PubMed HealthSearch

Biomedical subjects

M Harboe

Publications and source records attributed to M Harboe.

At least 19 recordsLinked to original sources

[Gerhard Henrik Armauer Hansen--still of current interest].

Armauer Hansen described his first observation of Mycobacterium leprae on 28 February 1873 in detail. His discovery of the leprosy bacillus was the result of a logical process in which distinct stages can be clearly seen. The first step was clinical, and established criteria for leprosy as "a specific disease". The second stage was epidemiological. Contrary to the main view favouring a genetic basis, these observations convinced him of the infectious nature of the disease. The third stage was the search for the bacillus. His basic observations were reproduced, as documented. Armauer Hansen afterwards tried to fulfill Koch's postulates, in order to prove that the bacillus was the causative agent of leprosy. His second main contribution is the work on leprosy control, emphasizing isolation to contain the infection. The subsequent decline of leprosy in Norway differs from the trend in many developing countries, where leprosy has remained an important public health problem. In recent years Norwegian medical research has made significant contributions to leprosy research, in the field of epidemiology in Bergen, and through immunological studies of the disease originating from the Armauer Hansen Research Institute in Addis Ababa, Ethiopia.

History, 19th Century

Heparin-binding properties of vitronectin are linked to complex formation as illustrated by in vitro polymerization and binding to the terminal complement complex.

Vitronectin (VN, complement S-protein) is a multifunctional protein which participates in cell adhesion, coagulation, fibrinolysis, and protection against complement lysis. VN is incorporated into several complexes, such as the terminal complement complex and thrombin-antithrombin III, and is bound to plasminogen activator inhibitor 1. The present study showed that purified VN spontaneously forms polymers of approximately 1000 kDa with a Stokes radius of 10 nm. The polymers are to a varying extent stabilized by disulfide bonds, but are quite stable even after reduction and alkylation, indicating the importance of noncovalent bonds. Plasma VN circulates mainly as a 65/75-kDa monomer containing a cryptic heparin-binding site which is exposed upon a conformational change induced by different stimuli, such as coagulation, heating, adsorption to surfaces, or exposure to acids, urea, or other denaturating agents. In the present study, VN was demonstrated to expose its heparin-binding site and its conformationally dependent 8E6 epitope when incorporated into the terminal complement complex. We suggest that exposure of the heparin-binding site and a putative hydrophobic binding site of VN are linked events dependent upon the same conformational change. In vivo, complex formation probably induces the heparin-binding site. Such a link might also explain why purified heparin-binding VN spontaneously forms polymers. The heparin-binding site may be involved in the elimination of multimolecular complexes containing VN.

Antibodies, Monoclonal

Protein antigens of mycobacteria studied by quantitative immunologic techniques.

Crossed immunoelectrophoresis has great resolving power in the demonstration of immunogenic constituents of mycobacteria. The pattern with multiple precipitate lines is highly reproducible and allows precise identification of components. After the isolation of individual proteins, immunologic specificity combined with molecular weight determination and N-terminal amino acid sequencing should be used to ensure consistent identification in different laboratories. Simultaneous quantification of individual proteins in sonicates of washed bacilli and culture fluids permits the determination of a localization index, which indicates whether the proteins are cytoplasmic constituents or actively secreted. Several "new," actively secreted proteins have recently been defined, and the role of these proteins in the interaction between the bacilli and the infected host is discussed.

Animals

The 38-kDa protein of Mycobacterium tuberculosis: a review.

This review illustrates that the 38-kDa protein is one of the most important antigens of Mycobacterium tuberculosis. It is actively secreted but partly attached to the surface of the mycobacterial cell by a lipid tail that may also be responsible for binding of carbohydrate to the protein. It is a major constituent of M. tuberculosis culture fluid after growth on the synthetic Sauton medium and occurs in bacille Calmette-Guérin in far lower concentrations. The protein induces B and T cell responses with high specificity for infection with M. tuberculosis and is a prime candidate for development of new diagnostic reagents for tuberculosis.

Animals

A family of cross-reacting proteins secreted by Mycobacterium tuberculosis.

Cross-reactions between five proteins actively secreted by Mycobacterium tuberculosis were studied by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting, and ELISA using polyclonal rabbit antisera and mouse monoclonal antibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT51 protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross-react extensively, each of the components containing component-specific as well as cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. N-terminal sequence studies revealed striking homology at the amino acid level between 85A, 85B, 85C and MPT51. MPT64 showed less homology. In addition, striking homology was demonstrated between two different stretches within the 85B sequence and indicated between three stretches within the MPT64 molecule. Thus, a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. MPT64 may also belong to this family.

Amino Acid Sequence

Identification and characterization of epitopes shared between the mycobacterial 65-kilodalton heat shock protein and the actively secreted antigen 85 complex: their in situ expression on the cell wall surface of Mycobacterium leprae.

Both mycobacterial hsp65 and the actively secreted antigen 85 complex of 30-kDa region proteins are considered to be major immune targets in mycobacterial diseases. In this study, by using a novel series of monoclonal antibodies (MAbs) directed to these antigens, we identified and partially characterized three unique epitopes (Rb2, Pe12, and A2h11) that are shared between mycobacterial hsp65 and the individual components of the antigen 85 complex. Dot blot assays with native purified proteins revealed that all three MAbs are strongly bound to hsp65 and antigens 85A (MPT44) and 85B (MPT59), while a weak reaction or no reaction was found with antigen 85C (MPT45). Immunoblotting showed that MAb Rb2 reacted strongly with both hsp65 and the antigen 85 complex proteins, whereas MAbs Pe12 and A2h11 reacted strongly with the former but weakly with the latter. Moreover, these MAbs did not react with other closely related MPT51 and MPT64 secreted proteins. Further characterization of these epitopes was performed by using recombinant fusion and truncated proteins of Mycobacterium bovis BCG hsp65 (MbaA) and the M. leprae 30- and 31-kDa antigen 85 complex fusion proteins. In hsp65, Rb2-Pe12- and A2h11-reactive epitopes were found to reside in the C-terminal region of amino acid residues 479 to 540 and 303 to 424, respectively. In the M. leprae 30- and 31-kDa antigen 85 complex, all three epitopes were located in an N-terminal region of amino acid residues 55 to 266, one of the known fibronectin-binding sites of the M. leprae antigen 85 complex. Comparison of these MAb-reactive amino acid sequence regions between mycobacterial hsp65 and the components of the antigen 85 complex revealed that these regions show certain amino acid sequence identities. Furthermore, by immunoperoxidase and immunogold ultracytochemistry, we demonstrated that Rb2-, Pe12-, and A2h11-reactive epitopes are expressed both on the cell wall surface and in the cytosol of M. leprae bacilli within the lesions of lepromatous leprosy patients and in M. leprae-infected armadillo liver tissue.

Amino Acid Sequence

Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface.

Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.

Animals

The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis.

The large number of different proteins synthesized by the mycobacterial cell are currently classified and studied in terms of groups of proteins with certain common properties such as physical and chemical characteristics, function, and localization in the mycobacterial cell. Proteins that are actively secreted during culture on synthetic media represent a particular group of great current interest. At least eight proteins secreted by Mycobacterium tuberculosis have been isolated and characterized to various extents. The genes coding for five proteins secreted from M. tuberculosis and/or Mycobacterium bovis BCG have been cloned and sequenced. All of them contain typical signal sequences. The proteins of the antigen 85 complex, which form the main subject of this review, are often the most common proteins in M. tuberculosis culture fluid. The constituents denoted 85A, 85B, and 85C are encoded by three genes located at different sites in the mycobacterial genome and show extensive cross-reactivity as well as homology at amino acid and gene levels. The proteins differ slightly in molecular mass in the 30- to 31-kDa region, and all of them are fibronectin-binding proteins, but the significance of the latter observation and the role of these proteins in mycobacterial physiology and interaction with the infected host remain to be elucidated. The antigen 85 complex proteins are strongly immunogenic in natural and experimental mycobacterial infections in terms of both induction of antibody synthesis and T-cell-mediated reactions. The well-recognized difference in the efficacy of live and dead mycobacterial vaccines should be considered in relation to the group of secreted antigens. After inoculation, live bacteria in vaccines such as BCG multiply in the host, probably releasing several constituents belonging to the class of secreted proteins and hence resulting in more efficient stimulation of the immune system. Secreted mycobacterial antigens are expected to be of particular significance in induction of various immune responses that are responsible for development of protective immunity in some individuals and for clinical symptoms and complications of the ensuing disease in others.

Amino Acid Sequence

Formation of C5a during cardiopulmonary bypass: inhibition by precoating with heparin.

A novel enzyme immunoassay based on direct detection of C5a by a monoclonal antibody (C17/5) specific for a neoepitope exposed in C5a/C5adesArg was used to measure in vivo and in vitro C5a formation during cardiopulmonary bypass. In vivo, we observed a significant threefold to fourfold increase in patient plasma C5a/C5adesArg levels from baseline values (5.6; 1.6 to 12.9 ng/mL) (median and range) up to 42 hours postoperatively (17.5; 6.5 to 46.0 ng/mL) when two different uncoated cardiopulmonary bypass circuits were used. Coating of the extracorporeal circuit with end-point-attached heparin completely abolished C5a formation in vitro during circulation of blood through the circuit for 120 minutes. The C5a concentration (median and range) was 3.2 (2.6 to 15.9) ng/mL at the start and 3.1 (2.7 to 15.0) ng/mL at the end of the experiment. In the uncoated setups the corresponding C5a concentrations were 10.1 (6.2 to 17.5) and 19.7 (13.1 to 24.3) ng/mL. Finally, heparin-coated cardiopulmonary bypass circuits were examined in vivo. C5a levels did not increase significantly during the cardiopulmonary bypass period in the heparin-coated group in contrast to the uncoated group, but the postoperative increase in C5a levels was similar in the two groups. We conclude that heparin coating improves biocompatibility by completely abolishing C5a formation in vitro. The discrepancy between the in vitro and the in vivo findings is probably related to the complicated biological turnover of C5a.

Antibodies, Monoclonal

Interpreting complicated chromatographic patterns.

GC-MS, HPLC, automatic amino acid analysis, high-resolution two-dimensional electrophoresis and capillary electrophoresis are suitable for the multicomponent analysis of body fluids and tissues. Manual interpretation of the complex metabolite and protein profiles thereby obtained is usually difficult, except in the case of metabolic disorders, where major deviations from the normal profiles often are observed. Implementation of multivariate data analysis makes it possible to retrieve diagnostic information that otherwise may be overlooked, as shown in this report where patients with leprosy have been examined. Urine samples were analysed by ion-exchange chromatography and by GC-MS to obtain profiles of amino acids and organic acids. Qualitative and quantitative information on 68 metabolites were then analysed by principal components analysis (PCA) and by partial least square models (PLS). Three different PLS dimensions were found (cross-validation) corresponding to controls (persons without leprosy), paucibacillary and multibacillary leprosy.

Amino Acids

A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis.

A series of mycobacterial antigens were quantified by immunoelectrophoresis, enzyme-linked immunosorbent assays, or SDS-PAGE with immunoblotting using antisera against purified mycobacterial antigens. The antigens showed a characteristic distribution profile. Some had a marked quantitative dominance in the culture fluid while others had a marked dominance in sonicates of whole washed bacilli. The majority of the antigens tested could thus be located and grouped as either secreted or cytoplasmic in terms of a localization index (LI) which is described. A 5-week-old Mycobacterium tuberculosis culture fluid preparation with a low degree of lysis was valuable in the delineation of localization indexes. The various secreted antigens showed a great span in LI values, from 5 to 1000. This variation may express different degrees of secretion efficiency or differences in tendency to adhere to the bacterial surface. The identification of proteins as extracellular or cytosolic according to their LI values was in agreement for cultures of M. tuberculosis with a low degree of lysis and cultures of M. bovis BCG and M. bovis AN-5 with significant lysis of the bacterial cells.

Antigens, Bacterial

Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis.

Five actively secreted proteins (MPT32, MPT45, MPT51, MPT53, and MPT63) and the MPT46 protein were purified to homogeneity from Mycobacterium tuberculosis culture fluid and compared with proteins previously purified by ourselves and other investigators. Antisera were obtained by immunization of rabbits with all of the newly isolated proteins identified to be immunogenic. Two-dimensional electrophoresis of culture fluids obtained each week for 2 to 10 weeks of culturing of M. tuberculosis revealed characteristic changes, permitting identification of two distinct groups of proteins being actively secreted from the mycobacterial cells or appearing later in the culture fluids as a result of the release of soluble proteins from the cytosol after lysis of bacteria. The N-terminal amino acid sequences of five MPTs were shown to be identical to those of proteins previously isolated by other investigators and given different designations, and five new sequences are given. These sequences and the use of the antisera may serve to identify these proteins with mycobacterial constituents isolated by other investigators. The previously identified but not isolated MPT45 protein was shown to correspond to the C component of the antigen 85 complex. The 27-kDa MPT51 protein was demonstrated to cross-react with the three components of the antigen 85 complex, and the N-terminal amino acid sequences of MPT51 and MPT59 showed 60% homology. This finding and the extensive cross-reactivity between the components of the antigen 85 complex may indicate that there is a family of closely related secreted proteins in mycobacteria.

Amino Acid Sequence

Genetic and immunological analysis of Mycobacterium tuberculosis fibronectin-binding proteins.

Recombinant phage clones, TB1 and TB2, were selected from a Mycobacterium tuberculosis lambda gt11 DNA expression library by screening with a polyclonal antiserum raised against the antigen 85 complex of Mycobacterium bovis BCG. Analysis of recombinant DNA inserts and expressed fusion proteins showed that two new genes had been isolated. The product of clone TB2 was identified as a member of the 30/31-kDa antigen 85 complex. Restriction enzyme analysis showed that this gene differs from previously cloned members of this antigen complex, with detailed serological analysis indicating that it may encode the 85C component. Antisera raised against the expressed product of clone TB1 recognized a 55-kDa protein in M. tuberculosis extracts. The 55-kDa protein also has fibronectin-binding activity and, like the 30/31-kDa family, is a prominent target of the antibody response in patients with mycobacterial disease. Although the clones were selected by using the same antiserum, detailed analysis by serology and by DNA hybridization showed that they represent two quite distinct types of fibronectin-binding activities expressed by M. tuberculosis. Further analysis of the fibronectin-binding antigens of M. tuberculosis may provide important insights into their role in mediating the interaction with the host immune system.

Antibodies, Monoclonal

C3 activation products, C3 containing immune complexes, the terminal complement complex and native C9 in patients with rheumatoid arthritis.

Complement activation products, C9 and C3-containing circulating immune complexes (CIC), were evaluated in plasma and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and osteoarthritis. C3 activation products and the fluid phase terminal complement complex were considerably elevated in SF from RA patients reaching levels five- to eighttimes that in plasma, consistant with a local activation of the whole cascade in the joints. The results emphazise the importance of detecting C3 activation by neoepitope expression instead of single fragment determinations. The concentration of native C9 was lower in synovial fluid compared with plasma, consistant with the excessive local complement activation. Increased CIC levels which correlated with the degree of complement activation were also found in the SF from the RA patients.

Adult

Purification and characterization of the 30,000 dalton native antigen of Mycobacterium tuberculosis and characterization of six monoclonal antibodies reactive with a major epitope of this antigen.

The 30,000 dalton native antigen of Mycobacterium tuberculosis is a major constituent of this organism and is secreted into culture medium. We purified this antigen by ammonium sulfate precipitation, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography to yield a single 29 to 30 kd component. The first 20 N-terminal amino acid sequence was determined and found to be identical to that reported for M. bovis alpha-antigen. Immunoelectrophoresis studies demonstrated the purified 30,000 dalton antigen to be immunologically identical with antigen 6 and antigen 85B. The 30,000 dalton native antigen was a potent skin test antigen in sensitized guinea pigs. Six immunoglobulin G1 murine monoclonal antibodies against the 30,000 dalton antigen were generated. By enzyme-linked immunosorbent assay and western immunoblotting, all six monoclonal antibodies reacted with the 30,000 dalton antigen, perhaps with the same epitope. When used with culture filtrates of other mycobacteria, the monoclonal antibodies demonstrated reactivity with M. gordonae and M. kansasii and to a lesser extent with M. avium and M. scrofulaceum.

Amino Acid Sequence