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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 organisms (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 second host which also acts as a vector.



  report

cale

The three key words of our Unit are "Immunophysiology" and "Intracellular Parasitism" : they assess 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 and renewed 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 mouse mononuclear phagocytic leukocytes in vitro : host cells rapidly a) invaded by Leishmania metacyclic promastigotes, b) where they differentiate as amastigotes (E. Fontan, T. Lang, E. Prina, S. Abdi ® 15/07/02, 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 molecule identified in our laboratory, 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 L. amazonensis survival.

The phagocytic leukocytes belong to related lineages, the mononuclear phagocyte one and the dendritic leukocyte one. 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 a pool of parasite-reactive CD4 T lymphocytes primed in mice once those have been exposed to the intradermal delivery of more or less invasive parasites : a special attention is given to other CD4 and CD8 T lymphocytes activated after metacyclic promastigote delivery. The studies with dendritic leukocytes are performed in collaboration with Nathalie Winter (Unité de Génétique mycobactérienne).

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 (J.-C. Antoine, S. Goyard depuis le 1/07/02, T. Lang, M. Lebastard, G. Marignac  ® 15/07/02, 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 non T non B 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, models of Leishmania major or L. amazonensis infection in C57Bl/6 or BALB/c mice have been established : they combine two main features of natural transmission : low dose (10, 100 to 1000 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the "transient" or irreversible dermal lesion could be dissociated into two distinct phases. The initial "silent" phase 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 C57Bl/6. In BALB/c mice inoculated with either L. major or L. amazonensis, the irreversible lesions which occur are assessing very complex processes under active study focusing on the draining lymph node as well as the parasite-loaded cutaneous sites. 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 they are able to reach. 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. Transgenic Leishmania major expressing different genes encoding proteins tractable by fluorescence or luminescence readout assays, are constructed, as well as Leishmania major with a new suicide gene (collaboration with P. Kaminski, Unité de Chimie organique)

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, T. Lang, M. Lebastard, H. Saklani ; P. Buffet, N. Jolly CRVBm/CMIP, E. Bourreau, G. Prévot, P. Launois, I.P. Guyane, G. Milon ).

Seven 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). Indeed in the late case, this site was priviledged for screening the protective immunogenic properties of LACK-transgenic Listeria monocytogenes delivered intragastrically. 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 very stimulating partnership to extend within the context of the Centre Médical de l'Institut Pasteur and the International Network of Institut Pasteur.

Photo1: Section of infected macrophage recovered from active cutaneous lesion loaded with Leishmania amazonensis (BALB/c mouse). PV : Lumen of parasitophorous vacuole ; L : Leishmania Bar: 1 m m. (from Thierry Lang).

Keywords: Leishmania spp., Immunophysiology, Intracellular Parasitism, Parasite developmental program, Parasite transmission, Parasite invasiveness



  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

GOYARD Sophie, Chargée de recherche IP,goyard@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

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

Activity Reports 2002 - Institut Pasteur
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