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Michael J Brennan

Publications and source records attributed to Michael J Brennan.

At least 19 recordsLinked to original sources
PubMed2006

PE_PGRS proteins are differentially expressed by Mycobacterium tuberculosis in host tissues.

Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues.

Adaptation, Physiological↗
PubMed2006

A PE protein expressed by Mycobacterium avium is an effective T-cell immunogen.

Infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes.

Amino Acid Sequence↗
PubMed2006

Variable expression patterns of Mycobacterium tuberculosis PE_PGRS genes: evidence that PE_PGRS16 and PE_PGRS26 are inversely regulated in vivo.

Evaluation of expression of 16 PE_PGRS genes present in Mycobacterium tuberculosis under various growth conditions demonstrated constitutive expression of 7 genes, variable expression of 7 genes, and no expression of 2 genes. An inverse expression profile for genes PE_PGRS16 and PE_PGRS26 was observed to occur in macrophages and in mice infected with M. tuberculosis. Variable expression of PE_PGRS proteins could have implications for their role in the immunopathogenesis of tuberculosis.

Antigens, Bacterial↗
PubMed2005

Expression of the PE_PGRS 33 protein in Mycobacterium smegmatis triggers necrosis in macrophages and enhanced mycobacterial survival.

Research on mycobacteria-specific PE_PGRS genes indicates that they code for cell surface proteins that may influence virulence and the infection of host cells by mycobacteria. In the studies presented here, we have expressed the PE_PGRS 33 gene in a non-pathogenic fast-growing Mycobacterium smegmatis strain and demonstrated that it survives better in macrophage cultures, in vitro as well as in mice after intraperitoneal administration, than the parental strain containing the vector only or a strain expressing only the PE domain of PE_PGRS 33. In macrophages, enhanced colonization by the M. smegmatis expressing PE_PGRS 33 was associated with macrophage aggregation and clearance of macrophage monolayers, visible cell necrosis and significantly greater levels of TNF (TNF-alpha) in the cultures compared with controls. The presence of macrophage cell necrosis was confirmed by measurement of significantly greater levels of lactate dehydrogenase and nucleosomes in the supernatants of the macrophage cultures infected with M. smegmatis expressing PE_PGRS 33. Antibodies directed against TNF partially reduced cytolysis, suggesting that this cytokine is critical but not sufficient for the observed macrophage necrosis and enhanced mycobacterial survival. These results extend earlier observations, which suggested that PE_PGRS proteins may have a role in the pathogenesis of mycobacterial disease and that there may be a specific role for these proteins in influencing host cell responses to infection.

Animals↗
PubMed2005

New live mycobacterial vaccines: the Geneva consensus on essential steps towards clinical development.

As the disease caused by Mycobacterium tuberculosis continues to be a burden, which the world continues to suffer, there is a concerted effort to find new vaccines to combat this problem. Of the various vaccines strategies, one viable option is the development of live mycobacterial vaccines. A meeting with researchers, regulatory bodies, vaccines developers and manufactures was held to consider the challenges and progress, which has been achieved with live mycobacterial vaccines (either modified BCG or attenuated M. tuberculosis). Discussion led to the production of a consensus document of the proposed entry criteria for Phase I clinical trials of candidate live mycobacterial vaccines. The vaccine must be characterised thoroughly to prove identity and consistency, as clinical trial lots are prepared. In pre-clinical studies, greater protective efficacy as well as improved safety potential relative to BCG should be considered when assessing potential vaccine candidates. A standard way to measure the protective efficacy to facilitate comparison between vaccine candidates was suggested. Additional safety criteria and verification of attenuation must be considered for attenuated M. tuberculosis. Two non-reverting independent mutations are recommended for such vaccines. When entering Phase I trials, enrollment should be based upon an acceptable characterisation of the study population regarding mycobacterium status and exclude HIV(+) individuals. BCG could be used as a comparator for blinding during the trials and to properly assess vaccine-specific adverse reactions, while assays are being developed to assess immunogenicity of vaccines. The proposed criteria suggested in this consensus document may facilitate the movement of the most promising vaccine candidates to the clinic and towards control of tuberculosis.

BCG Vaccine↗
PubMed2005

Finite element prediction of wave motion in structural waveguides.

A method is presented by which the wavenumbers for a one-dimensional waveguide can be predicted from a finite element (FE) model. The method involves postprocessing a conventional, but low order, FE model, the mass and stiffness matrices of which are typically found using a conventional FE package. This is in contrast to the most popular previous waveguide/FE approach, sometimes termed the spectral finite element approach, which requires new spectral element matrices to be developed. In the approach described here, a section of the waveguide is modeled using conventional FE software and the dynamic stiffness matrix formed. A periodicity condition is applied, the wavenumbers following from the eigensolution of the resulting transfer matrix. The method is described, estimation of wavenumbers, energy, and group velocity discussed, and numerical examples presented. These concern wave propagation in a beam and a simply supported plate strip, for which analytical solutions exist, and the more complex case of a viscoelastic laminate, which involves postprocessing an ANSYS FE model. The method is seen to yield accurate results for the wavenumbers and group velocities of both propagating and evanescent waves.

Acoustics↗
PubMed2004

Expression and purification of recombinant methylated HBHA in Mycobacterium smegmatis.

The Heparin-Binding Haemagglutinin (HBHA) is a mycobacterial adhesin involved in the dissemination of Mycobacterium tuberculosis from the site of primary infection and a potential candidate for the development of a new vaccine against tuberculosis. Methylation of HBHA is a novel post-translational event that imparts important immunological properties to the protein. Since recombinant HBHA expressed in Escherichia coli is not methylated, we investigated the possibility of producing recombinant methylated HBHA in fast growing mycobacteria for use in immunological and biochemical studies. The complete coding sequence of HBHA was cloned in the plasmid pMV206, under the control of a strong promoter (hsp60) or its own promoter. The constructs generated were electroporated into Mycobacterium smegmatis and the recombinant strains obtained were analyzed for the presence of the HBHA protein using the anti-HBHA monoclonal antibodies D2 and E4. Our results indicate that expression of high amounts of intact protein can be toxic for the mycobacteria, that methylated HBHA can be obtained in M. smegmatis only when using a promoter sequence weaker than hsp60 and that the expression of the complete structural gene is required in order to obtain methylated HBHA. We constructed a recombinant M. smegmatis strain (pMV3-38) that expresses a histidine-tagged methylated HBHA that can be easily purified. The use of fast-growing strains of M. smegmatis to obtain significant amounts of purified HBHA protein within a short timeframe, should be an effective strategy for the evaluation of a new HBHA-based vaccine candidate for tuberculosis.

Hemagglutinins↗
PubMed2004

Methylation-dependent T cell immunity to Mycobacterium tuberculosis heparin-binding hemagglutinin.

Although post-translational modifications of protein antigens may be important componenets of some B cell epitopes, the determinants of T cell immunity are generally nonmodified peptides. Here we show that methylation of the Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) by the bacterium is essential for effective T cell immunity to this antigen in infected healthy humans and in mice. Methylated HBHA provides high levels of protection against M. tuberculosis challenge in mice, whereas nonmethylated HBHA does not. Protective immunity induced by methylated HBHA is comparable to that afforded by vaccination with bacille Calmette et Guérin, the only available anti-tuberculosis vaccine. Thus, post-translational modifications of proteins may be crucial for their ability to induce protective T cell-mediated immunity against infectious diseases such as tuberculosis.

Animals↗
PubMed2004

Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure.

Identification of the novel PE multigene family was an unexpected finding of the genomic sequencing of Mycobacterium tuberculosis. Presently, the biological role of the PE and PE_PGRS proteins encoded by this unique family of mycobacterial genes remains unknown. In this report, a representative PE_PGRS gene (Rv1818c/PE_PGRS33) was selected to investigate the role of these proteins. Cell fractionation studies and fluorescence analysis of recombinant strains of Mycobacterium smegmatis and M. tuberculosis expressing green fluorescent protein (GFP)-tagged proteins indicated that the Rv1818c gene product localized in the mycobacterial cell wall, mostly at the bacterial cell poles, where it is exposed to the extracellular milieu. Further analysis of this PE_PGRS protein showed that the PE domain is necessary for subcellular localization. In addition, the PGRS domain, but not PE, affects bacterial shape and colony morphology when Rv1818c is overexpressed in M. smegmatis and M. tuberculosis. Taken together, the results indicate that PE_PGRS and PE proteins can be associated with the mycobacterial cell wall and influence cellular structure as well as the formation of mycobacterial colonies. Regulated expression of PE genes could have implications for the survival and pathogenesis of mycobacteria within the human host and in other environmental niches.

Antigens, Bacterial↗
PubMed2004

The mycobacterial heparin-binding hemagglutinin is a protective antigen in the mouse aerosol challenge model of tuberculosis.

The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a approximately 0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

Aerosols↗
PubMed2004

Tuberculosis vaccine development: research, regulatory and clinical strategies.

In the past decade, while the global tuberculosis (TB) epidemic has continued to devastate mankind, considerable progress has nevertheless been made in the development of new and improved vaccines for this ancient disease. Recombinant bacillus Calmette-Guerin strains, DNA-based vaccines, live attenuated Mycobacterium tuberculosis vaccines and subunit vaccines formulated with novel adjuvants have shown promise in preclinical animal challenge models. Three of these vaccines are being evaluated at present in human clinical studies, and several other vaccine preparations are being targeted for clinical trials in the near future. Although the preclinical characterisation and testing of new TB vaccines has clearly led to exciting new findings, complex regulatory and clinical trial design issues remain as a challenge to TB vaccine development. This report reviews some of the exciting advances in TB research that have led to the development of new TB vaccines, and addresses the unique regulatory and clinical issues associated with the testing of novel anti-TB preparations in human populations.

Adult↗
PubMed2002

Development of combinatorial chemistry methods for coatings: high-throughput optimization of curing parameters of coatings libraries.

An automated analytical system has been implemented for the high-throughput optimization of processing conditions such as curing parameters in fabrication of UV-cured automotive organic protective coatings. Selection of optimum process conditions of combinatorial arrays of coatings is essential to correlate the high-throughput screening and conventional processes and to achieve the desired physical properties of coatings. For monitoring of curing conditions of each coating in the array, a viscosity-sensitive fluorophore 4,4'-bis(2-benzoxazolyl)stilbene was incorporated into coating formulations. This fluorescence tagging approach permitted us to combine a gradient temperature heater and a UV curing system with the full capabilities of our high-throughput screening system, including generation of spectroscopic data and its analysis. This investigation demonstrated the possibility of rapid decoupling of temperature and radiation effects in curing of UV-curable coating formulations by using multiple coatings and process conditions at once. While the system described here was implemented for high-throughput optimization of temperature conditions of radiation curing of arrays of organic protective coatings for automotive applications, this system can be further applied for a variety of other applications where optimization of process parameters can be studied in situ or off-line using optical spectroscopic tools.

Journal Article↗
PubMed2002

Development of combinatorial chemistry methods for coatings: high-throughput screening of abrasion resistance of coatings libraries.

Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8 x 6 arrays of coating elements that are discretely deposited on a single plastic substrate. Each coating element of the library is 10 mm in diameter and 2-5 microm thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the coating materials undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at a single or multiple abrasion conditions followed by the high-throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0-30% haze. System precision of 0.1-2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. Although the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied for a variety of other applications for which materials ranking can be achieved using optical spectroscopic tools.

Journal Article↗
PubMed2002

The PE multigene family: a 'molecular mantra' for mycobacteria.

The PE multigene family of Mycobacterium tuberculosis is remarkable in that it is composed of approximately 100 highly homologous genes that are found only in mycobacteria. Early evidence suggests that proteins encoded by certain members of this gene family could be present in the mycobacterial cell wall, impact antigen-presentation pathways and the ensuing host immune responses, and also provide a mechanism for generating antigenic diversity in mycobacteria.

Amino Acid Sequence↗
PubMed2002

Enhanced bacterial virulence through exploitation of host glycosaminoglycans.

Present in the extracellular matrix and membranes of virtually all animal cells, proteoglycans (PGs) are among the first host macromolecules encountered by infectious agents. Because of their wide distribution and direct accessibility, it is not surprising that pathogenic bacteria have evolved mechanisms to exploit PGs for their own purposes, including mediating attachment to target cells. This is achieved through the expression of adhesins that recognize glycosaminoglycans (GAGs) linked to the core protein of PGs. Some pathogens, such as Bordetella pertussis and Chlamydia trachomatis, may express more than one GAG-binding adhesin. Bacterial interactions with PGs may also facilitate cell invasion or systemic dissemination, as observed for Neisseria gonorrhoeae and Mycobacterium tuberculosis respectively. More-over, pathogenic bacteria can use PGs to enhance their virulence via a shedding of PGs that leads to there lease of effectors that weaken the host defences. The exploitation of PGs by pathogenic bacteria is thus a multifaceted mechanistic process directly related to the potential virulence of a number of microorganisms.

Animals↗
PubMed2002

Active vibroacoustic control with multiple local feedback loops.

When multiple actuators and sensors are used to control the vibration of a panel, or its sound radiation, they are usually positioned so that they couple into specific modes and are all connected together with a centralized control system. This paper investigates the physical effects of having a regular array of actuator and sensor pairs that are connected only by local feedback loops. An array of 4 x 4 force actuators and velocity sensors is first simulated, for which such a decentralized controller can be shown to be unconditionally stable. Significant reductions in both the kinetic energy of the panel and in its radiated sound power can be obtained for an optimal value of feedback gain, although higher values of feedback gain can induce extra resonances in the system and degrade the performance. A more practical transducer pair, consisting of a piezoelectric actuator and velocity sensor, is also investigated and the simulations suggest that a decentralized controller with this arrangement is also stable over a wide range of feedback gains. The resulting reductions in kinetic energy and sound power are not as great as with the force actuators, due to the extra resonances being more prominent and at lower frequencies, but are still worthwhile. This suggests that an array of independent modular systems, each of which included an actuator, a sensor, and a local feedback control loop, could be a simple and robust method of controlling broadband sound transmission when integrated into a panel.

Acoustics↗
PubMedDate not supplied

The tuberculosis vaccine challenge.

Although antibiotic treatments for tuberculosis are available, because of re-infection, drug resistance, AIDS, and economic reasons, it is unlikely that we will be able to control the global spread of tuberculosis without an effective vaccine. A number of new candidate vaccines for tuberculosis are under development and some are being evaluated for safety in normal human subjects in clinical trials. Additional vaccine candidates have been shown to be safe and effective when administered prior to infection in animal models. However, in areas of the world where tuberculosis is endemic, up to two thirds of the population are already infected with Mycobacterium tuberculosis, and it is unlikely that a new pre-exposure vaccine would have a substantial impact on disease for decades. In contrast, a vaccine that could be delivered to individuals already infected could reduce the disease burden. At this time, it is unclear whether the new TB vaccines can be safely and effectively used in populations already infected with M. tuberculosis, immunized with BCG vaccine or infected with HIV. This presents a major challenge to pre-clinical testing and clinical evaluation as well as eventual uptake of the new TB vaccines into areas of the world that are most at risk for tuberculosis.

Animals↗