Biomedical subjects

Gilles Marchal

Publications and source records attributed to Gilles Marchal.

13 recordsLinked to original sources

PubMed2006

Similar functional activity of dendritic cells recruited to the mesenteric lymph nodes of newborn and adult mice after the rectal delivery of Mycobacterium bovis BCG.

BCG rectal administration to newborn and adult mice induced protective immune responses against tuberculosis. BCG reaches the sub-epithelial site and the draining mesenteric lymph nodes (MLNs), and dendritic cells (DC) could be recruited to these sites. Using polarized Caco-2 epithelial cells, we showed that BCG translocates epithelial cells to basolateral compartment. Delayed in newborn BALB/c mice, an important recruitment of CD11c+ DCs, was documented in the rectal lamina propria and the MLNs during the first two weeks after rectal BCG delivery. In MLNs, two major DC subtypes were observed: conventional DCs (cDCs) (B220-) and plasmacytoid DCs (pDCs) (B220+). CIRE, mouse DC-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) is predominantly expressed on pDCs and at a higher level on pDCs from the adult compared to newborn MLNs. cDCs with a higher capacity to induce the proliferation of naïve CD4+ T cells than pDCs, triggered CD4+ T cells to produce interferon-gamma whereas pDCs triggered them to release interleukin-10. Both DC subtypes equilibrates T cells as a source of microbicidal/microbiostatic signals and those acting as source of counter-inflammatory signals, preventing tissue damage and/or accelerating tissue repair. Thus, rectal delivery of BCG could be a safe and efficient route of vaccination against tuberculosis.

Administration, Rectal↗

PubMed2006

Synthesis and biological evaluation of bicyclic nucleosides as inhibitors of M. tuberculosis thymidylate kinase.

Herein we describe the synthesis and conformational analysis of a series of bicyclic thymidine derivatives and their evaluation as inhibitors of thymidine monophosphate kinase from Mycobacterium tuberculosis (TMPKmt), based on previously discovered bicyclic sugar nucleosides. With a K(i) value of 2.3 microm, 1-[3-aminomethyl-3,5-dideoxy-2-O,6-N-(thiocarbonyl)-beta-D-ribofuranosyl]thymine emerged as the most potent TMPK inhibitor of this series. Moreover, this promising compound displays inhibitory potency against Mycobacteria cultures with an IC(99) value of 100 microg mL(-1), thus promoting TMPKmt for the first time as a validated target for further inhibitory design. Attempts to rationalise the observed structure-activity relationship (SAR) involving molecular modelling and conformational analysis are described.

Binding Sites↗

PubMed2005

Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity.

Interactions between dendritic cells and Mycobacterium tuberculosis, the aetiological agent of tuberculosis in humans, are thought to be central to anti-mycobacterial immunity. We have previously shown that M. tuberculosis binds to human monocyte-derived dendritic cells mostly through the C-type lectin DC-SIGN (dendritic-cell-specific intercellular molecule-3-grabbing non-integrin)/CD209, and we have suggested that DC-SIGN may discriminate between mycobacterial species through recognition of the mannose-capping residues on the lipoglycan lipoarabinomannan of the bacterial envelope. Here, using a variety of fast- and slow-growing Mycobacterium species, we provide further evidence that mycobacteria recognition by DC-SIGN may be restricted to species of the M. tuberculosis complex. Fine analyses of the lipoarabinomannan molecules purified from these species show that the structure and amount of these molecules alone cannot account for such a preferential recognition. We propose that M. tuberculosis recognition by DC-SIGN relies on both a potential difference of accessibility of lipoarabinomannan in its envelope and, more probably, on the binding of additional ligands, possibly including lipomannan, mannose-capped arabinomannan, as well as the mannosylated 19 kDa and 45 kDa [Apa (alanine/proline-rich antigen)] glycoproteins. Altogether, our results reveal that the molecular basis of M. tuberculosis binding to DC-SIGN is more complicated than previously thought and provides further insight into the mechanisms of M. tuberculosis recognition by the immune system.

Bacterial Adhesion↗

PubMed2005

A nose-only apparatus for airborne delivery of Mycobacterium tuberculosis to mice: calibration of biological parameters.

Mycobacterium tuberculosis is the main cause of tuberculosis and is still a public health concern worldwide. This mycobacterium is transmitted through aerosols from human beings suffering from pulmonary tuberculosis to susceptible persons. To study this natural route of infection, we designed a new nose-only aerosol apparatus--system of aerosolisation of microorganisms (SAM)--in a carefully designed biohazard facility. For safety reasons, Mycobacterium smegmatis was first used to calibrate several parameters, such as inoculum density, atmospheric conditions (i.e. hygrometry) and particle size distribution. We present evidence that our apparatus is totally adapted to airborne delivery; the particle size of generated aerosol ranges from 1 to 7 microm, which is ideal for an infection by inhalation. We found that 99% of generated particles (<7 microm) could be retained by the respiratory tract, and among these particles, 62-79% (<3.3 microm) were able to reach pulmonary compartments. The next step was to simultaneously challenge 48 mice with M. tuberculosis in a highly reproducible way. We showed that a moderate dose (4 log10 colony-forming units (CFU) per mice) of M. tuberculosis was capable of causing progressive lung pathology and death in mice 30 days post-aerosolisation. Therefore, our apparatus, once calibrated, is easy to handle, safe, and can be used with any pathogen, which is spread by aerosol.

Aerosols↗

PubMed2005

Alpha-galactosylceramide-induced iNKT cells suppress experimental allergic asthma in sensitized mice: role of IFN-gamma.

Allergic asthma is a multifaceted syndrome consisting of eosinophil-rich airway inflammation, bronchospasm, and airway hyper-responsiveness (AHR). Using a mouse model of allergic asthma, we previously reported that invariant NKT (iNKT) cells increase the severity of this disease. Herein, we demonstrate that a single i.v. injection of alpha-galactosylceramide (alpha-GalCer), 1 h before the first airway allergen challenge of OVA-sensitized mice, abrogates elicitation of AHR, airway eosinophilia, IL-4 and IL-5 production in bronchoalveolar lavage fluid, and specific anti-OVA IgE antibodies. Further, alpha-GalCer administered intranasally also strongly inhibited the major symptoms of asthma in sensitized and challenged mice. Alpha-GalCer treatment induces iNKT cell accumulation in the lungs, and shifts their cytokine profile from pro-asthmatic IL-4 to a protective IFN-gamma production. The role of IFN-gamma from iNKT cells in protection was shown by adoptive transfer of sorted iNKT cells from OVA-sensitized and alpha-GalCer-treated mice which protected immunized recipients from manifesting asthma by an IFN-gamma-dependent pathway. Our findings demonstrate for the first time that alpha-GalCer administered locally inhibits asthma symptoms, even in predisposed asthmatic mice, through an iNKT cell- and IFN-gamma-dependent pathway.

Administration, Intranasal↗

PubMed2005

Contribution of transmembrane tumor necrosis factor to host defense against Mycobacterium bovis bacillus Calmette-guerin and Mycobacterium tuberculosis infections.

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

Animals↗

PubMed2003

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis.

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

Animals↗

PubMed2003

Characterization of a putative type IV aminophospholipid transporter P-type ATPase.

The P-type ATPases comprise a well-studied family of proteins involved in the active transport of charged substrates across biological membranes. Starting from a mouse bone marrow-derived macrophage cDNA library and using a signal peptide trapping strategy, we identified a new P-type ATPase family member. We characterized the genomic structure of this gene, named Atp10d, as well as its human counterpart. The presence of P-type ATPase consensus motifs and phylogenetic analysis showed that this gene is a member of the type IV, putative amphipath transporters subfamily. We showed that this gene is expressed in kidney and placenta. We also found that the C57BL/6 strain carries a constitutive stop codon in the sequence of Atp10d exon 12, whereas 14 other inbred mouse strains show an uninterrupted reading frame at this location. This mutation in C57BL/6 should lead to a non-functional protein, suggesting that this gene may not be essential. We discuss the involvement of the Atp10d gene in the fat-prone phenotype of the C57BL/6 strain and its physical mapping within a QTL associated with HDL-cholesterol levels.

Adenosine Triphosphatases↗

PubMed2003

Enhanced cloning efficiency of mouse bone marrow macrophage progenitors correlates with increased content of CSF-1 receptor of their progeny at low oxygen tension.

Mononuclear phagocytes are located in every tissue of metazoan organisms. In this extravascular space, they are designated as macrophages and are known to sense and process many signals including the local oxygen tension (PO2), which ranges from 150 mmHg at the lung apices to around 40 mmHg in mixed venous blood and most organs, and to less than 10 mmHg in tissues where long-term and dynamic remodeling processes occur. Most tissue macrophages survive and maintain their differentiated status within an environment bathed by colony-stimulating factor (CSF)-1 through the CSF-1 receptor, encoded by the Csf1r gene. In order to investigate the mRNA expression profile of macrophages as a function of PO2, we developed an in vitro model in which monocyte-derived macrophages were generated from mouse bone marrow progenitor cells grown and maintained under low (36 mmHg) or atmospheric (142 mmHg) PO2, in the presence of L929-conditioned medium (L-CM) as a source of CSF-1. We show that CSF-1-reactive C57BL/6 bone marrow cells displayed an increased cloning efficiency under a PO2 of 36, compared with 142 mmHg. Furthermore, we provide evidence of the overexpression of both CSF-1 receptor protein and mRNA by mouse monocyte-derived macrophages generated from bone marrow under low PO2.

Animals↗

PubMed2003

Dendritic cells recruited to the lung shortly after intranasal delivery of Mycobacterium bovis BCG drive the primary immune response towards a type 1 cytokine production.

We showed in a previous study that the intranasal (i.n) delivery of bacille Calmette-Guérin (BCG) to BP2 mice (H-2q) inhibits eosinophilia and bronchial hyperreactivity in a mouse model of asthma. The present work has been performed to characterize the leucocyte lineages recruited to the lungs of mice after i.n. delivery of BCG and potentially involved in the polarization of T lymphocytes. The different antigen-presenting cells (APC) recruited to bronchoalveolar lavage (BAL) and to lung tissue of mice shortly after the delivery of BCG were analysed in parallel as well as their capacity to drive the immune response towards a T helper type 1 cytokine production. Alveolar macrophages (AM) from the BAL were CD11c+, F4/80+ and CD11b-, and in the lung tissue two major populations of potential APC were detected: one CD11c-, F4/80+, CD11b+ and I-Aq- was identified as interstitial macrophages (IM) and a second expressing CD11c+ and I-Aq+ antigens, negative for CD11b and F4/80 markers as leucocytic dendritic cells (DC). Freshly isolated DC up-regulated CD11b and CD40 antigens after overnight culture, but remained negative for CD8alpha antigen, suggesting a myeloid origin. Lung DC which produced high amount of interleukin (IL)-12 were potent inducers of naive CD4+ T lymphocyte priming, as assessed by interferon-gamma (IFN-gamma) production by these naive CD4+ T cells. Lung explants recovered long term after BCG delivery produced sustained levels of IFN-gamma. Our results suggest that AM and particularly DC by secreting IL-12 shortly after BCG delivery induce the long-term persistence of IFN-gamma-secreting T cells percolating in BCG-loaded lung tissue.

Administration, Intranasal↗

PubMed2003

Synthesis, degradation, and antimicrobial properties of targeted macromolecular prodrugs of norfloxacin.

Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only when it is released in its native form. Also, targeting by using mannose as a homing device is required to achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its native form in vivo, can be transformed into an active drug by targeting.

Animals↗

PubMed2002

[Mycobacterium tuberculosis and its host].

Human beings are the only hosts of Mycobacterium tuberculosis. Owing to a peculiar architecture and composition of the cell wall, M. tuberculosis presents characteristic properties (acid fastness, high hydrophobicity). Molecules, proteins and polysaccharides, present on the cell surface play a key role in the rapid bacterial phagocytosis by macrophages. M. tuberculosis is able to inhibit the intracellular bactericidal mechanisms. Potent thymodependent immune responses control bactericial growth through inflammatory reactions.

Humans↗

PubMed2002

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha.

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.

Animals↗