Diversité et Immunogénicité des protéines salivaires de Culicidae

Eviter la piqûre de moustiques vecteurs en utilisant des mesures antivectorielles reste le meilleur moyen de se protéger des maladies vectorielles. La salive de moustique peut induire une réponse anticorps (Acs) spécifique chez l hôte qui pourrait être utilisé pour définir l'efficacité de ces mesures de protection antivectorielle. L objectif de notre projet était d évaluer la possibilité d utiliser cette réponse Acs anti-salive de moustiques pour mesurer l exposition à des espèces spécifiques de moustiques ainsi que d identifier des marqueurs d exposition. Nous nous sommes tout d abord assurés de l absence de différences intraspécifiques entre différentes colonies de moustiques, une condition indispensable pour pouvoir observer des différences au niveau de l espèce. Par ailleurs, nous avons mis au point un protocole pour préserver les échantillons salivaires dans des conditions de terrains non optimales. A partir de ces expérimentations préliminaires, nous avons évalué la diversité du répertoire protéique salivaire de quatre espèces d Anopheles par des différentes approches, et montré une spécificité de genre et d espèce aussi bien au niveau protéique qu antigénique. Enfin, nous avons montré une évolution spatio-temporelle de l intensité de la réponse Acs anti-salive ainsi que sa spécificité de genre et d espèce, chez des individus exposés à différents niveaux à Ae. caspius. Ces résultats souligne la possibilité de caractériser des antigènes salivaires spécifiques de genre et d espèces qui peuvent avoir un intérêt pour mesurer le contact hôte/vecteur au niveau individuel, le risque de transmission de maladies vectorielles ou l efficacité des mesures antivectorielles.The primary mean to protect individuals from arthropod-borne diseases is the prevention of bites from infected arthropods which could be achieved by vector control strategies. Mosquito saliva could induce a specific antibody response in exposed individuals that could be used to assess the effectiveness of anti-vector measures. The aim of this study is to assess the possibility to use anti-mosquito saliva antibody responses in order to evaluate the exposure to specific species of vectors and to identify salivary protein candidates that can be used as immunological markers of exposure. We first verify the lack of intraspecific differences among several mosquito colonies which is essential to further observe potential differences at the species level. Moreover, a convenient storage method was developed to preserve salivary samples in non optimal condition on the field. Based on these preliminary results, we evaluated the salivary gland protein repertory diversity among four Anopheles species using complementary approaches and we shown a genus and species specificity at the protein and antigen level. At least, a spatio-temporal evolution of anti-saliva antibody responses was shown according to the Aedes caspius density using sera of differentially exposed individuals. The specificity of this response was also reported at the genus and species level. All together, these results suggest the feasibility to characterize genus and species specific salivary antigens which could be used as immunological markers of exposure to evaluate host/vector contacts, the risk of vector-borne disease transmission or the effectiveness of anti-vector strategies.AIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

Plasmodium falciparum proteome changes in response to doxycycline treatment

<p>Abstract</p> <p>Background</p> <p>The emergence of <it>Plasmodium falciparum </it>resistance to most anti-malarial compounds has highlighted the urgency to develop new drugs and to clarify the mechanisms of anti-malarial drugs currently used. Among them, doxycycline is used alone for malaria chemoprophylaxis or in combination with quinine or artemisinin derivatives for malaria treatment. The molecular mechanisms of doxycycline action in <it>P. falciparum </it>have not yet been clearly defined, particularly at the protein level.</p> <p>Methods</p> <p>A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite <it>P. falciparum </it>following doxycycline treatment. A comparison of protein expression between treated and untreated protein samples was performed using two complementary proteomic approaches: two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and isobaric tagging reagents for relative and absolute quantification (iTRAQ).</p> <p>Results</p> <p>After doxycycline treatment, 32 and 40 <it>P. falciparum </it>proteins were found to have significantly deregulated expression levels by 2D-DIGE and iTRAQ methods, respectively. Although some of these proteins have been already described as being deregulated by other drug treatments, numerous changes in protein levels seem to be specific to doxycycline treatment, which could perturb apicoplast metabolism. Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to confirm this hypothesis.</p> <p>Conclusions</p> <p>In this study, a specific response to doxycycline treatment was distinguished and seems to involve mitochondrion and apicoplast organelles. These data provide a starting point for the elucidation of drug targets and the discovery of mechanisms of resistance to anti-malarial compounds.</p

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

OBJECTIVE: The main objective of this study was to assess the influence of gas mixtures on in vitro Plasmodium falciparum growth and 50% inhibitory concentration (IC(50)) for chloroquine. METHODS: The study was performed between February 2004 and December 2005. 136 Plasmodium falciparum isolates were used to evaluate gas mixtures effect on IC(50 )for chloroquine by isotopic microtest. The oxygen effect on asexual blood cycle of 3D7 and W2 clones was determined by thin blood smears examination and tritiated hypoxanthine uptake. RESULTS: From 5% O(2 )to 21% O(2 )conditions, no parasiticide effect of O(2 )concentration was observed in vitro on the clones 3D7 and W2. A parasitostatic effect was observed during the exposure of mature trophozoïtes and schizonts at 21% O(2 )with an increase in the length of schizogony. The chloroquine IC(50 )at 10% O(2 )were significantly higher than those at 21% O(2), means of 173.5 nM and 121.5 nM respectively (p < 0.0001). In particular of interest, among the 63 isolates that were in vitro resistant to chloroquine (IC(50 )> 100 nM) at 10% O(2), 17 were sensitive to chloroquine (IC(50 )< 100 nM) at 21% O(2). CONCLUSION: Based on these results, laboratories should use the same gas mixture to realize isotopic microtest. Further studies on comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol to survey malaria drug resistance

Assessment of exposure to Plasmodium falciparum transmission in a low endemicity area by using multiplex fluorescent microsphere-based serological assays

Background: The evaluation of malaria transmission intensity is a crucial indicator for estimating the burden of malarial disease. In this respect, entomological and parasitological methods present limitations, especially in low transmission areas. The present study used a sensitive multiplex assay to assess the exposure to Plasmodium falciparum infection in children living in an area of low endemicity. In three Senegalese villages, specific antibody (IgG) responses to 13 pre-erythrocytic P. falciparum peptides derived from Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, Csp and Pf11.1 proteins were simultaneously evaluated before (June), at the peak (September) and after (December) the period of malaria transmission, in children aged from 1 to 8 years. Results: Compared to other antigens, a high percentage of seropositivity and specific antibody levels were detected with Glurp, Salsa1, Lsa3NR2, and Lsa1J antigens. The seropositivity increased with age for all tested antigens. Specific IgG levels to Glurp, Salsa1, Lsa3NR2, and Lsa1J were significantly higher in P. falciparum infected children compared to non-infected and this increase is significantly correlated with parasite density. Conclusion: The multiplex assay represents a useful technology for a serological assessment of rapid variations in malaria transmission intensity, especially in a context of low parasite rates. The use of such combined serological markers (i.e. Glurp, Lsa1, Lsa3, and Salsa) could offer the opportunity to examine these variations over time, and to evaluate the efficacy of integrated malaria control strategies

Platelets Alter Gene Expression Profile in Human Brain Endothelial Cells in an In Vitro Model of Cerebral Malaria

Platelet adhesion to the brain microvasculature has been associated with cerebral malaria (CM) in humans, suggesting that platelets play a role in the pathogenesis of this syndrome. In vitro co-cultures have shown that platelets can act as a bridge between Plasmodium falciparum-infected red blood cells (pRBC) and human brain microvascular endothelial cells (HBEC) and potentiate HBEC apoptosis. Using cDNA microarray technology, we analyzed transcriptional changes of HBEC in response to platelets in the presence or the absence of tumor necrosis factor (TNF) and pRBC, which have been reported to alter gene expression in endothelial cells. Using a rigorous statistical approach with multiple test corrections, we showed a significant effect of platelets on gene expression in HBEC. We also detected a strong effect of TNF, whereas there was no transcriptional change induced specifically by pRBC. Nevertheless, a global ANOVA and a two-way ANOVA suggested that pRBC acted in interaction with platelets and TNF to alter gene expression in HBEC. The expression of selected genes was validated by RT-qPCR. The analysis of gene functional annotation indicated that platelets induce the expression of genes involved in inflammation and apoptosis, such as genes involved in chemokine-, TREM1-, cytokine-, IL10-, TGFβ-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM

Relationship between Exposure to Vector Bites and Antibody Responses to Mosquito Salivary Gland Extracts

Mosquito-borne diseases are major health problems worldwide. Serological responses to mosquito saliva proteins may be useful in estimating individual exposure to bites from mosquitoes transmitting these diseases. However, the relationships between the levels of these IgG responses and mosquito density as well as IgG response specificity at the genus and/or species level need to be clarified prior to develop new immunological markers to assess human/vector contact. To this end, a kinetic study of antibody levels against several mosquito salivary gland extracts from southeastern French individuals living in three areas with distinct ecological environments and, by implication, distinct Aedes caspius mosquito densities were compared using ELISA. A positive association was observed between the average levels of IgG responses against Ae. caspius salivary gland extracts and spatial Ae. caspius densities. Additionally, the average level of IgG responses increased significantly during the peak exposure to Ae. caspius at each site and returned to baseline four months later, suggesting short-lived IgG responses. The species-specificity of IgG antibody responses was determined by testing antibody responses to salivary gland extracts from Cx. pipiens, a mosquito that is present at these three sites at different density levels, and from two other Aedes species not present in the study area (Ae. aegypti and Ae. albopictus). The IgG responses observed against these mosquito salivary gland extracts contrasted with those observed against Ae. caspius salivary gland extracts, supporting the existence of species-specific serological responses. By considering different populations and densities of mosquitoes linked to environmental factors, this study shows, for the first time, that specific IgG antibody responses against Ae. caspius salivary gland extracts may be related to the seasonal and geographical variations in Ae. caspius density. Characterisation of such immunological-markers may allow the evaluation of the effectiveness of vector-control strategies or estimation of the risk of vector-borne disease transmission

Etude de la chimiosensibilité et du polymorphisme génétique des populations de plasmodium falciparum à Dakar au cours de la saison de transmission 2002

BORDEAUX2-BU Santé (330632101) / SudocPARIS-BIUM (751062103) / SudocPARIS-Bib. Serv.Santé Armées (751055204) / SudocSudocFranceF

Les chondroïtines sulfates de type A (rôle dans l'adhésion à l'endothélium microvasculaire et aux syncytiotrophoblastes placentaires des hématites parasitées par Plasmodium falciparum)

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Coping with stress, fatigue, and sleepiness during medical studies: Experience of the French military medical school

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