each α1,3-FucT in CHO cells. Immunophenotypic analysis of CHO transfectants indicated that FucT-VII synthesizes sLex and CLA but not Lex or VIM-2. Lex and CLA were generated by both FucT-IV and -IX. FucT-IV and FucT-IX differed in their ability to synthesize VIM-2, FucT-IX being less efficient than FucT-IV. The role of FucT-IX in regulating selectin-dependent rolling was assessed under hydrodynamic flow conditions. P-selectin-dependent interactions were transient and occurred at high velocities (median: 497.95 1,µm/s, n=96). In contrast, much slower rolling velocities were observed on E-selectin (median: 7 µm/s, n=64). The recruitment of CHO-C2F9PSGL-1 and CHO-C2F7PSGL-1 cells was similar on E-selectin (mean ± SEM: 127.44 ± 4.38, n=5 vs 151.16 ± 3.16 cells/min/mm2, n=5). In the other hand, CHO-C2F4PSGL-1 cells were less efficiently recruited on E-selectin (54.20 ± 2.13 cells/min/mm2, n=5). This results indicate that FucT-IX is involved in the biosynthesis of Lex, VIM-2 and CLA and that it confers E-selectin binding activity to CHO cells. By contrast to FucT-IV and -VII, FucT-IX had a minor role in regulating P- and L-selectin-dependent rolling on CHO transfectants. The frequent expression of FucT-IX in myeloblasts suggests that it may participate with FucT-IV and -VII in regulating E-selectin-dependent cell migration into tissues. Finally, this thesis work was extended to the identification of the cytoplasmic proteins interacting with cytoplasmic domain of PSGL-1 that may be involved in transducing intracellular signals. We planned to identify these intracellular ligands of PSGL-1 by using the double hybrid technique and already confirmed that syk and N-moesin bind to the cytoplasmic domain of PSGL-1. Additional PSGL-1 ligands will be sought by the same technique using a CD34+ stem cell library as pray. RESUME DESTINE A UN LARGE PUBLIC : L'adhésion et la migration leucocytaire sont nécessaires à de nombreux processus cellulaires comme la régulation de l'hématopoïèse, mais aussi dans la pathogenèse de l'artériosclérose, des maladies inflammatoires et de la métastatisation des cellules cancéreuses. Les molécules impliquées constituent depuis peu des cibles pour la thérapie du cancer. La migration leucocytaire vers un site d'inflammation dépend de mécanismes complexes, se déroulant en plusieurs étapes, nécessitant l'interaction séquentielle de molécules d'adhésion leucocytaires et endothéliales. Ainsi, chronologiquement, suite à un stimulus inflammatoire, les leucocytes « roulent » sur les cellules endothéliales, sont activées, s'arrêtent et traversent la paroi endothéliale (diapédèse) pour migrer dans les tissus environnants inflammés selon un gradient chimiotactique. La première étape de roulement met en jeu deux molécules principales : PSGL-1 (P-Sélectine Glycoprotéine Ligand-1) du coté des leucocytes et les sélectines du coté de l'endothélium de la paroi vasculaire. L'interaction entre ces deux molécules nécessite des décorations de ces protéines par des sucres, des résidus sulfates et des acides sialiques. Le sucre essentiel à la liaison demeure le fucose qui est attaché aux protéines grâce à des enzymes de la famille des fucosyltransferases. Actuellement, neuf fucosyltransférases humaines ont été identifiées et désignées sous FucT-I à IX. La FucT-IX, dernière fucosyltransférase clonée, a un faible degré d'homologie avec les autres fucosyltransférases mais sa séquence est extrêmement conservée entre les espèces. Ceci traduit son importance par une forte résistance à la pression évolutive. L'examen de son expression au sein de 120 cas de leucémies aiguës a mis en évidence son comportement atypique. En effet, alors que les autres FucTs sont toujours présentes, la FucT¬IX ne s'exprime que dans un cas sur deux en moyenne avec une préférence plus importante pour les leucémies myéloïdes. Ainsi, une étude plus approfondie de cet enzyme à mis en évidence sa capacité à induire une interaction cellulaire plus spécifique de la E-sélectine. Elle décore non seulement des protéines de surface, mais aussi certainement les glycolipides constituant la membrane cellulaire.

Protective effects of maternal nutritional supplementation with lactoferrin on growth and brain metabolism.

Background:Intrauterine growth restriction (IUGR) is a major risk factor for both perinatal and long-term morbidity. Bovine lactoferrin (bLf) is a major milk glycoprotein considered as a pleiotropic functional nutrient. The impact of maternal supplementation with bLf on IUGR-induced sequelae, including inadequate growth and altered cerebral development, remains unknown.Methods:IUGR was induced through maternal dexamethasone infusion (100 μg/kg during last gestational week) in rats. Maternal supplementation with bLf (0.85% in food pellet) was provided during both gestation and lactation. Pup growth was monitored, and Pup brain metabolism and gene expression were studied using in vivo (1)H NMR spectroscopy, quantitative PCR, and microarray in the hippocampus at postnatal day (PND)7.Results:Maternal bLf supplementation did not change gestational weight but increased the birth body weight of control pups (4%) with no effect on the IUGR pups. Maternal bLf supplementation allowed IUGR pups to recover a normalized weight at PND21 (weaning) improving catch-up growth. Significantly altered levels of brain metabolites (γ-aminobutyric acid, glutamate, N-acetylaspartate, and N-acetylaspartylglutamate) and transcripts (brain-derived neurotrophic factor (BDNF), divalent metal transporter 1 (DMT-1), and glutamate receptors) in IUGR pups were normalized with maternal bLf supplementation.Conclusion:Our data suggest that maternal bLf supplementation is a beneficial nutritional intervention able to revert some of the IUGR-induced sequelae, including brain hippocampal changes.

Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes.

The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible C. albicans (O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the CDR1, CDR2, CaMDR1, and FLU1 transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (P = 0.05 at 48 h with an inoculum of 10(3) CFU/ml) and a more rapid fungicidal effect of FLC-CY (P = 0.05 at 24 h with an inoculum of 10(3) CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (P = 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (P = 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a "dual-hit" mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by CDR1, CDR2, CaMDR1, and FLU1 genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.

Astrocytic αVβ3 integrin inhibits neurite outgrowth and promotes retraction of neuronal processes by clustering Thy-1.

Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to α(V)β(3) integrin in trans eliciting responses in astrocytes. Nonetheless, whether α(V)β(3) integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of α(V)β(3) integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous α(V)β(3) integrin restricted neurite outgrowth. Likewise, α(V)β(3)-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(-) CAD cells. In differentiating primary neurons exposed to α(V)β(3)-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, α(V)β(3)-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by α(V)β(3) integrin. Binding of α(V)β(3)-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, α(V)β(3)-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that α(V)β(3) integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.

DNA vaccine encoding nucleocapsid and surface proteins of wild type canine distemper virus protects its natural host against distemper.

Canine distemper virus (CDV), a member of the genus Morbillivirus induces a highly infectious, frequently lethal disease in dogs and other carnivores. Current vaccines against canine distemper consisting of attenuated viruses have been in use for many years and have greatly reduced the incidence of distemper in the dog population. However, certain strains may not guarantee adequate protection and others can induce post vaccinal encephalitis. We tested a DNA vaccine for its ability to protect dogs, the natural host of CDV, against distemper. We constructed plasmids containing the nucleocapsid, the fusion, and the attachment protein genes of a virulent canine distemper virus strain. Mice inoculated with these plasmids developed humoral and cellular immune responses against CDV antigens. Dogs immunized with the expression plasmids developed virus-neutralizing antibodies. Significantly, vaccinated dogs were protected against challenge with virulent CDV, whereas unvaccinated animals succumbed to distemper.

Genome-wide association study identifies loci influencing concentrations of liver enzymes in plasma.

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function.

Cardiovascular drug interactions with tyrosine kinase inhibitors

Imatinib mesylate, a selective inhibitor of tyrosine kinases, has excellent efficacy in the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumour (GIST). Inducing durable responses and achieving prolonged survival, it has become the standard of care for the treatment of these diseases. It has opened the way to the development of additional tyrosine kinase inhibitors (TKIs), including sunitinib, nilotinib, dasatinib and sorafenib, all indicated for the treatment of various haematological malignancies and solid tumours. TKIs are prescribed for prolonged periods and are often taken by patients with - notably cardiovascular - comorbidities. Hence TKIs are regularly co-administered with cardiovascular drugs, with a considerable risk of potentially harmful drug-drug interactions due to the large number of agents used in combination. However, this aspect has received limited attention so far, and a comprehensive review of the published data on this important topic has been lacking. We review here the available data and pharmacological mechanisms of interactions between commonly prescribed cardiovascular drugs and the TKIs marketed at present. Regular updating of the literature on this topic will be mandatory, as will the prospective reporting of unexpected clinical observations, given the fact that these drugs have been only recently marketed.

Systems analysis of MVA-C induced immune response reveals its significance as a vaccine candidate against HIV/AIDS of clade C.

Based on the partial efficacy of the HIV/AIDS Thai trial (RV144) with a canarypox vector prime and protein boost, attenuated poxvirus recombinants expressing HIV-1 antigens are increasingly sought as vaccine candidates against HIV/AIDS. Here we describe using systems analysis the biological and immunological characteristics of the attenuated vaccinia virus Ankara strain expressing the HIV-1 antigens Env/Gag-Pol-Nef of HIV-1 of clade C (referred as MVA-C). MVA-C infection of human monocyte derived dendritic cells (moDCs) induced the expression of HIV-1 antigens at high levels from 2 to 8 hpi and triggered moDCs maturation as revealed by enhanced expression of HLA-DR, CD86, CD40, HLA-A2, and CD80 molecules. Infection ex vivo of purified mDC and pDC with MVA-C induced the expression of immunoregulatory pathways associated with antiviral responses, antigen presentation, T cell and B cell responses. Similarly, human whole blood or primary macrophages infected with MVA-C express high levels of proinflammatory cytokines and chemokines involved with T cell activation. The vector MVA-C has the ability to cross-present antigens to HIV-specific CD8 T cells in vitro and to increase CD8 T cell proliferation in a dose-dependent manner. The immunogenic profiling in mice after DNA-C prime/MVA-C boost combination revealed activation of HIV-1-specific CD4 and CD8 T cell memory responses that are polyfunctional and with effector memory phenotype. Env-specific IgG binding antibodies were also produced in animals receiving DNA-C prime/MVA-C boost. Our systems analysis of profiling immune response to MVA-C infection highlights the potential benefit of MVA-C as vaccine candidate against HIV/AIDS for clade C, the prevalent subtype virus in the most affected areas of the world.

Concentrations sériques de protéines placentaires au 1er trimestre dans des grossesses monofoetales et multiples après FIV : implications pour le dépistage de la trisomie 21 [First trimester serum concentrations of placental proteins in singleton and multiple IVF pregnancies: implications for Down syndrome screening]

First trimester biochemical trisomy screening is based on serum concentrations of pregnancy-associated plasma protein A (PAPP-A) and human chorionic gonadotrophin (hCG). Our aim was to confirm previously suggested modifications in serum marker concentrations after in vitro fertilisation (IVF) and embryo transfer (ET), and to assess the need of establishing normal medians for trisomy screening in these. We compared 56 singleton pregnancies obtained after ET (of which 40 in gonadotrophin stimulation cycles) with 120 gestation-matched spontaneous controls. For multiple pregnancies, 17 treated cycles were compared with 25 controls. The levels of PAPP-A, hCG, and pregnancy-specific β1-glycoprotein were determined and compared between treated and spontaneous pregnancies. Serum PAPP-A levels were reduced in pregnancies achieved after gonadotrophin-stimulated IVF and ET, and this was more pronounced in earlier gestational stages. SP1 followed the same trend, while hCG tended to be increased, and this not only in pregnancies obtained from gonadotrophin-stimulated but also from oestrogen supported cycles, and with a more pronounced effect in the later gestational ages examined here. Decreased PAPP-A together with increased hCG concentrations produce falsely elevated results in first trimester Down syndrome screening, but we do not recommend the establishment of normal medians for IVF pregnancies due to the variations in stimulation protocols.

LIGHT/HVEM/LTβR interaction as a target for the modulation of the allogeneic immune response in transplantation.

The exchange of information during interactions of T cells with dendritic cells, B cells or other T cells regulates the course of T, B and DC-cell activation and their differentiation into effector cells. The tumor necrosis factor superfamily member LIGHT (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) is transiently expressed upon T cell activation and modulates CD8 T cell-mediated alloreactive responses upon herpes virus entry mediator (HVEM) and lymphotoxin β receptor (LTβR) engagement. LIGHT-deficient mice, or WT mice treated with LIGHT-targeting decoy receptors HVEM-Ig, LTβR-Ig or sDcR3-Ig, exhibit prolonged graft survival compared to untreated controls, suggesting that LIGHT modulates the course and severity of graft rejection. Therefore, targeting the interaction of LIGHT with HVEM and/or LTβR using recombinant soluble decoy receptors or monoclonal antibodies represent an innovative therapeutic strategy for the prevention and treatment of allograft rejection and for the promotion of donor-specific tolerance.

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD.

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.

What influence do anticoagulants have on oral implant therapy? A systematic review.

OBJECTIVES: This systematic review aims to assess the risks (both thromboembolic and bleeding) of an oral anticoagulation therapy (OAT) patient undergoing implant therapy and to provide a management protocol to patients under OAT undergoing implant therapy. MATERIAL AND METHODS: Medline, Cochrane Data Base of Systematic Reviews, the Cochrane Central Register of Controlled Trials and EMBASE (from 1980 to December 2008) were searched for English-language articles published between 1966 and 2008. This search was completed by a hand research accessing the references cited in all identified publications. RESULTS: Nineteen studies were identified reporting outcomes after oral surgery procedures (mostly dental extractions in patients on OAT following different management protocols and haemostatic therapies). Five studies were randomized-controlled trials (RCTs), 11 were controlled clinical trials (CCTs) and three were prospective case series. The OAT management strategies as well as the protocols during and after surgery were different. This heterogeneity prevented any possible data aggregation and synthesis. The results from these studies are very homogeneous, reporting minor bleeding in very few patients, without a significant difference between the OAT patients who continue with the vitamin K antagonists vs. the patients who stopped this medication before surgery. These post-operative bleeding events were controlled only with local haemostatic measures: tranexamic acid mouthwashes, gelatine sponges and cellulose gauzes's application were effective. Post-operative bleeding did not correlate with the international normalised ratio (INR) status. In none of the studies was a thromboembolic event reported. CONCLUSIONS: OAT patients (INR 2-4) who do not discontinue the AC medication do not have a significantly higher risk of post-operative bleeding than non-OAT patients and they also do not have a higher risk of post-operative bleeding than OAT patients who discontinue the medication. In patients with OAT (INR 2-4) without discontinuation, topical haemostatic agents were effective in preventing post-operative bleeding. OAT discontinuation is not recommended for minor oral surgery, such as single tooth extraction or implant placement, provided that this does not involve autogenous bone grafts, extensive flaps or osteotomy preparations extending outside the bony envelope. Evidence does not support that dental implant placement in patients on OAT is contraindicated.

Perinuclear Mlp proteins down-regulate gene expression in response to a defect in mRNA export

Résumé Une caractéristique des cellules eucaryotes est le confinement du matériel génétique (ADN/DNA) dans le noyau. Pour décoder cette information, un ARN messager (mRNA) est d'abord transcrit sous forme d'un ARN prémessager (pré-mRNA). Ce-dernier doit subir plusieurs étapes de maturation pour aboutir à une particule ribonucléoprotéique (mRNP) qui sera exportée vers le cytoplasme et traduite en protéine. La protéine de levure Mex67p et son homologue humain TAP sont des récepteurs d'export médiant la translocation du mRNP au travers des complexes du pore nucléaire (NPC). Mex67p/TAP ne se lient pas directement au mRNA, mais nécessitent la présence de protéines adaptatrices, telles que Yra1p et son homologue humain REF1. Afin d'identifier de nouveaux facteurs impliqués dans l'export des mRNPs ou de nouvelles fonctions pour Yra1p, nous avons effectué un crible génétique avec un mutant thermosensible de Yra1p, GFP-yra 1 -8. Ce mutant présente un défaut d'export des mRNAs et une diminution des niveaux de transcrits du gène rapporteur LacZ ainsi que de certains transcrits endogènes. Nous avons trouvé que la perte de Mlp2p, ou d'une protéine hautement similaire, Mlp1p, restaure la croissance du mutant GFP-yra1-8 à température restrictive. Mlp1p et Mlp2p sont des protéines nucléaires, dont l'homologue humain est TPR. Les Mlp (myosin¬like proteins) ainsi que TPR forment des structures filamenteuses ancrées aux NPC. Bien que la fonction des Mlp ne soit pas clairement définie, un rôle dans la biogenèse et la surveillance des mRNPs a été récemment proposé. Notre étude montre que la perte des Mlp, non seulement restaure la croissance de GFP-yra1-8, mais augmente aussi les niveaux des transcrits LacZ et facilite leur apparition dans le cytoplasme. Des expériences d'immunoprécipitations de la chromatine révèlent que Mlp2p diminue le taux de synthèse du transcrit LacZ dans GFP-yra1-8. Des analyses du transcriptome montrent que Mlp2p réduit aussi les niveaux d'une population de transcrits endogènes dans le mutant. Finalement, des localisations in situ suggèrent que la transcription du rapporteur LacZ a lieu à la périphérie du noyau, à proximité des Mlp. Ainsi, les protéines Mlp pourraient préférentiellement diminuer la transcription de gènes exprimés à la périphérie nucléaire. Nous montrons aussi que Yra1p interagit génétiquement avec Nab2p une protéine liée au mRNA et impliquée dans son export, mais non avec d'autres protéines également impliquées dans l'export des mRNAs. Les résultats obtenus soutiennent un modèle où les protéines Yra1p et Nab2p sont nécessaires à l'arrimage des mRNPs sur la plate-forme des Mlp. Si ces signaux manquent ou sont défectueux, les mRNPs ne peuvent pas poursuivre leur trajet vers le canal central du NPC. Ce bloc induirait par la suite une diminution de la transcription d'une population de gènes potentiellement localisée à la périphérie nucléaire. Dans son ensemble, cette étude suggère que les protéines Mlp établissent un lien entre la transcription de certains mRNAs et leur export au travers du pore nucléaire. Summary A hallmark of the eukaryotic cell is the packaging of DNA in the nucleus. To decode the genetic information, a messenger RNA (mRNA) is first synthesized as a pre-mRNA molecule, which undergoes different maturation steps resulting in an mRNP (messenger RNA ribonucleoprotein), which can be actively transported to the cytoplasm and translated into a protein. Yeast Mex67p and its human homologue TAP are export receptors mediating mRNP translocation through the nuclear pore complex (NPC). The recruitment of Mex67p/TAP to mRNA is mediated by mRNA export adaptors of the evolutionarily conserved REF (RNA and Export Factor binding) family: yeast Yra1p and human REF1. To uncover new functions of Yra1p or new factors implicated in mRNA export, we performed a genetic screen with a themiosensitive (ts) yra1 mutant, GFP-yra1-8. This mutant exhibits mRNA export defects and a decrease in the levels of LacZ reporter and certain endogenous transcripts. We found that the loss of Mlp2p, or the related Mlp1p protein, substantially rescues the growth defect of the GFP-yra1 -8 mutant. Mlp1p and M1p2p are large non-essential proteins, homologous to human TPR, proposed to form intra-nuclear filamentous structures anchored at the NPC. Their role is not clearly defined, but they have been implicated in mRNP biogenesis and surveillance. Our study shows that loss of Mlp proteins not only restores growth of GFP-yra1-8, but also rescues LacZ mRNA levels and increases their appearance in the cytoplasm. Chromatin immunoprecipitation and pulse chase experiments indicate that Mlp2p down-regulates LacZ mRNA synthesis in GFP-yra1-8. DNA micro- array analyses reveal that Mlp2p also reduces the levels of a subset of cellular transcripts in the yra1 mutant strain. In situ localizations suggest that LacZ transcription occurs at the nuclear periphery, in close proximity to Mlp proteins. Thus, Mlp proteins may preferentially down-regulate genes expressed at the nuclear periphery. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlp proteins rescues the growth defect of yra1 and nab2, but not other mRNA export mutants. The data support a model in which Nab2p and Yra1p are required for rnRNP docking to the Mlp platform. Lack of these signals prevents mRNPs from crossing the Mlp gate. This block may then negatively feed-back on the transcription of a subset of genes, potentially located at the nuclear envelope. Overall, this study suggests that perinuclear Mlp proteins establish a link between mRNA transcription and export.

Poxvirus vector-based HIV vaccines.

PURPOSE OF REVIEW: In this review, we will provide the scientific rationale for the use of poxvirus vectors in the field of HIV vaccines, the immunological profile of the vaccine-induced immune responses, an update on the current use of poxvirus vector-based vaccines in HIV vaccine clinical trials, and the development of new modified poxvirus vectors with improved immunological profile. RECENT FINDINGS: An Ad5-HIV vaccine was tested in a phase IIb clinical trial (known as the Step trial). Vaccinations in the Step trial were discontinued because the vaccine did not show any effect on acquisition of infection and on viral load. After the disappointing failure of the Step trial, the field of HIV vaccine has regained enthusiasm and vigour due to the promising protective effect observed in the phase III efficacy trial (known as RV-144) performed in Thailand which has tested a poxvirus-gp120 combination. SUMMARY: The RV-144 phase III has provided for the first time evidence that an HIV vaccine can prevent HIV infection. The results from the RV-144 trial are providing the scientific rationale for the future development of the HIV vaccine field and for designing future efficacy trials.

Prediction of free imatinib concentrations based on total plasma concentrations in patients with gastrointestinal stromal tumours.

AIM: Total imatinib concentrations are currently measured for the therapeutic drug monitoring of imatinib, whereas only free drug equilibrates with cells for pharmacological action. Due to technical and cost limitations, routine measurement of free concentrations is generally not performed. In this study, free and total imatinib concentrations were measured to establish a model allowing the confident prediction of imatinib free concentrations based on total concentrations and plasma proteins measurements. METHODS: One hundred and fifty total and free plasma concentrations of imatinib were measured in 49 patients with gastrointestinal stromal tumours. A population pharmacokinetic model was built up to characterize mean total and free concentrations with inter-patient and intrapatient variability, while taking into account α1 -acid glycoprotein (AGP) and human serum albumin (HSA) concentrations, in addition to other demographic and environmental covariates. RESULTS: A one compartment model with first order absorption was used to characterize total and free imatinib concentrations. Only AGP influenced imatinib total clearance. Imatinib free concentrations were best predicted using a non-linear binding model to AGP, with a dissociation constant Kd of 319 ng ml(-1) , assuming a 1:1 molar binding ratio. The addition of HSA in the equation did not improve the prediction of imatinib unbound concentrations. CONCLUSION: Although free concentration monitoring is probably more appropriate than total concentrations, it requires an additional ultrafiltration step and sensitive analytical technology, not always available in clinical laboratories. The model proposed might represent a convenient approach to estimate imatinib free concentrations. However, therapeutic ranges for free imatinib concentrations remain to be established before it enters into routine practice.