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T M Shinnick

Publications and source records attributed to T M Shinnick.

At least 55 records · Page 3Linked to original sources
PubMed1992

Encystation of Giardia lamblia leads to expression of antigens recognized by antibodies against conserved heat shock proteins.

During in vitro encystation, Giardia lamblia expresses several stage-specific proteins which are recognized in immunoblots by antisera raised against antigens from three different pathogens. The antigens belong to two different families of conserved stress proteins: (i) HSP60 purified from Legionella pneumophila and recombinant HSP60 from Mycobacterium bovis BCG and (ii) recombinant HSP70 from Plasmodium falciparum.

Animals↗
PubMed1992

Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis.

A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns.

Bacterial Typing Techniques↗
PubMed1991

Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.

Animals↗
PubMed1990

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.

Amino Acid Sequence↗
PubMed1990

Epitopes of the mycobacterial heat shock protein 65 for human T cells comprise different structures.

T cell recognition of foreign antigens is a result of a ternary complex between T cell receptor, nominal peptide and major histocompatibility complex molecule. It has been proposed that the nominal peptide, which is presented by accessory cells to T cells, has a characteristic structure which can be predicted on the basis of physicochemical criteria. To further study this aspect, we stimulated T cells from normal human blood donors with synthetic peptides (each of approximately 15 amino acids in length) from the heat shock protein 65 of Mycobacterium tuberculosis-M. bovis. We found that while the characterization of certain epitopes follows commonly used predictions, other epitopes cannot be predicted by known methods.

Amino Acid Sequence↗
PubMed1990

Rapid and sensitive detection of Mycobacterium leprae using a nested-primer gene amplification assay.

By using a set of four nested oligonucleotide primers, a two-step polymerase chain reaction assay for the detection and identification of Mycobacterium leprae that does not require the use of radioactivity labeled hybridization probes was developed. The nested-primer procedure amplified a 347-base-pair product from M. leprae genomic DNA. No amplification products were produced from DNAs of 19 other Mycobacterium species, 19 non-Mycobacterium species, mouse cells, or human cells. Minor amplification products were observed with three additional Mycobacterium species, i.e., "M. lufu", M. simiae, and M. smegmatis. These products were easily distinguished from the M. leprae product by size and restriction enzyme cleavage patterns. The assay could amplify the 347-base-pair product from samples containing as little as 3 fg of M. leprae genomic DNA--the amount of DNA in a single bacillus. The assay also amplified target sequences in crude lysates of M. leprae bacilli isolated from tissue biopsy specimens from infected animals and humans. The entire assay, from sample preparation to data analysis, can be completed in less than 8 h.

Animals↗
PubMed1988

Epitopes of the Mycobacterium tuberculosis 65-kilodalton protein antigen as recognized by human T cells.

A synthetic peptide approach has been used to identify the epitopes recognized by clonal and polyclonal human T cells reactive to the recombinant mycobacterial 65-kDa protein Ag. Three of the four epitopes identified were recognized as cross-reactive between Mycobacterium tuberculosis and Mycobacterium leprae, although their amino acid sequence in two of three cases was not identical. The peptide (231-245) defining an epitope recognized as specific to the M. tuberculosis complex contains two substitutions compared with the homologous M. leprae region of which one or both are critical to T cell recognition. The reactive T cell clones showed helper/inducer phenotype (CD4+, CD8-), and secrete IL-2, granulocyte-macrophage-CSF, and IFN-gamma upon Ag stimulation. The same clones display cytotoxicity against macrophages pulsed with the relevant peptides or mycobacteria.

Amino Acid Sequence↗
PubMed1988

The Mycobacterium tuberculosis 65-kilodalton antigen is a heat shock protein which corresponds to common antigen and to the Escherichia coli GroEL protein.

Monoclonal hybridoma antibodies directed against a 65-kilodalton (kDa) mycobacterial protein could detect similarly sized antigens in many other bacterial species. In Pseudomonas aeruginosa, the cross-reacting protein corresponded to a 62-kDa antigen that has been called Common Antigen. The mycobacterial 65-kDa antigen and Common Antigen are similar in that both (i) are highly immunoreactive molecules, (ii) contain species-specific and genus-specific epitopes in addition to the broadly cross-reactive epitopes, (iii) can be isolated as homomultimers of greater than 240 kDa, and (iv) have similar amino acid compositions. In Escherichia coli, the cross-reactive protein corresponded to the GroEL protein. Both the GroEL protein and the mycobacterial 65-kDa protein are expressed as heat shock proteins.

Antigens, Bacterial↗
PubMed1988

Serological responses of patients with lepromatous and tuberculoid leprosy to 30-, 31-, and 32-kilodalton antigens of Mycobacterium tuberculosis.

Sera from patients with lepromatous and tuberculoid leprosy were examined in immunoblot assays for antibodies to Mycobacterium tuberculosis culture filtrate antigens. Antibodies to 30- and 31-kilodalton proteins were present in 88 and 81%, respectively, of 16 patients with lepromatous disease and absent in 16 patients with tuberculoid disease. Antibodies to a 32-kilodalton protein were found in 12 and 38% of lepromatous and tuberculoid patients, respectively. These reactivities may be useful for distinguishing lepromatous and tuberculoid leprosy.

Antibodies, Bacterial↗
PubMed1987

The 65-kilodalton antigen of Mycobacterium tuberculosis.

The immune response of the host to the antigens of Mycobacterium tuberculosis plays the key role in determining immunity from infection with as well as the pathogenicity of this organism. A 65-kilodalton (kDa) protein has been identified as one of the medically important antigens of M. tuberculosis. The gene encoding this antigen was isolated from a lambda gt11-M. tuberculosis recombinant DNA library using monoclonal antibodies directed against the 65-kDa antigen as the specific probes. The nucleotide sequence of this gene was determined, and a 540-amino-acid sequence was deduced. This sequence was shown to correspond to that of the 65-kDa antigen by constructing a plasmid in which this open reading frame was fused to the lacZ gene. The resulting fusion protein reacted specifically with the anti-65-kDa protein antibodies. A second long open reading frame was found downstream of the 65-kDa antigen gene which could encode a protein of 517 amino acids. This putative protein contained 29 tandemly arranged partial or complete matches to a pentapeptide sequence.

Amino Acid Sequence↗
PubMed1986

The neuronal identifier element is a cis-acting positive regulator of gene expression.

A middle-repetitive DNA element termed the identifier (ID) sequence, located in introns of postnatal-onset neuronal-specific genes, is transcribed in early postnatal rats by RNA polymerase III (Pol III) specifically in the brain. We show that these Pol III transcripts, although brain specific in vivo, are also expressed in immortalized rodent cell lines in culture. We demonstrate that the ID sequence can act as a positive regulator or enhancer of RNA polymerase II gene expression in cell lines that express these RNAs but not in primary cells in which Pol III transcription of the ID sequence is absent. Thus, ID elements may be positive regulators of postnatal-onset neuronal-specific gene expression.

Animals↗
PubMed1986

A chemically synthesized peptide which elicits humoral and cellular immune responses to mycobacterial antigens.

Monoclonal antibodies directed to Mycobacterium bovis BCG (BCG) and to M. tuberculosis H37Rv (H37Rv) were used in conjunction with affinity chromatography to prepare a mycobacterial component which was designated BCG-a. A synthetic peptide antigen was prepared based on the amino acid sequence of BCG-a and was designated BCG-a-P. Significant immunological similarities were found between BCG-a-P and antigens in extracts of BCG and H37Rv but not between BCG-a-P and antigens of nontuberculous mycobacteria. An enzyme-linked immunosorbent assay detected antibodies to BCG-a-P in sera from rabbits that had been immunized with BCG and H37Rv sonicates. In Western blot analysis, antibodies to BCG-a-P reacted to 10,000-molecular-weight components of extracts of BCG and H37Rv. Delayed cutaneous hypersensitivity reactions to BCG-a-P were elicited in guinea pigs immunized with sonicates of BCG and H37Rv but were weak or nonexistent in unimmunized animals or in animals immunized with sonicates of nontuberculous mycobacteria. This study points out the feasibility of using monoclonal antibodies to prepare and characterize synthetic mycobacterial peptides with a potential for immunodiagnostic purposes.

Animals↗