Cutinase-like proteins of Mycobacterium tuberculosis: characterization of their variable enzymatic functions and active site identification.

Discovery and characterization of novel secreted enzymes of Mycobacterium tuberculosis are important for understanding the pathogenesis of one of the most important human bacterial pathogens. The proteome of M. tuberculosis contains over 400 potentially secreted proteins, the majority of which are uncharacterized.

Influence of BCG vaccine strain on the immune response and protection against tuberculosis.

The Bacille Calmette-Guérin (BCG) vaccine has been used for more than 80 years to protect against tuberculosis. Worldwide, over 90% of children are immunized with BCG, making it the most commonly administered vaccine, with more than 120 million doses used each year. Although new tuberculosis vaccines are under investigation, BCG will remain the cornerstone of the strategy to fight the worsening tuberculosis pandemic for the foreseeable future.

Immunological diversity within a family of cutinase-like proteins of Mycobacterium tuberculosis.

Secreted proteins of Mycobacterium tuberculosis play key roles in the assembly of the mycobacterial cell wall, with many being major targets of the host immune response. To date, meaningful characterization of a significant proportion of this important group of proteins is lacking. Among the group of putatively secreted proteins of M.

Comparison of IFN-gamma responses to mycobacterial antigens as markers of response to BCG vaccination.

An increase in interferon-gamma (IFN-gamma) production to Mycobacterium tuberculosis purified protein derivative (Mtb PPD), as measured in the cultured diluted whole blood assay, is one indicator of a protective immune response to BCG vaccine. We have explored the potential for this assay to be improved by measuring IFN-gamma responses to more defined antigens of M. tuberculosis (short-term and mid-term culture filtrates, ESAT-6, 38 kDa), Mycobacterium bovis (MPB70), M.

Inactivation of the Mycobacterium tuberculosis fadB4 gene results in increased virulence in host cell and mice.

The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism, a subset of which are required for virulence. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, is up-regulated during infection of macrophages and is predicted to protect the bacterium from the hostile environment of the host cell. In order to determine if fadB4 plays a role in the virulence of M.

Improved protection against disseminated tuberculosis by Mycobacterium bovis bacillus Calmette-Guerin secreting murine GM-CSF is associated with expansion and activation of APCs.

Modulating the host-immune response by the use of recombinant vaccines is a potential strategy to improve protection against microbial pathogens. In this study, we sought to determine whether secretion of murine GM-CSF by the bacillus Calmette-Guérin (BCG) vaccine influenced protective immunity against Mycobacterium tuberculosis. BCG-derived GM-CSF stimulated the in vitro generation of functional APCs from murine bone marrow precursors, as demonstrated by the infection-induced secretion of IL-12 by differentiated APCs, and the ability of these cells to present Ag to mycobacterium-specific T cells.

Early predictors for developing allergic disease and asthma: examining separate steps in the 'allergic march'.

Background: Sensitization and symptoms of allergic disease are strongly correlated, but little is known about the early clinical precursors of the development of allergen sensitization in childhood. The aim of this study was to identify these predictors, and to examine separately the effect of early sensitization on subsequent wheeze, asthma, rhinitis and eczema.

Methods: In the Childhood Asthma Prevention Study, children with a family history of asthma were assessed for allergen sensitization, total serum IgE, wheeze, asthma, eczema and rhinitis at ages 18 months and 5 years.

Effects of DNA- and Mycobacterium bovis BCG-based delivery of the Flt3 ligand on protective immunity to Mycobacterium tuberculosis.

The control of intracellular pathogens such as Mycobacterium tuberculosis is dependent on the activation and maintenance of pathogen-reactive T cells. Dendritic cells (DCs) are the major antigen-presenting cells initiating antimycobacterial T-cell responses in vivo. To investigate if immunization strategies that aim to optimize DC function can improve protective immunity against virulent mycobacterial infection, we exploited the ability of the hematopoietic growth factor Fms-like tyrosine kinase 3 ligand (Flt3L) to expand the number of DCs in vivo.

IL-5 T-cell responses to house dust mite are associated with the development of allergen-specific IgE responses and asthma in the first 5 years of life.

Background: Allergen-specific T(H)2-like cytokine responses are considered to be important in sensitization and allergic diseases.

Objective: To examine the profile of house dust mite (HDM) stimulated T-cell cytokines and their relationship to allergic disease in children over the period of the first 5 years of life.

Methods: Subjects with a family history of asthma who were enrolled antenatally in the Childhood Asthma Prevention Study and had skin prick tests, clinical evaluation for asthma and eczema, and in vitro assessment of lymphocyte cytokine responses to HDM extract performed at ages 18 months (n = 281), 3 years (n = 349), and 5 years (n = 370).

The genetic control of susceptibility to Mycobacterium tuberculosis.

Mycobacterium tuberculosis is one of the most successful human pathogens, surviv ing in latent foci of infection in one third of humanity, yet causing lung necrosis in sufficient individuals to ensure its transmission. Each stage of the host response to M. tuberculosis is under genetic control, including the initial encounter with mycobacteria by macrophages, epithelial cells and dendritic cells in the lung, induction of the inductive T cell response, and killing by activated macrophages within granulomas.

Life and death in the granuloma: immunopathology of tuberculosis.

During tuberculosis (TB) infection, the granuloma provides the microenvironment in which antigen-specific T cells colocate with and activate infected macrophages to inhibit the growth of Mycobacterium tuberculosis. Although the granuloma is the site for mycobacterial killing, virulent mycobacteria have developed a variety of mechanisms to resist this macrophage-mediated killing. These surviving mycobacteria become dormant, however, if host cellular immunity or the signals maintaining granuloma structure wane, or if mycobacteria resume replication, leading to reactivation of TB.

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.

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses.

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain.

A polymorphism in the P2X7 gene increases susceptibility to extrapulmonary tuberculosis.

Rationale: Genetic variation influences susceptibility to clinical tuberculosis (TB). Activation of the P2X(7) receptor on human macrophages induces killing of mycobacteria. We have identified polymorphisms in the P2X(7) gene that markedly reduce this killing.

Secretion of functional monocyte chemotactic protein 3 by recombinant Mycobacterium bovis BCG attenuates vaccine virulence and maintains protective efficacy against M. tuberculosis infection.

A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCG(MCP-3)) was developed. Mice vaccinated with BCG(MCP-3) displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCG(MCP-3) was equivalent to that with control BCG; however, immunodeficient mice infected with BCG(MCP-3) survived significantly longer than mice infected with the control BCG strain.

Contribution of L-alanine dehydrogenase to in vivo persistence and protective efficacy of the BCG vaccine.

The tuberculosis (TB) vaccine strain Mycobacterium bovis BCG is unable to utilise alanine and this deficiency is thought to inhibit the growth of the vaccine in vivo and limit vaccine efficacy. In this report we demonstrate that L-alanine catabolism can be conferred on BCG by introduction of the gene encoding L-alanine dehydrogenase (Ald) of Mycobacterium tuberculosis. Restoration of Ald activity did not change the in vivo growth of BCG in macrophages or mice, and protection against aerosol M.

Differential requirements for soluble and transmembrane tumor necrosis factor in the immunological control of primary and secondary Listeria monocytogenes infection.

The relative contributions of transmembrane tumor necrosis factor (memTNF) and soluble tumor necrosis factor (solTNF) in innate and adaptive immunity are poorly defined. We examined the capacities of wild-type (WT) mice, TNF-/- mice, and memTNF mice, which express only transmembrane TNF, to control primary and secondary Listeria monocytogenes infections. Soluble TNF was not required for induction or maintenance of protective immunity against a low-dose (200-CFU) Listeria infection.

Genetic susceptibility to mycobacterial disease in humans.

Mycobacterial disease remains a serious global health problem. Tuberculosis causes more than 2 million deaths a year, and leprosy is still a cause of severe disability in many parts of the world. As a result of the study of individuals with marked susceptibility to usually nonpathogenic mycobacteria, as well as case-control studies with candidate genes and genome-wide screens of affected populations, there is substantial evidence for the role of genetic factors in the susceptibility to mycobacterial disease.

Plasmid interleukin-23 (IL-23), but not plasmid IL-27, enhances the protective efficacy of a DNA vaccine against Mycobacterium tuberculosis infection.

Protection against intracellular pathogens such as Mycobacterium tuberculosis requires the development of Th1-like T-cell responses. This in turn is dependent on the pattern of cytokine produced from dendritic cells (DCs) after infection. Three heterodimeric cytokines, interleukin-12 (IL-12), IL-23, and IL-27, as well as IL-18, contribute to the differentiation and expansion of naive CD4(+) T cells.

A Thr357 to Ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs ATP-induced mycobacterial killing by macrophages.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.

Independent protective effects for tumor necrosis factor and lymphotoxin alpha in the host response to Listeria monocytogenes infection.

Although the essential role of tumor necrosis factor (TNF) in resistance to Listeria monocytogenes infection is well established, the roles of the related cytokines lymphotoxin alpha (LTalpha) and lymphotoxin beta (LTbeta) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTalpha, and LTbeta in the host response to Listeria. To overcome the lack of peripheral lymph nodes in LTalpha(-/-) and LTbeta(-/-) mice, bone marrow chimeras were constructed.

Gene dosage determines the negative effects of polymorphic alleles of the P2X7 receptor on adenosine triphosphate-mediated killing of mycobacteria by human macrophages.

Background: Stimulation of the P2X7 purinergic receptor (P2X7) in bacille Calmette-Guerin (BCG)-infected human macrophages with extracellular adenosine triphosphate (ATP) leads to pore formation and killing of mycobacteria. We examined the effect of polymorphisms in the P2X7 gene (P2X7) on the capacity of macrophages to kill mycobacteria.

Methods: Polymorphisms and mutations in P2X7 were identified by both DNA sequence analysis and determination of uptake of ethidium by time-resolved flow cytometry.

Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons.

Nerve growth factor (NGF) has been shown to promote survival and function of cholinergic neurons in the basal forebrain in various models of neuronal degeneration in rodents and primates. We examined whether a regulatable in vivo expression system can control the survival of cholinergic neurons after injury, using a tetracycline-regulated promoter ("tet-off" system) to modulate lentiviral NGF gene delivery. Two weeks after lesions to cholinergic neurons, significant cell rescue (65+/-8% neuron survival; P<0.

Sensitivity of human gamma interferon assay and tuberculin skin testing for detecting infection with Mycobacterium tuberculosis in patients with culture positive tuberculosis.

Setting: Nine university-affiliated chest clinics in Australia.

Objective: To evaluate the sensitivity of a whole blood human gamma-interferon assay (HGIA, QuantiFERON-TB) for specific T lymphocyte responses and Tuberculin skin testing (TST) for the detection of Mycobacterium tuberculosis infection in subjects with culture-proven M. tuberculosis disease (TBCP).

Epitope-tagging vectors for the expression and detection of recombinant proteins in mycobacteria.

New tools are required to study the growing number of uncharacterised genes derived from genome sequence projects that are specific to bacterial pathogens such as Mycobacterium tuberculosis. We have developed a series of vectors that permit the specific detection of recombinant proteins expressed in mycobacterial species. Gene expression in these vectors is driven by the strong hsp60 promoter of Mycobacterium bovis BCG and detection of expressed products is facilitated by C-terminal fusion of residues 409-419 of the human c-myc proto-oncogene.