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Biomedical subjects

B Gicquel

Publications and source records attributed to B Gicquel.

At least 19 recordsLinked to original sources
PubMed2007

Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain.

The Mycobacterium tuberculosis phoP mutant strain SO2 has been shown previously to be more attenuated than Mycobacterium bovis bacillus Calmette-Guérin (BCG) and confers protective immunity against tuberculosis in mice and guinea pig models. In this study we have investigated the survival and immunological responses of Balb/c mice infected with the M. tuberculosis SO2 strain. All Balb/C mice survived intratracheal infection with M. tuberculosis SO2 strain under conditions where all the mice infected with the parental M. tuberculosis MT103 had died after 9 weeks. Infection of Balb/c mice with M. tuberculosis SO2 was associated with comparatively lower levels of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha and higher levels of inducible nitric oxide synthase (iNOS) during the late stage of infection, when compared with M. tuberculosis MT103 infection. The delayed-type hypersensitivity (DTH) response against M. tuberculosis culture filtrates was similar in mice infected with either the M. tuberculosis phoP SO2 strain or M. tuberculosis MT103. The protective efficacy of M. tuberculosis SO2 was compared with M. bovis BCG when delivered subcutaneously to groups of Balb/C mice. Following intratracheal challenge with M. tuberculosis H37Rv, protection was generated by 60 days post-challenge in mice vaccinated with either vaccine. At day 120 post-challenge the levels of protection were still significantly greater when compared with the non-vaccinated control group. The levels of protection conferred by vaccination with M. tuberculosis SO2 or with M. bovis BCG were similar, as measured by granuloma coalescence and pneumonia in addition to growth reduction of M. tuberculosis H37Rv.

Animals↗
PubMed2006

Effect of phthiocerol dimycocerosate deficiency on the transcriptional response of human macrophages to Mycobacterium tuberculosis.

The control of mycobacterial infections is dependent on the finely tuned synergism between the innate and adaptive immune responses. The macrophage is the major host cell for Mycobacterium tuberculosis and the degree of virulence of mycobacteria may influence the initial macrophage response to infection. The cell wall molecule, phthiocerol dimycocerosate (DIM), is an important virulence factor that influences the early growth of M. tuberculosis in the lungs. To explore the basis for this effect we have compared the early gene response of human THP-1 macrophages to infection with virulent M. tuberculosis and the DIM-deficient DeltafadD26 M. tuberculosis strain using microarrays. Detailed analysis revealed a common core of macrophage genes, which were rapidly induced following infection with both strains, and deficiency of DIM had no significant effect on this initial macrophage transcriptional responses. In addition to chemokines and pro-inflammatory cytokines, the early response genes included components of the Toll-like receptor signalling, antigen presentation and apoptotic pathways, interferon response genes, cell surface receptors and their ligands, including TNF-related apoptosis inducing ligand (TRAIL) and CD40, and other novel genes. Therefore, although fadD26 deficiency is responsible for the early attenuation of the growth of M. tuberculosis in vivo, this effect is not associated with differences in the initial macrophage transcriptional response.

Animals↗
PubMed2006

Mycobacterium bovis BCG as a delivery system for the RAP-1 antigen from Babesia bovis.

Babesia bovis is the causative agent of babesiosis, a tick-borne disease that is a major cause of loss to livestock production in Latin America. Vaccination against Babesia species represents a major challenge against cattle morbidity and mortality in enzootic areas. The aim of this study was to evaluate the capacity of Bacille Calmette-Guerin (BCG) to deliver the rhoptry associated protein (RAP-1) antigen of B. bovis and to stimulate specific cellular and humoral immune responses in mice. Two of five mycobacterial expression vectors efficiently expressed the antigen. These constructs were subsequently studied in vivo following three immunization protocols. The construct with the greatest in vivo stability proved to be the one that induced the strongest immune responses. Our data support the hypothesis that specific T lymphocyte priming by rBCG can be employed as a component of a combined vaccine strategy to induce long-lasting humoral and cellular immune responsiveness towards B. bovis and encourage further work on the application of rBCG to the development of Babesia vaccines.

Animals↗
PubMed2006

The use of mutant mycobacteria as new vaccines to prevent tuberculosis.

Given the variable protective efficacy generated by Mycobacterium bovis BCG (Bacillus Calmette-Guérin), there is a concerted effort worldwide to develop better vaccines that could be used to reduce the burden of tuberculosis. Rational attenuated mutants of Mycobacterium tuberculosis are vaccine candidates that offer some potential in this area. In this paper, we will discuss the molecular methods used to generate mutant mycobacteria, as well as the results obtained with some of these strains, in terms of attenuation, immunogenicity and level of protection, when compared with the conventional BCG vaccine in diverse animal models. Tuberculosis vaccine candidates based on safe and live mycobacterial mutants could be promising candidates.

Animals↗
PubMed2006

Identification of Nudix hydrolase family members with an antimutator role in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacterium tuberculosis and Mycobacterium smegmatis MutT1, MutT2, MutT3, and Rv3908 (MutT4) enzymes were screened for an antimutator role. Results indicate that both MutT1, in M. tuberculosis and M. smegmatis, and MutT4, in M. smegmatis, have that role. Furthermore, an 8-oxo-guanosine triphosphatase function for MutT1 and MutT2 is suggested.

Gene Deletion↗
PubMed2005

[Laboratory diagnosis of mycobacterial infections].

Every 10 seconds, one person in the world dies of tuberculosis (TB). It is estimated that one third of the world's population is latently infected with Mycobacterium tuberculosis. The proportion of multidrug-resistant strains of M. tuberculosis is increasing at an alarming rate in some parts of the world linked in part with the human immunodeficiency virus epidemic. For these reasons, TB remains a major public health problem, both in less-developed countries and in many industrialized countries, with 8-10 million new cases and 2 million deaths yearly in the world. Clinical, radiological and histological signs are not specific for tuberculosis or for other mycobacterial infections and allow only a presumptive diagnosis. In the same way, the tuberculin skin test is useful if the reaction is strong or phlyctenular because this test depends on various factors as previous BCG vaccination, contact or primary infection and host immune responses. The diagnosis of mycobacterial infection is proved only when bacilli are present in biological samples. Nevertheless, only 50% of cases in adults and 30% in infants have a positive bacteriological result. It seems necessary to develop new methods for a rapid and efficient diagnosis to optimize the therapy and the control of the epidemic. Laboratory testing in the mycobacterium field is experiencing more changes today than ever before. Determining what assays will be most useful to the clinician is a challenge, and acceptance of the new technology is under discussion. Progress in future will be linked probably to the progress of the genomic area. However the incidence rate is higher in less-developed countries, it is also important to develop now techniques possible to use in these countries. This review focuses on the current state-of-the-art resources useful for accurate and rapid laboratory diagnosis of mycobacterial infections.

Bacteriological Techniques↗
PubMed2005

Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant.

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette-Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis.

Animals↗
PubMed2002

Detection and isolation of equine herpesviruses 1 and 4 from horses in Normandy: an autopsy study of tissue distribution in relation to vaccination status.

Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive horses between vaccinates and unvaccinates was similar. Both viruses were detected in all tissues of both groups; in particular, lymph nodes draining the respiratory tract, nasal epithelium and nervous ganglia [i.e. trigeminal ganglia (TG)], which represent the main positive sites for EHV-1 and EHV-4. In vaccinated animals, the nervous ganglia (i.e. TG) were less frequently positive than in unvaccinated animals. Detection of positive TG was strongly correlated to the presence of EHV-1 in nasal epithelium.

Animals↗
PubMed2002

Plasmidic versus insertional cloning of heterologous genes in Mycobacterium bovis BCG: impact on in vivo antigen persistence and immune responses.

Bivalent recombinant strains of Mycobacterium bovis BCG (rBCG) expressing the early regulatory nef and the structural gag(p26) genes from the simian immunodeficiency virus (SIV) SIVmac251 were engineered so that both genes were cotranscribed from a synthetic operon. The expression cassette was cloned into a multicopy-replicating vector, and the expression levels of both nef and gag in the bivalent rBCG(nef-gag) strain were found to be comparable to those of monovalent rBCG(nef) or rBCG(gag) strains. However, extrachromosomal cloning of the nef-gag operon into a replicative plasmid resulted in strains of low genetic stability that rapidly lost the plasmid in vivo. Thus, the nef-gag operon was inserted site specifically into the BCG chromosome by means of mycobacteriophage Ms6-derived vectors. The resulting integrative rBCG(nef-gag) strains showed very high genetic stability both in vitro and in vivo. The in vivo expression of the heterologous genes was much longer lived when the expression cassette was inserted into the BCG chromosome. In one of the strains obtained, integrative cloning did not reduce the expression levels of the genes even though a single copy was present. Accordingly, this strain induced cellular immune responses of the same magnitude as that of the replicative rBCG strain containing several copies of the genes.

Animals↗
PubMed2001

Culture at high density improves the ability of human macrophages to control mycobacterial growth.

The mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 x 10(4) cells/well, but increasing the number of cells to 2 x 10(5) cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 x 10(4) cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.

Cell Adhesion↗
PubMed2001

Analysis of stress- and host cell-induced expression of the Mycobacterium tuberculosis inorganic pyrophosphatase.

BACKGROUND: The gene encoding the inorganic pyrophosphatase (PPase) of the intracellular pathogen Legionella pneumophila is induced during intracellular infection, but is constitutively expressed in Escherichia coli. The causative agent of tuberculosis, Mycobacterium tuberculosis, contains a well conserved copy of PPase. We sought to determine if expression of the M. tuberculosis PPase is regulated by the intracellular environment. RESULTS: A strain of Mycobacterium bovis bacille Calmette-Guérin (BCG) was constructed in which the Aequoria victoria green fluorescent protein (GFP) is controlled by the promoter of the M. tuberculosis ppa gene. After prolonged exposure of the recombinant BCG strain within murine bone-marrow-derived macrophages, there was no observed increased activity of the ppa promoter. Furthermore, there was no change in promoter activity after exposure to various stress stimuli such as reduced pH, osmotic shock, nutrient limitation or oxidative stress. CONCLUSIONS: These results suggest that macrophage induction of ppa is not a general phenomenon among intracellular pathogens.

Animals↗
PubMed2001

Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier.

Among the few characterized genes that have products involved in the pathogenicity of Mycobacterium tuberculosis, the etiological agent of tuberculosis, are those of the phthiocerol dimycocerosate (DIM) locus. Genes involved in biosynthesis of these compounds are grouped on a 50-kilobase fragment of the chromosome containing 13 genes. Analysis of mRNA produced from this 50-kilobase fragment in the wild type strain showed that this region is subdivided into three transcriptional units. Biochemical characterization of five mutants with transposon insertions in this region demonstrated that (i) the complete DIM molecules are synthesized in the cytoplasm of M. tuberculosis before being translocated into the cell wall; (ii) the genes fadD26 and fadD28 are directly involved in their biosynthesis; and (iii) both the drrC and mmpL7 genes are necessary for the proper localization of DIMs. Insertional mutants unable to synthesize or translocate DIMs exhibit higher cell wall permeability and are more sensitive to detergent than the wild type strain, indicating for the first time that, in addition to being important virulence factors, extractable lipids of M. tuberculosis play a role in the cell envelope architecture and permeability. This function may represent one of the molecular mechanisms by which DIMs are involved in the virulence of M. tuberculosis.

Base Sequence↗
PubMed2001

High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology.

The worldwide threat of tuberculosis to human health emphasizes the need to develop novel approaches to a global epidemiological surveillance. The current standard for Mycobacterium tuberculosis typing based on IS6110 restriction fragment length polymorphism (RFLP) suffers from the difficulty of comparing data between independent laboratories. Here, we propose a high-resolution typing method based on variable number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 human minisatellite-like regions of the M. tuberculosis genome. MIRU-VNTR profiles of 72 different M. tuberculosis isolates were established by PCR analysis of all 12 loci. From 2 to 8 MIRU-VNTR alleles were identified in the 12 regions in these strains, which corresponds to a potential of over 16 million different combinations, yielding a resolution power close to that of IS6110-RFLP. All epidemiologically related isolates tested were perfectly clustered by MIRU-VNTR typing, indicating that the stability of these MIRU-VNTRs is adequate to track outbreak episodes. The correlation between genetic relationships inferred from MIRU-VNTR and IS6110-RFLP typing was highly significant. Compared with IS6110-RFLP, high-resolution MIRU-VNTR typing has the considerable advantages of being fast, appropriate for all M. tuberculosis isolates, including strains that have a few IS6110 copies, and permitting easy and rapid comparison of results from independent laboratories. This typing method opens the way to the construction of digital global databases for molecular epidemiology studies of M. tuberculosis.

Bacterial Typing Techniques↗
PubMed2001

Effects of Mycobacterium bovis BCG on the development of allergic inflammation and bronchial hyperresponsiveness in hyper-IgE BP2 mice vaccinated as newborns.

Asthma may result from excessive Th-2 response in children not previously exposed to Th-1-inducing infections. We tested the hypothesis that BCG vaccination in Th-2-susceptible newborn BP2 mice blocks allergic inflammation and bronchial hyperreactivity (BHR). Ten day-old BP2 mice received 10(5) CFU of BCG 1173P2 intranasally (IN), and 6, 10 or 14 weeks thereafter were sensitized with 100 microg ovalbumin (OVA) in aluminium hydroxide twice subcutaneously (SC) at 1 week interval, and challenged 1 week after the second sensitization with 10 microg OVA IN. Compared to OVA-challenged unvaccinated mice, those that received BCG 8 weeks before challenge developed intense bronchial inflammation, BHR, and high IgE titers. Inflammation involved T cells, macrophages, dendritic cells and was accompanied by increased levels of Interleukin-5 (IL-5) in the bronchoalveolar lavages (BAL). However, animals challenged 16 weeks after BCG vaccination did not develop BHR nor bronchial hypereosinophilia, and showed reduced IgE levels. Bronchial infiltration by immunocompetent cells was also significantly reduced. Increased levels of gamma-interferon (IFN-gamma) after in vitro stimulation of tracheo-bronchial lymph node cells accompanied this blockage, but levels of IL-5 remained high. We demonstrate that 16 weeks after vaccination with BCG in newborn BP2 mice which have a high Th-2 background, allergic inflammation and BHR were blocked, even though a clear Th-1 shift was not achieved.

Animals↗
PubMed2001

An essential role for phoP in Mycobacterium tuberculosis virulence.

Two-component regulatory proteins function in bacteria as sensory and adaptive factors in response to a wide range of environmental stimuli. Some two-component systems, such as PhoP/PhoQ, control transcription of key virulence genes essential for survival in host cells in diverse intracellular bacterial pathogens, including Salmonella sp., Shigella sp. and Yersinia sp. In this study, we have disrupted the phoP gene from Mycobacterium tuberculosis, which codes for a putative transcription regulator factor of the two-component system PhoP/PhoR. The phoP mutant strain exhibited impaired multiplication when cultured in mouse bone marrow-derived macrophages. However, the mutation did not appear to affect survival of the organisms adversely inside macrophages. The mutant strain was also attenuated in vivo in a mouse infection model, with impaired growth observed in the lungs, livers and spleens. The results suggest that the phoP gene is required for intracellular growth of M. tuberculosis but is not essential for persistence of the bacilli.

Animals↗
PubMed2001

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

Animals↗
PubMed2001

Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems.

Linear plasmids were unknown in mycobacteria until recently. Here, we report the complete nucleotide sequence of 23-kb linear plasmid pCLP from Mycobacterium celatum, an opportunistic pathogen. The sequence of pCLP revealed at least 19 putative open reading frames (ORFs). Expression of pCLP genes in exponential-phase cultures was determined by reverse transcriptase PCR (RT-PCR). Twelve ORFs were expressed, whereas no transcription of the 7 other ORFs of pCLP was detected. Five of the 12 transcribed ORFs detected by RT-PCR are of unknown function. Sequence analysis revealed similar loci in both M. celatum pCLP and the Mycobacterium tuberculosis chromosome, including transposase-related sequences. This result suggests horizontal transfer between these two organisms. pCLP also contains ORFs that are similar to genes of bacterial circular plasmids involved in partition (par operon) and postsegregational (pem operon) mechanisms. Functional analysis of these ORFs suggests that they probably carry out similar maintenance roles in pCLP.

Amino Acid Sequence↗