Mechanisms governing lentivirus integration site selection.

An essential step of the life cycle of retroviruses is the stable insertion of a copy of their DNA genome into the host cell genome, and lentiviruses are no exception. This integration step, catalyzed by the viral-encoded integrase, ensures long-term expression of the viral genes, thus allowing a productive viral replication and rendering retroviral vectors also attractive for the field of gene therapy. At the same time, this ability to integrate into the host genome raises safety concerns regarding the use of retroviral-based gene therapy vectors, due to the genomic locations of integration sites. The availability of the human genome sequence made possible the analysis of the integration site preferences, which revealed to be nonrandom and retrovirus-specific, i.e. all lentiviruses studied so far favor integration in active transcription units, while other retroviruses have a different integration site distribution. Several mechanisms have been proposed that may influence integration targeting, which include (i) chromatin accessibility, (ii) cell cycle effects, and (iii) tethering proteins. Recent data provide evidence that integration site selection can occur via a tethering mechanism, through the recruitment of the lentiviral integrase by the cellular LEDGF/p75 protein, both proteins being the two major players in lentiviral integration targeting.

Immune response mechanisms and protection against "Plasmodium falciparum" and "Plasmodium berghei"

Malaria is one of the most important tropical and infectious diseases causing many deaths and enormous social and economic consequences, particularly in the developing countries. Despite of widely use of anti-malaria drugs and insecticide, the development of successful vaccines constitutes one of the main strategies to control malaria transmission. Several proteins expressed from blood stage such as merozoite surface proteins (MSP] or liver stage as circumsporozoite protein (CSP) are shown to be the targets of immune responses in humans and in animals. Thus, several studies have illustrated that natural infection and laboratory immunizations of humans and animals with Plasmodium sporozoite (SPZ) and its derivate-proteins (peptides) can elicit protection and control of parasite infection. However, a clear understanding of immune response against defined Plasmodium proteins should be the prerequisite conditions before any development of appropriate vaccines. In this order, our study focused on the immune responses to MSP2 (dimorphic and C-terminal fragments) in human and mice; and the mechanisms by which mouse infected hepatocytes present Plasmodium antigens to CD8+ T-cells to induce protective immunity in mice.¦The first part of this work shows that infected hepatocytes can present Plasmodium antigens to PbCSP-specific CD8+ T-cells and induce a protective immunity in mice. Here, this was addressed in vivo and showed that the infected hepatocytes were able of stimulating of primed-and naive-CD8+ T-cell clones and induced fully protective immunity against SPZ challenge. The role of infected hepatocytes in antigen presentation was illustrated here by their graft into immuno-deficient mice and depletion of cosspresenting dentritic cells (DCs) that are known to have key role in the activation of CD8+ T-cells during the liver cycle stage of Plasmodium.¦The second part of this project concerned the fine specificity of Ab responses regarding D and C regions of the two allelic families of MSP2 (3D7 and FC27). Covering of the two regions by overlapping-20 mers led to delineate the epitopes in the different endemic areas and different age groups of donors. The major epitopes characterizing D or C regions were conserved in different endemic areas (P12/P13 and P15/P16 for the 3D7-D, P23/24 and P25/26 for the FC27-D; P29/P30 for the C region). This offers thus, the possibility of a multi-epitope vaccine design including the major epitopes from the two domains of the two allelic MSP2 families. On the other, the 20 mers, particularly some major epitopes of the 3D7-Dregion (P12, P13 and P16) belonged to the epitopes that presented a high probability to be associated with protection in the children group [1 to 5 year-old). In addition, D and C LSP purified Abs (pAbs) recognized merozoite derived polypeptides and native proteins. A crossreactivity activity of homologous pAbs against the heterologous was also illustrated between the two allelic MSP2 parasites. Finally, the functional analysis of D regions pAbs showed an inhibition of Plasmodium falciparum growth suggesting the functional biological activity of the D region pAbs in the control of malaria.¦The last part of this project aimed the evaluation of the immunogenicity of the D and C region LSPs of the two allelic MSP2 families in the presence of adjuvants for the possible use in clinical trial study in humans. The MSP2 LSP mixture showed that D and C were immunogenic and defined limited epitopes (whose intensity of immune responses) depending on the adjuvants and mouse strain for the D regions. The major epitopes characterizing the C region were usually conserved in different strains of mouse and adjuvants used. Furthermore, the single region (either with D or C) immunization of mice confirmed the immunogenicity and the presence of their limited epitopes. We concluded that the possibility to finely delineate in animals the immune responses to antigens might help to select optimal antigen/adjuvant combinations to be tested later in clinical trials. Thus, formulation of glucopyranosyl-lipid A stable emulsion, GLA-SE (toll like receptor (TLR) 4 agonist) and its different combination (CpG: TLR9 agonist and GDQ: LR7 agonist) with MSP2 LSP was better than with alum, montanide ISA 720 (Mt) and virosome. Immunization of mice with allelic LSP did not show a crossreactivity between the two allelic MSP2 parasites unlike as humans, suggesting that the crossreactivity could be acquired during natural infection of the population who are usually exposed to both allelic parasite forms (3D7 and FC27).¦Nevertheless, similar epitope of D (P12, P13 and P25) and C (P29) regions have been found both in mice and human. This offers an opportunity to compare their epitopes in naïve immunized donors with LSPs and naturally infected populations in the endemic areas.

Interest of qualitative and quantitative profiling of endocannabinoids for evaluation of their implication in drug addiction process

Introduction: In the middle of the 90's, the discovery of endogenous ligands for cannabinoid receptors opened a new era in this research field. Amides and esters of arachidonic acid have been identified as these endogenous ligands. Arachidonoylethanolamide (anandamide or AEA) and 2-Arachidonoylglycerol (2-AG) seem to be the most important of these lipid messengers. In addition, virodhamine (VA), noladin ether (2-AGE), and N-arachidonoyl dopamine (NADA) have been shown to bind to CB receptors with varying affinities. During recent years, it has become more evident that the EC system is part of fundamental regulatory mechanisms in many physiological processes such as stress and anxiety responses, depression, anorexia and bulimia, schizophrenia disorders, neuroprotection, Parkinson disease, anti-proliferative effects on cancer cells, drug addiction, and atherosclerosis. Aims: This work presents the problematic of EC analysis and the input of Information Dependant Acquisition based on hybrid triple quadrupole linear ion trap (QqQLIT) system for the profiling of these lipid mediators. Methods: The method was developed on a LC Ultimate 3000 series (Dionex, Sunnyvale, CA, USA) coupled to a QTrap 4000 system (Applied biosystems, Concord, ON, Canada). The ECs were separated on an XTerra C18 MS column (50 × 3.0 mm i.d., 3.5 μm) with a 5 min gradient elution. For confirmatory analysis, an information-dependant acquisition experiment was performed with selected reaction monitoring (SRM) as survey scan and enhanced produced ion (EPI) as dependant scan. Results: The assay was found to be linear in the concentration range of 0.1-5 ng/mL for AEA, 0.3-5 ng/mL for VA, 2-AGE, and NADA and 1-20 ng/mL for 2-AG using 0.5 mL of plasma. Repeatability and intermediate precision were found less than 15% over the tested concentration ranges. Under non-pathophysiological conditions, only AEA and 2-AG were actually detected in plasma with concentration ranges going from 104 to 537 pg/mL and from 2160 to 3990 pg/mL respectively. We have particularly focused our scopes on the evaluation of EC level changes in biological matrices through drug addiction and atherosclerosis processes. We will present preliminary data obtained during pilot study after administration of cannabis on human patients. Conclusion: ECs have been shown to play a key role in regulation of many pathophysiological processes. Medical research in these different fields continues to growth in order to understand and to highlight the predominant role of EC in the CNS and peripheral tissues signalisation. The profiling of these lipids needs to develop rapid, highly sensitive and selective analytical methods.

The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis.

The fatty acid oxygenation up-regulated 2 (fou2) mutant in Arabidopsis thaliana creates a gain-of-function allele in a non-selective cation channel encoded by the Two Pore Channel 1 (TPC1) gene. This mutant genetically implicates cation fluxes in the control of the positive feedback loop whereby jasmonic acid (JA) stimulates its own synthesis. In this study we observed extensive transcriptome reprogramming in healthy fou2 leaves closely resembling that induced by treatment with methyl jasmonate, biotic stresses and the potassium starvation response. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA-inducible proteins. In agreement with these analyses, epistasis studies performed by crossing fou2 with aos indicated that elevated levels of JA in fou2 are the major determinant of the mutant phenotype. In addition, generation of fou2 aba1-5, fou2 etr1-1 and fou2 npr1-1 double mutants showed that the fou2 phenotype was only weakly affected by ABA levels and unaffected by mutations in NPR1 and ETR1. The results now suggest possible mechanisms whereby fou2 could induce JA synthesis/signaling early in the wound response. In contrast to fou2, transcriptome analysis of a loss-of-function allele of TPC1, tpc1-2, revealed no differential expression of JA biosynthesis genes in resting leaves. However, the analysis disclosed reduced mRNA levels of the pathogenesis-related genes PDF1.2a and THI2.1 in healthy and diseased tpc1-2 leaves. The results suggest that wild-type TPC1 contributes to their expression by mechanisms somewhat different from those affecting their expression in fou2.

Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration.

The molecular mechanisms underlying lymphocyte extravasation remain poorly characterized. We have recently identified junctional adhesion molecule-2 (JAM-2), and have shown that antibodies to JAM-2 stain high endothelial venules (HEVs) within lymph nodes and Peyer patches of adult mice. Here we show that mouse lymphocytes migrate in greater numbers across monolayers of endothelioma cells transfected with JAM-2. The significance of these findings to an understanding of both normal and pathologic lymphocyte extravasation prompted us to clone the human homologue of JAM-2. We herein demonstrate that an anti-JAM-2 antibody, or a soluble JAM-2 molecule, blocks the transmigration of primary human peripheral blood leukocytes across human umbilical vein endothelial cells expressing endogenous JAM-2. Furthermore, we show that JAM-2 is expressed on HEVs in human tonsil and on a subset of human leukocytes, suggesting that JAM-2 plays a central role in the regulation of transendothelial migration.

Molecular mechanisms and effects of a rasgap-derived peptide on metastatic progression

Cancer is the second leading cause of mortality worldwide. Cancer progression leads to metastasis formation, which accounts for more than ninety percent of cancer-related death. Metastases are more difficult to be surgically removed because of their invasive behavior and shape. In addition, during their transformation journey, they become more and more resistant to anticancer drugs. Significant improvements have been achieved in therapy against cancer in recent years but targeting the metastatic cascade remains the Achilles heel of the cure against cancer. A First step in the metastatic process is the escape of cancer cells from the primary tumor site. This involves an increase in cell motility and the concomitant ability to clear a path through the extracellular matrix. From a therapeutic point of view, inhibition of cell migration is a logical approach to develop anti-metastatic drugs. Our lab previously developed a cell permeable peptide derived from a caspase-3-generaied fragment of the RasGAP protein called TAT-RasGAP317-326. This peptide efficiently and specifically sensitizes cancer cells to chemotherapy- and radiotherapy-induced ceil death, which allows decreasing the anticancer drug doses and eventually their associated side- effects. In the present study we discovered that TAT-RasGAP317.326 also increases cell adhesion which was associated with inhibition of cell migration and invasion into the extracellular matrix. The ability of TAT-RasGAP317.326 to increase ceil adhesion involves the dramatic depolymerization of actin cytoskekton together with redistribution of focal adhesions. We found that the inhibitory effects on migration were mediated by a RhoGAP tumor and metastasis suppressor cailed DLC1 (Deleted in Liver Cancer 1). Moreover. DEC 1 was found to be a direct RasGAP-interacting protein and this interaction requires the RasGAP tryptophan 317 residue, the very first RasGAP residue of TAT-RasGAP317.326. We then evaluated the roie of RasGAP fragments in the in vivo metastatic cascade. We found that breast cancer cells overexpressing the parental RasGAP fragment, to which the TAT-RasGAP317.326 peptide belongs, have a markedly decreased ability to form lung metastases. Unfortunately, we were not able to recapitulate these an ti-metastatic effects when TAT-RasGAP317.326 was injected. However, we later understood that this was due to the fact that TAT-RasGAP317.326 was not properly delivered to the primary tumors. Further work, aimed at better understanding of how TAT-RasGAP317.326 functions, revealed that the ten amino acid TAT-RasGAP317.326 peptide could, be narrowed down to a three amino acid TAT-RasGAP317.329 peptide while keeping its sensitizer activity. In parallel, investigations on the RasGAP-DLCl binding indicated that the arginine linger of the DLC1 GAP domain is required for this interaction, which suggests that TAT-RasGAP317.326 modulates the GAP activity of DLC1. Additional work should be performed to fully elucidate its mechanism of action and render TAT-RasGAP317.326 usable as a tool to fight cancer on two fronts, by improving chemotherapy and preventing metastatic progression. - Le cancer est la deuxième cause de mortalité dans le monde. La formation de métastases est la dernière étape de la progression cancéreuse et représente plus du nonante pour cent des morts induites par le cancer. De par leur morphologie et comportement invasifs, ii est difficile d'avoir recours à la chirurgie pour exciser des métastases. De plus, les cellules cancéreuses en progression deviennent souvent de plus en plus résistantes aux drogues anticancéreuses. Ces dernières années, des avancements significatifs ont contribué à l'amélioration de la lutte contre le cancer. Néanmoins, pouvoir cibler spécifiquement la cascade métastatique demeure cependant le talon d'Achille des thérapies anticancéreuses. Une première étape dans ie processus métastatique est l'évasion des cellules cancéreuses du site de la tumeur primaire. Ceci requiert une augmentation de la motiliié cellulaire couplée à la capacité de se frayer un chemin au sein de la matrice extracelluiaire. D'un point de vue thérapeutique, inhiber la migration cellulaire est une approche attrayante. Notre laboratoire a développé un peptide, nommé TAT-RasGAP317.326 dérivé d'un fragment qui est lui-même le résultat du clivage de la protéine RasGAP par la caspase-3. Ce peptide est capable de pénétrer les cellules cancéreuses et de les sensibiliser spécifiquement à la mort induite par la radiothérapie et la chimiothérapie. La finalité des effets de ce peptide est de pouvoir diminuer les doses des traitements anti-cancéreux et donc des effets secondaires qu'ils engendrent. Dans cette étude, nous avons découvert que TAT-RasGAP317.326 augmente l'adhésion des cellules et inhibe la migration cellulaire ainsi que l'invasion des cellules à travers une matrice extracellulaire. La capacité de TAT-RasGAP317.326 à induire l'adhésion repose sur ia dépolymérisation du cytosquelette d'actine associée à une redistribution des points d'ancrage cellulaire. Nous avons découvert que l'inhibition de ia migration par TAT-RasGAP317.326 nécessitait la présence d'un suppresseur de tumeur et de métastases appelé DLC1 (Deleted in Liver Cancer l), qui par ailleurs s'avère aussi être une protéine RhoGAP. De plus, nous avons aussi trouvé que DLC1 était un partenaire d'interaction de RasGAP et que cette interaction s'effectuait via l'acide aminé tryptophane 317 de RasGAP. qui s'avère être le premier acide aminé du peptide TAT-RasGAP317.326. Nous avons ensuite évalué le rôle joué par certains fragments de RasGAP dans le processus de métastatisation. Dans ce contexte, des cellules de cancer du sein qui sur-expriment un fragment de RasGAP contenant la séquence TAT-RasGAP317.326 ont vu leur potentiel métastatique diminuer drastiquerment. Malheureusement, aucun effet anti-métastatique n'a été obtenu après injection de TAT-RasGAP317.326 dans les souris. Cependant, nous avons réalisé rétrospectivement que TAT-RasGAP317.326 n'était pas correctement délivré à la tumeur primaire, ce qui nous empêche de tirer des conclusions sur le rôle anti-métastatique de ce peptide. La suite de cette étude visant à mieux comprendre comment TAT-RasGAP317.326 agit, a mené à la découverte que les dix acides aminés de TAT-RasGAP317.326 pouvaient être réduits à trois acides aminés, TAT-RasGAP317.329, tout en gardant l'effet sensibilisateur à la chimiothérapie. En visant à élucider le mode d'interaction entre RasGAP et DLC1, nous avons découvert qu'un acide aminé nécessaire à l'activité GAP de DLC1 était requis pour lier RasGAP, ce qui laisse présager que TAT-RasGAp317.32c, module i'activité GAP de DLC1. Des travaux supplémentaires doivent encore être effectués pour complètement élucider les mécanismes d'action de TAT-RasGAP317.326 et afin de pouvoir l'utiliser comme un outil pour combattre le cancer sur deux fronts, en améliorant les chimiothérapies et en inhibant la formation de métastases.

Angiotensin II is an endogenous neurotransmitter for rat and human mesenteric resistance blood vessels

Angiotensin II (Ang II) is one of the most potent vasoconstrictors. We document here the innervation of rat and human mesenteric resistance arteries (MRA) by angiotensinergic neurons of the rat and human sympathetic coeliac ganglia. Angiotensinogen (Ang-N)-mRNA and angiotensin converting enzyme-mRNA but no renin-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia. In the same extracts, cathepsin D-mRNA was detected: This protease also cleaves Ang I from Ang-N and could therefore account for the generation of neuronal Ang peptides in the absence of renin. In situ hybridization confirmed the presence of Ang-N-mRNA in the cytoplasm of rat coeliac ganglia. By using solid-phase extraction, high performance liquid chromatography and subsequent radioimmunoassay, Ang II and its metabolites were detected in rat and also in human coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia neurons and their projections innervating MRA. In addition, segmental angiotensinergic innervation of MRA was also observed. By means of confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings could indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally in MRA.

Quantitation of endogenous mouse mammary tumor virus superantigen expression by lymphocyte subsets.

Superantigens (SAg) encoded by endogenous mouse mammary tumor viruses (Mtv) interact with the V beta domain of the T cell receptor (TcR-V beta). Presentation of Mtv SAg can lead to stimulation and/or deletion of the reactive T cells, but little is known about the quantitative aspects of SAg presentation. Although monoclonal antibodies have been raised against Mtv SAg, they have not been useful in quantitating SAg protein, which is present in very low amounts in normal cells. Alternative attempts to quantitate Mtv SAg mRNA expression are complicated by the fact that Mtv transcription occurs from multiple loci and in different overlapping reading frames. In this report we describe a novel competitive polymerase chain reaction assay which allows the locus-specific quantitation of SAg expression at the mRNA level in lymphocyte subsets from mouse strains with multiple endogenous Mtv loci. In B cells as well as T cells (CD4+ or CD8+), Mtv-6 SAg is expressed at the highest levels, followed by Mtv-7 SAg and (to a much lesser extent) Mtv-8,9. Consistent with functional Mtv-7 SAg presentation studies, we find that Mtv-7 SAg expression is higher in B cells than in CD8+ T cells and very low in the CD4+ subset. The overall hierarchy in Mtv SAg expression (i.e. Mtv-6 > Mtv-7 > Mtv 8,9) was also observed for mRNA isolated from neonatal thymus. Furthermore, the kinetics of intrathymic deletion of the corresponding TcR-V beta domains during ontogeny correlated with the levels of Mtv SAg expression. Collectively our data suggest that T cell responses to Mtv SAg are largely controlled by SAg expression levels on presenting cells.

Evolution of asexuality via different mechanisms in grass thrips (thysanoptera: Aptinothrips).

Asexual lineages can derive from sexual ancestors via different mechanisms and at variable rates, which affects the diversity of the asexual population and thereby its ecological success. We investigated the variation and evolution of reproductive systems in Aptinothrips, a genus of grass thrips comprising four species. Extensive population surveys and breeding experiments indicated sexual reproduction in A. elegans, asexuality in A. stylifer and A. karnyi, and both sexual and asexual lineages in A. rufus. Asexuality in A. stylifer and A. rufus coincides with a worldwide distribution, with sexual A. rufus lineages confined to a limited area. Inference of molecular phylogenies and antibiotic treatment revealed different causes of asexuality in different species. Asexuality in A. stylifer and A. karnyi has most likely genetic causes, while it is induced by endosymbionts in A. rufus. Endosymbiont-community characterization revealed presence of Wolbachia, and lack of other bacteria known to manipulate host reproduction. However, only 69% asexual A. rufus females are Wolbachia-infected, indicating that either an undescribed endosymbiont causes asexuality in this species or that Wolbachia was lost in several lineages that remained asexual. These results open new perspectives for studies on the maintenance of mixed sexual and asexual reproduction in natural populations.

Passive lipoidal diffusion and carrier-mediated cell uptake are both important mechanisms of membrane permeation in drug disposition.

Recently, it has been proposed that drug permeation is essentially carrier-mediated only and that passive lipoidal diffusion is negligible. This opposes the prevailing hypothesis of drug permeation through biological membranes, which integrates the contribution of multiple permeation mechanisms, including both carrier-mediated and passive lipoidal diffusion, depending on the compound's properties, membrane properties, and solution properties. The prevailing hypothesis of drug permeation continues to be successful for application and prediction in drug development. Proponents of the carrier-mediated only concept argue against passive lipoidal diffusion. However, the arguments are not supported by broad pharmaceutics literature. The carrier-mediated only concept lacks substantial supporting evidence and successful applications in drug development.

Sorting mechanisms regulating membrane protein traffic in the apical transcytotic pathway of polarized MDCK cells.

The transcytotic pathway followed by the polymeric IgA receptor (pIgR) carrying its bound ligand (dIgA) from the basolateral to the apical surface of polarized MDCK cells has been mapped using morphological tracers. At 20 degreesC dIgA-pIgR internalize to interconnected groups of vacuoles and tubules that comprise the endosomal compartment and in which they codistribute with internalized transferrin receptors (TR) and epidermal growth factor receptors (EGFR). Upon transfer to 37 degreesC the endosome vacuoles develop long tubules that give rise to a distinctive population of 100-nm-diam cup-shaped vesicles containing pIgR. At the same time, the endosome gives rise to multivesicular endosomes (MVB) enriched in EGFR and to 60-nm-diam basolateral vesicles. The cup-shaped vesicles carry the dIgA/pIgR complexes to the apical surface where they exocytose. Using video microscopy and correlative electron microscopy to study cells grown thin and flat we show that endosome vacuoles tubulate in response to dIgA/pIgR but that the tubules contain TR as well as pIgR. However, we show that TR are removed from these dIgA-induced tubules via clathrin-coated buds and, as a result, the cup-shaped vesicles to which the tubules give rise become enriched in dIgA/pIgR. Taken together with the published information available on pIgR trafficking signals, our observations suggest that the steady-state concentrations of TR and unoccupied pIgR on the basolateral surface of polarized MDCK cells are maintained by a signal-dependent, clathrin-based sorting mechanism that operates along the length of the transcytotic pathway. We propose that the differential sorting of occupied receptors within the MDCK endosome is achieved by this clathrin-based mechanism continuously retrieving receptors like TR from the pathways that deliver pIgR to the apical surface and EGFR to the lysosome.

The alpha 1a and alpha 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action.

In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.

Results of the 2nd scientific workshop of the ECCO (III): basic mechanisms of intestinal healing.

The second scientific workshop of the European Crohn's and Colitis Organization (ECCO) focused on the relevance of intestinal healing for the disease course of inflammatory bowel disease (IBD). The objective was to better understand basic mechanisms, markers for disease prediction, detection and monitoring of intestinal healing, impact of intestinal healing on the disease course of IBD as well as therapeutic strategies. The results of this workshop are presented in four separate manuscripts. This section describes basic mechanisms of intestinal healing, identifies open questions in the field and provides a framework for future studies.