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Biomedical subjects

G Milon

Publications and source records attributed to G Milon.

At least 19 recordsLinked to original sources
PubMed2003

Increased production of interferon-gamma by Leishmania homologue of the mammalian receptor for activated C kinase-reactive CD4+ T cells among human blood mononuclear cells: an early marker of exposure to Leishmania?

A prospective study was undertaken to define early predictive immunological marker(s) of exposure to Leishmania in naïve subjects who have never been exposed to any Leishmania and who were also free of any cutaneous leishmaniasis lesions. These naïve subjects could have been exposed to Leishmania in a rain forest where Leishmania guyanensis and their natural vectors and mammalian host are cocirculating. The production of interferon (IFN)-gamma in response to the Leishmania homologue of the mammalian receptor for activated c kinase (LACK), a candidate for vaccine against leishmaniasis was analysed. At the end of their stay in the rain forest, LACK-specific CD8+ T cells were detected in subjects whose peripheral blood mononuclear cell (PBMC) produced IFN-gamma in response to soluble Leishmania antigens (SLA) and in those whose PBMC remained unresponsive to SLA. However, LACK-specific CD4+ T cells were detected only in PBMCs from individuals who became IFN-gamma responders to SLA. In subjects whose PBMC became positive to SLA, LACK-reactive CD4+ T cells producing high level of IFN-gamma were detectable before the SLA-reactive IFN-gamma producing CD4+ T cells, suggesting that the former readout assay could be used as an early predictive immunological marker of exposure to Leishmania in subjects who remained disease free.

Animals↗
PubMed2002

Brain engraftment of autologous macrophages transduced with a lentiviral flap vector: an approach to complement brain dysfunctions.

Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.

Animals↗
PubMed2002

Development of mixed Th1/Th2 type immune response and protection against Mycobacterium tuberculosis after rectal or subcutaneous immunization of newborn and adult mice with Mycobacterium bovis BCG.

Cell-mediated immunity plays a key role in containing the growth of Mycobacterium tuberculosis in the host. The induction of an antibody response or a mixed cell-mediated and humoral response is frequently associated with tuberculosis disease or a decrease in the ability to control M. tuberculosis load. We recently reported the induction of similar immune responses and protection by rectal, subcutaneous (SC) or intradermal administration of Mycobacterium bovis BCG in adult mice, guinea pigs and macaques. The rectal immunization, which did not induce the side-effects associated with parenteral routes (axillary adenitis) and which could be used to reduce the risks of viral transmission associated with unsafe injections in the developing world, was analysed and compared in newborn and adult BALB/c mice. The rectal and SC immunization induced, in mice immunized as newborns or as adults, a mixed T helper 1/T helper 2 (Th1/Th2) immune response; however, particularly in adult mice, after SC administration of BCG, the level of Th2 immune response is significantly higher than it is by the rectal route. Six months after immunization with BCG, rectal and SC delivery induced similar levels of protective immunity against a virulent challenge with M. tuberculosis strain (H37Rv) in mice immunized as adults, but the rectal BCG delivery triggered stronger protection than the SC delivery if mice were immunized as newborns.

Administration, Rectal↗
PubMed2001

Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host.

We studied the sequence of cellular events leading to the dissemination of Listeria monocytogenes from the gut to draining mesenteric lymph nodes (MLNs) by confocal microscopy of immunostained tissue sections from a rat ligated ileal loop system. OX-62-positive cells beneath the epithelial lining of Peyer's patches (PPs) were the first Listeria targets identified after intestinal inoculation. These cells had other features typical of dendritic cells (DCs): they were large, pleiomorphic and major histocompatibility complex class II(hi). Listeria were detected by microscopy in draining MLNs as early as 6 h after inoculation. Some 80-90% of bacteria were located in the deep paracortical regions, and 100% of the bacteria were present in OX-62-positive cells. Most infected cells contained more than five bacteria each, suggesting that they had arrived already loaded with bacteria. At later stages, the bacteria in these areas were mostly present in ED1-positive mononuclear phagocytes. These cells were also infected by an actA mutant defective in cell-to-cell spreading. This suggests that Listeria are transported by DCs from PPs to the deep paracortical regions of draining MLNs and are then transmitted to other cell populations by mechanisms independent of ActA. Another pathway of dissemination to MLNs was identified, probably involving free Listeria and leading to the infection of ED3-positive mononuclear phagocytes in the subcapsular sinus and adjacent paracortical areas. This study provides evidence that DCs are major cellular targets of L. monocytogenes in PPs and that DCs may be involved in the early dissemination of this pathogen. DCs were not sites of active bacterial replication, making these cells ideal vectors of infection.

Animals↗
PubMed2000

An immunomodulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major.

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.

Animals↗
PubMed2000

A natural model of Leishmania major infection reveals a prolonged "silent" phase of parasite amplification in the skin before the onset of lesion formation and immunity.

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 dermal lesion could be dissociated into two distinct phases. The initial "silent" phase, lasting 4-5 wk, favored establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histopathologic changes 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 killing of parasites in the site. The onset of immunity/pathology was correlated with the appearance of cells staining for IL-12p40 and IFN-gamma in the epidermal compartment, and an expansion of T cells capable of producing IFN-gamma in the draining lymph node. Parasite growth was not enhanced over the first 4.5 wk in anti-CD4-treated mice, SCID mice, or C57BL/6 mice deficient in IL-12p40, IFN-gamma, CD40 ligand, or inducible NO synthase. These mice all failed to ultimately control infection in the site, but in some cases (anti-CD4 treated, IL-12p40-/-, CD40 ligand-/-, and SCID) high dermal parasite loads were associated with little or no pathology. These results extend to a natural infection model a role for Th1 cells in both acquired resistance and lesion formation, and document the remarkable avoidance of this response during a prolonged phase of parasite amplification in the skin.

Animals↗
PubMed2000

The outcome of Leishmania major experimental infection in BALB/c mice can be modulated by exogenously delivered iron.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L. major infection in BALB / c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron-loaded mice. Importantly, the "protective" effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L. major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.

Animals↗
PubMed2000

Distinct innate and acquired immune responses to Leishmania in putative susceptible and resistant human populations endemically exposed to L. (Viannia) panamensis infection.

Mechanisms of constitutive and acquired susceptibility/resistance to Leishmania Viannia panamensis (L. (V ) p.) were investigated in endemically exposed human populations presenting either recurrent disease (putative susceptible) or subclinical infection (clinically resistant). Cutaneous delayed type hypersensitivity response to leishmanin was significantly lower among individuals experiencing recurrent leishmaniasis than among those whose skin test converted without developing the disease. Monocyte derived macrophages from individuals with recurrent disease were more permissive in vitro to the entry of parasites than macrophages from subclinically infected individuals. In vitro proliferation of CD4 and CD8 T lymphocytes in response to intracellular amastigotes was significantly lower among individuals with a history of recurrent disease compared with subclinically infected individuals. Linear regression analyses revealed a strong direct relationship between the production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 by peripheral blood mononuclear cells (PBMC) from resistant (subclinically infected) individuals and no correlation in the production of these cytokines by PBMC from individuals who experienced recurrent disease. The results provide evidence of differences in the innate and acquired responses to Leishmania according to the outcome of the natural infection. These findings support the feasibility of identifying the immunological bases of innate and acquired resistance through studies in naturally exposed human populations.

Adolescent↗
PubMed2000

Macrophage subsets harbouring Leishmania donovani in spleens of infected BALB/c mice: localization and characterization.

The purpose of the current study was to characterize parasite-containing cells located in spleens of BALB/c mice infected with Leishmania donovani. In particular, expression of MHC class II molecules by these cells was examined to determine whether they could potentially act as cells capable of immunostimulating Leishmania-reactive CD4+ T lymphocytes. To this end, an immunohistological analysis of spleens taken at various time points after infection was undertaken. Using this approach, we observed, in the red pulp, the formation of small cellular infliltrates containing heavily infected macrophages that could be stained with the monoclonal antibodies MOMA-2 and FA/11. All of them expressed high levels of MHC class II molecules. Parasites were also detected in the white pulp, especially in MOMA-2+, FA/11+ and MHC class II+ macrophages of the periarteriolar lymphocyte sheath and in MOMA-2+ marginal zone macrophages. Infected cells were further characterized by fluorescence microscopy after their enrichment by adherence. All infected mononuclear cells recovered by this procedure could be stained with MOMA-2 and FA/11 and thus very probably belonged to the mononuclear phagocyte lineage. Furthermore, all of them strongly expressed both MHC class II as well as H-2M molecules, regardless of the time points after infection. Analysis of the parasitophorous vacuoles (PV) by confocal microscopy showed that these compartments were surrounded by a membrane enriched in lysosomal glycoproteins lamp-1 and lamp-2, in macrosialin (a membrane protein of prelysosomes recognized by FA/11) and in MOMA-2 antigen. About 80% of the PV also had MHC class II and H-2M molecules on their membrane. Altogether, these data indicate that in the spleens of L. donovani-infected mice, a high percentage of amastigotes are located in macrophages expressing MHC class II molecules and that they live in PV exhibiting properties similar to those of PV detected in mouse bone marrow-derived macrophages exposed to a low dose of interferon gamma (IFN-gamma) and infected in vitro.

Animals↗
PubMed2000

Transient IFN-gamma synthesis in the lymph node draining a dermal site loaded with UV-irradiated herpes simplex virus type 1: an NK- and CD3-dependent process regulated by IL-12 but not by IFN-alpha/beta.

Our previous studies have shown that UV-inactivated, non-replicating herpes simplex virus type 1 (UV-HSV-1) triggers early and transient synthesis of IFN-alpha/beta in the mouse regional lymph node when delivered upstream (i.e. in the ear dermis). In this study, it is demonstrated, by use of a quantitative RT-PCR readout assay, that IFN-gamma mRNA expression was rapidly and transiently upregulated in draining lymph nodes when UV-HSV-1 was delivered in the ear dermis of C57Bl/6 mice. An increased number of IFN-gamma-producing cells was also detected in the lymph node by flow cytometric analysis. Two different subsets of cells, namely DX5(+) NK cells and CD3epsilon(+) T cells, accounted for this early IFN-gamma synthesis. Prompt upregulation of IFN-alpha and IL-12p40 mRNA was also recorded. We took advantage of IFN-alpha/beta-receptor knockout and wild-type 129 mice to study a potential role of IFN-alpha/beta in the signalling pathway leading to IFN-gamma transcription/translation. IFN-gamma mRNA upregulation still occurred in IFN-alpha/beta-receptor(-/-) mice, showing that IFN-alpha/beta was dispensable. The use of IL-12-neutralizing antibodies, prior to UV-HSV-1 delivery, confirmed the major role played by IL-12 in the early/transient IFN-gamma burst.

Animals↗
PubMed2000

Leishmania major reaches distant cutaneous sites where it persists transiently while persisting durably in the primary dermal site and its draining lymph node: a study with laboratory mice.

So far, studies of Leishmania persistence in mice have used injections of parasites administered either intravenously in the tail vein or subcutaneously in the footpad. These routes poorly reflect the natural conditions when the sandfly delivers metacyclic promastigotes intradermally. In this study B10D2 and BALB/c mice were inoculated within the ear dermis with 10(4) Leishmania major metacyclic promastigotes. The parasite load was monitored by quantitative PCR in different tissues from the dermal inoculation site to distant tissues. The two sites of multiplication and persistence of parasites were the site of L. major inoculation and the draining lymph node (DLN), with a different pattern in the two mouse inbred lines. These two organs were the only sites harboring parasites 12 months postinoculation, with the DLN of BALB/c mice harboring around 10(7) parasites, a stable load from months 3 to 12. In these two sites, 8 and 12 months after inoculation, interleukin 4 (IL-4), gamma interferon, and inducible nitric oxide synthase transcripts parallel the parasite load while IL-10 transcript levels remain high. In addition, at early time points until month 3, parasite DNA was also detected in distant tissues such as the contralateral noninoculated ear or the tail skin, indicating that blood was at least transiently disseminating the parasites. In contrast, L. major DNA in liver, spleen, and femoral bone marrow remained sporadic in mice of both lines. This study is discussed within the framework of Leishmania transmission from the vertebrate host to the sandfly vector, a complex process still poorly understood.

Animals↗
PubMed2000

Listeria monocytogenes as a short-lived delivery system for the induction of type 1 cell-mediated immunity against the p36/LACK antigen of Leishmania major.

Listeria monocytogenes has been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models. Susceptibility of BALB/c mice to Leishmania major infection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands for Leishmania homologue of receptors for activated C kinase). Experimental vaccination with LACK can redirect the differentiation of CD4(+) T cells towards the Th1 pathway if LACK is coadministrated with IL-12. As IL-12 is known to be induced by L. monocytogenes, we have tested the ability of a recombinant attenuated actA mutant L. monocytogenes strain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible to L. major, respectively. After a single injection of LACK-expressing L. monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-gamma)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains. These primed IFN-gamma-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after an L. major footpad challenge. Although immunization of BALB/c mice with LACK-expressing L. monocytogenes did not change the course of the infection with L. major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls. Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells, L. monocytogenes can also be used as a live recombinant vector to favor the development of potentially protective IFN-gamma-secreting Th1 CD4 T lymphocytes.

Animals↗
PubMed1999

Transmission stages of Plasmodium: does the parasite use the one same signal, provided both by the host and the vector, for gametocytogenesis and sporozoite maturation?

Among the microorganisms that strictly depend upon other organisms (hosts or vectors) for achieving their life cycle, protozoan and metazoan parasites have been often primarily distinguished through the major pathogenic processes they could induce. A variety of different mechanisms linked to parasitism can indeed systemically (e.g. Plasmodium falciparum) or locally (e.g. Toxoplasma gondii) induce important alterations of tissue homeostasis. But more than obvious pathogenicity, it is the capacity to be transmitted that is essential for parasite survival and there is increasing evidence that certain parasites can achieve their life cycle to the point of transmission in the absence of clinically detectable processes. For this, constitutive microenvironments of the host or vector can be exploited. Moreover, parasites are sometimes able to highjack effectors of the host's immune response towards conditioning the microenvironments which are permissive to differentiation of transmissible developmental stages. Based on a few examples taken from studies on the transmission stages of Leishmania, Toxoplasma and Plasmodium, we have here attempted to formulate a few hypothesis on the biology of the transmission stages of P. falciparum, i.e. on gametocytogenesis and sporozoite maturation. As discussants, we may have been somewhat dwarfed by issues evoked by the organizers of this meeting in the title of the session, i.e. 'Vector-parasite-man interactions'!... In reaction, we may have taken refuge in somewhat over-selective comments, biased by the objects of our personal research....

Animals↗
PubMed1998

Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.

Galectin-3, also known as the macrophage marker Mac-2, is a member of a family of structurally related animal lectins that exhibit specificity for beta-galactosides. In order to investigate the role of galectin-3 in acute inflammation, we have compared the number of leucocytes present in the peritoneal cavity of wild type and galectin-3 null mutant mice after intraperitoneal (i.p.) injection of thioglycolate broth. At day 1 after injection, we found no difference in the recruitment of mononuclear phagocytes and granulocytes to the peritoneal cavity. However, 4 days after thioglycolate injection, galectin-3 mutant mice exhibited a significantly reduced number of recoverable granulocytes compared to wild-type animals. As mutant granulocytes did not exhibit an accelerated rate of apoptosis and their uptake by macrophages appeared to be unaffected by the mutation, the phenotype described here suggests that galectin-3 participates in an additional level of control during the resolution of acute inflammation.

Acute Disease↗
PubMed1998

Permissiveness of human monocytes and monocyte-derived macrophages to infection by promastigotes of Leishmania (Viannia) panamensis.

During natural infections, Leishmania is in contact with a variety of mononuclear phagocytic cells in different tissues, including resident macrophages and monocytes mobilized to the site of infection from the bone marrow and blood circulation. Because the functional capabilities of fully differentiated macrophages and blood monocytes differ, the outcome of infection by Leishmania may depend upon the stage of differentiation of the host cells. To address this question, we evaluated Leishmania panamensis infection of (1) the human promonocytic/histiocytic cell line U-937 before and after induction of differentiation by phorbol myristate acetate; (2) fresh human peripheral blood monocytes; and (3) macrophages derived from monocytes by differentiation in vitro. Based on the percentage of cells infected and the number of parasites per cell, macrophages derived from monocytes or by induction of differentiation of U-937 cells were significantly more permissive to infection by stationary-phase L. (Viannia) panamensis promastigotes than monocytes. Increasing time and maturation in culture prior to exposure to infective promastigotes was associated with the increased permissiveness of differentiated macrophages to infection (P<0.05). The percentage of cells infected and number of amastigotes per cell increased with time postinfection for both monocytes and macrophages but remained significantly greater for macrophages. The increased expression of CD68, CD16, and lysozyme, and decreased expression of peroxidase by macrophages cultured for 5 days in vitro compared with fresh monocytes, whether adherent or in suspension, supported the distinct maturation status of these cells.

Animals↗
PubMed1997

Quantitation of messenger RNA by competitive RT-PCR: a simplified read out assay.

A competitive RT-PCR method that permits reliable quantification of minute amounts of reverse-transcribed mouse lymph node mRNA is described. Using this technique, an absolute number of cDNA copies ranging from 10(3) to 10(5) can be determined, with a precision superior to 25%. The standard templates described in the present study permit the quantitation of beta-actin, IFN gamma, IL2, IL3, IL4, IL10, IL12 (p40 subunit), TGF beta 1, inducible nitric oxide synthase, ELAM-1, VCAM-1, and ICAM-1 mouse mRNA. The expression of a particular transcript is normalized to an arbitrary number of actin transcripts. The standard templates and wild-type cDNA have nearly identical sequences, but they can be distinguished by unique restriction sites. Known amounts of these standard templates, are co-amplified with serial dilutions of the cDNA derived from the mRNA of interest. Oligonucleotide primer pairs possessing 3' octamers found infrequently in the mouse genome (< or = 0.26 x 10(-6)) are used to amplify sequences, chosen to contain no GC stretches longer than 8 (PCRare software) (Griffais et al., 1991). Samples of each PCR product are digested separately with restriction endonucleases unique either for the wild-type or the standard amplicon. The quantitation of the test product and the standard product is easily carried out following their electrophoresis in an ethidium bromide-stained agarose gel.

Actins↗
PubMed1997

Early production of IL-4 in susceptible mice infected with Leishmania major rapidly induces IL-12 unresponsiveness.

Previous results have documented a burst of IL-4 mRNA that peaks in draining lymph nodes of susceptible BALB/c mice 16 h after infection with Leishmania major. The importance of this early IL-4 response in subsequent Th2 cell maturation is supported by observations showing that 1) neutralization of IL-4 at the initiation of infection or 2) administration of IL-12, which results in an inhibition of the 16 h IL-4 mRNA burst, inhibits Th2 cell development. However, both treatments are effective in hampering Th2 cell development only if given at a time when IL-4 has been produced for <48 h. At this time after infection, lymph node CD4+ T cells from BALB/c mice no longer respond to IL-12. This IL-12 unresponsiveness is prevented in mice treated with anti-IL-4 Abs at the initiation of infection. Finally, the inhibition of Th2 development in BALB/c mice treated with anti-IL-4 Abs at the onset of infection results from maintenance of IL-12 responsiveness, since it requires IL-12. Together, these results reveal a narrow window of time, between 16 h and <48 h after infection, during which IL-4 produced rapidly in BALB/c mice renders T cells unresponsive to IL-12, allowing their differentiation toward the Th2 phenotype.

Animals↗