PubMed Health⌕ Search

Biomedical subjects

M Harboe

Publications and source records attributed to M Harboe.

At least 19 recordsLinked to original sources
PubMed2006

Design of a complement mannose-binding lectin pathway-specific activation system applicable at low serum dilutions.

Recently we showed that alternative pathway (AP) amplification was responsible for more than 80% of specific classical pathway-induced terminal pathway activation under physiological conditions. The present study aimed to design a system for specific lectin pathway (LP) activation applicable at low serum dilutions with a fully functional AP. Comparison between activation of normal human serum (NHS), a mannose-binding lectin (MBL) homozygous D/D-deficient serum, and sera deficient in C1q and C2, all diluted 1 : 2, was essential to document optimal conditions for LP specificity. Mannan on the solid phase of enzyme-linked immunosorbent assay (ELISA) plates was used for activation, showing 0.5 microg mannan/well to give optimal conditions because at this concentration a good signal was preserved for C4 and TCC deposition in NHS, whereas the C3 deposition observed in C2-deficient serum at higher mannan concentrations reached nadir at 0.5 microg/well, indicating a lack of direct AP activation under these conditions. Pooled NHS and C1q-deficient serum gave the same degree of C4 and terminal complement complex (TCC) deposition, whereas deposition of these products was not obtained with MBL-deficient serum. Reconstitution with purified MBL, however, restored the depositions. A blocking anti-MBL monoclonal antibody (mAb) completely abolished the complement deposition, in contrast to a non-inhibiting anti-MBL mAb. Activation of C2-deficient serum induced C4 deposition similar to NHS, but negligible deposition of C3 and TCC, confirming the lack of direct activation of AP. Thus, this assay is unique in being LP-specific at low serum dilution and thus particularly suitable to study LP activation mechanisms and the role of AP amplification under physiological conditions.

Antibodies, Monoclonal↗
PubMed2004

Female genital tuberculosis in Ethiopia.

OBJECTIVES: To study the occurrence of female genital tuberculosis (FGTB) in Ethiopia and to compare the different methods available for its diagnosis. METHODS: Biopsy or curettage samples from 25 clinically suspected cases of FGTB were investigated with histopathology, smear microscopy, TB culture and PCR for mycobacteria. HIV status was determined by ELISA. CD4:CD8 ratio was evaluated by flow cytometry. RESULTS: Among the 25 clinically suspected patients investigated, only one was AFB smear positive, three were culture positive, seven were histology positive and 12 were positive by PCR (a total of 16 positives). Samples taken from the fallopian tube were more frequently positive than those from the endometrium. CONCLUSIONS: The results showed that FGTB is a significant clinical problem in Ethiopia. A combination of PCR with the other available methods was found to be the best alternative to achieve sufficient sensitivity and specificity for the diagnosis of FGTB in this setting.

Adolescent↗
PubMed2004

Immunodominant B-cell epitope in the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis cross-reacting with glutathione S-transferase.

The TB1-5 76C monoclonal antibody raised against a synthetic 60-mer peptide in the N-terminal part of the Mce1A mammalian cell entry protein of Mycobacterium tuberculosis has previously been shown to react with a linear epitope in the KRRITPKD region, residues 131-138 in Mce1A, and to cross-react with Mce1F. Six additional monoclonal antibodies raised against the same peptide were also shown to cross-react with Mce1F. Four of them reacted with a linear epitope in the same area, indicating that this area is immunodominant but showed distinct differrences in fine specificity. Two monoclonal antibodies did not react with synthetic peptides from this region on the solid phase in enzyme-linked immunosorbent assay, indicating greater influence of conformation on reactivity. None of the monoclonal antibodies reacted with 14-mer synthetic peptides from the corresponding area in Mce2A, Mce3A, Mce4A, M. avium, M. smegmatis or M. leprae. The reaction pattern of the monoclonal antibodies was analysed in relation to our model of the Mce1A molecule (AK Das et al. Biochem Biophys Res Commun 2003;302:442-7). The epitope is located on the surface of Mce1A, at the distal beta-strand-loop region in the beta-domain supporting its potential role in promoting uptake of M. tuberculosis in host cells. Monoclonal antibody TB1-5 19C cross-reacted with glutathione S-transferase of Schistosoma japonicum containing a PKE triplet. Monoclonal antibody TB1-5 76C gave a major band at about 44 kDa in Western blotting of M. tuberculosis sonicate, whereas polyclonal rabbit anti-Mce1A peptide antibodies reacting with the extended TTPKNPTKRRITPKDVI area of Mce1A showed a distinct band above the 160 kDa molecular mass standard.

Amino Acid Sequence↗
PubMed2004

The quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation.

Complement activation with formation of biologically potent mediators like C5a and the terminal C5b-9 complex (TCC) contributes essentially to development of inflammation and tissue damage in a number of autoimmune and inflammatory conditions. A particular role for complement in the ischaemia/reperfusion injury of the heart, skeletal muscle, central nervous system, intestine and kidney has been suggested from animal studies. Previous experiments in C3 and C4 knockout mice suggested an important role of the classical or lectin pathway in initiation of complement activation during intestinal ischaemia/reperfusion injury while later use of factor D knockout mice showed the alternative pathway to be critically involved. We hypothesized that alternative pathway amplification might play a more critical role in classical pathway-induced C5 activation than previously recognized and used pathway-selective inhibitory mAbs to further elucidate the role of the alternative pathway. Here we demonstrate that selective blockade of the alternative pathway by neutralizing factor D in human serum diluted 1 : 2 with mAb 166-32 inhibited more than 80% of C5a and TCC formation induced by solid phase IgM and solid- and fluid-phase human aggregated IgG via the classical pathway. The findings emphasize the influence of alternative pathway amplification on the effect of initial classical pathway activation and the therapeutic potential of inhibiting the alternative pathway in clinical conditions with excessive and uncontrolled complement activation.

Antibodies, Monoclonal↗
PubMed2004

Female genital tuberculosis in Ethiopia.

UNLABELLED: To study the occurrence of female genital tuberculosis (FGTB) in Ethiopia and to compare the different methods available for its diagnosis. METHODS: Biopsy or curettage samples from twenty-five clinically suspected cases of FGTB were investigated with histopathology, smear microscopy, TB culture and PCR for mycobacteria. HIV status was determined by ELISA. CD4:CD8 ratio was evaluated by flow cytometry. RESULTS: Among the 25 clinically suspected patients investigated, only one was AFB smear positive, three were culture positive, seven were histology positive and 12 were positive by PCR (a total of 16 positives). Samples taken from the fallopian tube were more frequently positive than those from the endometrium. CONCLUSIONS: The results showed that FGTB is a significant clinical problem in Ethiopia. A combination of PCR with the other available methods was found to be the best alternative to achieve sufficient sensitivity and specificity for the diagnosis of FGTB in this setting.

Adolescent↗
PubMed2003

Nasal carriage of Mycobacterium leprae DNA in healthy individuals in Lega Robi village, Ethiopia.

The number of registered leprosy patients world-wide has decreased dramatically after extensive application of WHO recommended Multiple Drug Therapy (MDT). The annual number of new cases has, however, been almost unchanged in several populations, indicating that the infection is still present at community level. Nasal carriage of Mycobacterium leprae DNA was studied in Lega Robi village in Ethiopia. MDT had been applied for more than ten years, and 718 residents over 5 years old were eligible for the study. During the first survey nasal swab samples were collected from 664 (92.5%) individuals. The results of a Peptide Nucleic Acid-ELISA test for M. leprae DNA interpreted by stringent statistical criteria were available for 589 (88.7%) subjects. Thirty-five (5.9%) individuals without clinical signs of leprosy were positive for M. leprae DNA. Seven PCR positive individuals lived in a household where one or two other members were also positive for M. leprae DNA. During a second survey 8 (46%) of 175 interpretable PNA-ELISA tests were positive. Of 137 individuals tested twice, only two were positive on both occasions whereas 10 were PCR positive only once. The study confirms the widespread distribution of M. leprae DNA in healthy individuals. The feasibility of curbing possible transmission of subclinical infection needs further consideration.

Adolescent↗
PubMed2003

T- and B-cell epitopes in the secreted Mycobacterium bovis antigen MPB70 in mice.

MPB70 is a soluble secreted protein highly expressed in Mycobacterium bovis and strains of bacille Calmette-Guérin (BCG); as such, it is a candidate for subunit and DNA vaccines against tuberculosis. MPB70 was screened for T-cell epitopes in four different inbred mouse strains. Major histocompatibility complex (MHC) H-2b-expressing mice (C57BL/6) secreted interferon-gamma (IFN-gamma) after stimulation with peptides from the regions 1-20, 41-50, 81-110, 121-150 and 161-193 of the MPB70 sequence. H-2db mouse (B6D2) splenocytes secreted IFN-gamma after stimulation with some of the same peptides, whereas H-2d mice (BALB/c and DBA/2) did not secrete IFN-gamma upon stimulation with the peptides. Sera from H-2db mice immunized with native MPB70 in incomplete Freund's adjuvant (IFA), mpb70 DNA or live BCG Moreau were found to contain antibodies against the native MPB70 antigen. H-2db mice immunized with native MPB70 in IFA exhibited high titres of peptide-reactive immunoglobulin G1 (IgG1) antibodies, whereas DNA-immunized mice reacted with IgG2a antibodies against some of the same peptides. As some of the epitopes recognized by mouse T and B cells have previously been found to stimulate immune responses in humans, cattle and rabbits, we conclude that these epitopes may be good general epitopes for the stimulation of T- and B-cell responses and candidates for a DNA vaccine with a broad applicability.

Amino Acid Sequence↗
PubMed2002

Generation of antibodies to the signal peptide of the MPT83 lipoprotein of Mycobacterium tuberculosis.

The mpt83 gene (Rv2873) encodes the exported MPT83 lipoprotein of Mycobacterium tuberculosis. The corresponding identical mpb83 gene of Mycobacterium bovis is expressed to varying extents in different substrains of M. bovis Bacille Calmette Guerin (BCG), BCG Tokyo and BCG Moreau being high producers and BCG Danish 1331, a low producer of the MPB83 protein. Immunization with the 13-mer N-terminal part of the signal peptide of MPT83, MINVQAKPAAAASC, coupled to keyhole limpet haemocyanin (KLH) through the added C-terminal cysteine resulted in rapid antibody formation monitored by enzyme-linked immunosorbent assay (ELISA) with free immunizing peptide on the solid phase. In ELISA, with four 20-mer overlapping peptides covering the N-terminal part of the MPT83 sequence, three polyclonal rabbit antisera reacted only with the N-terminal peptide. Antigenic signal peptide could not be detected in sonicates of BCG Tokyo and BCG Moreau. After SDS-PAGE and blotting, the antibodies reacted with sonicates of recombinant Escherichia coli containing the entire mpt83 gene including the signal sequence, but not with the 22 kDa form of native MPB83 purified from BCG culture filtrate. In partition chromatography the recMPT83 partitioned in the water phase while 26 kDa MPB83 in BCG culture filtrate partitioned in the lipid phase confirming that lipidation at the N-terminal cysteine residue occurs after the splitting of the polypeptide chain by signal peptidase II.

Amino Acid Sequence↗
PubMed2002

Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis.

In addition to the previously cloned Mce1A and Mce1E genes of the Mce1 operon of Mycobacterium tuberculosis (Ahmad et al. Scand J Immunol 1999;50:510-8), Mce1B, Mce1D and Mce1F were cloned and expressed as glutathione-S-transferase (GST) fusion proteins in recombinant Escherichia coli. Polyclonal antibodies against a predicted B-cell epitope of each of the Mce1 proteins of M. tuberculosis were produced by immunizing rabbits with synthetic peptides coupled to keyhole limpet haemocyanin. These antibodies reacted specifically with the corresponding fusion protein, except for GST-Mce1F. A mouse monoclonal antibody, TB1-5 76C, raised against a synthetic 60-mer peptide corresponding to the residues 106-165 in the N-terminal part of Mce1A, reacted strongly with GST-Mce1A. The antibody cross-reacted with GST-Mce1F, but not with the other recombinant GST-Mce1 fusion proteins or free GST. Bioinformatic analysis revealed only slight homology between Mce1A and Mce1F, along the length of the polypeptide chains. Higher homology was found between the residues 106-165 of Mce1A and the residues 347-406, further into the mature Mce1F polypeptide chain. There was a striking, localized homology, indicating that the epitope reacting with the monoclonal antibody TB1-5 76C may be narrowed to the KRRITPKD region, the residues 131-138 in Mce1A corresponding to the residues 372-379 in Mce1F. This was confirmed in enzyme-linked immunosorbent assay, showing binding of TB1-5 76C to a 17-mer synthetic peptide containing the KRRITPKD sequence.

Amino Acid Sequence↗
PubMed2001

Novel monoclonal antibodies against major antigens of Mycobacterium bovis.

MPB70 and MPB83 are among the most characteristically exported proteins defining a strongly expressed phenotype of Mycobacterium bovis. These proteins are known to be homologous to osteoblast-specific factor 2. By in vitro culture of mycobacteria they appear to have a limited species distribution and to be relatively specific for M. bovis. Virtually identical genes are however, present in Mycobacterium tuberculosis. In order to facilitate further research into the immunobiology of these proteins and their potential application for differential diagnosis of tuberculosis as a result of M. bovis, we describe the reactivities of 20 monoclonal antibodies (MoAbs) to these proteins. Immunizing with bovine PPD generated 10 MoAbs. These antibodies reacted preferentially with the soluble MPB70 antigen using reducing conditions in SDS-PAGE with western blotting. Ten MoAbs were generated by immunizing mice with fractions derived from a whole cell sonic extract of M. bovis. These antibodies reacted preferentially with the surface exposed MPB83 lipoglycoprotein.

Animals↗
PubMed2001

Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis.

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.

Animals↗
PubMed1999

Cloning, expression and significance of MPT53 for identification of secreted proteins of Mycobacterium tuberculosis.

Based on our N -terminal amino acid sequence of MPT53 and a deduced DNA sequence, we searched for the corresponding gene in the Mycobacterium tuberculosis genomic sequence at the Sanger centre, localizing mpt53 close to mpt70 and mpt83. The gene was cloned and expressed, followed by purification of MPT53 to homogeneity from recombinant M. smegmatis culture fluid. In MPT53 there is 60 % identity with the active site of thioredoxin of M. tuberculosis (MPT46) with two cysteins in a CXXC motif, but MPT53 could not serve as an alternative substrate for thioredoxin reductase. Testing for IgM and IgG1 anti-MPT53 in cattle sera showed that MPT53 is immunogenic following natural and experimental infection with M. bovis. Cloning of mpt53 represents cloning of the last of the 10 proteins originally defined as "secreted proteins" of M. tuberculosis and M. bovis based on determination of their "Localization index" (LI) (J Gen Microbiol 1991;137 : 875-84). The need for a precise definition of the term "secreted protein" is discussed. So far we have observed full concordance between occurrence of an LI value indicating secretion of a protein and occurrence of a signal sequence in the corresponding gene. Signal sequence independent protein secretion in mycobacteria may occur for a limited number of proteins and remains to be established.

Amino Acid Sequence↗
PubMed1999

The mammalian cell entry operon 1 (mce1) of mycobacterium leprae and mycobacterium tuberculosis.

The genome project on Mycobacterium tuberculosis H37Rv has revealed four mammalian cell entry (MTmce1-4) operons putatively involved with entry and survival of mycobacteria in host cells. A homologous operon to the MTmce1 operon was identified in cosmid B983 of Mycobacterium leprae. By comparison with M. tuberculosis, several mutations, or sequencing errors, were predicted at specific sites causing frame shifts in the MLyrbE1A, MLyrbE1B and MLmce1D genes. Using targeted sequencing, sequence errors were identified. The corrected MLmce1 operon sequence appears to be highly homologous to the MTmce1 operon, and similarly encodes eight potential genes. Thus, both M. tuberculosis and M. leprae mce1 operons may be functional and involved in host cell targeting.

Base Sequence↗
PubMed1999

Cloning, expression and immunological reactivity of two mammalian cell entry proteins encoded by the mce1 operon of Mycobacterium tuberculosis.

The DNA segments corresponding to two members of the mammalian cell entry operon 1 (mce1) encoding Mce1A and Mce1E proteins were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reaction, cloned and subcloned into pGEM-T and pGEX-4T-3 vectors, respectively, and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) of Schistosoma japonicum as the fusion partner. The recombinant proteins appeared as major cellular proteins in SDS-PAGE gels at the expected molecular mass of 68 kDa and 64 kDa for GST-Mce1A and GST-Mce1E, respectively. The identity of each fusion protein was confirmed by reactivity with anti-GST antibodies in Western immunoblots. The fusion proteins were purified to near homogeneity by affinity chromatography, and purified Mce1A and Mce1E, free of the fusion partner, were recovered following specific proteolytic cleavage of the GST portion by thrombin protease. Purified Mce1E appeared as a single band of 38 kDa, whereas purified Mce1A tended to exist in degraded as well as aggregated forms of different sizes. The fusion proteins, free GST and monomeric Mce1A and Mce1E reacted in Western immunoblots with antibodies in pools of human sera from six to 11 tuberculosis patients. Similar analysis showed the presence of antibodies to GST and Mce1A, in pools of human sera from M. bovis BCG-vaccinated healthy subjects. When pure Mce1E was blotted against individual sera, antibodies in 4/10 sera from tuberculosis patients reacted, whereas no reaction was seen with 10 individual sera from M. bovis BCG-vaccinated healthy subjects. However, when the same sera were tested for reactivity to the purified preparation of Mce1A, 8/10 sera from both tuberculosis patients and M. bovis BCG-vaccinated healthy subjects showed positive reactivity. These findings demonstrate that both Mce1A and Mce1E are expressed and immunogenic during natural infection with M. tuberculosis.

BCG Vaccine↗