Charlotte Behr

French National Centre for Scientific Research, Lutetia Parisorum, Île-de-France, France

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Publications (47)182.87 Total impact

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    ABSTRACT: Malaria induces potent activation and expansion of the Vγ9Vδ2 subpopulation of gamma-delta T-cells, which inhibit the Plasmodium falciparum blood cycle through soluble cytotoxic mediators, abrogating merozoite invasion capacity. Intra-erythrocytic stages efficiently trigger Vγ9Vδ2 T-cell activation and degranulation through poorly understood mechanisms. P. falciparum blood stage extracts are known to contain phosphoantigens able to stimulate Vγ9Vδ2 T-cells but how these are presented by intact infected red blood cells (iRBCs) remains elusive. Here we show that, unlike activation by phosphoantigen-expressing cells, Vγ9Vδ2 T-cell activation by intact iRBCs is independent on butyrophilin expression by the iRBC and contact with intact iRBC is not required. Moreover, blood stage culture supernatants proved as potent activators of Vγ9Vδ2 T-cells as iRBCs. Bioactivity in the microenvironment is attributable to phosphoantigens, as it is dependent on the parasite DOXP pathway, on Vγ9Vδ2 TCR signaling and on butyrophilin expression by Vγ9Vδ2 T-cells. Kinetic studies showed that the phosphoantigens were released at the end of the intraerythrocytic cycle at the time of parasite egress. We document exquisite sensitivity of Vγ9Vδ2 T-cells, which respond to a few thousand parasites. These data unravel a novel framework, whereby release of phosphoantigens into the extracellular milieu by sequestered parasites likely promotes activation of distant Vγ9Vδ2 T-cells that in turn exert remote anti-parasitic functions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Infection and immunity 07/2015; DOI:10.1128/IAI.00446-15 · 4.16 Impact Factor
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    ABSTRACT: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized by a high TNF/IL-10 ratio. Whether this TNF/IL-10 imbalance results from an intrinsic incapacity of SMA patients to produce IL-10 or from an IL-10 unresponsiveness to infection is unknown. Monocytes and T cells are recognized as the main sources of TNF and IL-10 in vivo, but little is known about the activation status of those cells in SMA patients. The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLA-DR+ and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA) used as surrogate of unspecific monocyte and T cell stimulant. The number of circulating pigmented monocytes was also determined. Monocytes and T cells from SMA and CM patients showed similar activation profiles with a comparable decreased HLA-DR expression on monocytes and increased frequency of CD69+ and HLA-DR+ T cells. In contrast, the acute-phase IL-10 production was markedly decreased in SMA compared to CM (P = .003) and UM (P = .004). Although in SMA the IL-10 response to LPS-stimulation was larger in amplitude than in CM (P = .0082), the absolute levels of IL-10 reached were lower (P = .013). Both the amplitude and levels of TNF produced in response to LPS-stimulation were larger in SMA than CM (P = .019). In response to PHA-stimulation, absolute levels of IL-10 produced in SMA were lower than in CM (P = .005) contrasting with TNF levels, which were higher (P = .001). These data reveal that SMA patients have the potential to mount efficient IL-10 responses and that the TNF/IL-10 imbalance may reflect a specific monocyte and T cell programming/polarization pattern in response to infection.
    Malaria Journal 08/2012; 11(1):253. DOI:10.1186/1475-2875-11-253 · 3.49 Impact Factor
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    ABSTRACT: Fulani ethnic group individuals are less susceptible than sympatric Mossi ethnic group, in term of malaria infection severity, and differ in antibody production against malaria antigens. The differences in susceptibility to malaria between Fulani and Mossi ethnic groups are thought to be regulated by different genetic backgrounds and offer the opportunity to compare haematological parameters, Tregs and γδT cell profiles in seasonal and stable malaria transmission settings in Burkina Faso. The study was conducted at two different time points i.e. during the high and low malaria transmission period. Two cross-sectional surveys were undertaken in adults above 20 years belonging either to the Fulani or the Mossi ethnic groups 1) at the peak of the malaria transmission season and 2) during the middle of the low malaria transmission season. Full blood counts, proportions of Tregs and γδ T cells were measured at both time-points.As previously shown the Fulani and Mossi ethnic groups showed a consistent difference in P. falciparum infection rates and parasite load. Differential white blood cell counts showed that the absolute lymphocyte counts were higher in the Mossi than in the Fulani ethnic group at both time points. While the proportion of CD4+CD25high was higher in the Fulani ethnic group at the peak of malaria transmission season (p = 0.03), no clear pattern emerged for T regulatory cells expressing FoxP3+ and CD127low. However CD3+γδ+ subpopulations were found to be higher in the Fulani compared to the Mossi ethnic group, and this difference was statistically significant at both time-points (p = 0.004 at low transmission season and p = 0.04 at peak of transmission). Our findings on regulatory T cell phenotypes suggest an interesting role for immune regulatory mechanisms in response to malaria. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities.
    BMC Research Notes 01/2012; 5:76. DOI:10.1186/1756-0500-5-76
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    ABSTRACT: The control of Plasmodium falciparum erythrocytic parasite density is essential for protection against malaria, because it prevents pathogenesis and progression toward severe disease. P falciparum blood-stage parasite cultures are inhibited by human Vγ9Vδ2 γδ T cells, but the underlying mechanism remains poorly understood. Here, we show that both intraerythrocytic parasites and the extracellular red blood cell-invasive merozoites specifically activate Vγ9Vδ2 T cells in a γδ T cell receptor-dependent manner and trigger their degranulation. In contrast, the γδ T cell-mediated antiparasitic activity only targets the extracellular merozoites. Using perforin-deficient and granulysin-silenced T-cell lines, we demonstrate that granulysin is essential for the in vitro antiplasmodial process, whereas perforin is dispensable. Patients infected with P falciparum exhibited elevated granulysin plasma levels associated with high levels of granulysin-expressing Vδ2(+) T cells endowed with parasite-specific degranulation capacity. This indicates in vivo activation of Vγ9Vδ2 T cells along with granulysin triggering and discharge during primary acute falciparum malaria. Altogether, this work identifies Vγ9Vδ2 T cells as unconventional immune effectors targeting the red blood cell-invasive extracellular P falciparum merozoites and opens novel perspectives for immune interventions harnessing the antiparasitic activity of Vγ9Vδ2 T cells to control parasite density in malaria patients.
    Blood 11/2011; 118(26):6952-62. DOI:10.1182/blood-2011-08-376111 · 10.43 Impact Factor
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    ABSTRACT: Human T cells are usually considered to contribute to fast-acting local immune responses. Their somewhat limited T cell receptor (TCR) diversity implies that large subsets of T cells share the capacity to respond to the same restricted set of antigens, rather than showing the fine specificity toward extremely diverse antigens, as is characteristic of T cells. This has been well demonstrated for V9V2 T cells, particularly in non-human primate models. However, much less is known about the other subsets of T cells, herein collectively called V2 neg T cells. Most of these cells express the V1 chain, some express the V3 chain, and very few express the four remaining V chains (V4 to V8). All these V chains can be associated with any of the six V chains (V2, 3, 4, 5, 8, 9). V2 neg T cells are mainly located in epithelial tissues and the spleen, and are barely found in the circulation in normal physiological conditions. This tissue localization has limited their analysis. Establishment of murine models is difficult since murine and human T cell populations vary greatly. For example, the equivalent of murine dendritic epithelial T cells (DETC) does not exist in humans, and conversely, the equivalent of human V9V2 T cells is present only in primates. Therefore, human V2 neg T cells have mostly been examined during pathological situations where their circulating levels are increased. Like V9V2 T cells, V1 and V3 T cells have been shown to be involved in widely diverse pathological contexts, such as infection, cancer, auto-immunity, and inflammation. This suggests that T cells respond to a variety of altered microenvironments induced by these situations. It is acknowledged that T cells can recognize ubiquitous stress-induced conserved antigens in their native form, and altered-self or foreign ligands presented on non-polymorphic molecules in total independence of classical MHC molecules. Since V2 neg T cells can recognize broadly distributed antigens and are localized at the interface with the outer environment within epithelial tissues, V2 neg T cells can act as a first line of defense in the surveillance of body integrity and microorganism infections. Nevertheless, V2 neg T cells can also display effector/memory phenotypes similar to conventional MHC-restricted T cells. This suggests an ability to mount long-lasting anamnestic immunity similar to conventional T cells. Here, we will review what is currently known about V2 neg T cells highlighting the pathological situations where they expand. We will also discuss what is known concerning the cellular and molecular mechanisms of their activation and their effector functions.
    The Open Immunology Journal 10/2009; 2(2):106-118. DOI:10.2174/1874226200902020106
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    ABSTRACT: gammadelta T cells recognize stress-induced autoantigens and contribute to immunity against infections and cancer. Our previous study revealed that Vdelta2-negative ((neg)) gammadelta T lymphocytes isolated from transplant recipients infected by cytomegalovirus (CMV) killed both CMV-infected cells and HT29 colon cancer cells in vitro. To investigate the antitumor effects of Vdelta2(neg) clones in vivo, we generated hypodermal HT29 tumors in immunodeficient mice. Concomitant injections of Vdelta2(neg)clones, in contrast to Vdelta2(+) cells, prevented the development of HT29 tumors. Vdelta2(neg) clones expressed chemokine C-C motif receptor 3 (CCR3) and migrated in vitro in response to chemokines secreted by HT29 cells, among which were the CCR3 ligands macrophage inflammatory protein-1delta and monocyte chemoattractant protein-4. More importantly, a systemic i.p. treatment with Vdelta2(neg) clones delayed the growth of HT29 s.c. tumors. The effect of in vivo gammadelta T-cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-CCR3 antibody. gammadelta T-cell passive immunotherapy was dependent on the cytotoxic activity of the gammadelta effectors toward their targets because Vdelta2(neg) clones were not able to inhibit the growth of A431 hypodermal tumors. Our findings suggest that CMV-specific Vdelta2(neg) cells could target in vivo cancer cells, making them an attractive candidate for antitumor immunotherapy.
    Cancer Research 05/2009; 69(9):3971-8. DOI:10.1158/0008-5472.CAN-08-3037 · 9.28 Impact Factor
  • C. Behr · M. Pizon · P. Legembre
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    ABSTRACT: Le récepteur de mort CD95 appartient à la famille du récepteur tumor necrosis factor (TNF). Ce récepteur est retrouvé muté et non fonctionnel dans les souris Lpr et Lprcg et chez les patients atteints d’autoimmune lymphoproliferative syndrome de type Ia (ALPS). Ces mutations du récepteur CD95 bloquent le signal apoptotique et entraînent, chez le patient comme chez la souris, une lymphoprolifération, des adénopathies, une splénomégalie, une accumulation d’une population lymphocytaire T CD4−CD8− et de l’auto-immunité. Alors que CD95 a été impliqué, dans un premier temps, dans la contraction du nombre de lymphocytes T activés lors de la réponse antitumorale ou infectieuse, il semble en fait jouer un rôle dans la tolérance périphérique et l’élimination des lymphocytes activés de manière chronique par les antigènes du soi de faible affinité. Le ligand de CD95, le CD95L, est exprimé à la surface des lymphocytes T activés et des cellules natural killer (NK) où il joue un rôle important dans l’élimination des cellules tumorales et infectées. L’engagement de CD95 par le CD95L déclenche l’activation de cystéines protéases appelées caspases (cysteine aspartyl-specific proteases). Une de ces caspases, la caspase-12 est retrouvée sous une forme longue (active et ancestrale), principalement dans certaines populations d’origine africaine, et interviendrait dans l’atténuation de la réponse inflammatoire. La pression de sélection responsable de la conservation par ces populations de la forme longue de la caspase-12 reste, à ce jour, inconnue. The death receptor CD95 belongs to the tumor necrosis factor (TNF) receptor super family. Lpr and Lprcg mice and patients suffering from autoimmune lymphoproliferative syndrome (ALPS) type Ia exhibit mutated and non-functional CD95 alleles. The patients and the mice display similar phenotypes such as lymphoproliferation, adenopathy, splenomegaly, accumulation of double negative T lymphocytes (B220+CD4−CD8−) and the production of autoimmune antibodies. Although CD95 was initially thought to be involved in the elimination of T lymphocytes activated by tumoral or infectious antigens, recent studies have shown that in fact CD95 plays a pivotal role in peripheral tolerance and the elimination of lymphocytes chronically stimulated by self-antigens. The cognate CD95 ligand, CD95L, is expressed at the membrane of activated lymphocytes and natural killer cells upon which it plays a pivotal role in the elimination of transformed and infected cells. The engagement of CD95 leads to the activation of a family of cysteine proteases termed caspases and the subsequent induction of the apoptotic phenotype. Among these caspases, the long form of caspase-12 has been found only in certain African populations, wherein it may play an essential role in the attenuation of the inflammatory response. To date, the selection pressure responsible for the conservation of the long form of the caspase-12 in these populations remains unknown.
    Journal africain du cancer / African Journal of Cancer 05/2009; 1(2):104-109. DOI:10.1007/s12558-009-0020-5
  • C. Behr · M. Pizon · P. Legembre
    Journal africain du cancer / African Journal of Cancer 01/2009; 1(2):104-109.
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    ABSTRACT: The Pf72/Hsp70-1 antigen is a major target in the naturally acquired immunity against Plasmodium falciparum malaria. We carried out an extensive analysis of the responses to several epitopes on the least conserved C-terminal domain, according to the mode of sensitization: malaria infection or immunization with different immunogens. We found significant differences in the panel of B-cell epitopes recognized by animal models including primates, and by humans sensitized by natural infection. We focused the analysis on one epitope that is unique to Plasmodium species. It is specifically recognized by a monoclonal antibody that mediates the killing of infected hepatocytes in vitro. We produced a polymeric multiple antigenic peptide (MAP) form of this sequence, which enabled us to identify a new B-cell epitope not detected by ELISA with linear peptides. The polymer was strongly recognized by sera from monkeys or humans sensitized by natural infection, whereas the monomer was not. We modelled the three-dimensional structure of the Pf72/Hsp70-1 sequence, using known Escherischia coli DnaK structures as a template. This predicted that the corresponding region would form a loop in the native antigen. The results presented here suggest that the MAP strategy is also particularly useful as a means of obtaining suitable synthetic models for conformation-dependent epitopes.
    Parasite Immunology 07/2008; 22(11):535 - 543. DOI:10.1046/j.1365-3024.2000.00333.x · 1.85 Impact Factor
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    ABSTRACT: Epidemiological data point to an increased risk of HIV-1 mother-to-child transmission in pregnant women with malaria, by unknown mechanisms. We show here that surface binding of a recombinant Plasmodium falciparum adhesin to chondroitin sulphate A proteoglycans increases HIV-1 replication in the human placental cell line BeWo, probably by a P. falciparum adhesin-induced long-terminal repeat-driven TNF-alpha stimulation. This suggests that placental malaria could increase the risk of HIV-1 transmission in utero.
    AIDS (London, England) 04/2008; 22(6):785-7. DOI:10.1097/QAD.0b013e3282f560ee · 6.56 Impact Factor
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    Retrovirology 01/2008; 5(Suppl 1). DOI:10.1186/1742-4690-5-S1-P9 · 4.77 Impact Factor
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    ABSTRACT: T cells are thought to play a critical role in cerebral malaria pathogenesis. However, available evidences are restricted to rodent models in which V beta specific T cell expansion has been associated with neurological syndrome suggesting involvement of superantigens or dominant antigens. Using flow cytometry, we studied the peripheral V beta T cell repertoire of Ghanaian children with cerebral malaria, uncomplicated malaria and asymptomatic control children, to look for either expansion or deletion of specific V beta associated with cerebral malaria. At admission, the general pattern of the repertoire of the patients was very similar, with no major distortion compared to the control group a part a significant increase of the frequency of the V beta 21.3 subset correlating with disease severity and attributed to the CD4 subset. During convalescence very limited fluctuations were observed including a significant decrease of the V beta 21.3 subset and increase of the V beta 20 subset, a subset not detected at admission. The remarkable stability of the V beta repertoire observed in acute malaria either cerebral or uncomplicated argues against the idea that cerebral malaria would result from a T cell-mediated inflammatory shock syndrome driven by some dominant super-antigenic activity(ies). The significance of the reproducible increase of the CD4+V beta 21.3T cell subset deserves further investigations.
    Microbes and Infection 10/2007; 9(11):1252-9. DOI:10.1016/j.micinf.2007.05.019 · 2.73 Impact Factor
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    ABSTRACT: Respiratory distress (RD), a symptom of underlying metabolic acidosis, has been identified as a major risk factor for mortality in children with severe malaria in Africa, yet the molecular mediators involved in the pathogenesis of RD have not been identified. We studied circulating levels of mediators of inflammation--including the cytokines tumor necrosis factor (TNF)- alpha and interleukin (IL)-10; the chemokines macrophage inflammatory protein (MIP)-1 alpha , MIP-1 beta , and IL-8; and the immune activation marker neopterin--in children with RD, severe malarial anemia (SMA), cerebral malaria (CM), and uncomplicated malaria (UM). Children with RD had significantly higher plasma levels of TNF- alpha , IL-10, and neopterin and a significantly higher TNF- alpha : IL-10 ratio than those without RD. In addition, the results demonstrated that, relative to UM, CM was associated with increased levels of TNF- alpha and decreased levels of MIP-1 alpha , whereas SMA was associated with decreased levels of IL-10. Circulating levels of neopterin were inversely correlated with hemoglobin, whereas levels of MIP-1 beta were positively correlated with parasitemia. We conclude that distinct clinical presentations of severe malaria are associated with specific patterns of inflammatory mediators. In particular, we show, to our knowledge for the first time, that patients with malaria and RD have a strong and unbalanced proinflammatory response that may be involved in the pathogenesis of the underlying metabolic acidosis.
    The Journal of Infectious Diseases 12/2006; 194(10):1438-46. DOI:10.1086/508547 · 5.78 Impact Factor
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    ABSTRACT: The Saimiri sciureus monkey is a well-established host for experimental studies with human malaria parasites. During the course of iterative inoculations with Plasmodium falciparum parasitised red blood cells (RBC), anti-RBC alloantibodies were detected in the sera of two of eight Saimiri monkeys. These anti-RBC antibodies were further used to investigate RBC phenotypes in 35 colony-reared Saimiri monkeys by flow cytometry. Three RBC phenotypes (named I-III) were observed. Their distribution was I (86%), II (11%) and III (3%). Using the Palo Alto FUP-2 strain, a variant P. falciparum line insensitive to hyperimmune serum and the passive transfer of anti-RBC alloantibodies, a dramatic drop in parasite growth was documented in an incompatible monkey.
    Microbes and Infection 07/2005; 7(7-8):983-9. DOI:10.1016/j.micinf.2005.03.032 · 2.73 Impact Factor
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    ABSTRACT: The neotropical primate squirrel monkey is used in many areas of biomedical research including neuroendocrinology, immunology and infectious diseases. However, research has been hampered by the lack of immunological tools for this primate. A series of 67 commercially available monoclonal antibodies to human CD antigens or cytokines were tested on Saimiri mononuclear cells and the specificity was assessed by double staining using flow cytometry. Monoclonal antibodies defining the main mononuclear cells subsets (monocytes, B, T, including CD4 and CD8 T cells) as well as activation markers have been identified. The conditions to specifically identify the various cell subsets using two color flow cytometry and establish their relative proportions have been set-up. We also have established normal values of the main circulating mononuclear cell subsets for adult Saimiri sciureus monkeys from the breeding unit of Institut Pasteur in French Guiana. The distribution between spleen, blood and lymph nodes has been compared. These tools allow documenting the phenotype of most Saimiri mononuclear cell subsets and assessing their activation level. This opens new perspectives for vaccinology and immunopathology research in this experimental non-human primate host, in particular for malaria research.
    Journal of Immunological Methods 03/2005; 297(1-2):61-71. DOI:10.1016/j.jim.2004.11.019 · 2.01 Impact Factor
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    ABSTRACT: In endemic areas, clinical manifestations of Plasmodium falciparum infection range from asymptomatic parasitaemia to life-threatening severe syndromes. Immune differences that could account for this disparity are poorly understood. Using tight criteria to classify patients into non-overlapping clinical categories, we showed that cerebral malaria and severe anaemia were distinct immunological syndromes and that a proper quantitative description of cytokine profiles in the various clinical groups is essential to the understanding of the activation of immunocompetent cells. Due to the limited size of paediatric blood samples, we chose to measure cytokine mRNA using real-time RT-PCR. We showed that RT efficiency displayed intra-and intergenic variations that have to be taken into consideration for reliable absolute RNA quantification when comparing clinical cases. We thus developed a SYBR Green I-based real-time RT-PCR method using synthetic external RNA standards specific for each gene. Absolute RNA quantification is achieved by reverse transcribing known copy numbers of this RNA standard in parallel with cellular RNA. Strictly specific primers were designed to allow the quantification of any RNA in the same thermocycling parameters for future automation. Our method gave similar results for a lower cost when compared with TaqMan, and led to reproducible and reliable absolute RNA quantification. We validated it in vitro on naïve PBMC stimulated by LPS and ex vivo on PBMC from malaria patients. This new method raises the unprecedented possibility to compare cytokine mRNA levels between different clinical groups and is a powerful tool to further study the inflammation processes associated to malaria.
    Journal of Eukaryotic Microbiology 03/2005; 52(2). DOI:10.1111/j.1550-7408.2005.05202003_3_2.x · 3.22 Impact Factor
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    ABSTRACT: Human T γδ lymphocytes, bearing Vγ9 Vδ2 chains, constitute a small proportion (1–5%) of the lymphocytes in the blood. These cells recognize nonpeptidic molecules such as phosphoantigens, intermediary metabolites products by microorganisms, including parasites. These antigens are recognized in the absence of molecules CMH. Our previous results have shown that Plasmodium falciparum synthetize phosphoantigen, intermediary products of the Rohmer pathway. These phosphoantigens are able to induce the proliferation of T γδ lymphocytes, producing pro-inflammatory cytokines and providing a parasitotoxic activity against parasitized red blood cells. Also, during the acute phase of infection, these lymphocytes increase both in percentage and numbers. Today, the biological role of T γδ lymphocytes remains poorly understood. Indeed, these lymphocytes are found in primates but there is no equivalent in rodents. We decided to study this population in the infection model Plasmodium falciparum/spleen intact Saimiri scirieus. In this monkey, we found a population of TVγ9 (representing 0.2–1% of total lymphocytes in the peripheral blood) which react with phosphoantigens as do human TVγ9 cells. Activation kinetics of these cells in infected monkeys show an early activation occuring on the 6th day of infection, while the CD4 T activation starts at day 10. Also, we observed an important increase of numbers and percentages of these TVγ9. Their maximal peak coincides with the beginning of parasite clearance. Together, these data associated with parasitotoxic activity in vitro argue for a role of TVγ9 cells in parasitemia control, particulary during primary infection.
    Journal of Eukaryotic Microbiology 03/2005; 52(2). DOI:10.1111/j.1550-7408.2005.05202003_3_13.x · 3.22 Impact Factor
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    ABSTRACT: Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency. To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1beta, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-gamma, MIF, TGF-beta1 and TNF-alpha mRNA. We showed that the beta-2 microglobulin (beta2-MG) gene was suitable for data normalisation since the level of beta2-MG transcripts in naive PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-beta1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-beta1 mRNA in response to LPS. The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications.
    BMC Immunology 02/2005; 6(1):5. DOI:10.1186/1471-2172-6-5 · 2.25 Impact Factor
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    ABSTRACT: If a number of cytokines and growth factors that have been characterized from human cells were investigated in non-human primates, results from such approaches would allow the development of assays to detect and quantitate cytokines in experimental models. Tumor necrosis factor-alpha (TNF-alpha) is an important pluripotent cytokine which plays a crucial role in host defense. As yet, no complete molecular data have been reported for the squirrel monkey TNF-alpha. Polymerase chain reaction (PCR) primers were used to trace introns, by comparing product sizes obtained using cDNA and genomic DNA as templates. The genomic DNA is composed of four exons and three introns with 1793 nucleotides. The corresponding cDNA is 702 nucleotides and phylogenetic analysis showed that the Saimiri sciureus was most closely related to that of the genus Aotus, a new-world primate, compared to old-world primates (genus Macaca and Papio). The deduced TNF-alpha protein consists of 233 amino acids with 82% identity to human, 95% to new-world monkeys and 79% to old-world monkeys. The cloned TNF-alpha cDNA will be useful to quantitate TNF-alpha at the mRNA level.
    Veterinary Immunology and Immunopathology 04/2003; 92(1-2):37-43. DOI:10.1016/S0165-2427(03)00018-7 · 1.75 Impact Factor

Publication Stats

1k Citations
182.87 Total Impact Points

Institutions

  • 2011–2015
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2012
    • University of Bordeaux
      Burdeos, Aquitaine, France
  • 2002–2005
    • Pasteur Institute of India
      Raj Nilgiri, Odisha, India
  • 1991–1998
    • Institut Pasteur
      • Department of Parasitology and Mycology
      Lutetia Parisorum, Île-de-France, France