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  Director : MILON Geneviève (gmilon@pasteur.fr)


  abstract

 

Leishmania spp., protozoan parasites, circulate between mammalian hosts and hematophagous sandflies, named vectors. Once inoculated in the upper dermis of mammalian hosts (rodents, dogs, humans,…), they are phagocytosed by professional phagocytic leukocytes and they establish long-term interactions assessed by either pathogenic processes, designated as leishmaniasis, or asymptomatic parasitic processes. Whatever the level of analysis of these unique parasite-host interactions (in vivo, ex vivo, in vitro), our objectives are to decipher the mechanisms underlying asymptomatic and/or pathogenic and repair processes (especially their immune components) as well as the parasite and tissue-dependent processes underlying the transmission of Leishmania spp. from mammalian host to the vector.



  report

cale

The three key words of our Unit are "Immunophysiology" and "Intracellular Parasitism" : they assesss the framework of our scientific activities as well as our long-term commitment to share our knowledge and expertise within and outside our Institute.

In any natural habitat, there are continual encounters and interactions between microorganisms and other unicellular or multicellular organisms named "hosts". Bacteria (e.g. Listeria monocytogenes, LM), a microorganism whose dominant biotope is the soil), protozoan parasites (e.g. Leishmania spp.), when encountering such hosts establish respectively short-term (LM) or long-term interactions (Leishmania spp.), with respect to the life time of the hosts. We do spend time and efforts to design the relevant models with which to decipher the different discrete steps the invasive microorganisms whether they are opportunistic (LM) or parasitic (Leishmania spp.) do trigger/exploit/subvert once delivered in their mammalian hosts. Since many years, we address many questions for better understanding the sequential discrete steps of the transient or prolonged cross-talks the microorganisms establish in their hosts especially with their multifocal immune system. When Leishmania is concerned, another important question is addressed, namely where and how — in the mammalian hosts — do Leishmania shape the optimal tissual microenvironment which allows the genetic program to be re-set for its transmission to the blood-pool feeder sandfly, which acts as its second host, named a vector.

 

The mononuclear phagocytic leukocytes : host cells rapidly a) invaded by Leishmania metacyclic promastigotes, b) where they differentiate as amastigotes : what are the sequential discrete processes tractable to quantitative and qualitative analysis in vitro ? (N. Courret : 1/10/01 ® 31/12/01, E. Fontan, T. Lang, E. Prina, S. Abdi : 5/11/01® , J.-C. Antoine).

The establishment of Leishmania in mammals depends on the differentiation of metacyclic promastigotes into amastigotes within macrophages. The kinetics of this process was examined using mouse macrophages infected with metacyclic promastigotes of L. amazonensis. The presence of amastigote characteristics, including large lysosome-like organelles called megasomes, stage-specific antigens, high cysteine protease activity and sensitivity to L-leucine methyl ester, was followed over a 5-day period. Megasomes were observed at 48 h but probable precursors of these organelles were detected at 12 h post-infection (p.i.) The promastigote-specific molecules examined were down-regulated within 5 to 12h after phagocytosis whereas the amastigote-specific antigens studied were detectable from 2 to 12-24 h. An increase in the cysteine protease activity and in sensitivity to L-leucine methyl ester of the parasites was detected from 24 h. The data indicate that at 48 h p.i., parasites exhibit several amastigote features but that complete differentiation requires at least 5 days. The biogenesis of megasomes or of megasome precursors and the rise in cysteine protease activity correlate quite well with the capacity of parasites to internalize and very likely degrade host cell Major HistoCompatibility (MHC) molecules. The fact that internalization by the parasites of host cell molecules occurs very early during the differentiation process argues for a role of this mechanism in parasite survival.

The phagocytic leukocytes belong to three related lineages, the mononuclear phagocyte lineage and the two dendritic leukocyte lineages. During the last year, mouse dendritic leukocytes derived from bone marrow progenitors have been used as host cells of either metacyclic promastigotes or recently isolated amastigotes. These phagocytic leukocytes loaded with live parasites are the relevant cells with which to monitor the pool of parasite reactive CD4 and CD8 T lymphocytes primed in mice once those have been exposed to the intradermal delivery of more or less invasive parasites : a special attention will be given to CD4 and CD8 T lymphocytes activated after metacyclic promastigote delivery.

 

The cutaneous tissue : a versatile tissual microenvironment where Leishmania establish long-term parasitic processes and from where they are transmitted to their second host, the hematophagous sandfly (collaboration with P. Volf & coll., Prague) (N. Courret : 1/10/01 ® 1/12/01, S. Lakhal : 1/10/01 ® 31/12/01, T. Lang, M. Lebastard, G. Marignac : 3/12/01 ® , L. Nicolas, H. Saklani, G. Milon).

Secondary lymphoid organs are sites of "immunological integration", where many circulating leukocytes including T, B lymphocytes, as well as migratory dendritic leukocytes could be stopped within highly organized microenvironments. The critical portals for leukocyte entry are the high endothelial venules (HEVs) of lymph nodes, and the afferent lymphatic vascular bed for which new reagents are now available. When we decided to design a model for deciphering the features of the intracellular parasitism driven by Leishmania at the tissual and loco-regional levels, our framework was shaped by a perception of integrative immunophysiology. Thus, a model of Leishmania major infection in C57Bl/6 mice has been established that combines two main features of natural transmission : low dose (100 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the "transient" dermal lesion could be dissociated into two distinct phases. The initial "silent" phase, lasting 4-5 weeks, favoured establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histopathologic change in the site. The second phase corresponds to the development of a lesion associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis and was coincident with the reduction of the parasite load in the site. The onset of pathology was correlated with the presence of cells staining for IL12p40 and IFNg in the epidermal compartment, and an expansion of T cells capable of producing IFNg in the draining lymph node. Parasite growth is not enhanced over the first 4-5 weeks whatever the gene disruption of the mice under study. The onset and stable maintenance of repair processes were correlated with the presence of CD4, CD8 and dendritic leukocytes whose properties have to be further defined, especially in the healed ear where parasites are persisting at a stable number (@ 1000 parasites/ear), as well as in the distant cutaneous tissues.

Indeed, at early time points, from week 2 until month 3, parasite DNA was also detected in distant tissues such as the contralateral non-inoculated ear or the tail skin, indicating that blood was at least transiently the compartment through which the parasites were delivered. In contrast, L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice whatever their genotypes. This study is processed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood. This model will be precious for delineating at the tissual, cellular and molecular levels the key processes which reprogram the amastigotes towards the developmental stage which is transmissible and pre-adapted to the vector.

 

LACK, a protein common to many species of Leishmania, and a source of peptides with affinity for mouse and human class I and class II Major HistoCompatibility Complex molecules : what are the functional properties of the T lymphocytes reactive to the LACK peptide AA 158-173 ? ? (J.-C. Antoine, N. Courret 1/10/01 ® 31/12/01, T. Lang, M. Lebastard, H. Saklani, G. Milon ; P. Buffet, CRC, I.P., E. Bourreau, P. Launois, I.P. Guyane).

Six years after the publication of the elegant screen used to isolate the gene fragment which specifies the synthesis of LACK, this parasite protein is still under active study in different settings. As far as our Unit is concerned, LACK has been used as a "model tool" to monitor the onset of T lymphocyte reactivity within the draining lymph node, as well as in the blood compartment, after parasite delivery in the dermal site (and exceptionally in the subcutaneous site). In addition, more recently, it was possible to detect and characterize the naive or activated phentoype of IFN¡ and IL10 producing CD4 and CD8 T lymphocytes in human subjects before and following Leishmania guyanensis exposure. These studies assessed a partnership to extend within the context of the Clinical Research Center and the International Network of Institut Pasteur.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BRULE Chantal, IP - cbrule@pasteur.fr

ANTOINE Jean-Claude, DR2 CNRS, jantoine@pasteur.fr

FONTAN Elisabeth, Chargée de recherche IP, efontan@pasteur.fr

LANG Thierry, Chargé de recherche IP, tlang@pasteur.fr

MILON Geneviève, Chef de laboratoire IP, gmilon@pasteur.fr

NICOLAS Luc, Chargé de recherche IP, lnicolas@pasteur.fr

PRINA Eric, Chargé de recherche IP, eprina@pasteur.fr

ABDI Sofiane, Master of Sciences – sabdi@pasteur.fr

COURRET Nathalie, postdoc – ncourret@pasteur.fr

LAKHAL Sami, Ph.D. (Tunis University) - slakhal@pasteur.fr

MARIGNAC Geneviève, Master of Sciences – marignac@pasteur.fr

LEBASTARD Maï, Ingénieur IP - lebastar@pasteur.fr

MAILLET Christine, Agent de laboratoire IP - cmaillet@pasteur.fr

MARANGHI Eddie, Technicien d’animalerie IP – edma@pasteur.fr

SAKLANI Hélène, Technicienne supérieure IP - hesak@pasteur.fr

SEBASTIEN Karim, Technicien d’animalerie IP - ksebas@pasteur.fr


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