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Biomedical subjects

M Harboe

Publications and source records attributed to M Harboe.

At least 109 records · Page 6Linked to original sources
PubMed1987

Purification, partial characterization, and identification of a skin-reactive protein antigen of Mycobacterium bovis BCG.

An immunogenic and skin-reactive protein called P64 was purified from Sauton zinc-deficient culture filtrate of Mycobacterium bovis BCG by using successively hydrophobic chromatography on phenyl-Sepharose, ion exchange on DEAE-Sephacel, and molecular sieving on Sephadex G-200. The final P64 preparation was found to be homogeneous based on several analyses. Protein P64 was a constituent of BCG cells since it was present in soluble cellular extract from normally grown BCG cells. It represented 8 to 9% of the soluble proteins of the extract and appeared as the major soluble protein antigen of BCG. This protein was found to have a molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in molecular sieving it eluted at a volume corresponding to a molecular weight of 246,000. An abnormal UV spectrum was observed for this protein. Its amino acid composition showed an abundance of acidic amino acids (or their amides). Aromatic amino acids represented only 3% of the total amino acid residues. The NH2-terminal amino acid sequence of this protein (10 amino acids) was determined. Its sugar content measured with the phenol-sulfuric acid test was lower than 0.3% (wt/wt.) Isolated P64 was tested by various crossed-immunoelectrophoresis techniques and was shown to correspond to antigen 82 in the reference system for BCG antigens. The protein antigen P64 elicited a delayed cutaneous reaction in guinea pigs sensitized with either living or heat-killed BCG. Its potency in skin reaction was, respectively, two- and threefold that of the BCG purified protein derivative. The two types of sensitization used for skin test reactions promoted significant immunoglobulin G antibody production against the protein antigen P64 in guinea pigs 7 weeks after sensitization.

Amino Acid Sequence↗
PubMed1987

Characterization of the secreted antigens of Mycobacterium bovis BCG: comparison of the 46-kilodalton dimeric protein with proteins MPB64 and MPB70.

Western blot analysis showed that the 46-kilodalton (kDa) dimeric protein antigen secreted in large amounts by some daughter strains of Mycobacterium bovis BCG corresponded to protein MPB70 present in long-term culture filtrates of the Japanese substrain. The 46/23-kDa antigen is the most abundant protein in supernatant from a 5-day culture but is masked by leaked products in old culture supernatants. No similarities were found between the 46-kDa protein and MPB64, a protein with the same strain distribution, or with the antigen of similar molecular mass recognized by monoclonal antibody SA1.D2D.

Antibodies, Monoclonal↗
PubMed1987

Antibodies to a synthetic analog of phenolic glycolipid-I of Mycobacterium leprae in healthy household contacts of patients with leprosy.

Fifty-four household contacts of lepromatous patients, 39 household contacts of tuberculoid patients, and 99 control persons were examined with an enzyme-linked immunosorbent assay for their antibody responses to phenolic glycolipid-I (PGL-I) of Mycobacterium leprae using a synthetic analog (PGL-ISA) with the same terminal sugar epitope, namely, O-(3, 6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(alpha-L-rhamnopyranosyl )-(1----9)-oxynonanoyl-BSA. This study was conducted in the Gurage area of Ethiopia in 15 households with a leprosy patient and 15 matched control households. Household contacts with more than 1 year of exposure to a lepromatous patient had antibodies to PGL-ISA significantly more often (19 of 34 persons) than did household contacts with less than 1 year of exposure to a lepromatous patient (4 of 20 persons), household contacts of tuberculoid patients (8 of 39 persons), and persons without exposure to leprosy in the household (33 of 99 persons). No significant association was found between the prevalence of antibodies to PGL-ISA in the household contacts and disease activity in the lepromatous index patients at the time of examination; nor was there a significant association between antibody responses and age or sex of the contacts. The increased prevalence of antibodies to M. leprae antigen in healthy persons with more than 1 year of contact with a lepromatous patient provides further evidence that subclinical infection in leprosy is common, and is related to the type of leprosy in the index patient. The fact that antibodies to PGL-ISA were detected in one third of the persons without household exposure to leprosy emphasizes the necessity to always include comparable controls from the same endemic area in studies of leprosy contacts.

Adolescent↗
PubMed1986

Complement activation in rheumatoid arthritis evaluated by C3dg and the terminal complement complex.

Complement activation was evaluated in plasma and synovial fluid from patients with rheumatoid arthritis. The activation fragment C3dg and the fluid-phase terminal complement complex were used as indicators of initial and terminal activation, respectively. Considerable activation of the whole complement cascade was demonstrated in most synovial fluid samples and in one-third of the plasma samples. No correlation was found between the level of activation products in synovial fluid and the level in plasma. Therefore, both compartments must be examined in order to evaluate local and systemic complement activation in rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed1986

Properties of proteins MPB64, MPB70, and MPB80 of Mycobacterium bovis BCG.

The immunogenic proteins MPB64 and MPB80 of Mycobacterium bovis BCG were purified to homogeneity and compared with MPB70. MPB70 and MPB80 showed a similar distribution in substrains of BCG, both being present in high concentrations in culture fluids of BCG substrain Tokyo, BCG Moreau, BCG Russia, and BCG Sweden and in only very small amounts in BCG Glaxo, BCG Tice, BCG Copenhagen, and BCG Pasteur. In various physicochemical properties MPB70 and MPB80 were closely similar, but MPB80 had a distinctly lower pI value. The N-terminal amino acid sequence was identical for the first 30 residues. In reactions with anti-MPB70 antibodies and delayed-type hypersensitivity skin reactions, MPB70 and MPB80 also had very similar properties. These results show that MPB70 and MPB80 are two closely similar forms of the same gene product, and postsynthetic changes probably explain the observed differences. By contrast, MPB64 had a higher molecular weight. The N-terminal amino acid sequence showed no homology with MPB70, and these two proteins showed no immunologic similarity. MPB64 and MPB70 showed only very restricted cross-reactivity with other species of mycobacteria but cross-reacted with Nocardia asteroides. The similar occurrence in eight different substrains of BCG indicated that the two proteins are influenced by similar control mechanisms, but in contrast to MPB70, MPB64 occurred in sufficient concentration in two strains of Mycobacterium tuberculosis to give a distinct spot in two-dimensional polyacrylamide gel electrophoresis of their culture fluids.

Amino Acid Sequence↗
PubMed1986

Purification and characterization of two protein antigens from the heterogeneous BCG85 complex in Mycobacterium bovis BCG.

The heterogeneous BCG85 complex is a major component of BCG culture fluid. BCG85A and BCG85B were purified by combining ammonium sulphate precipitation with chromatography on hydroxyapatite, DEAE-Sephacel and phenyl-Sepharose columns. Twenty percent of BCG85B was recovered. The chromatographic separation procedures were monitored by fused rocket immunoelectrophoresis. The BCG85 complex was found to consist of three antigens, which were heterogenous with regard to electrophoretic mobility, molecular weight (MW), hydrophobic and immunological properties. They were designated A, B and C in increasing order, according to their electrophoretic mobilities. Thus BCG85A had the lowest electrophoretic mobility, BCG85C the highest. The MW of BCG85A was found to be 31,000, while BCG85B had a slightly lower MW, 29,000, as determined by SDS-PAGE. The antigenic relationship between the components was evaluated by crossed immunoelectrophoresis and double diffusion, and reactions of partial identity between the antigens were found. The BCG85 complex occurs in far lower concentration in sonicates of BCG than in culture fluid.

Antigens, Bacterial↗
PubMed1986

MPB59, a widely cross-reacting protein of Mycobacterium bovis BCG.

The MPB59 protein of Mycobacterium bovis BCG was purified to homogeneity from culture fluid of BCG substrain Tokyo, and characterized by biochemical and immunological techniques. The molecular weight was 28,000, determined by SDS-polyacrylamide gel electrophoresis, and the pI value was 5.3. The N-terminal amino acid sequence was determined for 32 steps and showed no significant homology with MPB64, MPB70 or MPB80. By crossed immunoelectrophoresis, MPB59 was found to belong to the BCG antigen 85 complex and identified as corresponding to the 85B component of this complex. The protein cross-reacted extensively with other species of mycobacteria, and induced a marked humoral immune response in armadillos and monkeys during development of systemic mycobacterial infection after inoculation with Mycobacterium leprae.

Amino Acid Sequence↗
PubMed1986

Effect of nephritic factor on C3 and on the terminal pathway of complement in vivo and in vitro.

Plasma samples from patients with nephritic factor (NeF) were examined for their C3 converting activity. C3, C3dg, C5 and the fluid phase terminal complement complex (TCC) were quantified. All patients had evidence of C3 activation with low plasma C3 and high C3dg. Some patients had normal C5 and normal TCC levels, and thus no evidence of terminal pathway activation in vivo; others, with slower C3 conversion in vitro, had low C5 levels with TCC either elevated or in the upper normal range, suggesting in vivo activation of the terminal pathway. These observations were confirmed by in vitro experiments using purified NeFs. It is concluded that considerable activation of C3 may occur in vivo without a simultaneous activation of the terminal pathway, and that NeF is heterogeneous with regard to its ability to activate complement.

Adult↗
PubMed1985

Cell markers in stored blood.

Patients subjected to major injury and major operations often receive large quantities of stored blood. In units of whole blood the most common cell markers of lymphocytes were studied when the blood had been stored for 1 week. The blood was studied both before and after passing through a microfilter. The reduction in the total white blood cell count was substantially due to a decrease in the number of granulocytes. The percentage of lymphocytes forming E-rosettes was reduced from 69 to 17 per cent following 24 hours of storage, while the percentage of lymphocytes exhibiting non-specific esterase activity was not reduced after 1 week. The proportion of lymphocytes with surface-bound immunoglobulin (Ig) was increased. This increase was due to secondary Ig binding to lymphocytes carrying Fc receptors. The proportion of lymphocytes forming EA-rosettes was unaltered throughout the study. There was little loss of cells after filtration.

Blood Preservation↗
PubMed1985

Cross-reactivity of antigens from the cytoplasm and cell walls of some corynebacteria and mycobacteria.

Leprosy-derived corynebacteria (LDC) are non-acid-fast organisms isolated from leprosy lesions in humans. In this study 20 antigens of native LDC cytoplasm were identified by immunoelectrophoresis, and autoclaving yielded the M1 component, which strongly cross-reacted with antigen 60 of Mycobacterium bovis BCG (bacille Calmette-Guérin) and antigen 7 of Mycobacterium leprae. The polysaccharide moiety of M1 was immunologically related to the LDC cell wall polysaccharide previously characterized as arabinogalactomannan. The latter polysaccharide competitively inhibited the formation of immune complexes by labeled M1 and antisera to the LDC cell wall; cytoplasm and wall polysaccharides from other bacteria produced lower-level inhibition. In a radioimmunoassay with 125I-labeled antigen 7 of M. leprae, sera from patients with leprosy and antisera to the LDC cell wall yielded overlapping curves. Sera from patients with tuberculoid leprosy and those from patients with lepromatous leprosy afforded different levels of inhibition in this radioimmunoassay; this result indicated a difference in antibody specificity in the two forms of leprosy. In conclusion, the cell wall polysaccharide of LDC corresponds to the main thermostable cytoplasmic antigen M1, which strongly crossreacts with sera from patients with leprosy and, more specifically, with antigen 7 of M. leprae.

Antigens, Bacterial↗
PubMed1985

Characterization of the specificity of monoclonal antibodies against Mycobacterium tuberculosis by crossed immunoelectrophoresis.

The specificity of monoclonal antibodies against Mycobacterium tuberculosis was determined by crossed immunoelectrophoresis. Radiolabelled, non-precipitating monoclonal antibody was incorporated in the top gel together with unlabelled polyvalent, precipitating anti-bacillus Calmette-Guérin (BCG) antibody. Inclusion of labelled antibody in individual precipitate lines was demonstrated by autoradiography. Each monoclonal antibody was localized in a single precipitate line: antibody TB73 reacted with BCG antigen 56; TB71 and TB72 reacted with BCG antigen 78; and TB78 reacted with BCG antigen 82. The technique permits precise determination of the specificity of monoclonal antibodies at the level of reactivity with native immunogenic components of complex microorganisms without prior antigen purification.

Animals↗
PubMed1985

Monoclonal antibodies recognizing a neoantigen of poly(C9) detect the human terminal complement complex in tissue and plasma.

The terminal complement complex (TCC), consisting of C5b, C6, C7, C8, and C9, contains neoantigens that are absent from the individual native components. Neoantigens are present both in the membrane-bound (MAC) and the fluid-phase (SC5b-9) complex. The present study describes production of monoclonal antibodies against neoantigens of both forms of the TCC. A convenient screening and detection system, based mainly on enzyme-linked immunosorbent assays, crossed immunoelectrophoresis with autoradiography, and affinity chromatography with subsequent sodium dodecyl sulphate-polyacrylamide gel electrophoresis including immunoblotting, is described in detail. Two monoclonal antibodies were specific for a neoantigen located in the poly(C9) moiety of the TCC. One of these antibodies, MCaE11, was used for immunohistochemical detection of MAC in tissue and for quantification of the fluid-phase TCC in ethylenediaminetetraacetic acid plasma.

Antibodies, Monoclonal↗