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  Director : Pierre-André CAZENAVE (cazenave@pasteur.fr)


  abstract

 

The scientific activities of the Infectious Immunophysiopathology Unit are centered on the study of mechanisms controlling the physiology of the host immune system and those leading to immunopathologies following parasitic infections and diseases, namely Chagas' disease and Malaria. These studies are particularly grounded on more fundamental research on immune repertoires.



  report

cale

TcPRAC eukaryotic proline racemase eukaryote — a mitogen secreted by (P. Minoprio and collaborators).

We pursued the molecular, biochemical and functional analyses of the first eukaryotic proline racemase, TcPRAC (TcPA45), protein released by the protozoan parasite Trypanosoma cruzi, responsible for Chagas' disease. TcPRAC is a T-cell independent B-cell mitogen, largely implicated in polyclonal lymphocyte activation and consequently in the mechanisms of parasite evasion and persistence inside the host. We showed that TcPRAC mitogenic activity is dependent on the integrity of the enzyme active site. Furthermore, the parasite proline racemase was shown to be important to the parasite differentiation from non-infective to infective forms, as well as to parasite virulence to host cells. We defined a protein signature capable of identify putative proline racemases of several microorganisms of medical and agricultural importance. In collaboration with the Structural Biochemistry Unit we recently obtained the crystallographic structure of TcPRAC. Ongoing studies aim at designing enzyme inhibitors to be used in therapeutic approaches. Web site : http://www.pasteur.fr/recherche/unites/tcruzi/minoprio/minoprio.html

Studies on the diversity and plasticity of the Immune system (A. Six, D. Rueff-Juy, P.A.Cazenave).

One important activity of the laboratory concerns the improvement of new technologies able to explore the diversity and the plasticity of T- and B- cell repertoires represented by antigen specific Ig- and T-cell receptors. These studies focus on the study of repertoire alterations in the context of mouse and human Leishmaniasis and Malaria. The Immunoscope technique allows the definition of qualitative images of TCRb repertoires and as such the description of CDR3 sizes for each Vb gene fragment used in response to infections. ISEApeacks, a software developed in the laboratory, permits the computerized analysis of a large number of samples and reveals recurring oligoclonal TCR expansions. By this technology we showed that the expansion of TCR Vb8+ cells is polyclonal in B10.D2 mice that develop cerebral malaria (CM+) upon infection with P.berghei ANKA. Moreover, the quality of alterations of TCR profiles observed in CM+ mice is different from that obtained when using CM- infected individuals that do not develop cerebral syndromes. Present experiments intend to determine the relationship between oligoclonal expansions observed in the blood of human patients that develop CM+ and the presence of a particular T-cell population in the brain. In parallel, we studied the plasticity of B cell compartments (B cells and immunoglobulins) in extreme situations where the VH and VL germ line repertoire is very reduced. For this purpose, we developed a mouse model that is an important tool to analyze the evolution of the B-cell repertoire : B6.T15i/JH-.k-.lSEG, only expressing l2 and lx immunoglobulin light chains (k "knock-out" deletion of l1-l3 locus ) and " knock-in"  transgenic for VH 15 (gene encoding the IgH heavy chain anti-phosphorylcholine). The utilization of appropriate antibodies in vivo offers the capacity to sort B-lymphocytes pertaining to groups B1-a, B1-b or B2 in different lymphoid compartments and to follow their dynamics after appropriate cell transfers to immunodeficient mice submitted to different stimuli.

Immunophysiopathology and genetic studies of Plasmodium infection.

These studies concentrate on the recognition of protective and pathogenic responses following mouse model infections with Plasmodium species, namely P. berghei ANKA (pathogenic responses) and P. yoelii (protective reponses), or more or less severe natural human infections with Plasmodium falciparum.

Cellular responses implicated in the protection against parasite liver stages (S.Pied, J.Roland, P.A.Cazenave). These studies implicate the analysis of cellular mechanisms involved in the establishment of anti-malarial immunity in the liver. We showed that C57BL/6 mice infected with P . yoelii sporozoites or with P . yoelii infected red cells present an ‘unconventional' expansion of T-cells characterized by the phenotype NK1.1+TabCD4-CD8-. These NKT cells, mainly CD1d-restricted, bear specific anti-parasite activity since they are able to inhibit the development of parasite infection in the liver. We are presently undertaking a better characterization of the functional role of these cells, as well as their putative role in the regulation of conventional T-cell responses, particularly those implicating protective CD8+ T-cells. In parallel, we showed that NK T cells are important effectors of immunity against P. yoelii either by cytolytic activities towards infected targets, or by their interaction with other cellular effectors by the release of cytokines. Comparative studies of P. yoelii infection using RAG-2, gc KO (lacking B, T and NK lymphocytes) or RAG-2 (lacking NK cells) mouse models revealed that NK cells may control parasite burden and infection without inducing sterile immunity (collab. with the Cytokines and Lymphoid Development Unit). It would seem that NK cell activities following infection results from a balance between activation and inhibitory signals mediated by specific cell surface receptors. The expression analysis of Ly49 family receptors, been them involved with activation or inhibition, originated a repertoire of NK cells after infection that was then studied during different time points using C57BL/6 mouse model. The evolution of NK cell repertoire revealed in the spleen and in the liver an independent compartmentalization of NK cells responding to infection. Present analyses of cytokines produced by NK cells and putative NK cytotoxicity are been done.

Immune responses implicated in the pathogenesis of malaria — the mouse model (S.Pied, P.A.Cazenave). Several factors are at the origin of the mechanisms leading to cerebral malaria, namely red cell sequestration in the brain, down regulation of adhesion molecules and/or the pathogenic role of cytokines. The precise participation of the immune responses in this pathology is not known. We showed that mouse developing cerebral malaria after infection with sporozoites or P. berghei ANKA infected red cells present a preferential expansion of T cells in the brain, particularly those of phenotype CD8+, CD69+, CD25+, CD44hi and CD62L-, presenting a restricted utilization of the TCRVab repertoire. A systematic analysis of these cells showed that they express ICAM-1 and LFA-1 adhesion molecules and secrete INF-g and TNF-a cytokines, known to play a critical role in cerebral malaria. The implication of these cells in the genesis of cerebral malaria was corroborated by the absence or critical reduction of pathology observed when using mice depleted of CD8+ or TCR Vb8+ T cells.

Human studies (S.Pied, P.A.Cazenave). We studied the cytokine profiles observed in plasmas obtained from children of less than 5 years old presenting different forms of P. falciparum infection and cerebral syndromes. The results showed a significant correlation between the levels of IL-10 and parasitemia, while the severity of malaria correlated with increased levels of TNF; however, asymptomatic patients also presented comparable levels of TNFa revealing that this cytokine is not a good marker of pathology in the studied population. Furthermore, we started a multiparametric analysis of autoantibody repertoires in these patients.

Genetic determinism of resistance against malaria using the marine infection with P. berghei ANKA (P.A.Cazenave, S.Pied). The previous description of inbred mouse strains resistant to cerebral malaria (CM- phenotype) allowed us by appropriate genetic crosses with C57BL/6 mouse (CM+ susceptible phenotype) to determine a genetic association of the CM- phenotype with two distinct regions respectively on chromosomes 1 and 11. We discovered an unusual phenotype correlated with resistance to hyperparasitemia (HP- phenotype) that is associated to three chromosomal regions, namely 4, 9 and 11. Appropriate congenic strains are been derived over the C57BL/6 background.

Keywords: Immune system, immunopathology, B- and T- cell repertoires, proline racemase, Chagas’ disease, mitogen, cerebral malaria



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  personnel

  Office staff Researchers Scientific trainees Other personnel
  POSTE Isabelle, IP (iposte@pasteur.fr) MINOPRIO Paola, Senior Associate Professor, Chef de Laboratoire, IP, (pmm@pasteur.fr)

PIED Sylviane, Associate Professor, CR1 CNRS, (spied@pasteur.fr)

ROLAND Jacques, Associate Professor, CR1 CNRS, (jroland@pasteur.fr)

RUEFF-JUY Dominique, Senior Associate Professor, DR2 CNRS, (rueffjuy@pasteur.fr)

SIX Adrien, Assistant Professor, MC Université Pierre et Marie Curie (Adrien.Six@pasteur.fr)

BABAY Besma, PhD student (bbabai@pasteur.fr)

BLANC Anne-Laurence, DEA student (alblanc@pasteur.fr)

CHAMOND Nathalie, PhD student (nchatond@pasteur.fr)

FERRANDIZ Maria, Etudiante DEA (encarnit@pasteur.fr)

GOYTIA Maira, DEA student (mgoytia@pasteur.fr)

GUIYEDI Vincent, PhD student (guidyvin@pasteur.fr)

SOULARD Valérie, PhD student (valerie@pasteur.fr)

BARBIER Eliane, Research Engineer IP (ebarbier@pasteur.fr)

BERNEMAN Armand, Research Engineer IP (berneman@pasteur.fr)

COATNOAN Nicolas, Senior Technicien IP (coatnoan@pasteur.fr)

COSSON Alain, Qualified Technicien IP (acosson@pasteur.fr)

CORRE Jean-Philippe, SeniorTechnicien IP (jphc@pasteur.fr)

DRAPIER Anne-Marie, Senior Engineer CNRS (amdrapie@pasteur.fr)

GORGETTE Olivier, Senior Technicien IP (ogorgett@pasteur.fr)

SELLIER Christèle, Qualified Technicien CNRS (csellier@pasteur.fr)

VOEGTLE Danièle, Senior Engineer CNRS (dvoegtle@pasteur.fr)


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