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M J Colston

Publications and source records attributed to M J Colston.

At least 91 records · Page 5Linked to original sources
PubMed1991

The antibody repertoire to proteins of Mycobacterium leprae. Genetic influences at the antigen and epitope level.

The effects of both H-2 and non-H-2 genes on the antibody response to the proteins of Mycobacterium leprae were investigated. Using a B10 series of congenic mice we found that the repertoire of antibody responses was under H-2 gene control, and that non-H2 genes were also involved. By Western blotting, differences in the number and m.w. of proteins recognised by mice of different genetic background were apparent. Such differences were also reflected in the total antibody response to a soluble extract of M. leprae (M. leprae sonicate), as measured by ELISA. Concentrating on one particular Ag, the 65-kDa heat shock protein, we found that all strains of mice developed antibodies following immunization with the purified recombinant protein, although there was a continuous distribution in the titer of antibodies obtained, with differences between individual strains indicating both H-2 and non-H-2 effects. Using a library of overlapping peptides based on the amino acid sequence of this protein, we have mapped the B cell epitopes in the different strains of mice. H-2 genes had no effect on the structure and number of epitopes recognized, although this was influenced by non-H-2 genes. There was a high level of concordance between actual epitopes recognized and those predicted by calculations of antigenic index, and B cell epitopes were located in similar positions to previously determined T cell epitopes.

Amino Acid Sequence↗
PubMed1991

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.

Antigens, Bacterial↗
PubMed1991

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.

Amino Acid Sequence↗
PubMed1991

The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction.

Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue.

Animals↗
PubMed1991

Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product.

A fragment of Mycobacterium tuberculosis DNA containing recA-like sequences was identified by hybridization with the Escherichia coli recA gene and cloned. Although no expression was detected from its own promoter in E. coli, expression from a vector promoter partially complemented E. coli recA mutants for recombination, DNA repair, and mutagenesis, but not for induction of phage lambda. This clone produced a protein which cross-reacts with antisera raised against the E. coli RecA protein and was approximately the same size. However, the nucleotide sequence of the cloned fragment revealed the presence of an open reading frame for a protein about twice the size of other RecA proteins and the cloned product detected by Western blotting (immunoblotting). The predicted M. tuberculosis RecA protein sequence was homologous with RecA sequences from other bacteria, but this homology was not dispersed; rather it was localized to the first 254 and the last 96 amino acids, with the intervening 440 amino acids being unrelated. Furthermore, the junctions of homology were in register with the uninterrupted sequence of the E. coli RecA protein. Identical restriction fragments were found in the genomic DNAs of M. tuberculosis H37Rv and H37Ra and of M. bovis BCG. It is concluded that the ancestral recA gene of these species diversified via an insertional mutation of at least 1,320 bp of DNA. Possible processing mechanisms for synthesizing a normal-size RecA protein from this elongated sequence are discussed.

Amino Acid Sequence↗
PubMed1990

Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones.

Adjuvant arthritis in rats is induced by a T cell clone specific for amino acids 180-188 of the mycobacterial 65-kD heat-shock protein, and synovial T cell responses to this same Ag have been noted in human arthritis. We have isolated 65-kD Ag-specific T cell clones from synovial fluid mononuclear cells of a patient with acute arthritis, which, unlike the corresponding PBMC, showed a marked proliferative response to the 65-kD Ag. Using synthetic peptides corresponding to the whole sequence of the 65-kD Ag, all the clones were shown to recognize an epitope present in the first NH2-terminal peptide (amino acids 1-15), with no response to the adjacent peptide (amino acids 6-22) or to any other peptide. The complete dominance of this epitope in the response to the 65-kD Ag was shown by documenting responses to the peptide in PBMC obtained after recovery from the arthritis. This epitope, like that recognized by the rat arthritogenic T cell clone, is in a portion of the 65-kD sequence that is not conserved between bacteria and eukaryotes, so that in this case, joint inflammation could not be attributed to bacteria-induced T cell clones cross-reacting with the self 65-kD Ag.

Antigens, Bacterial↗
PubMed1990

The specific 18-kilodalton antigen of Mycobacterium leprae is present in Mycobacterium habana and functions as a heat-shock protein.

A mAb previously thought to be specific for Mycobacterium leprae has been found to cross-react with a cultivable mycobacterium, Mycobacterium habana TMC5135. The epitope is present on a protein of identical molecular mass (18 kDa) in both species. When M. habana is subjected to heat shock, expression of the protein is significantly increased, whereas other forms of environmental stress do not increase its expression. Since immunization of mice with M. habana results in protection against infection with M. leprae, the possibility of using a molecular genetic approach to investigate the role of this protein in protective immunity is raised.

Antibodies, Bacterial↗
PubMed1990

A mycobacterial 65-kD heat shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic.

A recombinant (r)65-kD protein from Mycobacterium leprae, at levels far in excess of those present in whole mycobacteria, was unable to induce arthritis. Even when combined with a synthetic adjuvant, CP20961, to mimic the peptidoglycan adjuvant component of the mycobacterial cell wall, the r65-kD protein failed to induce arthritis. Pretreatment with as little as 1 microgram r65-kD protein protected rats against arthritis induced by M. tuberculosis, but this r65-kD protein was markedly less able to protect against arthritis induced by the synthetic adjuvant, CP20961, or type II collagen. The r65-kD protein appears, therefore, to produce an antigen-specific protection against arthritis induced by bacterial cell walls containing the 65-kD protein. Such protection can be overcome, however, by arthritogenic T lymphocytes, suggesting that protection occurs by preventing clonal proliferation of autoreactive T lymphocytes that are induced by the adjuvant properties of mycobacterial cell walls. How the r65-kD protein abrogates this particular adjuvant activity, and the nature of the arthritogenic self antigen(s), remain to be elucidated.

Animals↗
PubMed1990

Identification, sequencing, and expression of Mycobacterium leprae superoxide dismutase, a major antigen.

The gene encoding a major 28-kilodalton antigen of Mycobacterium leprae has now been sequenced and identified as the enzyme superoxide dismutase (SOD) on the basis of the high degree of homology with known SOD sequences. The deduced amino acid sequence shows 67% homology with a human manganese-utilizing SOD and 55% homology with the Escherichia coli manganese-utilizing enzyme. The gene is not expressed from its own promoter in E. coli but is expressed from its own promoter in Mycobacterium smegmatis. The amino acid sequences of epitopes recognized by monoclonal antibodies against the 28-kilodalton antigen have been determined.

Amino Acid Sequence↗
PubMed1990

Analysis of variation in batches of armadillo-derived Mycobacterium leprae by immunoblotting.

Several batches of cell-free extracts of armadillo-derived Mycobacterium leprae were analyzed by SDS-PAGE and by immunoblotting with monoclonal antibodies. The presence or absence of protease inhibitors had a profound effect on the protein antigens, particularly the 65-kDa antigen. In the absence of protease inhibitors, there were both quantitative and qualitative differences between the different batches of M. leprae extracts.

Animals↗
PubMed1990

Purification of recombinant Mycobacterium leprae 65-kilodalton antigen and murine antibody responses to intravenously administered recombinant protein.

Recombinant Mycobacterium leprae 65-kilodalton antigen has been purified by a combination of differential solubility, ion-exchange and hydrophobic interaction chromatography, and a method for the quantification of the antigen in large scale protein preparations developed. By injecting large amounts of the recombinant antigen intravenously into mice, a limited degree of specific suppression of antibody-response has been induced.

Animals↗
PubMed1990

A longitudinal study of the incidence of leprosy in a hyperendemic area in Zaire, with special reference to PGL-antibody results. The Yalisombo Study Group.

Between 1984 and 1988, yearly surveys for leprosy were done among the 1500 people living in a previous leprosy segregation village in Zaire. In 1984 lepromin tests and phenolic glycolipid (PGL) antibody tests were done in a significant part of the population. The prevalence of the disease at that time was 16.1%, the proportion of multibacillary cases was 11.3% overall and 22% among active cases. Prior to 1984, 23% of paucibacillary cases and 56% of multibacillary cases had presented themselves spontaneously to the Leprosy Service. The exposure to the infection is uniform, but there is a suggestion of family clustering of cases. In spite of a rapidly bactericidal treatment of all known cases in 1984 and thereafter, the annual incidence of 0.34% did not decrease during the 4 years of the study. The PGL antibody test did not contribute to the diagnosis, classification or prognosis of the disease.

Age Factors↗
PubMed1989

Determination and evolutionary significance of nucleotide sequences near to the 3'-end of 16S ribosomal RNA of mycobacteria.

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

Base Sequence↗
PubMed1989

Schwann cells co-cultured with stimulated T cells and antigen express major histocompatibility complex (MHC) class II determinants without interferon-gamma pretreatment: synergistic effects of interferon-gamma and tumor necrosis factor on MHC class II induction.

Schwann cells (SC) do not express major histocompatibility complex (MHC) class II antigens under normal culture conditions. SC can, however, be induced in vitro to express MHC class II molecules by exposure to high concentrations of interferon-gamma (IFN-gamma) and can present antigens to antigen-specific T cell lines. In the present study immunohistochemical labeling showed that most SC (greater than 90%) prepared from rat neonatal sciatic nerves expressed MHC class II molecules when cultured together with mycobacterial antigen and T cells, and as a consequence were able to function as antigen-presenting cells in lymphoproliferation assays, without requiring pretreatment with IFN-gamma. Antigen or T cells alone were ineffective in stimulating MHC class II expression and induction of class II molecules was MHC restricted, requiring the presence of syngeneic T cells. Addition of monoclonal antibody DB1, directed against IFN-gamma to co-cultures of SC and T lymphocytes stimulated with antigen, prevented the induction of MHC class II antigen on SC. When SC were incubated with recombinant (r)IFN-gamma alone, up to 50% of SC showed positive labeling for MHC class II antigen. This level of expression was enhanced to greater than 80% when recombinant tumor necrosis factor (rTNF) was also added. rTNF alone had no effect, and addition of DBI antibody inhibited the synergistic effects of rTNF on MHC class II expression. The effects of rIL 4 were also investigated but neither rIL 4 alone nor rIL 4 in combination with rIFN-gamma induced MHC class II expression by SC. These results show that in the presence of sensitized T lymphocytes and antigen, SC do not require pretreatment with exogenous rIFN-gamma to express MHC class II antigens and function as antigen-presenting cells. T cell-derived TNF and IFN-gamma appear to act as mediators of the T cell-induced expression of MHC class II by SC.

Animals↗
PubMed1989

Vaccination of mice against Mycobacterium leprae infection.

Intradermal immunization with killed Mycobacterium leprae renders mice immune to infection with viable M. leprae. This protection is long lasting and systemic in that immunization in the left flank results in protection in both the left and right footpads. Immunization with Mycobacterium vaccae was ineffective in protecting mice against M. leprae infection, while Mycobacterium bovis BCG provided partial protection. Mycobacterium habana TMC 5135 (now known as Mycobacterium simiae) was found to be as effective as M. leprae in protecting mice against footpad infection.

Animals↗