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Leishmania donovani induit un défaut d’acidification du phagosome

Leishmania donovani induit un défaut d’acidification du phagosome

Laval (Québec) H7V 1B7, Canada. albert.descoteaux@iaf.inrs.ca NOUVELLE > La leishmaniose viscérale (Kala-azar) est causée par le protozoaire Leishmania donovani, un parasite qui infecte les macrophages du foie, de la rate et de la moelle osseuse. Cette infection est chronique et risque d’être mortelle si elle n’est pas traitée. Leishmania est transmis à l’humain par voie sanguine sous sa forme promastigote lors d’un repas sanguin de la mouche des sables du genre Lutzomyia ou Phlebotomus. Une fois chez l’hôte mammifère, le parasite est internalisé par des macrophages. Il altère alors le processus normal de phagocytose et mène sans opposition sa différenciation en amastigote. La forme amastigote, qui est résistante à l’arse- nal microbicide du phagolysosome, peut alors se répliquer et infecter d’autres macrophages. Il n’existe pas de vaccin efficace pour prévenir la leishmaniose. De plus, la résistance du parasite aux agents pharmacologiques réduit gran- dement l’efficacité des médicaments conventionnels.
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In vitro effects of purine and pyrimidine analogues on Leishmania donovani and Leishmania infantum promastigotes and intracellular amastigotes

In vitro effects of purine and pyrimidine analogues on Leishmania donovani and Leishmania infantum promastigotes and intracellular amastigotes

1 Université de Lyon, Université Claude-Bernard Lyon I, ISPB-Faculté de Pharmacie, Lyon, France; 2 Institut de recherche pour le développement (IRD), UMR InterTryp IRD/CIRAD, campus international de Baillarguet, Montpellier, France Abstract Inhibition of parasite metabolic pathways is a rationale for new chemotherapeutic strategies. The pyrimidine and purine salvage pathways are thus targets against Leishmania donovani and L. infantum, causative agents of visceral human leishmaniasis and canine leishmaniosis. The antiproliferative effect of the pyrimidine analogues Cytarabine and 5-fluorouracil and of the purine analogues Azathioprine and 6-mercaptopurine was evaluated in vitro on the promastigote and the intracellular amastigote stages of the parasite. Cytarabine and 5-fluorouracil were the best inhibitors against promastigotes, whereas 5- fluorouracil and aza- thioprine displayed the best efficacy against the amastigote stage. The ultrastructural study showed an important cytoplasmic vacuolization and with azathioprine and 5-fluorouracyl, a mitochondrial swelling and appearance of autophagosome-like struc- tures. Alterations of the kinetoplast were also observed with 5-fluorouracil, all these damages eventually resulting in an autol- ysis process that triggered the subsequent death of the intracellular parasites.
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Multifaceted population structure and reproductive strategy in Leishmania donovani complex in one Sudanese village.

Multifaceted population structure and reproductive strategy in Leishmania donovani complex in one Sudanese village.

Leishmaniases are worldwide vector-borne diseases of humans and domestic animals, caused by protozoan parasites of the genus Leishmania. These parasitic infections are a serious public health problem, with about 350 million persons at risk and 2,357,000 new cases per year [1]. The genus Leishmania totals approximately 20 described species causing human infections (reviewed in [2]) with a wide variety of clinical symptoms: cutaneous, visceral, mucocutaneous, mucosal and post-kala-azar dermal (PKDL) leishmaniases. Visceral leishmaniasis is the most severe form of the disease, which can be lethal if it goes untreated. It is the most widespread leishmaniasis form, especially in India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil [1,3,4]. In this study, we focused on human and canine samples collected in Sudan, where visceral leishmaniasis is endemic in the eastern and southern parts of the country and has claimed the lives of thousands of people [5]. Visceral leishmaniasis is mainly caused by species from the Leishmania donovani complex [6]. Multilocus enzyme electrophoresis
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Synthesis and biological evaluation against Leishmania donovani of novel hybrid molecules containing indazole-based 2-pyrone scaffolds

Synthesis and biological evaluation against Leishmania donovani of novel hybrid molecules containing indazole-based 2-pyrone scaffolds

2-Pyrone is an important class heterocyclic scaffold. Due to its pharmacophore properties, synthesis of hybrid derivatives bearing 2-pyrone moiety has attracted much attention; most of the compounds display strong biological activities such as cytotoxic, antibiotic and antifungal. 13-16 It is also known that some pyrones exhibit antileishmanial activity. 17,18 In that context, we planned to explore the possibility of introducing a 2-pyrone into indazole ring. We wish to report here the reductive coupling of N-alkyl-6(5)-nitroindazoles and 4-hydroxy-6- methyl-2-pyrone in order to obtain indazole/pyrone hybrid compounds and their inhibition activity against the axenic and intramacrophage amastigotes of Leishmania donovani.
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Innate Immune B Cell Activation by Leishmania donovani Exacerbates Disease and Mediates Hypergammaglobulinemia

Innate Immune B Cell Activation by Leishmania donovani Exacerbates Disease and Mediates Hypergammaglobulinemia

reported to generate decreased antibody responses ( Pasare and Medzhitov, 2005; Kasturi et al., 2011 ). The role of B cell TLR signaling in antibody production has mainly been studied in models of autoimmune diseases. TLR7 and 9 were shown to contribute importantly to the production of anti-nuclear antibodies in various models of lupus-like disease ( Christensen et al., 2006; Han et al., 2015 ). In contrast, little is known about the role of B cell TLR signaling in infectious dis- eases. MyD88 appears to be required for preventing lethal dissemination of commensal bacteria during colonic damage caused by dextran sulfate sodium ( Kirkland et al., 2012 ). In this model, MyD88 was essential for the production of immunoglob- ulin M (IgM) and complement by B cells. TLR9, TLR7, and MyD88 expression in B cells was also shown to be involved in substan- tially enhancing T cell-dependent germinal center immunoglob- ulin G (IgG) responses following inoculation with virus-like particles ( Hou et al., 2011 ). During Salmonella typhimurium infec- tion, however, MyD88 in B cells was mainly associated with cytokine production and led to disease susceptibility via inter- leukin-10 (IL-10) induction ( Neves et al., 2010 ). We have also pre- viously reported that B cell-derived IL-10 production following Leishmania donovani infection was dependent on MyD88 expression in B cells ( Bankoti et al., 2012 ).
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Immunostimulatory properties of dendritic cells after Leishmania donovani infection using an in vitro model of liver microenvironment.

Immunostimulatory properties of dendritic cells after Leishmania donovani infection using an in vitro model of liver microenvironment.

Because of its location and function, the liver is continuously exposed to a wide range of antigens. Pathogenic microorganisms must be eliminated while a large number of dietary or commensal organism antigens and hepatic metabolites must be tolerated. Therefore, the liver has developed a specialized immune system that favours tolerance rather than immunity and liver dendritic cells (DCs) act as a major cell subtype in promoting this response. Our work aimed to examine if such immunologic hyporesponsiveness has an impact on the control of the hepatic burden of Leishmania donovani, a protozoan parasite that grows in liver and spleen tissues after infection (called visceral leishmaniasis in South America and Mediterranean basin, and Kala Azar in South East Asia). We first modelized an original model of hepatic DCs and infected them with Leishmania donovani. In contrast to control DCs, infection of hepatic DCs restored the alterate capacity of non-infected liver DCs to stimulate allogeneic T cell proliferation and IFNc secretion. Such characteristics were recently shown to favour granuloma formation in mouse liver. This research provides an explanation for the observation that Leishmania parasite growth is controlled in the liver via an efficient granuloma response.
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Changes in Lipid and Fatty Acid Composition During Intramacrophagic Transformation of Leishmania donovani Complex Promastigotes into Amastigotes

Changes in Lipid and Fatty Acid Composition During Intramacrophagic Transformation of Leishmania donovani Complex Promastigotes into Amastigotes

Lipids Introduction Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis (VL) in humans and of canine leishmaniasis in dogs. Leishmania is an intracellu- lar pathogen, whose establishment depends on its success- ful internalization and multiplication inside macrophages, its mammalian host cell. The Leishmania life cycle is divided into two phases, each of them involving a differ- ent stage, the promastigote stage in the insect vector and the amastigote stage inside the host’s macrophages. The promastigotes inoculated during the blood meal of the hematophagous sandfly are phagocytosed by endocytosis and undergo a transformation into amastigotes within a parasitophorous vacuole of phagolysosomal origin. This process is a vital step in the Leishmania life cycle and could be pivotal in the research for new treatments against
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Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani

Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani

Distributed under a Creative Commons Attribution - NonCommercial - ShareAlike| 4.0 Haplotype selection as an adaptive mechanism in the protozoan pathogen Leishmania donovani Pablo Prieto Barja, Pascale Pescher, Giovanni Bussotti, Franck Dumetz, Hideo Imamura, Darek Kedra, Malgorzata Domagalska, Victor Chaumeau,

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Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

Genetic polymorphism kDNA minicircle sequence A B S T R A C T Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani. In addition to fatal VL, these parasites also cause skin diseases in immune-competent and -suppressed people, post-kala azar dermal leishmaniasis (PKDL) and HIV/VL co-infections, respectively. Genetic polymorphism in 36 Ethiopian and Sudanese L. donovani strains from VL, PKDL and HIV/VL patients was examined using Ampli fied Fragment Length Polymorphism (AFLP), kDNA minicircle sequencing and Southern blotting. Strains were isolated from di fferent patient tissues: in VL from lymph node, spleen or bone marrow; and in HIV/VL from skin, spleen or bone marrow. When VL and PKDL strains from the same region in Sudan were examined by Southern blotting using a DNA probe to the L. donovani 28S rRNA gene only minor differences were observed. kDNA sequence analysis distributed the strains in no particular order among four clusters (A – D), while AFLP analysis grouped the strains according to geographical origin into two major clades, Southern Ethiopia (SE) and Sudan/Northern Ethiopia (SD/NE). Strains in the latter clade were further divided into subpopulations by zymodeme, geography and year of isolation, but not by clinical symptoms. However, skin isolates showed signi ficantly (p < 0.0001) fewer polymorphic AFLP fragments (average 10 strains = 348.6 ± 8.1) than VL strains (average 26 strains = 383.5 ± 3.8).
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Activité des MAPKs et de NF-KB chez le macrophage
infecté par Leishmania donovani

Activité des MAPKs et de NF-KB chez le macrophage infecté par Leishmania donovani

Nous avons ensuite choisi des macrophages dérivés de la moelle osseuse (BMM) de souris BALB/c comme modèle d'infection. Les macrophages, traités ou non à l'IFN-y, ont ét[r]

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Modulation des radeaux lipidiques et des propriétés de fusion des phagosomes par le lipophosphoglycane du parasite intracellulaire Leishmania donovani

Modulation des radeaux lipidiques et des propriétés de fusion des phagosomes par le lipophosphoglycane du parasite intracellulaire Leishmania donovani

Finalement, nous avons aussi identifié sur le phagosome plusieurs protéines reconnues comme étant présentes dans les radeaux lipidiques de la membrane plasmique, telles que la flotiÏline[r]

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Study of Leishmania pathogenesis in mice : experimental considerations

Study of Leishmania pathogenesis in mice : experimental considerations

investigate the pathogenic diversity, the impact of the host genetic background and of the Leishmania genotypes, animal models are widely used. Classically, in infected animals, parasite-activated CD4 + T cells rapidly proliferate in the lymph nodes, differentiate and secrete specific cyto- kines. Th1 cells secrete IL2, IFNγ and TNFα, leading to macrophage activation and parasite elimination. On the other hand, the Th2 response is associated with IL4, IL5 and IL13 production and with parasite proliferation (for review see [11]). When studying a newly isolated strain, the experimental settings have to be carefully designed and several parameters must be taken into account. The object- ive of this review is to summarize results on the pathogenic mechanisms in mice infected by Leishmania spp. We will focus on the two main clinical forms: visceral leish- maniasis (VL) and cutaneous leishmaniasis (CL). We will first describe the experimental data on the influence of the genetic background in mouse models of VL and CL caused by Leishmania donovani and L. infantum and of CL caused by L. major, L. mexicana and L. tropica. Then, data obtained in mouse models of VL by L. infantum and of CL by L. major will be reviewed, particularly: (i) the immune cells involved and the associated-immune response and (ii) the parameters (mouse and parasite genotypes, parasite dose and inoculation route) that influence the infection outcome.
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Case Report: Leishmania and HIV Co-Diagnosis: How to Understand Medical History?

Case Report: Leishmania and HIV Co-Diagnosis: How to Understand Medical History?

pattern. During the late stages of the course of HIV infection the specific antibodies are lost and can lead to a weakly reactive immunoblot ( 2 ). Leishmaniasis is a sand fly-borne protozoan parasitic disease. Visceral leishmaniasis (VL) is the disseminated form of Leishmania donovani or infantum infection with potential severe complications (cachexia, hepatic dysfunction, hemorrhages and - almost always without proper treatment - death). Leishmaniasis is considered as one of the 13 “core” neglected tropical diseases in the world with 50 000 to 90 000 estimated cases of VL each year worldwide ( 3 , 4 ). In 2015, 90% of new VL cases reported to WHO occurred in 7 countries (Brazil, India, Ethiopia, Kenya, Somalia, South Sudan and Sudan) ( 5 ).
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Antimony resistance and environment: Elusive links to explore during Leishmania life cycle

Antimony resistance and environment: Elusive links to explore during Leishmania life cycle

In the anthroponotic forms of leishmaniasis, like Leishmania donovani and most foci of Leishmania tropica, parasites are trans- mitted from patients to patients by the bite of a sandfly. In zoo- notic forms of leishmaniasis, that include different cutaneous or mucocutaneous leishmaniasis, parasites are transmitted to pa- tients by sandflies from a reservoir host, represented by domestic or wild mammals. In this context, the main chemotherapeutic pressure that Leishmania will encouter will be during the treatment of individuals, with the exception of zoonotic leishmaniasis caused by L. infantum. In this visceral form of the disease, the therapeutic pressure is applied on a population of parasites that is not trans- mitted, because humans are generally not considered as a reservoir for L. infantum, with the exeption for HIV/Leishmania co-infection ( Molina et al., 1999 ). Dogs are the main domestic and peridomestic reservoir host of L. infantum. In the occidental part of the Mediter- ranean (France, Italy, Spain, Portugal. . .), dogs are treated with the same drugs used to treat human leishmaniasis, while this is not supposed to occur. Infected dogs never achieve parasitological cure, even when their health improves. This, in conjunction with the high percentage of animals that will relapse and repeatedly be treated with the same compound, makes antimony resistant parasites selection and transmission possible ( Campino and Maia, 2012 ). Contradictory results have been obtained in the very few studies made to evaluate the susceptibility to antimonials of L. infantum strains isolated from treated and untreated dogs. While
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Multilocus microsatellite typing of Leishmania and clinical applications: a review

Multilocus microsatellite typing of Leishmania and clinical applications: a review

(Leishmania) infantum and Leishmania (Leishmania) donovani belong to the species complex Leishmania donovani, while Leishmania (Leishmania) tropica and Leishmania (Leish- mania) major constitute two distinct species complexes. Numerous Leishmania species have been identified and the current classification is based on isoenzyme typing using mul- tilocus enzyme electrophoresis (MLEE) [ 46 ]. MLEE, consid- ered by the World Health Organization as the reference method for strain identification, separates Leishmania strains into groups through identification of their enzymatic patterns, so-called zymodemes. Genotypes were therefore identified indirectly, meaning that nucleotide substitution might not be detected by MLEE, leading to a low discrimination power. So, for detailed population genetics studies, it is essential to use genetic markers with high discriminatory potential. Analy- sis of highly variable, codominant microsatellite markers is a reliable alternative genotyping method. Microsatellites are repeated motifs of about 1–6 non-coding nucleotides found in all eukaryotic and prokaryotic genomes [ 28 ]. They are Mendelian codominant and neutral markers (not affected by natural selection) [ 28 ]. The mutation rate of microsatellites is often quoted in the range of 10 3 –10 4 per locus per genera- tion [ 19 , 20 ]. The genetic variation at many microsatellite loci is characterized by high heterozygosity and the presence of multiple alleles which makes microsatellite sequence modifica- tions particularly useful for studying differences between closely related organisms [ 20 , 57 ]. Consequently, the analysis of microsatellite sequence variation is an important tool for population genetic studies for many species [ 20 , 56 , 57 ]. More- over, multilocus microsatellite typing (MLMT) yields consis- tently reproducible results that are potentially exchangeable among laboratories [ 57 ]. MLMT has been used to identify, dis- criminate, and characterize geographically distributed popula- tions of strains of Leishmania even at the intra-zymodeme level [ 14 , 44 , 57 ]. This review aims (i) to draw an inventory of the existing microsatellite markers, (ii) to explore the asso- ciation between MLMT genotypes and clinical variations on the one hand, and drug-resistant strains in endemic foci on the other, and (iii) to discuss the use of this tool in case of several leishmaniasis episodes in patients.
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Les facteurs solubles sécrétés par les cellules B en réponse à Leishmania infantum et leurs effets sur la réplication du VIH-1

Les facteurs solubles sécrétés par les cellules B en réponse à Leishmania infantum et leurs effets sur la réplication du VIH-1

33 Abstract Visceral leishmaniasis is caused by the protozoan parasites Leishmania infantum and Leishmania donovani. This infection is characterized by an uncontrolled parasitization of internal organs which, when left untreated, leads to death. Disease progression is linked with the type of immune response generated and a strong correlation was found between disease progression and serum levels of the immunosuppressive cytokine IL 10. Other studies have suggested a role for B cells in the pathology of this parasitic infection and the recent identification of a B-cell population in humans with regulatory functions, which secretes large amounts of IL-10 following activation, have sparked our interest in the context of visceral leishmaniasis. We report that incubation of human B cells with Leishmania infantum amastigotes resulted in upregulation of multiple cell surface activation markers and a strong dose-dependent secretion of IL-10. Cell sorting experiments allowed us to identify the IL-10-secreting B cell subset (i.e. CD19 + CD24 + CD27 - ). The parasite-mediated IL-10 secretion was shown to rely on the
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Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

Results Leishmania major promastigotes impair the recruitment of LC3 to phagosomes in a GP63-dependent manner Growing evidence suggests that LAP contributes to augmenting the microbicidal and immune functions of phagosomes [ 18 , 21 – 23 , 25 ]. Given the ability of Leishmania promastigotes to interfere with phagolysosomal biogenesis, we sought to investigate the impact of Leishmania promastigotes on LAP during their internalization by macrophages. Because the GPI-anchored zinc-metalloprotease GP63 interferes with phagolysosomal biogenesis and function [ 7 , 11 ], it was of interest to determine the potential impact of this molecule on LAP. To this end, we included a L. major GP63-deficient mutant (Δgp63) and its complemented counterpart (Δgp63 +gp63) in our study. Recruitment of LC3 to membranes requires the lipidation of cytosolic LC3-I to form membrane-bound LC3-II [ 27 ]. We first assessed by Western blot whether inter- nalization of L. major promastigotes led to the lipidation of LC3 in bone marrow-derived mac- rophages (BMM). We observed that WT parasites induced a rapid and transient conversion of LC3-I to LC3-II ( Fig 1A ). Furthermore, GP63-expressing (WT and Δgp63+gp63) and Δgp63 parasites promoted conversion of LC3-I to LC3-II to a similar extent ( Fig 1B ), indicating that GP63 is not responsible for this conversion. In addition, expression of the autophagic cargo receptor sequestasome (p62/SQSTM1) increased with time upon infection independently of GP63 ( Fig 1C ) and was sensitive to cycloheximide. We next determined whether internaliza- tion of L. major promastigotes was accompanied by the recruitment of LC3 to phagosomes. To this end, we incubated macrophages with WT, Δgp63, or Δgp63+gp63 promastigotes and we examined the intracellular distribution of LC3 by confocal immunofluorescence microscopy. We detected LC3 on less than 10% of phagosomes containing WT promastigotes ( Fig 2A and 2B ). By contrast, we detected LC3 on a significantly higher percentage (~20%) of phagosomes containing Δgp63 parasites ( Fig 2A and 2B ). As expected, phagosomes containing the Δgp63 +gp63 parasites were similar to WT phagosomes with respect to the presence of LC3. In all cases, the optimal time point for the recruitment of LC3 to phagosomes containing Leishmania promastigotes was 1 hour after the initiation of phagocytosis. Recruitment of LC3 to phago- somes was previously shown to be preceded by recruitment of Beclin-1 [ 18 ] and was shown to be involved in LAP of Burkholderia pseudomallei [ 28 ]. In the case of L. major, we did not detect recruitment of Beclin-1 to phagosomes ( Fig 2C ). Collectively, these results suggest that L. major promastigotes interfere with recruitment of LC3 to phagosomes through a GP63-depen- dent mechanism.
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No evidence of interspecific genetic exchange by multi-locus microsatellite typing between Leishmania Killicki and Leishmania Major in a mixed focus of cutaneous Leishmaniasis in Southeast Tunisia

No evidence of interspecific genetic exchange by multi-locus microsatellite typing between Leishmania Killicki and Leishmania Major in a mixed focus of cutaneous Leishmaniasis in Southeast Tunisia

Copyright: ©2017 Harrabi M, et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract Sixty-four Leishmania samples were isolated from patients in several villages in the Tataouine governorate, southeast Tunisia. This region is known to be a mixed focus of human cutaneous leishmaniasis caused by

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Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania

Novel insights into genome plasticity in Eukaryotes: mosaic aneuploidy in Leishmania

Introduction Leishmaniasis is one of the world’s most neglected diseases, causing a broad spectrum of diseases (ranging from asymptomatic to lethal) in 98 countries on four continents. An estimated 350 million people are considered at risk of contracting leishmaniasis, with approximately 59 000 deaths due to visceral leishmaniasis per year. Mortality and morbidity from leishmaniasis worldwide show a worrying increasing trend; and there is a lack of potent, cost-effective and safe therapy (den Boer et al., 2011, Croft et al., 2011). Leishmaniases are due to protozoa of the genus Leishmania, digenetic parasites that are transmitted to mammals by the bite of an infected insect vector, a Phlebotomine sand fly. During its lifecycle, the parasite alternates between (i) motile promastigotes that live extracellularly in the digestive tract of the vector and develop into non-
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Implication des Galectines-3, 7 et 9 dans la réponse immunitaire contre le parasite Leishmania major

Implication des Galectines-3, 7 et 9 dans la réponse immunitaire contre le parasite Leishmania major

Parasites et cellules Les souches de Leishmania sont maintenues en croissance dans un milieu SDM modifié et supplémenté avec 10% de FBS (Thermo Scientific), 5g/ml d’hémine (Sigma) ainsi que 100g/ml de pénicilline (Gibco), 100g/ml de streptomycine (Gibco) et 2M de glutamine (Fisher Scientific). Ils sont cultivés à température de la pièce dans flasques de 25cm 2 (Corning) placés dans un agitateur rotatif. Sauf avis contraire, les parasites utilisés sont des promastigotes en phase de croissance stationnaire ; c’est-à-dire qu’ils sont en culture depuis environ 7 jours. Les expériences effectuées in vivo sont réalisées avec des promastigotes fraichement issus d’amastigotes. Les amastigotes sont obtenus suite à l’injection de promastigotes en phase stationnaire dans le coussinet plantaire de souris BALB/c. Environ 5 à 7 semaines post infection, les souris sont sacrifiées et les coussinets plantaires sont récupérés afin d’isoler les amastigotes. L’isolation débute par le bris mécanique des tissus par l’emploi d’un pilon et d’un treillis métallique (Sigma). Les macrophages (contenant les amastigotes) sont ensuite lysés dans un homogénisateur à tissus (Pyrex) et les amastigotes recueillis sont lavés avec du PBS puis cultivés à température de la pièce comme mentionné précédemment afin qu’ils se transforment en promastigotes. Afin de s’assurer qu’ils conservent un maximum de virulence, ces promastigotes sont utilisés suite à un maximum de trois passages dans les cas des expériences faites chez la souris.
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