Imaging malaria sporozoites in the dermis of the mammalian host

The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. the steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo.Inst Pasteur, Unite Biol & Genet Paludisme, F-75724 Paris 15, FranceWeb of Scienc

Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis

Analyse cellulaire et moléculaire des effets du facteur de nécrose tumorale sur la mobilité et la pathogénicité du parasite Entamoeba histolytica

L agent étiologique de l amibiase, Entamoeba histolytica, atteint 50 millions de personnes, entraînant 100 000 décès par an. Lors de l amibiase, une réponse inflammatoire, orchestrée par le facteur de nécrose tumorale (TNF), se développe chez l hôte. La mobilité de E. histolytica est essentielle pour le processus invasif. Notre travail a analysé l incidence du TNF sur la migration du parasite, sur la résistance du trophozoïte envers cette cytokine et sa relation avec la protéine majeure d adhe sion : la lectine galactose/N-acétyl-D-galactosamine (Gal/GalNAc). Nous avons démontré que le TNF humain est chimioattractant pour E. histolytica. La migration chimiotactique du parasite vers le TNF nécessite l adhérence via la lectine Gal/GalNAc et la mise en place d une signalisation et d une polarisation des éléments du cytosquelette différente de celle de la mobilité. Dans le modèle d abcès hépatique, l absence de signalisation via la lectine Gal/GalNAc conduit à la perte de sécrétion du TNF et à un retard de la mise en place de l apoptose de cellules de l hôte. L analyse de la transcription de gènes du parasite, dans un milieu homogène de TNF, montre que la stratégie de survie du parasite, consisterait d une part à développer une réponse au stress oxydatif et d autre part, à diminuer l internalisation du TNF et augmenter la dégradation protéique. Ces résultats soulignent pour la première fois un dialogue moléculaire direct entre le TNF et E histolytica, qui modulerait la physiopathologie de l amibiase.The etiological agent of amoebiasis, Entamoeba histolytica, leads to 50 million clinical cases and 100,000 deaths per year. During amoebiasis, an inflammatory response, orchestrated by the tumour necrosis factor (TNF), is developed by the host. The motility of E. histolytica is essential for the invasive process. Our work analysed the incidence of TNF on the migration of the trophozoite, on the parasite s resistance towards this cytokine and its relation with the major adherence protein: the galactose/N-acetyl-D-galactosamine (Gal/GalNAc) lectin. We have demonstrated that human TNF is chemoattractant for E. histolytica. The parasite s chemotactic migration towards TNF requires adherence via the Gal/GalNAc lectin, the activation of a signalling pathway and the polarisation of cytoskeletal elements different to those for motility. In the hepatic abscess model, the absence of signalling via the Gal/GalNAc lectin, leads to the loss of TNF secretion and a delay in the initiation of host cell apoptosis. The analysis of parasite gene transcription, in a homogenous TNF medium, shows that the parasite s survival strategy would consist firstly in developing a response to an oxidative stress and secondly in diminishing the internalisation of TNF and in enhancing protein degradation. These results underline for the first time a direct molecular dialogue between TNF and E. histolytica, which could modulate the physiopathology of amoebiasis.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Human Tumor Necrosis Factor is a Chemoattractant for the Parasite Entamoeba histolytica

In an analysis of the molecular factors triggering amebiasis, we investigated the chemotaxis of Entamoeba histolytica toward tumor necrosis factor (TNF) in vitro, using quantitative imaging techniques. Our findings enabled us to propose a hitherto unknown role for TNF as a chemokinetic and chemoattractant agent for this parasite

Chemotaxis of Entamoeba histolytica towards the pro-inflammatory cytokine TNF is based on PI3K signalling, cytoskeleton reorganization and the GalactoseN-acetylgalactosamine lectin activity

International audienceEntamoeba histolytica is the protozoan parasite responsible for human amoebiasis. During invasive amoebiasis, migration is an essential process and it has previously been shown that the pro-inflammatory compound tumour necrosis factor (TNF) is produced and that it has a migratory effect on E. histolytica. This paper focuses on the analysis of parasite signalling and cytoskeleton changes leading to directional motility. TNF-induced signalling was PI3K-dependent and could lead to modifications in the polarization of certain cytoskeleton-related proteins. To analyse the effect of TNF signalling on gene expression, we used microarray analysis to screen for genes encoding proteins that were potentially important during chemotaxis towards TNF. Interestingly, we found that elements of the galactose/N-acetylgalactosamine lectin (Gal/GalNAc lectin) were upregulated during chemotaxis as well as genes encoding proteins involved in cytoskeleton dynamics. The alpha-actinin protein appeared to be an important candidate to link the Gal/GalNAc lectin to the cytoskeleton during chemotaxis signalling. Dominant negative parasites blocked for Gal/GalNAc lectin signalling were no longer able to chemotax towards TNF. These results have given us an insight on how E. histolytica changes its cytoskeleton dynamics during chemotaxis and revealed the capital role of PI3K and Gal/GalNAc lectin signalling in chemotaxis

The pharmacology of effort-related choice behavior: Dopamine, depression, and individual differences

This review paper is focused upon the involvement of mesolimbic dopamine (DA) and related brain systems in effort-based processes. Interference with DA transmission affects instrumental behavior in a manner that interacts with the response requirements of the task, such that rats with impaired DA transmission show a heightened sensitivity to ratio requirements. Impaired DA transmission also affects effort-related choice behavior, which is assessed by tasks that offer a choice between a preferred reinforcer that has a high work requirement vs. less preferred reinforcer that can be obtained with minimal effort. Rats and mice with impaired DA transmission reallocate instrumental behavior away from food-reinforced tasks with high response costs, and show increased selection of low reinforcement/low cost options. Tests of effort-related choice have been developed into models of pathological symptoms of motivation that are seen in disorders such as depression and schizophrenia. These models are being employed to explore the effects of conditions associated with various psychopathologies, and to assess drugs for their potential utility as treatments for effort-related symptoms. Studies of the pharmacology of effort-based choice may contribute to the development of treatments for symptoms such as psychomotor slowing, fatigue or anergia, which are seen in depression and other disorders

The management of acute venous thromboembolism in clinical practice - study rationale and protocol of the European PREFER in VTE Registry

Background: Venous thromboembolism (VTE) is a major health problem, with over one million events every year in Europe. However, there is a paucity of data on the current management in real life, including factors influencing treatment pathways, patient satisfaction, quality of life (QoL), and utilization of health care resources and the corresponding costs. The PREFER in VTE registry has been designed to address this and to understand medical care and needs as well as potential gaps for improvement. Methods/design: The PREFER in VTE registry was a prospective, observational, multicenter study conducted in seven European countries including Austria, France Germany, Italy, Spain, Switzerland, and the UK to assess the characteristics and the management of patients with VTE, the use of health care resources, and to provide data to estimate the costs for 12 months treatment following a first-time and/or recurrent VTE diagnosed in hospitals or specialized or primary care centers. In addition, existing anticoagulant treatment patterns, patient pathways, clinical outcomes, treatment satisfaction, and health related QoL were documented. The centers were chosen to reflect the care environment in which patients with VTE are managed in each of the participating countries. Patients were eligible to be enrolled into the registry if they were at least 18 years old, had a symptomatic, objectively confirmed first time or recurrent acute VTE defined as either distal or proximal deep vein thrombosis, pulmonary embolism or both. After the baseline visit at the time of the acute VTE event, further follow-up documentations occurred at 1, 3, 6 and 12 months. Follow-up data was collected by either routinely scheduled visits or by telephone calls. Results: Overall, 381 centers participated, which enrolled 3,545 patients during an observational period of 1 year. Conclusion: The PREFER in VTE registry will provide valuable insights into the characteristics of patients with VTE and their acute and mid-term management, as well as into drug utilization and the use of health care resources in acute first-time and/or recurrent VTE across Europe in clinical practice. Trial registration: Registered in DRKS register, ID number: DRKS0000479