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D B Young

Publications and source records attributed to D B Young.

At least 37 records · Page 2Linked to original sources
PubMed2001

Enhanced antimycobacterial response to recombinant Mycobacterium bovis BCG expressing latency-associated peptide.

With a view to exploring the role of transforming growth factor beta (TGF-beta) during mycobacterial infection, recombinant clones of bacillus Calmette-Guérin (BCG) were engineered to express the natural antagonist of TGF-beta, latency-activated peptide (LAP). Induction of TGF-beta activity was reduced when macrophages were infected with BCG expressing the LAP construct (LAP-BCG). There was a significant reduction in the growth of LAP-BCG in comparison to that of control BCG following intravenous infection in a mouse model. The enhanced control of mycobacterial replication was associated with an increase in the production of gamma interferon by splenocytes challenged during the acute stage of infection but with a diminished recall response assessed after 13 weeks. Organ weight and hydroxyproline content, representing tissue pathology, were also lower in mice infected with LAP-BCG. The results are consistent with the hypothesis that TGF-beta has a detrimental effect on mycobacterial immunity. While a reduction in TGF-beta activity augments the initial response to BCG vaccination, early bacterial clearance may adversely affect the induction of a long-term memory response by LAP-BCG.

Animals↗
PubMed2001

Mycobacterium tuberculosis 19-kilodalton lipoprotein inhibits Mycobacterium smegmatis-induced cytokine production by human macrophages in vitro.

Vaccination of mice with Mycobacterium vaccae or M. smegmatis induces some protection against M. tuberculosis challenge. The 19-kDa lipoprotein of M. tuberculosis, expressed in M. vaccae or M. smegmatis (M. smeg19kDa), abrogates this protective immunity. To investigate the mechanism of this suppression of immunity, human monocyte-derived macrophages (MDM) were infected with M. smeg19kDa. Infection resulted in reduced production of tumor necrosis factor alpha (TNF-alpha) (P < 0.01), interleukin-12 (IL-12) (P < 0.05), IL-6 (P < 0.05), and IL-10 (P < 0.05), compared to infection with M. smegmatis vector (M. smegV). Infection with M. smeg19kDa and with M. smegV had no differential effect on expression of costimulatory molecules on MDM, nor did it affect the proliferation of presensitized T cells cocultured with infected MDM. When MDM were infected with M. smegmatis expressing mutated forms of the 19-kDa lipoprotein, including non-O-glycosylated (M. smeg19NOG), nonsecreted (M. smeg19NS), and nonacylated (M. smeg19NA) variants, the reduced production of TNF-alpha or IL-12 was not observed. When the purified 19-kDa lipoprotein was added directly to cultures of infected monocytes, there was little effect on either induction of cytokine production or its inhibition. Thus, the immunosuppressive effect is dependent on glycosylated and acylated 19-kDa lipoprotein present in the phagosome containing the mycobacterium. These results suggest that the diminished protection against challenge with M. tuberculosis seen in mice vaccinated with M. smegmatis expressing the 19-kDa lipoprotein is the result of reduced TNF-alpha and IL-12 production, possibly leading to reduced induction of T-cell activation.

Antigens, Bacterial↗
PubMed2001

Genetic diversity among Mycobacterium bovis isolates: a preliminary study of strains from animal and human sources.

Mycobacterium bovis has the broadest host range of species in the Mycobacterium tuberculosis complex and is responsible for disease in humans and diverse animal species. We report on genotypic differences at multiple loci among 13 isolates derived from a range of human and animal infections. All isolates were classified as M. bovis by phenotypic analysis but could be subdivided into five distinct genotypes based on polymorphisms at the pncA and oxyR loci, the status of the RD5 deletion region, and the spoligotype pattern. These findings suggest the existence of a spectrum of strains with genotypic characteristics between those of M. tuberculosis and M. bovis.

Aged↗
PubMed2000

Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.

A novel in vitro whole blood model was developed to study human antimycobacterial immunity. Recombinant reporter mycobacteria were used to enumerate the bacteria, and interactions between host immune cells and mycobacteria were studied using whole blood rather than cell fractions. The ability of healthy tuberculin-positive and tuberculin-negative individuals to restrict mycobacterial growth was compared. Growth of luminescent mycobacteria was significantly lower in blood samples of tuberculin-positive individuals than in blood samples of tuberculin-negative individuals (P=.005). Restricted mycobacterial growth was associated with significantly higher production of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma (P=.01 and.004, respectively). Inhibition of the TNF-alpha and IFN-gamma response pathways by neutralizing monoclonal antibodies increased mycobacterial growth in whole blood. This model is the first functional assay in which individual variations in cell-mediated immunity are shown to correlate with differences in ability to control mycobacterial growth. It provides a new tool for studying human mycobactericidal mechanisms and, potentially, for the evaluation of improved vaccines.

Adult↗
PubMed2000

Comparison of Mycobacterium tuberculosis genomes reveals frequent deletions in a 20 kb variable region in clinical isolates.

The Mycobacterium tuberculosis complex is associated with a remarkably low level of structural gene polymorphism. As part of a search for alternative forms of genetic variation that may act as a source of biological diversity in M. tuberculosis, we have identified a region of the genome that is highly variable amongst a panel of unrelated clinical isolates. Fifteen of 24 isolates examined contained one or more copies of the M. tuberculosis-specific IS6110 insertion element within this 20 kb variable region. In nine of the isolates, including the laboratory-passaged strain H37Rv, genomic deletions were identified, resulting in loss of between two and 13 genes. In each case, deletions were associated with the presence of a copy of the IS6110 element. Absence of flanking tri- or tetra-nucleotide repeats identified homologous recombination between adjacent IS6110 elements as the most likely mechanism of the deletion events. IS6110 insertion into hot-spots within the genome of M. tuberculosis provides a mechanism for generation of genetic diversity involving a high frequency of insertions and deletions.

Carboxylic Ester Hydrolases↗
PubMed2000

High potassium intake inhibits neointima formation in the rat carotid artery balloon injury model.

Recently, we reported that elevated extracellular potassium concentration in vitro inhibited proliferation and migration of vascular smooth muscle cells, formation of free radical compounds by macrophages, and reduced platelet sensitivity to agonists. In the present study we analyzed the effects of long-term, in vivo elevation of extracellular potassium concentration resulting from changes in dietary potassium intake on the vascular response to injury. The rat carotid artery balloon injury model was employed in 70 adult Sprague Dawley rats assigned to three groups. Beginning 14 days before surgical placement of the carotid lesion and continuing until death, the animals were fed diets containing either low (0.1% potassium, n = 25), normal (1.5% potassium, n = 19), or high potassium (4.0% potassium, n = 26). Fourteen days postsurgery the animals were killed and the arteries were analyzed to determine quantitatively the ratio of neointimal to medial area. Dietary potassium had a significant effect on arterial plasma potassium concentration (one-way analysis of variance, P < .01). Group mean and standard errors were 4.26+/-0.12 mmol/L for the low-potassium group, 5.22+/-0.19 mmol/L for normal, and 5.80+/-0.23 mmol/L for the high-intake group. Increases in dietary potassium attenuated neointima formation significantly (P < .05, one-way analysis of variance), with the mean ratio of neointimal area to medial area being 0.447+/-0.106 for the low-intake animals, 0.384+/-.116 for normal, and 0.240+/-.046 for the high-intake group. These results are consistent with a hypothesis that a high level of potassium intake is effective in inhibiting neointima formation in vivo.

Animals↗
PubMed2000

Increased potassium concentration inhibits stimulation of vascular smooth muscle proliferation by PDGF-BB and bFGF.

The effects of changes in extracellular potassium concentration on the rate of vascular smooth muscle cell proliferation stimulated by cytokines and serum were analyzed in vitro. To analyze the DNA synthesis response, cells from swine coronary artery were grown in DMEM medium containing 3, 4, 5, or 6 mmol/L potassium together with 20 ng/mL platelet-derived growth factor BB (PDGF-BB), 25 ng/mL basic fibroblast growth factor (bFGF), or 5% fetal bovine serum (FBS), with [methyl 3H] thymidine added, for 24 h. Proliferation responses were analyzed in cells grown in medium with potassium concentrations of 3, 4, 5, or 6 mmol/L, together with either 20 ng/mL PDGF-BB, 25 ng/mL bFGF, or 5% FBS, for 7 days, then harvested and counted. Highly significant inverse relationships were observed between potassium concentration and 3H-thymidine incorporation stimulated by each of the three agonists (P < .01 for each, ANOVA), and between potassium concentration and proliferation (all P < .01, ANOVA). Elevation of potassium concentration within the physiologic range inhibits vascular smooth muscle cell DNA synthesis and proliferation.

Animals↗
PubMed2000

Tumour necrosis factor-alpha production in human alveolar macrophages: modulation by inhaled corticosteroid.

Using an ex vivo alveolar macrophage model, the hypothesis that inhaled preparations of corticosteroids might have important anti-inflammatory effects on cells of the peripheral airway was tested. The tumour necrosis factor (TNF)-alpha-inducing potential of three glycolipid preparations from nonpathogenic (arabinofuranasyl lipoarabinomannan (LAM (Ara-LAM)) and virulent (mannase LAM (ManLAM)) mycobacteria and Gram-negative bacteria (lipopolysaccharide (LPS)), in primary alveolar macrophage preparations was investigated. A novel inhaled chlorofluorocarbon (CFC)-free preparation of beclomethasone dipropionate (hydrofluoroalkane 134a (HFA)-BDP) with increased peripheral lung deposition was investigated for its ability to modulate glycolipidinduced TNF-alpha production by human alveolar macrophages, in comparison with a CFC-containing preparation and placebo. Compared to the basal TNF-alpha bioactivity of 0.72 ng x mL-1 (geometric mean), the TNF-alpha bioactivity in the macrophage preparation increased following incubation with LPS (138 ng x mL-1, p<0.001), AraLAM (12.6 ng-mL-1, p<0.001) and ManLAM (1.42 ng x mL-1, p=0.02). HFA-BDP, administered in vivo, significantly reduced LPS- and ManLAM-induced TNF-alpha production by alveolar macrophages cultured ex vivo. No change in glycolipid-induced TNF-alpha production was observed following in vivo administration of CFC-BDP or HFA-placebo. This is the first demonstration of an immunomodulatory effect on alveolar cells of corticosteroid delivered via metered dose inhaler. The present findings suggest that alveolar deposition of beclomethasone dipropionate is capable of modulating the inflammatory potential of the alveolar macrophage population.

Administration, Inhalation↗
PubMed2000

Bacille Calmette-Guérin (BCG)-associated inflammation and fibrosis: modulation by recombinant BCG expressing interferon-gamma (IFN-gamma).

Immunization with existing BCG vaccines has failed to confer consistent protection against tuberculosis. One of the ways to improve the efficacy of BCG is by enhancing its ability to induce a type-1 T cell response. However, this approach carries the risk that enhanced immunoreactivity may exacerbate tissue pathology associated with vaccination. The aim of the present study was to determine whether use of a recombinant BCG expressing IFN-gamma (BCG-IFN) would result in an alteration in the pattern of inflammation and local tissue fibrosis. A murine intravenous BCG infection model was used in which there was a time- and dose-dependent increase in the weight and number of granulomas in the liver. Infection was associated with increased inflammatory activity in the liver, as shown by the increase in expression of inducible nitric oxide synthase (iNOS) assessed by immunochemistry and by measurement of specific mRNA, and in fibrosis measured by hydroxyproline content of the liver and percentage of granuloma cells staining positively for type 1 procollagen. Infection with BCG-IFN resulted in a reduction in organ weight and bacterial load on day 21 compared with infection with control BCG transformed with vector alone (BCG-plasmid). By day 21, there was also a reduction in iNOS mRNA and iNOS+ cells in granulomas in mice infected with BCG-IFN compared with infection with BCG-plasmid, and a similar reduction in both total number of granulomas and liver hydroxyproline content. These results demonstrate that the granulomas in the areas of mycobacterial infection are active sites of both inflammation and fibrosis, and that the local expression of IFN-gamma by the recombinant BCG results in more efficient bacterial clearance which is accompanied by a reduction in tissue pathology.

Animals↗
PubMed2000

The 19-kD antigen and protective immunity in a murine model of tuberculosis.

The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.

Animals↗
PubMed2000

Deletion of the 19kDa antigen does not alter the protective efficacy of BCG.

Expression of the Mycobacterium tuberculosis 19kDa lipoprotein in saprophytic mycobacteria has been found to reduce their ability to prime a protective response to subsequent virulent challenge in the mouse model. The present study was designed to test whether 19kDa expression has an analogous detrimental effect on the efficacy of BCG vaccination. In contrast to the results in saprophytes, neither overexpression of the 19kDa antigen, nor deletion of the endogenous 19kDa gene altered the ability of BCG to protect against M. tuberculosis challenge in a mouse model.

Animals↗
PubMed2000

Current tuberculosis vaccine development.

Information derived from the complete genome sequence of Mycobacterium tuberculosis makes it possible to develop a range of new vaccine candidates. Strategies currently under investigation include construction of whole cell live attenuated mycobacterial vaccines, as well as the use of individual antigens delivered by a variety of subunit vaccination procedures. Fundamental questions associated with the rational design, preclinical testing, and future application of new tuberculosis vaccines are reviewed.

Animals↗
PubMed2000

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

Amino Acid Sequence↗
PubMed2000

Identification of a Mycobacterium tuberculosis gene that enhances mycobacterial survival in macrophages.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis. To identify M. tuberculosis genes required for intracellular survival within macrophages, an M. tuberculosis H37Rv plasmid library was constructed by using the shuttle vector pOLYG. This plasmid library was electroporated into Mycobacterium smegmatis 1-2c, and the transformants were used to infect the human macrophage-like cell line U-937. Because M. smegmatis does not readily survive within macrophages, any increased intracellular survival is likely due to cloned M. tuberculosis H37Rv DNA. After six sequential passages of M. smegmatis transformants through U-937 cells, one clone (p69) was enriched more than 70% as determined by both restriction enzyme and PCR analyses. p69 demonstrated significantly enhanced survival compared to that of the vector control, ranging from 2.4- to 5.3-fold at both 24 and 48 h after infection. DNA sequence analysis revealed three open reading frames (ORFs) in the insert of p69. ORF2 (1.2 kb) was the only one which contained a putative promoter region and a ribosome-binding site. Deletion analysis of the p69 insert DNA showed that disruption of ORF2 resulted in complete loss of the enhanced intracellular survival phenotype. This gene was named the enhanced intracellular survival (eis) gene. By using an internal region of eis as a probe for Southern analysis, eis was found in the genomic DNA of various M. tuberculosis strains and of Mycobacterium bovis BCG but not in that of M. smegmatis or 10 other nonpathogenic mycobacterial species. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that all M. smegmatis eis-containing constructs expressed a unique protein of 42 kDa, the predicted size of Eis. The expression of this 42-kDa protein directly correlated to the enhanced survival of M. smegmatis p69 in U-937 cells. These results suggest a possible role for eis and its protein product in the intracellular survival of M. tuberculosis.

Amino Acid Sequence↗
PubMed2000

Human herpesvirus 8 LANA interacts with proteins of the mSin3 corepressor complex and negatively regulates Epstein-Barr virus gene expression in dually infected PEL cells.

The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL). To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein. SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter. Glutathione S-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR. The corepressors bound to the amino-terminal 340-amino-acid domain of LANA. In transient expression assays, this same domain of LANA mediated repression when targeted to a 5xGal4tk-CAT reporter as a GAL4-LANA fusion. PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV). We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels. In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters. Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid. In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter. The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.

Antigens, Viral↗
PubMed2000

Inhibition of vascular smooth muscle cell migration by elevation of extracellular potassium concentration.

The effect of potassium on the migration of vascular smooth muscle cells was analyzed in media made with extracellular potassium concentrations of 3, 4, 5, and 6 mmol/L. The migration of cultured porcine coronary artery cells was stimulated with platelet-derived growth factor (PDGF)-BB. In the first study, cells were exposed to PDGF-BB at concentrations of 0, 10, or 20 ng/mL for 5 hours with the use of a Boyden chamber. Cells were quiescent overnight in 0.5% fetal bovine serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 4 mmol/L. With increasing potassium concentration, migration was significantly inhibited (P<0. 02, 2-way ANOVA). In the cells exposed to 10 ng/mL PDGF-BB, migration ranged from 500+/-86% to 294+/-44% (value in wells with 0 ng/mL PDGF-BB and 4 mmol/L potassium concentration=100%) in medium containing 3 to 6 mmol/L extracellular potassium concentration (P<0. 03). Long-term potassium exposure was investigated in cells grown in 5% serum in Dulbecco's modified Eagle's medium with an extracellular potassium concentration of 3, 4, 5, or 6 mmol/L for 3 to 4 weeks. Migration was assessed with 0 or 20 ng/mL PDGF-BB. Migration was significantly inhibited by the elevation of extracellular potassium concentration (P<0.01, 2-way ANOVA). With 20 ng/mL PDGF-BB, the migration rates ranged from 152+/-11% in medium with 3 mmol/L potassium to 69+/-5% in 6 mmol/L potassium (P<0.01). Increases in extracellular potassium concentration within the physiological range significantly and directly inhibit vascular smooth muscle cell migration.

Animals↗
PubMed2000

Diagnostic assays for leprosy based on T-cell epitopes.

To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.

Epitopes, T-Lymphocyte↗