Biomedical subjects

Peter Andersen

Publications and source records attributed to Peter Andersen.

72 records · Page 4Linked to original sources

PubMed2003

Human T-cell responses to the RD1-encoded protein TB27.4 (Rv3878) from Mycobacterium tuberculosis.

In recent years, there has been considerable focus on the discovery and characterization of proteins derived from Mycobacterium tuberculosis leading to the identification of a number of candidate antigens for use in vaccine development or for diagnostic purposes. Previous experiments have demonstrated an important immunological role for proteins encoded by the RD1 region, which is absent from all strains of bacillus Calmette-Guérin (BCG) but present in the genomes of virulent M. bovis and M. tuberculosis. Herein, we have studied human T-cell responses to the antigen encoded by the putative open reading frame (rv3878) of the RD1 region. Immunoblot analysis revealed that rv3878 was expressed and the native protein was designated TB27.4. Immunological evaluations demonstrate that TB27.4 elicits a prominent immune response in human tuberculosis patients with a dominant region in the C-terminal part of the molecule. In contrast, very limited responses were seen in M. bovis BCG-vaccinated donors. This study therefore emphasizes the diagnostic potential of proteins encoded by the RD1 region.

Amino Acid Sequence↗

PubMed2003

Gamma interferon responses induced by a panel of recombinant and purified mycobacterial antigens in healthy, non-mycobacterium bovis BCG-vaccinated Malawian young adults.

We have previously shown that young adults living in a rural area of northern Malawi showed greater gamma interferon (IFN-gamma) responses to purified protein derivatives (PPD) prepared from environmental mycobacteria than to PPD from Mycobacterium tuberculosis. In order to define the mycobacterial species to which individuals living in a rural African population have been exposed and sensitized, we tested T-cell recognition of recombinant and purified antigens from M. tuberculosis (38 kDa, MPT64, and ESAT-6), M. bovis (MPB70), M. bovis BCG (Ag85), and M. leprae (65 kDa, 35 kDa, and 18 kDa) in >600 non-M. bovis BCG-vaccinated young adults in the Karonga District of northern Malawi. IFN-gamma was measured by enzyme-linked immunosorbent assay (ELISA) in day 6 supernatants of diluted whole-blood cultures. The recombinant M. leprae 35-kDa and 18-kDa and purified native M. bovis BCG Ag85 antigens induced the highest percentages of responders, though both leprosy and bovine tuberculosis are now rare in this population. The M. tuberculosis antigens ESAT-6 and MPT64 and the M. bovis antigen MPB70 induced the lowest percentages of responders. One of the subjects subsequently developed extrapulmonary tuberculosis; this individual had a 15-mm-diameter reaction to the Mantoux test and responded to M. tuberculosis PPD, Ag85, MPT64, and ESAT-6 but not to any of the leprosy antigens. We conclude that in this rural African population, exposure to M. tuberculosis or M. bovis is much less frequent than exposure to environmental mycobacteria such as M. avium, which have antigens homologous to the M. leprae 35-kDa and 18-kDa antigens. M. tuberculosis ESAT-6 showed the strongest association with the size of the Mantoux skin test induration, suggesting that among the three M. tuberculosis antigens tested it provided the best indication of exposure to, or infection with, M. tuberculosis.

Adolescent↗

PubMed2003

PPE protein (Rv3873) from DNA segment RD1 of Mycobacterium tuberculosis: strong recognition of both specific T-cell epitopes and epitopes conserved within the PPE family.

Proteins encoded by DNA segment RD1 of Mycobacterium tuberculosis have recently been demonstrated to play important roles in bacterial virulence, vaccine development, and diagnostic reagent design. Previously, we characterized two immunodominant T-cell antigens, the early secreted antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP10), which are encoded by the esx-lhp operon in this region. In the present study we characterized a third putative open reading frame in this region, rv3873, which encodes a PPE protein. We found that the rv3873 gene is expressed in M. tuberculosis H37Rv and that the native protein, Rv3873, is predominantly associated with the mycobacterial cell or wall. When tested as a His-tagged recombinant protein, Rv3873 stimulated high levels of gamma interferon secretion in peripheral blood mononuclear cells isolated from tuberculosis (TB) patients, as well as from healthy tuberculin purified protein derivative-positive donors. In contrast to other RD1-encoded antigens, Rv3873 was also found to be recognized by a significant proportion of Mycobacterium bovis BCG-vaccinated donors. Epitope mapping performed with overlapping peptides revealed a broad pattern of T-cell recognition comprising both TB-specific epitopes and epitopes also recognized by BCG-vaccinated donors. The immunodominant epitope (residues 118 to 135) for both TB patients and BCG-vaccinated individuals was found to be highly conserved among a large number of PPE family members.

Adult↗

PubMed2003

Genomic approach to identification of Mycobacterium bovis diagnostic antigens in cattle.

Differential delayed-type hypersensitivity skin testing with tuberculin purified protein derivatives from Mycobacterium bovis and M. avium is the standard for diagnosing bovine tuberculosis. However, improved tests based on defined, specific antigens are urgently needed. In the present study, a combination of bioinformatics, molecular biology, and bovine models of infection were used to screen mycobacterial proteins for their potential as diagnostic reagents which could be used in a whole-blood assay for diagnosis of tuberculosis. Initial screening of 28 proteins selected in silico and expressed as recombinants in Escherichia coli indicated that CFP-10, ESAT-6, TB27.4, TB16.2, TB15.8, and TB10.4 induced strong gamma interferon responses in experimentally infected cattle. A more thorough investigation over time in two groups of animals infected with a high (10(6) CFU) and a low (10(4) CFU) dose of M. bovis revealed that, for both groups, the strength of the in vitro response to individual antigens varied greatly over time. However, combining the results for ESAT-6, CFP-10, and TB27.4, possibly supplemented with TB10.4, gave sensitivities at different infection stages close to those obtained with M. bovis purified protein derivative. Importantly, while responsiveness to ESAT-6 and CFP-10 correlated strongly for individual samples, the same was not the case for ESAT-6 and TB27.4 responsiveness. The results suggest that combinations of specific antigens such as these have great potential in development of optimized diagnostic systems for bovine tuberculosis.

Animals↗

PubMed2003

Selecting the components for a safe and efficient tuberculosis subunit vaccine--recent progress and post-genomic insights.

Prophylactic vaccination against tuberculosis with BCG gained much of the credit for the decline of TB in Europe. However, with TB resurgent in many parts of the world, better vaccines are urgently needed. To improve on BCG, a rapid, rational approach to vaccine discovery is needed. Fortunately, advances in the fields of molecular biology and computer science have spawned new disciplines: Genomics, Proteomics and Transcriptomics are transforming the ways in which candidate vaccine antigens are discovered. In this review, we discuss how these new approaches have accelerated the pace of antigen discovery and vaccine development, and highlight some of the most promising new candidate vaccines and vaccine targets.

Animals↗

PubMed2002

An enteric helminth infection protects against an allergic response to dietary antigen.

Although helminths induce a polarized Th2 response they have been shown, in clinical studies, to confer protection against allergies. To elucidate the basis for this paradox, we have examined the influence of an enteric helminth infection on a model of food allergy. Upon Ag challenge, mice fed peanut (PN) extract plus the mucosal adjuvant cholera toxin (CT) produced PN-specific IgE that correlated with systemic anaphylactic symptoms and elevated plasma histamine. PN-specific IgE was not induced in helminth-infected mice fed PN without CT. Moreover, when PN plus CT was fed to helminth-infected mice, both PN-specific IgE and anaphylactic symptoms were greatly diminished. The down-regulation of PN-specific IgE was associated with a marked reduction in the secretion of IL-13 by PN-specific T cells. When helminth-infected PN plus CT-sensitized mice were treated with neutralizing Abs to IL-10, the PN-specific IgE response and anaphylactic symptoms were similar to, or greater than, those seen in mice that receive PN and CT alone. Taken together, these results suggest that helminth-dependent protection against allergic disease involves immunoregulatory mechanisms that block production of allergen-specific IgE.

Administration, Oral↗

PubMed2002

In vivo 1H2O T2+ measurement in the human occipital lobe at 4T and 7T by Carr-Purcell MRI: detection of microscopic susceptibility contrast.

A high-resolution spin-echo imaging method is presented (called CP-LASER) which exploits the spin refocusing capability of an adiabatic Carr-Purcell (CP) pulse sequence to measure apparent 1H2O transverse relaxation (T2+) and generate contrast based on microscopic tissue susceptibility. High-resolution CP-LASER images of the human occipital lobe were acquired at four different echo times from six subjects at 4T and eight subjects at 7T to investigate the effect of magnetic field strength (B(0)) and the CP interpulse time (tau(cp)) on T2+. Susceptibility contrast was identified and T2+ was quantified for long tau(cp) (>10 ms) and short tau(cp) (7 ms at 4T and 6 ms at 7T) in gray matter, white matter, and cerebral spinal fluid. The 1H2O relaxation rate constants (1/T2+) of gray and white matter each increased approximately linearly with field strength and T2+ was inversely related to tau(cp). The average T2+ value of gray matter was 19% and 9% smaller than that of white matter at 4T and 7T, respectively. These results are consistent with higher levels of compartmentalized ferritin and increased blood volume in gray matter compared to white matter in this region of the brain.

Body Water↗

PubMed2002

Proton T2 relaxation study of water, N-acetylaspartate, and creatine in human brain using Hahn and Carr-Purcell spin echoes at 4T and 7T.

Carr-Purcell and Hahn spin-echo (SE) measurements were used to estimate the apparent transverse relaxation time constant (T2) of water and metabolites in human brain at 4T and 7T. A significant reduction in the T2 values of proton resonances (water, N-acetylaspartate, and creatine/phosphocreatine) was observed with increasing magnetic field strength and was attributed mainly to increased dynamic dephasing due to increased local susceptibility gradients. At high field, signal loss resulting from T2 decay can be substantially reduced using a Carr-Purcell-type SE sequence.

Aspartic Acid↗

PubMed2002

Zoomed functional imaging in the human brain at 7 Tesla with simultaneous high spatial and high temporal resolution.

Functional neuroimaging in the human brain using noninvasive magnetic resonance methods has the potential of providing highly resolved maps of neuronal activation. Decreasing the voxel size and obtaining simultaneously high temporal resolution is a major challenge and is mainly limited by sensitivity. Here, signal-to-noise gains at high magnetic fields (7 Tesla) and an optimized surface coil setup are combined with a novel approach for zoomed functional imaging in the visual cortex. For echoplanar imaging, the acquisition time and segmentation was shortened fourfold by using a reduced field-of-view. An adiabatic outer-volume suppression method, BISTRO, was used to obliterate signal outside the area-of-interest achieving effective suppression even for inhomogeneous B1-fields. A single-shot acquisition was performed at submillimeter resolution in the human brain, while simultaneously maintaining a high temporal resolution of 125 ms. Functional studies with and without field-of-view reduction were performed. Activation and percent change maps were compared with respect to spatial extent, t values and percentage changes of the BOLD contrast. The detection of functional activation was found to be equal within the inter-series variability for the two acquisition schemes. Thus, single-trial BOLD responses were detected for the first time robustly at a 500 x 500 microm2 in plane and 250 ms temporal resolution, significantly expanding the possibilities of event-related functional imaging in the human brain. The magnetization transfer effect induced by the outer-volume suppression pulses was investigated and found to be increased during neuronal activity.

Algorithms↗

PubMed2002

Tuberculosis vaccine development.

If the end of the 20th century saw the flowering of genomics, the beginning of the 21st century is witnessing an equally rapid blossoming of proteomics. These twin technologies have had a dramatic influence on the study of tuberculosis (TB), and nowhere more so than in the development of new TB vaccines. Only a few years ago, it was generally accepted that clinical trials of new TB vaccines would not take place for at least a decade. However, the first trials are now scheduled to start within months. Much TB vaccine research has thus shifted from developing new vaccines, to selecting the best vaccines for human use from the increasing number of effective new candidates becoming available. This selection is being eased by the improved animal models that have been developed over the past few years and by our increasing understanding of immunity to TB.

Animals↗

PubMed2002

Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family.

The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the esat-6 family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the Mycobacterium tuberculosis complex, including BCG, and M. kansasii, whereas other atypical mycobacteria had either one (M. avium, M. intracellulare, and M. marinum) or none (M. scrofulaceum, M. fortuitum, and M. szulgai) of the genes. The fine specificity of the T-cell response to the three closely related esat-6 family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.

Amino Acid Sequence↗

PubMed2002

Responses of bovine WC1(+) gammadelta T cells to protein and nonprotein antigens of Mycobacterium bovis.

WC1(+) gammadelta T cells of Mycobacterium bovis-infected cattle are highly responsive to M. bovis sonic extract (MBSE). In mycobacterial infections of other species, gammadelta T cells have been shown to respond to protein and nonprotein antigens, but the bovine WC1(+) gammadelta T-cell antigenic targets within MBSE require further definition in terms of the dominance of protein versus nonprotein components. The present study sought to characterize the WC1(+) gammadelta T-cell antigenic targets, together with the role of interleukin-2 (IL-2), in the context of M. bovis infection. This was achieved by testing crude and defined antigens to assess protein versus nonprotein recognition by WC1(+) gammadelta T cells in comparison with CD4(+) alphabeta T cells. Both cell types proliferated strongly in response to MBSE, with CD4(+) T cells being the major producers of gamma interferon (IFN-gamma). However, enzymatic digestion of the protein in MBSE removed its ability to stimulate CD4(+) T-cell responses, whereas some WC1(+) gammadelta T-cell proliferation remained. The most antigenic protein inducing proliferation and IFN-gamma secretion in WC1(+) gammadelta T-cell cultures was found to be ESAT-6, which is a potential novel diagnostic reagent and vaccine candidate. In addition, WC1(+) gammadelta T-cell proliferation was observed in response to stimulation with prenyl pyrophosphate antigens (isopentenyl pyrophosphate and monomethyl phosphate). High levels of cellular activation (CD25 expression) resulted from MBSE stimulation of WC1(+) gammadelta T cells from infected animals. A similar degree of activation was induced by IL-2 alone, but for WC1(+) gammadelta T-cell division IL-2 was found to act only as a costimulatory signal, enhancing antigen-driven responses. Overall, the data indicate that protein antigens are important stimulators of WC1(+) gammadelta T-cell proliferation and IFN-gamma secretion in M. bovis infection, with nonprotein antigens inducing significant proliferation. These findings have important implications for diagnostic and vaccine development.

Animals↗

PubMed2002

Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis.

The efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of the Mycobacterium avium complex were found to block BCG activity completely.

Acyltransferases↗

PubMed2002

Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis.

Immunity against Mycobacterium tuberculosis depends largely on activation of cell-mediated responses, and gamma interferon has been shown to play a crucial role in this process in both humans and animal models. Since the lung is normally the organ in which infection is initiated and is the major site of pathology, immune responses in the lung play a significant role in restricting initial infection with M. tuberculosis. The aim of the present study was to stimulate efficient immunity in the lung by targeting the gut mucosa. Detoxified monophosphoryl lipid A (MPL) has been shown to be a relatively nontoxic adjuvant which efficiently promotes the induction of type 1 responses when it is given by the traditional subcutaneous route. We have therefore compared subcutaneous immunization of mice to oral immunization by using a model subunit vaccine carrying two immunodominant proteins from M. tuberculosis, in combination with MPL-based adjuvants. While less effective when used to prime a response, a heterologous priming and boosting vaccination strategy employing oral boosting induced significant systemic type 1 responses which equaled and surpassed those attained by subcutaneous immunization protocols. Moreover, the increased immune responses observed correlated with the induction of substantial protection against subsequent aerosol infection with virulent M. tuberculosis at levels comparable to, or better than, those obtained by multiple subcutaneous vaccinations. These results demonstrate that booster vaccinations via mucosal surfaces, by combining efficient subunit vaccines with the potent adjuvant MPL, may be an effective method of addressing some of the shortcomings of current vaccination strategies.

Acyltransferases↗

PubMed2002

Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins.

The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

Alanine Dehydrogenase↗

PubMed2002

Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients.

Diagnosis of latent Mycobacterium tuberculosis infection is considered essential for tuberculosis control but is hampered by the lack of specific reagents. We report that strong recognition of tuberculosis complex-specific antigen ESAT-6 by healthy household contacts of tuberculosis patients correlates with the subsequent development of active tuberculosis during a 2-year follow-up period.

Antigens, Bacterial↗

PubMed1998

A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10).

The early secreted antigenic target 6 kDa protein (ESAT-6) is a potent T-cell protein antigen synthesized by Mycobacterium tuberculosis. Its corresponding gene (esat-6) is located in RD1, a 10 kb DNA region deleted in the attenuated tuberculosis vaccine strain Mycobacterium bovis BCG. The promoter region of M. tuberculosis esat-6 was cloned and characterized. A new gene, designated lhp and cotranscribed with esat-6, was identified. Moreover, computer searches in the M. tuberculosis genome identified 13 genes related to the lhp/esat-6 operon, defining a novel gene family. The transcription initiation sites of the lhp/esat-6 operon were mapped using M. tuberculosis RNA. The corresponding promoter signals were not recognized in Mycobacterium smegmatis, in which transcription of lhp/esat-6 is initiated at different locations. The M. tuberculosis lhp gene product was identified as CFP-10, a low-molecular-mass protein found in the short-term culture filtrate. These results show that the genes encoding CFP-10 and ESAT-6 are transcribed together in M. tuberculosis and that both code for small exported proteins.

Amino Acid Sequence↗

PubMedDate not supplied

TB vaccine strategies--what is needed to solve a complex problem?

An estimated 2 billion people are latently infected with Mycobacterium tuberculosis, the majority of which are already BCG vaccinated and repeatedly sensitized to mycobacterial strains from the environment. To be successful in the high endemic regions, any future TB vaccine strategy will have to be tailored in accordance with the resulting complexity of the TB infection and anti-mycobacterial immune response. In this review we will discuss some of the most advanced attempts to address this challenge.

Humans↗