Anopheles salivary gland proteomes from major malaria vectors.

BACKGROUND: Antibody responses against Anopheles salivary proteins can indicate individual exposure to bites of malaria vectors. The extent to which these salivary proteins are species-specific is not entirely resolved. Thus, a better knowledge of the diversity among salivary protein repertoires from various malaria vector species is necessary to select relevant genus-, subgenus- and/or species-specific salivary antigens. Such antigens could be used for quantitative (mosquito density) and qualitative (mosquito species) immunological evaluation of malaria vectors/host contact. In this study, salivary gland protein repertoires (sialomes) from several Anopheles species were compared using in silico analysis and proteomics. The antigenic diversity of salivary gland proteins among different Anopheles species was also examined. RESULTS: In silico analysis of secreted salivary gland protein sequences retrieved from an NCBInr database of six Anopheles species belonging to the Cellia subgenus (An. gambiae, An. arabiensis, An. stephensi and An. funestus) and Nyssorhynchus subgenus (An. albimanus and An. darlingi) displayed a higher degree of similarity compared to salivary proteins from closely related Anopheles species. Additionally, computational hierarchical clustering allowed identification of genus-, subgenus- and species-specific salivary proteins. Proteomic and immunoblot analyses performed on salivary gland extracts from four Anopheles species (An. gambiae, An. arabiensis, An. stephensi and An. albimanus) indicated that heterogeneity of the salivary proteome and antigenic proteins was lower among closely related anopheline species and increased with phylogenetic distance. CONCLUSION: This is the first report on the diversity of the salivary protein repertoire among species from the Anopheles genus at the protein level. This work demonstrates that a molecular diversity is exhibited among salivary proteins from closely related species despite their common pharmacological activities. The involvement of these proteins as antigenic candidates for genus-, subgenus- or species-specific immunological evaluation of individual exposure to Anopheles bites is discussed.

Comparison of thoracic wall behavior in large animals and human cadavers submitted to an identical ballistic blunt thoracic trauma.

BACKGROUND: Several models of ballistic blunt thoracic trauma are available, including human cadavers and large animals. Each model has advantages and disadvantages regarding anatomy and physiology, but they have not been compared with identical ballistic aggression. METHODS: To compare thoracic wall behavior in 40-kg pigs and human cadavers, the thorax of 12 human cadavers and 19 anesthetized pigs were impacted with two different projectiles at different speeds. On the thoracic wall, the peak acceleration, peak velocity, maximal compression, viscous criterion, and injury criteria (e.g. abbreviated injury scale and number of rib fractures) were recorded. The correlations between these motion and injury parameters and the blunt criterion were compared between the two groups. The bone mineral density of each subject was also measured. RESULTS: The peak acceleration, the peak velocity and the viscous criterion were significantly higher for the pigs. The AIS and the number of rib fractures were significantly higher for human cadavers. The bone mineral density was significantly higher for cadavers, but was, for the two groups, significantly lower than for 30-year-old human. CONCLUSION: The motion of the pig's thoracic wall is greater than that of the human cadaver, and the severity of the impact is always greater for human cadavers than for pigs. In addition, pig bone is more elastic and less brittle than older human cadaver bone. Due to the bone mineral density, the thoracic wall of human adults should be more rigid and more resistant than the thoracic wall of human cadavers or pigs.

Adult-brain-derived neural stem cells grafting into a vein bridge increases postlesional recovery and regeneration in a peripheral nerve of adult pig.

We attempted transplantation of adult neural stem cells (ANSCs) inside an autologous venous graft following surgical transsection of nervis cruralis with 30 mm long gap in adult pig. The transplanted cell suspension was a primary culture of neurospheres from adult pig subventricular zone (SVZ) which had been labeled in vitro with BrdU or lentivirally transferred fluorescent protein. Lesion-induced loss of leg extension on the thigh became definitive in controls but was reversed by 45-90 days after neurosphere-filled vein grafting. Electromyography showed stimulodetection recovery in neurosphere-transplanted pigs but not in controls. Postmortem immunohistochemistry revealed neurosphere-derived cells that survived inside the venous graft from 10 to 240 post-lesion days and all displayed a neuronal phenotype. Newly formed neurons were distributed inside the venous graft along the severed nerve longitudinal axis. Moreover, ANSC transplantation increased CNPase expression, indicating activation of intrinsic Schwann cells. Thus ANSC transplantation inside an autologous venous graft provides an efficient repair strategy.

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins.

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-ß-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

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.

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.

Implication of haematophagous arthropod salivary proteins in host-vector interactions.

The saliva of haematophagous arthropods contains an array of anti-haemostatic, anti-inflammatory and immunomodulatory molecules that contribute to the success of the blood meal. The saliva of haematophagous arthropods is also involved in the transmission and the establishment of pathogens in the host and in allergic responses. This survey provides a comprehensive overview of the pharmacological activity and immunogenic properties of the main salivary proteins characterised in various haematophagous arthropod species. The potential biological and epidemiological applications of these immunogenic salivary molecules will be discussed with an emphasis on their use as biomarkers of exposure to haematophagous arthropod bites or vaccine candidates that are liable to improve host protection against vector-borne diseases.

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-, TGFß-, death-receptor-, and apoptosis-signaling. Overall, our results support the hypothesis that platelets play a pathogenic role in CM.

Hypertonic saline hydroxyethylstarch restores right ventricular-arterial coupling after normovolemic hemodilution in piglets.

BACKGROUND: Normovolemic hemodilution is known to inhibit hypoxic pulmonary vasoconstriction. How the coupling between the pulmonary arterial (PA) circulation and the right ventricle (RV) is affected by normovolemic hemodilution and by the composition of replacement solutions remains unknown. Therefore, the effects of isotonic and hypertonic saline hydroxyethylstarch solutions on the pulmonary circulation and RV, in control and hypoxic conditions, were compared. METHODS: Anesthetized piglets (n = 14) were equipped with manometer-tipped catheters in the RV and main PA and an ultrasonic flow probe around the main PA. The pulmonary circulation was assessed by pressure-flow relations and vascular impedance, RV afterload by effective arterial elastance (Ea), RV contractility by end-systolic elastance (Ees), and RV-PA coupling by the Ees/Ea ratio. Measurements were done in control (Fio2 0.40) and hypoxic (Fio2 0.12) conditions before and after acute normovolemic hemodilution with either 20 ml/kg isotonic saline hydroxyethylstarch (hydroxyethylstarch 130/0.4 6% in NaCl 0.9%, Voluven, Fresenius-Kabi, Sevres, France) or 5 ml/kg hypertonic saline hydroxyethylstarch (hydroxyethylstarch 200/0.5 6% in NaCl 7.2%, HyperHES, Fresenius-Kabi) solutions. RESULTS: Hypoxic pulmonary vasoconstriction was associated with proportional increases in Ea and Ees and did not affect RV-PA coupling. Hemodilution attenuated the hypoxic response. Hemodilution with isotonic saline hydroxyethylstarch did not affect the RV-PA coupling, whereas hemodilution with hypertonic saline hydroxyethylstarch increased Ees and the Ees/Ea ratio. CONCLUSION: In experimental normovolemic hemodilution, both in control and in hypoxic conditions, RV-PA coupling is unaffected by isotonic saline hydroxyethylstarch but improved by hypertonic saline hydroxyethylstarch, mainly because of an increase in RV contractility.

A multiplex assay for the simultaneous detection of antibodies against 15 Plasmodium falciparum and Anopheles gambiae saliva antigens.

BACKGROUND: Assessment exposure and immunity to malaria is an important step in the fight against the disease. Increased malaria infection in non-immune travellers under anti-malarial chemoprophylaxis, as well as the implementation of malaria elimination programmes in endemic countries, raises new issues that pertain to these processes. Notably, monitoring malaria immunity has become more difficult in individuals showing low antibody (Ab) responses or taking medications against the Plasmodium falciparum blood stages. Commonly available techniques in malaria seroepidemiology have limited sensitivity, both against pre-erythrocytic, as against blood stages of the parasite. Thus, the aim of this study was to develop a sensitive tool to assess the exposure to malaria or to bites from the vector Anopheles gambiae, despite anti-malarial prophylactic treatment. METHODS: Ab responses to 13 pre-erythrocytic P. falciparum-specific peptides derived from the proteins Lsa1, Lsa3, Glurp, Salsa, Trap, Starp, CSP and Pf11.1, and to 2 peptides specific for the Anopheles gambiae saliva protein gSG6 were tested. In this study, 253 individuals from three Senegalese areas with different transmission intensities and 124 European travellers exposed to malaria during a short period of time were included. RESULTS: The multiplex assay was optimized for most but not all of the antigens. It was rapid, reproducible and required a small volume of serum. Proportions of Ab-positive individuals, Ab levels and the mean number of antigens (Ags) recognized by each individual increased significantly with increases in the level of malaria exposure. CONCLUSION: The multiplex assay developed here provides a useful tool to evaluate immune responses to multiple Ags in large populations, even when only small amounts of serum are available, or Ab titres are low, as in case of travellers. Finally, the relationship of Ab responses with malaria endemicity levels provides a way to monitor exposure in differentially exposed autochthonous individuals from various endemicity areas, as well as in travellers who are not immune, thus indirectly assessing the parasite transmission and malaria risk in the new eradication era.

Specific antibody responses against membrane proteins of erythrocytes infected by Plasmodium falciparum of individuals briefly exposed to malaria.

BACKGROUND: Plasmodium falciparum infections could lead to severe malaria, principally in non-immune individuals as children and travellers from countries exempted of malaria. Severe malaria is often associated with the sequestration of P. falciparum-infected erythrocytes in deep micro-vascular beds via interactions between host endothelial receptors and parasite ligands expressed on the surface of the infected erythrocyte. Although, serological responses from individuals living in endemic areas against proteins expressed at surface of the infected erythrocyte have been largely studied, seldom data are available about the specific targets of antibody response from travellers. METHODS: In order to characterize antigens recognized by traveller sera, a comparison of IgG immune response against membrane protein extracts from uninfected and P. falciparum-infected red blood cells (iRBC), using immunoblots, was performed between non exposed individuals (n = 31) and briefly exposed individuals (BEI) (n = 38) to malaria transmission. RESULTS: Immune profile analysis indicated that eight protein bands from iRBC were significantly detected more frequently in the BEI group. Some of these antigenic proteins were identified by an original immuno-proteomic approach. CONCLUSION: Collectively, these data may be useful to characterize the singular serological immune response against a primary malaria infection in individuals briefly exposed to transmission.

Intrathoracic pressure impulse predicts pulmonary contusion volume in ballistic blunt thoracic trauma.

BACKGROUND: Blunt thoracic trauma including behind armour blunt trauma or impact from a less lethal kinetic weapon (LLKW) projectile may cause injuries, including pulmonary contusions that can result in potentially lethal secondary complications. These lung injuries may be caused by intrathoracic pressure waves. The aim of this study was to observe dynamic changes in intrathoracic hydrostatic pressure during ballistic blunt thoracic trauma and to find correlations between these hydrostatic pressure parameters (especially the impulse parameter) and physical damages. METHODS: Thirty anesthetized pigs sustained a blunt thoracic trauma. In group 1 (n = 20), pigs were protected by a National Institute of Justice class III or IV bulletproof vest and shot with 7.62 NATO bullets. In group 2 (n = 10), pigs were shot by an LLKW. Intrathoracic pressure was recorded with an intraesophageal pressure sensor and three parameters were determined: intrathoracic maximum pressure, intrathoracic maximum pressure impulse (PI(max)), and the Pd.P/dt(max), derived from Viano's viscous criterion. Relative right lower lung lobe contusion volume was also measured. RESULTS: Different thoracic loading conditions were obtained. PI(max) best correlated with relative pulmonary contusion volume (R² = 0.64 and p < 0.0001). This result was homogenous for all experiments and was not related to the type of chest impact (LLKW-induced trauma or behind armour blunt trauma). CONCLUSIONS: The PI(max) is a good predictor of pulmonary contusion volume after ballistic blunt thoracic trauma. It is a useful criterion when the kinetic energy record or thoracic wall displacement data are unavailable, and the recording and calculation of this physical value are quite simple on animals.

Recombinant antibodies specific for the Plasmodium falciparum histidine-rich protein 2.

Early diagnosis and appropriate treatment are key elements of malaria control programs in endemic areas. A major step forward in recent years has been the production and use of rapid diagnostic tests (RDTs) in settings where microscopy is impracticable. Many current RDTs target the Plasmodium falciparum histidine-rich protein 2 (PfHRP2) released in the plasma of infected individuals. These RDTs have had an indisputably positive effect on malaria management, but still present several limitations, including the poor characterization of the commercial monoclonal antibodies (mAbs) used for PfHRP2 detection, variable sensitivity and specificity, and high costs. RDT use is further limited by impaired stability caused by temperature fluctuations during transport and uncontrolled storage in field-based facilities. To circumvent such drawbacks, an alternative could be the development of well-characterized, stabilized recombinant antibodies, with high binding affinity and specificity. Here, we report the characterization of the cDNA sequences encoding the Fab fragment of F1110 and F1546, two novels anti-PfHRP2 mAbs. FabF1546 was produced in the Escherichia coli periplasm. Its properties of binding to the parasite and to a recombinant PfHRP-2 antigen were similar to those of the parental mAb. As the affinity and stability of recombinant antibodies can be improved by protein engineering, our results open a novel approach for the development of an improved RDT for malaria diagnosis.

Plasmodium falciparum proteome changes in response to doxycycline treatment.

BACKGROUND: The emergence of Plasmodium falciparum 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 P. falciparum have not yet been clearly defined, particularly at the protein level. METHODS: A proteomic approach was used to analyse protein expression changes in the schizont stage of the malarial parasite P. falciparum 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). RESULTS: After doxycycline treatment, 32 and 40 P. falciparum 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. CONCLUSIONS: 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.

Salivary gland protein repertoire from Aedes aegypti mosquitoes.

Diseases caused by arthropod-borne viruses are a significant threat to the health of human and animal populations throughout the world. Better knowledge of the molecules synthesized in the salivary gland and saliva of hematophagous arthropods could be of use for improving the control of pathogen transmission. Recently, a sialome analysis of three Aedes aegypti mosquito colonies (PAEA, Rockefeller, and Formosus) carried out in our laboratory allowed us to identify 44 saliva proteins. Of these secreted proteins, none was exclusively expressed in one colony, suggesting that expression of salivary proteins is highly conserved across populations. In another study, we reported that some of these salivary proteins could be used as the genus-specific markers for travelers' exposure to mosquito vectors. Here, comparison of salivary gland protein profiles between these same three Ae. aegypti colonies was performed using the one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) difference gel electrophoresis method. As observed at the saliva level, no significant differences were detected between these three colonies. The salivary gland protein repertoire from the Ae. aegypti mosquito was analyzed using a proteomic approach. One hundred and twenty proteins were identified in these salivary glands representing the largest description of the Ae. aegypti salivary gland protein catalog. We succeeded in identifying 15 secreted proteins, some of which have already been reported as being involved in blood feeding. A comparison of the proteins identified between the salivary glands and the sialome is discussed.

Platelet-endothelial cell interactions in cerebral malaria: the end of a cordial understanding.

Cerebral malaria is an acute encephalopathy evolving from an infection with Plasmodium falciparum which kills more than one million people each year. Brain tissues from patients who died with cerebral malaria revealed multifocal capillary obstruction by parasitised red blood cells, platelets, and leukocytes. Many studies are unified in their proposal of two major hypotheses consisting of cell adhesion to the brain endothelium and excessive immune stimulation resulting in further vascular inflammation, prothrombotic cell activation, mechanical obstruction of cerebral capillaries and, consequently, blood-brain barrier disruption. Platelets and endothelial cells communicate on multiple levels. Infection-induced changes in platelets and endothelial cells occur in cerebral malaria, resulting in their concomitant activation, increased interactions between these two cell types, and a secondary procoagulant or hypercoagulable state. Here we review evidence for these mechanisms and highlight the possible role of platelets as effectors of endothelial damage in cerebral malaria. A better understanding of the complex regulation of these various interactions between brain endothelial cells and platelets in the context of cerebral malaria may prove useful in the development of new approaches to the treatment of this disease.

In vitro isolation of neural precursor cells from the adult pig subventricular zone.

In order to improve cell therapy techniques, we have characterized a multipotent neural precursor cell isolation technique from the subventricular zone of adult pig brain. The pig is a non-primate species that is immunologically closest to human. The proliferative zone of this neurogenic structure was first localized in situ in the pig brain by Ki-67 immunohistochemistry, as a ventral subfield of the Nissl-stained subventricular zone. For in vitro cultures, the striatal forebrain was sampled from deeply anaesthetized adult pigs and SVZ tissue explants were immediately microdissected out and dissociated in the appropriate medium. Primary cell culture in the presence of EGF and bFGF allowed growth of spherical masses that exhibited sustained growth and self-renewal capacity through six subsequent passages. Molecular characterization using reverse transcription-polymerase chain reaction (RT-PCR) showed that expanded pro-differentiating neurospheres expressed markers of proliferation, neural stem cells, and committed neural progenitors. After growth factor removal, the spheres became adherent and were shown to contain the three neural cell lineages by triple immunocytofluorescence and confocal microscopy. The present protocol therefore allowed for in vitro expansion of pig brain primary cells that display capacities for proliferation, self-renewal, and multipotency, i.e., the cardinal features of multipotent neural precursor cells.

High prevalence and fixation of Plasmodium vivax dhfr/dhps mutations related to sulfadoxine/pyrimethamine resistance in French Guiana.

Plasmodium vivax isolates from French Guiana were studied for the presence of mutations associated with sulfadoxine/pyrimethamine (SP) drug resistance. Ninety-six blood samples were collected from 2000 to 2005 from symptomatic malaria patients. SP drug resistance was predicted by determining point mutations in the dihydrofolate reductase (pvdhfr) and dihydropteroate synthase (pvdhps) genes. All samples showed mutant genotypes in both genes with a prevalence > 90% for the 58R, 117N, 382C, and 383G. A new mutation (116G) in pvdhfr was found at a frequency of 3.3%. Six different pvdhfr/dhps multilocus genotypes were observed with the predominance of the quintuple mutant-type 58R/117N/173L-382C/383G (59.3%). No significant differences were observed between the prevalence of haplotypes and the year of collection. Our results indicate that, in this area, the fixation of SP drug-resistant parasites in the P. vivax population is stable.

Platelet microparticles: a new player in malaria parasite cytoadherence to human brain endothelium.

Cerebral malaria (CM) is characterized by accumulation of circulating cells within brain microvessels, among which platelets play an important role. In vitro, platelets modulate the cytoadherence of Plasmodium falciparum-parasitized red blood cells (PRBCs) to brain endothelial cells. Here we show for the first time that platelet microparticles (PMPs) are able to bind to PRBCs, thereby transferring platelet antigens to the PRBC surface. This binding is largely specific to PRBCs, because PMPs show little adherence to normal red blood cells. PMP adherence is also dependent on the P. falciparum erythrocyte membrane protein 1 variant expressed by PRBCs. PMP binding to PRBCs decreases after neutralization of PRBC surface proteins by trypsin or after treatment of PMPs with a mAb to platelet-endothelial cell adhesion molecule-1 (CD31) and glycoprotein IV (CD36). Furthermore, PMP uptake is a dynamic process that can be achieved by human brain endothelial cells (HBECs), inducing changes in the endothelial phenotype. Lastly, PMPs dramatically increase PRBC cytoadherence to HBECs. In conclusion, our study identifies several mechanisms by which PMPs may participate in CM pathogenesis while interacting with both PRBCs and HBECs. PMPs thereby provide a novel target for antagonizing interactions between vascular cells that promote microvascular sludging and blood brain barrier alteration during CM.