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Biomedical subjects

G Milon

Publications and source records attributed to G Milon.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mice heterozygous for a deletion of the tumor necrosis factor-alpha and lymphotoxin-alpha genes: biological importance of a nonlinear response of tumor necrosis factor-alpha to gene dosage.

The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections. To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles. In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts. This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop. Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses. These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.

Alleles↗
PubMed1997

The fate and persistence of Leishmania major in mice of different genetic backgrounds: an example of exploitation of the immune system by intracellular parasites.

Leishmania spp. are intracellular protozoan parasites that are delivered within the dermis of their vertebrate hosts. Within this peripheral tissue and the draining lymph node, they find and/or rapidly create dynamic microenvironments that determine their ultimate fate, namely their more or less successful expansion, and favour their transmission to another vertebrate host though a blood-feeding vector. Depending on their genetic characteristics as well as the genetic make-up of their hosts, once within the dermis Leishmania spp. very rapidly drive and maintain sustained T cell-dependent immune responses that arbitrate their ultimate fate within their hosts. The analysis of the parasitism exerted by Leishmania major in mice of different genetic backgrounds has allowed us to recognize some of the early and late mechanisms driven by this parasite that lead to either uncontrolled or restricted parasitism. Uncontrolled parasitism by Leishmania major characterizing mice from a few inbred strains (e.g. BALB/c) is associated with the expansion of parasite reactive Th2 CD4 lymphocytes and results from their rapid and sustained activity. In contrast, restricted parasitism characteristic of mice from the majority of inbred strains results from the development of a polarized parasite-specific Th1 CD4 response. This murine model of infection has already been and will continue to be particularly instrumental in dissecting the rules controlling the pathway of differentiation of T cells in vivo. In the long run, the understanding of these rules should contribute to the rational development of novel immunotherapeutic interventions against severe infectious diseases.

Animals↗
PubMed1997

Listeria monocytogenes in laboratory mice: a model of short-term infectious and pathogenic processes controllable by regulated protective immune responses.

The mammalian immune system is an integrated network of tissue, leukocytes and effector and regulatory molecules. All these components operate i) to maintain the proper structure of and processes expressed by each tissue. and ii) to protect the hosts from those microorganisms that generally invade them as part of their life cycle. Among the invading microorganisms. Listeria monocytogenes (L. monocytogenes) can persist as a live organism independent of the host, and is, thus, able to drive short-term infectious and pathogenic processes that are controlled by integrated innate and adaptive protective immune responses driven by CD8 and CD4 type 1 T lymphocytes acting on non-T non-B leukocytes. Although the effector functions and the fine specificity of T lymphocytes have been more and more characterized, an understanding of the precise regulation of both leukocyte traffic and T-lymphocyte migration depends on knowledge of the early tissue distribution of L. monocytogenes, points that are addressed in this review.

Animals↗
PubMed1997

The biology of macrophages.

The main properties of the mononuclear phagocytic system (MPS) are summarized, focusing on their relevance within the framework of the steady-state and the inducible functions of the mammalian immune system, more specifically the immune system of the laboratory mouse, a reference vertebrate which remains the best studied. A peculiar attention is given to the rationale underlying the generation of so-called specific tools and reagents whose use is promoted to characterize this lineage, whatever the level under study, i.e. tissular, cellular, or subcellular levels. As one lineage among other lineages of the hemopoietic system, the MPS is characterizable by constitutive and inducible phenotypic and functional markers whose combination is unique for a given tissular micro-environment. Considering our present understanding of the innate and adaptive immune system functions, some of the properties of the MPS are discussed in relation with properties of another recently recognized hemopoietic lineage, namely the dendritic leukocyte system.

Animals↗
PubMed1996

A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in laboratory mice.

We describe a new method to recover and study cells present in the dermis of mouse ear at homeostasis or after intradermal injection of disturbing agents (lipopolysaccharide or Listeria monocytogenes). The ears either left untreated or inoculated were handled and processed as culture explants of the dorsal and ventral leaflets, their dermal sides being spread on a buffered medium. Within this medium emigrate/sediment, with different kinetics: neutrophils, mononuclear phagocytes, dendritic leucocytes, T lymphocytes expressing either gamma delta or alpha beta TCRs, and other minor subsets, the identification of which deserves more relevant reagents: they are likely to be NK, mast cells, eosinophils and their local progenitors. All the major subsets were identified through a combination of immunocytochemical and flow cytometry labeling. Two examples illustrating the advantages and limitations of this new method are given: either 1 microgram of LPS or 10(4) Listeria monocytogenes were injected within the ear 48, 24, 12, 6, 3 h before ear explant culture. This ear explant culture has been further compared to the ear sheet treatment with collagenase/disease for three cell populations, the epidermal dendritic leucocytes, the gamma delta epidermal T cells as well as the alpha beta T cells recirculating within the steady state dermis. This method provides the first evidence of the existence of recirculating T CD4 lymphocytes in the mouse dermis.

Animals↗
PubMed1996

The outcome of the parasitic process initiated by Leishmania infantum in laboratory mice: a tissue-dependent pattern controlled by the Lsh and MHC loci.

Human visceral leishmaniasis is mainly due to intracellular protozoan parasites of the Leishmania donovani complex, i.e., L. donovani and L. infantum (or L. chagasi). A mouse model has been established to monitor 1) the parasitic process initiated by L. infantum in three tissues they invade, and 2) parameters of the acquired immune response they trigger. Mice congenic at the Lsh locus and mice of inbred strains differing at the MHC locus have been inoculated by the i.v. route with L. infantum. The parasitic process has been evaluated by the follow-up of the parasitic load in the liver, the spleen, and, for the first time, in the bone marrow using a very sensitive limiting dilution assay. As previously established for L. donovani, the early outcome of L. infantum is also under the control of the Lsh locus in the liver; genes of the MHC complex are involved in the development of the subsequent acquired immune response. "Cure" or "noncure" haplotypes are the same for the two species of Leishmania; as far as the cure haplotype is concerned, whatever the tissues being screened, the parasites are never totally cleared, although the liver is the tissue in which the best parasite load reduction is achieved. Through immunostaining, it was established that sialoadhesin-positive stromal bone marrow macrophages contain parasites; such long-lived mononuclear phagocytes could be the host cells where the parasite can find "safe targets" unreactive to the dominant effector immune mechanism triggered by the replicative stage of the parasites.

Alleles↗
PubMed1996

Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.

Animals↗
PubMed1996

Activated mast cells release biological activities able to support eosinophil production from mouse hemopoietic precursors.

Mouse bone marrow cells cultured for 6 days in the presence of recombinant murine IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were used as a source of precursors responsive to eosinopoietins. They were further cultured for 7 days in the presence of either a combination of recombinant cytokines or supernatants of bone marrow-derived mast cells (BMMC) activated with either immunological or nonimmunological stimuli. Cytosmears of collected cells were analyzed for eosinophil contents and allowed to demonstrate that supernatants of passively sensitized BMMC support both total cell proliferation and eosinophil production, after various periods of incubation with monoclonal rat anti-mouse IgE antibodies (the 6HD5 mAbs). In contrast, a stimulation with 100 ng/ml dinitrophenylated bovine serum albumin (DNP-BSA) did not generate supernatants displaying such bioactivities. Low doses of methyl ester of L (but not D)-leucine or of the calcium ionophore A23187 also allowed the release of eosinopoietic bioactivities. In addition, immunoreactive IL-5, GM-CSF, and IL-3 were quantified in the BMMC supernatants. These results demonstrate that activated BMMC are able to effect eosinophil production.

Animals↗
PubMed1996

Molecular characterisation of ninein, a new coiled-coil protein of the centrosome.

We describe the cDNA cloning of ninein, a novel component of centrosomes. In the mouse, ninein is predicted to be an acidic protein (calculated pI of 4.8) with alternatively spliced forms of 245 kDa and 249 kDa that contain extensive regions of coiled-coil structure flanked by non-coiled ends. Other interesting features of this protein include an EF-hand-like domain, a potential GTP binding site and four leucine zipper domains. Specific polyclonal antisera were raised to two non-overlapping recombinant fragments of the protein and used to characterise the cellular distribution of ninein. Immunofluorescence and immunoelectron microscopy experiments with macrophage-like cells, Mm1, showed that ninein is localised specifically in the pericentriolar matrix of the centrosome. Studies with NIH3T3 fibroblasts demonstrated that ninein is associated with the centrosome throughout the cell cycle and can also be detected within nuclei at interphase. At mitosis ninein was also observed in association with the mitotic spindle. Immunocytochemical staining of mouse tissues showed that ninein was expressed in a heterogeneous fashion. Staining, if present, was always consistent with a centrosomal localisation and was never associated with nuclei. Ninein provides a new molecular tool for analysing the structure and function of the centrosome.

3T3 Cells↗
PubMed1996

Abnormal organisation of the splenic marginal zone and the correlated leukocytosis in lymphotoxin-alpha and tumor necrosis factor alpha double deficient mice.

In this study we further characterized the phenotype at the homeostasis of mice genetically deficient in Tumor Necrosis Factor-alpha and Lymphotoxin-alpha (LT-alpha TNF-alpha -/-). As initially observed in LT-alpha -/- mice, these mice are devoid of lymph nodes and Peyer's patches, while in their spleen the white and red pulp domains are no more detectable. In the blood the leukocytosis dominated by lymphocytosis is not solely due to the absence of lymph nodes. Indeed, this abnormality was shown to be correctable by the transfer of wild type bone marrow in the absence of lymph node. We now report that the metallophilic macrophages of the marginal zone are no more detectable with an antibody reactive to sialoadhesin, a macrophage restricted transmembrane molecule known to bind myeloid and lymphoid cells. The absence of sialoadhesin within the marginal zone, a critical domain for lymphocyte trafficking towards the white pulp suggests a possible cellular basis for the observed blood leukocytosis. In addition, in the peritoneal cavity of LT-alpha TNF-alpha-/- mice, the size of the resident leukocyte population is increased. By their amplitudes these leukocytosis are similar within the blood and the peritoneal compartments.

Animals↗
PubMed1995

Normal development and function of natural killer cells in CD3 epsilon delta 5/delta 5 mutant mice.

The CD3 epsilon polypeptide contributes to the cell surface display as well as to the signal transduction properties of the T-cell antigen receptor complex. Intriguingly, the distribution of CD3 epsilon is not restricted to T cells, since activated mouse, human, and avian natural killer (NK) cells do express intracytoplasmic CD3 epsilon polypeptides. CD3 epsilon is also present in the cytoplasm of fetal thymic T/NK bipotential progenitor cells, suggesting that it constitutes a component of the NK differentiation program. We report here that the genetic disruption of CD3 epsilon exon 5 alters neither NK cell development nor in vitro and in vivo NK functions, although it profoundly blocked T-cell development. These results support the notion that CD3 epsilon is dispensable for mouse NK cell ontogeny and function and further suggest that the common NK/T-cell progenitor cell utilizes CD3 epsilon as a mandatory component only when differentiating toward the T-cell lineage.

Amino Acid Sequence↗
PubMed1995

Transgenic mice expressing high levels of soluble TNF-R1 fusion protein are protected from lethal septic shock and cerebral malaria, and are highly sensitive to Listeria monocytogenes and Leishmania major infections.

Mice bearing a transgene coding for a soluble tumor necrosis factor receptor type 1 (TNFR1)-FcIgG3 fusion protein and placed under the control of the alpha-1-antitrypsin gene promoter were generated. Depending on the mouse line, blood levels of the protein ranged from 25 ng/ml to over 100 micrograms/ml; this level of expression was most often transmitted to the transgene-bearing progeny as a relatively stable feature. High-expressor mice were completely resistant to lipopolysaccharide-induced shock and lethality, including after D-galactosamine sensitization, and mice expressing about 1 microgram of the fusion protein/ml were partially (60%) protected. In contrast, mice expressing less than 0.1 microgram of the protein/ml were more sensitive than controls with respect to incidence and time of death, even though the biological activity of serum tumor necrosis factor (TNF) was partially neutralized. High-expressor mice of the adequate genetic background were markedly, although not completely, protected from death by cerebral malaria after injection with Plasmodium berghei. They were highly susceptible to Listeria monocytogenes, dying from bacterial dissemination after sublethal infection, and to Leishmania major, displaying severe, non-healing lesions after local infection. Under the same conditions, mice expressing about 1 microgram protein/ml were only partially sensitive to these last agents, compared to non-transgenic littermate mice which were fully resistant. These transgenic mice represent a model of permanent, complete or partial, impairment of TNF use, which compares favorably, for ease of breeding and for the range of effects, to mice bearing gene disruptions.

Animals↗
PubMed1995

Listeria monocytogenes: a live vector able to deliver heterologous protein within the cytosol and to drive a CD8 dependent T cell response.

After an introduction on the entry and lifestyle of Listeria monocytogenes within mammalian eucaryotic cells, this chapter gives a brief overview of murine experimental listeriosis. Among the main characteristics of this murine model of infectious/pathogenic processes initiated by a facultative intracellular bacteria, we point out two main recent advances. One relates to Listeria monocytogenes-induced production of cytokines as local, and transient signals able to direct the immune responses along a type 1 pathway of CD4/CD8 T cell differentiation. The other relates (a) to the recognition of L. monocytogenes-reactive CD8+ T lymphocytes as effectors able, once recruited within infected loci, to critically contribute to the complete clearance of the bacteria, and (b) to the recently recognized specificity of some of these CD8 lymphocytes in BALB/c mice. In this paper, we also briefly review (a) the readout assays presently used to monitor the outcome of the infectious/pathogenic processes and the related development and expression of immune responses induced by intravenous inoculation of wild-type virulent or attenuated L. Monocytogenes, (b) why all this information allows us to consider the use of L monocytogenes of attenuated virulence as relevant live recombinant vectors in order to deliver heterologous proteins to the class I processing and presentation pathway, and to induce CD8 T cells along the type 1 pathway.

Animals↗
PubMed1995

Immunobiology of experimental cutaneous leishmaniasis.

The study of the murine model of infection with Leishmania major is providing important insights into the understanding of the complex interactions between the host and intracellular pathogens. Using this model system, basic research is actively leading to the identification of host factors promoting or circumventing the development of immunity to L. major. Here, Geneviève Milon, Giuseppe Del Giudice and Jacques A. Louis review recent results related to the characterization of immunological host factors determining resistance and susceptibility to this parasite, and try to identify areas where further research is required for a better understanding of the complex events triggered by intracellular parasites within their hosts. Extrapolation to the human leishmaniases of the rapid advances made in this murine model of infection, should pave the way to the rational design of future immunoprophylactic and immunotherapeutic measures.

Journal Article↗
PubMed1995

Parasite-host relationships: in-situ study of Leishmania spp. in resistant and susceptible mice.

The host's skin is a critical tissue in the natural life cycle of the Leishmania spp. known to cause an 'asymptomatic' infectious process or cutaneous or visceral leishmaniasis in mammals. The dermis, once disturbed by the inoculation of infective parasites, becomes a site of dynamic events, the progression of which depends upon both host and parasite characteristics. Whatever the final site of the morbidity caused by the parasites, whether it be cutaneous, visceral or muco-cutanous, this site reflects the parasite and host's ability to create a pro- or anti-parasite micro-environment. The characteristics of this environment are now amenable to analysis in situ, as illustrated by the study of the cutaneous processes initiated by inoculation of Leishmania major in laboratory mice.

Animals↗
PubMed1995

Experimental models for leishmaniasis and for testing anti-leishmanial vaccines.

In public health terms, leishmaniases are diseases of humans and dogs, whereas, in epidemiological terms, Leishmania spp. are considered to represent infections of a wide variety of animals, which represent the natural reservoirs of the various parasite species involved. Humans and dogs (which may be considered secondary or 'accidental' hosts in the leishmanial life-cycle) often exhibit severe clinical signs and symptoms when infected, whereas reservoir hosts generally show a few, minor or no signs. This situation makes the definition of a suitable laboratory model a difficult one, since the various experimental hosts may behave either like a reservoir or an accidental host. This review discusses the concept of animal models for leishmaniases and provides a critical evaluation of the most common experimental models and their respective advantages and disadvantages. In this state-of-the-art review, particular emphasis is given to the value of using mouse, hamster, cotton-rat, dog and primate models, especially in the context of testing potential anti-leishmanial vaccines.

Animals↗
PubMed1995

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus.

Listeria monocytogenes spends most of its intracellular life cycle in the cytosol of the infected eucaryotic cells. Within this cellular compartment originates the endogenous pathway of antigen processing and presentation. We thus assumed that recombinant L. monocytogenes expressing an heterologous protein, the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV), should be able to induce antigen-specific CD8+ T cells in vivo. The LCMV nucleoprotein gene was inserted in phase with the sequence coding for the putative signal sequence of the hemolysin of L. monocytogenes in order to target its secretion into the cytosol of the infected cell. The ability of this recombinant bacterium to induce LCMV-reactive CD8+ T cells was then monitored in BALB/c mice. The immune status of the immunized BALB/c mice was studied on the seventh day after a single i.v. injection of a sublethal dose of the recombinant bacteria: (i) cytotoxic CD8+ T cells were detected in liver; (ii) using in vitro re-stimulation with PMA and ionomycin, secondary cytotoxic CD8+ T cells were detected in spleen; (iii) an early inflammatory reaction dependent on the presence of CD8+ T cells occurred in the footpad after intraplantar inoculation of live LCMV; and (iv) mice were protected against an otherwise lethal intracerebral LCMV challenge; the protection was accompanied by elimination of the virus. When the immune status of the immunized hosts was monitored for a longer period post-immunization, the balance between immune protection and immunopathology described for the anti-LCMV immune responses was observed; two phases of protection were detected, flanking a transitory phase of exacerbation of the lymphocytic choriomeningitis disease (weeks 2-5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗